R 67 1 S m o o t h m u s c l e c e l l s in v i v o and in v i t r o d u r i n g atherogenesis. E. Betz P r o l i f e r a t i o n and m i g r a t i o n of s m o o t h m u s c l e c e l l s (SMC) in the i n t i m a of a r t e r i e s are i n t e g r a l proc e s s e s in a t h e r o g e n e s i s . D u r i n g the d e v e l o p m e n t of a t h e r o m a s , m e d i a l SMC a n d i n t i m a l L a n g h a n s - c e l l s c a n not be o b s e r v e d d i r e c t l y in r i v e in p a t i e n t s so that v a r i o u s t y p e s of e x p e r i m e n t s h a v e b e e n introd u c e d to p r o d u c e a t h e r o m a s or f i b r o m u s c u l a r p r o l i f e r a t e s w i t h i n such a s h o r t time t h a t all p h a s e s of SMC r e s p o n s e s f o l l o w i n g a t h e r o g e n i c s t i m u l i c a n be f o l l o w e d . For the p r o d u c t i o n of local a r t e r y s t e n o ses h a l l o n i n g of a r t e r i e s , l o c a l t r a n s m u r a l e l e c t r i cal s t i m u l a t i o n s of an a r t e r y w a l l or c o l l a r i n g art e r i e s are f r e q u e n t l y u s e d t e c h n i q u e s in a n i m a l e x p e r i m e n t s . W h e n a r t e r y w a l l s are e x p o s e d to a t h e r o g e n i c s t i m u l i SMC m i g r a t e f r o m the m e d i a into the i n t i m a and p r o l i f e r a t e there. M a n y of t h e s e SMC c h a n g e f r o m a " c o n t r a c t i l e " into a " m e t a b o l i c " s t a t e . T h i s p h e n o t y p i c a l m o d u l a t i o n is a c c o m p a n i e d w i t h an i n c r e a s e of e n e r g y c o n s u m p t i o n and a c h a n g e in the p r o t e i n e x p r e s s i o n of the c e l l s ( 2 - D - G e l elektrepheresis). The m i g r a t i o n of SMC in r i v e has b e e n s t u d i e d his t o l o g i c a l l y in s e r i a l e x p e r i m e n t s . A f t e r r e a c h i n g the intima, SMC o r i e n t a t e t h e m s e l v e s p a r a l l e l to the d i r e c t i o n of b l o o d flow. In the c o u r s e of a few w e e k s t h e y a r r a n g e t h e m s e l v e s in a s p i r a l s h a p e d f o r m a r o u n d the v e s s e l l u m e n and b e c o m e c a p a b l e of b u i l d i n g a n e w i n t e r n a l e l a s t i c l a m i n a b e l o w the e n d o t h e l i a l lining. In S M C - c u l t u r e s on f l e x i b l e , h y d r e p h J l i z e d s i l i c o n e m e m b r a n s some c y t o s k e l e t a l c o m p o n e n t s , d u r i n g SMC o r i e n t a t i o n , c h a n g e t h e i r d i r e c t i o n d e p e n d i n g on the d i r e c t i o n of m e c h a n i c a l stress. M i g r a t i o n and p r o l i f e r a t i o n of SM~ can be i n h i b i t e d by v a r i o u s s u b s t a n c e s (e.g. h e p a r i n o i d s , some c a l c i u m a n t a g o n i s t s ) . - D e t a i l s of the a c t i o n of a n t i - m i g r a t o r y a n d a n t i p r o l i f e r a t i v e s u b s t a n c e s can b e s t be s t u d i e d in c e l l e u l t u r e s . For such studies c u l t u r e s of c e l l s o b t a i n e d f r o m n o r m a l and a t h e r i o s c l e r o t i c h u m a n a r t e r i e s and v e i n s have b e e n established.ln addition,SMC obtained from secondary s t e n o s e s a f t e r a n g i o p l a s t y h a v e b e e n c u l t u r e d . SMC f r o m p r i m a r y and s e c o n d a r y s t e n o s e s e x h i b i t cons i d e r a b l e d i f f e r e n c e s in t h e i r m o r p h o l o g y and t h e i r g r o w t h c h a r a c t e r i s t i c s . T h i s l e a d s to the c o n c l u s i o n that r e s t e n o s e s a f t e r a n g i o p l a s t i c t r e a t m e n t are c a u s e d by a s u b p o p n l a t i o n of SMC and t h a t t h e s e c e i l s do not e x i s t in all a t h e r o m a t o u s p l a q u e s . G r o w t h c h a r a c t e r i s t i c s of c e l l s in v i t r o d e v i a t e c o n s i d e r a b l y f r o m the in r i v e s i t u a t i o n so t h a t coc u l t u r e s y s t e m s w e r e b u i l t u p t h a t r e s e m b l e the in v i v o c o n s t r u c t i o n of an a r t e r y wall. The i n t e r n a l and the e x t e r n a l e l a s t i c l a m i n a e w e r e i m i t a t e d by polycarbonate filters. Filters with pore diameters of m o r e t h a n 5 ~m a l l o w SMC, s e e d e d on the filter, or SMC f r o m m e d i a e x p l a n t s to m i g r a t e t h r o u g h the p o r e s a n d to f o r m a t r a n s f i l t e r - p r o l i f e r a t e w i t h all c h a r a c t e r i s t i c s of an in r i v e f i b r o m u s c u l a r p l a q u e . A d d i t i o n of o x i d i z e d l o w d e n s i t y l i p o p r o t e i n s and m o n o c y t e s to the c u l t u r e m e d i a c a u s e a l i p i d cont a i n i n g p r o l i f e r a t e w i t h f o a m cells. It is d e m o n s t r a ted that c o n f l u e n t e n d o t h e l i a l c e l l s s e e d e d on one side of a f i l t e r i n h i b i t t r a n s f i l t e r m i g r a t i o n of SMC. If, in a s a n d w i c h - l i k e a r r a n g e m e n t ~ a d v e n t i tial t i s s u e is p l a c e d on the o u t e r side of a filter w i t h p o r e s of 0,2 ~m d i a m e t e r no c e l l s c a n p a s s the f i l t e r . H o w e v e r , t h e a d v e n t i t i a e x e r t s an i n h i b i t o r y a c t i o n on SMC m i g r a t i o n and on the m o d u l a t i o n from c o n t r a c t i l e into m e t a b o l i c SMC. In o r g a n c u l t u res of a r t e r y w a l l s , m e c h a n i c a l l e s i o n s of the e n d o thelium cause similar proliferative intimal reponses in v i t r o as has be s e e n in v i v o a f t e r a n g i o plasty. P h y s i o l o g i s c h e s I n s t i t u t I, U n i v e r s i t i t G m e l i n s t r . 5, D - 7 4 0 0 Tfibingen

T~bingen,

2 INTERACTION OF G PROTEINS WITH RECEPTORS THE MODEL OF RHODOPSIN AND TRANSDUCIN K.P. Hofmann Rhodopsin (Rh) and transducin (GO are now recognized as archetypes of the receptor and G-protein (PUG) families. Signal transduction in an PUG system starts with the agonist-induced transformation of R into an active state (R*) and formation of a complex in which GDP/GTP exchange in the G heterotrimer is catalysed [reviewed in ref. 1]. There is a close relation of rhodopsin, with the light-sensitive agonist 11-cis retinal, to the ligandactivated receptors [2]. Retinal, like other agonists, is held in a hydrophobic pocket formed by seven transmembrane helices. The four loops on Rh's cytoplasmic surface are plausible candidates for the interaction with the peripherally bound Gt. They interconnect the transmembrane stretches and a membrane anchor on the C-terminus. To investigate structure/function relations in Rh-Gt interaction, we combined peptide competition and site-directed mutagenesis with flash photolysis techniques. Monitors for active Rh, the Rh-Gt complex and activated Gt are available. Synthetic peptides from three of the cytoplasmic R loops compete against the interaction with Gt [3]. Thus, sites on these loops must be part of the interaction domain and may correspond to three sites identified on G, [4]. It was shown that the initial R-G-GDP collisional coupling does not yet involve the full interaction domain. It first induces the release GDP from G, which, in turn, enables by induced fit the transition into a stable 3-loop interaction [5]. Only two of the three interactive loops of R have to bind with Gt to form a stable complex. However, activation is more demanding than binding: mutants that bind only via two loops (one loop altered) fail to release activated G~-GTP and probably cannot bind GTP [6]. The specific loop interactions may not be the only mode of binding between Rh and Gt. In the intact isolated retina, the rate of Gt activation by a single actice Rh on a membrane exceeds 3000/s/R* at 33~ [7]. This value is at the diffusion limit and reflects very efficient collisional coupling, which may involve another more unspecific "capture" type of interaction that remains to be investigated. Besides Gt, other proteins, namely, Rh kinase and arrestin, interact with MII. For arrestin, we have recently shown that it undergoes, like Gt, conformational rearrangement when interacting with Rh [8]. In general, this may be a necessary step to activate the protein for its physiological function. In this context, it is interesting to note that certain aspects of the visual process (bleaching desensitization) require a second activity of rhodopsin besides Gt activation [9]. 1. Cbabre & Deterre (1989) Eur. J. Biochem. 1793, 255-266 2. Zhukovsky, E.A., Robinson, P.R. & Oprian, D.D. (1991) Science 251, 558-560 3. K6nig, B., Arendt, A., Mc Dowell, J.H., Kahlert, M., Hargrave, P.A., & Hofmann,K.P. (1989) Prec. Natl. Acad. Sci. USA 86, 6878-6882 4. Hamm, H.E., Deretic, D., Arendt, A., Hargrave, P.A., K6nig, B., & Hofmann, K.P. (1988) Science 241, 832-835 5. Kahlert, M., K6nig, B., & Hofmann, K.P. (1990) J. Biol. Chem. 265, 18928-18932 6. Franke, R.R., K6nig, B., Sakmar, T.P., Khorana, H.G., & Hofmann, K.P. (1990) Science 250, 123-125 7. Kahlert, M., & Hofmann, K.P. (1991) Biophys. J. 59, 375-386 8. Palczewski, K., Pulverm~iller, A., Buczylko, J., & Hofmann, K.P. (1991) J. Biol. Chem., in press 9. Kahlert, M., Pepperberg, D.R., & Hofmann, K.P. (1990) Nature 345, 537-539 Institut ffir Biophysik und Strahlenbiologie der Universit~t Freiburg Albertstrage 23, D-7800 Freiburg

R 68 3 MICROVASCULAR NETWORKS: EXPERIMENTAL AND SIMULATION ANALYSIS OF STRUCTURE AS RELATED TO FUNCTION P. Gaehtgens Quantitative correlations between the results of microcirculatory research and macrohemodynamic measurements at the whole organ level have generally not been very satisfactory. As an example, substantial disagreement exists between the predictions of total blood flow from measurements of flow in single capillaries, both under resting conditions and during functional or reactive hyperemia. As a result, hypotheses like that of regulated capillary recruitment have repeatedly been revived, although experimental evidence has remained negative. In this unsatisfactory situation, the concept of microvascular networks has been developed. In this concept, networks are considered to represent the basic functional units in which adaptation of vascular supply to demand is achieved, both acutely, by coordinated regulation of local hydraulic conductivities, and chronically, by an adaptive growth process. The network concept is functionally supported by the accumulating findings which demonstrate the existence of mechanisms coordinating vasomotor responses within vascular provinces to physiological stimuli by way of cell/cell conduction or biochemical interaction along the vessel tree. Propagated changes of vasomotor tone and communication between vessel segments including true capillaries appear to lead to responses involving entire network units rather than individual vessels. Due to complex architecture and topology, hemodynamic properties of microvascular networks are not directly predictable from morphological or hemodynamic measurements at the capillary level: In addition to geometrical features of each constituting microvessel segment, the connectivity of the numerous arterio-venous flow pathways must be known to derive a quantitative estimate of flow dispersion within the network which is an important determinant of exchange efficiency. Complete data sets have so far only been obtained for planar networks which showed considerable geometrical and topological non-symmetry. As a result, both model simulations and experimental observations demonstrate substantial perfusion heterogeneity which varies with vasomotor tone and blood rheological properties. This heterogeneity has also been observed at the macro-scale, i.e. on the basis of integrating parameters of transvascular exchange relative to changes of blood flow. So far, it is not clear whether this heterogeneity is physiologically controlled thus serving as a variable efficiency factor. Since differences of network design between vascular provinces suggest a close relationship between architecture and physiological function, it is of interest to relate network structure to principles of angiogenesis and vessel growth. This is of considerable relevance in view of the long-term adaptations of terminal vascular systems to tissue function, as occurs e.g. in response to physical activity or with age. Mathematical simulations have been used to generate vascular networks by utilizing algorithms representing different growth principles. After development of appropriate quantitative parameters, the architectural properties of simulated networks can be compared with those existing in vivo. The results of comparisons performed so far indicate that neither random terminal nor random segment branching models yield entirely satisfying predictions. Preliminary conclusions suggest that the capacity to generate newly forming vessel segments is not randomly distributed within the network; furthermore, arterial and venous trees cannot be simulated by the same algorithms, thus suggesting distinct differences in growth processes. Institut f/Jr Physiologie der Freien Universit~t Berlin, Arnimallee 22, D-1000 Berlin 33

4 Analysis of endothelin production in various tissues and its biological significance. Masashi Yanagisawa, M.D., Ph.D. Department of Pharmacology, Kyoto University Facul.ty of Medicine, Kyoto 606, Japan The first member of the endothelm (ET) family, ET-1, was originally identified as a 21-amino-acid potent vasoconstrictor peptide produced by vascular endothelial cells in culture. Subsequent studies demonstrated three separate ET-related genes in the human and other mammalian genomes, which encode for the three distinct isopeptides, ET-1, ET-2 and ET-3. eDNA cloning of ET isopeptides in human revealed that they are expressed in multiple tissues with distinct distribution patterns: while only ET-1 is detected in the vascular endothelium, ET-2 and/or ET-3, in addition to ET-1, are expressed in other tissues such as the brain, lung and kidney. ETs are produced via the proteolytic cleavage of =40-residue inactive intermediate called 'big ETs', catalyzed by a membrane-bound neutral metalloprotease(s) called 'endothelin converting enzyme'. This enzyme is sensitive to the protease inhibitor phosphoramidon, and apparently distinct from any other metalloprotease known. Apart from their potent and long-lasting vasoconstrictor/pressur activities, ETs possess a wide spectrum of non-vascular actions in various tissues. Furthermore, the existence of at least two subtypes of ET receptors with different selectivities to the three isopeptides has been demonstrated. We have recently cloned two distinct subtypes of ET receptor; in vascular tissues for example, the smooth muscle cells express the 'ETA receptor' which shows the rank order of affinity ET-1 = ET-2 >~ET-3, whereas the endothelial cells express the 'ETB receptor' with the affinity rank order of ET-1 = ET-2 = ET-3, which may mediate the ET-induced EDRF release. These ET receptor mRNAs are expressed also in other ET-producing tissues such as those listed above, suggesting the importance of ET family peptides as locally acting mediators. In cultured endothelial cells, the production of ET-1 is augmented by various chemical and mechanical stimuli, including thrombin, TGF-~, IL-1 angiotensin II, vasopressin, and increased shear rate. In contrast, EDRF attenuates ET-1 release from isolated porcine aorta. Our on-going studies in isolated perfused rat mesenteric arteries have shown that both immunoreactive ET-1 and ET-1 mRNA is significantly up-regulated in response to a low concentration (10-1~M) of vasopressin. Although this vascular bed shows no acute vasoconstrictive response to this dose of vasopressin, an extremely slow vasoconstriction is observed 3-6 hours after the initiation of the vasopressin challenge. Furthermore, the induction of ET-1 mRNA and the slow constrictor response are both completely abolished by concomitant administration of the mRNA synthesis inhibitor actinomycin D. These results suggest the possibility that endogenous de novo production of ET-I may mediate the slow constrictor response of the mesenteric vascular bed to circulating vasopressin.

5 ACTIVATION AND DESENSITIZATION OF GLUTAMATE RECEPTOR CHANNELS AFTER SHORT AGONIST PULSES IN ISOLATED PATCHES FROM HIPPOCAMPAL NEURONES D. Colquhoun, P. Jonas, and B. Sakmann Outside-out patches were isolated from the soma of CA3 and CA1 pyramidal cells of rat hippocampal slices. Pulses of L-glutamate (L-gin) of different length were applied. The 20-80% risetime for a change in offset current on switching from Ringer solution to a solution of low ionic strength was lower than 200 Its. The decay of the current after short agonist pulses was significantly faster than decay in the maintained presence of agonist due to desensitization. With 1 ms pulses of 1 mM L-ghi, the current rose to a peak within tess than 500 Its and decayed with a time constants of 1.37-2.75 ms in CA1 cell patches and 1.67-3.50 ms in CA3 cell patches at -50 mV membrane potential. With 100 ms pulses of L-glu, agonist-activated currents desensitized with a time constant of 7 - 14 ms for both cell types. Dose response curves of the peak current activated by L-glu showed half-maximal responses for 342 ItM with CA3 and 424 ItM with CA1 cell patches and Hill coefficients of 1.2 and 1.3, respectively. As measured in double pulse experiments, desensitization occurred even after 1 ms pulses. The fraction of desensitized channels directly after a 1 ms pulse was 55 and 60 %, respectively, and the time constant of recovery from desensitization was about 50 ms for patches from both cell types. The current-voltage relation of the peak current evoked by 1 ms pulses of 1 mM L-glu was almost linear in the voltage range -100 to +60 mV for both cell types and the time constants of current decay showed no sign of voltage dependence. The results indicate that 1 ms pulses of 1 mM Lghi mimic the main features of the excitatory postsynaptic currents in pyramidal cells of rat hippocampus. They suggest that desensitization of postsynaptic ghitamate receptor channels may occur even during a single synaptic event. Max-Planck-Institut fiir medizinische Forschung, Abteilung Zellphysiologie, W-6900 Heidelberg, Germany.

R 69 6

8

I N C R E A S E D A N T I E P I L E P T I C EFFECTS OF V E R A P A M I L LOW M g + + - I N D U C E D I N T E R I C T A L FIELD POTENTIALS HIGH EX~RACELLULAR K+-CONCENTRATION M. Pohl . H. Straub and E.-J. SDeckmann

ON IN

OF O D O R S E N S I T I V E , BY

INTRACELLULAR

CYCLIC AMP GATED Ca2+: A M E C H A N I S M

ION FOR

R E S P O N S E DESENSITIZATION F, Zufall*, S, Firestein and G.M. Shepherd

Low Mg++-induced epileptic field potentials (EFP) in h i p p o c a m p a l s l i c e s are b l o c k e d by t h e organic calcium antagonist verapamil (Pohl et al., E u r o p . J . N e u r o s c i . , 1991). A c o m p a r i s o n of the a n t i e p i l e p t i c e f f e c t s of v e r a p a m i l in neurons with normal and decreased membrane potentials w a s the aim of the p r e s e n t i n v e s t i g a t i o n s . Therefore, the experiments were carried out with normal and elevated K+-concentrations. Hippocampal slices ( 4 0 0 - 5 0 0 Bm; n = 3 6 ) f r o m guinea pigs were used. EFP were induced by omitting M g ++ f r o m a r t i f i c i a l cerebrospinal f l u i d (CSF). V e r a p a m i l (40 or 60 ~ m o l / l ) w a s added to CSF with normal (4 mmol/1) or elevated (8 mmol/1) K+-concentrations. Elevated K+-concentrations r e s u l t e d in (i) a s h o r t e n i n g of the l a t e n c y of E F P a b o l i t i o n and (ii) a prolongation of the depressive effects of v e r a p a m i l f o l l o w i n g its w i t h d r a w a l . The l a t t e r effect did not occur with normal K+-concentrati on of CSF during washing out period. The results show that the antiepileptic efficacy of t h e o r g a n i c c a l c i u m a n t a g o n i s t verapamil d e p e n d s on e x t r a c e l l u l a r K + - c o n c e n t r a t i o n s . It is assumed that changes of the m e m b r a n e potential are directly or indirectly involved. * Supported by Humboldt

INHIBITION CHANNELS

In response to odors vertebrate olfactory receptor neurons produce an inward ionic current which is the result of a G-protein coupled second messenger system utilizing cAMP to directly activate ion channels (Firestein, Zufall & Shepherd (1991) J. NeuroscL. in press; Zufall, Firastsin & Shepherd (1991) J. Neurosci., in press). Mechanisms underlying the termination of the odor response are less clear. During continuous presentation of the stimulus the odor current is transient with a decay time constant of 3s. The current remains transient even in the continous presence of cAMP indicating that the channel itself is an important site for response desensitization. However, in excised patches with low divalent solutions, the cAMP gated channel does not desensitize to saturating concentrations of cAMP ruling out the possibility that desensitization occurs at the cAMP receptor site. A clue to the possible source of desensitization comes from the result that removing Ca2+ from the bathing medium transforms the odor evoked current from transient to sustained. Consistent with this is the finding that intracellular Ca 2+ (Ca i) regulates the activity of the cAMP gated channel in inside-out patches. Raising Caifrom 100 nM to 3 pM decreases channel open probability from 0.6 to 0.09. This effect is reversible and does not require ATP. The decrease in mean open probability is correlated with an increase in channel closed time; neither open time nor channel amplitude are affected which is compatible with an alloste-

Foundation

tic action of Cai. Thus, the Ca2+ mediated regulatory function could ideally pro-

I n s t i t u t fur P h y s i o l o g i e d e r U n i v e r s i t ~ t M U n ster, Robert-Koch-Strafle 27a, W-4400 MUnster

vide a rapid negative feedback link for response desensitization. Section of Neurobiology, Yale University, 333 Cedar St., New Haven, CT 06510 and *Physiol. Inst. TUM, Biedersteinerstr. 29, D-8000 MiJnchen 40

9

7 REGULATION

OF FAST INACTIVATION

OF CLONED MAMMALIAN

Ik(A ) CHANNELS. J.Peter Ruppersberg Modulation

of

neuronal

of K + ~channels term

memory

molecular We

report

mediated proteins

but

has

here from

formation

and

plays yet

by

regulation

a role

been

in short-

studied

the

fast-inactivating composed RCK

Xenopus

at

a

and

of

Raw

protein

that

the

presence

of

families,

are

form spontaneously forms the

disulfide-bridges inactivation.

the

inactivation

of regulation

and might

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of

end of

gate.

glutathion

enable

by The

in absence

the N-terminal

reducing

break

when

regulated

disulfide-bridges.

agent and immobilize

the

K + currents

of K + channel-forming

oocytes,

break

disulfide-bridges

type

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by channels in

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excitability

potentially

level.

expressed

reducing

and M. Koenen

In the

fast

channel

play a role

in vivo

to link metabolism to excitability and to regulate excitability in specific membrane areas. Max-Planck-Institut

ffir medizinische

Abteilung Zellphysiologie, Heidelberg, FRG

Jahnstr.

Forschung, 29,

E F F E C T S O F L E U K O T R I E N E B4 O N T H E O U T W A R D C U R RENT OF CULTURED NEURONES AND ASTROCYTES M.Pekel, H.KSller, M.Siebler Inflammatory processes of the CNS, e.g. viral infection often led to reversible neurological defieites. Leukocytes and astrocytes are considered to participate in the immunological process by secreting immune mediators such as leukotrienes (1). In the present study we were interested on the effects of leukotrienes on membrane properties of neurons and astrocytes. Therefore we have investigated cultured neurones from embryonic rat neocortex (El5) in serum free medium (2). Astrocytes were prepared from new born rat neocortex (P1). By means of the patch clamp technique we performed whole cell recordings from cells after 6-12 DIV. In the voltage clamp mode we could evoke 4-AP sensitive and a TEA sensitive outward current (IA,IK). When adding Leukotriene B4 (LTB4) to the bath solution the resting membrane potential of the neurones decreased slowly after about 10 minutes. The membrane resistance was nearly constant or increased slightly. Application of LTB4 led to a decrease of the slow activating outward current in neurones and astrocytes, respectively, which could be partially washed out. The blocking effect of LTB4 on the outward current was dose dependent (100 to 400 nmol). Action potentials and sodium currents were not remarkably influenced by LTB4. We conclude from these results that LTB4 influence the membrane properties of neurones as well as of astrocytes which could lead to a neuronal dysfunction during inflammatory processes within the CNS. Departement of Neurology, University of Dfisseldorf Moorenstr.5, D-4000 Dfisseldorf 1, FRG Supported by SFB 194 (SI B7)

W-6900

1) Hartung HP, Toyka K (1987) Brain Res. 435:367-370 2) M/iller HW,Seifert W (1982) J.Neurosci.Res. 8:195-204

R 70 10 CHANGES IN EEG PATTERN AND NEURONES DURING THERMAL ANAESTHETIZED GUINEA-PIGS

12 R E S P O N S I V E N E S S OF RAPHE SKIN STIMULATION IN

P. Hinckel, M. R~sinq and K. Schr6der-Rosenstock Neurones in the nucleus raphe magnus (NRM), a circumscribed area in the lower brain-stem, have been shown to belong to the thermoafferent system. The response of 58 single units in the ~ to changes in abdominal, thoracic and leg skin temperature was recorded in 30 anaesthetized male gulnea-plgs (l,41g urethane per kg body weight i.p.). Additionally the EEG and the neck muscle electromyographic activity was monitored. Sixteen units were thermoresponsive (i0 warm, 2 cold, 4 atypical), 29 units were non-thermoresponsive and 13 units were questionable. The recorded units were not influenced by colonic core temperature, pain or other mechanical stimuli. Two stable cortical EEG states (activated and basic) and one transition EEG state could be defined. The EEG pattern changes correlated with the different temperature profiles of the animal, but the thermoresponslve neurones started to elevate firing rate without EEG pattern changes. All complete thermal responses were observed within an activated EEG state. It is concluded from these experiments that the EEG changes induced by thermal stimulation of the receptive skin fields in urethane anaesthesia are mediated by afferent pathways ascending in parallel to the thermal pathways branching off to the NRM cells. The data are in accordance with previous observations by Dickenson (J. Physiol. 273, 277293, 1977) showing unchanged thermal NRM responses in decerebrated rats. The results, however, don't support conclusions by Grahn et al. (Am. J. Physiol. 256, R840-R849, 1989). These authors reported, that no responses of lower brain-stem neurones were observed to changes in ambient temperature within an arousal state. Physiologisches Institut, Universit~t Giessen, Aulweg 129, D-6300 Giessen 1

VISUAL MOTION EVOKED POTENTIALS ARE LINEARLY CORRELATED W I T H THE VELOCITY OF RANDOM-DOT PATTERNS Th. P r o b s t A 40~ ~ large c o m p u t e r - g e n e r a t e d r a n d o m - d o t s t i m u l u s p a t t e r n was p o s i t i o n e d in t h e subject's (S's) left visual field with an e c c e n tricity of 4S ~ m e a s u r e d f r o m the c e n t e r of the display (paperwhite p h o s p h o r monitor}, its pixels (4.1S min o f arc) m o v e d horizontally back and f o r t h with c o n s t a n t a n g u l a r velocities o f 4 . 7 4 ~ 2.37~ o r t.19~ respectively. The interval b e t w e e n t h e o f f s e t o f one and the o n s e t of t h e n e x t m o v e m e n t period was 686ms. 21 healthy v o l u n t e e r s with n o r m a l or c o r r e c t e d n o r m a l vision (cont a c t lenses, no glasses} participated. They fixated a g r e e n l i g h t e m i t t i n g diode (LED} 20 min o f arc in d i a m e t e r t h r o u g h o u t the e x p e r i m e n t . T w o b r e a k s for a period of in each case tSs within t h e a v e r a g i n g p r o c e d u r e a s s i s t e d in t h e Ss' ability to a d e q u a t e l y fixate this t a r g e t . Mental a r i t h m e t i c s kept t h e i r vigilance on a high level. Motion e v o k e d biosignals w e r e picked up c o n t r a l a t e r a l l y b e t w e e n e l e c t r o d e positions 4 and 5 o f the Queens Square s t a n d a r d , i.e. Scm above the e x t e r n a l occipital p r o t u b e r a n c e and 7.Scm to t h e right o c c i p i t o - t e m p o r a l region r e f e r r e d to F z with A 2 c o n n e c t e d to g r o u n d (international 10-20-system}. 100 a v e r a g e s / t r i a l w e r e b a n d p a s s e d t h r o u g h O.S3Hz-3OHz. Every trial was r e p e a t e d once. The r e s u l t s were as follows: 1.) C o m p o n e n t latencies of t h e N 1 - P 2 - N 2 - c o m p l e x d e c r e a s e d linearly with increasing p a t t e r n velocity (p < 0.001, r = 0.91). 2.) Mean slope o f the l a t e n c y - v e l o c i t y - f u n c t i o n s was -11 m S / o / ~ . 3.) P e a k - t o - p e a k a m p l i t u d e N1/P 2 and P,,/N 2 increased linearly with increasing p a t t e r n velocity (p < O.00t, r~= 0.99), 4.) Mean slope o f t h e a m p l i t u d e - v e l o c i t y - f u n c t i o n s was 0.SS ~V/o/s. In conclusion, r a n d o m - d o t m o t i o n stimuli provide a good tool for s t i m u l a t i n g m o t i o n sensitive cells o f t h e visual s y s t e m w i t h o u t the d i s a d v a n t a g e o u s side e f f e c t of" c o n c u r r e n t s t i m u l a t i o n with luminance c o n t r a s t , [ n s t i t u t fiir Physiologische Psychologie, Lehrstuhl II, H e i n r i c h - H e i n e [Iniversit~it DiJssetdorf, H n i v e r s i t a t s s t r a B e 1, D - 4 0 0 0 Diisseldorf 1

11

13

SYNCHRONIZATION OF OSCILLATORY RESPONSES BETWEEN DIFFERENT ~REAS OF CAT VISUAL CORTEX

POSTURAL RESPONSES IN THE CAT AFTER APPLICATION BOTULINUM TOXIN TYPE A INTO THE TRICEPS MUSCLES. F.P. Kolb, G. Arnold, W.H. Fischer

A. K. Krciter, A. K. Engel~ P. K6nig and W. Singer Previously we demonstrated that oscillatory neuronal responses can synchronize across orientation columns in urea 17 of cat visual cortex. It was proposed that this synchronization may provide a mechanism for the formation of cell assemblies in visual cortex which encode objects. This hypothesis predicts that 1) simila~ oscillations should occur in othes visual areas and 2) there should be evidence for intcrareal synchronization. To test these predictions, we made simultaneous recordings of malti-unlt activity (MUA) from area 17 and PMLS in four adult cats. The oscillatory nature of the response and the strength of synchronization were quantified by computation of auto- and cross-correlation functions and subsequent fitting of damped sine wave functions to the correlograms. We obtained the following results: 1) 76% of the PMLS recordings (n=25) displayed an oscillatory modulation in a frequency range of 40-80Hs. 2) In 24 out of 46 eases tested, we observed a synchronization of oscillatory responses between area 17 and PMLS. This interareal synchsonisation wa% on average, weaker than that observed within area 17. 3) Response synchronization was observed more frequently and was stronger if ceils had overlapping receptive llelds. 4) No significant dependence of response syncl~onisation on the difference of orientation preferences has been found. Phase-locking was observed even in pairs whith orientation preferences differing by more than 45 degrees. 5) In two cases of MUA recordings with nonovedapping receptive fields, we obtained evidence that the interareal synchronization is sensitive to global stimulus features. If concurrently stimulated with a single long light bar, the cells were strongly synchronized. If a gap was inserted into the stimulus~ the inter-areal synchronization became weaker~ and it disappeared almost completely when the two stimulus f~agments moved into opposit direction. Our results are consistent with the hypothesis that oscillatory responses in a frequency range of 40-80Hz may be a general phenomenon in cat visual cortex.The data indicate that synchronization of oscillatory responses can occur between different areas of visual cortex. Similar to the intra-arcal interactions, the intcr-ascal synchronization seems to be sensitive to global features of the stimulus such as spatial continuity and coherence of motion. This mechanism may serve to encode relationships between ditfetent features of an object wich are represented in separate visual areas. Max-Plank-Institut flu Hirnforschung,Deutschordenstr. 46, W-6000 Frankfurt/Maln

OF

Posture is a multi level controlled event. The participation of the different motor areas is, however, still under discussion. A circumscript lesion in motor centers often results in a temporary disorder of postural adjustments. Using classically conditioned movements the postural adjustments have been analyzed before and after administration of Botulinum toxin. Cooperative cats were trained to stand still for a resting period on a 2x2 array of platforms (one paw on each platform). Following an auditory stimulus the animal had to perform a single step to a further frontally situated platform. The animal was rewarded by food if the step parameters fulfilled predefined criteria. The static center of gravity was calculated for the resting phase and for the time at which 20% of its bodyweight had been shifted to the fifth platform. The trajectory of the center of gravity was also constructed for the dynamic phase of the movement. After some weeks of training the cats were able to perform the conditioned step reliably. Although the dynamic characteristics varied considerably between cats, significant changes in the movement parameters could be observed as a result of training: the time between the onset of the tone and lifting the paw (reaction time) decreased to approximately 200ms; the time between lifting the paw and the time taken to transfer the bodyweight (step time) was reduced by about 30%; shortening of step time could also be deduced from an observed shortening of the length of the trajectory trace and by an increase in the step velocity. Botulinum toxin type A (15rig) was injected into the triceps muscles of the contralateral, i.e. the nonmoving, leg to elicit transient muscular weakness by blocking the transmitter release from peripheral cholinergic nerve endings. The necessary dose was comparable to that used clinically (e.g. 10- 15ng for the sternocleidomastoid muscle), and did not prevent the animal from performing the conditioned step. At the present stage of research only qualitative results can be described; however all the above mentioned characteristic parameters were changed. Institute of Physiology, University of Munich, Pettenkoferstr. 12, D-8000 MQnchen 2. This study is supported by the Deutsche Forschungsgemeinschaft (SFB 220, D9).

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16

CENTRAL NEURONAL H Y P E R E X C I T A B I L I T Y IN MIGRAINE DESCRIBED BY E E G - B R A I N - M A P P I N G R. Schellenberg, A. Schwarz, M. Thom, M. Schindler

EFFECTS OF THE EPILEPTOGENIC DRUG PENTYLENETETRAZOL ON RCKI-POTASSIUM CHANNELS (OOCYTE, XENOPUS

A new hypothesis describes, that patients with migraine plus aura experience a state of central neuronal h y p e r e x c i t a b i l i t y that predisposes them to develope spontaneous d e p o l a r i z a t i o n followed by suppression of neuronal function. Cerebral vasoc o n s t r i c t i o n may occur during a migraine attack. Vasoconstriction induced by serotonin can be effectively blocked by pretreatment with Mg ++. E E G - b r a i n - m a p p i n g was used to describe cerebrovascular resistance dependent changings of blood flow and m e t a b o l i s m and therefore the functional state of the brain. In migraine patients with aura a 16-channel quantitative EEG-analysis was performed using Fast Fourier T r a n s f o r m a t i o n and map computation. The patients have been divided in left (n=5) and right sided (n=8) pulsatory pain location. Recordings have been done during the acute attack and 1 hour after acute ergotamine medication (eyes closed). Results: In both groups there was a treatment independent significant beta-power increase at occipital and central regions. In the patients with left sided pain theta- and alpha-power was significantly less than in controls mostly at left temporal and both-sided occipital r e g i o n s . If left-sided h y p e r r e a c t i v i t y simultaneously influences left brain functions like abstract and rational thinking for example should be explored. In contrast right-sided pain is related with significant theta-power increase. This could be in correlation with blood flow and metabolic decrease and therefore possibly decreased intuitive and subjective behaviour.

M. Mad~a, M. Stocker, U. Mu~hoff, O. PongsandE.-J. Speckmann

In order to further analyse the functional significance of potassium cur rents in epileptogenesis, the effects of the epileptogenic agent pentylenetetrazol (PTZ) on a cloned voltage-dependent potassium channel was investigated. RCK1 potassium channels were expressed in oocytes of Xenopus laevis after injection of in-vitro transcribed RNA (0.3 - 1.0 ng per oocyte) from RCK1 cDNA sequence (Stiihmer et al., EMBO J., 8, 3235, 1989). Membrane currents were studied using the two-electrode voltage-clamp technique. Holding potential: -80 inV. Command potentials (Vc)" -70 to + 6 0 mV. Duration of Vc: 2 s. PTZ was administered in concentrations of 0.1 to 100 mmol/1 for up to 300 s. Under control conditions depolarizing V c elicited slowly inactivating outward currents (n=24). The amplitudes of the outward currents ranged between 0.4 and 1.6 IzA (Vc= -50 mV), 2.4 and 6.0/zA (Vc= -30 mV), and 8.0 and 11.5/zA (Vc= -10 mV). With application of PTZ the peak amplitudes of the outward currents were increased with V c between -50 and -40 mV and were decreased with V c more positive than -30 inV. The effects depended on the concentration of PTZ; threshold concentration was ca. 1 retool/1. The inactivation of the potassium current was enhanced in PTZ concentrations above 50 mmol/1. All effects of PTZ were reversible. The PTZ-induced change of the activation characteristics of the potassium currents counteracts depolarizations in the negative potential range and facilitates depolarizations in the more positive potential range. This represents in principle a gate function of the membrane.

Abteilung Pathophysiologie, Medizinische Akademie Magdeburg, Leipziger Str. 44, 0-3090 Magdeburg

Institut for Ph,/siologie, Universit~t M0nstor, Robert-Koch-Str. 27a, 4400 MOnster

15

17

LAEVlS}

FAST INACTIVATING POTASSIUM CHANNELS R E O P E N DURING R E C O V E R Y FROM INACTIVATION

REDUCTION OF GLUTAMATE A N D GABA REACTIONS BY THE EPILEPTOGENIC AGENT PENTYLENETETRAZOL (OOCYTES OF

J.Peter Ruppersberg

XENOPUS LAEVIS; INJECTION OF RAT BRAIN RNA) P. Bloms, U. Mul3hoff, M. Madeja, F. Spener, and E.-J. Speckmann

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in

channels

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fast-inactivating K +

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Max-Planck-Institut Abteilung

fur

medizinische

Zellphysiologie,

Heidelberg, FRG

Jahnstr.

Forschung, 29,

The effects of the epileptogenic agent pentylenetetrazol (PTZ) on reactions to the excitatory neurotransmitter glutamate (Glu) and to the i n h i b i t o r y n e u r o t r a n s m i t t e r GABA w e r e e x a m i n e d in oocytes of Xenopus laevis. For the expression of transmitter receptors, RNA was isolated from rat brain (guanidine/lithium chloride method after Cathala et al., DNA 2, 1983) and injected into oocytes (75 ng per cell). The s u b s t a n c e s (PTZ: 5 - 1 0 0 mmol/I; Glu: 2 0 0 p m o l / I ; k a i n a t e : 50 p m o i / I ; q u i s q u a l a t e : 50 # m o l / I ; GABA: 2 0 0 p m o l / I ) w e r e administered systemically. Membrane currents were recorded using a conventional voltage clamp technique (holding potential: -50 mV). (i) The Glu reaction was decreased and e v e n t u a l l y abolished with simultaneous applications of PTZ in increasing concentrations. A reduction d o w n to 60 and 0 % o f initial value was found with 5 and 100 mmol/I PTZ, respectively. (ii) Analogous interactions between PT7 and the agonists of Glu-subreceptors kainate and quisqualate were observed. (iii) The GABA reaction was reduced d o w n to 60 and 15 % of initial value with 5 and 100 mmol/I PTZ, respectively. The investigations show that the reactions to both the excitatory transmitter Glu and the inhibitory transmitter GABA are markedly reduced by the epileptogenic agent PTZ.

W-6900

Institut for Physiologie der Universit~it MOnster, Robert-Koch-Str. 27a, W4400 MOnster

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2O

C O N T R O L L E D SUPERFUSION OF T H E RAT SPINAL CORD IN VIVO: E F F E C T S OF N E U R O P E P T I D E S OR KAINIC ACID ON IMPULSE CONDUCTION, POSTSYNAPTIC P O T E N T I A L S AND P R O T O - O N C O G E N E EXPRESSION H. BECK, B. STELZER AND J. SANDK/JHLER We investigated the effect of spinally administered neuroactive substances on the impulse conduction in dorsal column fibers, the negative deflection of the cord dorsum potential (N-wave) which is the s u m m a tion of postsynaptic potentials, the activity of spinal dorsal horn neurons and the expression of the proto-oncogene c-los. Experiments were performed on deeply pentobarbital anesthetized rats. After lamlnectomy (L2-$2) a pool was formed on the lumbar spinal cord dorsum by means of a specially prepared silicon rubber. The pool was filled with approximately 5 #1 artifical cerebrospinal fluid (CSF), kainic acid (KA), substance P (SP) or somatostatin (SOM). KA (5 raM) reduced the N-wave amplitude to 14.1=t=9.1% of control but did not impair impulse conduction in dorsal column fibers. KA (0.3 pM) had no effect on the N-wave amplitude but increased the firing rate of dorsal horn neurons and induced the expression of c-los in neurons of laminae I to V as revealed by conventional immuncytochemical detection of FOS, the c-los protein product (perfusion of the animals one hour after superfusion). SP (1 pM) also increased the firing rate of nocieeptive dorsal horn neurons within 1 min after onset of supeffnsion and induced c-los expression as did" natural noxious skin stimulation. In contrast, SOM (10 pM) failed to induce e-los in the spinal cord. These results further challenge the role of somatostatin as an excitatory neurotransmitter of primary afferent nocieeptors (Sandkiihler et al., Neuroscienee 34(1990) 565-567). Depending on the concentration kainic acid m a y be used to selectively excite or block spinal cord neurons. Thus, the controlled superfusion of the rat cord dorsum provides a useful tool to study dose-dependent effects of intrathecally applied drugs and to evaluate neurotransmitter candidates at the single cell level. II. Physiologisches Institut, Univ. Heidelberg, Im Neuenheimer Feld 326, W-6900 Heidelberg. Supported by the DFG (SA 435).

SOMATOSTATIN-LIKE IMMUNOREACTIVITY IN REGIONS OF VISCERAL SENSORY PATHWAYS OF THE CAT D. Harder, C. Vahle-Hinz and K.-D. Kniffki Somatostatin (SOM) is a widely distributed neuropeptide which is thought to play a role in sensory processes. We investigated the distribution of SOM-like immunoreactivity (SOM-LI} in the brainstem and thalamus of the cat with special attention directed to regions of visceral sensory and somatic nociceptive pathways. Deeply anesthetized adult and juvenile cats were perfused transcardially with 4% paraformaldehyde and 0.2% picric acid. Serial frontal sections were reacted with an antibody against SOM (Incstar, 1:1500) using the peroxidase-antiperoxidase (PAP) method (2nd antibody: goat-anti-rabbit, Dakopatts; 3rd antibody: PAP complex, Dakopatts). In a caudo-rostral sequence of sections dense aggregations of immunopositive fibers, some with terminal-like endings, were observed in the marginal laminae of the dorsal horn and the caudal spinal trigeminal n., in the n. of the solitary tract and the parabrachial region including the n. cuneiformis. These nuclei are part of main visceral and somatic nociceptive pathways. Relatively dense immunostaining was seen in addition in other parts of the brainstem, such as the n. raphe, n. praepositus hypoglossi, n. locus coeruleus and the periaqueductal gray. At the thalamic level moderate immunoreactivity was only present in the central medial n., a part of the intralaminar nuclei. Single SOM-LI fibers were found near the midline, in the mediodorsal n., in the external medullary lamina ventral to the ventrobasal complex and in the zona incerta. The pattern of SOM-LI fibers found in the present material conforms with the presumed role of SOM in ascending visceral sensory pathways and appears to include those involved in somatic nociception as well. In viscero- and nociceptive regions of the lateral thalamus no SOM immunoreactivity was found. Whether this is due to a low concentration evading detection by immunohistochemical techniques or an actual absence of SOM in these regions has yet to be determined. Physiol. Institut Univ. W5rzburg, R6ntgenring 9, W-8700 WLirzburg

19

SIGNS OF PLASTICITY OF THE FUNCTIONAL NEURONAL N E T W O R K IN THE RAT SPINAL DORSAL HORN A. EBLEN-ZAJJUR AND J. SANDK/JHLER Lesions of the skin, e.g. by inflammation, have been shown to induce changes of the functional characteristics of single spinal dorsal horn neurons. Virtually nothing is known about the impact of peripheral lesions on the functional neuronal connectivity among dorsal horn cells. To address this question recordings were made from multiple neurons via a single electrode in the dorsal horn of the lumbar spinal cord of pentobarbital anesthetized rats. Single neuron discharges were identified using a template matching algorithm (Forster et al. J. Neurosci. Method. 31(1990)109) or by bidimentional cluster cutting. Possible functional connectivity among neurons was evaluated by cross-correlation (CC) and by joint impulse configuration scatter diagram (JICSD) (bin width 1 ms) of the neuronal discharges after stationarity test. Background activity was analysed during three periods of 10 min each at 20 min intervals. W i t h the skin intact the CCs and the JICSDs were stable and the CCs of 20/62 (32.3%) pairs of neurons had single broad peaks with width of more than 10 ms centered at zero delays. The CCs at only 2/62 (3.2%) pairs of neurons had bilateral sharp peaks (width < 4 ms and symmetrical delays _< 7 ms). With the glabrous skin at the ipsilateral hindpaw inflamed by radiant heat (46~ for 100s given three times at intervals of 20 rain, which caused the formation of a blister), CCs with bilateral peaks were found for 9/18 (50%) pairs of neurons, (width from 2 to 106 ms with symmetrical or asymmetrical delays of 2 to 80 ms). These results suggest that under normal conditions there is a predomin a n t common input via interneurons to neurons recorded at the same site in the spinal dorsal horn which is consistent with a parallel cascade processing model. With the skin inflamed signs of reverberating neuronal circuits were found. II. Physiologisches Institut, Univ. Heidelberg, Im Neuenheimer Feld 326, W-6900 Heidelberg. Supported by the DFG (SA 435) and DAAD.

21 HISTAMINE ENHANCES POPULATION SPIKE AMPLITUDE IN HIPPOCAMPAL SLICES OF WARM-ACCLIMATED AND OF HIBERNATING TURKISH HAMSTERS F.Gh. Nikmanesh, 11. Spangenberger and P. [gelmund Hypothalamic histaminergic neurons are discussed to be involved in thermoregulation. As the hippocampus is innervated by histamine-containing neurons

originating in the posterior hypothalamus, histamiasrgic modulation of hippocampal activity could also be related to temperature regulation and to hibernation. In this context, we investigated the effects of histamine on stimulus-induced field potentials in hippocampal slices (400 pro) prepared from warm-acclimated and from hibernating turkish hamsters as well as from rats. Slices were studied in an interface type chamber perfused with artificial cerebro-spinal fluid oxygenated with 95% 02/5% CO2. Each slice was tested at 37~ and 230C. Histamine (50 #M) was bath-applied for 15 rain. Field potentials evoked by submaximal stimulation of Schaffer collaterals/cnmmissural fibers at 30 s intervals were recorded in stratum pyramidale of area CA1. For test of statistical significance with Student's t-test, the amplitudes of six consecutive spikes were averaged during control, histamine application, and washout, respectively. In all experiments on hamster slices, the population spike amplitude was

significantly (p<0.05) increased by histamine application. In slices from warmacclimated hamsters, amplitude enhancement was 48%+31% (n=10) at 37~ and 23%+_8% (n=8) at 23~ In slices from hibernating hamsters, amplitudes were augmented by 123%+_39% (n=7) at 37~ and by 39%+_20% (n=ll) at 230C. Differences between hibernating and warm-acclimated hamsters just failed to reach statistical significance (p>0.05). The consistent amplitude increase in hamster slices is in contrast to our experiments on rat hippocampal slices, which only in 3 of 18 trials showed significant effects of histamine (2x enhancement, 1x depression). Inconsistent results with rat slices are also reported by others (c.f. Springfield and Geller, Cell. Mol. Neurobiol. 8:431:445,1988). As antagonistic effects of histamine on neuronal activity are mediated by two different receptor types, our data suggest - in contrast to the rat hippocampus a clear dominancy of the excitatory H 1 receptor in the CA1 region of turkish hamster hippocampus. The density or sensitivity of these histamine receptors might be modified with hibernation acclimation. Institut /fir Neurophysiologie der Universitdt zu Khln, Robert-Koch-Str. 39, D-5000 Khln 41, FRG Supported by the Deutsche Forsehungsgemeinschaft(Igl0/1-1)

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22 SYNCHRONEOUS ACTIVITY AND OSCILLATIONS TUBED HIPPOCAMPAL NETWORKS H.KSller, M.Siebler

IN CUL-

In p r i m a r y cultures of h i p p o c a m p a l neurones from embryonic rats 1 (E 18) spontaneous postsynaptic potentials and action potentials appeared after 4-6 day in vitro. Beside a r a n d o m distributed activity spontaneous depolarizations with durations up to 6 seconds could be recorded simultaneously in two neurones by means of the patch clamp technique. These repetitive synchroneous activities are supposed to be generated by synaptic events because (a) their appearance and durations are both independent of the m e m b r a n e potential, (b) the amplitudes of the underlying currents depend monotonically on the membrane potential and (c) they are reversed at the reversal potential of the excitatory postsynaptic potentials. Inhibition of the g l u t a m a t e r g i c synaptic transmission by kynurenic acid and the NMDA antagonist A P V led to a m a r k e d shortening of the long lasting depolarizations, whereas the blockade of the G A B A - m e d i a t e d inhibition by picrotoxin had no effect. These results provide evidence t h a t the ability of h i p p o c a m p a l neurones to generate synchroneous large depolarizations neither depend on highly ordered anatomical structures nor is it a characteristic feature of isolated h i p p o c a m p a l neurones, but they are rather generated by the self-organized neural network. Departement of Neurology, University of Dfisseldorf Moorenstr.5, D-4000 Dfisseldorf 1, F R G Supported by SFB 194 (SI B7) 1) Mfiller HW,Seifert W (1982) J.Neurosci.Res. 8:195-204

OCULARITY SPECIFIC SYNCHItONIZATION OF OSCILLATORY RESPONSES IN VISUAL CORTEX OF STRABISMIC CATS P. KSulg, A. K. Engel, S. LSwel and W. Singer We have recently shown that neurons in cat visual cortex display oscillatory responses in the range of 40-60 Hz which can synchronize over distances of up to 7 m m within area 17. This synchronization may serve to establish relations between features in different parts of the visual field. We have now investigated oscillatory responses in area 17 of strabismic cats, i.e. in a cortical substrate which almost completely lacks binocular neurons. We wondered wheter in these a~imals response synchronization primarily occurs between cells of similar ocular dominance (OD). We recorded multi-unit activity (MUA) to appropriately oriented moving light bars simultaneously from 4-6 spatially separate sites in cortical area 17 with a spatial separation of up to 2 mm. We used 6 adult cats in which divergent strabismus had been surgically induced at the age of 3 weeks. To determine the temporal relationship of the firing patterns~ we computed autoand erosscorrelation functions of the spike trains. Analysis of 202 recording sites gave the following results: 1) 90% of the responses were monocular or strongly dominated by one eye. We found as many cells responding to the operated eye as to the normal eye. 2) The occurrence of oscillatory responses was with 61% as frequent as in normal cats (68%). Responses to the normal and the operated eye behaved similarly. 3) 77% of MUA recordings with similar OD showed a response synchronization versus 82% for overlapping receptive fields in normal cats. 4) However only 28% of cells with different 0D showed a response synchronization. Our data support the hypothesis that the functional organization of area 17 is profoundly altered in strabismic cats: The finding that MUA responses preferentially synchronize between neurons driven by the same eye suggest that cells with different OD become functionally independent. As synchronization between spatially distributed neurons depends on cortico-cortical connections our data suggest that strabismus induces not only changes in thalamo-cortical but also cortico-cortical connectivity. Max-Plank-Institut fSr Hirnforschung, Deutschordenstr. 46, W-6OOOFrankfurt/Maln

23

25

TWO FREQUENCY BANDS OF OSCILLATORY NEURONAL ACTIVITY IN KITTEN VISUAL COItTEX Thomas B. Schlllen, Peter K6nig, Andreas K. Engel, and Wolf Singer

INTERNEMISPHERIC SYNCHRONIZATION OF OSCILLATORY NEURONAL I~ESPONSES IN CAT VISUAL CORTEX A. K. Engel, P. K~nig, A. K. Kreiter and W. Singer

By reason of theoretical considerations it has been suggested that the temporal structure of neuronal responses might play a role in visual processing in the brain. Recent studies have demonstrated stimulus-dependent osei]latory activity in several areas of cat visual cortex. It seems reasonable to assume that this oscillatory structure is at least to some degree an emergent network property of cortical interactions. Since in the developing brain the netwock's characteristics differ in several aspects from the adult, we were interested in investigating the structure of neuronal responses in still immature cortex. In this abstract we describe oscillatory activity found in Area 17 of the visual cortex of 4-5 week old kitten. In contrast to responses in adult c~t, stimulus-dependent oscillatory activity in kitten is found to fall into two frequency bands: one in the range of about 5-15 Hz, the other approximately between 30-40 Hz. Both frequency contents are present in multinnit spike recordings, local field potentials and spike-trlggered averaged field potentials. We recorded through single electrodes spike responses showing a superposition of both frequencies. In addition, data recorded simultaneously through two electrodes 5 m m apart document that the observed oscillation frequency is not global to the entire area. Both modes of oscillation could be found with each repeated presentation of a stimulus. Furthermore, both frequencies were present for different time windows during stimulus presentatlon~ indicating that the particular frequency contents was not correlated with the stimulus position in the receptive field. The observed differences of stimulus-dependent oscillatory activity in the visual cortex of kitten and adult cat support the assumption that developmental factors modify the behaviour of the underlying neuronal oscillator. Different frequency bands in kitten could facilitate the self-organization of visual areas during development.

Neurones in area 17 of cat visual cortex display oscillatory responses that can synchronize across spatially separate co]um~ in a stimulus-specific way. This synchronization is sensitive to features of the visual stimulus, such as spatial continuity of contours, similarity of orientation and coherence of stimulus motion. It was proposed that this synchronization provides a physiological substrate for early visual processes of scene segmentation and perceptual grouping. In the present study we investigated whether responses synchrouizatlon can also be observed between ceils located in different hemispheres and whether the temporal synchrony is established via callosal connections. We made simultaneous recordings of multi-unit activity from 17 in the left and right hemisphere close to the representation of the vertical meridian. Three adult cats were used in these experiments. We computed autoand cross-correlation functions of all spike trains. Interhemispheric interactions were analyzed in 128 cases. In 70% we observed a synchronization of oscillatory responses across the two hemispheres. On the average, the synchronization occurred with 0 ms phase lag. In two additional cats we lesioned the corpus callosum. In both experiments, interhemispherlc synchronization was abolished, whereas temporal correlation within area 17 of either side appeared normal. The results of this study demonstrate that neuronal discharges can be temporally correlated between homologous cortical areas of the two hemispheres representing the two visual hemifields, and that this interhemispheric synchronization is mediated by callosal connections. These results have several implications. First, they demonstrate directly that r connections can establish synchrony. Therefore it is likely that the previously described synchronization within and between cortical areas is also mediated by cortico-eortlcal connections. Moreover~ the results show that reciprocal connections can establish temporal correlation with zero phae lag despite of finite conduction delays. Max-Plank-Institut ~z Hiznfozschung~Deutschotdenstt. 46, W-6000 Frankfurt/Main

Max-Plank-Institut fur Hirnforschung~ Deutschordenstrafle 46, 6000 Frankfurt, FRG

R 74 26 S I M U L T A N I O U S L Y SCALP R E C O R D E D MEASURE, AMPLITUDE AND LATENCY)

28 V I S U A L P300 (AREA OF EACH HEMISPHERE

EFFECT OF PERINATAL D E X A M E T H A S O N E TREATMENT CONDITIONED TASTE A V E R S I O N IN RATS H. Schwarzberg, N. R o t h and K. Hiller

ON

T a g h a v y A, H. Hamer A l t h o u g h the g e n e r a t o r s of visual P300 in brain are still unknown, it has been shown that they are of different magnitude in each hemisphere in r e s p e c t to amplitude N250/P300. A r e a m e a s u r e has not yet been a p p l i e d to i n t e r h e m i s p h e r i c difference of visual P300-complex. We, therefore, s t u d i e d visual P 3 0 0 - c o m p l e x in 23 v o l u n t e e r s (17 m, 6 f; ~ = 46,1 ~ 13,1 y) w i t h no c o g n i t i v e disorders by f l a s h i n g 2 d i f f e r e n t kinds of l,i/s r a n d o m l y p r e s e n t e d c h e c k e r b o a r d p a t t e r n s (A: 16x16 caskets; B: 64x64). While c o u n t i n g the rare B stimuli (A/B = 4/1) the p o t e n t i a l s were s i m u l t a n i o u l s y d e r i v e d from Ol and 02 to Fz; Cz was ground. RESULTS: (i) N O i n t e r h e m i s p h e r i c latency of N250 and P300 was found. (2) For the amplitude N250/P300 t h e r e was a m a r k e d i n t e r h e m i s p h e r i c difference (right h e m i s p h e r e (RH): ~ = ii,i ~ 4,1 ~V; left h e m i s p h e r e (LH): ~ = 9,4 • 3,8 ~V). (3) The area of P300 b e h a v e d s i m i l a r l y but more promin e n t l y (RH: 286 ~ 109 ~V ms; LH: 213 ~ 97 ~V ms). Student-t-Test: t = 4,38 for amplitude and t = 5,47 for area (sign. niv. p < 0,001 in both). The area m e a s u r e m e n t b e t w e e n N250 and N400 c o m p r i s i n g P300 p r o v e d to be a b e t t e r index of interhemispheric d i f f e r e n c e b e c a u s e of avoiding the ambiguity of c h o o s i n g the proper P300 p e a k and c o u l d be more a p p r o p r i a t e in u n c o v e r i n g of i n t e r h e m i s p h e r i c differences in cerebral p a t h o p h y s i o l o g i c a l conditions than amplitudes.

The aim of the present study was to examine further behavioural effects of early postnatal dexamethasone. Male rats of an inbred W i s t a r strain were used in the experiment. The s e v e n - d a y - o l d rats were injected s u b c u t a n e u o u s l y with 1 mg/kg dexamethasone. One h u n d r e d days after dexamethasone treatm e n t the adult rats were used in the behavioural experiment of single choice conditioned taste aversion; the method has been described in detail by Bures et al. (Brain and Behaviour - Paradigms for R e s e a r c h in Neural Mechanisms. Wiley, Chichester, 1988). A p p l i c a t i o n of lithium chloride 30 min after sweet water intake resulted in a condit i o n e d taste aversion d u r i n g the retention test c a r r i e d out 48 hours later. The single dexamethasone treatment on the 7th postnatal day led to a significant attenuation of conditioned taste aversion in adult animals. While controls drank only 6.8 • 0.5 ml within 30 min, the liquid consumption was i0.0 • 0.7 ml in dexamethasone pretreated rats (p<0.01). These findings may be explained by d e x a m e t h a s o n e effects upon brain development which cause imp a i r m e n t of memory functions or, alternatively, decreased responsiveness to aversive stimuli.

N e u r o l o g i s c h e K l i n i k mit P o l i k l i n i k der FriedrichAlexander Universit~t Erlangen-NGrnberg, Schwab a c h a n l a g e 6, 8520 E r l a n g e n

Institut fGr Physiologie der M e d i z i n i s c h e n Akademie M a g d e b u r g , Leipziger Str. 44, 0-3090 Magdeburg, and -Institut fGr N e u r o b i o l o g i e und Hirnforschung, Brenneckestr. 6, 0-3090 Magdeburg, Germany.

27

29

WORD FREQUENCY AND AUDITORY PRIMING AFFECT ERP AND TASK PERFORMANCE DURING WORD RECOGNITION N. Roth 1, A. Heine 1, Th. Kbnig2, M. Knye 1 The effects of prime-target semantic distance and word frequency upon retrieval from long-term memory (LTM) were studied, using a task with repeated ultra-short stimuli (concrete nouns). Nine healthy volunteers participated in the experiment. In one task they had to decide whether a tachistoscepically projected word was identical with a prime word given by the experimenter, in the second task, word frequency was considered. Exposure times were individually adjusted to fit the following criterion: Recognition and match/mismatch jugdement should occur after 5 to 12 repetitions (ISI: 2.0 s) of the same word. Cumulative exposure times (CET) were calculated for each trial. After completion of the experiment, Ss had to rate the subjective word frequency as well as the semantic distance between the pairs of priming and target words used. Mean CETs and ERPs were then calculated with respect to (1) YES response = p r i m e and target identical, (2) NO(ld) response = !ong or (3) NO(sd) = short semantic distance between prime and target, and to rated word frequency, resp. CET were longer for NO(ld) than for YES and NO(sd) trials, indicating a semantic distance effect upon LTM scanning. Frequent words were identified significantly faster than rare ones. Prominent components of the ERP were N1/P2, P300, and LPC. Whereas the LPC varied between trials, i.e. successive presentations of each stimulus, P2 was significantly different between anterior and posterior locations. This component also discriminated between YES and NO(sd) and NO(ld) conditions. This adds support to the assumption that word processing may occur at early, preconsious stages, and that .both . primin . . and word frequency affect retrieval from LTM and sUmulus ldentli~icatlon. lInstitut ftir Neurobiologie und Hirnforschung, Brenneekestr. 6, OMagdeburg 5090 EidgenOssische Technische Hochschule, CH-8092 Ziirich

FLUPHENAEINE L O N G - T I M E - T H E R A P Y IN SCHIZOPHRENICS OPTIMIZED BY EEG-BRAIN M A P P I N G Angela Schwarz, R. Schellenberg, W. Knorr, M. Schindler Neuroleptic long-time-therapy with Lyogen R in chronic schizophrenics could be more effective to have an objective parameter to estimate an optimal time delay between the injections. The functional state of the brain is characterized by several e l e c t r o e n c e p h a l o g r a p h i c parameters. So the EEG turned out to be a practically useful tool for studying the functional states of the brain and also for diagnosing functional brain disturbances. EEG-mapping methods include more aspects than an efficient analysis of a stochastic fluctuating potential by statistical algorithms (time- and/or frequency domain). The topographical aspects are taken into consideration using EEG-mapping where the d i s t r i b u t i o n of statistical parameters of the EEG is represented by a coloured map. Patients: DSM-III diagnosed chronic schizophrenics of the p a r a n o i d - h a l l u c i n a t o r y type. At the beginning of the mapping study they have been free of m e d i c a t i o n for 4 weeks and w i t h typical symptomatology. Results: The typical signs of the schizophrenic EEG like reduced or lost occipital alpha-power at rest, increased frontal delta-power at rest, reduced alpha-power reactivity after activation and a disturbed laterality can he ~een very instructiv in the maps. After Lyogen these signs are changed in different time courses, dependent on the individual response of each patient. So the EEG-mapping increases the depot therapies efficiency using objective measured parameters of the brain function. Abt. Pathophysiologie, Med. Akad. Magdeburg, O-3090-Magdeburg, Leipziger Str. 44

R 75 30 C O N C E N T R A T I O N OF THE A N T I E P I L E P T I C DRUG V A L P R O A T E IN B L O O D A N D C O R T I C A L TISSUE OF RATS

32 LASER S C A N N I N G MICROSCOPY NEURONAL ACTIVITY

FOR

RECORDING

OF

Hiendl, R.; Rucker, F. and G. ten Bruggencate A n n e L~cke, T. Mayer, U. Altrup, A. Lehmenk~hler, E.-J. Speckmann B l o o d c o n c e n t r a t i o n s of v a l p r o a t e (VPA) r e f l e c t o n l y in p a r t its a n t i e p i l e p t i c e f f i c i e n c y . O n e r e a s o n for t h i s m i g h t be d i f f e r e n t m e a n b r a i n v e r s u s b l o o d concentrations. For further analysis the c o n c e n t r a t i o n of free V P A in the extracellular space of the cerebral cortex and in the sinus sagittalis of rats were m e a s u r e d c o n t i n u o u s l y by V P A - s e l e c t i v e m i c r o e l e c t r o d e s in v i v o (cf. Lehm e n k ~ h l e r et al., Eur. J. Neuro. Sci. Suppl. 3: 128, 1990). A c o n s t a n t a m o u n t of s o d i u m - V P A (0.6 m m o l / k g d i l u t e d in 0.5 ml R i n g e r solution) w a s i n j e c t e d into the V. femoralis (time of injections: 2 s to 5 min) of anesthetized and a r t i f i c i a l l y v e n t i l a ted rats. A f t e r i n j e c t i o n (duration ca. 30 s ) V P A in the e x t r a c e l l u l a r s p a c e a n d in t h e c e r e b r a l s i n u s r e a c h e d a p l a t e a u of ca. 0.4 mmol/l w i t h i n 15 m i n and r e m a i n e d s t a b l e for at l e a s t 30 min. A p e a k (maximum amplitude in tissue: 2 mmol/l, in blood: 12 mmol/l) p r e c e d e d t h e p l a t e a u . S h o r t e n i n g t h e injection time increased the peak value. The results show that nearly the same concentrations of free V P A were reached in the extracellular space of the cortex and in blood. I n s t i t u t f~r P h y s i o l o g i e , 4400 M~nster,

Robert-Koch-Str.

27a,

In order to investigate integrative processes in neuronal assemblies, we developed a photometric method utilizing voltage sensitive fluorescent dyes. The experimental setup allows simultaneous multisite recordings and consists of a laser, an aconsto-optical deflection system and an inverted microscope. The system has an optical resolution of 15 x 15 points. The sites for the optical recording on the preparation can be set randomly under visual control. The maximal overall sampling rate of 100 kHz can be disIributed over the number of channels. There is a choice between high spatial or high time resolution. Thus, with a smaller number of optical channels a higher sampling rate per channel is achieved or vice versa. The size of the recording area can be varied. By changing the objective, recording areas of 1000 x 1000, 550 x 550 or 250 x 250 microns are available. A video camera and an image processing hardware were installed to document the recording grid and the underlying structure of the preparation. Even with single sweep recording a sufficient signal to noise ratio was achieved. This is due to the high intensity of the laserlight and the use of the principle of ratio-measurement: the intensity of the excitation light is sampled simultaneously with the intensity of the fluorescent light. The ratio of fluorescent to excitation light is used to correct for the fluctuations of the excitation light. The combination of a very low numerical aperture for the laser excitation light path and a high numerical aperture for the fluorescence detection light path is optimal for slice preparations. These preparations are highly organized in the direction of the optical axis, so illumination with a small, nearly parallel laser beam is advantageous for the spatial resolution. A high numerical aperture in the light path of the detection allows high sensitivity. Data were obtained from hippocampal and neocortical slices (Rucker et al., this volume). Supported by the DFG, SFB220, TP Z Physiologiscbes Institut der Universi~'t Miinchen, Pettenkoferstrage 8 Miincben 2

33

31 IGF-I M O D U L A T E S Ca 2+ TIATED NEUROBLASTOMA V O L V E M E N T OF PROTEIN T. Kleppisch*, F.-J.

12,

CHANNEL CURRENT IN U N D I F F E R E N x GLIOMA CELLS. P O S S I B L E INKINAS~ C. # Klinz , J.Hescheler

T h e r e is emerging evidence that insulin and insulin-like growth factors (IGF) are neuroactive peptides. The underlying mechanisms of the IGF induced effects in the central nervous system remain to be elucidated. We studied the effect of IGF-I on Ca 2+ channel in neuroblastoma x glioma cells u s i n g the w h o l e - c e l l configuration of the patch-clamp technique. Ca 2+ channel currents recorded in u n d i f f e r e n t i a t e d control cells were mainly of a low-thresh o l d t r a n s i e n t type and showed a m e a n d e n s i t y of 5.9• p A / p F (n=42). Current density increased to 9.2• pA/pF (n=25) after 2 hours incubation w i t h IGF-I (0.2~g/ml) and declined thereafter to 6.0+0.6 pA/pF (n=16) within 24 hours. Stimulated Ca 2+ channel currents exhibited an enhanced amount of a d i h y d r o p y r i d i n e - s e n s i t i v e high-threshold slowly ina c t i v a t i n g type. Ca 2+ current stimulation by IGF-I was a b o l i s h e d by simultaneous incubation with the p r o t e i n k i n a s e C (PKC) inhibitors H-7 and staurosporin. Staurosporin and H-7 itself did not alter current density. Direct activation of PKC by phorbolesters m i m i c k e d the effect of IGF-I. M a x i m u m s t i m u l a t i o n of Ca 2+ channel current occured w i t h i n i hour and reversed thereafter~ Inactive phorb o l e s t e r did not stimulate the Ca 2+ channel current. The effects of IGF-I and phorbolesters w e r e not additive. We suggest that IGF-I may act as n e u r o a c t i v e peptide by modulation of the Ca 2+ channel function involving a PKC dependend step. * Institut f~r Physiologie der H u m b o l d t - U n i v e r s i t ~ t Berlin, Hessische Str. 3-4, 1040 Berlin, FRG # Institut f~r Pharmakologie der Freien U n i v e r s i t ~ t Berlin, Thielallee 69-73, i000 Berlin 33, FRG

SPREAD OF EPILEPTIFORM ACTIVITY IN RAT CORTICAL SLICES: PHOTOMETRIC MEASUREMENTS USING L A S E R SCANNING MICROSCOPY Rucker, F., I-Iiendl, R., Sutor, B. and G. ten Bruggencate Experimentally, epileptiform activity can be induced by different manipulations. By using a photometric multisite recording technique (see Hiendl et al., this volume), we investigated whether the spread of epileptiform activity depends on the mode of induction. Experiments were performed on rat neocortical slices. Field potentials were evoked by electrical stimulation in layer VI and recorded extracellularly using microelectrodes. For optical recordings, the slices were stained with the voltage-sensitive dye RH 414. Signals were sampled from 20 optical channels positioned within an area of lxl mm which covered all layers of the neocortex. The channels were arranged in 4 vertical rows consisting of 5 measuring points each. Upon application of the GABAA antagonist bicuculline (10 #M), stimulusrelated epileptiform activity developed within 10-20 min. With optical recordings, large changes in fluoresenee were observed indicating the synchronous depolarization of a large number of neurons. The latency of the optical response was shorter in the lower layers compared to that in the upper layers. However, the time to peak was similar in all layers. This resulted in a steeper rate of rise of the response in the upper layers. In addition, the amplitudes of the optical signals recorded in layers I-III were larger than those detected in deeper layers. The horizontal spread of activity was fast. There was no significant difference between the latency of optical signals recorded in the same layer at different horizontal positions. Following removal of Mg ions from the bathing solution, repetitive epileptiform discharges were observed. The spread of this activity was similar to that seen upon application of bicuculline. In contrast to the hippocampal slice preparation, it was not possible to induce epileptiform activity in neocortical slices by lowering the concentration of extracellular Ca ions to 0.2 mM or by raising the extracellular concentration of K ions up to 14 raM. These results suggest that epileptiform activity is triggered in the lower layers of the neocortex and amplified, by an unknown mechanism, in the upper layers. Furthermore, we conclude that in the models tested the spread of epileptiform activity is obviously independent from the mode of induction. (Supported by the DFG, SFB220, TPZ). Physiologisches Institut der Universitiit Miinchen, Pettenkoferstrasse 12, 8000 Miinchen 2.

R 76 34

36

CHOLINERGIC CONTRIBUTION TO SYNAPTIC TRANSMISSION IN MOUSE NEOSTRIATUM IN VITRO. B. SchlSsser, B. Sutor and G. ten Bmggencate

GABA INDUCED CURRENTS OF THE OT,~CTORY RULR

In a previous study, we demonstrated that excitatory synaptic transmission in the mouse neostriatum is mediated by excitatory amino ecids via both AMPAand NMDA-receptors (Schl6sser et al. Pfliigers Arch. 418, Rll, 1991). However, other pharmacological and electrophysiological studies suggested a predominantly eholinergic mechanism of generation of excitatory postsynaptic potentials in the mammalianneostriatum (see Nicoll et al. Physiol. Rev. 70, 513, 1990). We therefore investigated the cholinergie contribution to the generation of synaptic activity in the mouse neostriatum in vitro. Experiments were performed on neostriatal slices obtained from C57BL mice. Synaptic activity was evoked by intrastriatal stimulation and recorded extracellulady. All drugs were applied by addition to the bathing solution. At half-maximal stimulus intensities, evoked field potentials consisted of negative potentials (N1-N3) superimposed on a positive potential (P1). At concentrations of 1-10/zM, the acetylcholineesterase inhibitor eserine reversibly reduced the amplitude of the synaptically generated N2-component by 30-1-4.9% (mean + SEM, n=5). This effect was prevented by simultaneous application of the muscarinie antagonist atropine (0.5 #M). The nicotinic antagonists mecamylamine (0.1-30 #M) and d-tubocurarine (1-3 #M) produced either no effect or a slight increase (up to 15 %) in the amplitude of the N2-potential (n=7). In some experiments, the AMPA receptor antagonist CNQX (10 #M) was administered following the application of mecamylamine. In all cases, CNQX reversibly abolished synaptic activity. These results indicate that, under the given experimental conditions, endogenous acetylcholine is released upon intrastriatal electrical stimulation. The transmitter produces an inhibitory effect by acting on muscarinie receptors. This action might be due to a presynaptic inhibition of the release of excitatory transmitters and/or a postsynaptie hyperpolarization mediated via M2 eholinergie receptors. There was no evidence for a major contribution of nicotinic receptors to the generation of excitatory synaptic transmission in the mouse neostriatum. (Supported by the BMFT, "Morbus Parkinson und andere Basalganglien-Erkrankungen"). PhysiologischesInstitut derUniversit~tMfinchen, Pe~enkoferstrasse 12, 8000 Mfinchen 2

IN

NONSPIKING

INTERNEURONES

J. Bufler, F. Zufall, H. Distel* and H. Hatt Little is known about the function of interneurons in the vertebrate olfactory bulb. We have studied the physiological and morphological properties of the periglomerular cells (diameter 6-8 ~m), one main type of bulb interneurons, by using a thin slice preparation in combination with patch-clamp measurements and Lucifer Yellow staining. In current clamp recordings these neurons were characterized by their lack of action potentials upon depolarization. The I-V curve showed a strong outward rectification. Consistent with these results no Na + currents could be elicited in voltage clamp experiments. Two types of outward currents were distinguished: an inactivating and a non-inactivating K + current. The transient K + current could be selectively inhibited by adding i0 mM 3,4-Diaminopyridine (DAP) to the extracellular solution. This current was activable only from holding potentials more negative than -80 mV. About 70% of all investigated periglomerular cells (n=89) responded to the application of GABA and muscimol with an activation of Clcurrents (whole-cell recording). Two different types of Clcurrents could be distinguished by their different desensitization time constants (5s versus 20-30s). In most of the cells (n = 44) both of these currents were expressed. The currents were inhibited by bicuculline (I0 ~M) or picrotoxin (0.i mM). The addition of Zn zt (0.i mM) to the extracellular solution had no effect. The single channel currents activated by GABA (outside-out patch) showed a main conductance state of 24 pS and a mean open time of about 2 ms. Physiol. Institut der TU, Biedersteinerstr. 29, D-8000 Mfinchen 40 and *Institut f. Med. Psychol. der LMU, Goethestr. 29, D-8000 Mfinchen 2

35 PHASEOLUS LECTIN AS A RELIABLE LOCALIZED MARKER FOR NEURONAL RECORDING SITES. L.A. Sacher, L.M. Schindler*, F.P.Kolb*, J.A. BOttner-Ennever

37 S t a t i s t i c a l p r o p e r t i e s of n e u r o n a l n e t w o r k a c t i v i t y G.Rose, M.Pekel, H.KSller, M.Siebler

Several different techniques were used to mark the recording sites within the cat and rat cerebellar cortex. The aim of the electrophysiological experiments was to investigate the spread of excitation via a field potential anaylsis using a grid of recording points spaced 1001~m apart. Electrolytic lesions were not permisable because neuronal damage would invalidate the electrophysiological results. Small injections of horseradish peroxidase (DAB and TMB staining method) and Methylene Blue resulted in labeled areas of 50 - 1001~mand 250pm, respectively, and were not circumscript enough to reliably reconstruct the recording tracks or sites. In addition the tissue in the TMB experiments was brittle and led to frequent tears and artifacts. Pontamine Sky Blue, as a marker, was rejected because it failed to survive our histological treatment of the tissue and could never be detected. We found that the lectin Phaseolus vulgaris leucoagglutinin (PHA-L) provided the most localized and reliable marker under these conditions. The lectin was used in concentrations of 1-3% in the NaCI 3M filled recording micropipettes with a tip diameter of approximately 101~m.PHA-L was ejected by 1 - 5 time-controlled air pressure pulses of 2 kg/cm 2, depositing volumes of 0.1nl/pulse. After survival times between 10 minutes and 10 hours the brain was perfused, cut on the vibratome and treated with standard immunohistochemical procedures to visualize PHA-L (second method of Gerfen and Sawchenko, Brain Res., 290, (1984) 219-238). After detection of the marking spots, 15-50p.m diameter, the sections were photographed and counterstained for final analysis.

We have investigated the development of spontaneous activity of primary cultured hippocampal neurons from embryonic rats (E18). By means of the patch-clamp technique spontaneous postsynaptic potentials as well as action potentials could be recorded after a few days in vitro. The interspike intervals (ISis) were random with an exponentially distribution in the ISI-histogram. By improved graphical and statistical methods, (e.g.) modification of joint interval histograms, simple spike patterns or oscillations could be detected within a noisy background (Fig). N 3. 7 S l d i v

I

2~.2szd*~

?i]

....

~," . .'~ ISI-Ht,tz~e~

1~u .

N=)

The advantages of PHA-L as a marker are that it provides a highly localized and stable stain which is relatively easily visualized. In addition the anterograde transport away from the injection site, observed in some cases, was also an indication that the neurons at the recording/injection sites were intact.

We concluded from these results that spontaneous events were network generated. Therefore, statistical concepts were required to analyse further network properties. In our model each neuron is considered as an interface to the network. We propose to analyse the interactions between two arbitrary neurons by n-th order crosscorrelations from their statistical events. The dynamics of this interactions (plasticity) will be studied by application of different stimulation patterns.

Institutes of Neuropathology and Physiology*, University of Munich, Pettenkoferstr. 12, D-8000 MOnchen 2. This study is supported by the Deutsche Forschungsgemeinschaft (SFB 220, D8, D9).

Department of Neurology, University of Dfisseldorf Moorenstr. 5, D-4000 D/isseldoff 1, FI%G Supported by DFG SI 370/2-1

R 77

38

40

USING "Dir' IN LABELING OF THE SCIATIC NERVE AFTER NERVE TRANSECTION AND REGENERATION A. Kreischer and B. E. Nixdorf

REINNERVATION OF MUSCLE AND REORGANISATION OF SPINAL CORD MOTONEURONS IN MICE AFTER PROXIMAL NERVE SECTION (SCIATIC NERVE) K.A. Hartmann, D. Kleinebeckel, F.W. KluJJmann

The fluorescence tracer "DiI" labels neuronal processes in anterograde and retrograde direction in fixed tissue (Godement et al., Development 101,697713, 1987). The staining occurs due to a process of diffusion of dye along the plasma membrane or myelin sheath. It is supposed that sprouting axons on the level of a nerve injury are partly guided to inappropriate muscles. In the distal portion the sciatic nerve consists of a peroneal and a tibial fascicle. By using the carbocyanine dye "Dir' we tried to identify "misguided" axons from the peroneal fascicle to the tibial nerve. The right sciatic nerve was transected in 3 mice. After 6 months of regeneration EMG activity was recorded from the reinnervated tibialis anterior (TA) and the reinnervated gastrocnemins medialis (MG) muscle simultaneously during free running. Muscle coordination was essentially lost, i.e. "misguided" axons could be expected. On the left side there was coordinated muscle activity. Four weeks later the mice were anesthetized and perfnsed with 4% paraformaldehyde. The right sciatic nerve was removed 1 mm proximal and 5 mm distal to the nerve section, the left sciatic nerve was removed on the same level. At the proximal nerve stump the peroneal fascicle of the removed sciatic nerve was prepared, filled with a crystal of "DiI", and left in fixative for several months. The preparations were observed with an epifluorescence microscope equipped with a rhodamine filter set for viewing the orange-red "Dir' fluorescence. After a diffusion time of 4 months myelinated axons were labeled on a distance up to 3.6 mm. The pathway of axons could be identified proximal, within and distal to the site of the nerve transection. There was a pronounced fiber crossing from the peroneal fascicle to the tibial nerve. On the control side there was no fiber crossing. Such "misguided" axons may be in part a cause for the loss of muscle coordination.

Institut fiir Neurophysiologie der Universitt~t zu KOln, Robert-Koch-Str. 39, D-5000 KOln 41, FRG

The left sciatic nerve was sectioned in adult mice. Electromyograms (EMG) were recorded from the left tibialis anterior (TA) and its antagonistic medial gastrocnemius muscle (MG) during free running between the third day and 4 months postoperative. After EMG-recording the number and location of motonanrons supplying the TA were determined using horseradish peroxidase which was injected into the left and the right TA muscle. Seven days after nerve section first denervation potentials were recorded. Reinnervation began to appear after 18 days with an interindividual variability from day 18 to day 20. 21 days and more after nerve section muscle coordination between the TA and the MG was detoriated. Between day 3 and day 10 motoneurons could not be detected. Mean cell counts between day 13 and day 15 were about 2% of the control value. The stained motoneurons showed no preferred location within the cell column. Cell counts between day 16 and day 19 showed an increase and between day 20 and day 30 cell counts were about 77% of the control value. The animals which had been investigated 4 months after nerve section showed a rediminution to a mean of 55%. Compared with the mean of the longitudinal disribution of the TA cell column on the control side, the centre of the TA column after sciatic nerve regeneration was shifted 820)am +/- 350~um caudal. The results indicates a delay of about 5 days between the onset of muscle activities compared with the reorganisation of spinal cord motoneurons after sciatic nerve transsection. The considerable caudal displacement of the mean of the longitudinal distribution of the TA cell column after nerve regeneration is in agreement with the finding, that the TA muscle is reinnervated not only by appropriate nerve cells but also by neurons that formerly supplied other muscles. This is in accordance with the observed uncoordination of muscle activity.

Institut fiir Neurophysiologie der UniversiNit zu KOln. Robert-Koch-Str. 39, D-5000 KOln 41, FRG

39

41

REINNERVATION OF MUSCLE AND REORGANIZATION OF SPINAL CORD MOTONEURONS IN MICE AFTER DISTAL NERVE SECTION (PERONEAL NERVE) E. Hummers-Pradier, P. K l e i n e b e c k e l , F.W. Klu~mnnn

REINNERVATION OF SPLIT-SKIN GRAFTS IN MAN Ch. Weiss 1, H. Fruhstorfer 1, H. C. Friederich 2 and H. Winter s

The r i g h t deep p e r o n e a l n e r v e was s e c t i o n e d in a d u l t mice. Each day from t h e 1 s t t o t h e 1 5 t h , t h e n in 2 - d a y i n t e r v a l s up t o t h e 2 5 t h p o s t o p e r a t i v e day and a f t e r 1, 2 and 6 months, e l e c t r o m y o g r a m s were r e c o r d e d from t h e t i b i a l i s a n t e r i o r (TA) and t h e a n t a g o n i s t i c g a s t r o c n e m i u s m u s c l e in 2 of t h e 35 a n i mals. "Coordination f a u l t " was a s s e s s e d by c o m p u t e r - a i d e d evaluation. I m m e d i a t e l y a f t e r n e r v e s e c t i o n and a f t e r each EMG, t h e number and l o c a t i o n o f motoneurons s u p p l y i n g t h e TA were d e t e r m i n e d by i n j e c t i n g h o r s e r a d i s h p e r o x i d a n e i n t o b o t h TA m u s c l e s . C e l l c o u n t s on t h e l e s i o n s i d e were compared t o the contralateral control side. T h r e e days a f t e r n e r v e s e c t i o n , EMG-recordings showed e v i dence o f d e n e r v a t i o n . R e l n n e r v a t i o n began t o a p p e a r a f t e r 7 days, with large inter-individual variability. Coordination varied largely, w i t h a mean c o o r d i n a t i o n f a u l t o f 0 . 4 7 comp a r e d t o 0.025 in c o n t r o l a n i m a l s , and was n o t r e l a t e d t o t h e age o f t h e l e s i o n . (r=0.304). The mean c o n t r o l TA motoneuron pool c o n t a i n e d 133 n e u r o n s , c o n t r o l a n i m a l s showed b i l a t e r a l symmetry. I m m e d i a t e l y a f t e r n e r v e s e c t i o n , cell counts dec r e a s e d t o a b o u t 55% o f c o n t r o l v a l u e s , and c o n t i n u e d t o d i m i n i s h t o 4.5% on t h e 3 r d d a y . Between t h e 4 t h and t h e 25th day t h e r e was an a l m o s t l i n e a r i n c r e a s e up t o a maximum o f 138%. The l a t e r f o l l o w - u p s showed a r e d i m i n u t i o n t o a mean o f 46%. Motoneuron c o u n t s and c o o r d i n a t i o n were n o t r e l a t e d {r=0.016). These f i n d i n g s i n d i c a t e a c o n s i d e r a b l e d e l a y b e t w e e n t h e a n a tomical restoration o f c o n t i n u i t y and t h e o n s e t o f muscle function, a s w e l l a s an o v e r s h o o t s p r o u t i n g r e a c t i o n o f n e u rons, o n l y a b o u t h a l f o f which e s t a b l i s h definite connections, comparable to ontogenetic development. Institut ffir N e u r o p h y s i o l o g i e , K61n 41, D e u t s c h l a n d .

R o b e r t - K o c h - S t r . 39,

D-5000

Studies on sensory reinnervation of split-skin grafts have resulted in contradictory results (Hermanson, A. and Dalsgaard, C.-J., Med.Biol. 65:49 (1987); Waris, T. et al, Brit.J.Plast.Surg. 42:576 (1989)). As the observed difference in sensibility might be caused by the type of grafting, the quality of sensory reinnervation was studied in split skin grafted by two different methods. In 23 patients (mean age 56 years; range 28-79 years) split skin was grafted on the intact muscle fascia (Marburg group). In another 22 patients (mean age 52 years; range 30-74 years) the skin was grafted directly on the muscle after the fascia had been removed (Berlin group). The grafts had diameters of 5-10 cm and were situated only on arms and legs (Berlin group) or additionally on the trunc and the forehead (Marburg group). The interval between surgery and sensory examination was in all but one case 6 months to 5 years; in one patient the interval was 14 years. In all patients the following parameters were tested on the grafts and on the corresponding eontralateral skin: 1. tactile sensibility: touch threshold and two-point discrimination. 2. thermal sensibility: warm and cold thresholds. 3. nociception: thermal pain thresholds, quality of needle wheel stimulation, sensation and blood flow changes following histamine stimulation. In addition skin conductance was determined. On the grafted skin sensory thresholds were generally high or sensory modalities were completely missing. In the Marburg group sensory thresholds were mainly increased, whereas in most cases of the Berlin group sensory modalities were lacking. In most cases of both groups neurogenic inflammation was absent and the skin conductance was low. However, single patients of both groups showed a surprisingly good reinnervation of the transplanted skin. The reason for this finding is still unclear; but it is quite possible that characteristics of the grafted skin exert an influence on sensory reinnervation. 1)Institut fiir Physiologic und 2)Klinik ffir Hautkrankheiten der Philipps-Universitfit Marburg, Z}Klinik fiir Hautkrankheiten der Humboldt-Universitfit Berlin.

R 78 44

42 SYMPATHETIC AND AFFERENT NERVES OF THE RAT

NEURONES

PROJECTING

IN

FORELIMB

RESPONSES TO GRADED BARORECEPTOR STIMULI OF LUMBAR VASOCONSTRICTOR NEURONES SUPPLYING DIFFERENT VASCULAR BEDS IN THE CAT

R. Baron, W. J~nig and H. With

A. Boczek-Funcke,

The anatomy of the cervical sympathetic trunk was studied systematically in the rat. Details of the arrangements of w h i t e and g r a y rami c o m m u n i c a n t e s and s u p e r i o r cervical, middle cervical and stellate ganglia are given. Cell b o d i e s of a f f e r e n t and s y m p a t h e t i c n e u r o n e s w i t h axons projecting to skin and muscle of the rat forelimb were labeled r e t r o g r a d e l y w i t h horseradish p e r o x i d a s e (HRP) in order to study t h e i r numbers, s e g m e n t a l d i s t r i b u t i o n and l o c a t i o n q u a n t i t a t i v e l y . H R P w a s a p p l i e d to t h e n e r v e s supplying skeletal muscle (Ramus profundus of radial nerve, RP), hairy skin (N. cutaneus brachii lateralis of N. axillaris, CB) and to mixed nerves (median nerve, ME; ulnar nerve, UL; radial nerve, RA). All sensory and sympathetic cell bodies were located ipsilaterally. Sensory somata were commonly r e s t r i c t e d to two or three adjacent dorsal root ganglia (usually C6-7 for CB; C7-8 for ME; C7-TI for RA and RP; CS-TI for UL). Nearly all of t h e s y m p a t h e t i c s o m a t a w e r e l o c a t e d in t h e m i d d l e c e r v i c a l / s t e l l a t e ganglion (fusion of C6-T3). Some 0-0.4% lay in T4 and T5, none in the superior cervical ganglion. From the data, it is estimated that 400 sympathetic and 1050 afferent neurones project into CB, 1660 and 4050 into RA, 540 and 760 into RP, i010 and 3670 into ME, and 880 and 3040 into UL. T h e r e l a t i v e p r o p o r t i o n s of s y m p a t h e t i c a n d a f f e r e n t n e u r o n e s p r o j e c t i n g in t h e s e n e r v e s are s i m i l a r to d a t a obtained for rat hindlimb nerves.

It is known that a rise in carotid sinus pressure induces a d e c r e a s e in s y s t e m i c b l o o d p r e s s u r e w h i c h can be m a i n l y attributed to a diminution of total peripheral resistance. There are r e m a r k a b l e d i f f e r e n c e s of i n d u c e d r e f l e x b l o o d flow c h a n g e s b e t w e e n c u t a n e o u s and m u s c l e or s p l a n c h n i c vascular beds. We analysed the responses in vasoconstrictor n e u r o n e s s u p p l y i n g v a s c u l a r beds of the v i s c e r a and t h e hindlimb to graded carotid sinus harcreceptor stimulation. E x p e r i m e n t s w e r e p e r f o r m e d on c h l o r a l o s e - a n a e s t h e t i z e d , paralysed cats. The left carotid sinus was vascularly isolated and connected to a pressure reservoir, the right sinus nerve was cut. The carotid baroreceptors were stimulated by g r a d e d i n t r a s i n u s a l p r e s s u r e s of 120 to 250 mmHg. N e u r a l activity was recorded from axons of preganglionie sympathetic neurones projecting into the lumbar splanchnic nerves, functionally classified as visceral v a s o c o n s t r i c t o r (V~C) neurones, and from postganglionic neurones supplying hindlimb skeletal muscle and skin (MVC and CVC neurones, respectively). The decrease in neural activity during the first i0 seconds of each barorsceptor stimulus was analysed quantitatively. All W C and MVC neurones responded uniformly to stimulation of carotid baroreceptcrs whereas two groups of CVC neurones could be distinguished. There were no significant differences between the reactions of VVC, MVC and CVC 1 neurones. The responses of the CVC 2 neurones were significantly weaker. These results show that regional blood flow changes induced by stimulation of arterial baroreceptors correlate well with r e f l e x c h a n g e s of a c t i v i t y in v a s o c o n s t r i c t o r n e u r o n e s supplying the corresponding vascular beds.

Physiologisches Institut der Christian-Albrechts-Universit~t, Olshausenstr. 40-60, W-2300 Kiel

H.-J. H~blsr, W. J~nig and M. Michaelis

Physiologisches Institut der C h r i s t i a n - A l b r e c h t s - U n i v e r s i t~t, Olshausenstr. 40-60, W-2300 Kiel, F.R.G.

43

45

INFLUENCE OF ARTERIAL CHEMORECEPTOR STIMULATION BY ALMITRINE ON THE PLASMA LEVELS OF ACTH, CORTISOL, PRA, AND ALDOSTERONE IN ANAESTHETIZED CATS A.Honig, B.Wedler, H.Oppermann, S.Gruska, and M.Schmidt

INHIBITION OF ACTIVITY IN SYMPATHETIC TO AUGMENTED BREATHS IN THE RAT

Mammals react to well-tolerated acute whole-body high-altitude hypoxia with an increase of the plasma levels of ACTH and cortisol. It is a l s o known that acute whole-body hypoxic hypoxia has uncertain effects on plasma renin activity (PRA) but in most cases lowers plasma aldosterone levels thus leading to a suppression of the aldosteroneto-PRA ratio. But there are no systematic studies concerning a possibly existing reflex influence of the arterial chemoreceptors on the renin-aldosterone axis. In the experiments presented here we studied the effects of a pharmacological chemoreceptor stimulation by almitrine in b o t h c h e m o r e ceptor-intact and chemoreceptor-denervated, chloralosed, curarized and constantly ventitaled cats on the plasma concentrations of ACTH and eortisol as well as PRA and aldosterone. The chemoreceptor-intact animals reacted to the almitrine with a significant increase of both ACTH and cortisol. These responses were completely abolished by chemoreceptor deafferentation. Plasma renin activity (PRA) and plasma aldosterone rose with the time in both groups of experiments but were not influenced by arterial chemoreceptor stimulation. These data indicate that there exists a reflex influence of the peripheral arterial chemoreceptors on ACTH and cortisol release but not on the renin-aldosterone axis. Institute of Physiology and Clinic of Internal Medicine of the Ernst-Moritz-Arndt University of Greifswald, D-2200 Greifswald, Germany

NEURONES

IN PARALLEL

H.-J. H~bler, W. J~nig, M. Krummel and O. Peters It is w e l l k n o w n t h a t a n a e s t h e t i z e d rats breathing spontaneously show augmented inspiratory efforts at regular intervals. D u r i n g these augmented breaths a v a s o d i l a t a t i o n in rat hindlimb skeletal muscle has been observed (Marshall and Metcalfe, J. Physiol. 400, 15-27, 1988). In t h e p r e s e n t e x p e r i m e n t s t h e p a t t e r n of a c t i v i t y accompanying these augmented inspirations was analyzed in sympathetic postganglionic neurones supplying skeletal muscle and skin of the rat hindlimb. 16 r a t s (female, 240-260 g) a n a e s t h e t i z e d with p e n t o b a r b i t o n e (50 mg/kg) were used. In animals b r e a t h i n g room air single and multi-unit recordings were made using conventional techniques. Phrenic nerve activity was registered s i m u l t a n e o u s l y . Gasps occurred with an incidence of l-2/min during normal b r e a t h i n g f r e q u e n c i e s ( 7 0 - 8 0 / m i n ) . In p a r a l l e l to a n d f o l l o w i n g the a u g m e n t e d b r e a t h s the a c t i v i t y of n e u r o n e s s u p p l y i n g the v a s c u l a r beds of b o t h s k e l e t a l m u s c l e and hairy skin was depressed. Only one neurone, which showed an inspiratory peak in the periphrenic histogramme, was excited during augmented breaths. The other neurones displayed e x p i r a t o r y peaks of their activity. The m e a n arterial pressure increased during the gasp and decreased in the next expiration by about 5mmHg. These results provide evidence that the observed vasodilatation during augmented breaths is the result of an i n h i b i t i o n of sympathetic neurones supplying both skeletal m u s c l e and skin. A d d i t i o n a l l y t h e data m a y i n d i c a t e t h a t inspiratory neurones e x e r t an i n h i b i t o r y action on sympathetic or p r e s y m p a t h e t i c neurones in t h e r a t , presumably on the brainstem level.

Physiologischss Institut der Christian-Albrschts-Universit~t, Olshausenstr. 40-60, W-2300 Kiel, F.R.G.

R 79

46 THE INITIAL RESPONSE OF HEARTRATE DURING ORTHOSTAT!C AND MENTAL LOAD IN NORMOTENSIVEAND HYPERTENSIVE SUBJECTS W.-D.Kaiser, B,Peter, A.follnr and T.Pnller Introduction and Methods: Disturbances of the autonomic nervous system play a major role in the pathogenesis of prinary hypertension. To characterize the shortterm regulation via the autonomic nervous system during orthostmsis and mental iond in the hypertensive state, the i n i t i a l response of heart rate (HR) during the f i r s t i0 seconds of active (AO) and passive orthostasis {PO) and of mental arithmetic task CHA) were analysed in 28 nornntensive {NT) and 37 hypertensive subjects (HT). Additionally the respiratory sinus arrhythnim (RSA) during deep breathing was deternined. Results and Conclusions: i. In the whole group (NT and HT] the relationship between the HRresponses during different tests revealed a significant correlation between AO nnd MA after 3-~ s (p
48

TIM~

REQUIRED TO DETECT VISUAL MOTION AS A FUNCTION OF STIMULUS VELOCITY AND ECCENTRICITY L. Schlykowa, W.H. Ehrenstein*, C.R. Cavonius*

Choice reaction times (RTs) for detecting the direction of m o t i o n were measured for a small light spot (12' dia) viewed binocularly. The stimulus was presented foveally, or at 4, 8, 12, or 16 deg eccentricity along the vertical or horizontal meridian, at velocities of i, 2, 6 or 12 deg/s. M e a n RTs of 4 subjects decreased with increasing stimulus velocity from 461 to 352 ms (averaged over eccentricities a n d fields, p <.002); and increased with eccentricity from 357 to 445 ms (averaged over velocities and fields,p <.003). The increase in R T at 16 deg (peripheral R T minus foveal RT to the same stimulus) was greatest for stimuli in the upper visual field (108 ms) and least for those in the lower field (62 ms), compared to 78 ms for the left visual field and g0 ms for the right; differences were significant between upper and lower visual fields (p <.05), but not between left a n d right fields (p >.50). The difference between the foveal and peripheral RT decreased monotonically as velocity increased, being 110, 98, 72, and 58 ms (at 16 deg) for velocities of I, 2, 6, and 12 deg/s, respectively. There was no significant interaction between velocity and eccentricity

(p >.10). Our results are consistent with the fact that the motiononset VEP (compared to the pattern-reversal VEP) changes only moderately with eccentricity (Schlykowa et al., Perception 16: 272, 1987), as well as with the representational bias found in the topographic organization of area M T in monkey, which favors the lower visual field (Maunsell & Van Essen, J. Comp. Neurol. 266: 535, 1987).

I n s t i t u t for Pathophysiologie der Medizinischen Akademie Erfurt, Nnrdh~user Str. 7C, O-BOlD Erfurt

Carl-Ludwig-Instltut f~r Physlologle, Universlt~t Leipzig, Liebigstr. 27, 0-7010 Leipzig, Germany *Institut f~r Arbeltsphysiologie, W-4600 Dortmund I, Germany

47

49

RELATIONSHIP BETWEEN THE PERSONALITYPROFILE AND THE CARDIAC RESPONSE DURING ORTHOSTATIC AND NENTAL LOAD IN NORMOTENSIVE SUBJECTS AND PATIENTS WITH PRIMARY HYPERTENSION B.Peter and W,-O.Kmiser Introduction and Methods: There are no investigations about the inf!uence of different personality characteristics on the initial cardiac response patterns tn orthostmtic nnd psychic stress which are mediated via the autonomic nervous system. Therefore we correlated the personality characteristics(determined by means of the Freiburger Pers~nlichkeitsinventar - FPI) with the initial responses of heart rate [HR) and stroke vo!ume { SV) during the first 10 seconds of aktive (AO] and anssivm nrthostmsis (PO) as well am during mental arithmetic task [MA] in 24 nornotensive (NT) and 18 hypertensive subjects (HT). Resu!tm: The personality structure of the HT was characterized by the tendency to higher levels of nervousness, depression and affective l a b i l i t y in comparison with NT. The nervousness , the depression and the affective l a b i l i t y correlated in the f i r s t iO seconds to the HR response during MA only in HT ( r=-0,670, - 0,511 and - 0,510 respectively), but not in NT. INn aggresmivity and the e x c i t i b i l i t Y are positively correlated to the HR-reaction in PO during the f i r s t i0 seconds only in NT. In contrast other personality characteristics revealed significant correlations to the stroke volume responses: The sociability during PO only in NT (strongest relntionship after 7 seconds : r:-O,531; o<0,01 ), the inhibition and the frankness during PO only in HT { strongest corrnlatJon after i seconds with r=-0,627 and after 9 seconds with r=-O,693 respectively; p
EFFECT OF ADAPTATION ON THE PERCEIVED VELOCITY OF DRIFFING GRATINGS

!nstitut for Pmthophymiologie der Medizinischen Akademie Erfurt, Nordh~user Sir. 7C, O- 50!0 Erfurt

R. Miiller and M. W. Greenlee The perceived velocity of drifting gratings was measured as a function of contrast before and after adaptation to a high contrast grating drifting at variable rates. Test and reference gratings, each subtending 2 deg visual angle, were simultaneously presented (700 msec) 2 deg eccentric of a central fixation point. The gratings had a constant spatial frequency (2 c/deg), orientation (vertical) and drift direction (rightward). The contrast of the reference grating was constant (0.08) and that of the test grating varied from 0.02 to 0.64 in 11 steps. Three observers responded in a quasi-forced-choice procedure, which of the two gratings had a greater velocity. The point of perceptual equality was determined using a maximum-likelihood algorithm (Best-PEST) after 20 trials for stimuli grouped in blocks of 4 pairs. The perceptual equality determined before adaptation was compared to that measured after 100 sec adaptation to a 2 c/deg, drifting grating of 0.7 contrast which subtended 10 deg in the right visual field. The drift frequency of the adapting stimulus was varied from 1 to 32 Hz in octave steps to explore the selectivity of the adaptation effect. Perceived velocity increases monotonically with increasing test contrast, the slope of the function varying slightly over subjects. Adaptation at 2 Hz drift rate shifts this function by a factor of about 2-4 towards higher contrasts. The maximal adaptation effect occurs at 8 Hz adapting frequency and is less for lower and higher adapting frequencies. Comparisons are made between these results and Weber fractions for velocity discrimination for equal-contrast gratings, as well as contrast detection thresholds before and after adaptation.(Supported by DFG) Neurologische Universit~itsklinik,Abt. Neurophysiologie, W-7800 Freiburg, Carl-Ludwig-Institutf~irPhysiologieder Universit~t, O-7010Leipzig

R 80 50 EXTRAPOLATION OF VERTICAL VISUAL MOTION W.H. Ehrenstein, N. Yakimoff*, S. Mateeff*, J. Hohnsbein The aim of the present study was twofold: (i) to test whether the foveopetal preference in motion extrapolation that we found earlier for horizontal movement (Ehrenstein et al., PflOgers Arch. 415: R90, 1990) also holds for vertical motion, and (ii) to see whether extrapolation of upward motion would be equivalent to that of downward motion. A small light target moved upward or downward at 6 deg/s over 12 deg and was occluded over additional i0 deg along the vertical meridian. Subjects pressed a key to indicate the moment at which the target reached a stationary reference that was located randomly at one of seven positions, each 2 deg apart. For the first two positions alignment between the moving target and reference was visible, for the other five the target was occluded, so that virtual alignment had to be based on motion extrapolation 9 Fixation was either at the second reference position, at which the target disappeared (A), or it was at the actual position of the reference (B); hence extrapolation was always away from the fovea in A and always toward the fovea in B. No systematic differences in response times were found between conditions when the target was visible at the alignment. Extrapolation times, on the other hand, were consistently shorter for foveopetal and for downward motion (on average 104 and 68 ms, respectively) than for foveofugal and upward motion; this difference increased with extrapolakion distance9 The results indicate faster vertical motion extrapolation for foveopetal and for downward directions. Whereas the foveopetal preference relates _to a corresponding bias found for visual neurons in monkey parietal cortex, the downward preference might account for the fact that objects moving vertically do not remain at a constant speed but usually accelerate when falling down and decelerate when moving upward in everyday life. Supported by the DFG and by the Bulg. Commd tree of Scdence Institut fur Arheitsphysiologie, W-4600 Dortmund, Germany *Institute of Physiology, 1113 Sofia, Bulgaria

51 EFFECTS OF NITRIC OXIDE ON DARK VOLTAGE AND LIGHT RESPONSES OF RETINAL RODS K.-F. $chmidt, G.N. N611and Y. Yamamoto

52 FLUORIMETRIC EVIDENCE FOR CALCIUM/SODIUM IN X E N O P U S O L F A C T O R Y R E C E P T O R N E U R O N E S D. S c h i l d , S . I . C h o u d r y

EXCHANGER

When Xenopus olfactory neurones are stimulated by odours they usually respond via a second messenger chain including receptor proteins, G-proteins, adeuylate cyclase, cAMP, and opening of a cAMP-activated generator channels. These channels shows a permeability for mono- and divalent cations. 9 9 The mtracellular Ca 2 + -concentration has thus to be controlled in an effective mauner. Our recent patch clamp recordings from olfactory receptor cells (Schild and Bischofberger, 1991) could be well interpreted by assuming a Ca/Na transport. Specifically, an Ca-dependent outward current could be elicited by driving the Ca/Na- exchanger in the reversed mode thereby increasing Ca i. However, the exchanger has as yet not been shown in a more direct way. This was the goal of the project reported here. Olfactory receptor neurones of Xenopus were obtained by mechanically associating the olfactory mucosa in Ca/Mg-free solution. The cells were then incubated for 1 h in 100 M Fluo-3. The fluorescence was excited with an argon laser at 488 nm and measured at wavelengths higher than 515 nm (all optical measurements were done with a laserscan microscope). When the Na concentration in the bath was gradually decreased a gradual increase of Ca i was observed. The calcium increase clearly occurred in cilia, dendrite and soma 9 9 9 though it was problematic to assign absolute Ca 2 + -values to fluorescence intensities because fluo does not allow ratioing. Further, the existence of a Na/Ca exchanger was also demomstrated by incubating isolated cells as well as tissue with a polyclonal antibody (AB) provided by Dr. N. Cook, MPI Biophysik, Frankfurt. In the uudissociated tissue the AB bound mainly to the olfactory knobs. In order to exclude insufficient penetration of the AB into the tissue we also applied it to isolated ceils. Here the AB bound predominantly to olfactory+knobs and to the soma membrane. It is concluded that the intracellular Ca concentration in olfactory neurones is controlled in a spatially distinct Way suggesting that Ca 2 + might exhibit different effects in different compartments of the cell. Supported by DFG:SFB 236 Physiologisches lnstitut der Universit~t Humboldtallee 23~ 34 Gtttingen

5 3

A C H R O M A T I C COLOR DISCRIMINATION BASED ON CHOICE R E A C T I O N TIMES M. Miiller, W.H. Ehrenstein, J. I-Iamada*, C.R. Cavonius

Nitric oxide (NO) stimulates the guanylate cyclase and enhances the level of cGMP in cerebellum and vascular smooth muscle. In the central nervous system, NO is formed by the enzyme nitric oxide synthase from L-arginine. This enzyme has been immunohistochemically localized in autonomic nerve fibers in the retina and in other neuronal populations in the brain. Since the light-sensitive conductance of vertebrate photoreceptors situated in the plasma membrane of the outer segment is directly gated by cGMP, it is of interest to determine whether NO has an effect on the cGMP mediated membrane currents of intact photoreceptors. The turnover of cGMP in photoreceptors is related to two enzymes: a phosphodiesterase whose activity is controlled by a light dependent enzyme cascade and by a guanylate cyclase which is subject to regulation by the intracellular calcium level. The activities of these two enzymes determine the cytoplasmic level of cGMP which, in turn, regulates the membrane current and the membrane voltage of vertebrate photoreceptors. We have used the NO releasing substance sodium nitroprusside to alter the cGMP turnover in retinal rods. When the membrane voltage of these cells is recorded with patch-clamp pipettes in the whole-cell recording mode, an exchange of substances by diffusion between cytosol and the pipette filling solution can affect the cell's function. Loss and substitution of substances follow an exponential time course determined by both the molecular weight of the substance under study and the access resistance between pipette and cell. In retinal rods the diffusion-dependent loss of nucleotides is reflected by a slow hyperpolarization of the dark voltage and by alterations of the light responses. When the pipette medium was completed with 1 mM GTP the hyperpolarization was attenuated and decelerated, but not completely abolished. Addition of 1 mM ATP prevented the larger part of the alterations of the light responses. When sodium nitroprusside plus GTP was applied, the tendency of hyperpolarization disappeared and a stable dark voltage or even a slight depolarization was measured during the whole-cell recording period. Similar results were also obtained when GTP was given in combination with either EGTA or IBMX which are both known to interfere with the cGMP regulating enzymes in retinal rods. In addition to its effects on the dark current, an acceleration of the recovery phase of the light responses by sodium nitroprusside was also observed. Therefore it was concluded that sodium nitroprusside can activate the guanylate cyclase in photoreeeptors, as it has been demonstrated in other tissues. PhysiologischesInstitutder Justus-Liebig-Universit&t,Aulweg129,W-6300GieBen.FRG

Pairs of achromatic colors were presented on a computer-controlled Barco color monitor (Type HIREM). The stimuli were presented in square patches, subtending 1 x 1 deg at the retina; the inner edges of the square were 0.5 deg left and right from a fixation cross. Luminance ranged from 0.1 to 10 ed/m 2 in ten logarithmic steps, giving 45 stimulus pairs; the background was dark. On half of the trials the stimulus pairs differed in luminance, on the other half they were identical. 'Same' and 'different' trials were randomly intermixed. The subject had to press one key when the stimuli were the same and another key when they were different. Each possible stimulus pair was presented 90 times. Pooling all of the reaction times (RTs) to one luminance step, to two luminance steps, and so on, mean RTs from four subjects show a hyperbolic dependence on luminance separation: starting at 765 ms for the lowest Michelson contrast (C) of 0.25, dropping rapidly at first to 454 ms (C = 0,64) and then slowly to reach 399 ms (C = 0.98). Thus, RTs are highly sensitive to small differences between achromatic colors but they become less discriminative for larger differences. The data based on RT differ markedly from those based on category rating. With the latter we found an almost linear relationship between scaled value and stimulus separation. Our results imply that choice reaction times discriminate better at small stimulus contrasts whereas category ratings are superior in discriminating medium and large stimulus contrasts. * Supported by the Japanese Ministry of Education, Science and Culture. Institut fiir Arbeitsphysiologie, Ardeystr. Germany

67,

W-4600

Dortmund,

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54 THE E H R E N S T E I N I L L U S I O N 50 YEARS ON: EFFECTS ORIENTATION, CONTRAST POLARITY, AND GAP SIZE

56 OF

LINK

I O N C U R R E N T S O F ENZ..YMATICALLY I S O L A T E D R A B B I T R E T I N A L M U L L E R (GLIAL) C E L L S

Jiro Hamada Ehrenstein (1941) showed that if the point at which several dark lines cross is occluded by a disc, it appears lighter than the physically-identical background; conversely, if the lines are llght, the occluded crossing appears darker. Using psychophysical methods of limits and category judgment, I measured the strength of this illusion, induced by radial patterns of lines at every 45 deg azimuth on a 6-deg grey background disc, with gaps of 0.78, 1.57, or 2.37 deg between the inner ends of opposing lines. Contrast was either negative (dark line on grey background), positive (light on grey), or mixed, in which adjacent lines were alternately light and dark. Free fixation and alternation between the test pattern and a comparison field (a Munsell chip) was allowed. Irrespective of contrast polarity, the overall lightness of all patterns was judged to be lower than that of a control, which was the same grey background without lines. The effect of gap size depended on contrast polarity: with positive contrast the illusion was strongest for the small gap and decreased with increasing gap size; with negative contrast the illusion was weakest for the small gap and increased with gap size. With mlxed-contrast patterns the perceived brightness for all gap widths lay between the positive and negative contrast values. Orthogonal lines had more influence than oblique lines: when the horizontal and vertical were negative, the results more nearly resembled those with pure negative contrast; when they were positive, the results resembled those with positive contrast. The results suggest an interaction between two types of izthlbitlon in contrast perception: a global nonantagonistic and a local antagonistic mechanism. Supported by the Japanese Ministry of Education, Science, and Culture Instltnt ffir Arbeitsphysiologie, W-4600 Dortmund i, Germany

A. H e n k e , W. E b e r h a r d t a n d A. R e i c h e n b a c h Carl Ludwig Institute of Physiology, Leipzig U n i v e r s i t y , L i e b i g s t r . 27, D (O)- 7 0 1 0 -Leipzig, F R G . Enzymatically !solated Miiller cells of adult rabbit retinae were used to study the expression of voltage-dependent channels, by means of the whole-cell configt~ration ot the patchclamp techfiicme; Mfiller cells were isolated by incubation in a Ca 2+ free solution containing 1.5 mg/ml Nagarse R and by mechanical dissociation. The current response to voltage ramps showed, in all cells studied, distinct inward rectification which was eliminated by 5 mM Cs + or Ba + in the bath solution. T E A and Ba 2+ caused strong reduction - but no elimination - of all currents whereas in Cs + solutions, inward currents were selectively and completely eliminated. When current responses to voltage ramps were recorded in high K + solutions, a shift of the reversal potential occurred which was according to that expected from the NERNST equation for K + ions. In a~tdition to the inwardly rectifying current I~:am we found two different outward currents vxz. a delayed t~Efifier IVtDR~- which was blocked by 4-AP - and a rapidly inactivating-~cui'rent IA. In K + free soluuons, almost no currents could be evoked, with the exception of a NH4+ driven inward current activated by moderate depolarizations. The presence of functional receptors for glutamate and GABA was studied. Summarizing the results, isolated rabbit Mfiller cells show a relatively fiigt) resting membrane potential (-63.5 +/- 5 mV) and a reasonable input resistance (21.8 +/- 9.8 M~)~ and express several types of voltage-dependent K + channels and ftinctional receptors for neurotransmitters. In future experiments, we will compare the "standard set" of channels of freshly isolated adult cells with the channel expression of neonatal or cultured cells in order to find some rules for glial cell ion channel expression. SUPPORTED BY THE VOLKSWAGEN-STIFTUNG.

55 DOPAMINEALTERSDOSE-RESPONSECURVESOF GLUTAMATEINDUCEDCURRENTSFROMRETINALHORIZONTALCELLSOF THE PERCH(Perca fluviatilis) M. Kruseand K.-F. Schmidt Dopamine is known to enhance currents induced by glutamate agonists in horizontal cells (Knapp and Dowling Nature 325:93 ;1987). This enhancement is

mediated by a cAMP-dependent protein phosphorylation. The patch-clamp technique was employed to record whole-cell membrane currents from cultured horizontal cells. Cells were voltage-clamped at -50 mV and superfused with an extracellular solution either with or without dopamine. During the current recordings the standard medium could be exchangedby a similar one containing the agonists L-glutamate or kainate and the dye fast green. Agonist and dye slowly accumulated in the bath, and the concentration of dye at the cell under study was measured with the help of a photodiode as an indicator for the concentration of agonist. So it was possible to continuously measure the membrane current in relation to a given agonist concentration. Dose-response curves for currents elicited by kainate had a sigmoid shape and at a concentration of 0.1 mM maximum currents of about 1000 pA were obtained. Incubation with 0.2 mM dopamine for 2-5 rain before the application of kainate shifted the rise of the dose-responsecurve towards lower agonist concentrations, but did not increase the maximum currents. When L-glutamate in concentrations up to 3 mM was used as agonist, maximal currents of only about 250 pA were obtained. The dose-response curve for the glutamate induced current has a complex shape: a sigmoid-like increase of current with increasing agonist concentration is followed by a decrease of current at higher agonist concentrations.This effect can presumablybe attributed to the desensitizationof the glutamate receptors. When the cells were preincubated with dopamine the complex dose-response curve was replaced by a normal sigmoid curve and maximal currents of about 800 pA were obtained with glutamate. Glutamate induced membrane current from a horizontal cell clamped at

50 mV (lower trace) and extinction related voltage from the photodiode (upper trace) during accumulation of dye and agonist in the bath solution. Dopamine was not applied in this experiment. -

PhysiologisohesInstitut, Justus-Liebig-UnEversit&t,Aulweg 129,W-6300 Giel3en,FRG Mit Unterstgtzungder DFG(Sohm723/3.1)

57 THE EFFECT OF TEMPORAL FREQUENCY ON VISUAL ACUITY M A . Pak Visually evoked potentials (VEP) were used to determine the spatial contrast response function of the visual system and the visual acuity of the pigeon.The performance of the visual system in the spatial frequency domain (i.e the amplitude of VEP versus the spatial frequency) depends on the temporal frequency (pattern reversal frequency) of the visual stimulus. For description of this characteristic of the visual system, the contrast transfer function was measured at various temporal frequencies. The visually evoked responses were recorded using monopolar stainless steel electrodes inserted into the Stratum griseum superficiale of the optic tectum. The stimulus patterns were generated on a video monitor placed 75cm in front of the animal's eye perpendicular to the optic axis. The spatial contrast response function measured at 10% contrast has a maximum at a spatial frequency of O.5c/deg for the pattern stimulated at low temporal frequencies (0.5Hz and 2Hz), and at a spatial frequency of O.25o/deg for the pattern stimulated at relatively high temporal frequencies (4Hz and higher). In opposition to the highest resolvable spatial frequency of 15.5c/deg (corresponding to a visual acuity of 1.9 rain of arc) for the low temporal frequencies, the contrast transfer function supplies a low highest resolvable spatial frequency of 8c/deg (visual acuity 3.7 rain of arc) for the temporal frequencies 4Hz and higher. Physiologisches Institut, Abt. Neurophysiologie der HeinriohHeine-Universit&t DOsseldorf, 4000 DOsseldorf.

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EFFECTS OF ARTIFICIALLY INDUCED HYPOGLYCEMIA ON RETINAL AND CORTICAL POTENTIALS IN MAN Wolfgang Skrandies and Heike Heinrich

ACTIVATION OF NICOTINIC A C E T Y L C H O L I N E R E C E P T O R CHANNELS (nACHR) IN WILDTYPE, D E N E R V A T E D AND D Y S TR O P H IC (MDX) MOUSE MUSCLE

In the in vitro superfused cat eye preparation changes of neural reponses of the r e t i n a occur when the glucose concentration of the perfusing medium is altered over a wide range, We i n v e s t i g a t e d the effect of glucose level on the human v i s u a l system in vivo in the normal physiological range of blood sugar concentration. We stu died e i g h t h e a l t h y a d u l t s with normal vision who v o l u n t e e r e d to p a r t i c i p a t e after giving inform consent, and we p resent the d a t a of five subjects in whom blood sugar l e v e l s could be modified r e l i a b l y by i n t r a v e n e o u s infusion with insu lin or glucose. Luminance electroretinograms (LERG) were elicited with flashes of long and short w a v e length, and p a t t e r n ERG responses (P-ERG) to checkerboard r e v e r s a l stimuli of different c o n t r a s t were obtained s i m u l t aneously with cortical v i s u a l l y evoked p o t e n t i a l s (VEP). In addition, from all subjects a r e s t i n g EEG was obtained. The derived d a t a were r e l a t e d to blood sugar concentrations by correlation a n a l y s i s . Subjectively blurred vision at blood sugar v a l u e s below S5 mg/dl coincided with n o n - r e c o r d a b l e P-ERGs and VEPs. With glucose l e v e l s below 80% or above 100% of the normal va l ue the b - w a v e of the L-ERG was s i g n i f i c a n t l y l a r g e r t h a n with normal glucose levels, whereas the P-ERG displayed the opposite relationship: maximal amplitudes at normal glucose l e v e l s and a s i g n i f i c a n t amplitude decrease with glucose levels below or above normal. In a similar line the VEP amplitudes decreased s i g n i f i c a n t l y with glucose v a l u e s below 80% of the normal value. Component l a t e n c i e s of both the LERG and P-ERG showed an increase (i.e., a n e g a t i v e c o r r e l a t ion) with decreasing glucose levels. Our data i l l u s t r a t e that d i s t a l and proximal layers of the r e t i n a respond dif feren tly to c h a n g i n g blood glucose levels probably due to regional differences in r e t i n a l blood flow. M~x-Planck-Institute for Physiological and Clinical

D. K61t~en and Ch. Franke In our investigations of nAChR channels we have used enzymatically dissociated interosseus muscles of wildtype and mdx mice directly after dissociation or after keeping wildtype dissociated fibres 14 days in standard tissue culture (denervated fibres). Using the single channel recording patch clamp technique on wfldtype muscle a 48 pS channel with a me a n open time of 1.3 ms could be found. On denervated muscle fibres we found an embryonic like 30 pS channel with a mean open time of 2.1 ms. Both these channels exist in the sarcolemma of dystrophic (mdx) muscle: one with a conductance of 46 pS and a mean open time of 1.3 ms and the other with the lower conductance of 29 pS and a mean open time of 2.06 ms. The mdx mutant mouse carries the homolog gene responsible for Duchenne muscular dystrophy (DMD) in humans. D M D myotubes and mdx muscle fibres have an elevated intracellular C a z + concentration leading to an enhanced net degfgdation of rnuscle proteins. How the regulation of the intraceilular Ca ~T concentration is impaired by the mutation remains unclear. We propose a Ca z + leak into the cell via the Ca z + nAChR channels for we could show that the embryonic like channels desensitize less completely than the adult ones what results in a high sensitivity to low ACh concentrations. Institut fiir Physiologic der TU M Biedersteiner Str. 29, 8000 Mfinchen 40, F R G

Research, 6350 Bad Nauheim. F R G

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61

A G E N E T I C D E F E C T O F T H E T T X - S E N S I T I V E M U S C U L A R NA* CHANNEL CAUSES MEMBRANE DEPOLARIZATION AND WEAKNESS

REGULATION OF CHLORIDE CHANNELS IN HUMANMYOBALLS

Ch. Fahlke, E. Zachar, and R. R~del F.

Lehmann-Horn,

Ch.

Franke,

M. Koch*

Electrophysiological studies on skeletal muscle fibre segments from patients with periodic paral y s i s h a v e s h o w n t h a t t h e e p i s o d e s o f w e a k n e s s are caused by a sustained depolarization of the sarcolemma. In h y p e r k a ! e m i c p e r i o d i c p a r a l y s i s t h i s d e p o l a r i z a t i o n is c a u s e d b y n o n - i n a c t i v a t i n g sodium chann e l s w i t h r e d u c e d s i n g l e - c h a n n e l c o n d u c t a n c e , activated by slight membrane depolarization, e.g. w h e n e x t r a c e l l u l a r K § is i n c r e a s e d . T h e r e is s t r o n g e v i d e n c e t h a t t h i s a b n o r m a l f u n c t i o n a l s t a t e is c a u s e d b y a d e f e c t in t h e g e n e e n c o d i n g for t h e T T X - s e n s i t i v e N a § c h a n n e l of t h e m a t u r e s k e l e t a l muscle. Genetic linkage studies have shown the coupling between this Na + channel gene on chromos o m e 17 a n d a f f e c t e d m e m b e r s of f a m i l i e s w i t h h y perkalemic periodic paralysis. Neurologische Klinik der Technischen Universit~t M ~ n c h e n , M ~ h l s t r a B e 28, D - 8 0 0 0 M O n c h e n 80 Humangenetisches Institut der Universit~t Marburg

Using tight whole-cell recording as well as single channel recording in a double clamp configuration and in excised patches, we have characterized two types of chloride channels in human myoballs (Fahlke e t a ] , PflQgers Arch 418:R45, 1991). The two types have in commonthat they are open at the resting potential, and are distinguished by their different gating behaviour at highly positive potentials. Upon membrane depolarization one channel shows only activating kinetics, the other only inactivating kinetics. The activating whole-cell current becomesmore prominent the longer the duration of the experiment. This suggests the existence of physiologically regulating substances that might be washed out by dialysis through the pipette. The different kinetics of these currents provide the possibility for the study of the regulation of the conducting channels by means of the whole-cell recording technique. Intracellular application of GTP-~-S lead to an increase of the amplitude of the inactivating componentwhithin 30 min, whereas the activating component decreased. This effect did not depend on the presence of intracellular ATP and was not prevented by intracellular inhibitors of the protein kinases. This indicates that Gprotein-dependent channel phosphorylation is not reponsible for the observed alterations. Decrease of the activating and increase of the inactivating componentwere also seen after the extracellular application of a protein kinase C (PKC)-activating phorbol ester (phorbol 12-myristate 13acetate). The phorbol ester had no effect when H8, a specific blocker of PKC, or GDP-8-S were intracellularly applied. This indicates that PKC affects the Cl- channel via a G protein system. In 6 experiments on excised patches, we could so far not find that GTP-~-S produces the corresponding changes in the single channel activity. Possibly, the interaction between G protein and the chloride channels is not a direct one. Supported by the Deutsche Forschungsgemeinschaft (Ru-138/17). Abt. fQr Allgemeine Physiologie, Universit~t Ulm, D-7900 Ulm

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INTERLEUKIN 2 INHIBITS SODIUMCURRENTSIN HUMANMYOBALLS

BLOCKAGE OF K(ATP) CHANNELS IN MOUSE MUSCLE BY ATP-ANIONS AND BY ATP.Ca-, COMPLEXES

H. Brinkmeier, A. Kaspar, H. Wieth61ter*, and R. R~del

SKELETAL ATP-Mg-

S.Hehl and B.Neumcke

Interleukins play an important role in the immune response during inflammatory processes. Interleukin 2 ( l l 2 ) , a peptide hormone that stimulates p r o l i f e r a t i o n of T lymphocytes, reaches high tissue concentrations in inflammatory diseases such as Guillain-Barr~ syndrome and polymyositis, and in various muscular dystrophies (Isenberg et al, Clin Exp Immunol 63:450485, 1986). We have tested the effects of II 2 on the muscular Na+ channels in human myoballs voltage-clamped in the whole-cell configuration. Na§ currents were e l i c i t e d r e p e t i t i v e l y at 1Hz by depol a r i z i n g square pulses of 8 ms duration and going from -86 to -20 mV. The application of 500 U/ml II during such a series resulted in a reduction of the peak Nat current to 31 • I0 % (SD, n = 7) of the starting level. The e f f e c t occurred within < 10 s and was reversible upon washout in a similar time. l l 2 at 100 U/ml reduced the current to 75 • 0.07 % (n = 13). A control solution composed of our standard external f l u i d and the mixture of phosphate buffered saline, 0.01% acetic acid and 0.025 % BSA in wich II 2 is delivered, was i n e f f e c t i v e . The e f f e c t was not dependent on ATP or GTP since i t could be shown several times within more than i hr a f t e r seal formation. The addition of 5mM ATP and 5mM GTP to the pipette solution did not prevent the II 2 e f f e c t . A desensitation was not observed. The resuls suggest that II 2 can suppress c e l l u l a r e x c i t a b i l i t y of muscle cells by the i n h i b i t i o n of Na§ channels. I t is not clear whether II 2 binds to a specific II 2 receptor or d i r e c t l y to the Na§ channels or channel-associated structures. As interleukin 1 produces h y p e r e x c i t a b i l i t y of the nervous system, we conclude that interleukins and related factors may play a role in the modulation of nerve and muscle a c t i v i t y by c o n t r o l l i n g t h e i r e l e c t r i c a l e x c i t a b i l i t y and may be important in nerve or muscle diseases with changes in c e l l u l a r excitabil i t y . Supported by DFG Br 1139/I and Ru 138/17. Abt. fQr Allgemeine Physiologie, Universit~t Ulm, D-7900 Ulm and *Neurologische U n i v e r s i t ~ t s k l i n i k , D-7400 TUbingen

Flexor digitorum brevis muscles of adult mice were dissociated into single fibres with collagenase. Inside-out membrane patches were excised in Ringer and thereafter transferred to K+-rich internal solutions. Currents through single ATP-sensitive K channels [K(ATP) channels] were recorded at membrane potentials between -40 and -60 mV before, during and after ATP application in internal solutions of 160 mM KCI, i0 mM HEPES, pH 7.4 plus various concentrations of Ca, Mg. The strongest blockage of K(ATP) channels by internal ATP was observed without added Ca, Mg (relative openprobability p_u = 44 ~o at i0 ~M ATP), an inter. . medlate blockage wlth 2 mM Ca + 1 mM Mg (rel Po = 55 % at i0 ~M ATP) and the weakest blockage for 1 mM Mg + 1 mM EGTA (rel Po = 59 % at I00 ~M ATP). For each s o l u t i o n the concentrations of ATP 4-, ATP-H 3- anions and of the metal complexes ATP.Ca 2-, ATP'Mg 2-, ATP'K 3- were calculated from stability constants listed in Fabiato and Fabiato (J Physiol Paris 75: 463, 1979). It is concluded that the efficacy of K(ATP) channel blockage by internal ATP decreases in the sequence ATP 4- > ATP-Ca 2- >> ATP-Mg 2-. The prevailing ATP complex in intact muscle fibres will be the ATP-Mg 2- complex, and its weak blocking potency favours the opening of K(ATP) channels after a decline of the myoplasmic ATP concentration. I.Physiologisches Institut, Saarlandes, W-6650 Homburg/Saar. Supported by (SFB 246, A 6).

Deutsche

Universit~t

Forschungsgemeinschaft

63

65

AGONIST INDUCED MEMBRANE DEPOLARIZATION AND CALCIUM ENTRY IN CULTURED HUMAN CILIARY MUSCLE CELLS IS MEDIATED BY A COMMON NICKEL AND LANTHANUM SENSITIVE MECHANISM F. Stahl, A. Lepple-Wienhues, S. Berweck, B. Gebauer, G. Langenbeck-Groh, M. Wiederholt.

AN ACTIN FRAGMENT CONTAINING THE AMINO TERMINAL RESIDUES 1-44 ACTIVATES THE MAGNESIUM-DEPENDENT SI-ATPASE H. K6gler, A. Moir, J. C. R~egg

Acetylcholine and endothelin have been shown to increase calcium entry and to induce membrane depolarization in cultured human ciliary muscle cells [1,2,3]. We further investigated these effects using the calcium sensitive dye fura-2 and conventional intracellular microelectrodes. Endothelin-induced calcium ent[~ was reduced by addition of extracellular La 3 (5,10 -5 mol/l) (0.3• of control) and Ni 2+ (4 mmol/l) (1.6• of control). Acetylcholine-induced calcium entry was also reduced by La 3+ and Ni 2+ (22• and 11• of control, respectively). Both, endothelin and acetylcholine led to an initial transient hyperpolarization with a subsequent depolarization. The acetylcholineinduced depolarization was reduced in the presence of La 3+ and Ni 2+ (46• and 40• of control respectively). The depolarization induced by endothelin was also reduced by La 3+ to 38• of control. Verapamil (10 -5 mol/l) had no effect on both the calcium entry and the depolarization. Thus, calcium entry a n d membrane depolarization seem to be mediated b y a common La 3§ and Ni 2+sensitive but verapamil-insensitive mechanism. I. Biochem Biophys Res Commun 164:1031-39,1989. 2. Invest Ophthalmol Vis sci 31:2420-30,1990. 3. Pfl~gers Arch 415(Suppl.l): R76,1990. Supported by DFG Wi 328/11 Institut f. Klin. Physiologie, Klinikum Steglitz, FU Berlin, Hindenburgdamm 30, D-1000 Berlin 45.

des

The amino terminal region of actin seems to participate in the binding of myosin subfragment 1 (Sl) during cross-bridge cycling thereby activating the magnesium-dependent myosin ATPase. Thus, in an in-vitro assay using isolated Sl from rabbit skeletal muscle an actin fragment containing the amino terminal residues 1-44 mimicked this activating effect. It increased the magnesium-dependent Sl ATPase activity by a factor of 2.2 and 3.3 with peptide concentrations of 8.3 pM and 16.6 pM respectively. Sl concentration was 0.i pM. A control peptide (residues 82-119 of actin) had no effect. The acto-Sl ATPase on the other hand was inhibited by nearly 70% after addition of the peptide (16.6 pM), half-maximal inhibition occurring at a peptide concentration of 6.0 pM, whereas the control peptide had no effect. S1 concentration was 0.I pM, actin concentration 2.0 pM. At higher actin concentration larger peptide concentrations were required for inhibition suggesting that the peptide may compete with actin for its binding site on myosin. An involvement of the amino terminal region of actin in activation of myosin ATPase has also been suggested by van Eyk et al. (1991, in press) who found a =30% activation of S1 ATPase by a peptide containing residues actin 1-28. II. Physiologisches Institut der Universit~t, Im Neuenheimer Feld 326, 6900 Heidelberg

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Ca *§ A C T I V A T E D K + C H A N N E L S HUMAN SKELETAL MUSCLE H. Lerche,

S. Q u a s t h o f f ,

ON

C.

SARCOLEMMAL

Franke,

BLEBS

OF

F. L e h m a n n - H o r n

In the p r e s e n t s t u d y w e u s e d m u s c l e b i o p s i e s f r o m normal individuals. Even without enzymatic treatment a p r e p a r a t i o n a l l o w s o n e e a s y a c c e s s to t h e s a r c o l e m m a for p a t c h c l a m p i n g ( Q u a s t h o f f e t a l . , 1990). S i n g l e fibre s e g m e n t s w e r e m a x i m a l l y s t r e t c h e d a n d t h e n f i x e d in a K § r i c h s o l u t i o n (120 mM). B l e b s were formed spontaneously, ~articularly when the s o l u t i o n c o n t a i n e d C a ++ (10-'M). F r o m t h e s e b l e b s we got i n s i d e - o u t p a t c h e s . W e m e a s u r e d Ca *§ a c t i v a t e d K § c h a n n e l s in s y m m e t r i cal K+ r i c h s o l u t i o n s w h i c h h a d a c o n d u c t a n c e o f about 230 pS o v e r a p o t e n t i a l r a n g e of + 7 0 mV. A t h i g h ' i n t r a c e l l u l a r ' C a § (I0 -4 to 10 -3 M~, t h e o p e n p r o b a b i l i t y was n e a r l y i. T h e o p e n s t a t e w a s p e r manently interrupted by short closures of the channel. H y p e r p o l a r i s a t i o n t o m o r e t h a n -50 m Y d e c r e a s e d the o p e n p r o b a b i l i t y . A n o m i n a l l y Ca +* f r e e s o l u t i o n a p p l i c a t e d to t h e c y t o p l a s m i c s i d e of t h e p a t c h b l o c k e d the c h a n n e l . O n l y d u r i n g d e p o l a r i s a t i o n short openings were rarely registrated. Polymyxin B (50ug/ml) a d d e d ' i n t r e c e l l u l a r l y ' r e d u c e d t h e o p e n p r o b a b i l i t y to v e r y l o w v a l u e s . W e w i l l r e p o r t a b o u t f u r t h e r p h a r m a c o l o g i c a l s t u d i e s and p r o p e r t i e s o f the c h a n n e l at d i f f e r e n t C a §247c o n c e n t r a t i o n s . Neurologische Klinik der Technischen Universit~t MOnchen, M ~ h l s t r a S e 28, D - 8 0 0 0 M Q n c h e n 80

CONTRARY E F F E C T S OF K* AND A D R E N A L I N E ON C O N T R A C TIOlq AND C O N D U C T I O N V E L O C I T Y OF RAT M U S C L E S R.

KOseJer.

F.

Lange,

G.

l
To

o b t a i n more i n f o r m a t i o n a b o u t the i n f l u e n c e of elevated K ~ c o n c e n t r a t i o n and a d r e n e r g i c response during muscular activity both I<+ increase and adrenaline r e l e a s e were s i m u l a t e d in vitro. Thin bundles of m. e x t e n s o r d i ~ i t o r u m Iongus (EDL) and m. s o l e u s I S O L ) were p ~ e p e r e d end a d j u s t e d to ~ive maximum twitch tension. The conduction velocity tc.v.~ was c a l c u i a t e d from r e c o r d e d a c t i o n potentials of two e x t r a c e l l u l a r p l a t i n u m e l e c t r o d e s in a d i s t a n c e of 6 mm a l o n ~ the m u s c l e rabies. [noreasii~g I<+ of s bath s o l u t i o n from S to iO mM i n d u c e d a d r o p o# t w i t c h amplitudes by 28 +/- 4% (EDL, n=lOJ and of 25 +/- S% (SOL. n=6) after 20 man of i n c u b a t i o n . The t e t a n i c t e n s i o n was reduced by a b o u t 50% after iO mi;]. The c.v. decreased by 25% ~EDL~ and IS% LSI3L) after 20 man. Adrelaaiine [O.i to I0 IJM) it]creased the twitch a m p l i t u d e up to 50% end the c.v. up to 35% in beth muscle types after 20 man of e x p o s u r e . As I(e and a d ~ e n e i i n e i n d u c e d o p p o s i t e c h a n g e s [he simultaneous a p p l i c a t i o n or both s u b s ~ a n c e ~ was tested. ]n s o l u t i o n with i0 mM t( ~ a n d i0 ~J]'I a d r e n a l i n e n e i t h e r the d e p r e s s i o n or t w i t c h t e n s i o n nor the drop of the c.v. was i n f l u e n c e d r e m a r k a b l e 9 The decrease was the same as w i t h i0 mM l<+ w i t h o u t adrenaline but, a f t e r r e t u r ~ t o control s o l u t i o n the reversibility was f a c i l i t a t e d . The r e s u l t s suggest that high K+ concentration impaired electrical and mechanical p r o p e r t i e s of m u s c l e cells. AdDenaiine could not prevent these c h a n g e s but seemed to i m p r o v e the r e s t o r a t i o n p r o c e s s e s . B u n d e s s n s t a l t fi]r A r b e i t s m e d i a i n N o l d n e z s t r . 40-42. 0 - i 1 3 4 B e r l i n

67

69

FORCE-ELONGATION DEPENDENCE, TIME COURSE OF RELAXATION AND COLLAGEN CONTENTS OF TENDONS AND ILIO-TIBIAL FASCIAE

KINETICS OF INTRACELLULAR Ca2+-MOBILIZATION BY E X T R A C E L L U L A R A T P IN S M O O T H , M U S C L E C E L L S B.Kalthof, M.Bechem, L.Pott , and M.Schramm 2+ Ca -transients e v o k e d by e x t r a c e l l u l a r A T P h a v e b e e n s t u d i e d in s i n g l e v a s c u l a r s m o o t h m u s c l e c e l l s u s i n g a f a s t f u r a - 2 t e c h n i q u e (Bals e t a l . 1990, Cell Calcium 1!:385-396). The cells were isolated b y e x p l a n t i n g p i e c e s of m e d i a f r o m p o r c i n e a o r t a e a n d s u b c u l t u r e d for u p to 5 p a s s a g e s 9 B y b i n d i n g to t h e p u r i n e r g i c P 2 - r e ~ p t o r , ATP concentration-dependently (K = 2 x l 0 - M% increases 9 ~ D 9 -2+ 9 l n t r a c e l l u l a r Ca -concentratxon [Ca ]., r e a c h i n g m a x i m u m v a l u e s in e x c e s s of 2~M A s t h i s ~ C a Z ~ ] . - i n c r e a s e is n o t i n f l u e n c e d by extracellular ~a , it reflects release from intracellular stores, w h i c h w e r e i n s e n s i t i v e to c a f f e i n e or { ~ a n o d i n e . F o l l o w i n g s t i m u l a t i o n by ATP, t h e I C e - - i t - i n c r e a s e o c c u r e s w i t h a d i s t i n c t l a t e n c y t h a t is i n v e r s e l y r e l a t e d t o t h e . ~ P - c o n c e n t r a t i o n ~ t i m e to 80% e f f e c t 12 s at 1 0 - ~ M a n d 3 s at i0- M). As i o n o m y c i n a c t s w i t h i n less t h a n 1 s, t h i s d e l a y l i k e l y r e flects the activation process ra~er than diffusion of ATP. E v e n u n d e r ATP, t h e Ca b signal declines w i t h i n i man, i n d i c a t i n g a d e s e n s i t i z a t i o n m e c h a nism. A f t e r an A T P - f r e e p e r i o d of a b o u t i0 m a n o n e obtains the origi~l A T P - a n s w e r in t h e p r e s e n c e of extracellular Ca- . In its absence, repetitive a p p l i c a t i o n s of A T P r e s u l t in p r o g r e s s i v e d e c r e a s e of t h e a m p l i t u d e a n d d e l a y e ~ o n s e t of t h e C a - - t r a n s i e n t s . R e a d d i t i o n of C a " restores the normal response. These results indicate that ATP acts via Ca2+-release from intracellular stores, probably with I n s P 3 as s e c o n d m e s s e n g e r . H o w e v e r , as A T P a n d t h e f i l l i n g of t h e s t o r e s a f f e c t t ~ k i n e t i c s of t h e r e l e a s e in t h e s a m e way, a l s o C a ~ - - m e d i a t e d r e l e a s e p r o c e s s e s m i g h t be i n v o l v e d .

A. Rado~aj and H. Lorkovi6 Force-elongation dependence, time course of relaxation and collagen content of various tendon segments from mice and rabbit hindlimbs were measured. We found that segments that are bent around a hypomochlion have physiological properties that differ from those of straight segments. The straight segments have a stronger crimp and consist of fibrils that are more strongly interwoven than the bent segments. This kind of segments has a four times bigger cross section area, is less stiff at such elongations at which the crimp is not staightened out and is stiffer at greater elongations than the first kind of segments. The invivo tension in the straight segments is much smaller then the tension in the bent segments. Nevertheless the bent segments break at a slightly smaller force than the straight segments. Electron microscopy of the segment cross sections reveals much more tightly packed and thinner collagen fibrils in the bent segment. Force relaxation after abrupt elongation proceeds for both kinds of segments in three phases having different time constants. In a series of extensions, each followed by a 30 man recovery period at resting length, the time constants of all three phases become shorter. Significant differences between the relaxation courses of the two tendon types show up in the change of the base line force during the subsequent experiments. In the case of the straight segments the base line force decreases exponentially, as does the maximum force in the beginning of each relaxation. In the case of the bent segments the base line force remains constant. The same relaxation experiment series is made with collagen bundles taken out of the ilio-tibial fascia of children suffering from Duchenne muscle dystrophy. We found that both the longitudinal and transversal collagen bundles behaved as the straight tendon segments of mice. Abteilung fOx Allgemeine Physiologic, Universit~it [rim, I)-7900 Ulm, Germany

I n s t i t u t f~r P h a r m a k o l o g i e , B a y e r AG, W u p p e r t a l 9 I n s t i t u t f~r Z e l l p h y s i o l o g i e , U n i v e r s i t ~ t B o c h u m

R 85 70 E F F E C T S OF BRL 38227 ON WHOLE CELL CURRENTS IN VASCULAR SMOOTH MUSCLE Th. Noack, P. Deitmer, G. Edwards, A.H. Weston & K. Golenhofen Single cells from the rat portal vein were investigated using whole cell voltage clamp. In a calcium-free solution (1 mM EGTA), depolarizing voltage steps from a holding potential of -90 mV to 0 mV induced two major outward currents, one of which (ITo) inactivated to a steady current level within seconds. This steady current (INi) was also elicited by depolarization from -10 mV to 0 mV. When BRL 38227 (1 pM) was added to the bathing solution, currents at the holding potential (-10 mV) became noisy and were increased in the outward direction. This was more pronounced when the BRL 38227 concentration was elevated to 10 pM. Then, the magnitude of the current-voltage relationship increased, intersecting the control curve near the potassium equilibrium potential (EK), indicating the activation of a potassium current (IKco). The effect of BRL 38227 on ITO was studied using a holding potential of -90 mV. ITO was determined as the difference between the currents elicited by stepping from -90 mV and those elicited from -10 mV (INI ' IKCO). BRL 38227 (10 pM) inhibited a large part of ITO but had no effect on the v o l t a g e - d e p e n d e n c y of the steady-state activation curve. However, the net effect of BRL 38227 was always an increase of outward current at membrane potentials b e tw ee n EK and +10 mV. Both effects were antagonized by glibenclamide (10 pM), although the effect on ITO was smaller than on IKC O. Using stationary fluctuation analysis the single channel conductance Underlying IKcOWaS calculated to be 21 pS at 0 mV in physiological potassium concentrations. Supported by DFG (Go 130130-2). Physiologisches Inetitut der Universit~.t Marburg, DeutschhausstraBe 2, W-3550 Marburg.

71 ENDOTHELIUM DIFFERENTIALLYINFLUENCES THE RELAXING EFFECTS OF INHIBITORSOF TWO LOW Km CYCLIC AMP PHOSPHODIESTERASESIN RAT AORTA C. Lugnier, N. Komas,and J.C. Stoclet

72

DEXAMETHASONE PREVENTS L-ARGININE DEPENDENT I N D U C T I O N OF V A S C U L A R H Y P O R E A C T I V I T Y BY E N D O T O X I N / N V/VO, EX VIVO A N D / N VITRO G.A. Gray, D. Paya, I. Fleming and J.C. Stoclet Anti-inflammatory glucocorticoids such as dexamethasone (DEX) were recently shown to prevent activation by bacterial endotoxin (ETX) of nitric oxide (NO) synthesis from L-arginine in various tissues (Knowles et al., Biochem. Biophys. Res. Commun., 172: 1042-1048, 1990). The purpose of the present study was to examine the effect of DEX on ETX induced vascular hyporeactivity in various conditions which we have previously shown to involve activation of the L-arginine/NO pathway. Hyporeactivity to noradrenaline (NA) in anaesthetised rats receiving an infusion of ETX (10 m g "1 k g ' l h -1, i.v.) was prevented by pretreatment of rats with DEX (10 mg kg -1, i.p.). Ex vivo, in aortic rings (- endothelium) removed from rats 4h after saline or ETX (20 mg kg -1, i.p.) administration, DEX (5 m g kg-1, i.p.) prevented the rightward shift of the NA dose response curve (PD2 values of 8.03i-0.14 vs 7.41+0.19, P<0.05 respectively in untreated rats and 8.06+0.18vs 7.8+0.11, N.S. respectively in DEX pretreated rats) L-arginine induced a significant (P
73

MECHANISM OF THE CONTRACTIONS INDUCED BY FLUORIDE IONS IN RABBIT AORTA AND IN PORCINE CORONARY ARTERIES. H.Nguyen-Duong Fluoride ions (2 - 12 mM ) elicited in isolated rabbit aorta and porcine coronary arteries dose-dependent contractions in presence and absence of Cao. The Finduced contractions in Ca containing PSS (unaffected by organic Ca-antagonists and only very slowly relaxed by nitrates or Na-nitropruseide) were almost instantaneously and transiently relaxed by pH~manipulations, such as addition and removal of NH4CI or removal or reintroduction of CO2/HCO3- performed at a constant pH o. The transient relaxations were followed, with NH4+ still present in the bath, by extreme overshooting of the tension to levels approximating 50 % of high K+oinduced contractions (Fig.a). The strong enhancement of the phase 2 observed during F-induced contractions, which might reflect a stimulation of CF/HCO 3" exchange, can be attributed to the ability of F to replace CI- at the anion exchanger, inducing an intraceUularacidification by increasing the rate of HCO3"extrusion. Concentration-response curves for F-induced contractions were consequently shifted to the right and depressed by 10.4 M SITS, an inhibitor the anion-exchanger (Fig.b).

Two low Km cyclic AMP phosphodiesterases (PDEs): the cyclic GMP-inhibited PDE (PDE III) and the rolipram-sensitive PDE (P,DE IV), regulate cAMP levels in many tissues. To investigate their respective roles and tliat of endothelium in vascular relaxations, the effects of selective PDE inhibitors were studied on the PDE III and IV isolated by DEAE-sephacel chromatography from denudedendothelium rat aorta and on the relaxation of isolatedaortic rings, with and without functional endothelium precontrac,ted with noradrenaline (II~M). Nat aortic PDE III was selectively inhibitea in the ~M range by C1930, LY 195115 and milrinone and PDE IV was selectively inhibited by1 l.d~ rohpram or denbufylline. The selective PDE III inhibitors induced endothelium-inaependent relaxations (EC~ values in the presence and absence of functional endot~elium: 8 and 5 pMfor (31 930, 3 and 3 ~M for LY 195115 6 and 9 I~M for mitrinone, respectively). Surprisingly, selective PDE IV inhibitors induced relaxation only in the presence of functional e..ndothelium (ECso values, 30 pM for denbufylline and 152 pM for rolipram). N~-monomethyl L-arginine (300 I~M), an inhibitor of NO production, did not modify the relaxing effect of selective PDE III inhibitors but abolished the relaxing effects of selective PDE IV inhibitors. An inhibitor of soluble guanylate cyclase, methylene blue (10 ~M), reversed the relaxing effects of all the tested PDE inhibitors in all conditions. To further investigate the endothelium-dependent relaxing effects of selective PDE IV inhibitors, their effects were studied in the presence of drugs stimulating either adenylate cyciase (forskolin 3 I~M and moproterenol 0.1 p~M) or soluble guanylate cyclase (sodium nitroprusside SNP 1 nM) or inhibiting PDE III (milrmone 30 nM). These drugs were used at concentrations which produced 10% relaxation by themselves. In endotheliumdenuded rings, a relaxing effect of both denbufylline and rolipram was revealed in the presence of milrinone (ECso values 2 and 12 p.M, respectively) or SNP (ECs0 values 12 and 124 p.M respectively) but not in the presence of forskolin or isoproterenol. However in the presence of functional endothelium relaxations produced by denbufylline and rolipram were significantly potentiated by forskolin, Jsoproterenol, milrinone and SNP (respective EC~sovalues for denbufylline: 2, 2, 0.4 and 0.7 pM and respective ECsovalues for rolipram: 7, 13, 7 and 1 pM). These results show that PDE III inhibitors induce an endobhelium-independent relaxation, whereas PDE IV inhibitors modulate vascular tone only in the presence of a PDE III inhibitor or of guanylate cy,.claseactivators(EDRF and SNP). It is @roposedthat cyclic GMP potentiates cyclic AMP-mediatea relaxation and that inhloibon of PDE Illmay be involved in this phenomenon.

F-induced contractions seems thus to be due to cytoplasmic acidification resulting from a shift in the balance of a dual pH~ regulator system of the cell membrane, including both a Na+/H+-exchanger for extruding H+ and a Cl-/HCO3"-exchangerfor counteracting a displacement in alkaline direction. Supported by the Deutsche Forschungsgemeinschaft (Ng-1/1-1)

Laboratoire de,PharmacoLogie Cellulaire et Mo4cuaire, CNRS URA 600, niversit4 Louis ffasteur ae 8ttas~ourg, BP 24, 67401 IIIkirch, rrance

Dept.of General Physiology, University of UIm, 7900 UIm, Germany.

a

tq

b

PCA

/o /

I00- %

5%

9 SITS

10-"M

---Z~i/, K*50

N'~- 3O mM F-1 mM

2

4

6

,

,

8

12 mMF-

R 86

74

76

H I G H E R TRANSCAPILLARY FLUID LOSS IN ENDURANCE TRAINED SUBJECTS D U R I N G ORTHOSTATIC STRESS W. Hildebrandt, H. Schiitze, J. Stegemann

COMPARISON

Introduction. The effect of endurance training on orthostatic tolerance is discussed controversially in literature. Lower and unchanged orthostatic tolerance is reported. The present study compares quick as well as exspected slow bemodynamic changes during upright tilt of two groups (trained vs. normal). Methods. Two groups of male sports students (VO2max = 62,7+5,8 ml/min kg, n=9 vs. VO2max = 45,1+4,9 ml/min kg, n=9) performed an orthostatic test on a tilt table (I0 rain supine - 10 min upright - 10 min supine). The following parameters were measured : Heart rate (ECG), brachial blood pressure (Riva-Rocci), peripheral blood pressure (Finapres), right calf volume changes (impedance) and calf blood flow (venous occlusion technique). VO2max was determined separately during a bicycle ergometer test. Results. Both groups did not differ in total calf volume, calf circumference, body weight and height. During the orthostatic test, no syncopia occurred. Basic heart rate was lower in the trained group, b u t tilting induced changes in heart rate as well as in blood pressure were similar in both groups. In contrast, the transcapinary filtration rate in upright position as derived from calf volume changes was two times higher in the trained group during entire standing time (0.70ml/min 1 vs. 0.36ml/min 1). This occurs despite higher reduction of calf blood flow in that group. Conclusions. Endurance trained subjects have a higher loss of iutravasal fluid during orthostatic stress. This steady fluid loss estimated for both legs can amount up to 200 ml within 10 miu and occurs in addition to the fast blood shift. Whether or not this factor plays a limiting role in orthostatic tolerance depends on the fluid state before upright tilt.

H.-M. R e h ~ e l d t , Herr'mann

Physiologisches Institut, Deutsche Sporthochschule K61n, Carl-Diem-Weg 6, D - 5000 K61n 41, Germany

OF

INDIVIDUAL

CARDIOVASCULAR

TO STATIC AND D Y N A M I C M U S C L E T H E R M A L STRESS. AND M E N T A L L O A D D.

Peikert,

G.

RESPONSES

CONTRACTION.

Ca~fier.

H.

LOCAL

~all.

Previous studies on c a r d i o v a s c u l a r responses to mental load s u g g e s t that psychologic behavioural p a t t e r n s are r e l a t e d to the individual c a r d i o v a s c u lar r e a c t i v i t y of h e a l t h y subjects. Our study was i n t e n d e d to a n a l y s e the i n f l u e n c e of personality traits on cardiovascular activation d u r i n g d i f f e r e n t kinds of s t r e s s o r s i u o l u d i n ~ t o t a l m u s c u l a r work. The d o m i n a n t c o p i n g styles of 30 h e a l t h y m a l e students were deter mine~ u s i n g a q u e s t i o n n a i r e
Arbeitsmedizin. 0-1134 Berlin

75

77

CARDIOPUI~ONARY PAPJ~TERS DURING DYNAMIC WORK W I T H S~SLLL A N D ~ A N I~JSCLE ~ i S S E S I N D E P E N D E N C E ON GENDER AND AGE H. P r a u e n d o r f , E. P f i s t e r , U. K o b r y n , W. G e l b r i c h a n d B. H a r t m a n n ....

V E G E T A T I V E CARDIOVASCULAR CONTROL I N THE RANGE OF ENDURANCE L I M I T FOR DYNAMIC MUSCLE WORK E.

Pfister

Physical

T h e p u r p o s e of t h e e x p e r i m e n t s w a s to c h a r a c t e r i z e t h e bellavi011r of o s ~ d i o p u l m o n a r y parameters during o n e - a r m a n d t w o - a r m 0rar/~ing i n h e a l t h y , u n t r a i n e d z%sle sled f e m a l e s u b j e c t s b e t w e e n I % sold 4 9 y e a r s , The cardiopulmonary system reacts in youth in a similar kind as in adults but the regression lines of h e a r t r a t e a n d o x y g e n u p t a k e i n d e p e n d e n c e o n lead are the steeper the smaller the working m u s c l e m a s s is. T h e r e g r e s s i o n l i n e s o f h e a r t r a t e a n d o x y g e n u p t a k e of y o u t h a r e s m a l l a b o v e t h e corresponding ones of the adults. The physical endurance limit (maximal load which could be tolerated over hours without progressive fatigue) shows differences in dependence on gende~ age, and working muscle masses. In two-arm cranking male youth show a smaller mechanical performance and a higher oxygen uptake comparing with adult males. In one-arm cranking the oxygen uptake is higher as in adults. In female youth in two-arm cranking the mechanical performance and net efficiency are smaller as in adults but the oxygen uptake is equal. In one-arm cranking the cardiopulmonary reactions in female y o u t h a r e s i m i l a r to a d u l t s . T h e p h y s i c a l e n d u r a n ce l i m i t s i n w o m e n ( m e c h a n i c a l p e r f o r m a n c e , oxygen uptake) are generally smaller as in male subjects. These limits are smaller in one-arm cranking comparing with two-arm cranking but the reaction of systolic blood pressure in dependence of l o a d i s the h i g h e r the s m a l l e r t h e w o r k i n g m u s c l e m a s s is. Gesundheitszentrum Springpfuhl, siologie, Allee der Kosmonauten D-0-1140 Berlin-Ymmzahn

Abt. Arbeitsphy47, 2 P 96,

L.

and

8.

endurance

Hartmann limit

(PEL)

characterizes

the level of strain, w h i c h shall not be exc e e d e d in the a v e r a g e aT w o r k i n g shift to get s u f f i c i e n t r e c o v e r y uD to the f o l l o w i n g . In the l i t e r a t u r e was suPPosed, that the p h y s i cal PEL is c o n n e c t e d w i t h the e n d of vogel h e a r t r e g u l a t i o n or w i t h a m i n i m a l s i n u s a r r h y t h m i a , Our e x p e r i m e n t s i n c l u d e d 106 h e a l t h y y o u t h b e t w e e n 16 and 18 y e a r s [66 m a l e and 39 female), T h e r e Was f o u n d an e s s e n t i a l v o g e l i n f l u e n c e on the c o n t r o l of heart r a t e d u r i n g w o r k in the r a n g e of PEL. F u r t h e r m o r e i n t e r i n d i v i d u a l v a r i a b i l i t y of the heart r a t e s h o w s two r e g u l a t i o n 9 r o u ~ s e s o e o i a l l Y in rest and r e c o v e r y . I n s t i t u t for A r b e i t s m e d i z i n d e r M e d i z i n i s c h e n A k a d e m i e M a g d e b u r g . L e i p z i g e r Str. &&, 0 - 3 0 9 0 Magdeburg.

R 87

78

8O

NUTRITIONAL STATUS AND FITNESS IN THE MILITARY POPULATION. W. von Restorff, W. Klops, S. Zickgraf, and K.-H. Bach

EEG ACTIVATION E. H a n s e n , Th.

if nutrition supply exceeds daily energy demands the nutritional status will be of great importance for military readiness and general health and epidemiological aspects. Thus body fat content (BF %) was compared to a physical fitness test score in four groups: A consisting of 686 soldiers of all ages and ranks in various units in Koblenz, B and C consisting of 54 female and 442 male recruits, respectively. Group D consisted of 60 drafted recruits for parachutist basic training. Body weight (BW), body height (BH), circumferences of abdomen and hip (AC and HC) were measured and compared to calculated body fat content (BF) (4 skinfolds, according to Durnin, J.V.G.A. and Wormersley, J. : Brit.J.Nutr. 32 :77-97 (1974)) and to the score of the military physical fitness test (PFT). Linear regressions to age were calculated in group A. In group A both average BF and PFT-scores (although not available in all) are in a desirable range (Tab.l). As in this group statistical analyses couldn't detect differences in anthropometry of soldiers with or without PFT-scores data are given together. Regression analyses to age reveal that with aging BW, AC and BF increase while PFT-scores, however, improve (Tab. 2) . During basic training (12 weeks) groups B, C and D show an average drop in BF by 5 % and a considerable augmentation of fat free bodymass. Table i." Anthropometry in groups A, B, C, and D Age A BWA BHA AC A BFA PFT A BF B BF C BF D [y] [kg] [ c m ] [cm] [%] [aU] [%] [%] [%] Mean 24.9 77.8 179.8 84.8 18.3 3.6 27.6 16.6 15.5 SD 7.9 10.9 6.7 8.7 5.6 0.9 3.8 5.3 5.2 Min 18.0 51.3 157.2 64.0 5.7 1.0 19.7 7.2 7.7 Max 56.0 122.0 204.0 123.0 37.8 6.0 36.2 32.7 29.4 N 680 686 686 685 685 369 54 441 60 Table 2: Regression analysis (y = a + bx; r) BW BH AC HC BF PFT a 72.6 182.2 74.6 94.3 10.6 4.4 b .21 -.09 .41 .13 .31 -.04 r .15 -.ii .38 .08 .43 -.25 p <.05 n.s. <.05 n.s. <.05 <.05

T h e a i m of o u r s t u d y is to d e t e c t r e l a t i o n s b e t w e e n motor imagination and CNS activation by means of E E G p a r a m e t e r s . T e n s t u d e n t s in s p o r t s w e r e e x a m i n e d d u r i n g r e p e t i t i v e m e n t a l t r a i n i n g of s w i m m i n g s k i l l s . O u r s t u d y is b a s e d o n r e c o r d i n g s w i t h 16 E E G c h a n n e l s (10-20 system) a n d t h e c o m p u t a t i o n of spectral parameters of E E G b a c k r o u n d activity in five frequency bands. Power values are obtained f r o m E E G u n d e r r e s t i n g c o n d i t i o n s as w e l l as f r o m the recordings during the ideomotoric swimming p h a s e s . T h e y a r e c o m p a r e d for s i g n i f i c a n t d i f f e r e n ces. T h e r e s u l t s a r e d e m o n s t r a t e d in a t o p o g r a p h i c form. s i g n i f i c a n t d e c r e a s e s of t h e A l p h a - a n d T h e t a - p o w e r o f E E G in p a r i e t a l and occipital regions of both hemispheres in i m a g i n a t i o n p h a s e s a r e s h o w n . T h i s is i n t e r p r e t e d as t h e p a r t i c i p a t i o n of t h e v i s u a l cortex during spatial imagination. Repetition of the motor imagination during one session involves more cerebral regions in t h e n e u r o p h y s i o l o g i c a l p r o c e s s , w h i c h is s h o w n by a s i g n i f i c a n t d i m i n u t i o n of A l p h a - , T h e t a - a n d B e t a - l - p o w e r of t h e c e n t r a l a n d f r o n t a l areas, too. T h e s e c h a n g e s m a y b e i n t e r p r e t e d as a s p e c i a l and m o r e p r o n o u n c e d a c t i v a t i o n of r e g i o n s i m p l i c a t e d in t h e m o t o r system. Furthermore, c o n t i n u o u s r e c o r d i n g s of c a r d i a c a n d r e s p i r a t o r y a c t i v i t y w e r e m a d e d u r i n g all p h a s e s of session. In addition the students were asked to a s s e s t h e i r c u r r e n t i n t e r n a l s t a t e by m e a n s of t h e "Eigenzustandsskala" from Nitsch. The application of "Amthauer-Intelligenz-Struktur-Test" allows us to r e p o r t a b o u t t h e a b i l i t y to s p a t i a l i m a g i n a t i o n .

Ernst-Rodenwaldt-lnstitut, D-W-5400 Koblenz, Germany

ZInstSanBw KOB, Viktoriastr.

PATTERNS DURING MOTOR IMAGINATION Weiss, R. R o s t a n d L. B e y e r

Friedrich Schiller University, Institut logy, T e i c h g r a b e n 8, Jena, D - 0 - 6 9 0 0

79

81 EEG changes during s k i l l s in a p o p l e c t i c

Friedrich-Schiller-University, Physiology, D - O - 6 9 0 0 Jena, T e i c h g r a b e n 8

Institute

of

Physio-

11-13

CENTRAL NERVOUS ACTIVATION ACCOMPANYING MOTOR PERFORMANCE (A STUDY OF EEG-BACKGROUND ACTIVITY) L . B e y e r , T h . W e i 6 , E l l e n Hansen, R . R o s t Activation processes within the whole organism t a k e p l a c e in r e l a t i o n to p h y s i c a l load. E E G b a c k g r o u n d a c t i v i t y w a s a n a l y s e d in r e l a t i o n t o physical exercise and motor imagination. A significant decrease in the mean alphafrequency (MAF) after bicycle ergometer load and specific exercise in wrestling is interpreted as indication of higher CNS activation. Different changes above different cortical areas give the possibility to d i s t i n g u i s h b e t w e e n m o r e u n s p e c i f i c or s p e c i f i c activation processes. C h a n g e s in t h e E E G is s h o w n a l s o d u r i n g m o t o r imagination. S i g n s of a c t i v a t i o n a r e g i v e n b y d i m i n i s h e d p o w e r in t h e a l p h a - b a n d as w e l l as in t h e t h e t a - a n d b e t a - b a n d . T h e c h a n g e s in t h e occipital and parietal regions during a single p e r i o d of m o t o r i m a g i n a t i o n a r e c o m b i n e d w i t h the rising activity during repeated imagination periods interpreted as a more specific functional level, demanding the internal controlling circuits of m o t o r system during i d e o m o t o r i c m e n t a l p r a c t i c e s i m i l a r y to t h e s e of r e a l m o t o r acts.

of

mental practice patients

of

motor

T. W e i s s I, E. H a n s e n I, L. B e y e r I, R. R o s t I, Oh. N i c h e l m a n n 2, F. M e r t e n 2 ................................................ 15 a p o p l e c t i c p a t i e n t s u n d e r h o s p i t a l c o n d i t i o n s were requested to imagine their own arm movem e n t s of t h e h e a l t h y as w e l l as of t h e i m p a i r e d h a n d s i d e (mental p r a c t i c e ) . T h e m e n t a l p r a c t i c e was a part of a therapeutic treatment which consisted of physiotherapy (individual and in g r o u p s ) , w o r k i n g therapy, p s y c h o t h e r a p y etc. T h e mental practice sessions were performed once a day. E E G w a s r e c o r d e d f r o m 4 (C3-A2, C4-AI, P 3 A2, P4-AI) or 8 (C3, CZ, C4, P3, PZ, P4, O1, 02, reference - linked ear lobe electrodes) electrodes. Apoplectic patients show clear diminution of theta, a l p h a a n d b e t a - I p o w e r of E E G d u r i n g m o s t t e s t s i t u a t i o n s . T h e c h a n g e s of E E G p a r a m e t e r s f r o m r e s t to m e n t a l p r a c t i c e a r e d i f f e r e n t f o r t h e d i f f e r e n t p a t i e n t s . A n e f f e c t of r e p e t i t i o n of m e n t a l p r a c t i c e w a s n o t f o u n d g e n e r a l l y . T h e c h a n g e s of E E G p a r a m e t e r s w i l l b e d e m o n s t r a t e d b y e x a m p l e s of s i n g l e c a s e s a n d w i l l b e d i s c u s s e d in c o n n e c t i o n with the locus of insult as well as with neurological and psychological data. 1 2

F r i e d r i c h S c h i l l e r U n i v e r s i t y Jena, I n s t i t u t e of P h y s i o l o g y , T e i c h g r a b e n 8, JENA, D - 0 - 6 9 0 0 C l i n i c of G e r i a t r y , C l i n i c u m B e r l i n - B u c h

R 88 82

84

M O D I F I E D W I N G A T E TEST: C O N S T A N T PEDALLING FREQUENCY, V A R Y I N G BRAKING FORCE H.-V. Ulmer and N. Rybczynski .................................................. U s u a l l y the W I N G A T E TEST for maximal cycloergemetric p e r f o r m a n c e over 30s is p e r f o r m e d with v a r y i n g p e d a l l i n g frequencies and constant forces, including a great v a r i a t i o n of e f f i c i e n c y b e t w e e n 140 and 70 rpm. Therefore we m o d i f i e d a M O N A R K ergometer among other things as follows: Device for precise m o d u l a t i o n (by hand) of force with a quasi rectangular initial increase, linear r e g i s t r a t i o n of force by a spring balance. A c c e l e r a t i o n of the flyweel (increased inertia: 4,55 kg/m ~) by an electric motor. Test procedure: A c c e l e r a t i o n of flyweel until ca 110 rpm, after r e a c h i n g increase of force and begin of p e d a l l i n g simultaneously. D i m i n i s h i n g of force by hand so, that rpm remain constant over 30s. Continuous recording of rpm and force for later calculation of performance. Methods: 16 athletes (different endurance state), 5 repeated experiments each. Results: Peak performance: 1000 W • 16%, max: 1430 W = 16,3 W/kg. Mean p e r f o r m a n c e (over 30 s) : 710 W • 14,8% (max: 950 W = 10,8 W/kg). Intraindividual reproducibility: ca 6%. Typical performance sections were calculated over periods of 5 s, including fatigue index and decrease of performance. Conclusion: A l l t o g e t h e r the m o d i f i f i c a t i o n p r o o v e d to be practicable, enabling a better standardization, e s p e c i a l l y concerning efficiency.

Why do ankle edemas p r i m a r i l y d e v e l o p in w a r m environment? C. Stick, U. Hiedl, E. W i t z l e b

Sportphysiologische Abteilung, FB 26, Johannes Gutenberg-Universit~t, Saarstr. 21, W-6500 Mainz

U s i n g m e r c u r y in r u b b e r strain gauges low pass filt e r i n g of the p l e t h y s m o g r a p h i c recordings e n a b l e s to r e c o r d s l o w v o l u m e changes in the leg d u r i n g bicycling. By a p p l y i n g this technique reductions in calf v o l u m e d u r i n g cycle ergometrie were found (Funktio n s a n a l y s e b i o l o g i s c h e r Systeme 19, 187-193 (1989)). However, d e p e n d e n t edemas are p r i m a r i l y o b s e r v e d in the ankle region. T h e r e f o r e the previous findings are s u p p l e m e n t e d by v o l u m e m e a s u r e m e n t s w i t h the s t r a i n - g a u g e s p l a c e d near the ankle. M e a s u r e m e n t s w e r e done in 24 subjects d u r i n g m o t i o n l e s s s t a n d i n g for 12 min d u r a t i o n and cycle ergometer e x e r c i s e for 17 min (50 W at 50 rpm). The experiments w e r e perf o r m e d at different ambient temperatures of 20, 28 and 36 ~ C at 50% r e l a t i v e h u m i d i t y each. D u r i n g orthostasis the rate of the slow v o l u m e increase, that is largely due to the increase in the extrav a s c u l a r compartment, did not s i g n i f i c a n t l y differ at the three temperatures being 0.08%/min at 20 ~ C and 0.09%/min at 28 ~ and 36 ~ C respectively. D u r i n g cycling, however, the ambient temperature had a m a r k e d influence on the edema p r e v e n t i v e e f f e c t of m u s c l e e x e r c i s e w i t h the most p r o n o u n c e d e f f e c t at 20 ~ C. The m e a n rate of decrease were -0.028 and - 0 . 0 0 8 % / m i n at 20 ~ and 28 ~ C respectively. At 36 ~ C the v o l u m e did not change significantly. These results indicate that the volume changes in the ankle region d i f f e r o n l y in q u a n t i t y from those o b s e r v e d in the calf, but invariably are less pronounced. The results c o n f i r m that ankle edemas d e v e l o p in w a r m e n v i r o n m e n t b e c a u s e the muscle p u m p is less effective in p r e v e n t i n g edema. By constrast the rate of c a p i l l a r y filtration is only a f f e c t e d s l i g h t l y by temperature. Institut fur angew. Physiologie und m e d i z i n i s c h e Klimatologie, Olshausenstr. 40, D-2300 Kiel

85

83 EARLY ADJUS~4ENT OF PERIPHERAL BLOOD FLOW DURING A N D A F T E R I S O M E T R I C E X E R C I S E I N BXJMANS

D. Leyk, D. EBfeld, K. Baum, U. Hoffmann, J. Stegemann i0 healthy male volunteers carried out static e x e r c i s e in u p r i g h t b o d y p o s i t i o n at 90 ~ k n e e - j o i n t flexion. Extensions of the right foot had to be performed for is, 6s, 10s, 20s, 30s and 40s corres p o n d i n g to about 5% and 25% of m a x i m a l v o l u n t a r y c o n t r a c t i o n (MVC). In the femoral a r t e r y b l o o d f l o w was m e a s u r e d b e a t - b y - b e a t using a p u l s e d D o p p l e r device. At 5% M V C an initial, fast increase in b l o o d flow p a r a m e t e r s was followed b y a m a r k e d decrease. The p o s t - e x e r c i s e hyperemia was small showing no disc e r n i b l e effect of e x e r c i s e duration. At 25 % M V C the initial f l o w r e s p o n s e was augmented. Exercise levels w e r e r e a c h e d w i t h i n the first 5s. In c o n t r a s t to the exercise at 5% a steep i n c r e a s e of b l o o d flow o c c u r e d i m m e d i a t e l y a f t e r c e s s a t i o n of e x e r c i s e at 25% MVC. Additionally, the d u r a t i o n of the p o s t e x e r c i s e hyperemia was p o s i t i v e l y c o r r e l a t e d w i t h the e x e r c i s e duration.

POSTURAL EFFECTS ON THE RESPONSE OF CARDIAC OUTPUT AND BLOOD PRESSURE TO SHORT-TERM BARORECEPTOR STIMULATION H. Sch0tze, W. Hildebrandt, J. Stegemann Introduction. Investigations on short term baroreceptor control of cardiac function have so far mainly focussed on heart rate/RR-Interval. This study uses impedance cardiography as a beat-by-beat method to record stroke volume (SV) simultaneously with neck-suction induced RR-interval changes. Moreover, continuous non-invasive blood pressure readings are done to assess feed-back effects on transmural pressure. Method. Using a neck-chamber system for quick, R-wave-trlggered pressure changes, a beat-by-beat pressure profile was applied starting at +40mmHg, followed by a 7 step suction (-15mmHg) down to -65mmHg ('Eckberg profile'). RRintervals and the first derivative of thoracic impedance changes (tetrapolar 40 kHz device) were recorded for 7 repetitions of the pressure profile. These recordings were performed on 9 subjects in 5 tilting positions (-i0 ~ 0~ 30 ~ 60 ~ 90~ Additionally, blood pressure responses (Finapres) were compared in two positions (0~ 90 ~ in a different group of 16 subjects. Results. The well-known sigmoid RR-interval/ pressure relation reveals almost no postural influence. In contrast the SV response differs widely with position: when upright, SV is well maintained during suction, when horizontal or tilted head-down, there is an initial strong decrease followed by a subsequent increase. The resulting cardiac output is almost stable when upright, in supine subjects however, it is reduced by up to 40 %. Systolic blood pressure increased during suction in upright position, while mean and diastolic values decreased during suction. Conclusions. The response of cardiac output reveals a marked postural difference not reflected by heart rate. This is mainly due to a diffe~'ent inotropic response to neck-suction that cannot be explained by differences in preload alone. Direct inotropic drives as well as opposing interaction from baroreceptors responding to blood pressure changes may play a role.

The p r e s e n t results suggest that the m u s c u l a r perfusion kinetics at the onset of static e x e r c i s e is i n i t i a t e d by a d e c r e a s e in local venous p r e s s u r e ("muscle pump"). The subsequent flow values are influenced by p a s s i v e vessel c o m p r e s s i o n and changes in local v a s o m o t o r tone. A fast w i t h d r a w a l of m u s c l e compression, refillung of veins and a slow re- Physiologisches Institut, Deutsche SporthochschuIe K6in, a d j u s t m e n t of v a s o m o t o r tone d e t e r m i n e the post- Carl-Diem-Weg 6, D - 5000 K01n 41, Germany e x e r c i s e f l o w kinetics. P h y s i o l o g i s c h e s Institut der D e u t s c h e n Sporthochschule K61n, C a r l - D i e m - W e g 6, W-5000 K 6 1 n 41

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86 VASOPRESSIN (ADH) AND fl-ENDORPHIN LEVELS DURING AND AFTER PHYSICAL EXERCISE AND WARM WATER IMMERSION H.-Chr. Gunga, K. Kitsch, L. R6cker ADH and lactate were regarded as potent stimulators for the secretion of the fl-endorphines from the Proopiomelanocortin (POMC) ceils. Therefore experimental models were tested known to raise the ADH secretion (exercise, warm water immersion (WWI)) and the lactate production (exhaustive exercise). Applying these models is, however, followed by increases of the body temperature which might also influence the /3endorphine secretion. 8 healthy male subjects were exposed to exhaustive ergometer exercise, to WWI and to thermoneutral water immersion (TWI). The following parameters were measured: body weight, rectal and oral temperatures, blood lactate, ADI-I, fl-endorphine in the plasma. During exercise the levels of ~-endorphine, ADH, lactate, and body temperature increased as expected. WWI led to an increase of the ~endorphine and the ADH (p < 0.001) leaving lactate levels unchanged. TWI lowered ADH levels slightly leaving fl-endorphine and lactate levels unchanged. However, the body temperature increased slightly. From these results we conclude that indeed increased ADH levels stimulate the fl-endorphine secretion. The role of the body temperature remains undetermined, apparently only increases above 38~ are effective, whereas lactate has apparently no direct effect on the fl-endorphine secretion. Institut f~ir Physiologic der Freien Universitiit Berlin, Arnimallee 22, D-1000 Berlin 33

88 VARIATION IN RHYTHMICAL OSCILLATIONS OF THE PERIPHERAL PULSE PARAMETERS UNDER A DEFINED ANTIORTHOSTATIC STRESS V.

Sinz

and S.

H~uBler

The p e r i p h e r a l volume pulse achieved noninvasively and c o n t i n u a l l y show c h a r a c t e r i s t i c a l oscillations in the stationary state. They express rhythmical changes in the tonic of the vessels in the measured areas (finger, toe). The method of spectral analysis yields frequencies and the amount of this periods, their depandance from each o~her and their phases. In case of an antiorthostatic stress (patient position with the h e a d 8" l o w e r ) over 7 days the parameters of both the photoand t h e i m p e d a n c e v o l u m e - p u l s e measured i n a t i m e o f 10 m i n . p e r d a y showed an i n c r e a s e in t h e number o f o s c i l l a t i o n s for periods o f a b o u t 10 s and f o r t h o s e o f a b o u t 20 s and an i n c r e a s e of the oscillations w i t h 60 s p e r i o d s being registred from the 2rd to the 4th day. In this time the number of the circaminutes rhythm of the heart frequency oscillations increases. The o s c i l l a t i o n s of the photopuls amplitudes of circa 12 s had a maximum on the 4th and 5th day but the circaminutes rhythm h a v e a m i n i m u m . The g r a d e o f dependance between the pulse oscillations in a finger and a t o e was t h e l o w e s t on t h e 3 r d and 4 t h day in the minute range, the decoupling o f t h e IPO and PPG f i n g e r p u l s e s was r e g i s t r e d on t h e 6 t h and 7th day of the experiment. The c h a n g e s i n t h e f o r m of oscillations of the peripheral pulses is explained by the local and cranial volume and pressure changes yield by the antiorthostatic stress. Medizinische Akademie Dresden, Physiologie und P a t h o p h y s i o l o g i e , D-O 8019 D r e s d e n

87 CARDIOVASCULAR HEAD-UP

TILT

REACTIONS UNDER

THE

TO

DIFFERENT

CONDITIONS

OF

DEGREES

OF

NORMOXIA,

HYPOXIA AND HYPEROXIA A. S k a l w e i t and P. KreiBig Several investigations indicate that the arterial c h e m o r e c e p t o r s participate in the control of card i o v a s c u l a r function. The aim of the p r e s e n t study was to e x a m i n e the influence of inspiratory pO 2 u p o n the adaptation to different degrees of head up tilt. The investigations were performed on 41 h e a l t h y y o u n g m e n u s i n g noninvasive techniques. From the r e c o r d i n g s of ECG, PCG, ICG and s p h y g m o m a n o m e t r i c m e a s u r e d blood pressure, heart rate (HR), systolic, d i a s t o l i c and m e a n arterial blood p r e s s u r e (BPS, BPD, BPM) as well as relative changes in stroke v o l u m e (SVF), cardiac output (COF) and total peripheral resistance (TPRF) w e r e a n a l y z e d . W h i l e b r e a t h i n g normoxic, hypoxic (12% 02) or h y p e r o x i c (100% 02) gas m i x t u r e s the subjects u n d e r w e n t 30 ~ (n = 20) or 65 ~ (n = 21) of head-up t i l t i n g . U n d e r n o r m o x i a both tilting tests caused the w e l l k n o w n c h a n g e s of h e m o d y n a m i c p a r a m e t e r s . W h i l e r e l a t i v e changes in HR and SVF in the 65 ~ p o s i t i o n w e r e g r e a t e r than in the 30*- position, there w e r e no d i f f e r e n c e s in the decreases of COF and TPRF. V a r i a t i o n s in the inspiratory pO 2 didn't m o d i f y the p r i n c i p l e r e a c t i o n to orthostasls. But u n d e r hypoxia in c o m p a r i s o n with normoxia smaller increases in BP and TPRF were seen, possibly because of a h i g h e r starting-point of vascular sympathetic tone. The results support the hypothesis that c h a n g e s in the inspiratory pO 2 cause a shift in the w o r k i n g p o i n t of the baroreflex control of c a r d i o v a s c u l a r s y s t e m due to the interaction of c h e m o r e c e p t o r m e d i a t e d and peripheral local mechanisms . I n s t i t u t fur Physiologic, Humboldt- U n i v e r s i t ~ t Berlin, H e s s i s c h e Str. 3-4, O-1040 Berlin

zu

Institut Fetscherstr.

f~r 74,

89 CIRCASEPTAN RHYTHMS OF WATER BALANCE PARAMETERS 1N MAN DURING A 28 DAYS ISOLATION AND CONFINEMENT H.-Chr. Gunga 1, A. Maillet2, K. Kirsch1, H.-U. Balzer3, A. Hope4, L. R6cker 1. C. Gharib2 Isolating and confining a group of men imposes stress on the individuals but also allows to study certain body functions like the water turnover under constant environmental conditions. We therefore measured during isolation in 6 healthy male subjects dally the body weight (BW), fluid intake, urine output and 5 times during the study the transepidermal water loss (TEWL). Urine electrolytes were also measured. During the isolation two subjects lost body weight (-3%, -4%). One subject gained body weight (+2 %). The TEWL ranged between 20 to 40 % of the total fluid loss of the body. A close correlation appeared between fluid intake and total water output (r = 0.81). Fluid intake and urine output were found to increase constantly during the isolation. In addition, both parameters as well as the electrolytes sodium and potassium showed a strong circaseptan rhythmicity in the ftrst three weeks of isolation. Towards the end of the confinement nearly all these circaseptan rhythms disappeared. This study showed: 1. The water turnover in man could only be reliably established if urine output and the TEWL are known especially under stressful conditions. 2. The circaseptan rhythm plays a central role in the water balance in man. These rhythmicities can get lost during longer lasting stressful situations. llnstitut flir Physiologic der Freien Universit~t Berlin, Arnimallee 22, D-1000 Berlin 33; 2Dept. of Environmental Physiology; 3Dept. of Pathophysiology; 4Norwegian Underwater Technology Center A/S

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STRONG ACTIVATION OF LYMPHOKINE ACTIVATED KILLER (LAK) CELLS DURING WARM WATER IMMERSION (37.2 - 38.5~ H.-Chr. Gunza 1. K. KirschL L. R6cker 1, B. Thiele2

EFFECT OF BARORECEPTOR STIMULATION ON PAIN THRESHOLD IN NORMO- AND HYPERTENSIVES A. Kardos, C. Droste, M. W. Greenlee*, H. Rau ~ and H. Roskamm

Lymphokine activated killer (LAK) cells (CD3+/CD16/CD56+) and natural killer (NK) cells (CD3-/CD16/CD56+) play a central role in the immunological defense of the human body. The role of the body temperature stimulating their activation (fever), however, is less clearly understood. Therefore subjects were exposed to warm water immersion thereby raising skin and with a certain delay also the core temperature. LAK and NK cells were analysed using FACSean, Becton Dickinson. It was tested whether an increase of skin temperature during Warm Water Immersion can be regarded as a stimulus to activate these cells. 8 healthy, male volunteers were immersed in water up to the neck for one hour. Rectal and oral temperatures were measured. In this procedure, the skin temperature can be regarded as identical to the water temperature. The water temperature was continuously raised during the immersion from 36.9~ up to 38.5~ Between 37.2~ and 38.5~ a significant (p > 0.05) fourfold increase in the total amount of LAK cells and 10-fold expression of CD16/CD56 was observed indicating a strong cell activation. After finishing the warm water immersion, the number of LAK cells and expression of CDI6/CD56 decreased in line with a decrease of oral temperature. It seems indeed as if raising skin temperature activates the LAK cells. Practical consequences will be discussed. llnstitut fiir Physiologie der Freien Universifftt Berlin, Arnimallee 22, D-1000 Berlin 33; 2Diagnostisches Zentrum, Berlin, Germany

Electrocutaneous pain thresholds were determined prior to and d u r i n g b a r o r e c e p t o r stimulation in 16 h y p e r t e n s i v e and 13 normotensive male adults. Carotid baroreceptor stimulation was conducted using a ECG R-wave triggered, external neck-suction technique described by Rau et al. (1990). Pain thresholds for brief (10 msec) electrical impulses, delivered via an intracutaneous, finger-tip electrode, were d e t e r m i n e d in a 2-interval forcedchoice procedure using a maximum-likelihood algorithm (BestPEST). Measurements were performed between 11 am and 3 p m in all subjects to control for possible circadian effects. Before b a r o r e c e p t o r stimulation pain t h r e s h o l d s were significantly higher in hypertensives (t-test, p<0.05). Baroreceptor stimulation significantly increased pain thresholds in normotensives (p < 0.05), but had no effect on thresholds in hypertensive subjects. Heart rate was decelerated during baroreceptor stimulation and this effect was significantly greater in normotensives (p <0.001). The differential effect of carotid baroreceptor stimulation in normo- and hypertensives suggests that baroreceptor sensitivity differs between these subjects. As a result of prolonged elevated systemic blood pressure, hypertensives respond less to transient c h a n g e s in local carotid p r e s s u r e . Transient b a r o r e c e p t o r stimulation evokes an antinociceptive effect in normals but not in hypertensives suggesting that the baroreceptor set-point may be altered in chronic hypertension. Benedikt Kreutz Herz-Kreislauf Rehabilitationszentrum, D-7812 Bad Krozingen, Neurologische Universlt~itsklinik,D-7800 Freiburg and o Psychologisches Institut, Universit~t Tiibingen.

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Lokal dehydration and hyperhydration modulate cardiovascular responses to static exercise

CARDIOVASCULAR ADJUSTMENTS H Y P E R O X I A IN H E A L T H Y M E N P. K r e i B i g a n d A. S k a l w e i t

TO ACUTE

HYPOXIA

AND

K. Baum, D. Essfeld, D. Leyk, J. St0gemann Methods: Responses of mean blood pressure and heart rate to light static exercise of one calf w e r e d e t e r m i n e d in 13 h e a l t h y male subjects. Each subject p e r f o r m e d three exercise tests in supine body position w i t h 90 knee joint flexion. In each test, subjects had to counteract a spring-force (5-10 % of m a x i m a l v o l u n t a r y contraction) for 8 min. Prior to exercise the extracellular calf vol;ume was either d e c r e a s e d by a negative, local hydrostatic p r e s s u r e (calves about 40 cm above heart level) or increased b y venous congestions of 3 min a n d - 1 5 min duration (cuff inflated to 80 m m Hg). 3 min before exercise cuff p r e s s u r e was increased to 300 ram Hg to clamp the m a n i p u l a t e d volumes. Blood pressure and heart rate were d e t e r m i n e d by means of a non-invasive, continuous m e t h o d (Finapres) and standard ECG leads, respectively. Oxygen uptake was m e a s u r e d breath b y breath. Calf volumes w e r e recorded by means of w a t e r displacement. Results: Oxygen uptake remained at pre-exercise levels until the cuff was deflated indicating that e x c l u s i v e l y exercising calf muscles contributed to the cardiovascular response. The blood pressure responses to static exercise were found to be inv e r s e l y related to calf volume. Heart rate g e n e r a l l y increased during exercise but showed no simple r e l a t i o n s h i p to the m a n i p u l a t e d volume. Conclusion: Changes in extracellular volume affect the cardiovascular response to static exercise. D e h y d r a t i o n increases and hypohydration decreases the blood p r e s s u r e response while heart rate seems to be a d d i t i o n a l l y modulated b y the arterial baroreflex.

T h e i n f l u e n c e of an a c u t e n o r m o b a r i c h y p e r o x i a a n d hypoxia on the cardiovascular system was analyzed o n 41 h e a l t h y y o u n g men. During the i n s p i r a t i o n of h y p e r o x i c (100% oxygen) a n d h y p o x i c (12% oxygen) g a s m i x t u r e s t h e ECG, P C G a n d ICG w e r e r e c o r d e d and t h e h e a r t p e r i o d d u r a t i o n (HPD) a n d a s t r o k e v o l u m e f a c t o r (SVF) d e t e r m i n e d . T h e a r t e r i a l s y s t o l i c and d i a s t o l i c b l o o d pressure (BPS, B P D ) w a s m e a s u r e d noninvasively o n c e per minute by the method of R i v a R o c c i . The mean arterial blood pressure (BPM), a f a c t o r for t h e t o t a l p e r i p h e r a l r e s i s t a n c e (TPRF) a n d t h e c a r d i a c o u t p u t (COF) w e r e c a l c u l a t e d . U n d e r h y p e r o x i c c o n d i t i o n s the BPD, B P M ,TPRF a n d H P D w e r e increased, the COF d e c r e a s e d . In c o n t r a s t to h y p e r o x i a , in h y p o x i a BPD, B P M and T P R F r e m a i n e d u n c h a n g e d a n d H P D d e c r e a s e d . T h i s c a u s e d a r i s e in C O F d e s p i t e d i m i n i s h e d SVF. The results suggest that during hyperoxia the s y m p a t h e t i c o u t f l o w is d e c r e a s e d a n d d u r i n g h y p o x i a increased. But the local regulatory mechanisms may o v e r l a p t h o s e of the p e r i p h e r a l e f f e c t s of t h e a u t o n o m i c nerves. So a n e n h a n c e d p O 2 c a n l e a d t o a n i n c r e a s e in t h e v a s c u l a r t o n e a n d r e s p e c t i v e l y t o the described rise in T P R F , BPM and BPD. In a d d i t i o n , t h e v a s o d i l a t o r y e f f e c t of d i m i n i s h e d p O 2 under hypoxia may compensate the increased v a s c u l a r s y m p a t h e t i c activity. O n t h e o t h e r h a n d a d i f f e r e n c e in t h e l e v e l of t h e a u t o n o m i c a c t i v i t y of the h e a r t and b l o o d v e s s e l s m i g h t a l s o b e t a k e n into t h e c o n s i d e r a t i o n .

Physiologisches Institut schule K~in, BRD

I n s t i t u t fur P h y s i o l o g i e , H u m b o l d t - U n i v e r s i t ~ t B e r l i n , H e s s i s c h e Str. 3-4, O- 1040 B e r l i n

der

Deutschen

Sporthoch-

zu

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TESTING SENSOMOTORIC CAPACITY COMPUTER AIDED SYSTEM L.Beyer, M.Plietz, A.Steinbach,

96

BY

MEANS

OF

A

R.Rost,

Sensomotoric regulation of behavior is important in sports science as w e l l as in neurology and motor rehabilitation. To test movement performance t h e p r i n c i p l e of p u r s u i t tracking is a p p l i c a t e d in o u r s e l f - d e v e l o p e d s o f t w a r e system. Test persons will be r e q u i e r e d to follow a timevariant monitor defined movement (test function). As a parameter the integral of difference between test function and tracking movement was chosen. Performing sineshaped movements (0,3 u p t o 1,5 HZ; a n g l e c h a n g e s + / 18 ~ f r o m m i d d l e p o s i t i o n 120 ~ in e l b o w or k n e e joint) the quality of m o t o r output remains stable. Higher frequencies shows increasing integral differences and significant better performance of m o v e m e n t s in t h e e l b o w t h a n in t h e k n e e joint, which may be caused by different biomechanical conditions (body weight). Repetition of test demands leads not consequently to increasing sensomotoric performance. Our sensomotoric test may be applied in different tasks of m o t o r d i a g n o s t i c s a n d as sensomotoric task in different psychophysiological experiments. Friedrich-Schiller-University, Physiology, D - O - 6 9 0 0 Jena, T e i c h g r a b e n 8

Institute

of

CHANBES OF EMG-PARAMETERS AND CONDUCTION VELOCITY IN CONTRACTIONS WITH SUBMAXIMAL FORCE AT DIFFERENT TEMPERATURES R.Mucke, G . C a f f i e r During muscle contractions the surface electromyogram (EMG) i s influenced as well by local and central processes related to force and duration of contraction as by peripheral factors l i k e recording conditions and muscle temperature. The aim o f the study was to inquire into changes of amplitude and frequency parameters of EMG and muscular conduction v e l o c i t y (CV) at d i f f e r e n t muscle temperatures during nonfatiguing and f a t i g u i n g arm contractions with submaximal force. Five male students volunteered f o r this study. The temperature of the upper arm was changed by a c u f f flown through by water of controlled temperatures of O, i0, 20, 30, 40~ During the f i r s t hour of the experiments the subjects had to perform every f i v e minutes test contractions (TK) with 30% of maximum voluntary contraction force (MVC) and a duration of 3 seconds. The 13th TK was followed by a contraction with 40% MVC up to exhaustion. During 15 min of recovery f u r t h e r TK were performed. For calculation of mean power frequency (MPF), i n t e g r a t e d EMG (iEM6), and CV three EMOs of biceps brachii were recorded by a triple bipolar electrode located between the region of the endplates and the tendon. With decreasing temperatures a significant diminution of MPF and CV was observed in nonfatiguing test contractions during the f i r s t 60 minutes o f temperature application. Despite of unchanged f o r c e the iEMG showed a d r a s t i c decrease. At temperatures below 20~ the f a t i g u e r e l a t e d changes of EMG-parameters and CV in the exhausting contractions were neglected by the temperature e f f e c t . At c u f f temperature of 20~ we found the maximum time of sustaining contractions with 40% MVC. During recovery no sign of improvement of muscle properties could be observed by EMG parameters and CV a f t e r treatment with I0 and 20~ Humboldt-Universit&t Berlin~ C h a r i t ~ A r b e i t s m e d i z i n , PF 140, B e r l i n 0 - 1 0 4 0

95

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UNSPECIFIC AIRWAY HYPERREACTMTY INDUCED BY TOLUENE DIISOCYANATE IN AN ANIMAL MODEL OF OCCUPATIONAL LUNG DISEASE W. Marel~ B. Marczynsld, J. Potthast and X. Baur Diisocyanates, widely used in industry particularly for the production of polyurethanes, represent a growing problem in occupational medicine. Besides toxic responses, isocyanates induce sensitisation of exposed workers and may cause asthmatic effects or allergic alveolitis. In order to study the pathomechanisms of acute isocyanate induced lung injury, we developed an animal model using anaesthetised rabbits. Inhalation of toluene diisocyante (TDI) in the range of threshold limit value (TLV) (5, 10 and 30 ppb) four times for a period of 1 hour, performed in 3 groups of 4 rabbits, did not significantly alter airway resistance (R), dynamic elastance (Edyn), arterial pressure (Pa) or arterial blood-gas tensions (PaO2, PaCO2) within time of exposure. 8 animals inhaled TDI under the same conditions. In these experiments, before and after each TDI inhalation (lh), unspecific airway responsiveness to 2% Acetyleholine (ACH) aerosol, inhaled for 1 minute, was measured. Before inhalation of 10 ppb TDI, ACH challenge caused an increase in dynamic elastance (Edyn) from 20.2 _+ 2.9 to 27.4 +- 8.3 mmHg/dl VT. After 4 hours of TDI-inhalatinn, the amplitude of the bronchoconstrictory responses to ACH rose significantly (p < 0.005) to 3.6 times of the control value. Edyu increased after ACH challenge from 23.1 _+ 8.8 to 49.1 _+ 13.5 mmHg/dl VT. Similar changes in the amplitude of airway resistance were measured. After inhalation of 5 ppb TDI, no significant changes in airway reactivity were noticed, whereas the responses were further enhanced with 30 ppb TDI. In a control group of 6 animals, not undergoing TDI inhalation, the responses of Edyn and R to ACH aerosol inhalation did not significantly alter from one challenge to another. Conclusion: Diisocyanate atmospheres of threshold limit value of 10 ppb cause bronchial hyperreactivity within 4 hours of exposure in our rabbit model of occupational lung disease. ProfessionalAssociations'Research Institutefor OccupationalMedicine(BGFA) at the Ruhr-Universityof Bochum,Gilsingstr.14, D-4630"Bochumi

MUSCLE

Institut

for

FIBRE C O N D U C T I O N VELOCITY AND EMG POWER SPECTRA DURING ISOMETRIC CONTRACTIONS IN MAN

G. Caffier.

R.

Mucke,

D. Heinecke,

R.

Hinterthan

A l t e r a t i o n of m u s c l e fibre c o n d u c t i o n v e l o c i t y (CV) were t h o u g h t to be the m a i n r e a s o n for c h a n g e s of the m e a n power f r e q u e n c y ~MPF) of the power spectrum of the EMG signal. On the other hand, changes of d i s c h a r g e frequency, s y n c h r o n i z a t i o n , and rec r u i t m e n t order of motor units have b e e n discussed to a f f e c t the f r e q u e n c y c h a r a c t e r i s t i c s of EMG. In the p r e s e n t i n v e s t i g a t i o n MPP and CV w e r e measured during i s o m e t r i c biceps c o n t r a c t i o n s of different levels of force, fatigue, and a m b i e n t temperature. The aim of the study was to test w h e t h e r M B F and CV c h a n g e d in parallel d u r i n g d i f f e r e n t c o n d i t i o n s of muscle activity. EMGs were r e c o r d e d with surface e l e c t r o d e s in a b i p o l a r t r i p l e e l e c t r o d e c o n f i g u r a tion. CV was c a l c u l a t e d from time d e l a y of three EMG signals. Spectral a n a l y s i s was performed by a p p l y i n g the PET a l g o r i t h m . The r e s u l t s show that MPF and CV c h a n g e d n e a r l y in parallel with temperature when non-fatiguing cont r a c t i o n s were performed. D u r i n g f a t i g u i n g c o n t r a c tions. MPP d e c r e a s e d faster than CV. The e f f e c t was m a s k e d by t e m p e r a t u r e s of the m u s c l e s s u r f a c e b e l o w 20 ~ C which r e d u c e both MPF and CV in a similar way. A d i s s o c i a t i o n b e t w e e n MFF and CV was also o b s e r v e d in n o n - f a t i g u i n g c o n t r a c t i o n s of v e r y low l o a d ( 5 - 1 5 % MVC). The r e s u l t s support the hypothes i s that the shift of MPF is not due to c h a n g e s of CV alone. C h a n g e s of d i s c h a r g e frequency, synchronization, and r e c r u i t m e n t order of motor u n i t s may be a d d i t i o n a l m e c h a n i s m s . B u n d e s a n s t a l t fur A r b e i t s m e d i z i n N W l d n e r s t r . 40-42. 0 - 1 1 3 4 B e r l i n

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LACTIC ACID CONCENTRATIONS IN RACEHORSES (TRo'I-FERS) AFTER TRAINIG HEATS AND RACING H. Krzywanek, E. Mohr, A. Chrobok und M. Scharpenack

T H E S T A B I L I T Y O F M O T O R A C T I V A T I N G P A T T E R N IN H E A L THY SUBJECTS

Well conditioned racehorses (trotters) are normally trained only once or twice a week. On these days the horses have to go two heats. The other days of the week they only were jogged or had to go with low speed on the track. Usually these horses take part in races every 7 or 10 days. From 20 horses of two stables at the Berlin race track we have taken blood of the jugular vein after training heats as well as after races. Lactic acid concentration was measured immediately after the venipuncture with an Eppendorf- Lactat- Analyzer (ESAT 6661). Lactic acid concentrations were: 4,15 -+ 3,01 mmol/I 2 min after the first heat 7,19 _+ 2,85 mmol/I 2 min after the second heat 16,01 + 2,94 mmol/I 5 min after the race 16,55 _+3,16 mmol/I 10 min after the race [Differences betweenthe valuesof heatone and heattwo as well as between heattwo and racewerehighlysignificant (p<0,001)] It is astonishing that racehorses during training heats only reach lactic acid concentrations of 7,15 mmol/I (nearly twice the value of the anaerobic threshold) but tolerate much higher values during races. With regard to these results it should be discussed to modify training programs. Racehorses should be worked out in a way that higher acidosises are attained already in training.

Oh. Anders, Schumann, N.P., Scholle, H.Ch. F o r t h i s p i l o t s t u d y the E M G a c t i v i t y of t h e l u m bal back region was investigated during defined postures in 9 h e a l t h y subjects. U s i n g 16 m o n o p o l a r s u r f a c e e l e c t r o d e s (array of 4*4 e l e c t r o d e s , d i s t a n c e b e t w e e n e l e c t r o d e s 3 cm, s a m p l i n g r a t e 5 0 6 / s e c , t i m e c o n s t a n t 0,03 sec) t h e m y o e l e c t r i c a l a c t i v i t y w a s r e c o r d e d . T h e m e a s u r e d a c t i v i t y of every single electrode was transformed into s p e c t r a l power, by fast f o u r i e r t r a n s f o r m a t i o n (FFT). F r o m t h e s e d a t a a m a p - l i k e r e p r e s e n t a t i o n w a s c o m p u t e d b y l i n e a r i n t e r p o l a t i o n u s i n g t h e 4neighbours-method. D u r i n g the i n v e s t i g a t i o n t h e e l e c t r o d e array, s t a r t i n g a b o v e t h e s p i n a l c o l u m n w a s d i s p l a c e d 4 t i m e s w i t h one e l e c t r o d e r o w d i s t a n c e t o t h e left. It b e c o m e s e v i d e n t t h a t a h i g h intraindividual stability despite occuring interi n d i v i d u a l v a r i a b i l i t y of m u s c u l a r a c t i v a t i n g p r o c e s s e s exists. W h i l e p e r f o r m i n g i d e n t i c a l p o s t u r e s s t a b l e m o t o r i c p a t t e r n are evident. B y c o n s i d e r a t i o n of o v e r l a p p i n g i n v e s t i g a t i o n f i e l d s it is p o s s i b l e t o s h o w s y n e r g i s m s and a n t a g o n i s m s of t h e l o w e r b a c k m u s c u l a t u r e in g r e a t e r areas. B a s i n g o n t h e s e r e s u l t s it has to be i n v e s t i g a t e d , w h e t h e r a n d in w h i c h m a n n e r s u c h a p a t t e r n c a n be c h a n g e d b y d i s e a s e s of t h e m u s c u l o s k e l e t a l s y s t e m t o d e v e lop m o r e e f f e c t i v e t h e r a p y s t r a t e g i e s . Institut f~r Pathologische Friedrich-Schiller-Universit~t 3, 0 - 6 9 0 0 J e n a

Physiologie der Jena, L ~ b d e r s t r a B e

Inst. for Veterin~rphysiologie FU Berlin,Koserstr. 20, W- 1000 Berlin 33

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EXTRAPOLATION OF VISUAL MOTION: A LINEAR REGRESSION ANALYSIS OF INDIVIDUAL PERFORMANCE DIFFERENCES N. Yakimoff, W.H. Ehrenstein*, J. Hohnsbein*, S. Mateeff

EFFECT OF BREFELDIN A ON PROTEIN SECRETION IN

GUANOSINE-TRIPHOSPHATE MODULATES

THE INHIBITORY

PANCREATIC ACINAR CELLS The performance in extrapolating visual motion was investigated in ten subjects (5 female, 5 male). The target moved from left to right over 12 deg and then disappeared. Subjects pressed a key to indicate the moment at which the occluded target reached a stationary reference. Three extrapolation distances (4, 6, 8 deg) and target velocities (3, 6, 9 deg/s) were used and the resulting nine stimulus combinations were presented in random order. We tested the hypothesis that the relationship between the response time Tr, measured from the offset of motion to the moment of key press, and the actual time to arrival Ta of the concealed motion is linear: Tr = ~'T a +

e,

where the two parameters ~ (the slope) and 8 (the intercept) characterize an individual's performance. Linear regression analysis, applied to the T r data for each subject separately, showed high correlations (r) between T r and T a for all subjects (.980 < r < .999) All slope values and most of the intercepts were significantly different from zero. The regression lines of female observers had significantly lower slopes than those of males (p < .05), while their intercepts were similar to those obtained for males. The shortcomings of alternative approaches that describe the individual performance in terms of "errors" in relation to an "ideal" performance (T r = Ta) will be discussed. The present linear model may be useful to account for the fact that individuals differ in their ability to extrapolate motion in work and sport activities. (Supported by the Bulg.

S. Zeuzem, P. Zimmermann and I. Schulz Brefeldin A (BFA) causes rapid redistribution of Golgl proteins into the endoplasmic reticulum (ER), leaving no definable Golgi-apparatus, and blocks transport of proteins from the ER to distal secretory compartments of the cell (J. Lippincott-Schwartz et al., Cell 56: 801-813, 1989). Using pulse-chase experiments the present study shows that BFA (1/Ig/ml) inhibits basal and CCK-stimulated protein secretion in isolated pancreatic acinar cells by 65 -+ 6% and 84 -+ 5%, respectively. In isolated permeabilized cells higher concentrations of BFA (30 ~g/ml) were necessary to obtain inhibition of protein secretion. In parallel experiments protein secretion was stimulated by GTP (10-3 M). BFA had no inhibitory effect on protein secretion in the presence of GTP, indicating that BFA might act on a GTP-binding protein. Investigating the effect of BFA on small molecular weight GTP-binding proteins we observed that [a-32p]GTP binding to a 21 kDa protein in a subcellular fraction enriched in ER was increased in the presence of BFA. We conclude that this 21 kDa and possibly also other GTP-binding proteins may be the molecular target of brefeldin A in panereatic acinar cells.

Commi ttee of Sclence and b y the DFC)

Institute of Physiology, 1113 Sofia, Bulgaria *Institut f~r Arbeitsphysiologie, W-4600 Dortmund, Germany

Max-Planck-Institut ffir Biophysik, Kenuedyallee 70, D-6000 Frankfurt

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PHYSIOLOGICAL REGULATION OF PANCREATIC DUCTS

PROPERTIES OF THE BASOLATERAL M E M B R A N E ISOLATED CRYPTS FROM RABBIT DISTAL COLON

C. Pahl, I. Novak, R. Nitschke and R. Greger Pancreatic ducts secrete HCOs'-rich fluid, and a number of hormones, peptides and transmitters have been claimed to regulate this secretion. However, since such studies were performed on intact pancreas including acini and endocrine cells, it is not clear whether the control is exerted directly or indirectly on the ductal epithelium. Our preparation of isolated perfused pancreatic duct offers an opportunity to test the putative agonists and antagonists directly. The ducts were dissected from rat pancreas and perfused in vitro with physiological solutions at 37~ Individual cells were impaled with microelectrodes and the basolateral membrane potential (Vbl) w a s recorded with respect to the grounded bath. Secretin, the classical ductal agonist, depolarized Vbl from about -65 mV to -30 inV. From our previous studies of this leaky epithelium, we know that this depolarization is due to opening of luminal CI- channels via the cAMP transduction mechanism; CI- subsequently reenters the cell via a CI'/HCO 3" exchange and thus HCOs" is extruded into the lumen (1). The half-maximal dose of secretin was 0.04 nmol/l. Vasoactive intestinal polypeptide (VIP, 9 nmol/I) depolarized Vbl by 11 • mV (n =3). Cholecystokinin, a classical agonist of pancreatic acini causing them to secrete a CI- and enzyme-rich fluid, seems to have no effect on Vbl of ductal cells (n=2). Carbachol on the other hand, also an established acinar agonist, had a significant affect on duct cells. After application of carbachol (1-10 pmol/I) Vbl depolarized by 2 0 + 7 mV (n=6). The Vbl depolarization evoked by agonists tested was sustained and reversible. In another series of experiments on isolated perfused ducts, we measured the intracellular Ca2+-activity with the fura-2 fluorescence method. Our preliminary results show that the ducts have a relatively high resting Ca2+-activity. Carbachol transiently increased the Ca2+-activity, as estimated from the fluorescence ratio at 335/380 nm. In conclusion, this study shows that rat pancreatic ducts possess secretin, VIP and cholinergic receptors. The immediate questions the present study leads to are: (i) the role of Ca 2+ homeostasis in pancreatic ducts in general; (ii) what type of fluid is secreted by ducts following cholinergic stimulation. 1. Novak, l. and Greger,R. (1988) PflOgers Arch. 411:546-553 Physiologisches Institut, Albert - Ludwigs - Universitfit, Hermann - Herder Strasse 7, W-7800 Freiburg

E. Lohrmann and R. Greger The colonic crypt is thought to be the site of colonic CI'- and thus fluid secretion. The aim of our study was to obtain electrophysiological data from colonic crypts. Therefore, we developed an isolated perfused crypt preparation. Colonic crypts were dissected from distal colon of adult rabbits (> 2kg BW) with fine forceps without any pretreatment. The bottom part of the crypt (ca. 10/tm) was cut off. The crypts were perfused with the perfusion technique previously described for the thick ascending limb of Henle's loop (1) with the bottom side of the crypt at the perfusion side. Single cells were impaled from the basolateral (bath) side. Perfusions were performed with HCO~-containing Ringer-like electrolyte solutions. Geometrical parameters of 53 perfused crypts were: length 130+6 /~m, inner and outer diameter 14+1 and 5 4 + 2 pm. In 81 impalements basolateral potential was - 6 2 • inV. Elevation of bath K + from 5 to 20 mmol/I depolarized the cells by 24 + 2 mV (n =31 ). In H CO~-free solutions the basolateral membrane hyperpolarized by 5 • 1 mV (n = 11 ). Furosemide (10 -4 mol/I) led to a hyperpolarization by 6+1 mV (n=3). Bath verapamil (10 -4 mol/I) depolarized the cells reversibly by 3 5 • mV (n=3). Several compounds known to stimulate Cl'-secretion in mucosa preparations were tested: In a pilot experiment isoprenaline (10 -6 mol/I, bath) led to a depolarization by 11 mV. 1 mmol/I 8-CI-phenyl-thio-cAMP depolarized the cells by 2 2 • mV (n=4). Forskolin (10 -6 mol/I) led to a depolarization by 34• mV (n=6). Lowering of the bath CI--concentration led to a partial repolarization of the cell. In 2 of these experiments, by injecting current into the crypt lumen, a marked increase in fractional basolateral resistance could be measured. Surprisingly bumetanide up to 10-4 mol/I did not diminish the forskolin effect. PGE2 (10 -s mol/I) led to a depolarization of 2 7 + 7 mV (n=5). PGE2 (10 .7 mol/I) led to variable voltage changes ( 7 • mV, n=4). PGE2 (10 "8 and 10-9 mol/I) hyperpolarized by 4 • 1 (n=5) and 3• mV (n=4). Carbachol, a compound suggested to increase Cl'-conductance by increasing intracellular Ca2+-activity in the colonic carcinoma cell line HT29, hyperpolarized the crypt cells by 16• 1 mV (n=4). These preliminary data show the feasibility of voltage and resistance recordings in isolated perfused rabbit colonic crypts. (1) Greger R, Hampel W, Pfl0gers Arch 389:175-176,1981 Supported by DFG Gr 480/108; Physiologisches Institut, Albert-LudwigsUniversit~it, D-7800 Freiburg

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CELL VOLUME IN REGULATION OF HEPATIC METABOLISM D. Hfiussinger, C. Hallbrucker, S. vom Dahl, F. Lang

PURIFICATION OF A CYTOSOLIC FACTOR INHIBITING CL'CHANNELS T. Plusczyk, R. Sch/ifer and I. Schulz

Previous studies in this laboratory revealed that alterations of hepatocyte volume markedly modify hepatocellular metabolism. Swelling of liver cells is paralleled by stimulation of glycogen and protein formation, whereas shrinkage of liver cells is followed by stimulation of glycogen and protein degradation. The present study has been performed to test for the participation of cell volume in the regulation of hepatic proteinolysis by amino acids and hormones. Experiments have been performed in isolated perfused rat livers. Autophagic protein degradation has been determined from hepatic tracer leucine release, cellular water space by subtraction of inulin space from urea space, K + movements from the respective alterations of K + activity in the venous effluent. In some experiments intracellular amino acid concentrations have ben determined in shock frozen tissue. As a result, several amino acids (GLN, SER, GLY, PHE, PRO) lead to rapid swelling of liver cells, when added to the portal venous perfusate. The amino acid induced cell swelling is due to concentrative cellular uptake of the respective amino acid. Other amino acids (GLU) are without appreciable effect on liver mass. The antiproteolytic effect of GLN and GLY is fully accounted for by the amino acid induced cell swelling, and is quantitatively mimicked by the respective alterations of cell volume during reduction of extracellular osmolarity or inhibition of K + channels by 1 mmol/l barium. The antiproteolytic effect of PHE and PRO, on the other hand, is about twofold and threefold the value expected from cell swelling. Insulin (2 nmol/1) stimulates cellular K + uptake and increases liver cell volume, effects blunted by either, 0.1 mmol/l furosemide or 1 retool/1 amiloride, and abolished in the presence of both, furosemide and amiloride. Thus, insulin increases he~atocyte volume by stimulation of Na+,K+,2CI cotransport and Na /H + exchange in parallel to Na+/K + ATPase. Glucagon (lnmol/1) decreases the liver cell volume by release of K + and C1-. The antiproteolytic effect of insulin and the proteolytic effect of glucagon are fully accounted for by their effect on cell volume. In conclusion, alterations of liver cell volume play a decisive role in the regulation of hepatic proteolysis by amino acids and hormones. Department Internal Medicine, University of D-7900 Freiburg, Germany and Institute for Physiology, University of A-6010 Innsbruck, Austria

OF

Cl-channels are involved in secretagogue induced enzyme secretion from pancreatic acini. Zymogen granules (ZG) isolated from hormonal stimulated rat pancreatic aeim have an increased Cl'conductance as estimated by measuring the rate of decrease in optical density due to K + ionophore (vallnomyein) induced osmotic lysis (Fuller et. al. (1989) Pfliigers Arch. 415, 29-36). This CI" conductance has not yet been identified as a Cl-channel by electrophysiological methods. In patch clamp experiments hormonal regulated Cl'channels from epithelial cells were blocked by a cytosolic inhibitor (cyt. inh.) (Kunzelmann et. al. (1989) Pfltigers Arch. 415, 172-182). Looking for this cyt. inh. we prepared cytosol from rat pancreatic acini and tested it on the Cl'conductance of isolated ZG. The cytosol inhibited the Cl'conductance of isolated ZG and the inhibitory effect of the cytosol was abolished by heating (15 min at 95~ or by treatment with thermolysin or with trypsin. This suggests that the cyt. inh. is a peptide or a protein. The purification of this putative protein was carried out by hydrophobie interaction chromatography, immobilised cupper-ion affinity chromatography and anion exchange chromatography. Subsequently the molecular weight was estimated by gel filtration (Superdex 200 column) and by a 10% SDS-page. With both techniques we detected a protein of a molecular mass between 66 and 68 kDa, as a candidate for the cyt. inh. Therefore the cyt. inh. could be a heat sensitive protein of 66-68 kDa or a small molecule bound to another protein (albumin). Further experiments and purification are necessary to clarify the chemical nature and the pysiological or pathophysiological role of the cyt. inh. Max-Planck-Institut for Biophysik, Kennedyallee 70, D-6000 Frankfurt 70

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T H Y R O I D HORMONE STATUS U N D E R A P P L I C A T I O N OF A CALCIUM CHANNEL BLOCKER J. Wieberneit, Ch.-F. Wolf and F.S. Keck

E v a l u a t i o n of the serum retinol h o m o e o s t a s i s H.K.Biesalski, T.Gerlach The factors influencing storage and release of retinol from liver are not completely understood. Retinol is released from liver stores bound to retinol binding protein (RBP) in a strongly regulated process that aceunts for the constant amounts of retinol in normal circulation. The m e c h a n i s m involved of circulatory vitamin A homoeostasis is not known; it is assumed that s feedback signal from peripheral vitamin A target tissues is involved. To determine the kind of signal we carried out different experiments. Depletion of the vitamin A stores in rats showed that retinol plasma values remained unchanged untill the liver stores are nearly ~ompletely depleted. D u r i n g repletion with ll,12-JH-retinyl acetate liver vitamin A and plasma retinol levels increase initially in a similar way until plasma retinol reaches a plateau, whereas liver vitamin A stores grow continuously. During the increase of plasma retinol ape RBP also increases and remains constant after retinol reaches its concentrstion plateau. Even in severe h y p e r v i t a m i n o s i s A retinol remains at a constant level. It is known however, that d u r i n g impaired renal function an increase of plasma retinol becomes evident. C o n s e q u e n t l y we m e a s u r e d retinol and RBP in patients with renal i n s u f f i c i e n c y and detected increased plasma ape RBP levels. Purification of rat ape RBP and intra venous injection resulted in a dramatic increase of plasma retinol. From this results we assume that ape RBP is the peripheral signal for retinol release from the liver. Dept. of Physiol. Chemistry D u e s b e r g w e g 6, D 6500 MAINZ

C a l c i u m antagonists are k n o w n to affect certain endocrinological systems especially at high doses (De M a r i n i s et al., Horm. Res. 25:5, 1987). We s t u d i e d the influence of 3 months of nitrendipine (NIT) at different doses (24, 48, and 320 m g / k g b o d y weight, n=8) on t h y r o i d parameters in baboons with respect to s e r u m hormone levels and hepatic 5"-deiodinating a c t i v i t y (5"-DA, d e i o d i n a t i o n of thyroxine (T4) to t r i i o d o t h y r o n i n e (T3)) . While the lower doses of NIT d i d not exert any effect on the parameters investigated, high dose NIT decreased serum T 4 conc e n t r a t i o n s from 7.0• of the p l a c e b o treated control group to 4.7• ~g/dl (p<0.01). Thyroid s t i m u l a t i n g hormone values rose from 0.4• to 2.0• mU/l (p< 0.01), the total T 3 was uninfluenced (controls, 149• high dose group, 153• ng/ dl). Free hormones were c l o s e l y related to total values (T4, r=0.97, p<0.001; T3, r= 0.8, p<0.001), excluding b i n d i n g abnormalities. NIT caused an increase in 5"-DA from 1.88• to 2.48• ng T3/ m i n . m g m i c r o s o m a l p r o t e i n (p<0.05), w h i c h was due to a rise in the Vma x (from 2.5• to 3.3• p< 0.05) at unaltered values of the apparent K M (5.6• 0.7 and 5.2• 0.8 ~M, resp.). Stimulated 5'-DA was paralleled by an increase in microsomal protein content of the liver from 12.4• to 16.5• mg/g liver (p<0.05). In conclusion, these data suggest, that i. NIT impairs the c a l c i u m - d e p e n d e n t thyroidal T 4 secretion, ii. NIT increases h e p a t i c 5'-DA by e l e v a t e d enzyme content, iii. This may in part account for normal T 3 s e r u m levels.

and Pathobiochemistry, M e d i z i n i s c h e Klinik und Poliklinik, Abt. Innere Med. I, U n i v e r s i t ~ t Ulm, U l m / D o n a u

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ENDOTHELIN-I SYNTHESIS IN NONPIGMENTED CILIARY BODY EPITHELIAL CELLS A. Lepple-Wienhues, F. Stahl, J. Hensen, M. Becker and M. Wiederholt

PRESERVATION OF DIFFERENTIATED FUNCTIONS IN CULTURED GASTRIC MUCOSAL CELLS L Giebel and M. Schwenk

Recently we have shown a contractile response of ciliary muscle and trabeeular meshwork strips to endothelin-1 (ET-t) (htvest. Ophthalme1. Vis. Sci. 32:788, 1991). Using a commercial immunoassayi endothelin-1 was determined after extraction in bovine aqueous humoar, bovine serum, and the supernatant of cultured cells. l. Bovine aqueous humour contained 11.1 + 0.98 pg/ml ET-L The serum levels averaged to 5.0 +- 0.27 pg/ml (n=5, p<0.0t). 2. A virus-transformed cell line of human nonpigmented ciliary body epithelium produced ET-1. After subculturing the cells with the trypsin/ EDTA-method, the maximal ET-t-release was reached 5 days after seeding. The basal ET-l-release per area of the confluent ceil layer after 2 days of incubation with Dulbeceo's Minimal Essential Medium was 0.69 + 0.06 pg/cm2 (n=7). 3. 10% fetal calf serum stimulated the peptide release to t.94 +- 0.27 pg/cm2 (n=7, p<0.01). 4. Cycloheximide (10-Smol/1) decreased the serum-stimulated ET-1synthesis to 0.92 pg/cm2 (n=6, p<0.05) 5. Ceil lines of human ciliary muscle and bovine corneal endothelium showed no significant production of ET-1 under the same conditions. ET-1 synthesis of nonpigmented ciliary epithelial cells could be the source of this peptide found in aqueous humour. ET-1 may represent a humoral factor regulating aqueous humour dynamics. Supported by DFG Wi 328/11 Inst. f. Klin. Physiologie, Klinikum Steglitz der FU Berlin, Hindenburgdamm 30, t000 Berlin 45, FRG

Inla-oduction: Primary cultures of gastric mucosal cells of guinea pig form monolayers which are composed of mucous-, chief-, parietal- and submucosal ceils. It is presently unknown, how long these cell types maintain differentiated functions in culture: Methods: Mucosal cells were isolated by treatment of mucosal scrapings of guinea pig stomachs with collagenase/pronase. Cells were spread on tissue culture dishes in the presence of 10% FCS under 5% CO2. Selective functions of cells were demonstrated as follows: surface mucous cells=lectin from soybean; surface mucous cells and mucous neck cells-=lec~l from wheat germ or helix pomatia; chief cells=antipepsinogen polyclonal antibody; parietal cells=succinic dehydrogenase, carbonic anhydrase or janusgreen uptake; fibroblasts=antivimentin antibody; endothelial cells=uptake of LDL, phagocytes=uptake of latex beads, proliferative cells=DNA-labeling with BrdU. Fibronectin was detected with an antibody. Results: The freshly isolated cell suspension contained 47 % mucous cells, 20% chief cells, 28 % parietal cells, and 5% submucosal cells. In cocultures each cell type adhered to the culture dish with an efficiency of about 60%, and was integrated in the entire monolayer. Mucous- and chief- cells preserved their polarized granules during adhesion, but later (24 h) the granules were distributed within cells and after 3 days pepsinogen granules tended to disappear, while mucous granules persisted. Parietal ceUs adhered within 4-6 h, flattened after 1-2 days and remained on culture dishes for at least 8 days. Their succinic dehydrogenase and carbonic anhydrase activity remained high for at least 5 days. The share of proliferative epithelial cells increased from <1% (24h) to 25% (72h). A fibronectin-rich exlracellular matrix was deposited on culture dishes. Conclusions: Mucous-, chief- and parietal cells readily attach to culture dishes and preserve their differentiated behaviour for severaJ days. This is an important precondition for the use of such monolayers in long-term studies of regulatory functions in vitro. Dept. of Pharmacology, Medical School, D 3000 Hannover 61, Germany.

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A 120 pS K+-CHANNEL AS A POSSIBLE REGULATION SITE FOR. VIP-INDUCED K+-EFFLUX IN CACO-2 CELLS. S. Huber*, D. K61tgen, P. Heinz-Erian* und Ch. Franke

DIFFERENTIAL REACTIVITY OF CORTICAL AND JUXTAMEDULLARY GLOMERULA TO ADENOSINE-I AND ADENOSINE-2RECEPTOR-STIMULATION IN THE SPLIT HYDRONEPHROTIC RAT KIDNEY

Cells from the colonic tumor cell line Caco-2 are morphologically as well as physiologically highly polarized. They are proposed as an in vitro system for the investigation of secretagogue-induced CI--secretion. In order to assess fluxes of CI- and K+ during secretion we performed 1251--uptake measurement and determination of intracellular [K+] - concentration in these cells. Stimulation by 200 nM VIP led to an accumulation of cAMP and to a sustained decrease in the rate of 1251--uptake as compared to the controls. In the presence of 50/IM 4,4"-diisothiocyanato-stitbene-2,2"-disulfonic acid (D1DS), which did not affect the unstimulated, basal uptake of 1251- into the cells, the VIP-induced reduction in the rate of 1251--uptake was partially blocked pointing to an activation of DIDS-sensitive anion channels by VIP. Simultaneously stimulation by 200 nM VIP reduced the intracellular [K+]-concentration of the Caco-2 cells as determined by flame photometry. The results suggest a secretagogue-induced K+efflux which leads to a loss of intracellular KCI and water during secretion. This reduction in cell volume may account for the observed 1251--uptake phenomena. One possible way for the K+ to get out of the cell can be a K+-selective 120 pS channel we have recorded in patches excised from the medium facing membrane of Caco-2 cells. The mean open probability of this channel increased 100-200-fold if the membrane potential was depolarized from -80 mV to 0 mV. In the basolateral membrane of isolated colonic crypts a K+-channel with similar characteristics has been reported to be activated by cAMP and Ca ++ as assessed by cell attached patch clamp studies (DDF Leo and JD Kaunitz, J Membr Biol 110:19-28, 1989). Supported by DFG HA 1402 / 1-1 *Universit~,tskinderklinik, Lindwurmstr. 4, 8000 M0nchen 2 Physiologisches Institut der TU, Biedersteiner Str. 29, 8000 ML)nchen 40. 111 CHRONICALLY (= 8 0 m m H g )

M. S. Dietrich and M. Steinhausen with technical assistance by R. Dussel We examined the effects of locally applied adenosine receptor agonists on cortical and juxtamedullary glomerula, in order to test for a differential reactivity of these two types of glomerula, as suggested by Spielman et al. (Circulation Research 46, 449-456, 1980). Vascular effects of the adenosine-l-receptor agonist N6-cyclohexyl-adenosine (CHA) and the adenosine-2-receptor agonist N-ethylcarboxamideadenosine (NECA) were examined before and after angiotensin (Ang II) converting-enzyme-~CE)-inhibition by quinapril. CE-inhibition was undertaken to test for an interdependence of adenosine and Ang II, as we have already demonstrated for cortical glomerula and CHA (cf. Dietrich et al., Microvascular Research 41, 275-288r 1991). CHA produced a dose-dependent vasoconstriction and decrease of glomerular blood flow in cortical glomerula that was markedly attenuated by CEinhibition. However, CHA led to a lesser vasoconstriction and decrease of glomerular blood flow in juxtamedullary glomerula that was, on the contrary, not significantly influenced by CEinhibition. NECA led to a vasodilation and increase in glomerular blood flow in both cortical and juxtamedullary glomerula. Our findings confirm a differential reactivity of cortical and juxtamedullary glomerula to adenosine, and, additionally, an interdependence of adenosineand Ang II-induced vascular effects. I. Physiolog. Institut, INF 326; D-6900 Heidelberg Supported by DFG (Forschergruppe Niere)

113 LOWERED REDUCES

RENAL PERFUSION "RENIN - THRESHOLD"

PRESSURE

SEROTONINERGIC HEPATORENAL REFLEX RENAL FUNCTION Edda Tschernko, Irina 0tfl, D. H~ussinger, F. L a n g

REGULATING

Introduction: Acute r e d u c t i o n of renal p e r f u s i o n pressure (RPP) below a definite level (by about 95 mm Hg) increases renin release and plasma renin activity (PRA) (Kirchheim et al. , Pfl. Arch.405,127, 1985); ("Renin -threshold" (RTH)). In this study RTH was tested before and after 4 days of s e r v o - c o n t r o l l e d r e d u c t i o n of RPP to 80 mm Hg. Methods: 3 female beagle dogs, kept under standardized conditions (Na intake 5.5 mmol/(kg bw*day)) were e q u i p p e d w i t h an inflatable cuff above the renal arteries for servo-controlling RPP and catheters above and below the renal arteries. RPP was reduced in steps of i0 mm Hg down to 70 mm Hg for 30 min each, before (protocol i, PI) and after having servo-controlled RPP to 80 mm Hg for 4 days (protocol 2, P2). The increase in PRA due to reduction of RPP between Pl and P2 was compared. R e s u l t s : PI: PRA increased stepwise from 1-2 ng AII/ml/h (MABP=RPP) to more than 20 ng AII/ml/h at RPP=80 mm Hg and to more than i00 ng A I I / m l / h at RPP=70 mm Hg. P2: PRA at RPP=80 mm Hg was found within the range of P1 when MABP=RPP (< 5 ng AII/ml/h). Further red u c t i o n of RPP to 70 mm Hg y i e l d e d PRA increases of only i/i0 compared to RPP=70 mm Hg in PI. Conclusions: Chronic reduction in RPP reduces the pressure dependent RTH and the sensitivity of renin release severely.

According to previous observations from this laboratory, infusion of 2 tzmol/min glutamine into the superior mesenteric vein leads to a decrease of renal glomerular filtration rate (GFR) and urinary flow rate (V), whereas infusion of identical amounts of glutamine into the jugular vein does not significantly alter GFR or V. The present study has been designed to identify the mechanisms involved. To this end catheters were placed into the trachea, the right jugular vein, the superior mesenteric vein, the right femoral artery and the bladder of anaesthetized male Munich Wistar rats (160 - 220 g BW). The superior mesenteric artery was clamped. Inulin clearance was taken as a measure of GFR. The animals were infused at a rate of 20 /~l/min through the superior mesenteric vein (isotonic saline) and at a rate of 40/~i/min through the jugular vein (saline + 100 mmol/1 mannitol). Within 20 min., the infusion of glutamine (2/~mol/min, replacing equiosmolar NaC1) into the jugular vein (n = 7) led to a reduction of GFR by -34 __+7% and V by 39 _ 7%. Infusion of identical amounts of glutamine into the jugular vein (n = 4) did not significantly alter GFR ( + 2 + 4%) and V ( + 2 + 5%). Spinal transection at the thoracoeervical junction (n = 4) and transection of the hepatic vagal innervation (n = 4) abolished the effect of mesenteric glutamine on GFR (+3 _ 9%, and +12 ___ 13, resp.) and V (+18 + 12 and -3 __. 9, resp.). Following unilateral renal denervation (n = 5), the effect of mesenteric glutamine was abolished in the denervated kidney (GFR: -2 _ 8%, V: -7 _ 8%), but not in the intact kidney (GFR: -60 + 6%, V: -56 _ 8%). The effect of mesenteric glutamine was abolished upon simultaneous infusion of 20 nmol/1 methysergide (GFR: +5 _ 2%, V: + 7 ___ 6%, n = 6), and is mimicked by mesenteric infusion of serotonin (GFR: -31 _ 5%, V: -30 +_ 4%, n = 8). Taken together, these observations strongly suggest the existance of a serotoninergic hepatorenal reflex decreasing GFR and V.

AG exp. An~sthesie, 1000 Berlin 19

Institute for Physiology, University of A-6010 Innsbruck, Austria and Department Internal Medicine, University of D-7900 Freiburg, Germany

UKRV-C,

Spandauer

Damm

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LOCALISATION AND REGULATION OF THE METABOLISM OF SORBITOL IN THE INNER MEDULLA OF RAT KIDNEY R.W. Grunewald~, I.I. Weberz, R.K.H. Kinne2. Sorbitol is important for the osmoregulation of inner medullary collecting duct (IMCD) cells. Since sorbitol concentration in IMCD cells has been found to be higher than in total inner medulla (IM) in several investigations, one might conclude that components of sorbitol metabolism are differently distributed in the inner medulla. Therefore regulation and distribution of the enzymes (aldose reductase (AR) and sorbitol dehydrogenase (SDH)) were investigated. AR and SDH activities were determined photometrically in the homogenate of total renal inner medulla (IM) of male Wistar rats and freshly isolated IMCD cells during control conditions, diuresis, and antidiuresis. Diuresis (D) in rats was induced by 5% glucose in drinking water as only alimental supply. Antidiuresis (AD) was induced by deprivation of water for 3 days. Urine osmolarities were 12634-158 mosmol/I in C, 385---56 mosmol/I in D, 25864-305 mosmol/I in AD. AR activity was 2,3fold higher in IMCD cells than in total IM (IMCD 594-8 U/I, IM 254-4 U/I, p<0.001). SDH activity seemed to be lower in IMCD cells (0.37-+0.19 U/I) than in IM (0.87-+0.12 U/I; p<0.05). These results suggest that sorbitol synthesis mainly takes place inside whereas sorbitol degradation takes place outside IMCD cells. D and AD did not significantly influence this distribution. During AD AR activity was significantly increased in IMCD cells (1034-11 U/I, p<0.01) and total IM keeping the ratio (IMCD/IM) constant. SDH activity remained unaltered. During D AR activities were decreased in IMCD cells (41-+5 U/I; p<0.05) and IM with unchanged ratio. SDH activities were increased to 0.534-0.21 U/I in IMCD cells and 1.23-+0.48 U/I in IM - this difference, however, not being significant because of the low activity. These results suggest distribution of eorbitol synthesis and degradation to different compartments of the IM. This might be useful because of the different pH optima of both cytoplasmatic enzymes. It has to be further investigated whether the osmotic regulation of sorbitol metabolism can be proven in various cell types in vivo.

ENDOTHELIN INHIBITS TRANSEPITHELIAL TRANSPORT A N D DIFFERENTIATION OF RENAL (MDCK) CELLS. L. Wojnowski, B. GaBner, W. Steigner and H. Oberleithner Endothelins are a family of potent vasoactive peptidas that may regulate renal hemodynamics as well as transport function of the medullary portion of the kidney. Both synthesis and binding of endothelins were described in renal medulla. We investigated the effects of endothelin-1 (ET-I) on MDCK ceils, a cell line originally derived from renal collecting duct. The activity of transepithelial transport was assessed as the rate of dome formation in monolayers grown on solid support. Dome pH was measured by means of pH selective microelectrodes. Differentiation of monolayer cells was estimated as the peanut lectin (PNA) binding capacity of the apical membrane. After confluency was reached ceils were incubated for 12, 24 and 48 h in serum free medium at various concentrations of ET-1. Exposure to 10-r and 10-9 mol/l ET-1 reduced dome formation with a maximum at 24 h by 41 _+9% and 34 -+ 8% (n=4; p<0.02), respectively, whereas 10-n mol/1 ET-I had no effect. ET-1 (10-8 mol/l; 24 h) decreased dome pH from 7.52 + 0.02 to 7.36 -+ 0.03 (n=74; p<0.01) and reduced PNA binding by 20 + 4% (n=5; p<0.02). Moreover, ET-l (10 -s) completely blunted the increase in PNA binding (+54 -+ 9%; n=5) induced by 10-7 mot/1 aldosterone. ET-l reduced the growth rate of cells seeded at low density estimated at 24, 48, 72 and 96 h by 17, 22, 22 and 28%, respectively. We conclude that ET-I inhibits transepithelial transport and differentiation of MDCK ceils and thus may act as a local hormone regulating function of renal collecting duct. Physiologisches Institut der Universit~it Wiirzburg, Rfntgenring 9, D-8700 Wiirzburg

Sektion Nephrologie, Abteilung Innere Medizin IV, Universit&tsklinik UIm, Robert-Koch-StraSe 8, D-7900 UIm, FRG 2 Max-Planck-lnstitut for Systemphysiologie, Rheinlanddamm 201, D-4600 Dortmund, FRG

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RENAL HEMODYNAMICS AND TUBULAR FUNCTIONS D U R I N G HgCI,-INDUCED ACUTE RENAL FAILURE (ARF). STUDIES ON ISOLATED PERFUSED RAT KIDNEY (IPRK). B.Klanke, P.M.Sanz-Altamira, M.F.Wilks, H.Stolte HgClz-induced ARF produces glomerular and tubular damages in the kidney. In IPRK, the first functional changes have been observed at the hemodynamic level. By influencing this hemodynamic component with vasodilators, its consequences on tubular functions and the time course of events have been studied. Methods: IPRKs were reperfused with a complex medium (cellfree, 5% albumin) at a constant pressure. 20 ~M HgCI2 were added after 60 rain (=rriin 0) of perfusion. Vasodilators, when added, were given 10 min earlier or 90 min later. Vasodilators used were Na-nitroprusside 104M and Ca-antagonists (nifedipin 1 mg/1, verapamil 0.8 mg/l). Results: Three functionally distinct phases have been observed after addition of HgC1 (figure): a) Normal period, similar to controls, from mln 0 to rain 2 2 -+ 1. b) First fall in hemod>,namic parameters (renal perfusate flow, RPF and glomerular filtrauon rate, GFR) to about 3/4 of the controls, without changes in tubular functions, reaching a plateau (approx. duration: I5 rain). c) Second decrease in RPF and GFR accompanied by a fall in tubular sodium and glucose reabsorption .followed by a complete tubular failure. Vasodilators produced RPF ( m l / m l n / g kidney) renal hemodyn~ics, 2o .............. mainly RPF in phase c. tubularIn spite Offailurethis fact,couidthe ' i I .....................i.......... ~ : ' ~

.... __x__ . v~oan.

not be avoided. 0 50 zoo ~50 200 Conclusion: lime (mln) The partial improvement of hemodynamic alterations in HgCLinduced ARF does not produce any amelioration at the tubular level. Glomerular and tubular functions are separately attacked by HgC1r Abteilung ffir Nephrologie, Medlzinische Hochschule Hannover, Konstanty-Gutschow-Str.8, D-3000 Hannover 61.

EFFECT OF BRI~DYKXNXN (BE) ON THE INTR~CELLULl%R CALCIUM ACTIVITY AND THE MEMBRANE VOLTAGE OF HUMAN G L O M B R U L A R E P I T H E L I A L C E ~ L S (GEC} H. P a v ~ n s t ~ d t , F. Bengen , R. Fischer, M. Sp~th, R.

Greger-, P. Schollmeyer ................................................... Human GEC were grown in culture and characterized with an antibody which stains podocytes in kidney slices (antibody was a gift from Prof. W. Kriz, Heidelberg, Germany). The effect of BK on intracellular free calcium activity [Ca§ was investigated with the fura-2 method: BK (10 .9 - 10 .6 mol/l) caused a rapid increase of [Ca§247 followed by a plateau. This effect could be inhibfted by the BK 2 antagonist Hoe 140. The rapid increase of [Ca*§ induced by BK was still present in the absence of extracellular calcium, whereas the p l a t e a u was abolished. The effect of BK on the membrane voltage (PD) was investigated with the nystatin patch clamp technique. The patch pipettes contained a KCl/K-gluconate solution with 10 .5 mol/l nystatin. The restin~ PD of GEC was "42 • 1 mV (n = 39). BK (i0"-- mol/l) transiently hyperpolarized GEC by 8 • 3 mV (n = 5). The h y p e r p o l a r i z a t i o n was completely inhibited if the extracellular K + concentration was increased from 3.6 to 30 mmol/l (n = 5). The hyperpolarization was followed by a depolarization (7.4 ~ 2 mV, n=5) which was augmented in the presence of a low extracellular Cl- concentration of 30 mmol/l (12.8 • 3 mV, n=5). The data indicate that BK is capable to release [Ca§247 from intracellular stores and to activate a K + and a CI" conductance via a B ~ receptor in human GEC. Medizinische Universit~tsklinik Freiburg, A b t e i l u n g IV, HugstetterstraBe 55, 7800 Freiburg * Physiologisches Institut der Universit~t Freiburg, Hermann-Herder-Stra6e 7, D-7800 Freiburg

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EFFECT OF DEXAMETHASONE ON MEMBRANE CONDUCTANCES AND Na+/K+-ATPase ACTIVITY IN CULTURED A6 CELLS M. Granitzer, B. Lyoussi, J. Crabb6 and W. Nagel

RENAl p~AMINOHIPPURATE (PAIl) TRANSPORT SYSTEMS IN RAT AND BOVINE KIDNEY EXHIBIT SPECIES DIFFERENCES C. Sehmitt and G. Burckhardt

Apical and basolateral membrane conductances (ga and gb) as well as Na+/K+-ATPase activity (ATPase) were analyzed in A6 tissue cultures using standard microelectrode techniques and ouabain-inhibitable ATP hydrolysis. The tissues were incubated under control conditions and with 10-7M dexamethasone (dexa) for 24 hours. Control values of ga, gb and ATPase were 67#S/cm 2, 336#S/cm2 and 6.9 #M/mg prot.h, respectively. Dexa increased ga and gb by 602% and 342% (n=32). After dexa, ga was related with the short-circuit current (Isc) as a measure of Na + transport by a parabolic function, which results from a decrease in the EMF for Na + entry. Ise and gh were not significantly related. ATPase increased after dexa by 175%. ATPase was linearly correlated with Ise under both conditions, albeit with reduced slope after dexa. To keep the Ise at the unstimulated level, a subset of tissues was incubated with 4.106M amiloride along with dexa. This did neither affect the increases in g, (measured after wash-out of amiloride) nor ATPase. The raise of gb, in contrast, was completely prevented and was thus secondary to stimulation of transceUular Na + flux. The data indicate furthermore that the gain in gb was not related to K + conductance, but due to activation of voltagesensitive basolateral C1- channels, which can be inhibited with NPPB. Incubation with 10paM cycloheximide during dexa depressed the increase in ga and Ise only slightly, whereas stimulation of ATPase was completely abolished. This indicates that protein synthesis might not be mandatory for the control of apical Na + channels. Effective stimulation of Na + transport by corticoids, however, relates almost exclusively on the activation of apical Na + channels with minimal and/or secondary changes in other transport parameters.

Transport systems involved in proximal tubular secretion of organic anions were studied with basolateral and brush-border membrane vesicles isolated from rat and bovine kidneys. A single Krebs cycle intermediate, a-ketoglutarate, cis-inhibits and trans-stimulates [3H]PAH uptake into basolateral membrane vesicles from kidneys of both species indicating that PAH and this dicarboxylate share the transporter. In the presence of a Na + gradient (Na+out > Na+i~), c~-ketoglutarate in the medium stimulates PAI-I uptake into vesicles from both species proving the cooperation of (Na+)3-dicarboxylate cotransporter and PAH/c~-ketoglutarate exchanger during accumulation of PAH in proximal tubule cells in rat and bovine kidney. Surprisingly, Na + and a-ketoglutarate stimulate [3H]PAH uptake in brush-border membrane vesicles from bovine kidney indicating that (Na+)3-dicarboxylate cotransporter and PAH/a-ketoglutarate exchanger cooperate also in the luminal cell membrane. Moreover, a-ketoglutarate and probenecid interact with brush-border and basolateral PAH transport with similar kinetics suggesting the presence of identical transporters in both cell membranes. In contrast, a-ketoglutarate does not influence [3H]PAI-I uptake into brush-border membrane vesicles from rat kidney. Thus, in the rat, the luminal PAH transporter does not accept dicarboxylates and is clearly different from the PAH/a-ketoglutarate exchanger in the basolateral membrane. Different transporters in brush-border and basolateral membranes are consistent with the observed net secretion of PAH in rat kidney. Whether similar transporters present in brush-border and basolateral membranes of bovine kidney proximal tubule cells achieve net absorption or net secretion of PAH must be determined.

Dtpartement de Physiologic, Universit6 Catholique de Louvain, Av. Hippocrate 55, B-1200 Bruxelles, Belgium

Max-Planck-Institut f/Jr Biophysik, Kennedyallee 70, D-6000 Frankfurt/Main 70, F.R.G.

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POSSIBLE ROLE OF HIPPURATE IN ACID-BASE REGULATION Angelika Hackl, M. MNyusz, T. M~ilyusz, P. Wrigge and I-I. Sick

SERUM REDUCTION IN CULTURED RENAL EPITHELIAL CELLS: MORPHOLOGY, CELL GROWTH AND PEANUT LECTIN BINDING W. Steigner, B. Gassner, W. Pfaller and H. Oberleithner

It has been known for years that hippurate catalyzes renal tubular

ammoniagenesis but also via urea production.

Previous studies have shown that Madin-Darby canine kidney (MDCK) ceils which originate from the collecting duet of dog kidney, differentiate into principal ceils and intercalated cells of the alkali secreting B-type and the acid secreting A-type. Differentiation depends on cell age (passage number) and culture conditions as pH and osmolarity of the culture medium. In this study we investigated the effects of reduction of the fetal calf serum (FCS) from 10% to 2% in the culture medium on the development of MDCK cells during the first 10 splits after plating (two splits per week). Morphological studies have shown that cells in serum reduced medium change their shape from small, cuboid and dark cells into spindle shaped, bright and flat cells. Surprisingly, 8 to 14 days after plating the number of cells grown in 2% FCS medium is up to 1.9 times higher than in 10% FCS medium. Cells grown in 2% FCS increase their apical surface by 36% compared to the membrane surface of cells grown in control medium. This indicates partial multilayering of the epithelium. Furthermore, apical binding of peanut agglutinine (PNA) and wheat germ agglutinine (WGA) is increased in 2% FCS grown cells (+58+21% PNA and +23_+11% WGA binding, compared to control). We conclude that the reduction of FCS in the culture medium encoupies cell growth and increases the rate of spontaneous cell transformation. Supported by SFB 176, A6.

Physiologisches Institut der Universit~it Kiel and Bundesanstalt fiJr Milchforschung, 2300 Kiel

Physiologisches Institut, Universit~t Wiirzburg, R6ntgenring 9, 8700 Wiirzburg

ammoniagenesis (AG) from glutamine by acivating the enzyme rGT. Nevertheless, the significance of hippurate especially in chronic acidosis is still in dispute. Considering the chemical structure of hippurate we presumed that it could serve not only as catalyzer but also as substrate for renal AG either 1) indirectly via its glycine moiety or 2) directly by forming an azlacton which after condensation with an aldehyde spontaneously splits into NH3, fatty acid and benzoate. - Investigating this latter pathway we measured 15NH 3 production from 15N-labelled hippurate in normal and chronically acidotic rats in vitro using kidney slices incubated in 15N-hippurate (0. l m M - l m M ) . By mass spectrometry we clearly proved 15NH3 formation which increased significantly in chronically acidotic rats. Further investigations in vivo by 1) either systemic infusion via V. jugularis or 2) local infusion via A. renalis sinistra of 15N-labelled hippurate we found 1) 15NH3 in urine as well as in the liver, 2) enhanced urea-content in the urine. This would be a hint that hippurate may take part in systemic acid base regulation not only via

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THEORETICAL ASPECTS REGARDING THE EVALUATION OF INTRACELLULAR pH MEASUREMENTS (pHi) USING THE FLUORESCENT INDICATOR SNARFI/AM AND CONFOCAL LASER SCANNING MICROSCOPY (CLSM). N. Opitz, E. Dufau, B. B611ing, H. Acker As reported previously (PflOgers Arch. 418(1991), Suppl.1, R 81), using the pH indicator SNARF-I/AM and CLSM, 9 pH~ c a l l bration curves (Nigericin method) in human mallgn glloma cells (spheroids MG I18) varied considerably from one cell to another despite emission ratioing of the fluorescence signals. To achieve comparable results within a cell population, a normalization of the measured ratios of each individual cell had to be performed. However, with respect to measurements under physiological conditions, the choice of this peculiar emission ratio for normalization of all ratio data sets is strongly restricted to pH^ values, for which pH_:pH~. Since none of these pH:i valuesUis a priori known, u / . . . the experimentally determlned functional course of various emission ratios on the corresponding pHo values (monitored under physiol, cond.) was analyzed in comparison to the Nigericin curve by means of normalization independent formfactors. This analysis revealed that significant pHi regulation in human malign glioma cells occurs mainly in the alkaline pH range, enabling normalization of the data sets to emission ratios within the acidic pH^ range and, consequently, a correct determination of the ~hysiological pHi values. Evaluation shows that, for increasing pHn, pHi see6s to level out into a plateau with a PHi,max. value of approx. 7.3S (resting pHi about 7.1 for PHn:7.37), whereas pHi = pHo for pHe values <7.0. The functionaT course of pH~ on pH^ can be approximated assuming an i n t r a c e l l u l a r b u f f e r ' e f f e c t ~ Based on this approximation and, in addition, the theoretical Nigericin calibration curve (as derived previously), the functional course of the normalized emission ratios (monitored under physiol, cond.) can be derived. This result demonstrates the r e l i a b i l i t y of the Nigericin calibration procedure, in particular the v a l i d i t y of the assumption PHi=PHn, for quantitative pHi measurements with fluorescent pH ifidic~tors. Max-Planck-lnstitut for Systemphysiologie, Rheinlanddamm 201, 4600 Dortmund i, FRG

ON THE ELECTRICAL AND MOTOR ACTIVITY OF THE GUINEA-PIG DETRUSOR MUSCLE: PATTERNS, STRETCH AND TEMPERATURE EFFECTS M.Ch. Michailov, D. Martin, E. Neu, I. Prechter, E. Gornik

!

,

123 INACTIVATION OF ION CONDUCTANCES BY SHRINKAGE OF MADIN DARBY CANINE KIDNEY (MDCK) CELLS M. Steidl, M. Ritter, F. Lang As shown previously, cell swelling leads to transient activation of K + channels and sustained activation of unselective ion channels in MDCK cells. The subsequent release of intracellular ions thus accomplishes regulatory cell volume decrease. The present study has been performed to elucidate the effects of cell shrinkage on ion conductances of MDCK cells. To this end conventional electrophysiology and cellular cable analysis have been applied to determine cell membrane potential (PD), cell membrane resistance (Rm) and ion selectivity. Cell shrinkage by addition of 100 mmol/1 mannitol leads to a hyperpolarization of the cell membrane from -47 + 2 mV to -59 + 2 mV (n = 14), with little change o f K + selectivity and an increase of Rm (from 2.0 + 0.2 to 6.4 _ 0.1 kfl cm z) without significant change of Rc. Virtually the same effects are elicited by addition of 50 mmol/1 NaC1 to the extracellular fluid. The addition of 50 mmol/1 NaC1 to the extracellular fluid leads to a reduction of cell volume from 2.4 +__ 0.1 pl to 1.7 ___ 0.1 pl, followed by partial regulatory cell volume increase to 2.1 + 0.1 pl (n = 9). The regulatory cell volume increase is partially inhibited by furosemide (0.1 tzmol/1), and partially by dimethylamiloride (0.1 /~mol/1), and is completely abolished in the presence of both inhibitors. Cell shrinkage leads to a rapid (within minutes), sustained (12 hours) increase of intracellular K+: The estimated potassium equilibrium potential increases from -77 mV to 85 mV. The present observations indicate that cell shrinkage leads to a sustained hyperpolarization and parallel inhibition of K + and C1conductance. However, regulatory cell volume increase cannot be accomplished by inhibition of ion conductances alone, but requires the additional activation of Na+/H + exchange or Na + ,K + ,2C1- cotransport. /nstitut for Physiologie, Universi~t Innsbruck

Detrusor preparations generate various electrical and motor patterns which can be transformed by ions (Eeitr. Urol. !' 188, 1983; Proc. Int. Un. Physiolo Sci. I_~7, 529, 1989). An evaluation of the electrical activity, i.e. of membrane (MP) and action (AP) potential, frequency (F), duration (D), rate of rise (RR) and fall (RF) of spikes (S: 40%), bursts (B: 22%) and burst-plateaus (BP: 38%) is given (n=l12): MP

[mV]

AP

[mV]

F [Hz]

D [s]

S 42.0+3.5 47.6+7.3 0.25 +0.13 0.005-0.05 B 46.552.0 52.0T9.0 0.02 ~0.01 6.9 +3.4 BP 45.5T5.2 52.359.0 0.01850.02 9.9 ~3.0

RR [V/s] RF [V/s] 1.0-3.6

0.6 -2.1

0.01-0.1 0.06-0.1

Relaxed strips generated predominantly spikes and bursts, stretched strips bursts and burst-plateaus; after stretching of the strips (0.01 to over 0.05 N) more B and BP appeared. After cooling (37 to 30~ the BP were transformed into E, after warming (37 ~o 41~ the BP-frequency increased, but the EP-duration decreased. TTX (1 /tiM) had no influence. TEA (100 /uM-I raM) increased both BP-frequency and duration. Verapamil (0.1-0.5 /uM) transformed S into BP. The amplitudes of spontaneous phasic connractions (SPC) and of contractions after electrical nerve stimulation (CES: 10 and 100 Hz, 0.3 ms, 3 s) were augmented with an increase of tension (0.01 to over 0.i N) (isometric reg.) or diminished with an increase of loading (0.5 to 3.0 g) (isotonic reg.) and vice versa. Cooling (37 to 20~ verapamil and nifedipin (0.1-10 /uM) abolished, TEA (100 /uM-i mM) and chinidin (1-10 /nM) strongly augmented SPC and CES. Gadolinium (10-100 /tiM) had no uniform inhibitory and stimulatory effects. It is suggested that the transformation of the spike- into burst- and burst-plateau-activities is based on enhanced de~rusor excitability after stretching (K+/Ca++-mechanism) via activation of stretch dependent ionic channels (Kt, Ca++). GSF - Inst. f[~r Strahlenbiologie,

D - 8042 Nenherberg

125 "ESCAPE" FROM SODIUM RETAINING EFFECTS INDUCED CHRONIC REDUCTION OF RENAL PERFUSION PRESSURE CONSCIOUS DOGS. H.Wo Reinhardt, M.Corea, W.Boemke and R.Pettker Technical assistance: D.Bayerl, S.Molling ..................................................

BY IN

perfusion pressure activity of the renin-angiotensin-aldosterone s y s t e m ( R A A S ) w h i c h reduces sodium excretion. The aim of this study was to test the influence of long term reduction of R P P o n R A A S a n d c i r c a d i a n N a - b a l a n c e . Methods: 3 female Beagle dogs were instrumented with catheters above and below the renal arteries and with an inflatable cuff above the renal arteries f o r s e r v o - c o n t r o l l i n g RPP. (Kirehheim et al., Pfl.Arch.405, 127 (1985)). Dogs were kept under standardized conditions (sodium intake 5.5 mmol Na/(kgbw*day) (A,J.P, R274 (1990)). RPP was reduced to 80 mm Hg for 4 days. Urine was collect e d e v e r y 20 m i n . B l o o d s a m p l e s f o r h o r m o n e a n a l y sis were taken every 4 hours. Results: T h e R P P d e p e n d e n t a c t i v a t i o n of R A A S l a s t e d f o r 24 t o 36 h o u r s a n d w a s c o m b i n e d w i t h s e vere Na-retention (at l e a s t 70 % o f s o d i u m i n t a k e ) on the first day. Equilibration of the circadian Na-balance was achieved u n t i l d a y 3 of R P P r e d u c tion. After day 2 activity of R A A S w a s found in t h e r a n g e of c o n t r o l s . Conclusions: Stimulation of R A A S b y R P P r e d u c t i o n is a transient phenomenon. Circadian Na- balance became equilibrated after pressure dependent RAAS stimulation was no longer present. The reason for the almost complete interruption of the "pressure-RAAS-axis" is u n k n o w n . Short (RPP)

term reduction leads to an

AG exp. An~sthesie, i 0 0 0 B e r l i n 19

in r e n a l increased

UKRV-C,

Spandauer

Damm

130,

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LOCALIZATION AND QUANTIFICATION OF ANGIOTENSINII (All) RECEPTOR SUBTYPES IN FETALAND ADULT KIDNEYSOF MAN E. Fuchs1, M. Simon2, H.-J. Gr5ne2

BILE SALT TRANSPORT IN BRUSH-BORDER AND BASOLATERAL MEMBRANE VESICLES FROM RAT KIDNEY S. Dietrich-Esser, W. Kramer* and G. Burckhardt

AlI plays an Important modulatory role in renal hemodynamics. The recent development of angiotenstn receptor antagonists will allow Investigation of the effects of AII in renal pathophysiologic situations. The effects of AI[ on the kidney are transmitted by specific receptors. In order to better understand the action of angiotensin receptor antagonists in the human kidney we characterized AI[ receptors by in vitro autoradiography on fetal (FE) and adult (AD) tissue using 1251[SarLIles] AlI (12sI-All) and inhibited the binding with the specific antagonists DuP753 and PD123177. In AD kidneys (n=5), binding of 12SI-AI[ showed the following characteristics (M+_.SD):Large and small preglomerular vessels (KD 387 +__ 29 pM; BMax4t + 3fmol/mg), glomeruli (Ko 885 + 217 pM; BMax 35 48fmol/mg), medullary vascular bundles (KD 142 + 52 pM; BMax 7 + 2fmel/mg). PD123177 effectively displaced 1251-AIIin large preglomerular vessels only (Ki 13 nM), while DuP753 solely displaced the labeled Itgand in glomerula (~ 22 riM) and medullary vascular bundles (Ki 14 nM). In contrast to rat kidneys, the outer stripe of the outer medulla showed pronounced specific AII binding to the tubulointertitial and vascular structures.In FE kidneys (n=4), a diffuse 1251-Al[ binding could be demonstrated in the medulla (KD 36 4- 7 pM; BMax 25 .+_ 3fmol/mg) and In the cortex (KD 19 __+8 pM; BMax 7 4- 2fmol/mg). Both cortical (~ 79 nM) and medullary binding (K~25 nM) could only be displaced by Pet23177. Human AD kidneys express the type 2 receptor in pregtomerularvessels, the type 1 receptor on glomeruli and medullary vascular bundles. The type 2 receptor is the sole receptor type in FE kidneys and may mediate mitogenic effects. The renal effects of angiotensin receptor antagonists may vary according to their binding characteristics to the renal AII receptor subtype. 1German Primate Center, G6ttingen and 2Department of Pathology, University of G0ttingen, FRG

Bile salts are reabsorbed in rat kidney proximal tubules by a Na+-dependent process. To define the transport systems involved in absorption we measured [3H]taurocholate (TC) uptake into brush-border and basolateral membrane vesicles isolated from rat kidney. [3I-I]TC uptake into brushborder vesicles was stimulated by a Na + gradient (Na+out > Na+in). Unlabeled taurine- and glycine-conjugated bile salts in the medium inhibited [3I-I]TC uptake better than unconjugated bile salts, and dihydroxy bile salts inhibited better than trihydroxy bile salts. Extravesicular lactate, succinate, p-aminohippurate and sulfate did not inhibit [3H]TC uptake suggesting that the Na+-bile salt cotransporter is not identical with the Na+-coupled transporters for monocarboxylates, dicarboxylates and sulfate, and the p-aminohippurate/anion exchanger. [3H]TC "uptake" into basolateral membrane vesicles was sensitive to variations in the intravesicular space, not stimulated by Na +, and inhibited by a series of bile salts. Similar results, however, were obtained with asolectin liposomes indicating volume-dependent, bile salt-sensitive partitioning of [3H]TC into the lipid phase rather than transport into the vesicle interior. In addition, significant binding to proteins of basolateral membrane vesicles occurred as visualized by photoaffinity labeling of several protein bands with [3H]7,7-azo-TC. Abundant partitioning into the lipid phase and binding to proteins thus prevented definition of the bile salt transporter in the basolateral membrane. We conclude that a Na+-coupled bile salt transporter is present in the brush-border membrane. The transporter responsible for bile salt exit across the basolateral membrane remains to be defined by other methods. Max-Planck-Institut f. Biophysik, Kennedyallee 70, D-6000 Frankfurt 70, and *Hoechst AG, Postfach 80 03 20, D-6230 Frankfurt 80, F.R.G.

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B L O O D P R E S S U R E A N D R E N A L R E S P O N S E T O H Y P O X I A IN CONSCIOUS CAROTID BODY DENERVATED AND INTACT RATS R. B e h m l~,W.H, de Muinck Keizer, R. Rettig, Th. U n g e r

INFLUENCE OF ADH ON THE REGULATION OF VOLUME AND INTRACELLULAR NA, K, AND CL CONTENT IN THE RENAL INNER MEDULLARY COLLECTING DUCT (IMC.D) CELLS OF RAT. J.M. Grunewald u, R.W. Grunewald 1, R.K.H. Kinne ~.

It has been s h o w n that moderate hypoxia effects sodium and volume homeostasis in mammals. Moreover, evidence has cumulated that peripheral arterial chemoreceptors (PAC) which are stimulated in hypoxia have some influence on renal sodium excretion in h u m a n s a n d other mammals. The special effect of chemodeafferentat/on on renal response to hypoxia has not yet been elucidated in conscious rats. O u r study was designed to investigate the effects of normobaric hypoxia on kidney functions a n d systolic blood pressure in conscious carotid b o d y denervated and s h a m operated rats. T w e n t y adult male Wistar rats were unilaterally n e p h rectomized and chronically instrumented with venous, arterial and ureter catheters. Ten rats u n d e r w e n t carotid b o d y denervation (CBD) and ten s h a m operation (SO). All rats were consecutively exposed to normoxia (60 rain), hypoxia (30 rain in 12.5% O2) and normox/a (60 m/n). In SO, exposure to hypoxia resulted in significant decreases in arterial blood pressure (MAP) (106-+6 vs. 80-+21 mmHg), glomerular filtration rate (GFR) (814-+64 vs. 442-+113 ~I/g/min), renal blood flow (RBF) (5.13-+0.46 vs. 2.61-+0.54 ml/g/min), diuresis (32.1-+4.5 vs. 15.3-+4.9 ~I/g/min) and natriuresis (2.03-+0.71 vs. 0.92-+0.20 ~mol/g/min). These effects were significantly enhanced in CBD.. M A P (104-+7 vs. 56-+6 mmHg), G F R (891-+65 vs. 415-+136 ~I/g/min), R B F (5.00-+0.29 vs. 2.68-+0.83 ml/g/min), diuresis (35.8-+4.9 vs. 11.5-+3.7 ~I/m/n) and natriuresis (2.26-+0.7 vs. 0.62-+02 ~mol/g/m/n). These results suggest that P A C play an important role in the maintenance of sodium and volume homeostasis as well as the regulation of blood pressure a n d renal hemodynamics in conscious rats. The mechanisms b y which chemoreceptor activity exerts its effect on the kidney remains to clarify. Deutsches Institut fur Blur h o c h d r uc kf orsc h u n g und Pharmakologisches Inst/tut der Universit~t Heidelberg, Im Neuenheimer Feld 366, D-6900 Heidelberg, ~i Institut fur Physiologie der Universit~t Rostock

ADH plays an important role during the production of urine, while in the inner medulla collecting duct cells are exposed to rapid and extensive changes in osmolarity. Therefore the influence of ADH on the osmoregulation of IMCD cells of rat kidney was investigated. In 600 mOsm (isotonic; 268 mM NaCI) isolated IMCD cells were exposed to 300 mOsm (hypotonic; 118 mM NaCI) and to 900 mOsm (hypertonic; 418 mM NaCI). Cell volume was measured by determination of cell water via tritiated water, cell ion content was determined by electron microprobe analysis. Under hypotonic conditions after initial cell swelling volume regulatory decrease (RVD) was accelerated in presence of 10~ M ADH (RVD within 3 min vs. 7 min without ADH). The effect of ADH was mimicked by 10.4 M 8-BromocAMP and was prolonged by 5 mM barium, 20 mM Ouabain, and 1 mM SiTS indicating hormonal effects on a barium sensitive K channel, a ouabain sensitive Na/K-ATPase, and a SITS inhibitable anion exchanger. Under hypertonic conditions in presence of ADH or 8-Bromo-cAMP IMCD cells showed regulatory volume increase (RVI) after initial shrinkage (vs. constant decreased volume without ADH). The ion contents of Na (+95-+22%) and CI (+57-+7%) were elevated leading to increased concentrations whereas K concentration remained constant. RVI was inhibited by 0.1 mM bumetanide, 1 mM amiloride, and 1 mM SITS indicating participation of a Na/K/2CI cotransporter, cation exchanging, and anion exchanging systems in ADH effects on hypertonic volume regulation. These results suggest that ADH influences the osmoregulation of IMCD cells at least partly via altered regulation of ion transporting systems. 1Sektion Nephrologie, Abteilung Innere Medizin IV, Universit&tsklinik UIm, Robert-Koch-StraSe 8, D-7900 UIm 2Max-Planck-lnstitut for Systemphysiologie, Rheinlanddamm 201, D-4600 Dortmund.

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EFFECT OF CYCLOSPORIN A (CYA) ON L I P I D PEROXIDATION (LPO) IN VIVO AND IN VITRO IN RAT KIDNEY J. Hannemann and K. Baumann

STIMULATION OF PERIPHERAL. A R T E R I A L CHEMORECEPTORS D O E S NOT C H A N G E P R O X I M A L V O L U M E AND S O D I U M REABSORRTION OF THE KIDNEYS IN BORDERLINE HYPERTENSIVES C. L e d d e r h o s . W. Q u i e s ~ R. S a n c h e z . H, B r a u e r

C y A - i n d u c e d LPO was m e a s u r e d i n r e n a l t i s s u e in rive after treatment o f m a l e W i s t a r r a t s w i t h CyA (20 m g / k g BW, s . c . , once daily for 8 days) or in vitro in incubation experiments u s i n g CyA c o n c e n trations up t o 100 p g / m l medium and i n c u b a t i o n t i m e s up t o 3 h o u r s . LPO was m o n i t o r e d by e s t i m a ting the formation of malendialdehyde (MDA) u s i n g the thiobarbituric acid assay. In viva, CyA i n creased the MDA-content in renal cortical, slices from 36.2 to 46.6 nmol/g tissue, compared to control rats. In vitro, at the level of subcellular structures, CyA a t c o n c e n t r a t i o n s o f 3 p g / m l and higher caused a significant increase o f MDA p r o duction in microsomes. The C y A - i n d u a e d m i c r o s o m a l MDA p r o d u c t i o n was t i m e - and c o n c e n t r a t i o n - d e pendent. CyA had no i n f l u e n c e on t h e a s c o r b i c acid/Fe(II)-stimulated MDA p r o d u c t i o n in brush b o r d e r membrane f r a c t i o n s . Using renal cortical slices incubated in the CyA-containing medium, CyA in the range of concentrations tested, had no effect on t h e s l i c e - c o n t e n t o f MDA, p y r u v a t e - s t i mulated gluconeogenesis, content of glutathione (GSH) and p a r a a m i n o h i p p u r a t e (PAH)-accumulation. The r e s u l t s add f u r t h e r evidence to our recent suggestion that lipid peroxidation participates in cyclosporin A-induced nephrotoxicity. (Inselmann, G . , Hannemann, J . and Baumann, K . , Res. Commun. Chem. P a t h o l . Pharmacol. 68, 189-203, 1990).

In e x p e r i m e n t s p e r f o r m e d p r e v i o u s l y in n o r m o t e n s i v e s u b j e c t s u n d e r g o i n g w a t e r d i u r e s i s there were s o m e features relating to an inhibition of proximal v o l u m e and s o d i u m r e a b s o r p t i o n due to p h a r m a c o l o g i c s t i m u l a t i o n of p e r i p h e r a l a r t e r i a l c h e m o r e c e p t o r s . 7o check this fact Jn b o r d e r l i n e hypertensives (BHTJ we examined the belnaviour of lithium clearance, w h i c h has b e e n p r o p o s e d as an index ef fluid and sodium delivery from the proximal tubules, Jn 14 BHT u n d e r water diureeis during stimulation of their peripheral arterial c h e m o r e c e p t o r s by a l m i t r i n e b i e m e s y l a t e . There w e r e no d i f f e r e n c e s in f r a c t i o n a l excretion ef lithium between almitrine and control group indicating no c h a n g e s of fluid delivery to the distal nephron Jn BHT during chemoreceptor stimulation. The h a n d l i n g Of s o d i u m in the distal n e p h t o n in the almitrine group differed e ~ g n i f i c a n t l y f r o m that i n the eonti~ol group. Obviously chemoreceptor s t i m u l a t i o n e n h a n c e d the s o d i u m r e a b s o r p t i o n in the distal nephron, So that S o d i u m exoretlon became small and s m a l l e r w i t h d u r a t i o n of c h e m o r e c e p t o r stimulation~ Our results o b t a i n e d Jn BHT in r e l a t i o n to t h o s e of normotensives suggest that Proximal and dishal tubular functiens can be m o d i f i e d by peripheral arterial chemoreceptors differently each from another.

Abt. for Zellphysiologie, der Universit~t Hamburg, W-2009 Hamburg 13

Physiologisches Institut Grindelallee 117,

for Physiologie der Universitat RubenowstraBe 3. 0-2200 Greif'swa],d

133

131 GLYCINE-DEPENDENT TIME COURSE OF HYPOXIC IN I S O L A T E D T U B U L E S OF R A T R E N A L C O R T E X Klause,N.,

Inetitut Greifswaid.

*H.Gramberg,

W.Niedermayer,

SWELLING

and

*G.Gronow

Reported time spans necessary to induce a hypoxic disruptive s w e l l i n g in r e n a l c o r t i c a l cells vary f r o m 20 m i n u p to 1 hour. W e m e a s u r e d t h e r e l e a s e of intracellular m a r k e r s (LDH f o r c y t o p l a s m , GDH for m i t o c h o n d r i a , N A G for l y s o s o m e s , all in m U / m g protein) f r o m c o l l a g e n a s e - i s o l a t e d t u b u l a r s e g m e n t s of r a t r e n a l c o r t e x (n =24). T h e t u b u l i w e r e i n c u b a t e d at l o w o x y g e n t e n s i o n (PC2 < 1 ram Hg) in K r e b s - R i n ger-Bicarbonate (37~ a n d e i t h e r 5 m m o l /i g l y c i n e (G) o r 5 m m o l / l cycloleucine (C) w a s added: min: 5 I0 30 60 .................................................... LDH

C: 21.3• 48.8• 331.0• 369.0• G: 7.4• 7.6• 71.0• 135.0• GDH C: 1.83• 3.61• 16.7• 39.0• G: 0.99• 1.31• 5.2• 11.3• NAG C: 3.14• 6.91• 27.0• 58.6• G: 0.92• 0.95• 8.41• 19.2• It is concluded that severe cellular damage a l r e a d y o c c u r r e d a f t e r i0 min, i n d i c a t e d b y a m a r k e d 10ss of vital cellular constituents. Glycine significantly suppressed hypoxic cellular swelling, indicating a potential value for organ preservation. Abt. N e p h r o l o g i e * Physiologisches

i. Med. U n i v e r s i t ~ t s k l i n i k und Institut der Universit~t Kiel

Chenodeoxycholic acld and deoxycholic acid but not cholic acid or llthocholic acid are able to elevate kidney glntathlone in vivo E. Purucker and J. Lutz.

In t h r e e r a t - m o d e l s of c h r o n i c liver disease, i.e. c a r b o n t e t r a c h l o r i d e induced cirrhosis of the liver, porto-eaval shunting, and bile duct ligation, we recently observed an elevation of kidney reduced (GSH) and oxidized (GSSG) glutathione. The elevation was maximal in bile duct ligated animals accounting for +81% of controls for GSH and +38% for GSSG as soon as 6h a f t e r ligation. We h y p o t h e s i z e d that bile acids m i g h t induce these changes and therefore conducted the following study. Method: Male wistar rats (220-250 g) received a single 35 / I M / K G i.v. dose of either isotonic saline, tauroglycocholic acid (tg-C: a crude mixture of bile acids from ox bile), taurine or glyeine conjugates of cholic acid (tC, g-C), chenodeoxycholic acid (t-CDC, g-CDC), deoxycholic acid (t-DC, g - D C ) , or t a u r o l i t h o c h o l i c acid ( t - L C ) . A f t e r 4h the left k i d n e y was r e m o v e d u n d e r ether anesthesia, rinsed in i c e - c o l d isotonic NaCI, a n d immediately homogenized in 5%-5-sulfosalicylic acid. The content of GSH and GSSG were analyzed according to Griffith (1980). Results: Control values in the saline group (x+SD, n=8) accounted for 2.38 • 0.18 ~ M G S H / g ww and 178 • 14 nM GSSG/g. The changes induced by the various bile acids are listed in the table as percent of controls: t-C

g-C

GSH +44%

tg-C

+ 4%

-2%

t-CDC g-CDC t-DC +39%

+34%

+45%

+46%

g-DC

t-LC §

GSSG +28%

+26%

+31%

+48%

+49%

+68%

+63%

§

(all differences significant with p<0.01, except the values in cursive). Comment: 1) The different amidates of the bile acids exhibit an identical inductive potential on the renal tissue GSH and GSSG. 2) While no G S H i n c r e a s e is o b s e r v e d w i t h cholie a c i d a n d l i t h o c h a l i e a c i d , the d i hydroxylated bile acids (CDC, DC) cause a marked increase in GSH. With the exception of LC the GSSG increase is induced by all bile acids tested. 3) Thus, it can be concluded, that rather a direct stimulation of the GSHsynthesis than an adaptive GSH-increase after G S S G - f o r m a t i o n might be the reason for the observed GSH-elevation.

Physiologisches Institut der Universit~t, R0ntgenring 9, D-8700 WSrzburg

R 101

136

134 INHIBITION OF EPITHELIAL CHLORIDE E P I T H E L I A L CY'FOSOLS c.P. Hansen, B. Roch, K. Kunzelmann and R. Greger

CHANNELS

BY

Chloride channels of intermediate conductance showing outward rectification (ICOn) have been found in several epithelia. Recently, it has been shown that I c o n can be blocked by cytosol from several epithelia and placenta (1). In the present study we have tested whether the responsible cytosolic factor (CI) could be a member of the eicosanoid system. Icon in excised inside-out patches of HT:~9 cells was studied with the patch clamp technique. The following substances were tested: Mellitin and mepacrine (activator and inhibitor of the phopholipase A2), indomethacin and nordihydroguaiaretic acid (inhibitors of cyclooxygenase and lipoxygenase respectively), and the end products leukotriene D4 and prostaglandin E2. None of the tested substances had effects compatible with a direct regulatory role of eicosanoids on Icon. Consequently, CI is probably not an eicosanoid. In addition to inhibiton of Icon, cytosol from placenta reduces the baseline current in excised HT29 patches (1). To test whether this base-line current reduction was caused by inhibition of a chloride current, we have dissolved chloroform extracts of human placental cytosol in either NaCI or choline chloride. As expected I c o n is inhibited to the same extent by the two CIcontaining solutions. Furthermore, the baseline current is also reduced to the same extent by CI in NaCI and choline chloride, and therefore we suggest the existence of chloride channels with conductances which are too small to be identified as single channels, Separate experiments indicate that these chloride channels are not under direct control of Ca2+. Since reduced chloride secretion is a central pathogenic mechanism in cystic fibrosis (CF), we have speculated that CF epithelia might contain increased concentrations of CI. Therefore, we have prepared cytos01 from CF-PAC (CF epithelium) and HT;~9 (control) cells. Measurement of the cytosolic protein concentration indicates that the cytosols from both cell lines were diluted in the order of 10-15 times (with NaCI) as intended. Preliminary results show that both HT29 and CF-PAC cytosols inhibit Icon as well as base-line current in HT:z9 patches. The inhibitory potencies for CF-PAC cytosol and HT29 cytosol seem to lie in the same range. (1) K. Kunzelmann et al. PflOgers Archly 1991 (in press)

Na/K/CI-COTRANSPORT ACTIVITY IN NORMAL AND SHRUNKEN HUMAN AND FERRET RED CELLS IS REGULATED BY PROTEIN PHOSPHORYLATION AND DEPHOSPHORYLATION. H.MairNiurl and J.F.Hoffman. .

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Na/K/Cl-cotransport in human red ceils is known to be activated by cell shrinkage. We have shown that this activation is not caused by changes in the cellular concentrations of Na i, K i and Mg i. The present study was designed to further examine mechanisms involved in the regulation of cotransport activity by changes in cell volume. Freshly collected human red ceils were treated with nystatin to adjust Nai and Ki to 50 mM each in order to achieve Vr,~xof cotransport for these ions. Cotransport was measured as 22Na-effiux sensitive to 10 I.tM bumetanide in the presence of 0.1 mM ouabain. Ferret red cells were used unmodified (high Na-cells) and 22Na-uptake was measured instead of efflux. Flux media contained physiological ion concentrations (290 mosmol/1) with changes in the osmolarity being adjusted with sucrose. The maximum activation of cotransport in human red cells occurs at about 350 mosmol/1 and in ferret at about 450 mosmol/1. Even though increasing or decreasing Mg i (by use of a Mgo-buffering system and A23187) alters the Na/K/Cl-cotransport rate at normal ceil volume, the rate can always be stimulated by cell shrinkage. The protein phosphatase inhibitor okadaic acid (lgM) stimulates cotransport more than either Mgi or shrinkage, and neither of these two stimuli alone nor both in combination activate cotransport further in its presence. Pretreatment of red cells with ldnase inhibitors (K252a (10gM), staurosporine (0. lgM)) can reduce or completely inhibit cotransport in normal and shrunken red cells. Dibutyl-cAMP and phorbol ester (with or without increasing Cai) have no effect on cotransport. These results indicate that the phosphorylated state of the cotransporter and/or an associated protein determines the activity of the cotransport system. The results imply the involvement of a serine/threonine protein kinase and protein phosphatase type 1. Supported by NIH-grant HL-09906. Department of Cellular and Molecular Physiology, Yale Medical School, 333 Cedar Street, New Haven, CT 06511, USA.

Physiologisches Institut der Albert-Ludwigs-Universitfit, Hermann-Herder-Strasse 7, D-7800 Freiburg

135

137

CHARACTERIZATION OF CONDUCTIVE CHLORIDE TRANSPORT IN RENAL BRUSH-BORDER MEMBRANE VESICLES W. Krick, M. Vogt and G. Burckhardt

SPONTANEOUS MEMBRANE POTENTIAL OSCILLATIONS IN TRANSFORMED MADIN-DARBY CANINE KIDNEY (MDCK) CELLS H.-J. Westphale, B. GaBner, A. Schwab, and H. Oberleithner

As opposed to the luminal membrane of proximal tubule cells in situ are isolated brush-border membrane vesicles (BBMV) highly conductive for C1- as revealed from the following experiments. First, addition of C1- to a suspension of BBMV creates an inside negative C1--diffusion potential as visualized by the voltage-sensitive carhocyanine dye, DiS-C2(5), and by the stimulation of voltage-sensitive, Na+-coupled uptake of D-glucose. Second, an inside positive K+-diffusion potential enhanced C1- uptake measured with 36C1-, with an intravesicular C1--indicator, SPQ, or by recording salt transport-dependent volume changes of vesicles by alterations in light scattering. Using one or more of the aforementioned methods we found that the C1- (anion) conductance does not saturate with C1- concentrations up to 150 mM and accomodates the anions NO3- , SCN- > I- > Br-, C1- > F- > > SO42-, gluconate. Metabolic products such as lactate, pyruvate, and a-ketoglutarate did not influence C1transport suggesting that they do not share the anion conductance. Voltage-dependent C1- transport is half-maximally inhibited by -0.2 mM of the stilbene disulfonate, DIDS, and of the C1- channel blocker, NPPB. 20-100 /zg phosphatidylethanolamine, -choline, and -serine, and a series of C18 and C20 fatty acids did not influence C1- transport except for 100 #g/ml arachidonic acid which inhibited C1- conductance. Probenecid acted as a further inhibitor of CI- conductance when applied from the inside of the vesicles. Thus the brush-border membrane contains an anion conductance which is sensitive to C1--channel blockers and may be downregulated in the intact cell by a cytosolic inhibitor as reported earlier (Pflfigers Arch. 418: R81, 1991).

Hormones like aldosterone and growth factors stimulate plasma membrane Na+/H+ exchange leading to cytoplasmic alkalinization in renal target cells. Intracellular alkalosis reduces the H+ bonds between repressor proteins and DNA followed by the destabilization of nuclear chromatin. We observed that alkaline stress "per se" can induce malignant transformation of MDCK cells which resemble intercalated cells of renal collecting duct. Cells were kept in alkaline (pH 7.8) culture medium for 12 days. Within this period multiple "foci" appeared composed of spindle=shaped cells lacking contact inhibition and exhibiting poor adhesion to the culture support, typical characteristics of dedifferantiated tumor cells. "Foci" cells were cloned and grown in control medium (pH 7.4). They maintained their morphological characteristics indicating a stable pH-induced genetic transformation. Cells were fused with polyethylene glycol to giant cells and impaled with microelectrodes. In contrast to non-transformed giant MDCK cells the membrane potential showed spontaneous (temperature dependent) oscillations with a frequency of 1.4/min and an amplitude of about. 14 mV. Ca2+-free superfusion led to a dramatic decrease in frequency as well as in amplitude of spikes. The addition of the diuretic drug amiloride (lmM) reduced both spike frequency and spike amplitude, whereas superfusion with a Ba2+ (10mM) containing Ringer solution resulted only in a strong decrease of spike amplitude, but not of spike frequency. All maneuvres were reversible. We conclude that MDCK ceils can be transformed to malignancy by sustained alkaline stress indicated by an abnormal growth pattern and by the appearance of instable Ca2+ activated K+ channels. Amiloride could block the oscillating activity by intracellular acidification due to inhibition of Na+/H+ exchange and protonation of K+ channels.

Max-Planck-Institut fiir Biophysik, Kennedyallee 70, D-6000 Frankfurt/Main 70, F.R.G.

Dept. of Physiology, University of W0rzburg, ROntgenring 9, D-8700 Wi~rzburg.

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R 102

140

138 ON THE NATURE OF PD AND CALCIUM OSCILLATIONS IN RAS ONCOGENE EXPRESSING CELLS S. Waldegger, E. W611, H. Weiss, P. Died, K. Maly, H. Grunicke, F. Lang

K +- AND CI--CONDUCTIVE PATHWAYS ACTIVATED BY HYPOTONIC STRESS OF RAT HEPATOCYTES IN PRIMARY CULTURE F. Wehner

According to previous studies from this laboratory applying conventional electrophysiology, patch clamp and fluorescence measurements, NIH 3T3 fibroblasts expressing the ras oncogene unlike NIH 3T3 fibroblasts not expressing the oncogene respond to bradykinin with sustained oscillations of cell membrane potential (PD) due to oscillatory activation of calcium sensitive K + channels secondary to oscillations of intracellular calcium activity (Cai). The present study has been performed to elucidate the mechanisms accounting for the oscillations of PD and Cal. The PD oscillations do not require the presence of sodium, bicarbonate or chloride in the extracellular fuid and their amplitudes (- 35 _ 2 mV, n = 34) and peaks (-57 __. 2 mV, n = 34) are modified by alterations of extracellular potassium activity, as predicted for oscillations of potassium conductance. The frequency of the PD oscillations (1.17 ___ 0.O4/min, n = 34) is not sensitive to alterations of extracellular sodium, chloride, bicarbonate and potassium. The PD and Cal oscillations are abolished upon removal of extracellular calcium and the peak of the oscillations is significantly enhanced upon increase of extracellular calcium concentration from 1.2 to 5 retool/1. A reduction of extracellular calcium concentration from 1.2 to 0.2 retool/1 significantly decreases the frequency of the PD and Cal oscillations. The PD oscillations are not significantly modified by acute administration of 0.1 mmol/1 ouabain, 0.1 mmol/l cobalt, 0.1 mmol/1 gadolinum, 0.1 mmol/1 zinc, 10 /zmol/1 verapamil or 10 #mol/1 diltiazem. The oscillations, however, are abolished in the presence of 1O mmol/1 barium, 1 mmol/1 cadmium, 1 mmol/l lanthanum and 10 /~mol/1 nifedipine. Lower concentrations of nifedipine (1 #mol/1) do not significantly modify the oscillations. It is concluded that the oscillations are due to intermittent entry of extracellular calcium via a cadmium-, lanthanum - and nifedipinesensitive pathway and subsequent activation of barium sensitive K + channels. The calcium entry pathway is eventually inactivated by increase of intracellular calcium giving rise to the oscillations of Cal and PD.

There is mounting evidence that regulatory volume decrease in hepatocytes is mediated by K + and CI- exit through conductive pathways. Rat hepatocytes in confluent primary cultures were impaled with conventional microelectrodes to determine membrane effects of hypotonic stress. Reducing osmolarity by omission of 8 0 mM sucrose from the superfusate leads to a transient hyperpolarization of cell membranes (maximum after 5 rain) from -39,8 + 4.1 m e t e - 5 1 . 4 + 1.6 mV (n = 7) and from -18.9 + 1.3 mV to -36.1 + 3.6 mV (n = 6} in HCO3--containing and HCO3--free solutions, respectively. This hyperpolarization is completely blocked by 1 m M B a ++ (HCO 3- present).

Institutes for Physiology and Biochemistry, Innsbruck, Austria

In ion substitution experiments increasing K + from 2.7 to 27 mmol/I (HCO 3- present) depolarizes membrane voltage by 9.3 + 1,6 mV in normosmotic solutions. In hypoosmotic solutions this depolarization is increased to 19.5 + 1.1 mV at the time o f maximum hyperpolarization and decreases thereafter to 7 9 + 1.9 mV (n = 4). Decreasing CI- from 116.5 to 1.2 mmol/I (HCO 3- free) depolarizes hepatocytes by 23.9 + 3 . 0 mV in normosmotic solutions. In hypoosmotic solutions this effect is increased to 39.3 + 3.8 mV at maximum hyperpolarization and decreases again to 26.3 + 2.5 mV (n = 8). These results suggest parallel and transient activation of K +- and CI--conductive pathways upon hypotonic stress. K + will leave the cells together with CI" that in part electrically balances K + current. HCO 3- is probably not involved in regulatory volume decrease of rat hepatocytes. Max-Planck-lnstitut f0r Systemphysiologie, Rheinlanddamm 2 0 1 , D-46OO Dortmund

University of A-6010

139 Ecdysone induced intracellular alkalinization is a prerequisite for

gene

activation in Drosophila melanogaster salivary glands.

S. Wunsch, S. Schneider and H. Oberleithner Steroid hormone induced changes of intracellular ion composition are proposed to be an early regulating mechanism of gene activation. We used salivary glands

of Drosophila melanogaster as a model system in which gene activation, that is decondensation of chromatin, is seen as nuclear swelling. Previously, we showed that the moulting hormone ecdysone (Ec) hyperpolarizes the cell membrane potential and causes nuclear swelling. In this study we wanted to measure the influence of Ec on intracellular pH and the influence of pH on

geue activation. Glands of the third larval stage were superfused with a solution mimicking the hemolymph. Intracellular pH was measured with pH-electrodas and changes of nuclear volume were evaluated by video image analysis. Superfusion of the cells with Drosophila Ringer containing 5.10-6 mol/1 Ec leads to an intracellular alkalinization. Starting at a steady-state intracellular pH of 7.2-7.6, Ec induces within minutes a rise of cytoplasmic pH by 0.26 ~: 0.02 units (n=6). Superfusion of salivary glands for 60 minutes with acidic Ringer solution (pH=6.8) leads to a decrease of 16.9 • 3.9 % (n=14) in nuclear volume,

compared to the control volume at pH 7.15, whereas at pH 8.0 a small increase of nuclear volume of 4.2 • 1.4 % (n=43) is observed. Amiloride (10-s mol/1), a blocker of plasma membrane Na+/H + exchange, prevents Ec induced nuclear swelling most likely by intracellular acidification.

We postulate that Ec activates the Na+/H + exchanger leading to an intracellular alkalinization which is a prerequisite for steroid induced gene activation. Dept. of Physiology, Univ. Wilrzburg, Rontgenring 9, D-8700 Wilrzburg.

141 NH4+/NH3 EXPOSURES: CONTRIBUTIONS OF MEMBRANEMECHANISMS TO NH4+-TRANSPORT IN A SENSORY NEURON H. Moser, F. Fresser and N. Mair Experiments were performed in crayfish sensory neurones, using pH- and NH4+-selective microelectrodes (Fresser et al.u J Exp Biol~ 157, 227-241). In cells exposed to 20mM NH4+/NH3 salines for 3 min, changes of [NH4t]~ (intracellular ammonium concentration) were studl~ed in controls, during the presence of blockers and during ion substitutions~ Various blockers of K + channels were used at relatively high concentrations (50mM TEA, 13.SmM BaCI,~ 50aim CsCI, 3mM 4-aminopyridine and 10-6M apamine~. From these substances only 4-aminopyridine and CsCI substantially reduced [NH4+]i by about 12% a n d 20% i respectively. [NH4+]i remained unaffected, when Nachannels were blocked by tetrodotoxin (TTX). In contrast, inhibitors of anion-transport (0.1mM bumetanide; imM furosemide) affected [NH4e]i more effectively~ In furosemide [NH4+]~ was reduced by 50%. A similar reduction was f~und in Cl--free (isethionate and gluconate) salines. + + In Na -free (n-methyl-d-glucamine) salines, INN 4 & ]~ 9 Q , increased by about 10z. Stopplng pH i regulation, the effect could arise from acidification, which in turn results in an additional conversion of [NHA+]~ from [NH3]i, and/or from substitution of NH4+~fo~ Na + in the amiloride-sensitive Na+/H + exchange. In NH4+/NH~ exposed cells indirect (conversion from NH~) and d~rect mechanisms (transport of NH4 +) contribute to [NH4+~.o We could show that substantial amounts of [ N H 4 ~ i originate from transmembrane movements of N~4~-ions~ mediated by a transport system for which the presence of Cl- ions seems essential. Other pathways, such as cation channels~ and the pH i regulatin~ system contribute relatively little to direct [NH4 ~] entry. Supported by the FWF (project 6177)t Austria

Insto f. Zoologie, Technikerstr.25,

A6020 Innsbruck

R 103 144

142 CHARACTERIZATION OF AN AMILORIDE-SENSITIVE NA+-CHANNEL IN AN INVERTEBRATE

Mechanism of Intracellular Ca2+-Transients in HT29 cells R. Nitschke, J. Leipziger & R. Greger

(HIRUO0 MEDICINALIS) B. Dannenmaier,W.-M. Weber, W. Clauss Electrogenic

Na+-channels are welt known from different e p i t h e l i a l tissues

of a wide variety of vertebrate species. Their function is to manage Na+transport from the outside medium across the apical membrane to the cytosolic

side. Up to now only a few reports about Na+-channels in invertebrate epithelia have been published. in OUr experiments we used parts of the dorsal skin of the leech Hirudo medicinalis for measurements performed in Ussing chambers perfused with NaC[ringer. The leech skin with resistances of about 10 kQ/cm2 is a relatively tight epithelium compared to other epithelial tissues. The initial short circuit current (Isc) is in average 16 ~A/cm2. Removal of NB+ from the apical bath medium reduces ISC to about 60% of its original amount showing that other ions than NB+ are involved in the total exchange of charges across the membranes. Application of chloride-free solution to the apical bath causes a decrease of I close to zero. The NB+- conductance saturates with a concentration oafc 90 ~ in the apical solution (KM=5.9 mM). 40% of this Na+ current is inhibited by I00 #M amiloride on the apical side. The dependance of Na+-conductance from the amiloride concentration follows Michaelis-MentenKinetics (KIt4 #M, Vmax=90 #M). Thus leech skin Na+-channels are Less sensitive to amiloride than channels known from vertebrate epithelia. It is known from other tissues that intracellular cAMP stimulates Na+ uptake through amiloride sensitive channels. In leech skin, addition of cAMP or inhibitors of cAMP degradation (IBMX) which are able to permeate the membrane + nearly trebles iSC and increases amiloride sensitive Na -currents by 40 %. Using the method of current fluctuations ana[ys,s with a low-noise voltage clamp we found the channel density to be 5"106/cm2 with a single channel current of 2 pA. The selectivity for other ions as Li+, K+, Rb+, Cs+ and La3+ and the low affinities for amiloride strongly implies evidence that this Na§ behaves differently than vertebrate Na+-channels. This work was supported by EC and DFG Institut fbr Veterin~rphysiologie, Freie Universit~t Berlin, Koserstr. 20, W-1000 Berlin 33

ATP, carbachol (CCH) and neurotensin (NT) induce a rise in intracellular Ca2+-activity ([Ca2+] i) in HT29-cells, as we have shown recently with fura-2 measurements. The increase in [Ca2+] i consists of an initial peak (ATP, CCH, NT) and a following so-called plateau phase of elevated [Ca2+] i (only ATP and CCH) as long as the agonist is applied. We further investigated the nature of the Ca2+-transients. The ATP and CCH induced [Ca2+]i increases showed under extracellular Ca2+-free conditions only an initial release from intracellular Ca2+-stores. The normally following plateau phase was abolished under these conditions. The transient character of the NT induced (Ca2 +]i response in the absence or presence of extracellular Ca 2+ suggested an exclusive intracellular Ca2+-release mechanism. Using the fura-2 Mn 2+quenching technique we can show under low extracellular Ca 2+ (=;10 .6 tool/I) for the ATP-, CCH- and NT-induced Ca2+-transient in HT29-cells a simultaneous increase of [Ca2+][ due to intracellular Ca2+-reieases and a quench of the fura-2 fluorescence signal due to Mn2+-influx into the cell. These data confirm for all three agonists an early opening of Ca2+-influx pathways also permeable fur Mn 2+ during the onset of the Ca2+-transient. The dose dependence of [Ca2+] i during the plateau phase was measured for ATP and CCH. [Ca2+] i during the plateau phase was in the case of ATP reversely dependent on the agonist concentration. Whereas after at least 15 rain ATP-application (10 .5 and 10 .6 tool/I) a markedly elevated plateau in [Ca2+] i was reached, no plateau phase was seen at an ATP concentration of 10 -4 tool/I; [Ca2+]i decreased continuously within 10-15 rain to its resting value. These characteristic features of the Ca2+-transient could not be shown for CCH. After 1 h with CCH (10 -5 and 10 -4 tool/I) [Ca2+] i was still significantly higher than the resting [Ca2+]i, but slightly lower compared to the 15 min [Ca:~+]i-values. The intracellular store component of the ATP, CCH and NT-induced Ca 2+transient under Ca2+-free conditions could not be generated a second time within 10 min. This demonstrates, that the agonist sensitive Ca2+-stores were empty. However, prolonging the Ca2+-free periods to 30 rain a Ca 2+transient could be reindueed with ATP and with NT. We postulate an intracellular refilling mechanism, i.e. the intracellular agonist-released Ca 2+ is buffered for some time in a not agonist sensitive store and then retransported into the agonist sensitive pool.(Supported by DFG Gre 480/10) Physiologisches Institut II der Albert-Ludwigs-Universit~t Freiburg, HermannHerder-Str.7, 7800 Freiburg

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NEUROTENSIN INDUCES CI- and K + CONDUCTANCE IN COLONIC C A R C I N O M A CELLS R. Kubitz, M. Grolik, K. Kunzelmann, R. Greger

THE E F F E C T S OF C A L C I T R I O L DEFICIENCY ON THE K I N E T I C S OF C A L C I U M U P T A K E I N T O B R U S H B O R D E R M E M B R A N E V E S I C L E S F R O M PIG SMALL INTESTINE. R. Kaune, B. $ c h r 6 d e r and J. H a r m e y e r Brush Border membrane vesicles (BBMV) w e r e p r e p a r e d from d u o d e n a l s e g m e n t s of p i g l e t s s u f f e r i n g from p s e u d o v i t a m i n D - d e f i c i e n c y rickets, type I and f r o m c o n t r o l p i g l e t s . The r a c h i t i c p i g l e t s h a d an i n h e r i t e d d e f e c t of renal p r o d u c t i o n of c a l c i t r i o l [I,25(OH)~D~]. Ca z~ u p t a k e was s t u d i e d u s i n g the rapid filtration technique with icecold stop Buffer c o n t a i n i n g i m m o l / l EGTA. F r e e Ca 2~ c o n c e n t r a t i o n s ([Cat+i) in the i n c u b a t i o n m e d i u m w e r e a d j u s t e d by a d d i n g 0.5 m m o l / l EGTA. The i n i t i a l Ca ~* u p t a k e (up to 35 s) at low [Ca =~] (0.02-0.25 retool~l) was s a t u r a b l e . The a p p a r e n t V,~, of the initial Ca z' u p t a k e was s i g n i f i c a n t l y l o w e r in r a c h i t i c p i g l e t s (2.04 • 0.24 n m o l / ( m g ' 35s)) than in c o n t r o l p i g l e t s (3.63 • 0.i0). The a p p a r e n t K, was not i n f l u e n c e d by the rachitic condition (0.025 • 0 . 0 0 9 v e r s u s 0 . 0 2 9 • 0.002 m m o l / l ) . The d i f f e r e n c e B e t w e e n B o t h g r o u p s was m a i n t a i n e d o v e r at least 15 min in the p r e s e n c e of an i n s i d e n e g a t i v e K ~ d i f f u s i o n p o t e n t i a l and 0.03 m m o l / l [Ca ~ ] . The time c o u r s e of Ca Z+ u p t a k e s in the p r e s e n c e of a d i f f u s i o n p o t e n t i a l , f i t t e d to the e q u a t i o n : Y = A (I - e'kL), y i e l d e d a significantly lower k v a l u e for r a c h i t i c p i g l e t s than for c o n t r o l s (0.22 • 0.055 vs. 0.31 • 0 . 0 4 4 m i n "t, p < 0 . 0 5 ) . The A v a l u e s w e r e not d i f f e r e n t B e t w e e n B o t h g r o u p s . A d d i t i o n of 1 m m o l / l d , l - v e r a p a m i l to the v e s i c l e s u s p e n s i o n depressed v e s i c u l a r Ca ~+ u p t a k e in c o n t r o l p i g l e t s to the level p r e s e n t in r a c h i t i c piglets but had no e f f e c t in rachitic piglets. From varying verapamil concentrations B e t w e e n 0 and 1 m m o l / l the ICs~ was f o u n d to be 0.02 m m o l / l for c o n t r o l p i g l e t s . It is c o n c l u d e d that c a l c i t r i o l c a u s e s a s a t u r a b l e t r a n s p o r t mechanism for Ca :~ in b r u s h B o r d e r m e m b r a n e s , p r o b a b l y by increasing the n u m b e r of v e r a p a m i l sensitive transport components. Physiologisches Institut der T i e r ~ r z t l i c h e n Hochschule, B i s c h o f s h o l e r D a m m 15, D - 3 0 0 0 H a n n o v e r i.

.........................................................................................

The effects of the known cotransmitter neurotensin (NT) on HT29 colon carcinoma ceils were examined by a modified nystatin patch clamp technique. The patch pipettes were filled with a KCI/K-gluconate solution containing a low (_< 10 .5 tool/I) nystatin concentration. After obtaining a gigaseal nystatin slightly permeabilized the cell attached membrane, allowing furtheron the simultaneous recording of i) the cell membrane potential, ii) cell attached ion channels and iii) the input conductance of the cell attached membrane. The resting potential of HT29 cells was -58 + 2 mV ( n = 2 3 ) and was reversibly and transiently depolarized by 21 • 3 mV (n = 11) by NT at a concentration of 10 -9 mol/I. The effect of NT was dose dependend between 10 -11 and 10 -8 mol/I, with a concentration for half maximal depolarization of around 5 * 10 -lo mol/l. The depolarization was increased when neurotensin was applied in the presence of a low extracellular CI- concentration of 30 mmol/I and 15 mmol/I respectively. The depolarization was (mV) +35 • 8 (30CI, n = 3 ) compared to 18 • 4 (145CI) and + 3 7 • 3 (15CI, n = 6 ) compared to + 1 6 + 2 (145CI). These results indicate that the neurotensin induced depolarization is at least partially due to the activation of a CI- conductance. The depolarization was followed by a transient hyperpolarization of around 10-15 mV, most probably induced by the activation of K + conductance. An activation of a CI- channel with a conductance larger than 6-8 pS was not observed in these experiments. In contrast, an around 4 fold increase in the input conductance of the cell attached membrane was found, which was fully reversible. Again, this increase in the input conductance was dose dependend with an approximate K D of 10 -9 mol/I of neurotensin. These results indicate the activation of a low conductance (_< 6-8 pS) CI- channel by neurotensin. Physiologisches Institut der Albert-Ludwigs-Universit~it Freiburg, Hermann-Herder-Stral~e 7, D - 7 8 0 0 Freiburg

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MORPHOLOGICAL GAP JUNCTIONS

EPIDERMAL GROWTH FACTOR INHIBITS BOTH CHOLECYSTOKININ-OCTAPEPTIDE INDUCED INOSITOL 1,4,5-TRISPHOSPHATE PRODUCTION AND [CA2+] INCREASE IN RAT PANCREATIC ACINAR CELLS A. Pr6frock, A. Piiper, L. Eckhardt and I. Schulz

AND FUNCTIONAL CHARACTERISATION OF IN A B O N E C E L L C U L T U R E K. Schirrmacherl~ F. B r ~ m m e r Z ~ D.F. Hfilser z , O. T r a u h 3 , I. S c h m i t z 4 ~ E. W i n t e r h a g e r s , D. B i n g m a n n I It is w e l l k n o w n t h a t b o n e c e l l s "in v i v o " are int e r c o n n e c t e d w i t h e a c h o t h e r by g a p j u n c t i o n s . D u e to the difficult experimental access to these cells, the f u n c t i o n of s u c h i n t e r c o n n e c t i o n s , however, is w i d e l y u n c l e a r u n t i l now. T h e p r e s e n t stud y w a s s t a r t e d to a n a l y s e m o r p h o l o g y and function of t h e s e g a p j u n c t i o n s "in v i t r o " u n d e r c o n d i t i o n s which facilitate such experiments. Therefore, calvarian fragments of newborn rats were explanted onto collagen covered glasses in a m e d i u m c o n t a i n i n g f e t a l c a l f serum. When mineralization of the extracellular matrix started 3 - 6 w e e k s a f t e r explantation gap junctions were investigated: i) U l t r a s t r u c t u r a l studies showed gap junctions which mostoften were found between processes of neighbouring cells. 2) Immunostaining revealed connexine 43 antigen. 3) Injections of lucifer y e l l o w i n t o c e l l s (n=50) l e d to a s t a i n i n g of u p to 30 adjacent cells. 4) Membrane potential (MP) displacements by c u r r e n t i n j e c t i o n s typically were a c c o m p a n i e d by M P s h i f t s of 4-5 m V in the a d j a c e n t cells even if t h e y w e r e 100 p m r e m o t e f r o m the f i r s t one. T h e f i n d i n g s s h o w t h a t 43 kD g a p j u n c t i o n s are form e d in c u l t u r e s of b o n e c e l l s w h i c h m a y l e a d to a n e x t e n d e d n e t w o r k of f u n c t i o n a l l y c o u p l e d cells. Supported

b y D F G Bi 6 2 7 8 / 6 - 1 .

I I n s t i t u t ffir P h y s i o l o g i e ~ H u f e l a n d s t r . 55, W - 4 3 0 0 E s s e n i, FRG; 2 B i o l o g i s c h e s I n s t i t u t , W - 7 0 0 0 S t u t t g a r t 80; 3 I n s t i t u t ffir G e n e t i k , W - 5 3 0 0 Bonn; 4 I n s t i t u t ffir P a t h o l o g i e ( B e r g m a n n s h e i l ) ~ W - 4 6 3 0 B o c h u m ; S I n s t i t u t fur A n a t o m i e , W - 4 3 0 0 E s s e n 1

It has been shown that epidermal growth factor (EGF)stimulates both basal and cholecystokinin-octapeptide (CCK-OP) induced enzyme secretion from pancreatic acinar eeUs as a long term function (Logsdon and Williams, 1983 Am. J. Physiol. 244, G675-G682). In a number of different transformed cell lines EGF stimulates the production of IP3, whereas it has no effect in non-transformed cells (Hepler et al, 1987 J. Biol. Chem. 262, 2951-2956). The interaction of activated EGF receptors with G-proteins has also been demonstrated in different cell types (Johnson & Garrison, 1986 Proc. Natl. Acad. Sci USA 83, 2032-2036). Recently we have shown that EGF- and CCK-receptors interact with the same Gi-proteins, namely Gil, Gi2 and Gi3 in plasma membranes of rat pancreatic acinar cells (Pr6frock et al, 1991 Biochem. Biophys. Res. Commun. 175, 380-386). We assume that one, two or all three of these G i proteins are involved in the coupling of CCK-receptors with phosphollpase C (PLC), increase in inositol 1,4,5 triphosphate (IP3) and consequent Ca2+ release from IP 3 sensitive Ca 2 + stores. We have now investigated effects of EGF (100 ng/ml) on CCK-OP (0.3 #mol/1)-induced IP 3 production and on CCK-OP (1 nmol/1)-stimulated increase in intracellular [Ca2+]i in pancreatic acinar cells. Our data show that EGF inhibits both CCK-OP-induced increase in IP3 production and in [Ca2+]i. We assume that activation of Gi-proteins in the plasma membrane could be responsible for the inhibitory effects of EGF on CCKinduced PLC activation observed in pancreatic acinar cells. Max-Planck-Institut for Biophysik, Kennedyallee 70, D-6000 Frankfurt 70

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TRANSPORT SYSTEMS AT THE CELL MEMBRANE AND CELL VOLUME IN THE REGULATION OF CELL PROLIFERATION BY RAS ONCOGENE E. W611, M. Ritter, H. Weiss, S. Waldegger, I. Bichler, K. Maly, H. Grunicke, F. Lang.

EFFECTS OF GTPTS ON SMALL MOLECULAR WEIGHT GTPBINDING PROTEINS IN RAT PANCREATIC ACINAR CELLS

The present study has been performed to elucidate the involvement of altered transport across the cell membrane in the stimulation of cell growth. The experiments have been performed in 3T3 NII-I fibroblasts transfected with a transforming H-ras MMTV-LTR construct, expressing the ras oncogene, which is pointmutated at position 12, upon treatment with 1/zmol/l dexamethasone (+ras). Controls were transfected cells not expressing the oncogene (-ras) and non-transfected cells treated with dexamethasone (oras). Expression of the ras oncogene increases the activity of both, Na+/H + exchanger and Na+,K+,2C1 - cotransport. The concerted action of these transport systems leads to an increase of cell volume from 2069 ___ 91 fl to 2767 ___ 20 ft. On the other hand, in +ras but not in -ras and oras, bradykinin, bombesin or 0.5 % fetal calf serum produce sustained oscillations of cell membrane potential, due to intermittent activation of calcium sensitive K + channels by oscillations of intracellular calcium activity. These oscillations are abolished by 10 /zmol/1 nifedipine. Despite its inhibitory effect on cell membrane potential oscillations, nifedipine does not significantly modify the cell volume of +ras (2763 -I- 27 t ) or -ras (1954 _ 79 t ) cells. + m s grow in serum depleted media (replication factor [RF] in 24 h: 1.88 ___ 0.18), in contrast to -ms (RF: 1.11 _ 0.13). The growth of +ras is abolished in the presence of 0.1 mmol/1 dimethyl- amiloride and 0.1 mmol/l furosemide (RF: 1.11 + 0.12) as well as in the presence of 10 tzmol/1 nifedipine (RF: 1.13 _ 0.16), whereas the number of -ras is not significantly altered by neither dimethylamiloride and furosemide (RF: 0.83 + 0.09) nor nifedipine (RF: 0.97 ___ 0.06). In conclusion, the expression of ms oncogene leads to stimulation of cellular ion accumulation and subsequent increase of cell volume. Furthermore, in cells expressing the ras oncogene several mediators elicit oscillations of cell membrane potential and intracellular calcium. Both events are required for stimulated growth in those cells.

Small molecular weight GTP-binding (smg) proteins play a role in the regulation of intraceUular vesicle transport, secretion (H.R. Bourne et al. (I990) Nature 348:125-132) and in signal transduction (E.G. Lapetina et al. (1989) Proc. Nat. Acad. Scl. 86:3131-3134). We have detected seven different smgproteins with molecular masses between 18 and 27 kDa in subfractions of rat pancreatic acinar cells by the [~-32P]GTP'binding assay. Using monoclonal antibodies we have identified a 19 kDa smg-protein in the cytosol and in a fraction enriched in endoplasmic reticulum (ER) as the ADPribosylation factor (art'). Recently we have shown that stimulation of isolated pancreatic acinar cells with the secretagogues cholecystoldn-octapeptide (CCK-OP) reduced [c~-32p]GTP-binding to a yet unidentified 21/22 kDa smg-protein, whereas binding to yet unidentified 23 kDa snagproteins in a fraction enriched in endoplasmic reticulum (ER) was enhanced. Now we demonstrate that after stimulation of permeabilized acini with the non-hydrolyzable GTP analogue, GTPTS, which activates phospholipase C through heterotrimeric G proteins, the same effect as due to CCK-OP stimulation is observed. In addition GTPTS enhances GTPbinding to the 19 kDa arf-protein. In isolated membranes enriched in ER in the presence of cytosol direct addition of GTPTS causes apparent translocation of the 19 kDa protein (art) from the cytosol to membranes, but had no effect on other smg-proteins. This indicates that: 1.) the GTPTS effects on GTP-binding to 21/22 and 23 kDa smg-proteins in membranes isolated from stimulated acinar cells is indirect and is due to stimulation of PLC by activation of a coupling trimeric G protein. 2.) Since the GTPTS effect on the arf protein was observed not only in cells but also in isolated ER membranes the effect of GTPxS on the arf-protein is direct. It appears that association of arf/with the vesicle membrane is increased when the arf is in the GTP-boundstate.

Institutes for Physiology and Biochemistry, University of A-6010 Innsbruck, Austria

P. Zimmermann, S. Zeuzem, S. Schnefel & I. Schulz

Max-Planck-Institut fiir Biophysik, Kennedyallee 70, D-6000 Frankfurt 70

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152 INTERNAL PERFUSION OF CARDIOBALLS VIA PATCH PIPETTES DOES NOT EFFECTIVELY CONTROL SUBSARCOLEMMAL Na+ CONCENTRATION H.G. Glitsch and A. Tappe

STABLE GAP JUNCTIONAL COMMUNICATION BETWEEN BONE CELLS IN VITRO DURING CHANGES OF THE MICROMILIEU K. S c h i r r m a c h e r I, F . B r ~ m m e r z, D. B i n g m a n n I Little is known about the functional properties of gap junctions between bone cells. To get more informations, cultured bone cells derived from calvaria of n e w b o r n r a t s were studied electrophysiologically. (i) Parallel intraeellular recordings from neighbouring cells showed that amplitudes of s p o n t a n e o u s or s t i m u l a t e d m e m b r a n e p o t e n t i a l (MP) changes in o n e c e l l were constantly related to t h o s e in c o u p l e d c e l l s . (2) T h e c o u p l i n g factor of 24 pairs of cells ranged between 0.i and 0.7 (~= 0.47 • 0.18; n=48). (3) The coupling strength between both cells hardly depended on the MP. (4) Elevation of the intracellular cAMP level by extracellular administration of dibutyryl-cAMP (n=8) or caffeine (i a n d I 0 mmol/l; n=2) d i d not affect the electric coupling. (5) A r i s e of the calcium concentration in the bath up to 8 mmol/l affected the coupling strength only in 3 out of 8 cases. (6) Acidosis (pH < 6.6) and hypercapnia (pCOz > 80 mmHg) did not affect electric( n = 6) and dyecoupling (lucifer yellow; n= 8). (7) In the present study, only the antiepileptic drug carbamazepine (i00 Dmol/l; n=5) reduced electric coupling significantly. On the whole, gap junctional cultured bone cells proved various conditions. Supported

by

DFG

Bi

communication between to be stable under

278/6-1.

1

Institut fur Physiologie, Hufelandstr. W-4300 Essen i, F R G z Biologisches Institut, Pfaffenwaldring W-7000 Stuttgart 80, FRG

The Na pump current (Ip) of sheep Purkinje cardioballs was measured as K+-activated, dihydroouabain-sensitive outward current by means of whole cell recording (standard pipette solution (raM): 15 Na+, 120 K+, 20 Cs+, 11 Mg2+, 135 CI-, 30 OH-, 6 ATP, 6 EGTA, 36 HEPES; standard external solution (mM): 150 Na+, 2 Ba2+, 1.5 Mg2+, 0.9 Ca2+, 144.2 CI-, 20 OH-, 40 HEPES and either 5.4 K+ or TEA+; holding potential: - 2 0 mV; 30-33~ The external solution could be quickly changed via a multibarrelled pipette near the cell. Switching from a K+-free to a K+containing solution immediately evoked Ip that decreased (within~l min) from an initial maximum to a steady-state value. Both the Ip maximum and the Ip decline depended on the duration of preceding interval in K+free solution, on the external K+-concentration and on the Na+concentration in the pipette solution. The initial Ip maximum increased with increasing duration of the K+-free interval and reached a maximum after 2 min of K+-free superfusion whereas the steady-state value was independent of the duration of the interval in K+-free solution. The initial Ip overshoot above the steady-state increased with increasing external K+-concentration. Judging from Ip at steady-state or at the initial maximum half maximal Ip activation by external K+ occured at 1.4 or 2.1 mM K+, respectively. The maximal Ip amplitude activated by external K+ was two times larger in the latter case. Switching the Na+-concentration in the patch pipette from 15 mM to 50 mM reduced reversibly the overshoot, and shortened the Ip decline. The results suggest changes of Na+ at the intracellular pumping sites. Thus during whole cell recording the pipette solution does not effectively control the subsarcolemmal Na+ concentration of cardioballs. For corresponding findings with freshly isolated cardiac ceils see Bielen et al. (1991) Biochim. Biophys. Acta

55,

1065: 2 6 9 - 2 7 1 .

57,

Department of Cell Physiology, Ruhr-Universit~it,Postfach 102148, D-4630 Bochum, F.R.G, Supported by the DFG.

151

153

AMILORIDE-SENSITIVE NA-CHANNEL FROM HEN LOWER INTESTINE EXPRESSED [N XENOPUS

M O D U L A T I O N OF C A R D I A C O U T W A R D L Y RECTIFYING CHANNELS BY PROTEIN KINASE A A N D A G-PROTEIN I. Benz, U. Fr6be and M. Kohlhardt

OOCYTES

W.-M. Weber, C. Asher, H. Garty , W. Clauss

The amitoride-blockable Na+-specific channel plays a key role in sodium reabsorption in a l l vertebrates and even some invertebrates. I t s i n t e s t i n a l and renal a c t i v i t y i s known to be regulated by the d i e t a r y NaCl intake, presumably by modulating plasma aldosterone Levels. Oocytes of Xenopus laevis are a widely used system f o r the expression of foreign mRNA. Since these c e l l s have no endogenous a m i l o r i d e - s e n s i t i v e Na+-channel they ere p a r t i c u [ a r y suited for the expression of this transporter. Total and poly(A)+-RNA was isolated from hen lower intestine (colon and coprodeum). The animals used were fed either by low salt (LS, 0 % NaC[) diet, or by high salt diet (HS, 0.5 %, w/w NaCl). Stage V oocytes were selected, injected with the two matched RMA preparations (50 ng/oocyte) and tested for sodium conductance one or two days later. In Xenopus oocytes injected with mRNA prepared Of lower intestine of HS animals no detectable amiloridesensitive current could be seen. Current-voltage relationships measured from -150 mV to +30 mV were the same in the presence and absence of amiloride (100 #M) or with and without Na+ (substituted by TMA-CL). Similar IV-curves were recorded also in non-injected control oocytes. On the other hand oocytes injected with LS-RNA developed a large amiloride-blockabte current (eg. 200 nA at -150 mV or ~'00 nA at -100 mV). This current was also blocked by removal of sodium from the outer bath. IV-curves recorded in Na+-free (110 mM TNA-CI) solution were essentially the same as those measured in the presence of amiloride (100 #H) or benzamil (100 #M). No ami[oride-sensitive current could be detected even if external Na+ was substituted by K+, suggesting that the pathway expressed is highly Na+ specific. These data demonstrate that the avian intestine Na+-channel can be expressed and studied in Xenopus oocytes. Our results also provide the first evidence that transport regulation by the d i e t a r y MaCl intake occurs at the t r a n s c r i p t i o n a l Level. This work was supported by DFG (CI 63/7-1) and by the Segov-Pomereniae research fund.

kidney

L n s t i t u t fur Veterin~r-Physiologie, Freie U n i v e r s i t ~ t Berlin, Koserstr. 20, W-lOO0 B e r l i n 33, FRG and the Weizmann I n s t i t u t e of Science, 76100 Rehovot, Israel

K+

Cardiac K + channels comprise a family with distinctly heterogeneous properties. They are involved in setting the resting potential and controlling the shape of the cardiac action potential. Some of them are sensitive to 6vadrenergic stimulation and become activated by a cascade of molecular events with the formation of c A M P as a key reaction. The present inside-out experiments with neonatal rat cardiocytes identified a K + channel with outwardly rectifying properties under quasi-physiological, i.e. asymmetrical ionic conditions at membrane potentials positive to -40 mY. Pc steeply rose on shifting the membrane potential in the positive direction. Near Erev (which coincides with EK), however, Pc wa~ negligibly small. Topen showed a similar voltage dependence. Evidence was obtained for two kinetically different channel populations, one of them characterized by an open time of 1.70=F_0.10 ms end the other population by an open time of 0 . 9 5 + 0 . 1 9 ms (at -7 mV). Cytosolic ATP application (100 pmol/I; in the simultaneous presence of Mg + +) caused a severalfold increase of Po. No channel activation occurred when ATP was administered in the absence of Mg + + ions. Further evidence for a phosphorylation reaction as underlying the Pc increase was obtained in experiments with the catalytic subunit of protein kinase A (120-150u/ml). Moreover, the outwardly rectifying K + channel may be additionally controlled by a G-protein, since GTP-F-S (100 pmol/I) caused also a channel activation. Consequently, cardiac outwardly rectifying K + channels in situ can be expected to respond with an increased open probability under conditions leading to increased cAMP formation and increased G-protein activity. Physiological Institute of the University; D - W 7 8 0 0 Freiburg/Br., Germany

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INHIBITION OF PROTEIN-PHOSPHATASE AFFECTS INSULIN-DEPENDENT GLUCOSETRANSPORT IN M Y O C Y T E S

EFFECTS OF T H E CA-SENSITIZER E M D 57033 ON ISOLATED GUINEA-PIG C A R D I A C MUSCLE: INCREASE IN ISOMETRIC F O R C E WITH MINOR CHANGES IN E N E R G Y E X P E N D I T U R E T. Grog and J. Daut

S. Bauer, K. Schwarz, and J.C. Rfiegg

W. Hasselbach*,

G. Pfitzer

glucose transport system of calcium tolerant rat myocytes was studied by measuring the uptake of 2-desoxy-14C glucose into rat myocytes by a millipore filtering method. Both insulin (0.9 nM) and okadaic acid (0A, 1 ~M) enhanced the uptake rate compared to the control uptake. However, OA (i pM) in the presence of insulin (0.9 nM) delayed uptake by about 5 minutes (fig.) indicating a phosphorylation dependent regulation of insulin action. The

s

~0

~5~

Figure legend: Uptake of 2-desoxy [I14C] glucose (0.5 mM) into rat myocytes in the presence of insulin (~ OA (A), and OA in the presence of insulin ( ); x = control. Incubation buffer (in mM): NaCI 135, KCL 4.8, KH2P04 1.2, MgCI2 1.8, CaCI 2 0.i, HEPES 25, Albumin 5g/l, pH 7.4, 21~

II. Physiologisches Institut der Universit&t, Im Neuenheimer Feld 326, 6900 Heidelberg * Max-Planck-Institut ffir medizinische Forschung, Jahnstr. 29, 6900 Heidelberg

Ca-sensitizers increase the contractility of cardiac muscle by shifting the pCa-tension relation to the left. In order to assess the potential usefulness of this class of drugs for the treatment of heart failure we have studied the effects of the Ca-sensitizer E M D 57033 on contractile tension and on myocardial energy expenditure. Thin ventricular trabeculae (diameter 100-400/~m) were isolated from guinea-pig heart and superfused with physiological salt solution at 37 ~ Isometric tension was measured at a stimulation rate of 2 or 3 Hz. Application of 10 # M E M D 57033 increased isometric tension by 198 +- 48 % (mean -+ s 9 .; n = 4 ) within 20 min and increased the duration of a twitch by about 10 %. In the presence of 5 ~M E M D 57033 isometric tension was increased by 140 _+ 24 % (n=4). The effects of the Ca-sensitizer were compared to the effects of various concentrations of isoprenaline in the same preparations. With 50 nM isoprenaline isometric tension was increased by 245 % (n =7). In parallel experiments the rate of heat production of thin ventricular trabeculae was measured with a microcalorimetric technique as described previously (Dant & Elzinga, 1988, J. Physiol. 398, 259-275). In the presence of 50 nM isoprenaline contraction-related heat production was increased more than threefold. In contrast, 10/zM E M D 57033 increased contraction-related heat production only by 48 + 14 % in the steady state (n=7). Thus effect of the Ca-sensitizer on the rate of heat production was about four times smaller than its effect on the tensiontime integral. Resting heat production was unaffected by 10 ~M E M D 57033. The rate of heat production measured in the steady state is probably proportional to the rate of ATP hydrolysis (see Daut, Grog & Elzinga, 1991, Thermochimica Acta, in the press). Thus the ATPase rate (and the rate of oxygen consumption) required to produce a given tension was considerably reduced in the presence of E M D 57033. The mechanism by which this is achieved is still unknown. Our results support the idea that Ca-sensitizers could be potentially useful drugs for the treatment of heart failure. PHYSIOLOGISCHES INSTITUT D E R TECHN. UNIVERSITAT M U N C H E N , BIEDERSTEINER STR. 29, D-8000 M1)NCHEN 40

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CAFFEINE AS A TOOL FOR STUDYING SARCOPLASMIC RETICULUM FUNCTION IN VOLTAGE-CLAMPED GUINEA-PIG ATRIAL MYOCYTES USING INDO-1 FLUORESCENCE P. Lipp, G.Callewaert* and L. Pott

HEMODYNAMIC RELEVANCE OF PURINERGIC P2-RECEPTORS IN THE INTACT CORONARY SYSTEM B. F. Becker, L. M. Schwartz and E. Gedach

Caffeine (Cat') is a useful experimental tool for studying aspects of [Ca2+]iregulation in cardiac and skeletal muscle: upon extracellular application it rapidly opens SR release channels (Rousseau & Meissner, Am. J. Physiol. 256: H328-}/333, 1989); in its presence these channels maintain opening activity, resulting in a functional elimination of the SR. Furthermore, it is rapidly reversible. Recently O'Neill ct al. (J. Physiol. 425: 55-70, 1990) have reported that Car quenches fluorescence of indo-1 in ester-loaded myocytes. This quenching can be used to monitor [Caf]i. Using a fast superfusion device we have applied Caf under continuous recording of indo-1 fluorescence (405 and 485 nm) and Na+/Ca2+-exehange current to guinea-pig atrial myocytes. If the SR was repetitively challenged with brief (ca. 1 s) pulses of Caf (1-5 raM) in the absence of [Ca2+]o, the amplitude of Ca2+transients calculated I2om the fluorescence ratio (405/485 nm) decayed to zero within less than 10 s, as did the exchange current. Thereafter only the Carinduced quenching of the fluorescence signals was recorded. The ratio of the quenched to unquenched signal however, remained constant. A slow decay of the Ca2+-transients and INaCa was recorded during repetitive pulses of Caf in the presence of [Ca2+]o. Under this condition the decay of the Ca2+-transients was paralleled by a marked increase of their latency (with regard to the rise in [Cat]i) and a decrease of their rate of rise (fig. 1). Our data suggest that the rate of activation of release channels by Caf depends on the state of filling of the SR.

In the coronary vascular system two subclasses of P2-receptors, P2x and P~, have been proposed to exist on smooth muscle and on endothelial cells, respectively [BURNSTOCK G (1990) Ann N Y Acad Sci 603:1-18, 31-45], Activation of P~ causes contraction of vascular smooth muscle, and stimulation of P2y gives rise to endothelium-mediated relaxation. By definition, neither response is sensitive to inhibition by theophylline (Theo), the classical PCreceptor antagonist. To test the hemodynamic relevance of the Pa-receptors, the effects of two "non-hydrolyzable" ATP derivatives, ~,8- and g,~"-methylene-ATP (MeATP) on coronary flow were assessed in isolated perfused hearts of guinea pigs and rats. Both derivatives are more potent P2x-agonists than ATP and additionally act on P2y-recaptors. The measurements were conducted under steady-state conditions (5 min infusion) in the absence and presence of Theo (50/~M). In addition, catabolism of the infused nucleotides was determined during coronary passage. Results: Infusion of ~,8-MeATP (0.5-10/~M) had no effect on coronary flow, whereas 8,r-MeATP caused a concentration dependent rise in flow (+75% at 0.5 /~M). Theo strongly inhibited this dilatory effect, but did not unmask a constrictory component of action of either nucleotide. Similar results were obtained in both species tested. In the guinea pig, approx. 25% of the infused 8,F-MeATP (0.5 p,M) was degraded to AMP and adenosine during coronary passage; dephosphorylation of 0.5 p.M ,<,8-MeATP (to the ADP-derivative) amounted to less than 10%. In contrast to AMP (a Pl-agonist), ~,8-MeADP is vasoinactive. Conclusions: Neither P2x" nor P2y-receptors appear to be hemedynamically relevant in the intact coronary bed of guinea pig and rat, at least under conditions in which flow is determined largely by the small resistance vessels. In the ease of 8, 3~MeATP, intracoronary degradation to monophosphate and adenosine probably accounts for the theophylline-inhibitable dilatation.

~

~

Onsetof 1st (o) and 4th (o) Ca2+transient upon repetitivesuperfusion with Car-containing soln. (1 raM). The arrow marks the point of time when [Caf]i started to rise, as monitored by the quenching of the 405 nm fluorescence. Holding potential -50 mV; T=25 ~ Calibr. bars: 0.5 s; 0,25/xMCa2+.

Institut fiir Zellphysiologie Ruhr-Universit~it, Posffach 102148, 4630 Bochum *Laboratorinm voor Fysiologie, KUL B-3000 Leuven Belgium

Physiologisches Institut, Universit&t M0nchen, Pettenkoferstr. 12, 8000 M(inchen 2

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EFFECT OF ADENINE ON FUNCTION AND METABOLISM OF THE RAT HEART H.-G.Zimmer, A. Schneider

HYPOXIC VASODILATATION IN ISOLATED GUINEA-PIG HEART IS INHIBITED BY NANOMOLAR CONCENTRATIONS OF THE ANTIDIABETIC SULPHONYLUREA GLIBENCLAMIDE S. Cyrys and J. Daut

The hemodynamic and metabolic effects of adenine are not well characterized. We have therefore administered this purine base as continuous i.v. infusion (50 mg/kg/h) for 5 hours and have measured functional parameters and the metabolic effect of adenine alone and in combination with ribose (200 mg/kg/h) in control and isoproterenol (ISO)-treated rats. Adenine reduced heart rate (beats/min) from 336 • 14 (n=8) to 298 • 11 (n=7), LVSP (mmHg) from 134 • 3 to 113 • 3 and LV dp/dtma x from 11251 • 1691 to 8996 • 379. Mean arterial pressure (M~&P, m m H g ) and total peripheral resistance (TPR, m m H g x kg x min/ ml) were depressed from 118 • 3 to 95 • 3, and from 0.36 • 0.03 to 0.24 • 0.01, respectively. Cardiac output (CO, ml/kg/ min) was increased from 335 • 17 to 403 • 15. When rats had been treated with ISO (25 mg/kg, s.c.), adenine attenuated the increase in heart rate and LV dp/dtmax and aggravated the decline in LVSP, MAP and TPR. The ISO-induced increase in CO was not changed. The cardiac levels of ATP and of total adenine nucleotides (AN, ~moles/g) were depressed by ISO from 5.02 • 0.06 and 6.41 • 0.08 (n=12) to 3.51 • 0.13 and 4.83 • 0.15 (n=10), respectively. This decline was not affected by adenine (3.56 • 0.21; 4.98 • 0.22, n=7) or ribose (3.64 • 0.11; 5.13 ! 0.14, n=9). When adenine was administered together with ribose, the ISO-induced reduction was prevented (5.18 • 0.23; 7.24 • 0.30, n=7). Thus, adenine had negative chronotropic and inotropic as well as vasodilatory effects and attenuated the positive chronotropic and inotropic effects of 6-adrenergic stimulation. Only when the available pool of 5phosphoribosyl-l-pyrophosphate was elevated with ribose, the salvage of adenine was effective and prevented the ISO-induced decline of the cardiac ATP and AN pool. This study was supported by the Wilhelm Sander-Stiftung Physiologisches Institut, Universit~t MHnchen, Pettenkoferstr. 12, 8000 MHnchen 2

In 1970 the University Study Group. (USA) reported that in patients with adult-onset diabetes the mortality from cardiovascular causes was almost doubled in the group treated with tolbutamide as compared to a control group treated with placebo (Diabetes 19, Suppl. 2, 789-830). Antidiabetic sulphonylureas like telbutamide and glibenclamide increase insulin secretion by inhibiting ATP-sensitive potassium channels (K(ATP) channels) in pancreatic B-cells. Recently we have suggested that hypoxic vasodilatation in isolated perfused hearts may also be mediated by K(ATP) channels (Science 247, 1341-1344, 1990). In order to investigate possible cardiovascular side-effects of antidiabetic suphonylureas we have studied the effects of low concentrations of glibenclamide on hypoxic vasodilatation in isolated perfused guinea-pig heart. In hearts arrested by increasing the K ~- concentration of the perfusate to 15 mM, switching to hypoxic perfusate (Pn9 < 10 mmHg) increased coronary vascular resistance by at least 50%."In the presence of 5 nM glibenclamide the delay between application of hypoxic solution and half-maximal vasodilatation increased from 63 -+ 8 to 89 -+ 4 s (mean _ S.D.; n=4). In order to obtain an estimate of the concentration of glibenclamide which blocks 50% of the K(ATP) channels (Ki) we induced a sustained vasodilatation by perfusing the hearts with lemakalim, an opener of K(ATP) channels. Lemakalim (50-500 nM) caused a concentration-dependent decrease in coronary resistance. In the presence of glibenclamide the concentration-effect curve of lemakalim was shifted to the right. From a double reciprocal plot of the effect of lemakalim in the presence and absence ofglibenclamide .Ki was estimated to be about 10 nM. This is in the range of therapeutic glibenclamide concentrations in human plasma, and in the range of glibenclamide concentrations causing half-maximal inhibition of K(ATP) channels in mouse pancreatic B-cells (Ztinkler et al., NaunynSchmiedeb. Arch. Pharmacol. 337, 225-230, 1988). It appears possible that the increased mortality of patients treated with antidibetic sulphonylureas may be related to the impairment of hypoxic vasodilatation. PHYSIOLOGISCHES INSTITUT DER TECHN. UNIVERSIT~.T MUNCHEN, BIEDERSTEINER STR. 29, D-8000 MUNCHEN 40

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Conclusions: 1) Ischemia induces different effects on exocytotic noradrenaline release in rat and guinea pig heart. 2) Single factors of ischemia (energy depletion, acidosis) have opposing effects on exocytotic noradrenaline release. 3) A species related differential response to anoxia and acidosis may cause net increase or decrease of noradrenaline release in early ischemia.

THE CHRONOTROPIC EFFECTS OF ACETYLCHOLINE AND NORADRENALINE ARE RELATED TO RIGHT ATRIAL PRESSURE F. R o s s b e r 9 .................................................. Numerous s t u d i e s have shown t h a t t h e s p o n t a n e o u s r a t e o f i s o l a t e d h e a r t s i s d e p e n d e n t on i n t r a l u m i nal r i g h t a t r i a l pressure. This control of heart r a t e i s caused by s t r e t c h - s e n s i v i t y of sinoatrial region (!ntracardiac Bainbridge ~ffect, IBE).Obviously, t h e a d e q u a t e s t i m u ] u s f o r IBE i s t h e c o n centric distension of the atrium, linear stretch e . g . o f i s o l a t e d s i n u s p r e p a r a t i o n s - shows r e p r o ducible results rarely. T h e r e f o r e , t h e IBE was i n v e s t i g a t e d usin9 isolated right atrial preparations from rabbits. The h y d r o static filling p r e s s u r e a t a p e r f u s i o n r a t e o f 5-7 m l * m i n -1 was t h e r e s u l t o f t h e h e i g h t d i f f e r e n c e between t h e m i d d l e o f t h e a t r i u m and t h e end o u t f l o w . A f t e r 20 min o f p e r f u s i o n w i t h T y r o d e ' s s o l u tion the chronotropic response of acetylcholine (ACH) i n c o n c e n t r a t i o n s between 10 - 8 and 10 - 3 and noradrenaline (NA) i n c o n c e n t r a t i o n s between tO - 9 and 10 - 5 N was t e s t e d a t i n t r a ] u m i n a l pressures of O.1 kPa (10 mm H20) as w e l l as o f O.5 kPa ( 5 0 mm H20) . I n each c a s e t h e s p o n t a n e o u s r a t e was e s t i mated f o r t h e 11. m i n u t e i n a l l c o n c e n t r a t i o n s and both intraatrial pressures. As compared %o i n t r a l u m i n a l p r e s s u r e o f O.1 kPa a t 0 . 5 kPa t h e s l o p e o f d o s e - c e s p o n s e c u r v e s was c h a n ged s i g n i f i c a n t l y . D u r i n 9 p e r f u s i o n w i t h ACH t h e dose-dependent decrease in atrial r a t e was s t r o n g e r a t an i n t r a l u m i n a l p r e s s u r e o f 0 . 5 kPa. AC t h e p r e s s u r e o f O.1 kPa t h e d o s e - r e s p o n s e c u r v e s l o p e d more s t e e p l y by NA. Thus i n r a b b i t s t h e i n t r a l u m i n a l right atrial pressure determines the amount o f chronotropic r e s p o n s e c a u s e d by ACH and NA.

Innere Medizin III (Kardiologie) der Universit&t Heidelberg. Bergheimer Str. 58. D-6900 Heidelberg

Carl-Ludwig-Institut fQr Physiologie der Universitat Leipzig, Liebigstr. 27, D-O-7010 L e i p z i 9

EFFECT OF ISCNEMIA ON EXOCYTOTIC NORADRENALINE RELEASE IN ISOLATED GUINEA PIG AND RAT HEART M. Seyfarth, G. Richardt, M. Haass and A. Sch6mig In myocardial ischemia sympathetic activity is thought to play a crucial role in the pathogenesis of malignant arrhythmias. The effect of ischemia on sympathetic neurotransmitter release, however, remains controversial. In the present study the effect of global ischemia (10') and of single factors of ischemia (energy depletion, acidosis) on exocytotic noradrenaline release was investigated in rat and guinea pig isolated perfused hearts. Exocytotic release of endogenous noradrenaline (determined by HPLC) was evoked by electrical field stimulation (5V,6Hz, l min). Energy depletion was induced by anoxia and glucose free perfusion at constant flow. Acidosis was evoked by change of pH in the perfusion buffer from 7.4 to 6.0. Neuronal uptake was inhibited by desipramine (100 nmol/I) to prevent amine elimination and nonexocytotic release. All hearts were stimulated twice to compare basal ($1) and experimental ($2) condition. Noradrenaline release induced by $1 was 287+18 pmol/g and 140+8 pmol/g in guinea pig and rat heart, resp.. Results are expressed as $2/$1 ratio (mean + SEM, * p<0.05).

Control Ischemia Anoxia Acidosis Anoxia / Acidosis

Guinea Pig 0.98_+0.10 1.79+0.22 * 2.67+0.57 * 0.72+0.12 1.21_+0.24

Rat 0.98+0.07 0.31_+0.03 * 0.87+0.18 0.18_+0.05 * 0.26_+0.04 *

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OPERATING INACTIVATION PREVENTS CARDIAC Na + CHANNELS TO BE FUCKER-BLOCKED BY ANTIARRHYTHMIC DRUGS I. Benz and M. Kohlhardt

Improvement of postischemic fiber-shortening in dog hearts by infusion of glutathione: H. Krieter1, K. Schwarz2, S.F. Bauer~, U.B. Br0ckner3, J.C. R~egg2

Flicker blockade of voltage-dependent Na + channels occurs in the presence of many antiarrhythmic drugs and relies on a repetitive blocking and unblocking of the open pore. The essential requirement for flicker blockade seems to be removal of Na + inactivation. In order to stress the hypothesis that operating Na + inactivation makes Na + channels basically resistant against such an drug interaction, the influence of propafenone and quir)idine was studied in inside-out patch clamp experiments in neonatal rat cardiocytes with cooled Na + channels kept at 9~ Under these conditions, their open time is significantly prolonged and may attain values as in (-)-DPI-modified non-inactivating Na + channels. Propafenone (1-10 /Jmol/I) reduced NPo but left T o p e n unchanged. Even ultralong openings with a life time of 10 ms or longer proved drug-resistant in that they were not chopped into multiple transitions between a conducting and a non-conducting state. (-)-DPI-modified Na + channels showing a t - 4 5 mV and 9~ an open time of close to 10 ms responded in the expected fashion, i. e. became flicker-blocked by propafenone. Even quinidine (5/Jmol/I), suspected to possess a considerable affinity for open Na + channels, failed to provoke a flicker blockade if Na + inactivation is operative. A novel kinetic response to the presence of antiarrhythmic drugs was detected in F--treated Na + channels. Cytoplasmic F- retards Na + inactivation and can enable Na + channels to attain a second, long-lasting open state. The latter is drug sensitive in that propafenone hinders Na + channels to attain this particular conducting configuration. Supported by DFG (Ko 778/2-3) Physiological Institute of the University; D-W7800 Freiburg/Br.; Germany

Oxygen derived free radicals are probably playing a crucial role during repeffusion of the postischemic myocardium. Gfutathione (GSH, 2-5 mmol/L) has been shown to increase the contractile force of submaximaly Ca§ skinned cardiac fibers (SF Bauer et al.: Basic Hes.Cardiol: 1989 84:591 ). Here we.repo~ that in similar concentrations glutathion also improves segment-fiber-shortening of the postischemic aog heart. d In six anesthetized and mechanically ventilated, beagles the escending brancn of the leTt corona[y artery (LAD) was ligated for 30 min./en minutes prior to the re-opening the i.v. infusion of-GSSG was starteo and continued throughout the experiment. Myocardial fiberlength was measured by sonomicrometry regional blood flow was estimated by the m crosphere techn que. I:~ GSSG ,,0 Control I.VP mmEg

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As depicted above (Pressure-length diagrams, obtained 10 min after onset of reperfusion), the GSSG-treated animals showed an improved myocardial segment-fiber-shortening during the reperfusion period as compared to controls. Thus glutathione seems to ennance the contract e status of the post schemic myocaraium unser conditions of oxidative stress. 1 Jnst [ An~isthesiologie und Op, [ntensivmed]zJn - Klin. Fakult~t Mannheim der Univemi~t Heidelberg 2 II. Physiologisches Institut der Universit~t Heidelberg 3 SektJon Chirurgische Forschung - Chirurgie I - Univemit~t UIm

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THE INFLUENCE OF CYTOSOLIC Mg ++ ON SINGLE CARDIAC Na + CHANNELS I. Benz and M. Kohlhardt

I N T E R A C T I O N OF C A R D I A C VAGUS AND S Y M P A T H E T I C N E R V E AND C E N T R A L - D E N E R V A T E D RATS

Mg ++ represents the most abundant cytosolic cation in living cells and may, in physiologically relevant concentrations, significantly modulate the activity of L-type Ca + + channels (AGUS et al., Am.J.Physiol. 256, C452, 1989) and several K + conductances in heart muscle. In the present inside-out patch clamp experiments with neonatal rat cardiocytes, the influence of cytosolic Mg ++ variations on single cardiac Na + channels was studied. Exposing the cytoplasmic membrane surface to a Mg + +-free environment shortened the open state and reduced roDen to 62--+2% of the control value obtained at 5 mmol/I Mg + +i.-Other elementary channel properties including reopening and elementary current size remained unchanged. Mn ++ had a similar effect on the open state kinetics since switching from 5 mmol/I Mn + +i to 0 mmol/I Mn++i caused almost the same shortening of the conducting state. Internal Ni ++ changes proved ineffective, at least in a concentration range between 5 and 0 mmol/l. (-)-DPImodified non-inactivating Na + channels showed an identical response to internal Mg + + removal. Their exit rate from the open state is as sensitive to cytosolic Mg ++ variations as in normal Na + channels with intact inactivation. Topen reacted to a Mg++ i change from 0 to 5 mmol/I like Fclosed(1) to a Mg++ i change from 5 to 0 mmol/I, namely by a factor of 1.7. Tclosed2), however, which represents a long-lasting, non-conductive state eas ly detectable in non-inactivating Na + channels reacted to the change from 5 to 0 mmo]/I insignificantly, by a factor of 0 . 9 3 + 0 . 2 1 . This non-uniform sensitivity of channel kinetics suggest that, in modulating Na + channel gating, internal Mg + + may not only act by screening surface charges.

In a n a e s t h e t i z e d female w i s t a r rats the vagus n e r v e was p r e p a r e d in the n e c k region, d i s s e c t e d and its p e r i p h e r a l end stimulated. By c h a n g i n g the v o l t a g e of the s t i m u l a t i o n the s p a t i a l s u m m a t i o n ability could be p r o v e d and by a l t e r i n g the s t i m u l a t i o n f r e q u e n c y the temporal summation p r o c e s s could be measured. R e c o r d i n g the e l e c t r o c a r d i o g r a m and e s t i m a t i n g the amount of cardiac vagal c h r o n o t r o p i c i n h i b i t i o n v o l t a g e e f f e c t - c u r v e s as well as f r e q u e n c y - e f f e c t - c u r v e s could be obtained. A h i d d e n s y m p a t h e t i c effect was e l i m i n a t e d by a d m i n i s t r a t i o n of B l - s e l e c t i v e receptor b l o c k e r Cordanum. The following results were obtained: The effectiveness d e p e n d s on the basic action of the blocker. If the heart rate r e m a i n e d u n c h a n g e d , n o changes in cardiac e f f e c t i v e n e s s c o u l d be seen. Both, an increase as well as a d e c r e a s e of the heart rate c a u s e d a d i m i n u a t i o n of the vagus effect, but the inhibition process was always stronger after a heart deceleration. In order to e l i m i n a t e central nervous influence the aorta as well as b o t h venae cavae were ligated. Therefore blood c i r c u l a t i o n was limited to the lungs and to the heart. In w e l l c i r c u l a t e d hearts the spatial s u m m a t i o n p r o c e s s was a u g m e n t e d in c o m p a r i s o n to intact animals. A bad c i r c u l a t i o n led to a sooner e x h a u s t i o n of the temporal s u m m a t i o n process. I n c r e a s i n g or d e c r e a s i n g .the basic heart rate, the vagal c h r o n o t r o p i c e f f e c t always remained smaller. Our r e s u l t s p r o v o k e the a s s u m p t i o n that the c h r o n o t r o p i c vagal e f f e c t is a p e r i p h e r a l summation p r o c e s s and not a c e n t r a l r e g u l a t i o n event. Intracardiac interactions between the vagus and the s y m p a t h e t i c nerve exist in the intact rat and p e r h a p s in the c e n t r a l d e n e r v a t e d animal.

Supported by DFG (Ko 778/2-3) Physiological Institute of the University; D-W7800 Freiburg/Br., Germany

IN I N T A C T

Hiewald, G.-A. and H . K a s c h o w i t z

I n s t i t u t f~r P h y s i o l o g i e der F r i e d r i c h - S c h i l l e r - U n i v e r s i t ~ t J e n a , T e i c h g r a b e n 8, D - O - 6 9 0 0 Jena

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DIFFERENCES IN T H E COURSES O F H E A T O U T P U T IN LEFT A N D RIGHT VENTRICLES OF THE ISCHEMIC HYPOTHERMIA AND CARDIOPLEGIA

RAT

HEART

UNDER

INFLUENCE OF ADRIARYCIN - INDUCED CARDIOMYOPATHY ON CARDIOVASCULAR REGULATION IN NARCOTISIZED RABBITS

F. Bach, D. Singer, H.-J. Kuhn ~), and H.J. Bretschneider

T. Encke, ~. Flemming, D. Roloff, T. Wronski & E. Seeliger

To compare the metabolic behaviour of ischemic left ventricular (LV) and right ventricular (RV) myocardium, heat output was continuously monitored by microcalorimetry. Hearts from ether anaesthetized male rats were dissected either in a purely ischemic state or after perfusion with the histidine buffered cardioplegic solution H T K (Langendorff's technique; 5 min, 40-50ml). LV and R V slices were incubated in either H T K or Tutofusins and introduced into the microcalorimeter after a thermal equilibration period of 20 rain. Measurements were performed at 30, 25, and 20 oC, respectively. Typical decline of heat output can be described by the difference between the initial maximum and the final baseline values and the corresponding time period (lIW x gd,-~/min). Under pure ischemia these quotients are 3479/22, 2887/26, 2401/47 for the LV and 3054/32, 2042/44, 1789/63 for the RV, at 30, 25, and 20 oC, respectively. Under H T K protection the respective values amount to 4246/45, 3335/48, 2929/83 for the LV and 3713/37, 3299/43, 2879/69 for the R V (mean, n=?). Thus, under pure ischemia the metabolic decline is slower in the R V than in the LV. This is probably due to the fact that the thin R V slices are mostly supplied by the oxygen physically dissolved in the incubation medium. In both LV and RV, hypothermia retards the decline of ischemic metabolism with Q~o values around 3 (pure ischemia) or 2.5 (cardioplegia). Under cardioplegia, maximal heat output is higher in the LV as well as in the RV, indicating that at the start of measurement the metabolic rate had not fallen as far as under pure ischemia. However, the metabolic decline in the R V is steeper than under pure ischemia so that the final baseline is not reached later than in the LV. This effect may be explained by interactions between aerobiosis and cardioplegia in tissues partially supplied with oxygen by diffusion.

The aim of t h i s study was to characterize the model of Adriamycin- induced cardiomyopathy with regard to cardiovascular regulation. Rabbits received Doxorubicin (ADRIANYCIN, lmg/kg ~W i . v . ) twice weekly for 8 weeks. After a two week recovery period experiments were carried out; venous and a r t e r i a l pressures, l e f t ventricular parameters, heart rate, v e n t i l a t o r y frequency, etc. were measured. Stimuli were vagotomy, occlusion of the r i g h t common carotid artery, breathing pure oxygen, or a hypercapnic gas mixture, i . v . volume expansion, as well as combinations. Most of the basic values were d i f f e r e n t between Adriamyclntreated animals (A) and controls (C), eg. mean a r t e r i a l blood pressure was s i g n i f i c a n t l y lower in A (68,9+5,#mmHg) than in C (96,5+4,3l. However, the reactions to d i f f e r e n t s t i m u l i , l i k e carotid occlusion, hypercapnia and vagotmmy, were basic a l l y unchanged. Volume expansion caused a higher increase of a r t e r i a l pressures in A than in C, and led to inverse reactions between groups with regard to l e f t ventricular enddiastmlic and maximum central venues pressures. The a r t e r i a l pressure response to hyperoxia was stronger in A than in C. By combining two s t i m u l i , more differences between the groups occured. Carotid occlusion under hyperoapnic conditions caused a Kigher increase of d i a s t o l i c a r t e r i a l pressure in A than in C. Subsequent to volume expansion, maximum central venues pressure increased in A due to carotid occlusion, but decreased in C. Adriamycin changed the basic values to a large degree, however, cardiovascular regulation seems to be able to compensate for a wide range of the induced heart a l t e r a t i o n s . Strong differences are detectable only when stimuli are combined.

Zentrum Physiologie und Pathophysiologie a n d 1)Zentrum Anatomie der Universit~it, Humboldtallee 22, D-3400 GOttingen

Physiolog. I n s t i t u t , Msdizinische Fakult~t (Charite) der Humboldt- Universit&t , ~erlin 0-1040, Hesaische Sir. 3-4, D

Supported by the DFG, SFB 330

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Organprotektion

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GSttingen

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THE I N C R E A S E OF C A R D I A C O U T P U T BY I M M E R S I O N IN WATER M E A S U R E D BY T R A N S E S O P H A G E A L E C H O C A R D O I G R A P H Y . J. B8ckin$~ H. W i s k i r c h e n , G. Pals.

A L A S E R S C A T T E R I N G T E C H N I Q U E F O R I N V E S T I G A T I O N OF O S C I L L A T O R Y P H E N O ~ ] ~ A IN CULTURES 0F N E O N A T A L RAT HEART CELLS W. Linke, W. Boldt & M. W u s s l i n g

Objective. As indicated by theoretical considerations and radiological measurements, the r e d i s t r i b u t i o n of blood c a u s e d by i m m e r s i o n in w a t e r entails a volume stress on the heart. The extent of this s t r e s s is not known precisely e i t h e r in quantitative nor in q u a l i t a t i v e terms. We c a r r i e d out a pilot study on this to appraise the degree of risk for patients with cardiac diseases. Thirty m y o c a r d i a l h e a l t h y v o l u n t e e r s were i n v e s t i gated. A f t e r i m a g i n g the c a r d i a c c a v i t i e s of test subjects standing out of w a t e r as well as up to their neck in water, the s t r o k e volumes and the cardiac output were determined on the basis of s y s t o l i c and d i a s t o l i c v e n t r i c u l a r d i a m e t e r s . Results: Stroke Volume Cardiac Output not i m m e r s e d immersed not i m m e r s e d immersed 58 ml 83 ml 5.05 L/min. 6.39 L/min.

F r e q u e n c y and a m p l i t u d e of cultures of n e o n a t a l rat heart cells w e r e i n v e s t i g a t e d c o n t i n u o u s l y with a l a s e r o p t i c a l t e c h n i q u e f o r several days. A small group of h e a r t cells g r o w n on to the b o t t o m of a c u l t i v a t i o n f l a s k (age of the cultures at least 3 days) was s e l e c t e d f o r the i l l u m i n a t i o n with a l a s e r b e a m (He N e laser, 3 raW, b e a m d i a m e t e r 300 jam) and the s c a t t e r e d light w a s m e a s u r e d b y a CCD line. The set of the t e m p e r a t u r e w a s n o r m a l l y 37~ PC" s p~ Two w e r e u s e d f o r r e g i s t r a t i o n and computation. D a t a s a m p l e d e v e r y m i n u t e (amplitude) and every 5 m i n u t e s (frequency), r e s p e c t i v e l y were a n a l y s e d by Past F o u r i e r T r a n s f o r m s .

l.There is a m a r k e d i n c r e a s e in the volume of the h e a r t in p e r s o n s diving into deep water. 2.This i n v o l v e s all c h a m b e r s of the heart. 3.This i n c r e a s e leads to a d i s t i n c t i n c r e a s e in the s t r o k e volume and c a r d i a c output. Conclusions: The s t r e c h i n g of the left v e n t r i c l e a r i s i n g from i m m e r s i o n in w a t e r must be considered in p r e d a m a g e d hearts. On the other hand, i m m e r s i o n is also a prelaod t r a i n i n g even w i t h o u t physical exertion. C a s p a r N e i n r i c h Klinik,

D - 3 4 9 0 Bad D r i b u r g

F r e q u e n c y has been f o u n d to be r e l a t i v e l y constant during the p e r i o d of registration, o n l y i n s i g n i f i cant and m o s t l y i r r e g u l a r changes c o u l d be observed. To the c o n t r a r y c o n t i n u o u s m e a s u r e m e n t s of a m p l i t u d e showed p e r i o d i c changes w i t h p e r i o d l e n g t h s of m o s t l y 4 1/2 to 7 1/2 and about 12 hours. D e c r e a s e of pH f r o m 7.8 to 7,3 d u r i n g c u l t i v a t i o n as well as periodic e x t e r n a l f i e l d s t i m u l a t i o n of the w h o l e culture didn't i n f l u e n c e t h e s e rhythms. P e r i o d i c changes of a m p l i t u d e are not c o r r e l a t e d to the s o l a r day n i g h t cycle. R e s u l t s c o n c e r n i n g a m p l i t u d e suggest enzyme systems r e s p o n s i b l e f o r cell c o n t r a c t i o n to v a r i a t e t h e i r a c t i v i t y at constant i n t e r v a l s a l r e a d y at the level of a monolayer. J u l i u s - B e r n s t e i n - I n s t i t u t f G r P h y s i o l o g i e der M a r t i n L u t h e r - U n i v e r s i t ~ t H a l l e - W i t t e n b e r g , L e n i n a l l e e 6, D - 0 - 4 0 2 0 Halle/S.

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REGIONAL ISCHEMIA AND REPERFUSION: THE EFFECT OF MYOCARDIAL PROTECTION ON TItE INFARCT SIZE IN THE PIG U. Sunderdiek, M. Kantartzis*, X. Souvatzis, B. Peter +, J.D. Schipke

EFFECT OF INCREASES IN AORTIC AND CORONARY ARTERIAL PRESSURE AT CONSTANT CORONARY FLOW ON THE GEOMETRY OF THE LEFT VENTRICLE I. Stocks, J.D. Sehipke, G. Arnold

60 rain complete occlusion of a coronary artery and subsequent reperfusion induces 60 % myocardial infarction of the area at risk in the pig (Horneffer, Circulation 76:V-39, 1987). We investigated whether an additional global ischemia as necesseraly produced during cardiac surgery affects the amount of the irreversible myocardial injury. The left anterior descending coronary artery was occluded for 60 min in 13 anesthetized, thoracotomized pigs. The myocardium was made globally ischemic during additional 15 min cardiac arrest and protected either via hypothermia (30~ n = 6) or cardioplegia (n = 7). At the end of 180 rain reperfusion, the area at risk was delineated by means of Evans blue dye injected into the aortic root. The heart was then rapidly excised and the left ventricle sectioned into slices about 10 mm thick. For determination of the infarcted area, these slices were incubated in triphenyltetrazolinm chloride. Results: The planimetrie measurements showed an average of 36.0 __. 4.0% i (mean 4- SEM) infarct size of the area at risk. There was no significant difference between the two protection procedures (31.9 + 3.2 hypotharmia versus 39.59 -I- 4.3 % cardioplegia). Conelusinn: Our results demonstrate that the additional 15 rain global ischemia do not increase the amount of the infarcted myocardium. Either hypothermia or cardiac arrest or both conditions together seem to have a protective effect on the jeopardized myocardium such that the amount of the irreversibly injured myoeardium is even reduced compared with the amount of damage following immediate reperfusion. Inst. of Experimental Surgery, *Dept. of Thoracic- and Cardiovascular Surgery and +Animal Research Laboratory, University Diisseldorf, Moorenstr. 5, D-4000 Diisseldorf

Stepwise increases in aortic pressure are associated with increases in ventrieular function which persist even after ventricular diastolic volume has returned to its initial value (Artrep-effect). Likewise, increases in coronary arterial pressure are associated with improved ventricular function (garden-hose effect). In situ, alterations in aortic pressure are paralleled by corresponding alterations in coronary arterial pressure and coronary flow, i.e. oxygen supply. Thus, separation of the individual effects is hampered. It was the aim of the present study to investigate in the same experimental model the effect of isolated pressure increases on ventricular geometry (inner diameter and wall thickness; sonomicrometry). To circumvent increases in coronary flow, the viscosity of the perfusate was increased by admixing dextran. The experiments were performed on isolated, saline-perfused, working rabbit hearts. First, the coronary arterial pressure was increased from 60 to 80 mmHg in 28 interventions. This resulted in a decrease in the inner diameter from 9.67 + 0.21 (mean + SEM) to 9.43 + 0.21 mm and an increase in wall thickness from 6.05 _+ 0.09 to 6.18 + 0.08 ram. Coronary flow was kept constant. Next, the aortic pressure was increased from 60 to 80 mmHg in 18 interventions. The inner diameter increased from 9.61 _ 0.25 to 9.89 __. 0.23 mm and the wall thickness decreased from 6.12 __. 0.16 to 5.92 + 0.21 nun. Thereafter, the coronary arterial pressure was also increased to 80 mmHg. This time, the inner diameter decreased from 9.89 + 0.23 to 9.63 + 0.23 mm and the wall thickness increased from 5.92 __. 0.21 to 6.00 _ 0.21 ram. Again, coronary flow was maintained constant. Conclusion: Increases in coronary arterial pressure and in aortic pressure have opposing effects on ventricular inner diameter and on wall thickness. Increases in wall thickness and concomitant decreases in inner diameter in consequence to increases in coronary arterial pressure cannot be explained by increases in coronary flow but most probably by increases in vascular filling. Because effects owing to changes in oxygen supply were excluded, the Anrep-effect can be explained in part by the garden-hose effect for this experimental model. Institute of Experimental Surgery, University Diisseldorf, Moorenstr. 5, D-4000 Diisseldorf

171 USE OF MYOGLOBIN MEASUREMENTS DURING MYOCARDIAL REPERFUSION AFTER.REGIONAL ISCHEMIA AND CARDIAC ARREST IN THE PIG X. Souvatzis, U. Sunderdiek, M. Kantartzis*, J. Scheja +, J. D. Schipke Serum myoglobin concentration during acute occlusion of a coronary artery is reported to correlate with myocardial infarct size (Groth, Stand J Clin Lab Invest 47:599, 1987). On the other hand, the rapid increase in the myoglobin level is regarded to indicate suceessthl reperfusion of previously ischemic myocardium (Katus, Eur Heart J, 9:619, 1988). We investigated whether the peak concentration during reperfusion also correlates with infarct size or can predict functional recovery during reperfusion. In 13 anesthetized, thoracotomized pigs, the left anterior descending coronary artery was occluded for 60 min. Then, the heart was arrested for 15 rain under myocardial protection 01ypothermia (30~ or cardioplegia) with circulation maintained by means of a cardiopulmonary bypass. During the 180 rain reperfusion, left ventricular pressure (LVP) and dP/dt were continuously recorded, and blood was sampled from the femoral artery and the coronary sinus at fixed intervals. After the end of reperfusion, Evans blue and triphenyl-tetrazolium-chloride dye was used to distinguish infarcted areas within the areas at risk. RESULTS: The peak myoglobin concentration from coronary sinus blood correlated well with both the absolute infarcted myocardium (r=0.63) and the infarcted area normalized to left ventricular weight (r=0.85). There was no significant correlation between the peak myoglobin concentration and left ventricular function at the end of reperfusion (LVPmax: r=0.25; cardiac output: r=0.00; pressure-rate-product: r=0.30) except for dP/dtma x (r=0.60). CONCLUSION: Measurement of myoglobin concentration during reperfusion provides valuable information about successful reperfusion. In addition, it permits evaluation of the extent of irreversible myocardial damage, but it is of no explicit value in predicting ventricular function during reperfusion. Inst. of Experim. Surgery, *Dept. of Thoracic and Cardiovascular Surgery, +Dept. of Transfusion Medicine, University Diisseldorf, Moorenstr. 5, D-4000 Dfisseldorf

173 INFLUENCE OF STIMULATION-FREQUENCY AND EXERNAL IONS ON SHAPE AND AMPLITUDE OF CaZ+-TRANSIENTS OF CARDIOCYI'ES S.Y.Wang~ R. Meyer Stimulation frequency has a positive inotropie effect on heart. As inward Ca2+-current decreases with increasing stimulation frequency the positive inotropie effect seems to be a consequence of a higher Ca 2+load of the sarcoplasmic reticulum (SR). Because the internal Ca 2+concentration, [Caz+ ]i, is a more direct indicator for the Ca2+-release from the SR than the contraction, we measured Ca2+-transients at various stimulation frequencies and different external ionic conditions. In isolated adult ventricular myocytes of the guinea pig membrane pot e n t i a l and membrane currents were measured by whole ceil patch clamp. [Ca2+]i was monitored simultaneously by recording the furs-2 signal. Fura-2 salt was loaded via the patch pipette. Increasing stimulation frequency of action potentials (AP) causes differences in shape and peak amplitude of Ca2+-transients: At very low stimulation frequencies {0.2 Hz) in control solution (135 mM Na + 1.8 mM Ca 2+) Ca2+-transients have a slow rising phase, which last~ during the duration of the action potential (compare figure). The form of the Ca2+-transient resembles the form of a rested state contraction. At higher stimulation frequencies (0.5 Hz, compare figure) the Ca 2+transient has a steeper rising 2hase and declines nearly completely during the AP-duration. Peak CaZ+-concentration increases from 340 to 590 nM due to the change in stimulation freo~uency. Higher stimulation frequencies enlarge the amplitude of the CaZ+-transient further. Raising the external Ca 2+ from 1.8 to 10 mM changes the shape of the transient at 0.2 Hz comparable to a rise in stimulation frequency and at 0.7 Hz elevation of external Ca 2+ increases Ca2+-transients by 26 %. Reduction of external Na + by 50 % has only little influence on CaZ+-transients at stimulation frequencies below 0.5 Hz. A t higher stimulation frequencies the transients get somewhat higher and significantly prolonged. Prolongation can be interpreted as a sign of inhibition of Na+/CaZ+-exchange. Thus shape and amplitude of Ca2+-tran slants may depend mainly on the Ca2+load of the SR. Physiologisches Institut, Universitfit Bonn, Wilhelmstr.31, D-5300 Bonn 1

I300ms

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COMPARISON OF ENRICHMENT PROCEDURES FOR ISOLATED ADULT CARDIOCYTES M. Mfiller1, R:. Meyer 1, A. Dehn2 Every isolation procedure of adult cardiocytes delivers a mixture of rod-shaped and rounded hypercontracted cells. For all types of investigations, which allow a selection of individual cells like electrophysiology the percentage of rod-shaped cells is of minor interest, unless at low rates (<15%), where the yield is usually a sign for general damage of all cells. However the result of biochemical and statistical investigations is disturbed by rounded cells in the population. Thus different methods for specific enrichment of red-shaped cells were tested. Two completely different techniques were applied, eentrifugation and selective attachment. Separation of red-shaped cells by centrifugation was always carried out in a gradient, mainly percoll gradients. Both continous and discontinous gradients were used. Continous gradients had a linear (from 1.01 to 1.09 g/ml) or a S-shaped (from 1.01 to 1.14 g/ml) profile. The specific density of rod-shaped guinea pig cardiocytes proved to be 1.076 g/ml, the density of rounded ceils was between 1.033 and 1.051 g/ml. Thus the density of the discontinous graclients was chosen to be 1.06 g/ml in the upper layer and 1.085 g/ml in the Iower layer. All different percoll-gradients lead to a significant enrichment of red-shaped cells. The percentage of rod-shaped cells after eentrifugation depended more on the ratio of red-shaped/rounded cells in the original material than on the method of centrifugation. Starting with around 20 % red-shaped cells an enrichment to more than 65 % could be achieved in a one step cantrifugation. Starting above 40 % leads to a yield of more than 85 %. Although there were no significant differences in tb.e degree of enrichment between the different centrifugation techniques, the S-shaped gradient delivered the highest absolute amount of cells. Selective attachment was cmried out with laminin (1 /~g/cm2) and fetal calf serum (4 %, Piper et a1,1982, J Mol Cell Cardiol 14, 397-412). Both methods achieved a comparable degree of enrichment, which was also in the same range as in the case of centrifugation. But the absolute amount of rod-shaped cells was much higher in the case of laminin attachment than in the case of fetal calf serum. Starting with only 10 % rod-shaped cells an enrichment up to 90 % is possible if a centrifugation is followed by selective attachment. Iphysiologlsches Institut, Univ.Bonn, Wilhelmstr.31, D-5300 Bonn 1 2 KaliChemie, Pharma GmbH, D-3000 Hannover 1, Hans-g6ckler-Allee 20

Monitoring of Redox-State of Respiratory Enzymes and Myoglobin Oxygenation in the Working Rat Heart in Normoxia and Oxygen Deficiency. Zfindorf J., Tausehek D., Prank K., Ito K. 1, Nioka S.2, Kessler M., Chance B. 2 Iustitutfib-Physiologicund Kardiologieder Universit~tErlangen-Nfimberg,Waldstr.6, D-8520 Erlangen, 1BiophysicsDivisionResearchInstituteof AppliedElectricity, Hokkaido University,Sapporo060, Japan.2 Deparlmentof Biochemistryand Biophysics, Universityof P_r P.hil."adelphia,PA, USA. , ........ The cellular oxygen supply in the isolated, hemoglobin-free perfilsed, working rat heart can be determined by measurement of myoglobin oxygenation. However, for a precise analysis of mltochondrlal hypoxia and artoxia (pO2 < 0.01 Tort) redox-state of respiratory enzymes must be known. For an accurate estimation of physiological relationship between myoglobin oxygenation and mitochondrial redox-state two prerequisites are necessary: 1. The perfusion of all capillaries must be adequate.When disturbances in the distribution of capi/lary perfusion develop a certain number of capillaries become mlderperfused and thus produce partial hypoxia and]or anoxia of the myoeytes. 2. The spatial resolution of the applied sensor must be small enough in order to distinguish between normally and pathologically perfused supply units. Otherwise a sensor with a large catchment volume will lead to integrated signals thus mixing tissue values originating from well and badly perfnsed myocardinra. Such an integration of spatially distributed tissue signals will result in an apparent early reduction of respiratory enzymes. The experiments were performed using the model of the isolated, hemoglobin-free perfused, working rat heart. It was a modified version of the model, described by Tarek and Olders (Olders et al.: An Experimental Set-Up for the Blood Perfnsed Working Isolated Rat Heart, Oxygen Transport to Tissue XII, Edited by J, Piiper et al., Plenum Press, New York, 1990, 151-100). Heparine was injected into the vena femoralis five minutes before the heart was removed from the animal to prevent formation1 of microthrombi in the mierovessels of the myocardium, For measurements of the tissue remission spectra the EMPHO II was applied, using miero-lightgnide fibres with a diameter of 25@pro. The evaluation of the remission spectra obtained with different levels of tissue oxygenation revealed clear evidence that reduced eytochrome aa3 only is observed when myglobin oxygenation falls below 10 %. Integrated pO2 gradient fields recorded simultaneously under these conditions showed a concomitant increase in the number of pO2 values arround 1 Tort.

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A C h - L I K E EFFECTS OF A SERUM FACTOR IN G U I N E A - P I G A T R I A L MYOCYTES A R E MEDIATED BY A PERTUSSIS TOXIN SENSITIVE MECHANISM J. Hfiser, P. Lipp, M.C. Wellner and L. Port

NITRIC OXIDE SYNTHESIS INHIBITOR, N~-NITRO-L-ARGININE (LN~NA), ENHANCES MYOGENIC AUTOREGULAT1ON (MA) OF CORONARY VESSELS IN ISOLATED PERFUSED GUINEA PIG HEARTS. P. Bunsiricomchai, I-t.J.Drring, V.Hiller

We have communicated that in isolated guinea-pig atrial myocytes diluted sera (10-2 to 10-4) from various species (horse, guinea-pig, sheep, man) cause activation of an inward-rectifying K+-current, that is normally controlled by acetylcholine (IK(ACh); Hiiser et at., Pfliigers Arch. 418 Suppl. R90). Activation of this current could not be inhibited by muscarinie or pudnergic (AI) antagonists, in saturating concentrations, suggesting these receptors not to be involved. Using whole-cell voltage clamp and a device for rapidly switching between different superfusing solutions the mechanism of this effect was further investigated (T=24-27 ~ Pretreatment of the myocytes with Pertussis toxin (FIx, 2/tg/mi; 15-20 hours) resulted in a complete loss of sensitivity to ACh, adenosine and serum. This suggests that, like for the classical receptoragonists, a PTx-sensitive G-protein is involved in serum-induced activation of IK(ACh)(Szabo & Otero, Ann. Rev. Physiol. 5_.22e1990). Basal L-type Ca2+-current (ICa) was not affected by serum. Upon 13adrenergie stimulation using isoprenaline (10-7 M) the increased ICa was reversibly reduced by horse serum. A 100-fold dilution of semm corresponded to about 1 #M ACh. Like IK(ACh)-activation, the antagonistic effect of serum on I]-adrenergic augmentation of ICa was completely abolished in myocytes pretreated with PTx. This demonstrates that the signalling pathway activated by serum is mediated via Gi. The "muscarinic" effects of sera were not reduced by dialyis; no activity was found in the dialysing fluid. Other agonists that have been described as activators of cardiac IK(ACh) such as Phenylephrine (10-5 M) and somatostatin (3.10-6 M), in the present study did not induce a measurable current.

Various analogues of L-arginine have been shown to inhibit NO synthesis in vascular endothelial cells. Pohl et al. (Pflfigers Arch.415(Suppl.1), R62 (1990) demonstrated that, as a result of deficient NO liberation, MA of rabbit coronary vessels was increased after application of Na-Nitro-L arginine. The following experiments were done in oder to investigate LN'~NA, another inhibitor of NO synthesis. Electrically driven guinea pig hearts were perfused according to LANGENDORFF with modified KREBS-HENSELEIT solution. Contractile force (isovolumetrically), coronary flow (electromagnetically) and perfusion pressure (PP) were measured continuously and the collected data were stored on a PC. PP was increased stepwise by 10 mmHg from 40 to 90 mmHg. Myocardial function curves and pressure-flow (PF) curves were plotted from the data. Mean and maximum (diastolic) flow values were evaluated. In order to quantify autoregulation the slope of the PF curve (ml x rain -I x mmHg 4) and an index of autoregulation (AI) were calculated for the PP range of 50 - 70 mmHg and of 60 - 70 mmHg: AI = 1 (AF/~p) x (P(Fi). Increasing MA is characterized by a decrease of slope towards zero and an increase of AI towards 1. In control hearts during steady state (PP = 50 mmHg) contractile force amounted to 100 mmItg, mean coronary flow was 8.7 ml/min and diast. flow was 11.5 ml/min. During pressure load the slope of the PF curve was 0.12 and AI 0.55 (both for diastolic flow). Perfusion with 5 #mol/I LN~NA did not change myocardial contractile force but decreased the slope by 58% and increased AI by 44%. Diastolic coronary flow, measured during steady-state (PP = 50 mmHg), decreased only by 9%. Higher concentrations of LN~NA (20 and 30 ~mol/1) depressed contractile force by 10 or 27% and diast, flow by 19 or 22%; slope and AI exhibited no further alteration. The changes in MA calculated from mean flow data were less pronounced. Our results support the hypothesis that NO is a physiological antagonist of MA.

hasthut ftir Zellphysiologie, Ruhr-Universit/it, Postfach 102148, 4630 Boehum

Physiologisches Institut der Universit~t, Hermann-Herder-Str. 7 D-7800 Freiburg

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A CALCIUM AND ATP SENSITIVE NONSELECTIVE CATION CHANNEL IN THE ANTILUMINAL MEMBRANE OF RAT CEREBRAL CAPILLARIES

ROLE OF EDRF IN REGULATION OF CEREBRAL OXYGEN SUPPLY DURING ARTERIAL NORHOXIA AND HYPOXIA d.D. Noritz and d. Grote

R. Popp* and H. G6gelein 1 Rat cerebral capillaries were mechanically isolated from brain grey matter by homogenization. A 10 minutes enzymatic pretreatment of the basement membrane was necessary for using patch-clamp technique directly on the antiluminal membrane of these microvessels. In cell-excised (inside out) membrane patches a nonselective cation channel could be identified. This channel is about equally permeable to the monovalent cations Na + and K +. Chloride ions as well as divalent cations and large organic cations like N-methyl-D-glucamine are apparently impermeable. The nonselective cation channel has a conductance of 35+3 pS (n=9) in symmetrical 140 mM NaCI solutions and is activated by Ca2+ concentrations higher than 10-5 M and inhibited by ATP (500 #M) from the cytosolic membrane surface. In NaCI solution containing 1.3 mM Ca2+ the channels appeared mostly in clusters where each channel had a mean open-state probability of about 0,65. Drugs consisting of two phenyl rings linked by an amino bridge like flufenamlc acid (1O-10O/~M) and 5-chloro-2-(3-trifluormethylphenylamino)-3-nitrobenzoeic-acid (10-100 ~M) suppress channel activity. In addition the trivalent cation gadolinium (10-100/~M) also inhibits the nonselective cation channel. This channel type was also observed in endothelial cells of porcine cerebral capillaries, although with lower incidence than in rat. The functional role of the channel is not clear yet. In cell-attached membrane patches this channel was not detectable. It is likely, that this channel is only activated under pathophysiological conditions. * Max-Planck-Institut ftir Biophysik, Kennedyallee 70, D-6000 Frankfurt/Main 70, F.R.G.

Since the role of the endothelium in the hypoxie induced cerebral blood flow regOlation is being discussed, the effect of arterial hvpoxia on cerebral oxygen supply was studied after application of L-Nitroarginine, an inhibitor of the endothelium-dependent vasodilatation. The experiments were performed in rats, in which tissue PO2 of the brain cortex and diameters of pial arteries and veins were measured at normal and decreased arterial oxygen tensions. During a r t e r i a l n o r m o x i a i.v. bolus i n j e c t i o n of LNitroarginine (10 mg/kg b.w.) induced a left shift of the PO 2 histograms in the brain cortex and a significant decrease of mean tissue PO2 from 2R.9 to 23 mmHg. Simultaneously mean arterial blood pressure increased from 108 to 147 mmHg while no significant changes of pial vessel diameters could be observed. Arterial hypoxia (Pa02:40 mmHg) caused a pronounced decrease of brain tissue oxygenation, the mean tissue POB was found to be 10.8 mmHg. In addition, a significant vasodilatation occurred, the mean pial artery diameter increased up to 150 % o~ the initial value. In the control experiments comparable hypoxic conditions induced less pronounced decrease of mean cortical PO 2 to 11.4 mmHg. Following readjustment of arterial normoxia normal tissue PO2 histograms with a mean POB value of 27.5 mmHg were found. In the controls, however, mean tissue POB significantly increased above the initial level. The results demonstrate that the endothelium derived releasing factor plays an important role in the PO2 dependent regulation of cerebral blood flow and oxygen supply during arterial normoxia and arterial nypoxia. Physiologisches Institut der Universit~t Bonnj Nu~sllee 11, D-5300 Bonn

1Hoechst AG, Pharmaforschung, D-6230 Frankfurt/Main 80, F.R.G.

179

181

EFFECTS OF N~ (NOLAG) ON RAT PIALARTERIOLESIN S1TU A.A. Parsons*, Q. Wang +, L. Schilling, N.A. Lassen, and M. Wahl

INFLUENCE OF ENDOTHELIUM DERIVED RELAXING FACTOR ON LONGTERM BLOOD PRESSURE REGULATION IN CONSCIOUS DOGS J.E. Baumann, H. Ehmke, B. Nafz, H.R. Kirchheim, and P.B. Persson

We have previously characterized the effects of the nitric oxide (NO) synthase inhibitor, NOLAG, on rat isolated basilar artery (1). In the present study we have investigated the effects of NOLAG in rat pial arterioles in situ. Male Spragne-Dawley rats (220-250g) were anaesthetized with inactin (9mg/100g iv) and prepared for investigation of pial arterioles as described previously (2). Blood pressure, body temperature and arterial acid base status were maintained in the physiological range. The effects of NOLAG (10.5 - 10.3 M) on resting vessel diameter were investigated by microapplication and by 10 min cortical superfusion (104M). In addition, the effects of NOLAG (10"4M) on acetylcholine (ACh, 10"4M) and bradykinin (Bk, 10"6M) induced dilation were also investigated. Resting diameter of pial arterioles was between 20 - 50 I~m. Cortical superfusion and microappHcation of NOLAG alone (n = 4-10) did not significantly change diameter of pial arterioles. In the absence of NOLAG, BK induced a dilation of 19.6 -+ 6.0% (mean -+ SEM; n = 7), and ACh produced dilation of 13.8 -+ 2.0% (n = 7), respectively. Coadministration of NOLAG (10~M) and Bk did not affect Bk-induced dilation (19.6 -+ 2.5%, n = 7), however a significant inhibition of ACh induced dilation was observed (3.4 -+ 2.6%, n = 5). The inability of NOLAG to induce constriction of pial arterioles in situ is in contrast to its constricting action on rat basilar artery in vitro (1) and in situ (3). This result may therefore indicate that basal release of NO alone does not play a major role in maintenance of pial arteriolar diameter in situ. Furthermore, our results indicate a functional role for NO release in ACh- but not Bk-induced dilation of rat pial arterioles in situ. 1 Mackert, J. et al.: Int. J. Microclrc. Clin. Exp. 9:227, 1990 2 Wahl, M. et al.: Brain Res. 411:72-80, 1987 3 Faraci, F.: Amer. J. Physiol. 259:H1216-H1221, 1990 Physiol. Inst., Pettenkoferstr. 12, 8000 Mtinchen 2 *Supported by the Alexander von Humboldt Foundation +Supported by the Alfred Benzon Foundation, Denmark

Endothelium Derived Relaxing Factor (EDRF) is released by endothelial cells in response to changes of sheer-stress. Hitherto, the chronic effects of EDRFBlockade have not been clarified, furthermore a possible role in the phasic adjustment of blood pressure (BP) control has not been focused on. Thus, the influence of EDRF-blockade on the 24 hour BP profile was investigated in 5 conscious freely moving foxhounds. BP was continuously measured by a transducer located in the abdominal aorta (Konigsberg, USA). This signal was digitized and transmitted via a telemetry system (Glonner, FRG) to a receiver unit. The transducers were calibrated in vivo before the experiments by an aortic catheter. Heart rate (HR) was determined by measuring the time between the two largest dp/dt signals of BP. Spectral analysis of BP was made for the 432000 BP values obtained during each 24 hour period (sampling rate 5 Hz). 300 mg of L-Nitro-Arginine-MethyI-Ester (L-NAME) - a false substrate for EDRF generation - intravenously increased 24 hour BP by 16 +- 4 mmHg to 143 -+ 8 mmHg (P
Rl13 182 ATRIAL NATRIURETIC FACTOR (ANF) DOES NOT ALTER CAPILLARY FILTRATION OF SKIN AND SKELETAL MUSCLE IN HUMANS

T. ROLLEKE, S. BERK !=, J.O. ARNDT We have recently shown that ANF at doses of 0.1 ~g.min -1.kg body weight increased blood content in the splanchnic circulation at the expense of the heart while calf volume decreased (Thomas, Peters & Arndt, PflOgersArch. 418, R 105, 1991). Particularly, the last observation is at odds with others (L. Groban et aL, Am. J. PhysioL 259: H258-268, 1990) who found filtration rate to increase in forearm of humans though at ANF doses lower (0.005 p.g .min1 .kg body weight-1) than in our experiments. It was of interest, therefore, to explore how ANF would influence capillary filtration under our experimental conditions, i.e. in the presence of ANF plasma concentrations seen in hypervo]emia. In six healthy young men, capillary filtration coefficients as well as volume and blood flow of both foot and calf were determined by occlusion plethysmography before, during, and after ANF (bolus of 25 ~g and 30 min infusion 0.1 i~gmin -l'kg body weightl). Central venous pressure, arterial pressure, heart rate, and hematocrit were also recorded. The capillary filtration coefficients [103ml.minl'mm Hg't100 ml tissue volume1 , medians and ranges] were, for the foot, 3.2 (1.9-5.1) before, 3.3 (2.2-5.3) during, and 3.0 (1.7-5.5) after ANF-infusion, and, for the calf, 6.5 (4.7-12.5) 7.0 (3.5-13.3), and 7.4 (5.1-15.3), respectively. Because of strong intra- and interindividual variability filtration coefficients did not differ statistically. Still, foot .volume -0.3(-0.5+0.2)m1.100 ml tissue-1 as well as central venous pressure -3 (1.8-4.8)cm H20 decreased without consistent changes in blood flow and arterial blood pressure while hematocrit +3 (2-5)% and heart rate +19 (17-21)min-1 increased. ANF does not alter capillary filtration of skeletal muscle and skin when given at doses needed to attain plasma concentrations usually found in pathological states. Consequently, under such conditions, fluid loss in the microcirculation of skin and skeletal muscle can hardly account for the accompanying decrease in cardiac filling pressure and increase in hematocrit. Abteilung for experimentelle Anaesthesiologie der Heinrich-..Heine-Universit~it D0sseldorf~ Geb/iude 23.02.01, MoorenstraSe5, W-4000 DUSSELDORF1

184 CARDIAC HAEMODYNAMICRESPONSES TO AN ACUTE ARTERIAL PRESSURE RISE IN ATHEROSCLERSTIC RABBITS WITH IMPAIRED BARORECEPTOR REFLEX K.Wilfert and K.Drischel ............................................................. Baroreflex s e n s i t i v i t y is attenuated by atherosclerosis following a high-cholesterol diet in rabbits. The animals also show coronary stenosis and myocardial lesions. Under these conditions an acute elevation of systemic blood pressure might be a strong challenge to the adaptability of the heart. In the present study the haeeodynaeic adjustment of the heart to an acute pressure loading e l i c i t e d by angiotensin I I (0.4 ~g/kg.min for 15 ein) was investigated in rabbits with cholesterol-induced atherosclerosis (AR) in comparison to a control g r o u p (CR). The animals w e r e anaesthetized with urethane. Prior to the infusion the baseline values of the measured hsemodynmmic parameters did not show any s i g n i f i c a n t differences between AR and CR. The increase in aortic pressure and l e f t v e n t r i c u l a r systolic pressure induced by angiotensin is in AR some more pronounced during the whole period of infusion. In both groups the elevation of blood pressure is followed by a decrease of heart rate and cardiac output. In AR the drop of heart rate is smaller than in CR, In spite of this diminished baroreceptor mediated heart rate response, in AR the decrease of cardiac output reaches a s l i g h t l y g r e a t e r extent as compared to CR. In AR the f a l l of cardiac output is accomplished mainly by a decrease in stroke volume, whereas t h i s parameter remains unchanged in CR. Enddiastolic pressure rises in both groups of animals, in AR the increase tends to bs smaller than in CR. The results indicate that in AR the diminished baroreceptot-heart rate r e f l e x is widely compensated by a modified involvement of cardiac effector responses in the cardiocirculatory control. This mitered regulatory mechanism might cont r i b u t e to protect the heart from overloading in chronic cardiovascular desease due to atherosclerosis. I n s t i t u t ffir Physiologie~ Sir. 3/4, D-0-1040 Berlin

Humboldt-Universit~t~

Hessieche

183

185

ACTIVE VERSUS PASSIVE COMPONENTS OF THE VASCULAR RESPONSE IN HUMAN SKIN TO POSTURAL CHANGES H. Jeosen and P. Gaehtgens

CARDIOVASCULAR RESPONSES SODIUM CHLORIDE SOLUTION A. B r a t t s t r 6 m , E.Appenrodt, L. P h a r o w

In order to study the mechanisms contributing to vascular responses to changes of posture, Laser-Doppler flux (LDF) was measured in the palmar finger skin of 6 healthy probands. Transmural pressure (Ptn0 of the skin vessels was varied by combined variation of external pressure (Pext) and vertical height of the hand relative to heart level. Pext (range -120 to +180 mmHg) was applied in steps of 10 mmHg by compressed air or suction to the finger for periods of 1 min. Arterial blood pressure, heart rate, and respiration as well as LDF at a contraiateral site were recorded to exclude variations of systemic autonomic activity.

Intracerebroventricular (i.c,v.) administration of hypertonie NaC1 s o l u t i o n elevates blood pressure

The relationships between LDF and Pext obtained at 3 vertical hand positions were superimposed, if LDF was plotted as a function of the change of Ptm- A monotonous decrease of LDF to approx. 50 % of control was seen with elevated Ptm- LDF increased with reduction of Ptm by 2030 mmHg, but decreased towards zero upon further reduction. Upon release of positive Pext, reactive hyperemia occurred, while slow recovery of LDF was seen following release of external suction. The results are explained by passive vessel distension/compression inducing active adjustments of vascular tone. The magnitude of the active response can be deduced from the LDF values immediately following the release of Pext, when intravascular pressure is suddenly restored, but active tone is still maintained. As long as microvascular collapse does not occur, active adjustments of vascular tone override the effects of passive distension in the response to alteration of transmural pressure during a change of vertical hand position.

Institut ffir Physiologic der Freien Universi~t Berlin, Arnimallee 22, D-1000 Berlin 33

TO

CENTRALLY

APPLIED

A. B r a t t s t r g m , Jr.,

and

{BP) a n d h e a r t r a t e (HR) in a n a e s t h e t i z e d rats. A n impaired baroreflex ~dght contribute to these elevations. Therefore, in rats the barsrecepter h e a r t r e f l e x (BHR) w a s c h e c k e d b e f o r e a n d 15 m l n a f t e r t h e b e g l n n l n g of c o n t i n u o u s i.c.v, i n f u s i o n (0.5 pl/ m~n) of e i t h e r n o r m ~ t o n i c o r h y p P ~ o n i c (0.6 X; 1.0 M) N a C l s o l u t i o n . T h e B H R w a s t e s t e d b y e v a l u a t i n g t h e p r o l o n g a t i o n of t h e i n t e r - b e a t interval (IBI; resolution as high as 1 ms) in response to an arteficial EP rise which had been e v o k e d b y i.v. i n j e c t i o n of p h e n y l e p h r i a . T h e I B i s were plotted against the corresponding EP values and the slope of the correlation function was t a k e n t o i n d e x t h e r e f l e x s e n s i t i v i t y of t h e BILR. T h e m e a n s e n s i t i v i t y in t h e a n a e s t h e t i z e d r a t s w a s 0.6 ms/ ~ P~, d i s t r i b u t e d n o r ~ l l y
Medical School 0 - 3090 Magdeburg

Magdeburg,

R 114 188

186 BR]M]THELIN-I NTS

REDUCE

AnD

EB]MSTH~LIN-3

BL(XDD P ~ H ~ S U R E

AND

IB-FU~ED INTO HEART RATE

THE

RAT

A. B r a t t s t r ~ m . R. M i l l e r and W. D e J o n ~ Both endothelin-i (ET-1) and endothelln-3 lET-3) h a v e b e e n d e t e c t e d i n t h e c e n t r a l n e r v o u s system, as has m@eIA f o r ET-I and BT-3, and specific receptors for endothelins are present in discrete brain reEions , particularly i n t h e r e g i o n of t h e nucleus tractus solitaril (RTS), w h i c h r e l a y s t h e baroreceptor afferents. Bndothelins may act as neurotransmitters within the ~TS. Therefore, in anaesthetized ]mile u rats sm~ll amounts of BT--I o r ET-3 (i, i0, 50, i00 a n d 5 0 0 pg) w e r e infused into the NTS (0. ii p l / m i n ) f o r I0 mmln. Doses of 5 0 lO~ of E T - I or of I0 p g of E T - 3 e l i c i t e d m a x i m a l r e d u c t i o n s i n b l o o d p r e s s u r e (BP) of -1Y. 0 ~ 4 . 8 a n d - 1 4 . 2 ~ S . 2 ~mm E g r e s p e c t i v e l y a n d of h e a r t rate (HR) of - 8 0 . 8 ~ 1 . 5 and -54.4!9.7 beats/min respectively. Lower or higher doses elicited smaller reductions. These induced chan~es in cardiovascular parameters returned towards to pre--infusion levels on stopping the infusions. BT-3 seemd to be a little mre potent than ET-I at r e d u c i n g B P a n d HR. R e s p o n s e s t o i0 p g d o s e s o f B T - I a n d t o 1 P S d o s e s of E T - 3 w e r e r e p r o d u c i b l e . T h e c h a n g e s i n B P w e r e n e t s e c o n d a r y t o c h a n g e s in HR since only the latter responses were antagonized by pretreatment of t h e a n l m a l s w i t h Nmethyl-atroplne (Im~/kg). Maximal BP responses to i0 P E d o s e s of B T - I a n d H T - 3 w e r e a d d i t i v e w h i c h may indicate the presence of 2 receptors for endothelins in t h i s r e s p o n s e . Overall, this study shows that endothelins administered in fmol quantities locally into the RTS decrease BP and HR in a reversible manner and they suggest that these e f f e c t s m a y be d u e t o t h e a c t i v a t i o n 'of m o r e t h a n o n e t y p e of s p e c i f i c e n d o t h e l i n r e c e p t o r s . Inst. P h y s i o l . , )[edical S c h o o l ]~a~deburg, O-- 3 0 9 0 Magdeburg and ~rrell D e w Res. Inst., Strasbourg

EFFECTS O F C A P S A I C I N ON A O R T I C N E R V E S IN R A B B I T S R. S t a a k a n d R. O f t

SPONTANEOUS

ACTIVITY

OF

In 8 anaesthetized rabbits the influence of capsaicin on b a r o r e c e p t o r a o r t i c n e r v e a c t i v i t y (ANA) was studied. C a p s a i c i n (3SmM, d i s s o l v e d in olive oil) was a p p l i e d p e r i n e u r a l l y at t h e left aortic nerve distally from the bipolar recording electrod e s for 30 min. T h e n e u r o g r a m w a s fed in a c o m p u t e r and than peak-related events were extracted. Poststimulus time h i s t o g r a m s of t h e s e c r e a t e d events w e r e t r i g g e r e d b y t h e S - w a v e of t h e e c g (intracardial lead, T y p rS). T h e r e f o r e e a c h h e a r t p e r i o d d u r a t i o n (HPD) w a s d i v i d e d into 20 bins. Phasic disc h a r g e s w e r e a n a l y s e d in t h e interval f r o m b i n 3-8, t o n i c a c t i v i t y w a s c o u n t e d f r o m b i n 17-20. Aortic nerve a c t i v i t y (ANA) was d e c r e a s e d 20 min after application (p~0.01; s. table, X • Caps a i c i n i n d u c e d a r e d u c t i o n in p h a s i c a n d t o n i c ANA (ph. a n d ton. ANA; p ~ 0 . 0 1 ) . 20 m i n a f t e r wash out of capsaicin A N A a n d the t o n i c a c t i v i t y s h o w e d a m a r k e d t e n d e n c y to r e c o v e r y . C a p s a i c i n d i d n o t p r o duce s i g n i f i c a n t c h a n g e s in s y s t o l i c a n d d i a s t o l i c b l o o d p r e s s u r e (SBP, DBP). H P D w a s n o t a f f e c t e d .

ANA ph. ton. HPD SBP DBP

(I/s) A N A (%) A N A (%) (ms) (mmHg) (mmHg)

control 407.5• I00 i00 238.9• 1 0 2 . 3 • 6.2 72.3• 4.6

capsaicin 182.5• 4 3 . 6 • 8.9 44.0• 240.7• 1 0 2 . 6 • 7.6 7 3 . 0 • 5.9

w a s h out 276.6• 59.4• 82.2• 244.0• 9 6 . 5 ~ 7.2 6 6 . 2 • 4.9

Thus, results demonstrate that capsaicin affects the p h a s i c a n d t h e t o n i c s p o n t a n e o u s d i s c h a r g e s of a f f e r e n t b a r o r e c e p t o r n e r v e fibres. I n s t i t u t f~r P h y s i 0 1 o g i e d e r M e d i z i n i s c h e n A k a d e m i e M a g d e b u r g , L e i p z i g e r Sir. 44; M a g d e b u r g 0 - 3 0 9 0

187

189

THE REFLEX RESPONSE TO BARORECEPTOR STIMULATION UNDER CERTAIN CONDITIONS I N CHOLESTEROL FED RABBITS D.Roloff, T.Encke, B.Flemming, A.R01ke, E.Seeliger, a n d T. W r o n s k i ................................................... In p r e v i o u s s t u d i e s we f o u n d a n a l t e r e d cardiovasc u l a r r e f l e x r e s p o n s e p a t t e r n in cholesterol fed r a b b i t s a f t e r s t i m u l a t i o n of the c a r o t i d b a r o r e c e p tors. T h e a i m of t h i s s t u d y w a s to p r o o f t h e r e s u l t i n g h y p o t h e s i s t h a t the o v e r a l l c a r d i o v a s c u l a r ref l e x r e g u l a t i o n m o u l d be r e a l i s e d by changed control o f the e f f e c t o r s . T h e r e f l e x c a r d i o v a s c u l a r f u n c t i o n s of c a r o t i d bar o r e c e p t o r s t i m u l a t i o n w e r e s t u d i e d in cholesterol f e d r a b b i t s (CO) ( 2 g / d f o r 12 w e e k s ) and compared w i t h c o n t r o l g r o u p of a n i m a l s (NO) ( C h l o r a l o s e - u r e thane anaesthesia). The baroreceptor reflex functions were investigated under the conditions of hypoxia (8.5% oxygen), hyperoxia (100% o x y g e n ) , hypercapnia, volume expansion and perfusion of the isolated carotid sinus with venous blood. T h e b a r o r e c e p t o r r e f l e x r e s p o n s e f o r the mean arterial p r e s s u r e w a s l o w e r e d in CD breathing air. T h i s d e p r e s s i o n s h o w e d b o t h g r o u p s too d u r i n g perf u s i o n of the i s o l a t e d c a r o t i d sinus with venous blood. T h e g a i n in the r e f l e x r e s p o n s e of the p r e s s u r e v e l o c i t y p a r a m e t e r s of t h e l e f t v e n t r i c l e was h i g h e r in CD during hypercapnia. The baroreflex g a i n of t h e m i n i m a l central venous pressure was d i m i n i s h e d in CD d u r i n g h y p e r o x i a a n d hypercapnia. F r o m the r e s u l t s o b t a i n e d in t h e s e experiments we c o n c l u d e t h a t the c e n t r a l n e r v o u s output to the effectors reflex response pattern is altered in some parameters depending from the afferents of different receptors.

MECHANISMS OF REGULATION AND COUNTERREGULATION OF BLOOD PRESSURE FOLLOWING MESENTERIC TRACTION Ch.-F. Wolf, A. B r i n k m a n n , L . D u n t a s , J. W i e b e r n e i t , F.S. Keck, W. O e t t i n g e r a n d W. D. S e e l i n g

Institut d e r HUB,

f o r P h y s i o l o g i c , Med. F a k u l t ~ t ( C h a r i t Y ) H e s s i s c h e Str. 3-4, D - O - i O 4 0 B e r l i n

Mesenteric traction (MT) d u r i n g a b d o m i n a l surgery c a u s e s l o w e r e d b l o o d p r e s s u r e f o r i0 t o 30 m i n u t e s a n d e v e n t u a l l y a f a c i a l flush. T h e s y m p t o m s a r e a c companied by a release of prostacycline. (PGI.2) following the mechanically reduced local intestlnal blood circulation. B l o o d p r e s s u r e is r e c o n s t i t u t e d a f t e r 30 min. B e f o r e m a j o r a b d o m i n a l surgery (15 min) 25 o u t of 51 p a t i e n t s r e c e i v e d 400 m g I b u p r o f e n (IBU) i.v., 26 p a t i e n t s (Controls; C) r e c e i v e d a placebo. Apart from the hemodynamic parameters b l o o d p r e s s u r e (MAP (mm/Hg) a n d h e a r t r a t e (i/min), we determined plasmaconcentrations (pc) of r e n i n (ng/l), A N P (nM), a l d o s t e r o n e (pg/ml) a n d 6 - K e t o PGFIa (stable PGI2-metabolite; pg/ml) radioimmunologically. D a t a are g i v e n as m e a n • at 0, 5 a n d 30 m i n a f t e r MT. In C M T c a u s e d e n h a n c e d p c of 6-Keto-PGFla (65• ~ 2192• ~ 1195• and lowered blood pressure (118• ~ 95• ~ 110• T h e i n c r e a s e d p c of r e n i n a f t e r M T ( 1 5 . 1 • ~ 41.2• i n d u c e d e n h a n c e d p c of a l d o s t e r o n e ( 1 7 . 3 • 4.5 ~ ~ 1 6 4 . 9 • N o n e of t h e p a r a m e t e r s a b o v e w e r e a l t e r e d in t h e I B U - p r e t r e a t e d group. A N P p l a s m a c o n c e n t r a t i o n s d i d n o t r e a c t to M T n e i t h e r w i t h out (0.31• ~ ~ 0.26• nor with IBU-pretreatment (0.25• ~ ~ 0.32• n.s. vs. C). We c o n c l u d e t h a t i t h e i n h i b i t i o n o f t h e v a r i a t i o n of b l o o d p r e s s u r e b y I B U i n d i c a t e s an a c t i o n d e p e n d e n t to t h e cyclooxygenase, ii t h e r e c o n s t r u c t i o n of blood pressure is p r e d o m i n a n t l y mediated by the RAAS cascade, either directly or indirectly activated by prostaglandines. Medizinische Klinik und Poliklinik, Abt. I n n e r e Med. I, U n i v e r s i t ~ t Ulm,

Ulm/Donau

R 115 190

192

SPATIAL DISTRIBUTION OF OXYGEN SUPPLY UNITS IN HEART AND SKELETAL MUSCLE AND THEIR REGULATORY SIGNIFICANCE H6per, J., Kessler, M. Institut flit Physiologie und Kardiologie, Universit~t Erlangen-Nfirnberg, Waldstr. 6, D-8520 Erlan~en, German)' Systematic investigations of capillary blood flow and oxygen supply to tissue performed in heart and skeletal muscle reveal, that heterogenons distribution patterns of flow exsist in all muscle tissues. Such patterns are induced by the hcterogenous perfusion rates through terminal arterioles thus leading to formation of individual capillary supply units. Approximately 12 15 individual supply units form a cooperative group which lies within a distribution pattern of I-ibO2 between 50 - 100 % saturation and of pO2 between 5 - 80 m m Hg. The patterns of capillary flow, HbO2, tissue pO2 and MbO2 are rather homogeneous within eaeh individual capillary supply unit. Several physiological phenomena give an explanation for the existance of such functional structures: 1.Via the channels of high flow supply units with low peripheral resistance the mobile oxygen reserve (approx. 50 % of total blood flow) can be perfused more easily through the circulatory system (reduced energy consumption) under resting conditions. 2.The oxygenation of the "early warning" supply units with low flow channels lies within the 5 mmI-Ig range of the signal oxidases. These oxygen sensors can cause local regulatory changes in the distribution of flow patterns in between individual supply units of regional groups of eoopcrative supply units, when oxygen demand increases or PAO2 and]or microflow decrease. When intraeapillary HbO2 is monitored in beating heart or skeletal muscle of patients by recording 400 - 500 different values with light sensors moved across the tissue, integrated HbO2 gradients or gradient fields of regional groups of cooperative supply units are obtained. For a precise tissue monitoring in patients it is of utmost importance to analyze the functional state of tissue which is defined by the availability of microflow reserve, levels of oxygenation of cooperative supply units and the quantity of diminished 02 uptake rate, manifested by protective hypokinesia of myocardium. Additional spectrometric measurements of further local parameters such as hemoglobin concentration, oxygen content, oxygen uptake rate and microflow enable a multieomponent analysis of tissue supply.

INHIBITION OF N a + - H § EXCHANGE IN PORCINE BRAIN CAPILLARY ENDOTHELIAL CELLS BY EIPA AND HOE 694

A confocal laser scanning microscope and the pH sensitive fluorescence indicator 2',7'-bis-(2-carboxyethyl)-5(and-6)-carboxyfluorescein (BCECF) were used to monitor changes of the intraceilular pH (PHi) of capillary endothelial ceils isolated from porcine brain. Since commercially available eonfocal microscopes so far only provide a one-wavelength-excitation mode the absolute calibration of the intracellular pH was not possible. However, qualitative analysis could be performed. In the experiments endothelial cells freshly prepared as well as cells held in primary culture for up to 8 days were used. The mean fluorescence of small cell clusters (5to 50cells) was analysed. The brain capillary endothelial cells were acid-loaded using an NH4C1 prepuls. In a bicarbonate-free medium intraceliular pH recovered after removal of NH4CI from the external medium within 3 to 7 minutes. The pH i did not recover when external Na + was replaced by the organic cation tetramethylammonium. The Na + dependent pH i recovery could be completely blocked by 1 to 10/zM 5-(Nethyl-N-isopropyl)amlloride (EIPA). The benzoylguanidine derivative 3-methylsulfonyl-4-piperidinobenzoyl) guanidine hydrochloride (HOE 694) also showed to be a potent blocker of Na + - H + exchange. The effectiveness of this substance is comparable to the inhibition potency of EIPA. 1 to 10 #M HOE 694 inhibited pH i recovery completely, whereas 0.1 #M only caused a partial block of Na + - H + exchange.

191

193

PREVENTION OF CALCIUM AND CHOLESTEROL-OVERLOAD IN DISTAL BRANCHES OF THE ART. MESENTERICA SUP. OF SPONTANEOUSLY HYPERTENSIVE RATS (SHRs) BY NITRENDIPINE. M. Frey, F. Thimm, G. FleckensteinGrin, a. A. Fleckenstein. SHRs develop severe nodal arteriosclerotic lesions in the distal branches of the A.mesent.sup.during 21 months.The lesions could be classified according to the WHO stages I-III:Whereas the Ca content rose within 21 months from 1.2 (• g/kg dry weight to 38.3 (• g/kg, total cholesterol did augment from 4.5 (• g/kg d.w. to 9.2 (• g/kg d.w. However, with the help of nitrendipine (300 mg/kg/die) both the arteriosclerotic lesions and the changes in Ca as well as free and total cholesterol were completely prevented, Ca being most sensitive.

ADENOSINE-INDUCED PULMONARY VASOCONSTRICTION IS ATTENUATED BY THE ADENOSINE UPTAKE-INHIBITOR NITROBENZYLTHIOINOSINE (NBTI) IN ISOLATED PERFUSED RAT LUNGS V.Hiller, J.Bong, H.J.D6ring

50 4o

30

2-----

Content in glkg dry arterial t i s s u e weight

j Calcium -_.___~ Total Cholesterol ~ Free Cholesterol

n=21

Tn=17

/v'o /es/oz7 v/Lc/b /e / rl=15

n=17T I0

n = 20._~_. . . .

~ 4 r t e r / o - 0 healthy [ II s sLage." cleroZ/'c distal arteriosclerotic

Ill white segments

0 (no lesion) ~]~,~

Study Group for Calcium Antagonism, Physiol.Inst., Univ. Freiburg, Hermann-Herder-Str. 7, 7800 Freiburg

A. Schmid*, R. Popp*, H.C. Englert1, H.J. Lang1, W. Schoh 1, B.A. Sch61kens1

*Max-Planck-Institut far Biophysik, Kennedyailee 70 D-6000 Frankfurt/Main 70, F.R.G. 1Hoechst AG, Pharmaforschung, D-6230 Frankfurt/Main 80, F.R.G.

The effect of adenosine on pulmonary vessels is discussed controversely (Roepke et al., Exp Lung Res 17" 25, 1991): vasodilation as well as vasoconstriction Or both reactions dose-dependently, are reported. Our investigations were done in order to obtain further information about the obviously divergent actions of adenosine. Isolated rat lungs were negative-pressure ventilated (4 cm H20 endexpiratory / 13 cm H20 endinspiratory) and perfused with modified KREBSHENSELEIT solution with constant pressure (12 cm H20 ). Perfusion flow (PF) and air flow (AF) were measured continuously. Tidal volume (TV), dynamic compliance (Cayn) and lung conductance (G0 were calculated simultaneously with the data acquisition by means of a PC. Adenosine in a low concentration (100 nM) showed neither constriction nor dilation of pulmonary vessels and had no influence on the airway parameters. Higher concentrations (0.1 and 1 mM) decreased PF by 13% and 42% ; TV: 5% and 26%; C a n: 5% and 23%; GI: 10 and 37%. In contrast, N6-cyclopentyladenosine (CPA), a high-affinity ArAgonist (KD = 0.48 nM) showed no significant effects at concentrations of 10 pM to 100 nM and only a slight increase of PF with 10 and 30 #M. In adition, CPA, applied during preconstrietion with K + (20mM) and additional perfusion with forskolin (1/~M) (to raise intracellular c-AMP levels) exhibited no effects neither on PF nor on pulmonary mechanics. Preconstriction with K + alone, however, resulted in an increase of PF by 12% and 26% at the high CPA-eoncentrations of 10 and 30/zM without affecting pulmonary mechanics. Perfusion of NBTI (5 #M) attenuated the flow-decreasing effect of adenosine (1 mM) by about 80 % while the reduction of TV, Cdyn and G 1 was slightly intensified. There is evidence that adenosine-induced pulmonary vasoconstriction is not mediated by At-receptors. The vasodilation seen at high CPA concentrations may be due to an unspecific stimulation probably via A 2receptors. Our results indicate that adenosine may cause pulmonary vasoconstriction by an intracellular mechanism, possibly via P site. Physiologisches Institut der Universitat Freiburg, Hermann-Herderstr.7, D-7800 Freiburg

R 116 194 EFFECT OF DIFFERENT CA2+-ENTRY BLOCKERS ON VASCULAR TONE IN NEWBORN PIGLET - IN VITRO AND IN VIVO STUDIES R. Bauer, B. Louangsay, T. Graser, R.Schaller, U. Zwiener

196

Recently a growing awareness of the significance of hemodynamics in the pathogenesis of perinatal brain disorders has stimulated interest in the definition of factors which regulate brain perfusion during the perinatal period. Furthermore it has become apparent that regulation of cerebral blood flow is different in neonates than in adults. We investigated the influence of two types of Ca2+ entry blockers (1,4dihydropyridine derivative nimodipine and difluorinated piperazine derivative fiunarizine) on the vascular tone in vessel preparations (internal carotid artery and femoral artery) from 17 normal weight (NW) and 16 intra-uterine growth retarded (IUGR) newborn piglets (age:14-39 hours). The both Ca2+-entry blockers showed a selective dilating effect on the brain vessels (p < 0.05) which is also known in adults. The dilating effect was more effective by use of nimodipine in comparison to that by use of flunarizine (p < 0.01). The both animal groups investigated showed similar results. But in all cases the drug concentrations had to be used were much higher (by a factor of 10 to 100) than usual used in adult vessel preparations. In a second study the effect of nimodipine on the cerebral blood flow (CBF; measured by 133Xenon clearance) was studied in four IUGR piglets (the animal group with the most effective dilation response). We found a significant increase of CBF in the white matter by 70 % (p < 0.05) after administration of ulmodipine (1 [xg/kg i.v.) and but an only transient CBF elevation in the gray matter. Thus, these results suggest that there are functional voltage-dependent Ca 2+ channel receptors in the newborns, but there are important differences in sensitivity between the types of Ca 2+ channel receptors and in comparison to the known effectiveness in adults.

Hepatoma cells (Hap G2) are known to produce and secrete erythropoietin (Epo) in dependence on the oxygen partial pressure by an unknown oxygen sensing m e c h a n i s m where the participation of a heme protein is suggested (Goldberg et al., Science 242:1412-1414, 1988). For detection of heme proteins in these cells a photodiodespectrophotometer scanning a wavelength range between 400 and 650 nm (MCS 210, Zeiss) was adapted to a microscope by a light guide. Hap G2 cells grown as multicellular spheroids were placed in a superfusion chamber on the stage of the microscope and were viewed by a 40* objective after light from a halogen lamp passed the tissue. Reduced versus oxidized spectra (red/ox) of single spheroids induced by hypoxia in the superfusion bath (PO2=3 Torr) revealed characteristic absorption peaks~at 426, 445, 550, 560 and 605 nm representing the different cytochromes of the respiratory chain. Poisoning with KCN (1,5 mM) provoked also a reduction of the cytochromes in the respiratory chain, but additional hypoxia evoked a further reduction at 426 and 560 nm, indicating an extramitochondrial cytochrome b. Only severe anoxia induced by dithionite (I mM) led to a complete reduction of this cytochrome b hinting to a high 02 affinity of this heme protein. Since Epo production in the Hep G2 spheroids could be stimulated by hypoxia but not by cyanide (42.7 mU/mg protein-h versus 12 mU/mg protein-h), a cyanide insensitive cytochrome with a high 02 affinity not belonging to the respiratory chain might be a possible candidate for t h e assumed heme protein responsible for the oxygen sensing process in these cells. Max-Planck-Institut fur Systemphysiologie, R h e i n l a n d d a m m 201, D-4600 Dortmund 1

Institut ftir Pathologische Physiologic der Friedrich-Schiller-Universit/h Jena, LiSbderstral]e 3, 0-6900 JENA

195 MEASUREMENT OF PERICELLULAR OXYGEN TENSION AND ITS RELATION TO ERYTHROPOIETIN PRODUCTION IN A HUMAN HEPATOMA CELL CULTURE (HEP G2) M. Wolff, W. Jelkmann Oxygen dependent phenomena like erythropoietin (EPO) production have been investigated in cell cultures exposed to varied ambient O2 concentrations. However, the actual PO2 at the monolayer level remained unknown thus far as it depends on several variables, i.e. number and specific 02 consumption of the cells and diffusion properties of the culture system. The human hepatoma cell line HepG2 has been shown recently to produce Epo. But both increased and decreased Epo formation were measured when the 02 concentration in the incubation atmosphere was lowered. In the present study the production of immunoreactive EPO was related to the pericellular PO2 as measured by solid state type polarographic microeleetrodes. In addition, the oxygen consumption of adherent HepG2 cells was determined in a gas tight, stirred system. When confluent HepG2 cultures (0.5x106 cells and 0.5 ml medium per cm2) grown in common polystyrol dishes were incubated in air with 5% CO2, the PO2 at the cell level dropped to < 1 mmHg within 1 h after medium replacement. Hereby, the EPO production was stimulated to 269_+9 mU/mg cell protein in 24 h (mean+SD of 4 subcultures). When the cultures were exposed to gases containing 50, 70, and 95% 02 the respective pericellular steady state PO2 was <1, 12+7, and 201_+_24 mmHg and the rate of Epo production was progressively reduced (68%+96, 38%+7, 32%5:8 of respective air controls). Further experiments showed that culture dishes with a gas permeable FEPteflon bottom provide equilibration between ambient and pericellular PO2. The Epo production in these dishes at PO2= 7 mmHg 02 was t525:33 mU vs 42-1-6mU/mg cell protein in 24 h at 127 mmHg. The striking discrepancy between gas phase and pericellular PO2 under conventional conditions is due to a misproportion between a high respiration rate of HepG2 cells (6.4 nmol O21min.mg protein with PO2>15 mmHg, 1.4 nmol O2/min.mg protein with PO2<12 mmHg) and a limited 02 supply through the culture fluid layer. Thus, HepG2 cells have proved to be a useful model of Epo production. In studies using metabolically active cells it should be considered that the oxygen tension available for monolayers may be substantially different from the ambient PO2. Department of Physiology, University of Bonn, Nul]allee 11, D- 5300 Bonn 1

SPECTROPHOTOMETRIC CHARACTERISATION OF HEME PROTEINS IN E R Y T H R O P O I E T I N P R O D U C I N G H E P A T O M A C E L L S A. GSrlach, W. Jelkmann, B. BSlling, G. Holtermann, H. Acker

197 IMMUNOHISTOCHEMICAL LOCALISATION OF CARBONIC ANHYDRASE IV IN RAT SKELETAL MUSCLE S. Sender, G. Gros and W.S. Sly Slow skeletal muscles of the rat possess cytosolic carbonic anhydrase (CA) III, whereas fast muscles do not. Moreover, functional studies suggest the existence of an extracellular membrane-bound CA isozyme which may help to buffer H + ions and may support CO 2 uptake by the blood. Apart from muscle, an extracellular membrane-bound CA has been found in other organs, such as lung and kidney and was identified as CA IV. In order to study whether the extracellular CA in skeletal muscle is also CA IV, we performed immunofluorescence studies on acetone-fixed cryosections of slow soleus and fast extensor digitorum longus (EDL) using a polyclonal antibody against rat lung CA IV. All sections showed clear capillary staining. This result was further verified by applying two different techniques: a) Serial muscle sections were incubated either with anti-CA IV or anti-yon Willebrand-faetor (a capillary marker) or were processed for endothelial ATPase reaction. There was a good correlation between antibody-marked and ATPase-stained structures. b) A rat hindlimb was perfused with anti-CA IV to bind to the supposed intracapillary enzyme. Muscles were then dissected and cryosections were processed for immunostaining. They showed fluorescence associated with capillaries only. We therefore conclude that there is an extraeellufar CA I V within rat skeletal muscles that is associated with the capillaries and probably accessible from the capillary lumen. There might be other membrane-bound CA isozymes that are not shown with this specific antibody. Zentrum Physiologic, Mad. Hochschule, 3 Hannover 61 Dept. Biochem., St. Louis Univ., St. Louis, USA

Rl17 198

200

pH MICROELECTRODES WITH SHORT RESPONSE TIME S. Schreiber, K. Mtickenhoff, Y. Okada and P. Scheid

FUNCTION OF GUINF.A PIG AND HUMAN NEUTROPHILS IN THE PRESENCE OF ADENOSINE S. Zahler, B.F. Becker, P. Raschke and E. Gedach

pH-microelectrodes with fine tips, filled with liqnid ion exchanger, have been used, but their response time is usually rather long due to high input resistance and capacitance of the electrode and the amplifier. We have lowered the time constant in two steps: (1) We have decreased the input capacity, using the guarding technique (G.T.), achieved by applying a low-impedance bootstrap potential to the outside of the glass wall of the electrode, which was coated with a highly conductive silver fluid, and to the housing of the amplifier; (2) we have, further, linearized the frequency characteristics of the system, using an analog signal processing network (S.P.N.) (Mt~ckenhoff et al., 1986, NTG-Fachberichte 93, 47-53, VDE-Verlag). The dynamic response of the electrode system was studied using a step change of pH at the electrode tip, with a mechanical system, that allowed to change the pH within 1 msec. Typical 90% response time (T90) of a double barrelled microelectrode with 1.5 ]am tip diameter and 500 Gf~ resistance, were as follows: 49 sec without G.T. or S.P.N.; 2.4 sec with G.T. (no S.P.N.); 0.09 sec with both G.T. and S.P.N. For a double-barrelled microelectrode of 20 ]am tip diameter and 10 G ~ resistance, the corresponding typical values were: 0.12 sec, 0.04 sec, < 0.003 sec. These electrodes can be used to record rapid pH changes in biological tissues. Physiologisches Institut der Ruhr-Universit~t Bochum, D 4630 Bochum (Supported by the DFG, MU 594/3-1)

In recent studies we have shown that the isolated working guinea pig heart, perfused with homologous neutrophils (PMN), is a suitable model for studying the role of PMN in post-ischamic reperfusion injury [Raschke et al. 1991, Z f Kardiol 80 (Suppl.3):14]. However, the physiological and pharmacological properties of guinea pig neutrophils have been poody characterized to date. Adenosine (Ado) accumulates during ischemia (in our experiments the coronary effluent levels during repeffusion are about 100 nM) and reportedly has marked effects on human neutmphils, enhancing chemotaxis and inhibiting radical production [Cronstein et al. 1983, J Exp Med 158:1160-1177 and Rose et al. 1988, d Exp Med 167:11861194]. Consequently, the action of Ado (0,1 - 1 /.~M) on the release of oxygen radicals (luminol-dependent luminescence), aggregation (change of light transmission in a cell suspension) and chemotaxis (immigration rate of PMN into a nitrocellulose filter in a modified Boyden chamber) was assessed, comparing PMN of both species. The experiments were performed in the absence and presence of the stimulant FMLP (0.01-1/~M). In all three assays, addition of Ado to guinea pig cells had no consistant effect, irrespective of whether FMLP was present or not. Similady, coincubation of PMN with adenosine deaminase in order to remove any endogenous extracaUular Ado did not alter the responses to FMLP. Furthermore, the adenosine agonists NECA and PIA were without effect on radical formation by guinea pig PMN. In the case of human neutrophils, however, Ado suppressed radical production, but did not influence aggregation or, surprisingly, chemotaxis. Summary: Neutrophils of the guinea pig are apparently not responsive to adenosine, in contrast to human PMN in which adenosine inhibits radical production. Under our experimental conditions a chemotactic effect of adenosine on human neutrophils could not be demonstrated. Physiologisches Institut der Universit&t M0nchen, Pettenkoferstr. 12, 8000 MQnchen 2

199

201

HEMOLYSIS INDUCES RELEASE OF ENDOTHELIN FROM RAT ERYTHROCY'I'ES S. Pritze, I. Weide, B.A. Peskar and Th. Simmet

INFLUENCE OF VENTILATION ON TRANSPORT OF ISOTOPIC OXYGEN MOLECULES TO TISSUES AT REST. M. K6nen, H. Heller and K.-D. Schuster The different steps of oxygen transport from inspiratory gas to tissues exert their specific isotopic effects on the molecules 1602 and 160180, as these have different molecular weights. Those pathways which are limiting oxygen transport will contribute most to the overall fractionation effect of respiration, which is the percentage difference of overall conductances of 160 2 and 1 6 0 1 8 0 . Assuming a simple model of respiration, previous measurements of fractionation effects between both isotopic species suggested that oxygen transport is not limited by diffusion up to work loads of 250 W (Heller, H., K.-D. Schuster: Pfl~igers Arch 415 Suppl.1 (1990): R67). The aim of this study is to quantify the influence of ventilation on the overall fractionation effect between 1602 and 160180 and to prove the model above by further experimental data. Experiments were performed on 6 healthy subjects breathing air at rest. The overall fractionation effect was measured by mass spectrometry. It rose with increasing ventilatory rate in terms of a linear relationship to alveolar oxygen partial pressure (r = 0.83, n = 35) with a slope of 0.006 % mmHg-1 and an intercept nearly zero. From these results we concluded: (i) reducing respiration to an oxygen transport from alveolar space to tissues, the conductance is 0.89 % higher for 1602 than for 160180. This figure is independent of ventilatory rate. (ii) Since this figure is expected to depend on blood flow, diffusion and metabolic rate, it could become a useful parameter for studying these processes in vivo.

Endothelin (ET)-I is a potent vasoconstrictor peptide that has originally been isolated and sequenced from cultured porcine endothelial cells. Only recently we have shown that the acute oleic acid (OA)-induced lung injury, a commonly used experimental model of the adult respiratory distress syndrome, is accompanied by an increase in immuncreactive (ir) ET in rat plasma (NSAP 343 1991 344). In order to exclude blood cells as a possible source o1 origin, OA (0.5, 2.0 or 8.0 Id) was added to 1 ml of EDTA (5 mM) -anticoagulated whole rat blood incubated at 3TC for various time intervalls in vitro. Surpdsingly, after solid phase extraction radioimmunological analysis revealed a time-dependent and OA concentration-dependent generation of ir-ET which reached a plateau at 30 min and which remained stable up to 60 min (e.g. OA 8 itl/ml: 1284 + 295 pg/ml at 30 min; 1343 + 178 pg/ml at 60 min). Addition of OA to whole rat blood induced hemolysis as assessed by analysis of the hemoglobin (Hb) concentration in the plasma samples. Interestingly, free Hb correlated with the amount of ir-ET detected in the samples (r=0.6097, n=63, P<0.001) indicating that the ir matedal might originate from erythrocytes. This could be confirmed when whole rat blood was fractionated on Percoll gradients. The cells were homogenized with an ultrasonic device in phosphate-buffered saline and the homogenates were incubated at 37~ for up to 60 min. IroET could only be detected in the erythrocyte fraction but not in leukocytes containing all white blood cells nor in platelets. Fudhermore, these experiments showed a time-dependent increase of irET from 394 + 135 pg/5xl 09 erythrocytes directly after homogenization to a peak of 944 + 174 pg/5xl09 erythrocytes at 30 min indicating an at least partial enzymatic generation of ir-ET from a precursor molecule, most likely big ET. When hemolysis was induced in erythrocyte suspensions by NaCI of different concentrations (0.9%0.2% (wt/vol)) the concentration-dependent hemolysis was paralled by an increase of ir-ET after incubation at 37"C for 30 min..It could be demonstrated that the RIA developed in our lab shows no significant cross-reaction with rat Hb. Preliminary raverse phase HPLC analysis using an acetonitrile~ater gradient 30*/,-60% (vN) containing trifluoroacetic acid 0.1% (vh~) revealed that the ir material coelutes with the retention times of synthetic ET-1, ET-2, and possibly with big ET-1, while very small amounts migrated with ET-3. This is the first report on ET generation by blood cells. These findings might be of considerable importance for pathophysiological states involving hemolysis. Thus, apart from the indirect vasoconstrictor Hb which interacts with the vasodilatory NO/EDRF, erythrocytes also contain the direct vasoconstrictor ET. Dept. of Pharmacology, Ruhr-University, POBox 102148, D-4630 Bochum 1

Physiologisches Institut I der Universit/k Bonn, NussaUee 11, D-5300 Bonn 1

R 118

202

204

VENTILATORY PATTERN RESPONSES IN BORDERLINE HYPERTENSIVE MEN EVOKED BY ALMITRINE BISMESYLATE R. S & n c h e z , W. g u l e s , C. L e d d e r h o s , H. B r a u e r

RESPONSE TO CHANGING pH OF MEDULLARY NEURONS IN THE NEONATAL RAT Y. Okada, K. Mtickenhoff and P. Scheid

Almitrine bismesylate is an effective and specific stimulant of the peripheral arterial chemoreceptors (Pach). It is well documented that this drug causes augmented minute ventilation a n d tidal volume i n both young mammals as well in young humans being under normoxic conditions. Response of breathing rate is not significant. The aim of this work was to determine the differences of reactions of some parameters of external respiration between normotensive (13) and hypertensive (16) young persons after oral administering o@ a total dose of lO0 mg almitrine bismesylate. We hypothesized that hypertensive young males have no reactions of timing and drive components in the regulation of ventilation or at least have only very weak responses of these parameters because they show an augmented activity of Pach even under normoxic conditions The hypertensive group revealed a h i g h e r expired minute ventilation and a higher mean inepiratory flow under basal conditions. They did not show a significant reaction of these parameters after oral application of almitrine. In contrast to that the normotensive group reacted with a significant elevation of both parameters. Both groups did not show any reaction of the timing c o m p o n s n t s ( i n s p i r a t o r y time, e• time and fractional inspiratcry time). Our results lead to the conclusion that the bordeline hypertensive group has an increased resting drive of the Pach under normoxic conditions but they did not react significantly to oral application of almitrine.

The response of neurons in the ventrolateral medulla to changes in pH was investigated using the neonatal rat brainstem-spinal cord preparation. The isolated bralnstem-spinal cord preparation was continuously superfused with mock cerebrospinal fluid (CSF) at 27~ Control CSF was equilibrated at a CO 2 fraction (Fco~) of 0.02 and had a pH of 7.8. Respiratory activity was monitored by recording the compound phrenic activity from a C4 ventral root. Single-unit activity was recorded extracellularly from neurons in the ventrolateral medulla, using glass microelectrode. Phrenlc activity and neuronal activity during superfusion with control CSF were compared with activities obtained during superfusion with acidic CSF (pH 7.2, Fco2 0.08). The following types of neurons were recorded: (1) tonic neurons and (2) phasically bursting neurons, both without relationship to the respiratory cycle; (3) tonic neurons with a short inspiratory pause (tonic expiratory neurons) and (4) phase-spanning tonic neurons with inspiratory augmentation (tonic inspiratory neurons). The following responses were observed when changing from control to acidic CSF: Frequency of the phrenic activity increased. Most of the neurons of all types were suppressed; only some of the tonic inspiratory neurons were stimulated. Our experimental conditions do not allow to decide whether the pH effects observed constitute a direct pH sensitivity of the neurons or are due to pH sensitivity of their afferents. Physiologisches Institut der Ruhr-Unlversit~t Bochum, D 4630 Bochum

Institut for Physiologie der Universitit Greifswald Rubenowstr.3, 0 - 2 2 0 0 Greifswald

205

203 QUANTITATIVE EVIDENCE OF Na+/GLUCOSE SYMPORT AND N a + / K ACTIVITY IN PNEUMOCYTE TYPE II CELLS M.F. Horster, D.Dobrovic-Jenik and M.Sone

+ -

ATPase

Fluid absorption from the alveolar space in vivo is dependent on glucose in the alveolar fluid (1). To further characterize the cell type and the mechanism involved in alveolar solute transport, pneurnocyte type II cells (A #cells) were isolated from rat lungs in a pure preparation and grown to confluent monolayers in culture. The apical plasma membrane transport of ,~- methyl - D - glucoside (,,-MG), a nonmetabolizable hexose, was measured on day 4 in confluent A II cultures, and Na§ K§ activity was quantitated by enzymatic coupling of ATP hydrolysis to fluorescent NAD appearance (2). A//ceils in monolayer culture were identified by the tannic acid/polychrome stain of lamellar bodies (3). Sodium/glucose sympert of alveolar type II cells (apical rL - MG transport at 150 mM Na+ in the uptake medium) was (nmol/60 mini10 s cells) 50.2 t 5.4 (n=137 cultures). Uptake of glucose (14C~MG) in the presence of o~-MG(1.0 mM) and Na § (150 mM) reached steady-state values at 60 min; in the absence of Na§ mM), glucose uptake was less than 5% of the steady state value at all times between 10 and 90 min. When Na + in the uptake medium was lowered to one third (48 mM), glucose uptake changed less than 20%, while changes of Na§ 2 to 48 mM increased glucose uptake by a factor of about 20. The Na§ symport was substrate-dependent at concentrations of 0.1,1.0, and 5.0 mM of c~-MG (p<0.05). Initial glucose uptake (10 min)differs greatly (p<0.01) at these substrate concentrations, between 1.5 and 26.5 mM (n=209).The specific inhibition of glucose (14C-~-MG) uptake by phlodzin (10 -s M) in the medium, in the presence of c~-MG (1.0 mM) and of Na+ (150 mM ) was from 44.5 -+5.6 (n=38) to 11.8 -+ 2.2 (n=38) nmol/108cells / 60 min ( p<0.01); phloretin (10.5 M) in the apical medium had no effect on the Na + dependent glucose transport. The Na§ -K§ activity of All cens (n= 7 experiments) was 41.5 -+9.2 pmol/min/mg protein. These data characterize the alveolar epithelium by the sodium/glucose symport of its alveolar type II cells which may contribute to alveolar fluid balance and 1o the substrate supply for the synthesis of phosphatidylcholine. In addition, the Na § K§ ATPase activity of A II cells was quantltated and shown to be similar to that of pars recta cells of the renal proximal tubule. (1) BassetG, Crone O,Saumen G, J.PhysioL384:925-345,1987 (2) O'Neill RG, Dubinsky WP, Am.J.PhysioL 247:C814-C320,1984 (3) Mason RJ, Walker SR, Shields BA, Henson JE, Williams MC, Am.Rev.Respir.Dis.131:786-788,1985. Physiologisches Instltut der Unlversltfit Mfinchen,Pettenkoferstr.12, 8 Mfinchen 2

P L A S M A LEVELS OF DSIP IN INFANTS IN THE FIRST Y E A R OF LIFE AND SIDS RISK S. Scholle, G. Zwacka, R. Ekman, S. Glaser

The sudden infant death syndrome (SIDS) is the m o s t frequent caus e of death in the first year of l i f e a f t e r t h e n e o n a t a l p e r i o d . A n a g e dependent dysregulation of t h e r e s p i r a t o r y s y s t e m d u r i n g sleep may be responsible for the u n e x p l a i n a b l e death. In searching for abnormalities in respect to SIDS delta-sleep-inducing peptide (DSIP), a r e g u l a t o r y peptide with sleep promoting actions, was investigated in the first year of life in 4 groups of children: i. preterm infants (n=28), 2. infants w i t h a high mean apnea d u r a t i o n p o l y s o m n o g r a p h i c a l l y evaluated (n=31), 3. h e a l t h y f u l l - t e r m i n f a n t s (n=37) a n d 4. s i b l i n g s of S I D S - v i c t i m s (n=27). DSIP was r a d i o i m m u n o a s s a y e d in plasma. All infants were also p o l y g r a p h i c a l l y i n v e s t i g a t e d d u r i n g sleep. T h e r a t i o b e t w e e n quiet sleep and active sleep was determined. There was no age dependence of DSIP in the first year of life but an increase of the ratio q u i e t / a c t i v e sleep depending of maturity. The level of DSIP in healthy full-term infants was significantly higher (p<0.05) (median: 1.60 ng/ml, interquartile range: 0.64 ng/ml) than in p r e t e r m s (1.35; 0.33) and in infants with a h i g h m e a n apnea duration as a sign of a r e s p i r a t o r y d y s r e g u l a t i o n (1.31; 0.32). The results give a hint at the distinctive characteristics of the g r o u p s s e l e c t e d in r e l a t i o n to a p o s s i b l y e n h a n c e d risk of SIDS. Universit~ts-Kinderklinik, Jena.

Kochstr.

2,

0-6900

R 119 208

206 9 O BODY WEI(}Yr PATIO IS A ~ F ~ u I ~ O ~ O N

P A R A M ~ I ~ OF

W. Mueller-Klieser and P. Va%~-i

~he o~!~J~r~tiQn status of ~ mali~wk~ncies has been reviewed rec~rfcly by V a ~ e l et al. (CarcDerl~es. 49, 6449, 1989). ~]3e data cc~piled illustrate a large variability in the e~genaticn of patient t~Drs, yet tissue c~ggen tensicrs (pO2) were generally less in t~mDrs than in the no~mal tissue at the site of tLmDr growth. A ~ finding of recent studies cn tsmors in patients is the fact that c~ygen tensicrs in many, if not the majority of h ~ a n tumors are h i ~ than previous data derived from rodent tumors 07at~l et al., Cancer Res. 41, 2008, 1981). For crumple, a median p02 as high as 62 mmHg %~s registered in a bulky, stage T3 breast cancer. Moreover, c~r ter~icns did not correlate with histological grading er clinical staging of the canoers, whereas several st~iies cn rodent t ~ o r s have des~nstrated a negative correlation between tumor tissue pO2 and b x ~ r weight (Vaupel et al., 1981, s.a.). ~ suggests that tumor oxygenaticn may be evaluated based on the tumor weight to body w~ight (tw/hw) ratio to enable a ccmparisc~ between measur~m~nt~ in patients and in rodents. Mean pO2 values in rodent and h ~ n t~mDrs of various sizes were c~mpiled from the literature. A clear-cut negative oorrelation was ~ between the mean tissue p02 and the tw/bw ratio in all rodent malignancies investigated. Ox!~jen tensicr~ in human t~nors fell within this correlation cnly if advanced, bulky growth stages were ccrsidered. Early growth stages deviated ~cmsiderably from the correlation obtained for rodents. ~ u s , the overall c~r status of late stage malignancies in patients and of rodent t ~ D r s can be predicted from the tw/bw ratio. Rodent t ~ o r s as they are aooessible for pathq~hysiological investigaticns therefore represent cnly advanced stage malignancies of patients with r ~ to their metabolic milieu. Institute of Fnysiology and Pathq0hysiology, Mainz, Saarstrasse 21, D-6500 M~inz, Germany

University of

Hypoxia seems to be an important pathogenetic mechanism underlying the development of diabetic neuropathy. This disease predominantly affects sensory nerve fibres. In the present study, we investigated whether the differences in diabetes-related functional deficits of sensory and motor fibres can be explained by nerve hypoxia. Male Wistar rats (300-400 g) were anaesthetized with urethane for a laminectomy to expose the cauda equina. Excised ventral and dorsal roots were transferred to a threechambered plexiglass organ bath which allowed recordings of compound nerve action potential (CNAP) and extracellular d.c. potential. The isolated roots were superfused at 36~ with HEPES-buffered solutions containing 2.5 or 25 mM glucose. After a control period, the roots were exposed to hypoxia for 30 rain (Po2 < 3 mm Hg) and changes in CNAP and extracellular d.c. potential were recorded. In both ventral and dorsal roots incubated in 2.5 mM glucose, sensitivity to hypoxia and posthypoxic recovery were similar: A negative shift of the d.c. potential and a decrease in CNAP amplitude during hypoxia were followed by a rapid restoration after reoxygenation. Thereby, normalization of the d.c. potential was preceded by a transient positivity. In contrast, a strong difference in the sensitivity of ventral and dorsal roots to hyperglycaemic hypoxia was observed: In 25 mM glucose, ventral roots showed a marked resistance to hypoxia, whereas the response of dorsal roots remained unaltered. After hypoxia in high glucose, both ventral and dorsal roots failed to show a functional recovery. In conclusion, the different behaviour of sensory and motor fibres during hyperglycaemic hypoxia supports the hypothesis that hypoxia plays an important role in the pathogenesis of diabetic neuropathy. Supported by the DFG (SFB 220). Physiologisches Institut der Universitat Miinchen, Pettenkoferstr. 12, W-8000 Miinchen 2

209

207 THE I S C H E M I C THRESHOLD OF I N H I B I T I O N OF TEIN S Y N T H E S I S AND OF F A I L U R E OF ENERGY ING M I D D L E C E R E B R A L A R T E R Y O C C L U S I O N IN G. Mies, S. Ishimaru, Y. Xie, and K.-A.

DIFFERENCES IN SENSITIVITY TO HYPERGLYCAEMIC HYPOXIA OF ISOLATED RAT VENTRAL AND DORSAL ROOTS U. Schneider and P. Grafe

CEREBRAL PROSTATE F O L L O W RAT. Hossmann

The i s c h e m i c t h r e s h o l d s of p r o t e i n synthesis and energy state were d e t e r m i n e d 1 h, 6 h, a n d 12 h after m i d d l e ~ e r e b r a l a r t e r y (MCA) o c c l u s i o n in rats. Local c e r e b r a l b l o o d flow (CBF) and c e r e b r a l p r o t e i n synthesis (CPS) were m e a s u r e d by means of double t r a c e r a u t o r a d i o g r a p h y , and local ATP content by means of substrate-induced bioluminescence. Evaluations in a l i g n e d images were c a r r i e d out in cerebral cortex i p s i l a t e r a l to the M C A - o c c l u s i o n at the r e s o l u t i o n level of an image p i x e l of 75 p~n x 75 ~m. The ischemic t h r e s h o l d was d e f i n e d as the flow rate at w h i c h 50% of the p i x e l s e x h i b i t e d complete m e t a b o l i c suppression. One hour a f t e r M C A occlusion, the t h r e s h o l d of CPS was 55.3 • 12.0 m l / 1 0 0 g / m i n and that of e n e r g y failure 18.5 • 9.8 ml/100g/min. A f t e r 6 h (52.0 • 9.6 ml/100g/min) and 12 h (5.6.0 • 6.5 ml/100g/min) M C A occlusion, the t h r e s h o l d of CPS did not change but that of e n e r g y failure i n c r e a s e d s i g n i f i c a n t l y to 31.9 • 9.7 m l / 1 0 0 g / m i n (p < 0.05 c o m p a r e d to 1 h ATP t h r e s h o l d value; m e a n • SD) at 12 h f o l l o w i n g M C A occlusion. Therefore, the t h r e s h o l d of energy failure g r a d u a l l y a p p r o a c h e d that of p r o t e i n synthesis. Our results suggest that w i t h i n c r e a s i n g d u r a t i o n of ischemia, s u r v i v a l of b r a i n tissue is d e t e r m i n e d b y the high t h r e s h o l d of p e r s i s t i n g i n h i b i t i o n of p r o t e i n synthesis and not by the m u c h lower one of acute energy failure. If the ischemic p e n u m b r a c o r r e s p o n d s to the region in w h i c h CPS is s u p p r e s s e d and e n e r g y state is preserved, it follows that the size of the pen u m b r a d e c r e a s e s with the d u r a t i o n of ischemia. M a x - P l a n c k - I n s t i t u t ffir n e u r o l o g i s c h e Forschung, Abt e i l u n g f~r e x p e r i m e n t e l l e Neurologie; G l e u e l e r Str. 50, D-5000 K~in 41

CONTINUOUSREGISTRATIONOF DISSOLVEDGLUCOSEIN VITROANDIN THE SUBCUTANEOUSTISSUEINMAN. F. S. Keck, C. Meyerhoff and W. Kerner A device for the continuous registration of dissolved glucose was obtained by combining the microdialysis method (L6nroth et al., Am. J. Physiol. 253 (1987) E228-231) with a flow chamber, which measures glucose concentration amperometrically using the glucose-oxidase-H202 method. For that purpose, the outlet of a commercially available needle-type microdialysis probe (36ram x 0.6mm, polycarbonate-polyether membrane, molecular cut off 20,000 Dalton) was conducted to the flow chamber of the "Glucosensot Unitec Ulna" (Horm. recta. Res. 22 (1990) 382-384). In in vitro experiments, the glucose yield in the microdialysis outlet was linearly related to the concentration of the glucose standard solution (at least up to 300 mgldl, r > 0.94) and to the dialysing membrane area (varied by the factor 5.3, p = 0.99). The response time to instantaneous glucose concentration changes was hyperbolically related to the microdialysis perfusion velocity (varied between 1 and 25 ul/min) as was the relative recovery in the outlet. The microdialysis glucose yield was further linearly related to temperature changes (between 14 and 44 ~ of the standard solution by 1.32%/grd C (r = 0.99). When the glucose content in the microdialysis perfusion fluid was increased stepwise, unknown glucose concentrations could be determined by the corresponding increases and decreases of the glucose content in the microdialysis outlet (error < 5%). In in vivo experiments in type I diabetic patients, the microdialysis probe was inserted subcutaneously at the abdominal wall and the plasma glucose concentration was altered exogenously by means of the artificial pancreatic I~ cell (BiostatorR). Again a hyperbolic relationship between the relative recovery rate and the microdialysis perfusion velocity was found. When plasma glucose levels were clamped at about 80, 200, and again 80 mg/dl over 2 hrs. each in 5 diabetic patients, the subcutaneous glucose signal followed plasma glucose concentrations with a lag time of about 30 min (0.88 < r < 0.98), half of which has to be assigned to biological factors (e.g. exchange time between compartments). The sensitivity of the subcutaneous device can be calculated to 70 mg/dl plasma glucose content. Abt. Innere Medizin I, Medizinische Klinik und Poliklinik der Universit~it Ulm, Robert-Koch-Str. 8, D-7900 Ulm

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212

31 P NMR SPECTROSCOPY AND LASER DOPPLER FLO'k,~ETRY OF RAT

THE TOPOGRAPHICAL DISTRIBUTION OF EMG SPECTRAL PARAMETERS IN T H E H U M A N M A S S E T E R MUSCLE DURING DIFFERENT FUNCTIONAL STATES Schumann, N.P., Scholle, H.Ch. and Anders, Ch.

BRAIN AFTER GLOBAL ISCHEMIA O. Kloiber, M. H6hn-Berlage, T. Miyazawa, K. Beckhorst and K.-A. Hossman The effect of hemodynamie factors on the metabolic recovery of the brain after 30 min global isehemia was studied in rats submitted to bilateral carotid and vertebral artery occlusion (4-vessel-occlusion). ATP, PCr and pH were measured in a 4.7 T magnet by 31 P NMR spectroscopy, and blood flow by laser Doppler flowmetry. Vascular occlusion resulted in instantaneous arrest of cerebral blood flow. EEG became isoelectric after 15 • 7 s (mean + SD), terminal depolarization occurred after 1.4 • 0.4 min. Within 6 min of ischemia ATP and PCr disappeared~ Pi increased 13-fold and PH i declined by 0.5 • 0.08 units. In contrast to these stereotyped ischemic events, p0st-ischemic recirculation and recovery depended on the end-ischemie blood pressure. The recirculation delay (return of blood flow to control) ranged between 30 s and 29 min and correlated inversely with the end-ischemic blood pressure (r=0.81). The recovery time (return to 50% of control) of PCr ranged between 1 and 21 min and correlated positively with the reeirculationd delay (r=0.95). Despite this scatter, PCr returned to control in all but one animal within 3 h. ATP recovery also depended on the recirculation delay but it did not return to control when this delay was longer than 8 min. These data demonstrate that in the 4-vessel-occlusion model of rat post-ischemic recovery of energy metabolism depends strongly on the quality of blood recirculation which, in turn, is a function of end-isehemic blood pressure. Analysis of post-ischemie molecular processes in this model should therefore consider individual hemodynamics, particularly under conditions of drug therapy. Max-Planck-Institut f~r neurologische Forschung, Abteilung for experimentelle Neurologie, Gleueler Str. 50, D-5000 K61n 41

Electrophysiological, morphological and enzymeh i s t o c h e m i c a l r e s u l t s of t h e h u m a n m a s s e t e r m u s c l e refer t o a d i f f e r e n t i a t e d a c t i v a t i o n of separate motor r e g i o n s d e p e n d i n g o n the i n t e n d e d movement of t h e m a n d i b l e . To d e m o n s t r a t e and e v a l u a t e the t o p o g r a p h i c a l d i s t r i b u t i o n of m a s s e t e r E M G p a r a m e ters, t h e s p e c t r a l EMG m a p p i n g w a s used. During d e f i n e d f u n c t i o n a l c o n d i t i o n s (clench in r e l a t i o n t o t h e m a x i m a l b i t e force, latero- and m e d i o t r u s i o n a g a i n s t a d e f i n e d load, p r o t r u s i o n , r e t r a c t i o n ) a sixteen channel surface EMG was recorded monopolarly f r o m the m a s s e t e r muscle. T h e o r i g i n a l EMG c u r v e s w e r e v e r i f i e d on m o n i t o r in o r d e r to s e l e c t E M G i n t e r v a l s w i t h o u t artifacts. A f t e r p o w e r s p e c tral a n a l y s i s (FFT) of t h e s e intervals, spectral EMG parameters (total EMG power, band power, median frequency) w e r e c a l c u l a t e d and u s e d in a linear i n t e r p o l a t i o n p r o c e d u r e a n d in a n imaging p r o c e d u r e . A h i g h l y d i f f e r e n t EMG d i s t r i b u t i o n w a s found between the movements of t h e m a n d i b l e . During c l e n c h i n g in the i n t e r c u s p a l p o s i t i o n the lower third of t h e m a s s e t e r muscle shows the highest a c t i v a t i o n . The EMG d i s t r i b u t i o n during m e d i o t r u s i o n and p r o t r u s i o n is s i m i l a r t o t h a t of clenching, b u t t h e level is s i g n i f i c a n t l y lower. D u r i n g l a t e r o t r u s i o n and r e t r a c t i o n t h e m a x i m u m of t h e E M G p o w e r w a s f o u n d in the u p p e r t h i r d of the muscle. These different topographical structures of E M G p a r a m e t e r d i s t r i b u t i o n are w e l l explicable f r o m t h e m o r p h o f u n c t i o n a l p o i n t of view. T h e r e a r e some evidence that muscular dysfunctions influenc i n g t h e a c t i v a t i o n p a t t e r n can be c h a r a c t e r i z e d . I n s t i t u t f~r P a t h o l o g i s c h e P h y s i o l o g i e , F r i e d r i c h schiller-Universit~t, L~bderstr.3, D/O-6900 Jena

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T H E T O P O G R A P H Y OF M Y O E L E C T R I C A L A C T I V A T I O N O F M. B I C E P S B R A C H I I IN H E A L T H Y S U B J E C T S H.Ch. Scholle, Oh. Anders, S. Popp and N.P. Schumann

S E L E C T I V E S U P P R E S S I O N O F C A L C I U M D E P O S I T I O N IN AORTIC ATHEROMATA OF CHOLESTEROL-FED NEW ZEALAND RABBITS BY THE CALCIUM ANTAGONIST NITRENDIPINE. F. Thimm, M. Frey, G. F l e c k e n s t e i n - G r ~ n , A. Fleckenstein. C h o l e s t e r o l - f e d r a b b i t s (2 % c h o l e s t e r o l in d i e t o v e r a p e r i o d of 14-17 m o n t h s ) s h o w e d m a s s i v e i n c r e a s e s in f r e e (16,4-fold) a n d t o t a l (42,5fold) c h o l e s t e r o l c o n t e n t a b o v e normal. In c o n t r a s t , Ca r o s e b y o n l y 7,4 t i m e s as c o m p a r e d w i t h h e a l t h y c o n t r o l segments. T h e c a l c i u m a n t a g o n i s t n i t r e n d i p i n e (12-15 m g / k g b o d y w e i g h t ) p r o v e d to be u n a b l e to d i m i n i s h the c o n t e n t s in f r e e and t o t a l c h o l e s t e r o l if g i v e n for 14-17 m o n t h s in a d d i t i o n to the c h o l e s t e r o l - d i e t . Nitrendipine did not counteract plaque size and c h o l e s t e r o l c o n t e n t . In c o n t r a s t , n i t r e n d i p i n e p r e v e n t e d the r i s e in p l a q u e c a l c i u m c o m p l e t e l y .

By using a 16-channel-EMG-system the surface-EMG of M. b i c e p s b r a c h i i on t h e r i g h t a n d left hand s i d e of h e a l t h y s u b j e c t s w a s m o n o p o l a r l y r e c o r d e d . Basing on defined p a r t s of these EMG curves, parameters of s p e c t r a l EMG p o w e r w e r e calculated b y m e a n s of F a s t - F o u r i e r - T r a n s f o r m a t i o n . Considering t h e s e values, E M G - m a p s w e r e p r o c e s s e d by a linear interpolation technique (topographical pattern of m y o e l e c t r i c a l a c t i v a t i o n ) . In p e r s o n s investigated the m a x i m u m of s p e c t r a l EMG power (maximum of m y o e l e c t r i c a l a c t i v a t i o n ) d u r i n g 90grade-flexions by the elbow joint as w e l l as sup• of t h e f o r e a r m w a s f o u n d in the proximal caput-breve-region of M. biceps. During identical posture of e x t r e m i t i e s , but neutral p o s i t i o n of t h e f o r e a r m t h e m y o e l e c t r i c a l activation maximum changed the p o s t • in lateral direction. That means, caput breve et longum ( m e d i u m parts) w e r e s i m u l t a n e o u s l y a c t i v a t e d . In p r o n a t i o n p o s i t i o n the a c t i v a t i o n m a x i m u m changed a little bit more to latero-distal region of recording and the total spectral EMG power dis t i n c t l y i n c r e a s e s (a s i g n of s e l e c t i v e a c t i v a t i o n of c a p u t longum). T h e l o w e s t s p e c t r a l E M G p o w e r of all investigations c o u l d be d e m o n s t r a t e d during elbow joint flexions in n e u t r a l p o s i t i o n of the forearm. On the b a s i s of s u c h results a more differentiated diagnostic evaluation of motor control disturbances , especially neuromuscular d y s b a n l a n c e s s e e m s t o b e p o s s i b l e in future. Institut f~r Pathologische Physiologie, F r i e d r i c h - S c h i l l e r - U n i v e r s i t ~ t , L ~ b d e r s t r . 3, D/O-6900 Jena

T a b l e i: C a l c i u m a n d c h o l e s t e r o l c o n t e n t s in the a r c u s a o r t a e (mean v a l u e s e x p r e s s e d as g / k g d r y w e i g h t • SEM). CALCIUM

FREE CHOLESTEROL 2.0i0.3 n=6

TOTAL CHOLESTEROL 2.5• n=6

Controls

0.6• n=6

2 % Cholesterol

4.4• n=8

32.8• n=8

106.3• n=8

2 % Cholesterol+N• trend•

1.2• n=5

35.5• n=5

131.2• n=4

S t u d y G r o u p for C a l c i u m A n t a g o n i s m , P h y s i o l o g i c a l I n s t i t u t e , U n i v e r s i t y of F r e i b u r g , H e r m a n n - H e r d e r Str. 7, 7800 E r e •

R 121 214 REFLEX REACTION OF THE JOINT MUSCULATURE IN KNEE -AND H I P J O I N T INJURIES. G.Schier Joint injuries are combined often with atropical processes in pats of joint musculature. It should be proof, if this phenomenon is coupled with reflex changes in the respective muscles. With a pilot study was compared with a control group the reflex reaction of selected knee -and hip joint muscles as a result of suddenly removing the load after maximal voluntary ab -and adductiv contraction. It the following results: i. Joints with a good m u s c u l a r stabilisation bring about a d e f i n i t e l y reproducable reflex reaction pattern. 2. Joints with a bad muscular stabilisation show an increased v a r i a b i l i t y in the reflex reaction pattern. 3. Joint injuries are frequently coupled with a destruction in the reflex reaction pattern,i.e. atypical nonspecific reflex pattern. 4. With in joint injuries the central nerv inhibitions are p a r t i c u l a r y obvious. Despite these findings real reflex anomalies were observed only seldom. There was no direct relationship b e t w e e n the type of joint injury and the reflex reaction pattern. Fakult~t Sportwissenschaft, Fr.-Ludwig-Jahn-Allee 59, 0-7010 Leipzig

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301 CHARACTERIZATION SMOOTH MUSCLE F. Hofmann

OF CA2+-CHANNELS

IN V A S C U L A R

Smooth muscle cells contain two voltage dependent calcium channels, a low voltage activated T-type channel and a high voltage activated "L-type" channel. The ~i subunit of the "L-type" channel has been cloned. The deduced primary amino acid sequence is over 95 % identical with ~i subunit from cardiac muscle. Northern blot analysis shows that the smooth muscle ~I subunit is expressed in all tissues tested whereas the cardiac ~i subunit is only expressed in cardiac muscle. Injection of the cRNA for the u I subunits in Xenopus oocytes induces a L-type calcium current. Coinjection of the ~/6, and y subunits from skeletal muscle t o g e t h e r - w i t h these ~i subunits affects significantly the current density~ activation time and voltage dependence of steady state inactivation. Transfection of the u 1 subunit into CHO-cells induces L-type current which have most of the electrophysiological characteristics of the native channel. However, in contrast to the physiological channel, the expressed channels are not regulated by hormones, sofar. This suggest that regulation of these channels requires additional proteins. Institut fur Pharmakologie und Toxikologie der Technischen Universit~t M~nchen, Biedersteiner Strasse 29, W-8000 M~nchen 40

MECHANISMS OF ANTICALCINOTIC VASOPROTECTION WITH CALCIUM ANTAGONISTS. Fleckenstein-Gr~n G. u. Fleckenstein A. At present,the antiarteriosclerotic efficacy of calcium antagonists,first decribed in 1971,and subsequently analyzed in a multitude of experimental models on rats by FLECKENSTEIN and coworkers,is increasingly gaining interest. It is a well-known fact that arterial walls,altered by sclerotic processes, accumulate two main constituents:Lipids(particularly cholesterol) and calcium (Ca). However, since long,the accumulation of lipids has been incriminated as the primordial pathogenic factor, whereas the concomitant arterial Ca overload was considered a trivial phenomenon of secondary importance. Our own studies on the destructive power of excessive Ca uptake into the arterial walls started in 1970 using specific Ca antagonists as antiarteriosclerotic tools. In a variety of animal models both the crucial role of Ca overload in the pathogenesis of arteriosclerotic lesions (including loss of elastic distensibility) and the anticalcinotic vasoprotection by Ca antagonists could be demonstrated. We used light and electron microscopy, 4SCa and X4C cholesterol uptake measurements,Ca analyses with atomic absorption spectroscopy and cholesterol analyzes with gas chromatography.These experiments were carried out on rats intoxicated with dihydrotachysterol or vitamin D3 or kept under the long-term influence of risk factors such as nicotine,alloxan diabetes and hypertension (SHRs,salt-loaded DAHL-S and GOLDBLATT rats).In sharp contrast with Ca overload-induced forms of experimental arteriosclerosis,the feeding of New-Zealand rabbits with a cholesterol-rich diet produced in our studies a totally different type of arteriosclerotic damage. For instance,in cholesterol-fed New Zealand rabbits(2% cholesterol in diet), stenosing coronary lesions developed which were characterized by several peculiarities: i. Large accumulation of free and total cholesterol in the arterial walls, 2. only modest increase in mural Ca content above normal, 3. no clear inhibitory influence of Ca antagonists (verapamil, nitrendipine) on arterial cholesterol deposition, 4. soft consistency of cholesterol plaques in contrast with the stiffness of calcified arteriosclerotic vascular sections. In ageing human arteries,a steady increase in Ca content is a characteristic phenomenon. Interestingly,the natural age-dependent arterial Ca accumulation is enhanced in cases of severe diabetes,heavy smoking or hypertension.But most excessive degrees of Ca overload,correlating with the severity of structural damage,were found by us in human coronary artery plaques.Thus,in grade I plaques (according to WHO classification)the mural Ca content rose by 13 times,in grade II plaques by 24 times, and in grade III plaques by more than 80 times above the Ca content of healthy coronary segments in the same age group. In contrast,the average increase in free and total plaque cholesterol was rather modest (l.5-to 1.8-fold).Obviously,the formation of human coronary plaques is marked by a dramatic local uptake of Ca,whereas the analysis of the mural cholesterol content does not allow to distinguish arteriosclerotic from normal coronary artery segments. According to our experimental data,Ca antagonists directly interfere with an increased uptake of Ca into the vascular wall.This Ca overload may result from multifactorial disorders of vascular Ca metabolism under the influence of various risk factors. On the other hand,pure cholesterol atheromata induced by elevation of plasma cholesterol up to toxic levels(2,400 mg%)in cholesterol-fed New-Zealand rabbits,could in our experiments not be prevented by Ca antagonists(verapamil,nifedipine,nitrendipine). Stud. Group f.Ca Antagonism,Physiol. Inst., Freiburg

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TISSUE PROTECTION BY CALCIUM ANTAGONISTS W i n f r i e d G. Nayler

CHRONIC EFFECTS OF C A L C I U M A N T A G O N I S T S ON THE P R O G R E S S I O N OF A T H E R O S C L E R O S I S P.R. Lichtlen, W. Rafflenbeul, P. Nikutta, St. Jest and the INTACT-Study Group

The cardioprotective effect of the calcium antagonists is a complex phenomenon that needs to be considered in terms of immediate and long term benefits. Early during an ischaemic episode, at a time when reperfusion results in a "stunned" and therefore ultimately recoverable myocardium, cytosolic Ca 2~ rises. This early rise in cytosolic Ca2+ may trigger the cascade of events which culminates in irreversible injury - either by way of protease and phospholipase activation, by energy wastage associated with CaZ+-induced activation of the ATPases, or its effect on free radical production. The cause of the early rise in cytosolic Ca 2§ is uncertain. It precedes a marked depletion of the energy rich phosphate reserves and therefore cannot be due simply to failure of the ATP-dependent retrieval mechanisms. Possibly Ca ~§ entering in exchange for Na § , or Ca ~§ leaking from a damaged sarcoplasmic reticulum may be involved. Other changes which occur early during an ischaemic episode include externalization of the endothelin-i binding sites, and an altered distribution of other receptors. These changes in receptor distribution are indicative of changes in the organization of the sarcolemma which precede loss of m e m brane integrity. Laboratory and clinical studies have shown that the calcium antagonists confer protection under these conditions. The mode of action, however, is complex and involves energy preservation, attenuation of the early rise in cytosolic Ca 2§ , and, in some cases, an ability to attenuate membrane damage caused by lipid peroxidation. This protective effect of the calcium antagonists is, in some cases, stereospecific. Whilst the acute protective effects of the calcium antagonists may center around their ability to attenuate the early rise in cytosolic Ca 2§ , their long-term clinical usage undoubtedly benefits from their ability to slow atherogenesis. Department of Medicine, University of Melbourne, Austin Hospital, Heidelberg VIC 3084, Australia

Animal studies d e m o n s t r a t e d that c a l c i u m antagonists are able to significantly retard the evolution of a t h e r o s c l e r o t i c plaques. B a s e d on these experiences a study was performed in man (INTACT) a n a l y z i n g the progression of coronary artery disease by quantitated coronary angiography. 348 patients u n d e r w e n t 2 angiograms exactly at 3 years interval (175 on placebo, 173 on nifedipine, 80 mg/day); p r o g r e s s i o n was d e f i n e d either as an increase in the degree of stenosis by ~20% or transition to occlusion, or as development of n e w stenoses ~20% or new occlusions in segments previously a n g i o g r a p h i c a l l y normal. A f t e r 3 years, there was no d i f f e r e n c e b e t w e e n groups w i t h regard to pro- and r e g r e s s i o n in existing stenoses; however, there were fewer new lesions on nifedipine (144 on placebo vs. 103 on nifedipine -28%, p=0.034, Cochran's linear trend test) and also a trend to fewer patients w i t h new lesions on nifedipine (-18%, n.s.). In the meantime 3 other studies were published, a d m i n i s t e r i n g nicardipine in one and nifedipine in two, all d e m o n s t r a t i n g a significant reduction of new lesions, one also a reduction of p r o g r e s s i o n of e x i s t i n g lesions.- Due to the v e r y slow p r o g r e s s i o n of a t h e r o s c l e r o s i s (only 56% of I N T A C T - p a t i e n t s showing progression, 79% of them in new lesions, 31% only in old ones), clinical effects related to the p r e v e n t i v e treatment with nifedipine cannot be expected after 3 years, a follow-up of longer d u r a t i o n is necessary and on the way. D i v i s i o n of Cardiology, H a n n o v e r Medical School, Konstanty-Gutschow-Str. 8, D-3000 H a n n o v e r 61, FRG.

305 CA2+ CHANNELSANDTISSUESELECTIVITYOF CA2+ ANTAGONISTS

T. Godfraind In functional studies the term tissue selectivity is used to characterize an agent showing varying degrees of potency among tissues with a preferential action in a given one. Such a selectivity is well known in the fields of adrenoceptors and of other receptors. Does it exist in the pharmacology of Ca2+ channels? When examining the effects of calcium antagonists in various isolated preparations, several pharmacologicaI profiles may be delineated considering the following main determinants: 1. Characteristics of the drug : Ca channel selectivity (L,T,N type), Site of action within the channel, Selectivity window, Voltage and/or frequency dependency, Ratio Ca antagonistic/Ca agonistic action, Physicochemical properties (pKa, partition coefficient) Susceptibility to tissue metabolism 2. Properties of the tissue : Relative contribution of various Ca pools to final response, Density and types of Ca channels, Level of resting potential, Gating control of Ca channels Pharmacokinetic determinants 3. Characteristics of the stimuli : Level, duration and frequency of depolarization, Nature of activated ldnase(s) It is obvious that several factors are involved, which needs an extensive analysis. Therefore, this presentation will address some typical examples of this diversity including : a) the contribution in vascular and cardiac preparations of various Ca pools to final response, b) the influence of membrane potential on the interaction of some Ca2+ antagonists with their receptors, c) the evidence that inhibition by Ca 2+ antagonists of vascular contraction evoked by noradrenaline is related to their interaction with potential operated calcium channels, d) the evidence that the large difference in sensitivity between cardiac and vascular tissues, documented for a dihydropyridine such as nisoldipine, may be reIated to the kinetic parameters describing the interaction of Ca2+ antagonists with Ca2+ channels. Laboratoire de Pharmacologie, Universitd Catholique de Louvain, UCL 7350, Avenue E. Mounier 73, B - 1200 Brussels, Belgium.

R 124 306 Cellular processes in atherogenesis: targets of G. Schmitz and J, Hankowitz

307 Ca + +

antagonists

Atherosclerosis is characterized by the accumulation of lipids, Ca + + and extra-cellular matrix in the vessel wall. In the normal vessel wall and still at the stage of an early lesion, Ca + + is involved in regulatory processes, enhances low density lipoprotein (LDL) receptor binding, increases endothelial permeability, induces chemotaxis of macrophages and smooth muscle cells (SMC) and promotes the migration of these cells into the intima, and stimulates oxidative burst and the secretion of collagen and other components. At this stage Ca ++ antagonists may interfere with some of these processes either by modulation of signal transducers such as adenosine- and peripheral benzodiazepine receptors, the nucleoside transporter and multi-drug-resistance protein or by intercalating into cell membranes. Nifedipine and Nitrendipine arrest synchronized SMC in G /GI-Phase as monitored by flow cytometric cell cycle analysis leading to a reduced~ proliferation. Ca + + antagonists inhibit extracellular matrix synthesis, thereby diminishing cell adhesion and may be chemical modification of matrix-bound LDLs. Thus, less modified LDL are produced, which have to be catabolized by receptor mediated uptake or by phagocytosis in macrophages, leading to a reduction of foam cell production. Ca + § antagonists reduce LDL uptake in monocytes and acetyI-LDL uptake in macrophages. They also enhance the synthesis of membrane phospholipids, e.g. sphingomyelin, thereby increasing membrane turnover and fluidity. In addition, they interfere with radical metabolism, thereby reducing radical mediated cell damage. The resulting membrane protective effects may delay the progression of the lesions, improve the integrity of the cytoskeleton, and stabilize adjacent membranes. Various animal studies, and most recently human studies, show that the treatment with Ca ++ antagonists slow the progression of atheroeclerotic processes at the stage of eady lesions. The precise mechanism by which Ca ++ antagonists are antiatherogenic remains unclear. It seems that some of these effects are independent of these drugs on voltage operated Ca + + channels since only SMC express this type of Ca + § channels. A key target for future research is the characterization of Ca ++ antagonists induced side effects on speclfic signal transducing processes in cells involved in the atherosclerotic process. Institut for Klinische Chemie und Laboratoriumsmedizin, Franz--Josef-StrauS-Allee 1, D-8400 Regensburg

THE RENIN GENE IN HYPERTENSION: FROM MOLECULAR ANALYSIS TO HYPERTENSIVE TRANSGENIC RATS Detlev Ganten, MinAe Lee, Maike Sander, Jiirg Peters, Michael Bader, Martin Paul The development of potent inhibitors of the renin-angioteusin axis, such as renin and CE-inhibitors, angiotensin H antagonists has fostered a rapid expansion of our understanding of its pathophysiological significance and clinical relevance. This has gained additional interest due to the discovery of local tissue reninangioteusin systems in addition to the classical hormonal system. The existence of such intrinsic renin-angiotensin systems has now been confirmed by a number of recent studies demonstrating unequivocally expression of the genes for renin and angiotensinogen in a variety of tissues, e.g. brain, adrenal gland, heart, arterial wall, testis, ovary and kidney. To study the tissue-specific expression of components of the renin-angiotensin system (RAS) we have used transgenlc techniques. The use of specific gene constructs permits the localisation of expression in specific tissues. We have introduced the rat angioteusinogen gene into the mouse germllne and produced trausgenic mice. Results indicate that the trausgene is expressed within specific brain areas including the thalamus, the hypothalamus, the superior and inferior colliculi, the central perlaqueductal gray and the Purkinje cell layer and the granular layer of the cerebellar cortex. We have also established transgenic rats by introducing the mouse renin gene Ren2. Several independent lines of transgenic rats have been established and those animals which possess the mouse gene exhibit extreme hypertension. The segregation of the hypertensive phenotype with the presence of the trausgene in independent lines, indicates that expression of the mouse renin gene is responsible for the hypertension. Interestingly, these rats do not have abnormal levels of renin, angiotensin I or angiotensin H in their plasma and therefore overexpression of renin in the kidney does not account for the phenotype. These trangenic rats provide an attractive new experimental model for pharmacological and pathophysiologieal cardiovascular studies on the tissue renin-angiotensin system. We submit that different roles with respect to blood pressure regulation are realized by plasma RAS and by tissue RAS. The plasma RAS may be an acute regulator of vascular tone and electrolyte and volume homeostasis while the tissue RAS probably carries out more long-term functions on an autocrine or paracrine level. The inhibiton of the tissue RAS by inhibitors of the renin-angioteusin system may well be more important to the long-term therapeutic effects than their interference with the hormonal plasma reninangioteusin system. Hypertension is a chronic disease and treatment of mild to moderate hypertension does not warrant acute lowering of blood pressure. Thus, inhibition of the long-term effects of tissue angioteusin may be more desirable and important than the acute inhibitory effetes on the plasma RAS. As we learn more about the renin-angiotensin system and unravel an increasingly complex picture, we are presented with ever new questions and challenges to be addressed by both basic scientists and clinical investigators. Clearly, these and other studies show, that the renin-angiotensin system ist even more important for cardiovascular homeostasis and hypertension than was previously thought. Mullins JJ, Peters J, Ganten D. Fulmlnant hypertension in transgenic rats harbouring the mouse Ren-2 gene. Nature 1990;344:541-544 Schelling P, Fischer H, Ganten D. Angiotensin and cell growth: a link to cardiovascular hypertrophy? J Hypertens 1991;9:3-15 Ganten D, Lindpaintner K, Ganten U, Peters J, Zimmermann F, Bader M, Mullius J. Transgenic rats: New animal models in hypertension research. Hypertension 1991;17, No.6, Part 2:843-855

Lindpaintner K, Ganten D. The cardiac renin-angioteusin system An appraisal of present experimental and clinical evidence. Circ Res 1991;68, N0.4:905-921 German Institute for High Blood Pressure Research and Department of Pharmacology, University of Heidelberg, Im Neuenheimer Feld 366, 6900 Heidelberg

R 125 309

308 L O C A L I Z A T I O N OF C O M P O N E N T S ANGIOTENSIN SYSTEM F.A.O. M e n d e l s o h n

OF T H E R E N I N -

Q u a n t i t a t i v e in v i t r o a u t o r a d i o g r a p h y was u s e d to l o c a l i z e A n g II r e c e p t o r s and A C E in the brain, k i d n e y and c a r d i o v a s c u l a r system. The r a d i o l i g a n d u s e d to label A n g II r e c e p t o r s is the a n t a g o n i s t , 1 2 s I - [ S a r i , Ile B ] A n g II, and to label ACE, the potent selective inhibitor ~251-351A. In the c e n t r a l n e r v o u s s y s t e m and s p i n a l cord, t h e r e is as s t r i k i n g a s s o c i a t i o n of A n g II r e c e p tors w i t h a u t o n o m i c c o n t r o l centres, i n c l u d i n g the h y p o t h a l a m i c p a r a v e n t r i c u l a r nucleus, p a r a b r a c h i a l n u c l e u s , n u c l e u s of the s o l i t a r y tract, r o s t r a l and c a u d a l v e n t r o l a t e r a l m e d u l l a , s y m p a t h e t i c p r e g a n g l i o n i c n e u r o n e s of the s p i n a l cord, a n d sympathetic post-ganglionic neurones. M o s t of t h e s e areas r e c e i v e A n g II p o s i t i v e inn e r v a t i o n , s u g g e s t i n g i m p o r t a n t c e n t r a l roles of n e u r o n a l A n g II in r e g u l a t i n g f l u i d and e l e c t r o lyte b a l a n c e a n d a u t o n o m i c a c t i v i t y . A C E also occurs in m a n y of t h e s e sites, but has a w i d e r dist r i b u t i o n , s u g g e s t i n g a d d i t i o n a l roles in m e t a b o lism of o t h e r n e u r o p e p t i d e s . In the heart, A C E o c c u r s t h r o u g h o u t the m y o c a r d i u m at low c o n c e n t r a t i o n s and is h i g h e s t in the atria. C o r o n a r y v e s s e l s s h o w h i g h c o n c e n t r a t i o n s of ACE. The e n d o c a r d i u m has v e r y low levels of ACE, e x c e p t o v e r the v a l v e l e a f l e t s w h e r e e x t r e m e l y h i g h c o n c e n t r a t i o n s occur. A n g II r e c e p t o r s are f o u n d in the c o n d u c t i n g s y s t e m - the SA and AV n o d e s - as w e l l as a s s o c i a t e d w i t h c h o l i n e s t e r a s e p o s i t i v e n e r v e b u n d l e s and some i n t r a c a r d i a c g a n g l i a . A C E is not colocalized w i t h A n g II r e c e p tors at t h e s e sites, b u t o c c u r s in close p r o x i mity. L a r g e b l o o d v e s s e l s from a r a n g e of species, i n c l u d i n g humans, s h o w h i g h c o n c e n t r a t i o n s of A C E o v e r the l u m i n a l e n d o t h e l i a l cells, as is w e l l known. H o w e v e r , e q u a l l y h i g h c o n c e n t r a t i o n s of A C E are f o u n d in the a d v e n t i t i a , in k e e p i n g w i t h the f i n d i n g t h a t s t r i p p i n g the e n d o t h e l i u m does not a b o l i s h A n g I c o n v e r s i o n b y v e s s e l s in vitro. M u c h of the a d v e n t i t i a l A C E is a s s o c i a t e d w i t h v a s a vasora, w h i c h are e s p e c i a l l y r i c h in ACE. T h e s e s t u d i e s r e v e a l m u l t i p l e sites in the cent r a l n e r v o u s a n d c a r d i o v a s c u l a r s y s t e m s w h e r e loc a l l y - p r o d u c e d A n g II c o u l d exert i m p o r t a n t regul a t o r y i n f l u e n c e s on c i r c u l a t o r y c o n t r o l . D e p a r t m e n t of M e d i c i n e , A u s t i n H o s p i t a l , H e i d e l b e r g , V i c t o r i a 3084, A u s t r a l i a

VASCULAR STRUCTURE AND BLOOD AND AFTER TREATMENT WITH ACE M.J. M u l v a n y .

PRESSURE DURING INHIBTIONS

T h i s p a p e r r e v i e w s w o r k (i, 2, 3) c o n c e r n i n g the l o n g - t e r m e f f e c t s of a n t i - h y p e r t e n s i v e d r u g s on b l o o d p r e s s u r e and m e s e n t e r i c r e s i s t a n c e v e s s e l s t r u c t u r e ( m e d i a : l u m e n ratio) in s p o n t a n e o u s l y h y p e r t e n s i v e rats (SHR) a n d M i l a n h y p e r t e n s i v e rats (MHS), b o t h d u r i n g a n d a f t e r t r e a t m e n t . SHRs w e r e t r e a t e d from 4 to 24 wk w i t h A C E inhib i t o r s : p e r i n d o p r i l (1.5 mg/kg/day) or c a p t o p r i l (60 m g / k g / d a y ) ; or o t h e r drugs: h y d r a l a z i n e (25 m g / k g / d a y ) , i s r a d i p i n e (42 m g / k g / d a y ) or m e t r o p r o l o l (130 m g / k g / d a y ) . At 24 wk, 2 4 - h r m e a n b l o o d p r e s s u r e was 121 m m H g ( p e r i n d o p r i l ) , 137 m m H g (captopril), 140 m m H g ( h y d r a l a z i n e ) , 149 m m H g (israpidine) a n d 146 m m H g (metoprolol). C o n t r o l v a l u e s w e r e 177 m m H g (SHR) and 132 m m H g ( W i s t a r - K y o t o rats, WKY). M e d i a : l u m e n r a t i o was r e d u c e d to W K Y - l e v e l b y p e r i n d o p r i l (m:l = 4.4%), to m i d - w a y b e t w e e n 8HR- and W K Y - l e v e l s b y c a p t o p r i l h y d r a l a z i n e and i s r a d i p i n e (5,9%) b u t no a f f e c t e d b y m e t o p r o l o l (6,8%). W h e n t r e a t m e n t was d i s c o n tinued, low M B P (ca. 151 mmHg) p e r s i s t e d for 12 w k in the rats t r e a t e d w i t h the A C E i n h i b i t o r s , but r o s e r a p i d l y in t h e r a t s w h i c h h a d r e c e i v e d the o t h e r drugs. M e d i a : l u m e n r a t i o 3 to 12 wk a f t e r w i t h d r a w a l of drugs c o r r e l a t e d c l o s e l y w i t h the b l o o d p r e s s u r e e x p e c t e d from the SHR- a n d W K Y c o n t r o l v a l u e s . In a n o t h e r e x p e r i m e n t , w i t h the same p r o t o c o l , lower d o s e s of p e r i n d o p r i l w e r e g i v e n (0.4 a n d 0.8 mg/kg/day). At 24 wk, t h e r e was a d o s e - d e p e n d e n c e for b o t h b l o o d p r e s s u r e a n d m e d i a : l u m e n ratio. H o w e v e r , 12 wk a f t e r s t o p p i n g t r e a t m e n t , the b l o o d p r e s s u r e r e m a i n e d at W K Y levels in b o t h cases. By c o n t r a s t , in MHS, t r e a t m e n t w i t h p e r i n d o p r i l (0.4 a n d 1.5 m g / k g / d a y for the same p e r i o d of time, h a d s i m i l a r e f f e c t s on blood pressure and media:lumen ratio during treatment, b u t w h e n t r e a t m e n t was stopped, b l o o d p r e s s u r e rose r a p i d l y to c o n t r o l levels. The r e s u l t s s u g g e s t that t h e a b i l i t y of a t r e a t m e n t to c o n t r o l v a s c u l a r s t r u c t u r e d u r i n g t r e a t m e n t is not i t s e l f a p r e d i c t o r of t h e p e r s i s t e n t e f f e c t on b l o o d p r e s s u r e a f t e r w i t h d r a w a l of t r e a t m e n t . Danish Biomembrane Research Centre and Department P h a r m a c o l o g y , A a r h u s U n i v e r s i t y , 8000 A a r h u s C., Denmark

of

R 126 310 IMPROVEMENT OF PERIPHERAL CIRCULATION BY CHRONIC ACE-INHIBITION THERAPY SAFAR ME - Departement of Internal Medicine - H6pital Broussais Paris - France Converting enzyme inhibitors not only dilate small arteries but also alter the buffering function of large arteries, causing an increase in arterial diameter and compliance. Using non-invasive technics in hypertensive humans, it was shown that brachiat artery dilates following converting enzyme inhibition both acutely and in the long term, despite a substantial blood pressure redaction. A flow dependent mechanism could partly explain the arterial diameter enhancement. Since angiotensin II was unable to modify arterial diameter, several mechanisms as those related to autonomic blockade, bradykinin and prostaglandin could additionnaly influence brachial artery smooth muscle relaxation. Whereas the brachial artery constantly dilates following converting enzyme inhibition in hypertension humans, no comparable result was observed at the site of the common carotid artery. For that reason, in hypertensive rats, we used an original model of in situ carotid artery preparation to demonstrate the compliance enhancement prodoced by converting enzyme inhibition. Converting enzyme inhibition was shown to increase carotid compliance both in acute and long term situations independantly of pressure changes. Removing endothelium abolished the compliance enhancement. Although converting enzyme inhibition increased arterial compliance, the compliance increase did not reach the level obtained following total relaxation by potassium cyanide. However, with long term treatment, also compliance after total relaxation was improved, indicating that converting enzyme inhibition acted on both the functional and the structural component of the visco-elastic properties of the arterial wall. Additional morphometric studies confirmed the remodelling of the large artery wall which was observed after converting enzyme inhibition.

312 M E C H A N I S M OF E N D O T H E L I U M - D E P E N D E N T RELAXATIONS I N D U C E D BY ACE-INHIBITORS IN C O R O N A R Y ARTERIES. W. Auch-Schweik, C. Bossaller, M. Claus, K. Graf, M. Gr/ife, D. Hauth, E. Fleck. ACE-inhibitors may cause vasodilatation by reducing the degradation of bradyldnin, which stimulates the release of endothelium-derived relaxing factor(s). The present study was designed to elucidate other mechanisms, by which bradykinin can contribute to the vasodilatation in response to these drugs. Rings of isolated bovine coronary arteries with and without endothelium were mounted in organ chambers for measurement of isometric force. The effect of lisinopril (10"1~to 10-6 M) was investigated in rings contracted with PGF~ and stimulated with bradykinin or other vasodilators [in the presence of indomethacin (lff5 M)]. The drug caused concentrationdependent relaxations (half maximal effect at approximately 104 M, maximal relaxation (m.r.) at 10.7 M: 66_+8 %, n=28) in rings with endothelium stimulated with a threshold concentration of bradykinin (101~M causing 22_+5 % relaxation). The relaxations to lisinopril were independent from time of exposure to bradykinin (10, 30 or 60 min). The relaxing effect to lisinopril was not observed in rings without endothelium nor in rings with endothelium incubated with another endothelium-dependent vasodilators (acetylcholine 10-7 M, substance P 104 M, or ADP 104 M) or endothelium-independent vasodilators (SIN-1 10s M); it was attenuated by the inhibitors of nitric oxide synthetase (1-nitro-arginine, 104 M, m.r.: 9+_2%, n=14), guanylate cyclase (methyleneblue, llYs M, m.r. 42-+13%, n=10), and by the bradykinin2-receptor antagonists [(Thr 10-6 M, m.r. 15_+5 %, n=9]. Conclusion: Bradykinin (>10"1~ M) triggers the release of EDRF (nitric oxide) in response to the ACE-inhibitor lisinopril in isolated bovine coronary arteries. Klinik fiir Kardiologie/Innere Medizin. Deutsches Herzzentrum Berlin. Augustenburger Platz 1, 1000 Berlin 65

311

313

INHIBITORY NONADRENERGIC-NONCHOLINERGIC (NANC) NEUROTRANSMISSION IN VASCULAR TISSUE INVOLVES THE L-ARGININE-NITRIC OXIDE (NO)-CYCLIC GMP PATHWAY LJ. Ignarro, P.A. Bush, G.M. Buga, W.J. Aronson and J. Railer. UCLA School of Medicine, Los Angeles, California 90024. The precise mechanism of neurogenically-elicited relaxation of certain vascular smooth muscle beds is unknown. Electrical field stimulation (EFS) of isolated strips of human and rabbit corpus cavernosum causes marked vasodilation and penile erection attributed to activation of the NANC neuronal pathway. Male impotence can be temporarily overcome by local intracavernous injection of nitrovasodilators. The objective of this study was to elucidate the physiological role of NO in EFS-elicited NANC-mediated relaxation of corpus cavernosum in human and rabbit. EFS caused tetrodotoxinsensitive but indomethacin-insensitive relaxation of phenylephrineprecontracted strips of corpus cavernosum accompanied by formation of both cyclic GMP and NO in corporal smooth muscle. Acetylcholine caused endothelium-dependent,whereas EFS and authentic NO caused endothelium-independent relaxation. Relaxation, NO formation, and cyclic GMP accumulation were inhibited by the NO-synthase inhibitor Na-nitro-L-arginine (L-NNA), and this was reversed by L-arginine but not the enantiomer D-arginine. Hemoglobin and methylene blue inhibited, whereas the cyclic GMP phosphodiesterase inhibitor M&B 22,948 enhanced EFS-elicited relaxation. Authentic NO mimicked the effects of EFS and these actions were inhibited by hemoglobin and enhanced by M&B 22,948. EFS-elicited relaxation of corpus cavernosum could not be accounted for by VIP, adenosine, ATP, ADP, capsaicin-sensitive sensory neurotransmitters, or prostaglandins. These observations suggest that NO is responsible for inhibitory NANC neurotransmission in human and rabbit corpus cavernnsum that leads to smooth muscle relaxation and penile erection. The NO is either the NANC neurotransmitter or is synthesized in the vascular smooth muscle in response to release of an unknown NANC neurotransmitter.

REGULATION OF CORONARY ARTERY TONE DURING HYPOXIA BY THE ENDOTHELIUM. W. Auch-Schwelk, C. Bossaller, N. Nega, U. Bilsscher, and E. Fleck. The role of the endothelium in the regulation of vascular tone was investigated during hypoxia in isolated porcine and human coronary arteries (from heart transplantations). Rings with and without endothelium were mounted in organ chambers for measurement of isometric force. Hypoxia was induced by gassing the organ chambers with 95%N2/5%CO 2 instead of 95%Oz/5%CO 2 for 20 minutes. Rings with endothelium from both species which were contracted with prostaglandin F2= 10-6 M (PGF2~) showed contractions after approximately 7 minutes of hypoxia followed by a relaxation after 20 minutes. Only relaxations were observed in rings without endothelium contracted with PGFa=. The endothelium-dependent contraction could be prevented by inhibitors of endotheliumdependent relaxations (methylene blue 10-s M or 1-NC-monomethyl arginine 5x 10.4 M=I-NMMA), but not by indomethacin (10"s M). Quiescent rings from both species showed contractions during hypoxia, whether the endothelium was present or not. Like in the rings treated with PGF2~ the contractions in quiescent rings with endothelium could be inhibited by 1-NMMA, but not by indomethacin. In contrast contractions to hypoxia in rings without endothelium could be inhibited by indomethacin, but not by 1NMMA. These experiments demonstrate, that hypoxia causes contractions in isolated porcine and human coronary arteries in response to hypoxia. Two mechanisms may contribute to the contractions during hypoxia: a) in rings with endothelium the inhibition of basal release of the endothelium derived relaxing factor b) in rings without endothelium the generation of prostaglandins in the vascular smooth muscle. Klinik fox Kardiologie/Innere Medizin. Deutsches Herzzentrum Berlin. Augustenburger Platz 1, 1000 Berlin 65

R 127 314 Effects of the ACE-inhibitor captopril, the PDE-III-inhibitor enoximone and their combination on aortofemoral pulse wave velocity in healthy subjects.

316

1K. Breithaupt, 1C. de Mey, 2W. Schreck, 2H.A. Dieterich, 1G.G. Belz

A number of cell types including macrophages, neutrophils, cerebellar cells and endothelium are known to release nitric oxide (NO) from the terminal nitrogen atom of L-arginine. NO is known to regulate the vascular tonus as well as platelet function by activating the soluble guanylate cyclase. Therefore, the action of NO is traced back to an elevated cGMP level. Although several findings suggested that NO may have effects which were not mediated by cGMP. Recently, we showed that NO-generating agents like sodium nitroprusside (SUP) activated the ADP-ribosylation of an 39 kD l~r,ptein in several tissues measuring the incorporation of 9 z P labelled ADP-ribose from NAD. Further studies revealed that NO generated from an activated NO-synthase in cerebellum cytosol stimulates an ADP-ribosyltransferase. Addition of the known cofactors (arginine, 250 IxM; NADPH, 200 ~tM; tetrahydrobiopter!n; 0,5 ~M; calcium, 3 IxM and calmodulin, 10 Ixg/ml) to the cytosouc traction of cerebellum cytosol caused an increase m the ADP-ribosylation of the 39 kD protein up to 4-fold compared to control incubations without cofactors. This stimulation was inhibited by specific NO-synthase inhibitors like N-nitro-Larginine (1 mM) and N-methyl-L-arginine (5 mM). Thereby Nnitro-L-arginine is more effective than N-methyl-L-arginine. The same effects were observed usingcytosol of HL-60 cells which were differentiated with 1,25 % DMSO to granulocytes. Although the physiological significance of the ADP-ribosylation is still unknown, the ADP-ribosylation of a distinct protein may occur under physiological conditions and may explain recently described cGMP independent NO-effects.

Arterial compliance can be assessed by measurements of pulse wave velocity (pwv), high l'wv indicating low compliance and vice versa. Aorto-femoral l'wv (AFPWV) is an index for windkessel compliance (Wezler et al., Ergebn Physio141:313,1939). In a placebo-controlled, single-blind randomized cross-over study 10 fastin~ healthy males (mean age 26 y.) were studied 4 times, receiving either placebo (eL), a single oral dose of 100 mg enoximone (E), a single oral dose of captopril (C) or their combination (CE). AFPWVwas measured sphygmographically using a Boucke-Brecht sensor. The pre-dosing AFPWVwas 5.87, 5.79, 5.81 and 5.73 m/s for I'L, E, C and CE respectively. The time course of the changes of AFPWV and mean arterial pressure (MAP, mmHg) from the pre-dosing baseline is sulmnarized here below : AFeWV (m/s) MAP (mmHg) Time PL E C CE PL E C CE lh 0.06 0.06 -0.13 -0.31 -1 1 -2 -5 2h 0.00 -0.15 -0.05 -0.44 1 1 -3 -7 3h 0.02 -0.15 -0.07 -0.37 -1 -2 -1 -8 4h 0.03 -0.10 -0.05 -0.41 -0 -3 -1 -7 The PL-findings document the adequate within-study day reproducibility of the measurements. Analysis of overall c- and E- effects and their interaction (de Mey and Sparrow, Int J Clin Pharm Res 9:297, 1989) confirmed that both C and E significantly reduced AFPWV in healthy subjects. The c-effects appeared earlier (maximum at lh) whereas the E-effects appeared later (maximum at 2h) but lasted longer. There was no significant interaction, i.e. the c- and E-effects were merely additive. The observed effects can only partly be explained by the effects on MAP. Other mechanisms are likely to have been involved too, such as drug related reduction of the smooth muscle tonus in the aortic wall.

NO RELEASED BY NO-SYNTHASE STIMULATES ENDOGENOUS ADP-RIBOSYLTRANSFERASE S. Dimmeler and B. Brtine

AN

Faculty of Biology, Department of Biological Chemistry, Prof. Dr. V. Ullrich, University of Konstanz, 7750 Konstanz, FRG.

1 : Zeutrum ftir Kardiovaskul/ire Pharmakologie, 6500 Mainz 2" Merrell Dow Research Institute, 6090 Riisselsheim.

315 CALCIUM-INHIBITORY EFFECTS OF CICLETANINE ON VASCULAR SMOOTH MUSCLE IN COMPARISON WITH OTHER SMOOTH MUSCLE RELAXING AGENTS P. Deitmer, K. Golenhofen and Th. Noack Cicletanine (Cic) is a novel antihypertensive drug which, in addition to other effects, exerts an inhibitory effect on calcium currents in vascular smooth muscle cells under voltage clamp conditions (Noack et al., Pfltigers Arch. 418, Suppl. 1, R 49, 1991). We have now compared the effects of Cic with those of other relaxing agents, recording the mechanical activity in seven vascular smooth muscle preparations (portal vein of rabbit, rat and guinea-pig, aorta of rat and guinea-pig, iliae artery and ear artery of rabbit) and in several gastrointestinal and uterine preparations. Comparing the relaxant effects of Cic with those of the potassium channel opener BRL 34915 on two different potassium activations (K+ 20 / K+ 80 mmol/1) and using glibenclamide as antagonist to the effects of BRL 34915, clear differences between the two substances were observed. In all preparations Cic inhibited those components of activity which are sensitive to the calcium channel inhibitor nifedipine (10.6 mol/l), which is consistent with the results of Noack et al. (1991). Cic does, however, also inhibit with typical potency (half inhibition at a concentration around 10.4 tool/l) those nifedipine-resistantcomponentsin vascular smooth muscle which can be suppressed by sodium nitroprusside (SUP, 104 mol/l), for example in rabbit lilac artery, and even those nifedipine-resistant processes in some types of smooth muscle which cannot be suppressed by SUP (rabbit portal vein during strong stimulation with noradrenaline and guinea-pig uterus during strong stimulation with oxytocin). On the other hand Cic does not inhibit the nifedipine-resistant component in the acetylcholine-stimulatedactivity of guinea-pig fundus although SUP is very effective under these conditions. Altogether, Cic has a unique pattern of inhibitory action on vascular smooth muscle and can, in addition to the nifedipine-sensitive processes of calcium activation, also suppress the different types of nifedipine-resistant processes in smooth muscle, and in the latter part it has a clear preferential effect on vascular smooth muscle. This work was supported by the Deutsche Forschungsgemeinschaft(Go 130/30-2). Cicletanine was supplied by Intersan, Ettlingen. Department of Physiology, University of Marburg, Deutschhausstr. 2, W-3550 Marburg/Lahn, FRG

317 ENDOTHELIUM-DEPENDENT RELAXATION OF ARTERIES BY 5-HT RECEPTOR AGONISTS E. Glusa

PIG

PULMONARY

The aim of the present studies was to demonstrate the effects of 5-HT and analogues on isolated pig PUlmonary arteries with and without endothelium and to characterize the receptors accounting for. Ring segments (3 mm in length) from small side branches of the pulmonary artery from pig were contracted by PGF2~ ~3 ~mol/l). The integrity of the endothelium was assessed by the bradykinin (I0 nmol/l)-induced relaxation of PGF2_ -precontracted vessels. In pulmonary arteries wlh~h intact endothelium 5-HT caused a rapid reversible relaxation in the concentration range of 0.i-i00 nmol/l, at higher concentrations the contractile response prevailed. After mechanical removal of the endothelium relaxation was absent. Pre~ncubation of the vessels with methylene blue or N -nitro-L-arginine inhibited the relaxant effect. The relative agonist potency for relaxation was C~-methyl-5-HT > 5-HT > 5-methoxytryptamine > 5-carboxamidotryptamine > 8-OH-DPAT. Indomethacin, pindolol, phentolamine and ICS 205930 did not interfere with the relaxant effect of the 5-HT recept o r agonists. Ketanserin (i ~mol/l) inhibited only the contractile response, while mesulergine (i ~mol/l) blocked the relaxant as well as the contractile effect of 5-HT and ~-methyl-5-HT. The most potent antagonist for relaxation and contraction was methiotepin (0.i ~mol/l). The results suggest that 5-HT receptor agonists caused a biphasic effect on pulmonary arteries. Presumably, vasocontraction is due to activation of 5-HT 2 receptors. The 5-HT receptors at endothelial cells mediating the relaxant response via release of EDRF exhibit similarities to 5-HTIc receptors. Institute of Pharmacology and Toxicology, Medical Academy Erfurt, Nordh~user Str. 74, O-5010 Erfurt

R 128 318

32O

ENDOTHELIN PLASMA LEVEL IN CHD AND CHANGESBY PTCA

INVOLVEMENT OF cAMP IN B2-ADRENOCEPTOR - BUT NOT ANGIOTENSIN II RECEPTOR - MEDIATED FACILITATION OF NORADRENALINE RELEASE IN HUMAN ATRIUM. E. Schwertfeger, G. Fraedrich*, P. Sehollmeyer and L.C. Rump ............................................................... The aim of this study was to test B-adrenoeeptor and angiotansin (A) II receptor modulation of noradrenaline (NA) release from sympathetic nerves in human atria. Slices of human atrium (9.2 +_ 0.3 rag, n,~169) were incubated with JH-NA and electrically stimulated (10 Hz, 38 mA, 60 s) in superfusion chambers between two platinum electrodes. The slices stored radioactivity (210 000 + 6000 dpm/mg). The S-I outflow of radioactivity in the absence of drugs was 0.81 +_0.05 % of the total tissue radioactivity. 6-hydroxy-dopamine pretreatmant to destroy sympathetic nerve terminals, omission of Ca++ and addition of teSrodotoxin (1 #M) abolished S-I outflow of radioactivity. Separation of "H-NA from its metabolites showed ~at 82.6 _+3.1% (n=6) of the S-I outflow of radioactivity was intact 3H-NA. Thus, the S-I outflow of radioactivity was taken as an index of endogenous NA release. The 131, B2-adrenoceptor agonist isoprenaline (0.001-0.1 /~M) dose dependently enhanced NA release to maximally 146 + 9 % (n=7) of control. The concentration response curve of isoprenaline was shifted to the right by the 82adrenoceptor antagonist ICI 118551 (0.01-0.1 /~M; estimated pA 2 = 9.3) but not by the Bl-adrenoceptor antagonist atenolol (0.3-30 ~M). A I I (0.01 -1 #M) also dose dependently enhanced NA release to maximally 146 + 15 % (nffi6) of control and this concentration response curve was shifted to the right by the A II receptor antagonist saralasin (0.1 ~M; estimated PA2= 7.45). The cell-permeable cAMP analogue 8-bromo cAMP (300 /~M) in combination with the phosphodiesterase inhibitor 3isobutyl-l-methyl xanthine (IBMX, 90 /~M) enhanced NA release to 170 + 9 % (n=8) of control. In the presence of this saturating concentration of 8-bromo-cAMP the facilitatory effect of isoprenaline was inhibited but A I I still enhanced NA release. The data suggest that NA release from sympathetic nerves in human atria is facilitated through prejunctional 82-adrenoceptors and A I I receptors. Prejunctional 82-adrenoceptors but not A I I receptors seem to be linked to activation of an adenylate cyclase pathway.

F. Gradaus, D. Weilinz, B. Brisse Clinical studies gave evidence, that reduction of local myocardial perfusion in CHD may be important for the systemic vascular regulation. In this context the influence of changes in coronary perfusion caused by PTCA on endothelin plasma levels was determined. In 18 patients (16 m, 2 f, 56.1 y) undergoing successful PTCA, the basic level of endothelin i, 2 (peripheral vein, RIA, fmol/ml) was assessed (a = before PTCA, b = 2 days after PTCA). [coronary ~rteries: LAD = Left anterior descendant, RCx =Ramus circumflexus, RCA = Right coronary artery] Results:

Change in ET

(b - a ) :

I

LAD

I

RCx/RCA

(pos.)

[

5

I

1

no change: • 0.5 fmol/ml

]

3

[

1

decrease

[

0

1

8

increase

(neg.)

[Chi 2= 11.6 , p = 0.03; LAD compared with RCx/RCA] In patients with stenosis of LAD the mean ET plasma level increased from 4.0 to 5.9 fmo!/ml [p = 0.04], whereas patients with stenosis of RCA or RCx showed a decrease from 5.7 to 4.9 fmol/ml [p = 0.i0]. These data suggest that changes of local myocardial per[usion by PTCA lead to changes in endothelin plasma levels, which seem to depend on the location of coronary stenosJs . Medizinische Klinik C, Universit~tsklinik Albert-Schweitzer-StraBe 83, 4400 Miinster

Minster,

Universitfitsklinik Freiburg, Inhere Medizin IV und (*) Herz- und Gef/iBchirurgie, Hugstetter Str. 55, D-7800 Freiburg.

319

321 E F F E C T O F T H E E N D O T H E L I U M ON M E M B R A N E

P O T E N T I A L OF S M O O T H MUSCLES OF RAT AORTA

P. Krippeit-Drews, N. Morel

and T. Godfraind

The e n d o t h e l i u m is well k n o w n to m o d u l a t e the contractile beh a v i o u r of v a s c u l a r s m o o t h muscle cells (Godfraind et al., Clin Sci 68:653, 1985). However, it is n o t yet well established w h e t h e r a n d to w h a t e x t e n t t h e e n d o t h e l i u m derived r e l a x i n g factor ( E D R F ) c a n i n f l u e n c e t h e m e m b r a n e p o t e n t i a l of v a s c u l a r s m o o t h m u s c l e s (Taylor et al., Br J P h a r m a c o l 94:853, 1988). We c o m p a r e d m e m b r a n e p o t e n t i a l s of r a t a o r t a m e a s u r e d by two different m e t h o d s : c o n v e n t i o n a l microelectrode t e c h n i q u e (A) a n d 3 H - t e t r a p h e n y l p h o s p h o n i u m b r o m i d e (3H-TPP+) distribution (B; Morel & Godfraind, B r J P h a r m a c o l 102:467, 1991). N~n i t r o - L - a r g i n i n e (NO-Arg) w a s u s e d to block t h e p r o d u c t i o n of EDRF. M e m b r a n e potentials u n d e r control conditions were -63.4 _+ 1.3 m V (n=10, A) a n d -65.7 -+ 1.3 mV (n=10, B). F o r m e t h o d A, addition of NO-Arg (10 -4 mol/l) decreased the potential to -58.6 + 1.1 m V (n=10) w i t h i n 15.0 + 1.2 rain after onset of depolarization. W i t h m e t h o d B, t h e potential w a s depolarized to -60.9 _+ 1.0 m V (n=8) a f t e r a n i n c u b a t i o n t i m e of 30 m i n w i t h N O - A r g (10 -4 molfl). S u b s e q u e n t application of s u b s t a n c e s which, a t l e a s t i n p a r t , m i m i c t h e effects of E D R F led to a r e p o l a r i z a t i o n of the m e m b r a n e potential. It increased from -57.3 _+ 1.5 m V to -63.5 + 1.7 m V (n=4) a f t e r a d d i t i o n of linsidomine (SIN1, 10 -5 moU1) w i t h m e t h o d A a n d to -66.4 + 1.5 m V (n=10) by application of 8Br-cGMP (10 -4 moU1) w i t h m e t h o d B. The r e s u l t s obtained w i t h b o t h m e t h o d s are in good agreement. We therefore conclude t h a t m e t h o d B m i g h t be a useful tool w h e r e m e a s u r e m e n t s of m e m b r a n e potential w i t h microelectrodes are too difficult. F r o m our r e s u l t s we f u r t h e r conclude t h a t E D R F p e r m a n e n t l y hyperpolarizes t h e m e m b r a n e of s m o o t h m u s c l e s of r a t a o r t a by 4-10 mV.

Laboratoire de Pharmacologie, Universitd Catholique de Louvain , Av. E. Mounier 73, B-1200 Brussels (Belgium)

POTASSIUM ION ANTAGONISM AS A NEW PRINCIPLE THERAPY OF CIRCULATORY DISTURBANCES

OF

Elfriede Leniger-Follertl A s p r e v i o u s l y p u b l i s h e d ( L e n i g e r - F o l l e r t 1983) p o t a s s i u m i o n s e x e r t a d u a l e f f e c t on t h e t o n e s m o o t h m u s c l e c e l l s of c e r e b r a l a r t e r i a l a n d arteriolar vessels dependent on extracellular

potassium ion activity

of

( [ ~ * ~ . with increasing [K~

in t h e r a n g e of 0-20 m M t h e d i a m e t e r of c e r e b r a l arterial vessels increases until a maximal vasodilatation occurs and therefore microflow i n c r e a s e s a c c o r d i n g to t h e l a w of H a g e n - P o i s e u i l l e . Further i n c r e a s e of ~ 3 Q a b o v e 20 m M l e a d s t o t h e w e l l k n o w n d e p o l a r i z a t i o n of s m o o t h m u s c l e c e l l m e m b r a n e s a n d t h u s e f f e c t s an i n c r e a s i n g c o n s t r i c t i o n of c e r e b r a l a r t e r i a l m a c r o - a n d m i c r o v e s s e l s . A b o v e 50 m M ~ K § no-reflow phenomenon in t h e b r a i n occurs. T h e a i m of o u r s t u d i e s s i n c e 1983 w a s t o f i n d a pharmaceutical which can combate the circulatory disturbance and thus reopen the occluded vessels. W e w e r e s u c c e s s f u l in f i n d i n g a n a d e q u a t e p o t a s s i u m ion exchanger which can reestablish the normal p o t a s s i u m ion a c t i v i t y in t h e e x t r a c e l l u l a r space. Thus, t h i s n e w p r i n c i p l e of K ~ a n t a g o n i s m is s u i t a b l e f o r t h e t r e a t m e n t of a l l k i n d s of circulatory disturbances caused by the secondary K~ c o n s t r i c t i o n of v e s s e l s o c c u r r i n g a f t e r a n y p r i m a r y i s c h e m i c o r h e m o r r h a g i c event. Max

Planck-StraBe

68,

5800 H a g e n

R 129 322

323

LOCAL OXYGEN CONSUMPTION OF THE NORMAL AND ISCHEMIC SKIN OF PATIENTS WITH ARTERIAL OCCLUSIVE DISEASE

Functional role of NO-dependent cyclic G~IP synthesis in platelets

(AOD) Elfriede Leniqer-Follert Previously, we were able to show that local oxygen consumption of the brain cortex can be determined quantitatively from the slope of p02 decrease during a short period of complete ischemia, provided that local oxygen pressure was artificiallyx increased initially above the p02 where hemoglobin releases the chemically bound oxygen(Leniger-Follert, Eo, Pfl~gers Arch., 1977). The aim of the present investigations was to examine whether the above mentioned method can also be applied to patients suffering from AOD to determine the local oxygen rate of the ischemic skin. Experimental procedure: During inspiring of pure oxygen the time course of local cutaneous p02 was continously recorded on the skin with a multiwire surface electrode of the Clark type according to LHbbers and Huch.After a steady state of tissue p02 has been established the femoral circulation is completely arrested by a tourniquet. From the sloper of the p02 decrease oxygen consumption v is calculated according to v = ~ p02x~02xl00x60/At ~-02= solubility coefficient for oxygen Results and conclusions: l. By applying the described method it is possible to determine the local oxygen consumption of the normal and ischemic skin in patients, povided a sufficient perfusion is present. 2. v of the normal skin is about 0.2 ml 02 per i00 g and min. 3. ~ of the ischemic skin is significantly decreased in dependence on the extent of the AOD. 4~If local skin p02 does not increase during oxygen respiration this feature can be used in clinical investigations as a diagnostic criteria for insufficiency of microcirculation of the skin.

S. Moncada, The Wellcome Research Laboratories, Langley Court, Beckenham, Kent BR3 3BS, U.K. The synthesis of nitric oxide (NO) from L-arginine via a constitutive NO synthase enzyme occurs in endothelial cells and brain synaptosomes where it acts as the endogenous transduction mechanism for the stimulation of the soluble guanylate cyclase. Nitric oxide synthesis is also expressed by macrophages, vascular endothelial and smooth muscle cells and a number of other cells after activation by endotoxin or cytokines. The NO released by this inducible enzyme acts as a cytotoxic molecule for invading microorganisms and tumour cells and contributes to pathological vasodilatation and tissue damage. When platelets are stimulated to aggregate they also synthesize NO from L-arginine. The enzyme responsible for this synthesis resembles the constitutive NO synthase in endothelial cells and brain tissue in that it is Ca2+- and NADPH-dependent and can be inhibited by some analogues of L-arginine. Nitric oxide inhibits platelet aggregation and adhesion by stimulation of the soluble guanylate cyclase. Platelet aggregation in vivo is likely to be regulated by intraplatelet NO, as well as by NO and prostacyclin released from vascular endothelium. The combined action of these two mediators could result in a synergistic suppression of intracellular Ca2+ elevation and inhibition of platelet aggregation. Thus, the use of agents that increase the activity of the adenylate and guanylate cyclase offer interesting strategy for the development of effective anti-piatelet drugs.