R1

1

3

M U S C A R I N I C A C E T Y L C H O L I N E R E C E P T O R S AS M O D E L SYSTEMS F O R THE STUDY OF G P R O T E I N - C O U P L E D RECEPTOR FUNCTION J. Wess Muscarinic acetylcholine receptors were used as model systems to study how G protein-coupled receptors (GPCRs) function at a molecular level. The mechanisms governing receptor assembly and the selectivity of receptor/G protein interactions were studied by using a combined molecular genetic/pharmacological approach. To test the hypothesis that GPCRs consist of several independent folding units, the m3 muscarinic receptor was "split" in all three intracellular and all three extracellular loops. Coexpression studies in COS-7 cells showed that several of the resulting polypeptide pairs were able to form complexes that were capable of binding muscafinic ligands and activating G proteins. These data are consistent with the notion that GPCRs are composed of multiple structaral sabunits. Characteristically, each GPCR can recognize and activate only a distinct subset of the many structurally closely related G proteins expressed within a cell. To study the structural basis underlying this selectivity, the m2 (GUocoupled) and m3 (Gq-coupled) muscarinic receptors were used as model systems. By employing a combination of various different rnutagcnesis approaches, single amino acids responsible for this functional diversity were identified. To identify specific sites of contact between a receptor and its cognate G protein(s), the ability of a series of hybrid m2/m3 muscarinic receptors to interact with various wild type and mutant Ga-subunits was examined in transfected COS-7 cells. These studies showed that a very short region on the m2 muscarinic receptor can recognize the C-terminal five amino acids of Gc~o (Gcd). We could demonstrate that this interaction is essential for coupling selectivity and G protein activation.

MOLECULAR ASPECTS OF HISTAMINE RECEPTOR SIGNALLING R. Leurs, M.J. Smit, Ton ter Laak, E. Roovers and H. Timmerman The actions of histamine are mediated by three receptor subtypes, the H l, H2 and H3 receptors. In recent years the genes, encoding the H 1 and H2 receptor have been cloned and it appears that both receptors belong to the supergene family of G-protein coupled receptors. The availibility of the genes, encoding the two histamine receptor subtypes have greatly extended the possibilities for a molecular approach of histamine receptor signalling.The use of transfected cell lines have elucidated multiple signalling pathways for both receptor subtypes. Moreover, site-directed mutagenesis of the H2 receptor proteins revealed several structural features of the receptor proteins that are important for receptor signalling. Moreover, the use of transfected cell lines and various receptor mutants resulted in a detailed insights for the regulation of the H2 receptor function. Moreover, the use of mutant H l receptors revealed the importance of several amino acids in the fifth transmembrane domain for ligand binding. Extrapolating the known interaction of two serine residues in the fifth transmembrane domain of the beta-adrenoceptor with beta-agonists led us to examine the role of the amino acids in equivalent positions in the H lreceptor. For threonine 204 no functional role could be established. Yet, asparagine 207 is involved in the binding of the imidazole ring of histamine. Combining these results with a computer-modelling s t u ~ of the fifth transmembrane domain resulted in the identification of lysine2°'' as a putative, second interaction point for the imidazole ring of histamine. Mutating this residue confirmed this prediction. Yet, asparagine207 and lysine200 appear not to be involved in the interaction with all Hlagonists. Various residues in the third, fifth, sixth and seventh transmembrane are currently being investigated for their potential interaction with H 1 agonists.

Laboratory of Bioorganic Chemistry, NIH-NIDDK, Bethesda, MD 20892, U.S.A.

Leiden/Amsterdam Center of Drug Research, Division of Medicinal Chemistry, Department of Pharmacochemistry, Faculty of Chemistry, Vrije Univ~rsiteit, De Boelelaan 1083, 1081 HV Amsterdam

2

4

M O L E C U L A R BIOLOGY OF RECEPTOR ACTIVATION AND INACTIVATION

LH RECEPTOR MUTATIONS A.P.N. Themmen, R. Kraaij, H. Kremer* & H.G. Brunner* The family of glycoprotein hormones (luteinizing hormone, LH; folliclestimulating hormone, FSH; thyroid-stimulating hormone, TSH) bind to a subfamily of the GTP binding protein coupled receptors. This subfamily is characterized by a large extracellular domain that is encoded by 9 or 10 exons, which is involved in high affinity binding of the large (-30 kD) ligand. LH regulates steroid hormone production in the male and female gonads, and triggers ovulation in the female. We have investigated the possible role of LH receptor mutations in two syndromes that affect sex differentiation in males: familial male-limited precocious puberty (autosomal dominant, FMPP) and Leydig cell hypoplasia (autesomal recessive, LCH). FMPP, also called testotoxicosis, is characterized by precocious puberty, with concomitant phenomena including rapid growth, increased skeletal age and a large penis. LCH results in an opposite phenotype: 46XY individuals with feminized or ambiguous external genitalia, a blind-ending vagina and absence of breast tissue, but with inguinal testes complete with epididymis and vas deferens. In both syndromes, we have identified missense mutations in the transmembrane domain of the LH receptor. In the case of FMPP, all three identified mutations (MetS71-->lle, AspSZS-->Gly, Met39S->lle) resulted in an LH receptor protein that displayed constitutive activity, i.e. transfection of the cDNA into a heterologous cell system resulted in an increase of cAMP production in the absence of the ligand hCG, although the receptor was still responsive to hCG. The opposite was found in the case of LCH: the mutated LH receptor (Met593~Pro) displayed normal basal cAMP production but did not respond to hCG at all. These results indicate that LH receptor mutations are the cause of Leydig cell hyper- or hypoactivity observed in FMPP and LCH patients.

M. J. Lohse Signal transmission via G-protein-coupled receptors is a highly regulated process that is initiated by agonist binding, results in activation of Gproteins, and is terminated either by dissociation of the llgand or by a variety of intracellular processes that reduce receptor function. Receptor activation occurs by agonist binding to a defined set of amino acids within the receptor structure. The mode of (-)isoproterenol binding to the human 132-adrenergic receptor will be discussed as a prototype for such a binding and activation reaction. This binding occurs via at least 4 attachment points, which correspond to the NH3-function, the two catechol OH-groups and the stereospecific 13-OH group in isoproterenol. Interactions of the I~-OH group with the 6th transmembrane helix may be relevant both for stereoselective agonist recognition and for receptor activation.The transmission of such agonist binding to the cytosolic loops of the receptor, which interact with the G-protein, will be discussed. Receptor desensitization is triggered by phosphorylation of the receptor which can be catalyzed by the effector kinases, i.e. by protein kinase A and protein kinase C, or by specific kinases termed the B-adrenergic receptor kinases (BARK). The BARKs require specSfic membrane targeting by G-protein 13- and T-subunits. There appear to be specific interactions between some of these kinases and defined G-protein 13- and y-snbunits. Phosphorylation of receptors by the GRKs enhances the affinity of the receptors for a set of cytosolic inhibitor proteins called arrestins. Binding of an arrestin molecule to a phosphorylated receptor causes disruption of receptor/G-protein-coupling. The reversal of receptor desensitization seems to involve agonist-induced internalization of the receptors, followed by dephosphorylation and finally recycling to the cell surface. Regulation of signal transduction at the G-protein level can be exerted by phosducin, a cytosolic protein that also binds to G-protein 13- and l(subunits. Binding of phosducin to G-proteins is itself controlled via phosphorylation of phosducin by PKA. Phosducin can compete with 13ARK for G-protein binding and can thereby interfere with the desensitization process. Institute of Pharmacology, University of Wiirzburg, 97078 WiJrzburg.

Dept Endocrinology & Reproduction, Erasmus University Rotterdam, PO Box 1738, 3000 DR Rotterdam, The Netherlands, and *Dept Human Genetics, Academic Hospital Nijmegen, The Netherlands

R2 5 LOCALIZATION AND POSSIBLE FUNCTION OF NITRIC OXIDE SYNTHASE IN RAT CENTRAL AND PERIPHERAL NEURONS N. J. Dun, S. Y. Wu, S. L. Dun and U. F6rstermann Immunoreactivity to neuronal nitric oxide synthase (NOS) or isoform I was detected in neurons of the rodent brain, spinal cord and peripheral autonomic and sensory ganglia. In the brain, the heaviest concentration of NOS-IR neurons was noted in several pontine tegmental nuclei. In the hippoeampus, NOS-IR was detected in dentate hilar neurons and in neurons other than pyramidal neurons of the CA1 and CA3 areas. In the spinal cord, NOS-IR was present in sympathetic preganglionic neurons (SPNs), neurons of the superficial layers of dorsal horn and lamina X; it appeared to be absent in ventral horn motoneurons. Sympathetic ganglionic neurons in the paravertebral ganglia were surrounded by NOS-IR nerve terminals which had their origin in SPNs. A majority of sympathetic ganglionic neurons exhibited a low-to-moderate level of NOS-IR. NOS-IR was detected in some of the nodose and spinal ganglion cells. The presence of NOS-IR in SPNs provided an accessible mode to evaluate the function of NO in these synapses. Whole-cell patch recordings were made from antidromically identified SPNs in thoracolumbar spinal cord slices of 12-16 day old rats. Excitatory and inhibitory postsynaptic currents (EPSCs & IPSCs) evoked in SPNs were potentiated by bath application of L-arginine (L-arg, 300 #M) and sodium nitroprusside (SNP, 100/zM) and depressed by the NOS inhibitor NW-Nitro-L-arginine (30/zM). Prior treatment of the slices with hemoglobin (10/~M) blocked the potentiating effect of L-arg or SNP. Superfusing the slices with cyclic GMP (300/~M) in the presence of the phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine (750/xM) mimicked the potentiating effect of L-arg and SNP. The results suggest that NO released from SPNs may tonically potentiate excitatory as well as inhibitory synaptic responses in these neurons, probably by increasing excitatory and inhibitory transmitter release via a cyclic GMP-dependent mechanism.

7 NITRIC OXIDE AS A NANC NEUROTRANSMITTER IN THE GASTROINTESTINAL TRACT AND OTHER TISSUES H. Bult Part of the regulation of gastrointestinal motility is provided by nonadrenergic non-cholinergic (NANC) nerves. Stimulation of these elicits membrane hyperpolarization, called inhibitory junction potentials, and relaxation. The neurotransmitters that mediate the inhibitory NANC transmission have been a controversial subject for 30 years. Evidence to support the idea that nitric oxide (NO), in addition to vasoactive intestinal polypeptide and ATP may initiate NANC responses will be presented. A superfusion bioassay was developed to detect and characterize the inhibitory neurotransmitter released in response to electrical field stimulation of the canine ileocolonic junction and the rat gastric fundus. This technique provided evidence that NANC nerve stimulation causes the release of a vasorelaxant factor with pharmacological and physicochemical properties similar to NO. Its release is blocked by inhibitors of NO biosynthesis and enhanced by L-arginine. Its half life was comparable to that of NO, and enhanced by superoxide dismutase, its activity is abolished by haemoglobin. In addition, the transferable factor and authentic NO are similarly affected by the superoxide anion generator pyrogal!ol and by Lcysteine. On the other hand, S-nitrosothiols were different from the nitrergic factor and NO with respect to their pharmacological profile, indicating that NO, and not a nitrosothiol is released from the inhibitory NANC nerves. The release of the nitrergic factor is Ca2+-dependent and prejunctionally regulated by K+ channels and e2-adrenoceptors. Finally, immunohistochemicai evidence for the presence of NO synthase containing neurons in the myenteric plexus of the gastrointestinal tract and for the nitrergic innervation of smooth muscle in the genito-urinary system, trachea and some blood vessels (penile and cerebral) will be reviewed.

N. J. Dun, Dept. of Anatomy & Neurobiology, Medical College of Ohio, Toledo, OH 43614 USA

Univ. of Antwerp (UIA), Div. of PharmacoL, B-2610 Wilrijk, Belgium.

6

NITRIC OXIDE AS A MESSENGER IN LTP AND MEMORY FORMATION. G. Andrees B6hme The hippocampal synaptic plasticity phenomenon called long-term potentiation (LTP) is thought to be involved in certain forms of learning and memory, and depends upon activation of NMDA receptors. Because the diffusible intercellular messenger nitric oxide (NO) is also produced by neurons upon activation of NMDA receptors, it has been suggested that this molecule may play the role of a retrograde messenger during post-to-pre-synaptic signalling in LTP. We have demonstrated that this is indeed true by showing that blockade of endogenous NO-formation in rat hippocampal slices in vitro by exposition to NO-synthase (NOS) inhibitors or reduced haemoglobin blocks LTP. Moreover, we have shown that mimicking local NO formation by using NO-releasing compounds of various chemical structures produces a potentiation similar to electrically-induced LTP. In addition, this effect is dependent on pre-synaptic afferent activity. Behavioural experiments indicate that treatment of rats with the NOS-inhibitor Nco-nitro-L-arginine (L-NA) at systemic doses blocking LTP ex-vivo affect hippocampus-dependent learning tasks such as spatial and olfactory learning, but is inactive in learning test depending less on this brain structure such as shock-avoidance conditioning. Examination of the effects of L-NA given either before or after a learning task reveals that NO formation is necessary for the storage of new memories, but not for the recall of previously acquired information. These data suggest that NO is a physiological messenger involved in memory formation via its role in the process of hippocampal LTP. Rh6ne-Poulenc Rorer S.A, Neuroscience Research Program, Centre de Recherches de Vitry Alfortville, 94403 Vitry-Sur-Seine, France.

8 NITRIC OXIDE AND MIGRAINE E. Marcel van Gelderen and Pramod R. Saxena

The migraine syndrome is characterized by recurrent attacks of pulsatite and throbbing headache, often preceded by well defined neurosensory aura symptoms. These symptoms are associated with a reduction in cerebral blood flow and changes in neuronal activity. In contrast, the headache phase of migraine involves a dilatation of large extracerebral cranial arteries and arteriovenous anastomoses. The ensuing increase in amplitude of pulsations may account for the stimulation of perivascular sensory nerves to release neuropeptides and cause headache and other associated symptoms. Although direct evidence is still lacking, observations in man suggest that nitric oxide (NO) may be involved in the vascular events of migraine headache. Numerous stimuli can release NO by activating NO-synthase (NOS), which is expressed in endothelial and neuronal cells. Histamine stimulates NO production in isolated cranial blood vessels, enhances vascular pulsations and causes headaches in migraineurs. Similarly, nitrovasodilators, acting through NO release, induce a dose-dependent dilatation of temporal arteries with accompanying headache. Moreover, nitroglycerin-induced middle cerebral artery dilatations and headache are more pronounced in migraineurs relative to healthy subjects. Hence, migraineurs appear highly sensitive to both exogenous and endogenous NO. In addition, animal studies have shown that in the cranial circulation inhibition of NOS reduces blood flow through arteriovenous anastomoses. Likewise, under low anastomotic flow conditions, NO donors increase blood flow through arteriovenous anastomoses and enhance carotid pulsations. The unilateral nature of migraine headache suggests the involvement of neuronal pathways and immunohistochemical studies show the presence of nitroxidergic nerves surrounding cranial arteries. These nerves appear to originate from the sphenopalatine ganglion, since destruction of the ganglion abolishes both NOS-staining and vasodilator activity. Taken together, nitroxidergic nerves m~iy play an important role in the headache phase of migraine and NOS-inhibitors may be potentially effective in migraine. Department of Pharmacology, Erasmus University Rotterdam, P.O.box 1738, 3000 DR Rotterdam, The Netherlands.

R3

9

11

RECEPTOR- AND G-PROTEIN-MEDIATED REGULATION OF CALCIUM CHANNELS: IDENTIFICATION OF THE G PROTEINS INVOLVED. G. Schultz, F. Kalkbrenner, V. Degtiar, B. NOrnberg, B. Wittig.

TRANSDUCTIONAL CROSS-TALK AND CALCIUM SIGNALLING IN AIRWAY SMOOTH MUSCLE CELLS. H. Meurs, B.H. Hoiting, C.R.S. Elzinga, and J. Zaagsma. In airway smoothmuscle, stimulationof contractile receptors, such as muscarinic M3 and histamine H1 receptors, leads to activation of phospholipase C to generate the second messengers inositol 1,4,5-trisphosphate(IP3) and diacylglycerol (DAG). IP3 mobilizes Ca2+from intraceUular stores causing a rapid, transient rise in intracelhilar Ca2+concentration ([Ca2+]i) and initiation of contraction, while a sustained influx of Ca2+ and DAG-induced protein kinase C (PKC) activation are involved in tonic contraction. On the other hand, adenylyl cyclase (AC) activation and subsequent cAMP production and activation of protein kinase A (PKA) are involved in airway smooth muscle relaxation by 13-adrenoceptoragonists. Except that these transduction mechanisms lead to the functional responses by regulating the phosphorylationstate of myosin, there is increasing evidence for cross-talk between these signalling pathways. Thus, in isolated bovine tracheal smooth muscle (BTSM) cells, we demonstrated that both methacholine (MeCh)- and histamine (His)-induced C a 2+ mobilization and influx are concentration-dependently inhibited by the cAMPelevating agents isoprenaline (Iso), forskolin (Fors) and 8-Br-cAMP, when added prior to the contractile agonists. Since these agents had no or only minor effect on contractile agonist-induced PI metabolism, it was concluded that cAMP-activated PKA is mainly effective at a different level of the Ca2+homeostasis. The functional relevance of the observed interaction was indicated by a significant correlation between the Iso-mediated inhibition of contractile agonist-induced [Ca2+]i changes and the Iso-induced relaxation of BTSM contraction by these agonists. For Iso, but not for Fors or 8-Br-cAMP, the inhibition of MeCh-induced Ca2+ influx was markedly reduced when added after the contractile agonist, during the steady-state rise in Ca2+. The reduced inhibition could be reversed by the selective PKC inhibitor GF 109203X, indicating that MeCh-induced PKC activation may cause a reduced 13adrenoceptor function. This is in line with our previous observation of a significant relationship between the potency of various muscarinic agonists and histamine to induce PI metabolism and the potency of these agonists to reduce pD2 and Em~ values of Iso-induced relaxation. These results indicate that transductional cross-talk at the level of Ca2+ signalling in airway smooth muscle plays a major role in the functional antagonismbetweencontractile and relaxing stimuli.

Pertussis toxin(PT)-sensitive G proteins are involved in apparently direct stimulatory and inhibitory hormonal effects on voltage-dependent calcium channels (VDCC) in endocrine, neuronal and possibly other systems. Whereas stimulation of VDCC in endocrine cells is apparently mediated by G~2 and protein kinase C (stimulated within the concurrent PI response) (Gollasch et al., Proc. Natl. Acad. Sci. USA 90, 6265, 1993), Go is involved in inhibitions of endocrine (L-type) and neuronal (N-type) VDCC. Identification of the heterotrimeric G proteins involved in these modulations became possible by application of antisense oligonucleotides directed against the mRNA of G-protein subunits. Expression of G-protein a-, 13- or y-subunits was suppressed by microinjection of antisense oligonucleotides; two days after application, VDCC modulations were recorded by the whole cell modification of the patch clamp technique. Applying this antisense technique, a Go subform consisting of o~o213~y3 was found to be involved in the inhibitory somatostatin effect on VDCC in rat pituitary GH3 cells; the carbachol effect via muscarinic M4 receptors apparently involved c~0113ay4 (Kleuss et al., Science 259, 832, 1993). In contrast, the inhibitory galanin receptor apparently interacted with cto~ 13z72 and in addition (but less efficiently) with Ctol 133"/4. The assumption of specific roles of Go1 and Go2for coupling different receptors to VDCC was supported by the ability to reconstitute, in PT-pretreated cells, the carbachol and galanin effects and the somatostatin effect by Gcto~ and Gao2, respectively. Similar data were obtained in the rat insulinoma cell line, RINm5F, which in contrast to GH3 and other cell lines expresses far more Gc~o~than Gcto2. Here the same Go heterotrimers were found to be involved in the somatostatin and galanin effects as identified in the GH3 cells. These data show that Go~ and Go2 play different roles in the functional coupling of different sets of receptors and that the involvement of Go~ and Go= is independent of the relative amounts of Gc%~ and Gcto2 expressed, tn addition, the data clearly indicate that heptahelica[ receptors interact with specific G-protein heterotrimers. From preliminary studies it is likely that receptors interact with similar selectivity with G protein heterotrimers other than Go. Institut for Pharmakologie, Freie Universit~t Berlin, D-14195 Berlin, Thielallee 69-73, FRG

Department of Medicinal Chemistry and Molecular Pharmacology, University of Groningen, Ant. Deusinglaan2, 9713 AW, Groningen,The Netherlands.

10

12

CYCLIC NUCLEOTIDE-GATED CATION CHANNELS IN CENTRAL AND PERIPHERAL TISSUES Martin Biel, Xiangang Zong and Franz Hofmann

5-HT3 RECEPTOR LIGAND-GATED ION CHANNELS H.P.M. Vijverberg, A.R. Kooyman and J.A. van Hooft The serotonin 5-HT3 receptor, a ligand-gated ion channel, is expressed in neuronal cells, including periphel'al and central neurons and various neuronal cell lines. The ligand-binding profile of 5-HT3 receptors in the mouse neuroblastoma clone N1E-115 strongly resembles that of 5-HT3 receptors in the mammalian central nervous system. Agonist interaction, ion channel properties and modulation of the function of 5-HT3 receptors have been investigated in cultured N1E-115 cells using mainly electrophysiological techniques. Application of 5-HT3 receptor agonists to whole-cell voltage clamped N1E-115 cells evokes a transient ion current, which desensitizes in the continuous presence of low concentrations of the agonists. The endogenous monoamines dopamine and tryptamine are partial agonists of 5-HT3 receptors. The partial and full agonists act on the same population of 5-HT3 receptors, but induce different frequencies of, otherwise identical, single channel events. Allosteric modulation of the ligand-gated ion channel by the aromatic moiety of 5-HT, 5-hydroxyindole, leads to a reduction of agonist-induced desensitization of the 5-HT3 receptor. Conversely, small quaternary alkylammonium ions, resembling the cationic moiety of 5-HT3 receptor agonists, are competitive antagonists. Although the 5-HT3 receptor forms a large cation channel, which is permeable to a range of monovalent cations, its single channel conductance in excised membrane patches of neuroblastoma cells is very low. In cellattached patches exposed to 5-HT the same low as well as three higher conductance levels are observed. The four open levels appear to be subconductant states of the same ion channel and their relative probabilities of occurrence depend on cytoplasmic factors. Modulation of protein kinase activity of N1E-115 cells by stanrosporine and by the phorbol ester PMA shows that protein phosphorylation alters the conductance level of the 5-HT3 receptor Iigand-gated ion channel.

Cyclic nucleotide-gated (CNG) cation channels represent a class of nonselective cation channels that are directly activated by the binding of cGMP or cAMP. Initially, these channels have been characterized by electrophysiological means and molecular cloning in photoreceptor cells and olfactory neurons. There is now an increasing body of evidence that CNG channels may also play functional roles outside sensory systems. We have cloned and functionally expressed the full length eDNA of two CNG channels from rabbit aorta (rACNG) and bovine kidney (CNG3). Both channels display similar electrophysiological properties. However, rACNG is about 40 fold more sensitive to both cGMP and cAMP than CNG3. CNG3 is highly permeable for calcium over a broad range of voltage (-100 mV to +45 mV). Using a combination of northern blot analysis and PCR-based cloning we could show, that rACNG and CNG3 are expressed in several non-sensory tissues including brain. We now have cloned from bovine testis three splicing variants of a modulatory subunit (CNG4c-e, consisting of 939, 930 and 921 amino acids, respectively) that are likely to be associated with CNG3. CNG4c-e are not able to form a functional channel by their own. However, when coexpressed with CNG3 they modulate properties of the CNG3-indnced current including affinity to cyclic nncleotides, sensitivity to Ca2 + and channel blockers. Institut fttr Pharmakologie und Toxikologie, Technische Universit/it Mfinchen, Biedersteinerstr. 29, 80802 M~inchen, Germany.

Research Institute of Toxicology, Utrecht University, P.O. Box 80.176, NL-3508 TD Utrecht, The Netherlands

R4 13

15

ROLE OF T-LYMPHOCYTE DERIVED CYTOKINES IN AIRWAY INFLAMMATION AND HYPERRESPONSIVENESS IN A MURINE MODEL OF ALLERGIC ASTHMA A.J.M. Van Oosterhout1, E.M. HesseP, G. Hofman1, B. Van Esch~, H.F.J. Savelkoul2 and F.P. Nijkamp1 Airway inflammation and hyperresponsiveness (AHR) are two important features of patients with allergic asthma. It has been hypothesized that type 2 Th-lymphocytes which produce cytokines like IL4 and l i e are involved in these symptoms. Recently, we developed an animal model of allergic asthma in the mouse. Sensitization of mice with ovalbumin (OA) induces OA-speciflc IgE antibodies in the serum. Chronic inhalation of OA leads to development of AHR and airway inflammation with predominantly eosinophils. In the present study we investigated the involvement of IL4, IL5 and IFN? in these phenomena. BALB/c mice were sensitized with OA and treated before OA or saline inhalation (once a day for eight days) with antibodies to IL4, IL5, IFN? or control antibody. Twenty-four hours after the last inhalation challenge, the airway responsiveness to methacholine was measured in vivo and numbers of different leukocytes in bronchoalveolar lavage (BAL) fluid determined. In a separate group of animals, at various time-points after the last inhalation the levels of IL4, 11.5 and IFN't in cell-free BAL fluid were measured by ELISA. At six hours after the last OA inhalation, the levels of IL4 and IL5 in BAL fluid were significantly increased compared to saline challenged animals. IFN,/ could not be detected in the BAL fluid. Treatment with anti-IL5 completely inhibited eosinophll infiltration but not AHR. Treatment with anti-IFN? completely inhibited AHR but not eosinophil infiltration. Treatment with anti-IL4 did not affect either of these symptoms. These results demonstrate that IL5 is involved in eosinophil infiltration whereas IFN? is involved in development of AHR in this murine model of allergic asthma. It can be speculated that besides type-2 Th lymphocytes a different lymphocyte subtype producing IFN? may be involved in airway pathology in allergic asthma.

INTERLEUKIN-1 RECEPTORS AND SIGNAL TRANSDUCTION: MODERN CONCEPTS OF ANTI-INFLAMMATORY THERAPIES M.U.Martin and K.Resch The term Interleukin-1 (IL-1) comprises a small family of three polypeptide mediators of the immune system, two of which, I L - l a and IL-113, are potent pro-inflammatory cytokines, whilst the third member, IL-1Ra, is a naturally occuring receptor antagonist. IL-1 has local as weft as pronounced systemic effects, e.g. it induces fever and an acute phase response. IL-1 binds to two distinct plasma membrane receptors on a wide variety of target cells. The type I IL-1 receptor (IL-1RI) transduces signals into the cells by activating a cascade of protein kinases, of which one can be identified associating with the IL-1RI itself. The type II receptor (IL-1RII) seems to bind and remove the ligand, but a function in signal transduction has not been identified so far. Interference into the IL-1 / IL-1 receptor system is conceivable at several stages. Cytokine synthesis and release can be inhibited in the producing cell. Thus, the induction of IL-1 gene activation can be blocked by the use of CSAIDs, the transcription by glucocorticoids, and the release of IL-113 by inhibiting the IL-1 converting enzyme, ICE. Once released, bioactive IL-1 may be masked and cleared by neutralizing antibodies or soluble IL-1 receptor molecules. On the level of the target cell inhibition of receptor binding and triggering of signal transduction can be accomplished by applying recombinant IL-lra, which competes with IL-le or IL-113 for the binding site. A further mode of intervention at this point would be the application of blocking anti IL1RI antibodies. Finally, in the target cell itself, interference into the signal transduction mechanism is a possibility by using low molecular weight inhibitors of protein kinases which are specific for the IL-1 induced pathway. One would abrogate intracellular signal tranduction and amplification resulting in a lack of IL-1 -induced gene activation. Thus, the use of recombinant molecules or kinase inhibitors may open novel additional ways of anti-inflammatory therapies beyond glucocorticoids and NSAIDs.

i Dept. Pharmacology, Faculty Pharmacy, Utrecht University, P.O. Box 80.082, 3508 TB Utrecht 2 Dept. Immunology, Erasmus University, Rotterdam, The Netherlands

14 REGULATION AND MODULATION OF T CELL CYTOKINE PROFILES Martien L. Kapsenberg, Frank G.M. Snijdewint, Catharien M.U. Hilkens, Alies Snijders, Eddy A. Wierenga. The vigour of and the balance between specific cellular and humoral immune responses are determined largely by cytokines secreted by CD4 ÷ T helper (Th) lymphocytes after activation upon recognition of antigen presented by antigen-presenting accessory cells (AC). Subsets of Th cells secreting high levels of the Thl cytokine interferon (IFN)- 7 favour strong cellular responses, whereas high levels of the Th2 cytokines interleukin (IL)-4 and IL-5 favour humoral immunity characterized by the production of antigen-specific IgE (via IL-4) and eosinophilia (via IL5). Th cells with balanced levels of Thl and Th2 cytokines, induce mixed cellular and humoral (without IgE) responses that often act as less specialized immune activities. The level and the balance of Thl and Th2 cytokines is determined by various physiological factors, including early effects of soluble and membrane-bound molecules expressed by AC. Recent data imply tight interactive networks of multiple inducing, suppressing and feedback actions of the cytokines IL-12 and IFN-3, associated with Thl immune responses, and of the cytokines IL-4 and IL-10 combined with the eicosanoid PGE2 associated with Th2 immune responses. In vitro experiments with human cells stress that corticosteroids classdependently inhibit the production both of Thl- and Th2 immune response-associated cytokines. Interestingly, phosphodiesterase inhibitors may differentially modulate cellular and immediate immunity, because of their predominant inhibition of the production of Thl immune responseassociated cytokines. Dept. of Cell Biology and Histology, Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands

Molekularpharmakologie, Medizinische Hochschule Hannover, 30623 Hannover, Germany, FAX: + 4 9 511 532 4081

D-

16 THE ROLE OF CYTOKINES IN PROLIFERATION AND MIGRATION OF VASCULAR SMOOTH MUSCLE CELLS J. Fingerle Balloon catheter injury of the rat carotid artery is widely used as a model to study the role of cytokines in neointima formation in vivo. It was found that different cell biological mechanisms, governed by different cytokines, are involved in injury-induced intimal thickening. (1) Proliferation of smooth muscle cells (SMC) in the tunica media is dependent on the paracrine action of basic fibroblast growth factor (bFGF) released form dying SMC. In addition to local bFGF, endocrine activities from blood plasma seem to be important for SMC proliferation as judged from experiments with hypophysectomized rats. (2) SMC migration into the tunica intima is an absolute prerequisite for the onset of intimal thickening in rat carotid arteries. PDGF (platelet derived growth factor) from adhering platelets drives the migratory process most likely via the induction of proteases rather than via acting as a chemo-attractant. (3) The molecular mechanism driving intimal SMC proliferation is not yet understood. -y-IFN from T-lymphocytes, however, was shown to inhibit neointima formation most likely by inhibiting SMC proliferation. In summary: after arterial injury a network of cytokines activates and inhibits SMC migration and proliferation in a highly regulated paracrine and endocrine fashion. Pharma Division, Preclinical Research, F. Hoffmalm-La Roche ltd., CH-4002 Basel, Switzerland

1:15 17 HUMAN 0~2A-ADRENOCEPTORS: REGULATION M.C. Michel

19 TISSUE

DISTRIBUTION

AND

Three subtypes of %-adrenoceptors (AR) are recognized in man which are designated c~2A,c~2B,and ~2D-"In radioligand binding studies we have detected c~2A-adrenoceptors in human platelets, kidney, myometrium, and urinary bladder. While [3H]rauwolscine and ~ H]yohimbine detect only c~2A-AR in human kidney, [3H]RX 821002 (2-methox-idazoxan) also detects the additional presence of an ct2-AR subtype with high prazosin affinity, possibly C~c AR. a2A-AR expression in platelets and myometrium obtained from the same patients is well correlated. Therefore, human platelets are frequently used as a model system to study the regulation of human C~2A-ARin vivo. The expression density of platelet c~2A-AR can be regulated by endogenous catecholamines and by hereditary factors. Thus, expression is inversely correlated with plasma catecholamines in hemodialyis patients. In essential hypertensive patients treatment with nifedipine increases plasma catecholamines and reduces ~2A-AR expresssion, treatment with hydergine lowers plasma catecholamines and tends to increase ~2A-AR expression, while combined treatment does not affect either although all three treatment similarly lower blood pressure. A hereditary component of regulation has been shown by studies of normotensive children having one hypertensive or two normotensive parents and by twin studies. To fimher characterise this hereditary component we have used restriction fragment length polymorphism studies using genomic DNA digested by the Dra I enzyme. Patients with ~2A-AR alleles detected by this polymorphism differ with regard to a number of cardiovascular risk indicators related to glucose and lipid metabolism (e.g. HbA~, cholesterol) which can be regulated via %A-AR while parameters not regulated by ~2-AR (e.g. alkaline phosphatase) or by another subtype (e.g. plasma renin activity) were not different. Thus, such alMic polymorphism might be involved in the physiology of t~2A-AR effects in vivo.

HUMAN AND ANIMAL I'~-ADRENOCEPTORS: A NEW, MULTIMEMBERED RECEPTORFAMILY?

J. Zaagsma, R.E.P. de Boer and Ch. Hollenga There is now ample evidence both from pharmacological and structural studies that in addition to fl.- and l]z-adrenoceptors, a third g-adrenoceptor (BAR) subtype exists. The first evidence for it was obtained in rat white adipocytes, originally classified as g~. However, gl- and g2-selective agonists failed to confirm the Bruature and 1~1-, B2- as well as non-selective antagonists showed unusually low potencies an stereoselectivities on adipocyte BARs. Strong support for the atypical receptor nature was obtained in 1984 by Arch et al. with the introduction of lipolytically-salective g-agonists (like BRL 37344). In 1989, Emorine et al. cloned a gane for a human B-AR which after expression in CHO cells showed many atypical properties. The receptor protein, called B3-AR, had a structural homology of 51% and 46% with human g~- and I~2-ARs, respectively. The more recently cloned rat and mouse B3-ARsare structurally very similar to the human I~3-ARwith overall and transmembrane homologies of 80-82% and 93-94 %, respectively. B3-ARs are G,-eoupled to adenylyl cyclase like gc and ~-ARs. However, both in rat and human adipocytes, the relation between cAMP generation and lipolysis is much steeper when using a g3-seleetive agonist than with isoprenaline, suggesting that the B3-AR generated cAMP pool more efficiently activates the protein kinase A involved in triglyceride lipase activation. In recent years, it has become clear that B3-ARsare able to modulate a variety of organ functions. In the GI-tract they mediate relaxation of gastric fandus, jejunum, ileum, colon and oesophagus smooth muscle, they inhibit spontaneous mobility of jejunum and colon, and stimulate gastric acid secretion. In all cases, B~- or flz-ARs play an additional role as well. Both native and cloned B3-ARsappear to be less sensitive to desensitization than ill- and I~z-ARs. This regulation difference has been explained by the lack of phosphorylation sites for B-AR kinase and A-kinase in the carboxy terminal tail and the third cytoplasmic loop of the B3-AR. In addition, long-term regulation, involving changes in mRNA transcription, has been found to be different. Recent studies from our laboratory on human, rat and guinea pig gastrointestinal smooth muscle preparations, using a variety of newly developed fl3-selective agonists, have revealed marked differences in functional affinities and efficaeies. Since some B-AR antagonists also differentiated, the results provided evidence for differences in B3-AR recognition sites among the species: a multimemberedg3-adrenoceptor family?

Dept. of Medicine, University of Essen, 45122 Essen, Germany

Grnningen Utrecht Institute for Drug Exploration, Department of Medicinal Chemistry and Molecular Pharmacology, University of Groningen, Antonius Deusinglaan 2, 9713 AW Groningan, The Netherlands.

18

2O

RECEPTORS REGULATING NORADRENALINE RELEASE IN HUMAN TISSUES L.C. Rump Molecular genetics indicate that receptors of one class, e.g. o~adrenoceptors, may be structurally very similar across species but still have different pharmacological properties (Bylund et al. 1994, Pharm Rev 46: 121-136). The concept and classification of presynaptic receptors regulating noradrenaline release at peripheral neuroeffector junctions (Starke 1977, Rev Physiol Biochem Pharrnacol 77: 1-124) has been established mainly in isolated sympathetically innervated tissues of animal species. Thus, it is also necessary to assess and characterize prejunctional receptor function in human tissues. Human isolated blood vessels, renal cortex and right atrium were incubated with 3H-noradrenaline, electrically stimulated and the stimulation induced outflow of radioactivity was taken as an index of noradrenaline release (Rump et al. 1994, Circ Res 74: 434440). Activation of presynaptic ~2-adrenoceptors (cAMP-dependent) and angiotensin ATe-receptors (cAMP-independent) facilitates noradrenaline release in human renal cortex and atrium. Activation of bradykinin B2receptors to stimulate noradrenaline release in human atrium is only seen in the presence of captopril, which prevents breakdown of bradykinin. By comparison, the facilitatory effect of bradykinin in rat atrium is much greater than that in human atrium and, moreover, even detectable in the absence of captopril. Renal and cardiac sympathetic nerves possess inhibitory presynaptic dopamine De-receptors and c~2-adrenoceptors. The presynaptic o~2-adrenoceptors of human renal and cardicac sympathetic nerves operate as inhibitory autoreceptors and exhibit, in contrast to other species studied, pharmacological characteristics of an e~2c-adrenoceptor. Some of the observed differences may have implications for the development of drugs for hypertension and related clinical fields.

POSTJUNCTIONAL ALPHA-1 ADRENOCEPTORS IN THE ARTERIAL SYSTEM. RELATIONSHIP TO ADRENERGIC NERVES. Jo G.R. De Mey, Get Janssen and Roel Maas Alpha-1 adrenoceptors have been subdivided in binding sites exhibiting a high affinity and sites displaying a low affinity for prazosin (~IH and alL, resp.). Molecular biological evidence indicates that ~IH is actually a family of subtypes that can be further subdivided on the basis of pharmacological properties into ~IAand ~1B-adrenoceptors. In rats, findings in different types of artery with different degree of sympathetic innervation and observations following chemical sympathectomy of the animals, suggest that the occurrence of CtlA and OtlB on arterial smooth muscle correlates positively and negatively, resp., with the presence of adrenergic sympathetic nerves. We evaluated whether this is also the case in man. From patients undergoing coronary bypass grafting, segments of internal mammary artery (IMA) and of subcutaneous resistance arteries (CrA) were obtained. In IMA, catecholamine content was below the detection limit, no histological evidence for the presence of adrenergic nerves could be obtained and electrical field stimulation did not induce contractile responses. Ligand binding experiments with [3H]-prazosin (10-500 pM) revealed the presence of a single class of binding sites, all of which could be irreversibly blocked by 100 uM chloroethylclonidine (CEC) and all of which displayed a low affinity for (+)niguldipine. IMA contracted in response to phenylephrine. The sensitivity and maximal responsiveness to the ~l-agonist were reduced following CEC, but significant responses persisted. In CrA, adrenergic nerves could be visualized and electrical field stimulation induced contractile responses that could be blocked by guanethidine, 6-hydroxydopamine or phenoxybenzamine. Contractile responses of CrA to phenyIephrine were not modified after exposure to CEC. These findings suggest that in man, as in rats, adrenergic nerves influence the presence of alpha-1 adrenoceptor subtypes on arterial smooth muscle and that vasoconstrictor responses to alpha-1 adrenergic agonists may involve besides ctlA- and O~IB- also O~ILreceptors.

Innere Medizin IV, Univ. Klinik, Hugstetter Str. 55, D-79106 Freiburg

Department of Pharmacology and Cardiovascular Research Institute Maastricht, University of Limburg, P.O. Box 616, 6200 MD, Maastricht, The Netherlands.

R6 21

23

MOLECULAR BIOLOGY OF OPIOtD RECEPTORS V. H611t*, J. Kraus + , P. Mayer , S. Schulz , A. Zimprich +

OPIOIDS IN SYNAPTIC TRANSMISSION

Opioid drugs or endogenous opioid peptides exert their wide spectrum of effects on pain perception, mood and autonomic function by interacting with three pharmacologically distinct receptors termed g, and 5. This pharmacological concept of three subtypes of opioid receptors has recently confirmed by molecular cloning showing that the g-, 6- and K-receptors are encoded by three different genes. The receptors belong to the family of proteins with seven transmembrane domains which transduce their intracellular signals via G-proteincoupled pathways and mediate inhibition of adenylate cyclase, activation of potassium channels and/or inhibition of calcium channels through pertussis-toxin-sensitive mechanisms. Recently, we found evidence for a further degree of diversification of opioid receptors showing that the rat g-opioid receptor occurs in two alternatively spliced isoforms (rMOR1A and rMORB) which differ at the C-terminus. Analysis of the structure of the rMOR gene revealed the existence of at Least 5 exons. The expression pattern of rMOR1A and rMOR1B mRNA in the rat brain revealed that rMOR1A is the predominant isoform. The ratio of rMOR1A and rMOR1B mRNA levels varies indicating that a tissue specific splicing occurs in some brain regions. Both g-receptor isoforms, when stably expressed in CHO-K1 cells, show similar affinities to opioid compounds and are equally effective in inhibition of forskolin-induced cAMP formation and in stimulating phopholipase C. Studies measuring the inhibition of adenylate cyclase in cells that had been pre-exposed to the g-agonist DAMGO indicated that rMOR1B is much more resistant to agonistinduced desensitization than rMOR1A. This finding indicates that alternative splicing of the MOR gene may be a physiological mechanism to modulate tolerance development to g-opioids. *lnstitut for Pharmakologie und Toxikologie, Otto-von-Guericke Universit~it, Magdeburg +Physiologisches Institut, Universitfit, M0nchen, Germany

H. Pawelzik, G. Martin, R.A. Deisz and W. Zieglgdnsberger The activation of opioid receptors in the mammalian central nervous system commonly evokes an inhibition of spontaneous, chemically or synaptically induced neuronal discharge activity. Some of the excitatory responses result from a primary inhibitory effect on neighbouring neurons. Ultrastructural localization of opioid receptors indicate pre- and postsynaptic sites involving extrasynaptic and autoreceptors modulating both changes in the receptivity of target dendrites to other transmitters and transmitter release. The constitutive activity exhibited by opioid receptor activation was suggested to be driven by receptor phosphorylation. Opioids activate PTX-sensitive G-protein linked second messenger mechanisms and modulate the activity of ion channels. They reduce Ca z÷ influx (e,g. L-type Ca2+ channels) and enhance K ÷ conductance (e.g. delayed rectifier) in various structures including presynaptic terminals. There is evidence that the excitatory actions of naloxone are not simply due to an antagonism of an opioid-induced K ÷ conductance. /z-opioids also directly inhibit electrically evoked glutamatergic and GABAergic synaptic potentials. This action is not associated with changes in membrane potential or resting membrane resistance. In a recent study we found that the/z-receptor selective opioid agonist D-Ala2-N-Me-Phe4-Glytol enkephalin (DAMGO) decreased isolated AMPA/kainate- (EPSPA~r,)and increased NMDA (EPSPN) receptor-mediated excitatory postsynaptic potentials recorded in neocortical pyramidal neurons of the adult rat in vitro. After intracellular blockade of PKC the enhancing effect of the/xopioid on the EPSP N was abolished. There is evidence that in transfected GH3 cells a PTX sensitive G-protein mediated postsynaptic mechanism may be responsible for /z-opioid-induced inhibition of NMDA currents. In addition, the conductance of isolated inhibitory postsynaptic potentials (IPSPA and IPSPB) was decreased by the opioid receptor agonist most likely by a presynaptic reduction of GABA release. Such changes induced by opioid receptor agonists may also contribute to neuronal plasticity involved in the development of opiate tolerance and dependence.

Max-Planck-Institute of Psychiatry, Clinical Institute, Clinical Neuropharmacology, Kraepelinstr. 2, D-80804 Mi~nchen, Germany

22

24

ENDORPHINS AND ADDICTIVE BEHAVIOR Mlrjam A.F.M. Gerrits and Jan M. van Ree

M O R P H I N E - I N D U C E D P R O T R A C T E D CHANGES IN D O P A M I N E N E U R O T R A N S M I S S I O N PROCESSES IN RAT STRIATAL C O M P L E X A.N.M. S c h o f f e l m e e r One m o n t h after cessation of intermittent m o r p h i n e treatment, s e n s i t i z a t i o n towards the p s y c h o m o t o r effect of the opiate in rats was a s s o c i a t e d w i t h an e n h a n c e d r e a c t i v i t y of dopamine (DA) nerve endings and cholinergic interneurons in slices of the dorsal and ventral striatum to d e p o l a r i z a t i o n and activation of N M D A receptors. These n e u r o a d a p t i v e changes appeared to slowly build up strength in time after m o r p h i n e w i t h d r a w a l and to be a s s o c i a t e d with d e s e n s i t i z a t i o n of presynaptic D-2 DA receptors. The sensitivity of postsynaptic D-I receptors as well as the gene e x p r e s s i o n of dynorphin w i t h i n efferent GABA-ergic neurons of the striatal complex also g r a d u a l l y increased in time upon cessation of drug treatment. Studies in p r i m a r y cultures of rat striatal neurons r e v e a l e d that the adaptive increase in D-I receptor sensitivity is p o t e n t i a t e d by corticosterone. Moreover, sustained activation of inhibitory k a p p a - o p i o i d receptors in cultured DA neurons of the ventral mesencephalon, p r o f o u n d l y enhanced DA release upon d e p o l a r i z a t i o n or NMDA receptor activation, w h i c h is of p a r t i c u l a r interest in view of the role of dynorphin as e n d o g e n o u s kappa agonist. It is suggested that such p r o t r a c t e d adaptive changes in D A - r e l a t e d n e u r o t r a n s m i s s i o n processes in the brain play a crucial role in long-lasting behavioral s e n s i t i z a t i o n upon p r e v i o u s sporadic contact with drugs of abuse and its p o t e n t i a t i o n by stress, w h i c h is thought to enhance the individual vulnerability towards the acquisition of drug addiction.

It has been hypothesized that endogenous opioids are involved in experimental drug addiction. Evidence for this postulate has been provided by the effects of opioid antagonists on self-administration of addictive drags. Treatment with the opioid antagonist naltrexone (NTX) has been found to decrease alcohol consumption in monkeys. Results from studies with cocaine self-administration in rats showed that systemic treatment with NTX decreased cocaine intake during the initiation phase of self-admthistration. In fact, NTX caused a rightward shift in the dose-response curve for cocaine reward. Subsequent experiments demonstrated that NTX exerts its effect on initiation of cocaine self-administration through an action of the CNS. With regard to the local opioid systems in the brain, treatment with NTX in the ventral tegmental area mimicked the effect of systemic treatment with the drug, whereas local treatment with NTX in the terminal areas of the mesocorticolimbic system had no effect on cocaine self-administration. Thus, addictive drugs may affect opioid systems in the brain, e.g. causing release of endorphins, which may play a role in drag selfadministration. To gain insight into the alterations in endogenous opioids during drug self-administration experiments were performed at two time points, i.e. before and after a daily drag self-administration session. Firstly, 13-endorphin (BE) levels were measured in distinct areas of the brain. No changes in gE levels were found after a 5th daily session of cocaine intake, at the moment rats had injected their daily amount of the drug and as a consequence the desire for cocaine is low. On the next day just before a next session was scheduled, thus when the desire or craving for cocaine is assumed to be high, a decrease in gE levels in specific areas of the anterior limbic system was found. Interestingly a similar effect was present in animals self-administering heroin. Secondly, changes in endogenous opioid systems before and after a daily selfadministration session of animals offered cocaine or ethanol were investigated using an in vivo autoradiographic receptor occupancy technique. Marked changes in opioid receptor occupancy were observed in the subcortical brain after daily selfadministration in cocaine, but not in ethanol animals. Before a daily session, in both cocaine and ethanol animals the opioid receptor occupancy was changed in some restricted areas of the mesocorticolimbic system and of the thalamus, possibly reflecting release of endogenous opioids. These studies suggest that endogenous opioids, e.g. endorphins, in selected (limbic) brain areas are implicated in craving for addictive drugs. Department of Pharmacology, Rudolf Magnus Institute for Neurosciences, Utrecht University, Universiteitsweg 100, 3584 CG, Utrecht, The Netherlands

D e p a r t m e n t of Pharmacology, Free University, Medical Faculty, van der B o e c h o r s t s t r a a t 7, 1081 BT Amsterdam, The Netherlands.

R7 25

27

EFFECTS OF PURINE- AND P Y R I M I D I N E - N U C L E O T I D E S IN R A T M I C R O GLIA - E V I D E N C E F O R S E P A R A T E PURINO- AND P Y R I M I D I N O C E P T O R S

PAH/GLUTARATE COUNTERTRANSPORT IN PROXIMAL SEGMENTS OF RABBIT KIDNEYS. M. Kutzer, S. Meer, S. Haller, and J. Greven

S2

W. N6renberg, A. Cordes, P.J. Gebicke-Haerter, R. Frtihlich, G. El6hbaum, and P. Illes Both, extracellular ATP and UTP have important impacts on a wide range of physiologigal responses of cells. Effects of purines are mediated via specific purinoceptors either representing a class of ligand-gated cationic channels (e.g. Pz~-receptors) or G protein coupled The properties of tubular glutarate uptake and (e.g. PzY-) receptors, respectively. Of the latter type, the recently cloned P2~- (nucleotide) the coupling to PAH transport were studied on receptor is sensitive to both, the purine ATP and the pyrimidine UTP. However, lack of isolated nonperfused S 2 segments of proximal antagonism by purinoceptor antagonists against UTP as well as lack of crosstubules, microdissected from rabbit kidneys desensitizatiou between purine analognes and the pyrimidine led to the proposal that a Since the separate class of pyrimidinoceptors may exist (yon Kiigelgen et al., Naunyn-Schmiede- without the use of enzymatic agents. were totally collapsed, the tubular berg's Arch Pharnmcol 336:556, 1987). In the present work, we lmve investigated possible tubules effects of UTP and ATP in rat microgliat cells, the main source of brain macrophages. To glutarate uptake may be assumed to represent the transported across the basolateral this end, whole-cell patch clamp experiments were carried ont in cultured rat microglia, quantity The results show that the S segments pretreated with bacterial lipopolysaccharide (100 ng/ml; 12-24h), a maneuver known to membrane. drive these cells in an activated macrophage-like state. At potentials positive to the po- effectively accumulated 14C-glutarate (5~ ~M). The to bath 14C-glutarate concentration ratio tassium equilibrium potential (Er:), 2-MeSATP (2-MethylthioATP), ATP, ADP, and UTP cell concentration-dependently iaduced outward currents (ECso values at a holding potential of reached maximum values of about 20 after a 20 min period.. The tubular 14C-glutarate 0 mV were: 0.039, 1.65, 8.56, and 81.28 p/vl, respectively) most likely carried by K+ under incubation could be markedly depressed by these conditions. This view was supported by the finding that inclusion of Cs+ (a blocker of accumulation (5 mM) but not by probenecid (0.i mM), a wide variety of K+-chamlels) into the pipette abolished nucleotide effects. At a holding lithium potential near EK (-70 mV) UTP was ineffective. Hence, possible effects of UTP on P2×- which, however, inhibited tubular 3H-PAH (i ~M) receptor gated cationic channels, previonsly described in rat microglia (N6renberg et al., uptake. External PAH (0.i mM) stimulated efflux Br J Pharmacol 111:942, 1994), seem to be unlikely. The effect of UTP at 0 mV of 14C-glutarate from S 2 segments preloaded with disappeared, when either extracellular Ca2+ was renmved from the bathing medium or 14C-glutarate (50 ~M), and external glutarate GDP-fi-S (200 p/vl) was incladed in the patch pipette thereby characterizing the underlying stimulated tubular uptake of 3H-PAH (i ~M), channels as a class of Ca~+-activated K+-channels, the activation of which required a G providing evidence for glutarate-PAH protein triggered siglmlliug mechallism. The P2-antagonist reactive blue 2 (50 pNl) countertransport in proximal S 2 segments. The reduced outward currents elicited by either 2-MeSATP (0.3 ~ or ATP (100 pM) but did phorbol ester PMA (0.i ~M) stimulated PAH uptake not alter outward currents evoked by UTP (i00 pM). Aaother P2-receptor antagonist, but did not affect steady state cell to bath 14Csuramin (300 pM) also inhibited ATP (100 pM) effects but potentiated UTP (100 pM) glutarate concentration ratio nor the initial 14Ceffects possibly reflecting the inhibition of ecto-enzyme catalysed breakdown of nucleotideglutarate transport rate. Protein kinase C phosphates. UTP (1000 pM) applied for 10 rain induced an outward current which induced stimulation of basolateral PAH transport inactivates completely daring this period and also abolished the effects of a subsequent application of UTP (100 ~uVl).However, UTP (1000 p/vl) had only a minor effect on ATP may, therefore, not be due to an enhanced tubular (10 pM) induced outward currents. It is concluded, that rat microglia possess P2-receptors glutarate uptake. most likely of the P~y-subtype.In addition, a separate class of pyrinfidinoceptors may exist, of Pharmacology and Toxicology, the activation of which leads via G protein activatioa to the opening of Cae+-activated K+- Department RWTH Aachen, Wendlingweg 2, 52057 Aachen, FRG channels. Department of Pharmacology, University of Freiburg, Hermann-Herder Str. 5, 79104 Freiburg, Gennany

26

28

ADENOSINE A~ RECEPTOR MEDIATED CHANGES IN INTERNAL CA :+ AND PLASMA MEMBRANE CA 2÷ PUMPING IN DDT 1 MF-2 CELLS. A. Nelemans, H. Sipma, B. Fredholm Hand A. den Hertog.

CLONING AND CHARACTERIZATION OF A NOVEL GTPase (rag) PREDOMINANTLY EXPRESSED IN ADRENAL GLAND A. Schiirmann, A. Brauers, S.Ma6mann and H.G. Joost Two novel ras-related GTPases were cloned by PCR-ampIiflcation and cDNA library screening. We have recently employed a PCR approach using degenerate oligonucleotide primers matching the conserved PM1 and PM3 domain of the ARF-familiy. In addition to cDNA-fragments of the known ARFs and several unknown ARF-like isoforms, we have isolated a cDNAfragment which appeared to encode a GTP-binding protein but did not resemble any of the known subfamilies. Using this partial sequence as a probe, we isolated two full-length cDNA-clones comprising an open reading frame of 313 amino acids (clone A) or 341 amino acids (clone B), respectively, from a rat brain cDNA library. Since the two clones are nearly identical within the coding region, and clone B differs from clone A only in an insertion of 28 amino acids between the GTP-binding motifs PM2 and PM3, we designated the GTPases ragA and ragB. The amino acid sequences of both clones comprise 4 of the 6 known conserved GTP-binding motifs (PM1, 2, 3 and G1), the domains G2 and G3 being strikingly different from those of the ras-family. A multiple sequence alignment with other rasrelated proteins indicated that ragA and ragB cannot be assigned to any of the known subfamilies of the ras-homologues. They exhibit 52% identity with Gtrl, a putative GTP-binding protein from yeast presumably involved in phosphate transport and/or cell growth, mRNA of ragA was detected in most tissues and was predominantly expressed in adrenal gland, testis and ovary, ragB mRNA was similarly distributed among these tissues but was much less abundant than that of ragA. Recombinant glutathione-Stransferase (GST) fusion protein of ragA bound large amounts of GTP[S] in a specific and saturable manner, whereas ragB bound only small amounts of GTP. After labeling with [32p]-GTP GST-ragA and GST-ragB were able to hydrolyse GTP with the same kinetic constant. These data indicate that the two rag subtypes differ in their GDP/GTP-exchange, and that ragA and ragB generate functionally diverse isoforms.

Adenosine receptors are classified into different subtypes and coupled via GTPbinding proteins to a wide variety of effectors. The adenosine receptor signal transduction system has been well characterized in DDTI MF-2 smooth muscle cells. Adenosine A1 receptors inhibiting adenylyl cyclase (AC) and adenosine A2 receptors stimulating AC have been identified, while stimulation of adenosine A1 receptors also activates phospholipase C (PLC) leading to Ca2+ release from internal Ins(l,4,5)P3 sensitive stores and Ca2+ entry across the plasma membrane. A strong reduction of the adenosine and the A~ receptor agonist N 6cyclopentyladenosine (CPA) induced rise in [Ca~+]~ was observed after blocking Ca 2+ entry with La 3+, but not in the absence of extracellular Ca 2+. The effect was present at 22 °C as well as 37 °C, The difference in the CPA response could not be explained by changes in Ins(1,4,5)P3 formation in the presence of La 3+. In the absence of extracellular Ca 2+, the CPA induced Ins(1,4,5)P3 formation was reduced, also indicating that another factor than Ins(1,4,5)P3 determines the actual Ca 2+ response. A strong La3+ induced reduction of the CPA mediated rise in [CaZ+]~ was not observed after inhibiting plasma membrane Ca2+-ATPase by o-vanadate. The CPA response in the absence of extracellular Ca 2+ was not affected by o-vanadate. Apparently these pumps are not activated due to the low basal [Ca2+]~ under these circumstances. Unlike thapsigargin, an inhibitor of Ca2+-ATPase of internal stores, o-vanadate did not elicit a rapid increase in [Ca2+]j, showing that only plasma membrane pumping was affected, o-Vanadate has been shown to reduce also protein tyrosine phosphate phosphatase activity, which could stimulate tyrosine kinase mediated PLC activation. However, ovanadate and tyrphostin 25, a tyrosine kinase inhibitor, did not affect the CPA induced formation of Ins(1,4,5)P3 in DDT~ MF-2 cells. Thus, the large effects observed in the presence of La3÷ and o-vanadate on [Ca2+]i are explained by their action on the plasma membrane Ca2÷-ATPase. This implies that, besides Car+ entry and internal Ca2+ mobilization, these plasma membrane Ca2+ pumps also play an important role in the CPA induced rise in [CaZ÷]~. Groningen Institute for Drugs Studies (GIDS), Dept. Clinical Pharmacology, University of Groningen, Bloemsingel 1, 9713 BZ Groningen, The Netherlands and ~ Dept. Pharmacology, Karolinska lnstitutet, Stockholm, Sweden.

Institut fiir Pharmakologie und Toxikologie, Medizinische Einrichtungen der RWTH Aachen, Wendlingweg 2, D-52057 Aachen, Germany

R8 29

31

ARPI: A NOVEL RAS-RELATED GTPase WITH LIMITED SIMILARITY TO THE ARF-FAMILY. S. Maflmann, A. Schiirmann, and H.G. Joost In a PCR-approach with degenerate oligonucleotide primers matching the conserved PMI and PM3 domains ofth e ARF-family, followed by screening of rat and human tissue-eDNA-libraries, we cloned a novel ras-homologous GTPase. The deduced amino acid sequences of the human and rat eDNA-clones were highly homologous (97% identical amino acids, 88.3% identical nucleotides within the coding region) and comprise all 6 of the conserved motifs presumably involved in GTP-binding. The amino acid sequences exhibit a remote similarity to members of the ARF-family (33% identity with ARF1, 39% identity with ARL3), mainly restricted to he conserved sequence motifs PM1, PM2, PM3, GI, G2 and G3. Because of several differences to the ARF-family the protein was designated ARP1 (aft-related l~rotein). In contrast to ARF-proteins, ARP lacks the known myristoylation site, a glycin at position 2, and comprises an insertion of 8 amino acids between PM1 and PM2. Recombinant ARP was expressed as a GST-fusion protein in E.coli and showed specific and saturable binding of GTP[S]. Unlike ARF-proteins, recombinant ARP exhibited GTPase activity (kinetic constant: 0.093 rain-l) in the absense of tissue extracts or phosphotipids. Low mRNA-levels were found in all rat tissues examined (brain, heart, skeletal muscle, adipocy~es, liver, lung, spleen, intestine, thymus, and kidney). Western Blots with an ARP-specific antibody against the recombinant GST-fusion protein showed a 25 kDa protein in membranes from rat liver, testis, kidney and adipocytes, but not in heart. In fractionated 3T3-L1 cells, ARP1 was predominantly detected in the plasma membrane fraction, whereas ARF1 is predominantly located in the cytosol and in Golgi vesicles. Thus, the protein appears to be posttranslationally modified for membrane anchoring. Because of its unusual subcellular and tissue distribution, it is speculated that ARP 1 might play a role in tissue-specific, membrane-associated signalling.

C O M P E T I T I V E B U T DISCRIMINATORY ANTAGONISM BY O B I D O X l M E OF T H E A L L O S T E R I C ACTIONS OF ALCURONIUM, W84, and W D U O 3

lnstitut ftir Pharmakologie und Toxikologie, Medizinische Fakult~it der RWTH Aachen, Wendlingweg 2, D-52057 Aachen.

AT MUSCARINIC M2-RECEPTORS. C. Tr~nkle and K. Mohr

Alcuronium, the alkane-bis-ammonium compound W84 = hexane-l,6-bis (dimethyl-3'-phthalimidopropyl-ammonium bromide), and the bispyridinium derivative WDuo3 = 1,3-bis[4-(phthalimidomethoxyiminomethyl)pyridinium-l-yl] propane dibromide potently stabilize antagonist binding to M2-receptors. This ailostedc action is attenuated by obidoxime. In order to gain more insight into the events at the molecular level we analysed the concentration-dependency of the antagonistic action of obidoxime on the allosteric effects of alcuronium, W84, and WDuo3 in more detail. [3H]NMS dissociation (control t,/= ~ 4 min) was measured in porcine cardiac membranes (4mM Na2HPO4, lmM KH2PO4, pH 7.4, 23°C). The apparent rate constant of [3H]NMS-dissociation k. 1 was maximally reducedto < 2 % in case of the above-mentioned modulators and to 19 % in case of obidoxime. The concentrations inducing a halfmaximum reduction of k_1 were ECs0 = 7 nM for alcuronium, 44 nM for W84, 51 nM for WDuo3 and ECso = 40 pM for obidoxime. The concentration-effect-curve for the allosteric effect of obidoxime was shallow (nil=0.68). The potency of the allostedc modulators was determinedin the presence of various obidoxime concentrations (10010000 pM). Obidoxime evoked concentration-dependently a nearly parallel shift to the right of the concentration-effect-curve of all applied modulators. Analysis according to Arunlakshana & Schild yielded straight lines with slope factors of 1.11, 0.98 and 1.08 for alcuronium, W84, and WDuo3, respectively, which were not significantly different from unity. With slopes constrained to unity the following pA2-values were obtained for the antagonistic effect of obidoxime on the allosteric actions of the tested modulators: alcuronium = 4.05 + 0.12, W84 = 4.25 + 0.08, and WDuo3 = 3.58 + 0.08 (mean + SD, n=4). The pA2-values differed significantly (p < 0.05). In conclusion, the data are in line with a competitive antagonism by obidoxime of the allosteric actions of alcuronium, W84, and WDuo3 at the M2-receptor protein. Yet, the antagonistic potency of obidoxime varied depending on the allosteric modulator under investigation. This finding suggests that the allosteric modulators either utilize different points of attachment within the allosteric site or bind to topologically different sites. Pharmacology & Toxicology, Institute of Pharmacy, University of Bonn, An der Immenburg 4, 53121 Bonn, Germany

3O

32

RAS-DEPENDENT ACTIVATION BY PROTEIN KINASE C OF RAT GLUCAGON GENE TRANSCRIPTION W. Knepel, U. Ftirstenau, M. Schwaninger, R. Blume, and I. Keanerknecht

INFLUENCE OF THREE ALLOSTERIC MODULATORS OF MUSCARINIC MZRECEPTORS ON BINDING CHARACTER[STICS OF [3H]-PRAZOSIN AND [1251]-IODOCYANOPINDOLOL IN RAT CARDIAC TISSUE. M.Pfaffendorf, H.D. Batink, C. Tr~inkle 1, K. Mohr 1, P.A. van Zwieten The ligand binding to muscarinic receptors can be influenced by allosterie modulators. The most prominent feature of this interaction is a retardation of the antagonist dissociation from the main binding site. Since muscarinic receptors, like o- and &-adrenoceptors, are members of the G-protein coupled membrane receptor family the specificity of the allosteric effect for M 2receptors has to be established. For this purpose the effects of three well known allosteric modulators, alcuronium (ALC), W84 (N,N,N',N'-tetramethyk N,N'-bis-(3-phtalimido-propyl)-N,N'-hexane-1,6,-diyl-bis-ammonium dibromide), and gallamine (GAL) on the equilibrium binding and the dissociation of the al-adrenoceptor ligand [3H]-prazosin (PRAZ) and the r~-adrenoceptor ligand (-)-[t251]-iodocyanopindolol (ICYP) have been investigated. The membrane fraction of homogenized rat cardiac [eft ventricle was incubated for 45 min. with PRAZ, or for one hour with ICYP. At the end of the incubation period the equilibrium binding or, after the addition of an excess concentration of unlabelled phentolamine (c7, 10 pM) or (-+)-CGP 12,177 (l&, 10pM) the dissociation was determined in the presence or the absence of the allosteric modulators. Values are given as mean _+ SEM of 4-6observations. PRAZ showed a monophasic dissociation pattern with a half-life of 16 +- 1 rain in the absence and of 17 + 1, 8 -+ 1 and 13 -+ 1 rain. in the presence of 30/~M ALC, 30 pM W84 and 1000 pM GAL, respectively. In the presence of the allosteric modulators, the PRAZ equilibrium binding amounted to 36 -+ 2% (ALC), 93 _+ 2% (W84) and 42 + 2% (GAL) of control. Under all conditions investigated, the dissociation of ICYP was slow and after 3 hours more than 40% of the radioligand were still bound. Therefore, no reliable halflife could be calculated. However, at no time point the binding of ICPY in the absence and the presence of 30 pM W84 showed differences. In the presence of GAL and ALC, the dissociation curve of ICYP revealed an initial offset from the starting level followed by the major phase of dissociation being parallel to the control curve. The equilibrium binding of ICYP amounted to 92 _+ 6% (ALC), 83 -+ 8% (W84) and 91 _+ 5% (GAL) of control. In conclusion, ALC, W84, and GAL in concentrations known to be nearly maximally effective on [ 3H]-N-methylscopolamine dissociation, have no clear effect on the dissociation of PRAZ or ICYP. However, the equilibrium binding of PRAZ was considerably reduced by GAL and ALC, pointing to an inhibitory effect on radioligand association to a-adrenoceptors. A present, it remains to be established whether this effect is competitive or allosteric.

In order to maintain blood glucose levels within narrow limits, the synthesis and secretion of pancreatic islet hormones is controlled by a variety of nerval, hormonal and metabolic messengers that act through multiple signal transduction pathways. Glucagon gene transcription has been shown to be stimulated by cAMP and depolarization-induced calcium influx (Schwaninger et aL, J. Biol. Chem. 268:5168-5177, 1993). In this study, the effect of protein kinase C activation on glucagon gene transcription was investigated. The luciferase reporter gene was fused to 350 base pairs of the rat glucagon gene 5'-flanking region and transiently transfected into the glucagon-producing islet cell line c~TC2. Treatment with 12-O-tetradecanoylphorbol- 13-acetate (TPA) stimulated glucagon gene transcription, while 4ec-phorbol-12,13-didecanoate was inactive. The effect of TPA was enhanced by overexpression of protein kinase Cc~ and was synergistic with the stimulation by cAMP and depolarization-induced calcium influx. By 5'-deletions, 3'-deletions and internal deletion, the TPA-responsive element was mapped to the G2 element (from -165 to -200), that was also sufficient to confer TPA responsiveness to the truncated, nonresponsive glucagon promoter. TPA responsiveness of the G2 element was lost by mutating a sequence motif within G2 that bound a nuclear protein with HNF-3-1ike binding specificity in a gel shift assay. However, TPA responsiveness was also lost by mutating a separate sequence motif within G2 that bears similarity with binding sites of Ets family proteins. Like TPA, overexpression of activated Ras (Vl2Ras) stimulated glucagon gene transcription, whereas overexpression of a dominant negative mutant of Ras (N17Ras) abolished the effect of TPA. These data suggest that activation of protein kinase C stimulates glucagon gene transcription through the G2 element. The protein kinase C - activated mechanism may depend on Ras and an interaction between two transcription factors, an HNF-3-like factor and a protein recognizing sequences similar to binding sites of Ets family proteins. Department of Biochemical Pharmacology, University of G6ttingen, RobertKoch-Str. 40, D-37075 G6ttingen, Germany

Dept. Pharmacotherapy University of Amsterdam, Meibergdreef 15, 1 1 0 5 A Z Amsterdam, The Netherlands and Dept. Pharmacology, Inst. Pharmacy, University of Bonn, An der Immeoburg 4, 53121 Bonn, Germany

R9 33 PROMISCUITY IN G PROTEIN COUPLING FOR 5-ItT2C RECEPTORS A n j a Garritsen, Miranda H. van Triest, Eric Rovers a n d F r e d A. Dijcks Serotonin (5-HT) plays an important role in the etiology and treatment of depression. Targets for antidepressive drugs include the 5-HT~A and 5-HT2e receptors. It is known that the 5-HT~A receptor activates multiple second messenger systems, through both the ot and 13"/subunit of the heterotrimeric G protein. The 5-HT2c receptor couples to phosphoinositide (PI) hydrolysis via a pertussis toxin (PTX)-insensitive G protein, Gq. The possibility that the 5-HT2c receptor activates other signal transduction pathways was investigated. In NIH-3T3 cells transfected with the human 5-HT2c receptor, inositol phosphate formation increases 5-10 fold by addition of 5-HT with a pECs0 of about 7.5. Pretreatment with PTX resulted in a very small reduction in 5-HT-indueed PI turnover. G protein activation by 5-HT2c receptors was also studied by measuring the binding of the nonhydrolysable GTP analogue, [~sS]GTP'/S, to cell membranes. 5-HT induced a small increase in binding, but GTP)'S binding in these cells was completely abolished by PTX-treatment even though Gq is insensitive to PTX. This suggests that the effect represents coupling of the 5-HT2c receptor to Gi rather than Gq (Go is not present). The functional consequences of G~ coupling were investigated by studying the effects of 5-HT2c receptor activation on cAMP accumulation in intact cells. Interestingly, we observed an increase in cAMP followed by a decrease at higher 5-HT concentrations. The maximal response was observed at 10-7 M 5-HT. The inhibitory phase of this biphasic curve was completely eliminated by PTX treatment, revealing a regular sigmoidal curve for the stimulatory phase with a pECs0 of 7.3. The stimulatory phase could be eliminated by pretreatment with cholera toxin, revealing a monophasie inhibitory response (pECs0 6.4). The data indicate that in NIH-3T3 cells, the 5-HT2c receptor activates Gq as well as ~ and G,, but higher agonist concentrations are needed to observe the Grmediated effects, suggesting that this coupling is less efficient. Promiscuity in G protein coupling may be relevant for the molecular mechanism of action of drugs acting at the 5-HT2e receptor. Dept. Neuropharmacology, N.V. Organon, P.O. Box 20, 5340 BH Oss

35 PHARMACOLOGICAL ANALYSIS OF al-ADRENOCEPTORS IN RAT

AORTA: APPLICATIONOF A TWO-RECEPTOR MODEL P.H. Van der Graaf & P,R. Saxena

Previous analysisof competitiveantagonismof noradrenalinesuggestedheterogeneity of cq-adrenoceptorsin rat aorta (Van der Graafet aL, 1993). We now presentfurther evidence for this hypothesisobtained from a study of competitive antagonismof the selective (x~-adrenoceptor agonist, phenylephrine (PE). After removal of the endothelium, ring segmentsof rat aorta were mounted as described before (Van der Graaf et al., 1993). Tissueswere incubated for 90 min with 30 gM cocaine, 6 pM timolol and antagonist or vehicle. Of the seven antagonists investigated (Table 1), taglsulosin,prazosin,5-methylurapidil(5-MU),spiperoneand HV723 producedparallel rightward shifts of the PE E/[A] curves. Phentolamineand WB-4101, however, produced significantsteepening,inconsistentwith simple competitiveantagonism. In addition, the Schild plot slope parameters (b) for phentolamine and HV723 were significantly less than unity (Table 1). The data obtained with all antagonistswere fitted simultaneouslyto a two-receptormodel (Van der Graaf et al., 1993). The goodness-of-fitobtainedwith the two-receptormodelwas significantlybetter than that obtained with the one-receptormodel(Fro H=28.3 P<0.001) and two affinityestimates (pKm and pKBz)were obtained for each antagonist (Table 1). These estimateswere compared with reported antagonist affinities at rat cloned cqA, eqB and c%adrenoceptors(Laz et al., 1994;no data availablefor tamsulosinand HV723)and it was found that pKi valuesfor the e%-subtypecorrelatedwell with bothpKm (r~=l.00)and pKm (r~=0.94). In contrast, no significantcorrelationswere found between pKm or pKB2 estimates and pKi values for either c% or cqA-adrenoceptors. Althoughthe significant correlation (in=0.95) betweenpKm and pKB2estimates indicates that the model predictionsshouldbe interpretedwith caution, this study suggeststhe presence of two "otto-like" adrenoceptorsin rat aorta. Table 1 (data shown as mean +_ s.c.) Schild analysis pK B (pA2) b d.f. tamsuiosin 9.75+0.09 1.04-+0.05 33 phentolamine (7.6_+0.3) 0.74-+0.08* 28 prazosin 9.66_+0.i2 1.06-+0.10 44

WB-4101 5-MU spiperone

ITV723

Two-receptor model fit pKR2 PKm

10.0-+0.1 7.7_+0.2 9.8_+0.1 9.1_+0.1 8.88_+0.10 0.96_+0.09 25 7.59-+0.06 0.97_+0.04 22 7.8_+0.1 8.3_+0.1 7.97-+0.14 0.80_+0.13 25 8.2__+0.1 (8.4-+0.3) 0.83-+0.08* 30 *b significantly smaller than unity (P<0.05)

9.3_+0.2 6.2_+0.2 9.3_+0,2 8.4_+0.2 7.1_+0.2 7.5_+0.2 7.6__+0.2

Laz TM et al. (1994) Mol. Pharmacol.46: 414-422. Van der GraafPH et al. (1993) Br. J. Pharmacol. 110: 124P Departmentof Pharmacology,ErasmusUniversity,PO Box 1738, 3000 DR Rotterdam, The Netherlands

34

36

RELAXATION OF THE GUINEA-PIG ILEUM: AN I N VITRO MODEL FOR 5-HT7 RECEPTORS? H.O. Kalkman, F. Schneider

MECHANISM-BASED PHARMACOKINETIC-PHARMACODYNAMIC MODELLING OF BENZODIAZEPINES B. Tuk, J.W. Mandema and M. Danhof.

Affinity values obtained in a [125I]-LSD binding assay using membranes from guinea-pig ileum longitudinal muscle (Engel et al., J. Receptor Res. 4, 1984, Previously described concentration-EEG effect relationships of four 113-126), correlate remarkably well with published 5-HT 7 receptor affinities. benzodiazepine agonists, clobazam, flunitrazepam, midazolam and oxazepam, The receptor that is labelled by [12sI]-LSD in the ileum is probably involved were reanalyzed using a newly postulated mechanism-based model. The in smooth muscular relaxation (Kalkman et al., Eur. J. Pharmacol. 129, 1986, model, based on receptor binding theory, yields estimates for receptor affinity 139-145). The 5-HT7 receptor is characterized by a relative high affinity for (Kpd), intrinsic activity (C~pd)and the relationship between relative stimulus clozapine and for 8-OH-DPAT. We have now investigated the effects of these and EEG-effect. The for protein binding corrected outcome of K0d was compounds in the guinea-pig ileum. Ileum segments of 3 cm length were compared with in vitro receptor binding data determined at 37 ° C (yielding mounted in organ baths and pulsed with substance-P (s-P; 10-s M). Addition of 5-carboxamidotryptamine (5-CT) concentration-dependently reduced the estimates of I~) and pharmacodynamic estimates of potency (ECs0,vrz) as size of the s-P contraction. A pD2 value of 7.5 was calculated. This effect was derived in a model for anticonvulsant activity of benzodiazepines (the inhibited by clozapine (10-7 M; pA2 7.6). 5-HT and 8-OH-DPAT were, like pentylenetetrazol threshold model). The KpJK~ and KJECso.~rz ratios were 5-CT, agonists with pD2-values of 6.2 and 5.5, respectively. The agonist data 0.69 and 0.70 for clobazam, 0.45 and 0.41 for flunitrazepam, 0.33 and 0.27 correspond with pD 2 values for the activation of adenylate cyclase in cells for midazolam and 0.42 and 0.58 for oxazepam, indicating that mechanismexpressing the cloned 5-HT 7 receptor (reported by Lovenberg et al., Neuron based modeling of the data yields physioIogically relevant parameter 11, 1993, 449-458). Also the pA2-value obtained for clozapine corresponds estimates. Estimates of 0~vd were 0.8, 1.0, 0.9 and 0.9 for clobazam, with its published affinity for the 5-HT 7 receptor. Furthermore, clozapine blocked the relaxant response induced by 8-OH-DPAT, although a pA2 value flunitrazepam, midazolam and oxazepam respectively, which is in agreement could not be calculated. Taken together, these data suggest that relaxation of with what is known from in vitro data. s-P contracted longitudinal muscle of guinea-pig ileum by 5-HT compounds The outcome of the relationship between relative stimulus and effect is is mediated by the 5-HT 7 receptor subtype. More compounds need to be described using a cubic spline. Relative stimuli lower than 0.1 show no tested, however. increase in effect; relative stimuli higher than 0.2 show an almost linear increase with relative stimulus. Sandoz Pharma, Preclinical Research Dept, Building 360-405, Postfach, CH It is concluded that mechanism-based pharmacokinetic-pharmacodynamic 4002 Basel, Switzerland. modelling of the benzodiazepine EEG data yields pharmacological relevant data, making the model an alternative for existing empirical models. Leiden/Amsterdam Center for Drug Research, Division of Pharmacology, Sylvius Laboratory, PO Box 9503, 2300 RA Leiden.

RIO 37

39

R E G U L A T I O N OF GM-CSF RELEASE FROM H U M A N AIRWAY EPITHELIAL CELLS IN C U L T U R E

SUBCELLULAR DISTRIBUTION OF HISTAMINE IN HUMAN BLOOD MONOCYTES G. Zwadlo-Klarwasser, M. Vogts, W. Hamann and W. Schmutzler

H. Klapproth. K~ Rack6* and I. Wessler GM-CSF is released by a large variety of cells including epithelial ceils of human airways. This cytokine by activating macrophages and eosinophils and by increasing antigen presentation and expression of adhesion molecules may be involved in the pathogenesis of chronic airway" inflammation. Therefore we have investigated the effects of other cytokines and proinflammatory peptides on the release of GM-CSF from human bronchial epithelial (HBE) ceils in culture. Human bronchi were obtained from patients with lung cancer at thoraeotmny, Epithelial cells were separated by pronase treatment (24h, 4°C, 0.1%). Thereafter, these ceils could be collected by flushing the luminal side with cold PBS After centrifugation cells were plated on 24-well culture dishes and cultured with serum-free, hormone supplemented DMEM/F12 medium. HBE cells grew confluent within one week and were identified as epithelial cells by immunostaining with anti-pancytokeratin. Cells were cultured on unsupplemeuted DMEM/F12 medium for three additional days and thereafter exposed (24h) to cytokines and proinflammatory peptides [ILlfi (0.1-10 ng/ml), TNFcx (l, 10 ng/ml), IFN'~,(l, i0 ng/ml), LPS (1 ~tg/ml), bradykinin (BK, 1 nM-1 ~tM), CGRP (100 riM), substance P (SP. 1 gM)]. The GM-CSF content of the cell culture supernatant was meas.red by a commercial ELISA kit, A mixture of Illl3, TNFet, IFNy and LPS induced a more than 15-fold increase of GM-CSF release. Ill6 proved to be the strongest stimulus, 1 or 10 ug/ml Illfi enhanced GM-CSF release by a factor of 2 and more than 10, respectively. In expermients with indomethacin it was found, that blockade of cyclooxygenase activity enhanced the effect of a submaximal concentration of ILlfi. TNF~ (t-10ng/ml) and LPS (lgg/ml) were less effective than ILlfi; 1 ng/ml TNFoc increased GM-CSF release by about 50 % and 1 ~tg/ml LPS by about 40%. IFNy, CGRP and SP had no effect on the release of GM-CSF. BK (1 gM) doubled the release of GM-CSF into the supernatant, The present experiments show that cytokines (I111~, TNFoc) and the proinflammatory mediator BK enhance the GM-CSF release from human bronchial epithelial cells in culture. This effect may be mediated by a facilitated release or/and by an increased synthesis, i.e. an effect on gene expression. It is concluded that the human airway mucosa is a target for multiple mediators to control the release of the proinflammatory cytokine GM-CSF from these cells. Supported by the "Ministerium fOr Umwelt", Rhineland Palatinate, Germany" Department of Pharmacology, Univ. Mainz, Obere Zahlbacher Str. 67, D-55101 Mainz *Department of Pharmacology, Univ. Bonn, D-53113 Bonn, Germany

Recently we have shown that human blood monocytes contain substantial amounts of histamine which may be released on appropiate stimulation e.g. C5a. Here we studied the subcellular distribution of histamine and its synthetizing enzyme histidine decorboxylase (HDC) in blood monocytes isolated by two step density centrifugation as well as in the monocyte subsets 27E10 and RM3/1 purified by an immunomagnetic method (Miltenyi et at. 1990, Cytometry 11, 231). Histamine and HDC were measured in cell lysates including human umbilical endothelial cells as controls. HDC and histamine were only found in monocytes. The antiinflammatory monocyte subtype RM3/1 contained about 0.05 pg histamine/cell, the inflammatory subset 27E10 however about 0.10 pg/cell. Cell lysates were further fractionated in a discontinuous Percoll gradient. The fractions were characterized employing conventional cell compartiment marker enzymes. Histamine and HDC were colocalized in the low density cytosolic fraction associated with lactate dehydrogenase activity but not in other fractions. Dialysis of the cytosolic fraction using membranes of different pore size revealed that about 50% of histamine were connected to molecular weight material greater than 30 kD or less than 10 kd but not between 10 and 30 kD. These results show that histamine and its producing enzyme HDC are localized in the cytoplasms of monocytes. They also suggest that part of the histamine is bound to high molecular proteins of probably microsomal orign. Finally the data indicate that monocytes subsets vary in their histamine content emphasizing their different functional rotes. Institute of Pharmacology and Toxicology, Medical Faculty, RWTH Aachen, Wendlingweg 2, D-52057 Aachen

38

4O

INCREASED CYCLIC AMP LEVELS INHIBIT THE ADHESION OF NEUTROPHILS TO HUMAN BRONCHIAL EPITHELIUM

HISTAMINE MONOCYTES:

RELEASE FROM EFFECTS OF

HUMAN MAST CELLS AND B-CAROTENE AND RELATED

P.A.J. Henricks, P.G.M. BIoemen, M.C. van den Tweel, M.H.A. Kester, F. Engels, and F.P. Nijkamp.

COMPOUNDS W. Schmutzler, M. Gladis-Villanueva, T. Greven, G. Z w a d l o - K l a r w a s s e r

Bronchial epithelial cells express the cell adhesion molecule ICAM-I. The expression of ICAM-1 can be upregulated by cytokines and it mediates the binding of activated leukocytes (including neutrophils) via interaction with their adhesion molecules Mac-1 or LFA-1. The adhesive interaction between neutrophils or eosinophils with the epithelial layer is believed to be involved in the epithelial damage and the associated hyperreactivity found in the airways of asthmatic patients. Since neutrophils and epithelial cells possess 132-adrenergic receptors and ~2agonists are used by asthmatic patients therapeutically, we examined the effect of cyclic AMP elevating agents on the interactive adhesion between neutrophils and airway epithelial cells, Confluent monolayers of the human bronchial epithelial cell line BEAS2B were incubated with freshly isolated, Slchromium labeled, human neutrophils for 30 min at 37°C in the presence or absence of different cyclic AMP-elevating agents. Non-adhered cells were removed by gentle washes and the percentage of adhered neutrophils was determined. Basal adhesion of neutrophils to the epithelial cells was approximately 10%. The adhesion of the neutrophils to the BEAS-2B cells was increased to 30-40% when the neutrophils were activated with fMLP (10"8 M). This increased adhesion was inhibited dose-dependently by the cell-permeable cAMP analogues dibutyryl-cAMP and 8-bromo-cAMP. The non-selective I~-agonist isoprenaline and the long-acting ~-agonist salmeterol (10-t10 "s M) did not influence the fMLP-stimulated adhesion of neutrophils to the epithelial ceils. However, addition of the phosphodiesterase inhibitor IBMX to the incubation mixtures with the 13agonists resulted in significant inhibition of the adhesion. Therefore, a combined therapy resulting in elevated cyclic AMP levels for a prolonged period, might by effective in inhibiting the interaction between inflammatory cells and airway epithelium thereby preventing damage of the epithelium as often found in asthmatic patients.

M o n o c y t e s and lymphocytes in a d d i t i o n to b a s o p h i l s have been found to be important sources of h i s t a m i n e in b l o o d w h i c h can be r e l e a s e d by a n u m b e r of stimulants, e.g. A 23187, C5a, s u b s t a n c e P (Agents & A c t i o n s 41, C99, 1994). Since free radicals have been shown to o r i g i n a t e from m o n o c y t e s and to a c t i v a t e m a s t cells and this m i g h t be an important p r o c e s s in i n f l a m m a t o r y reactions we tested the effects of radical scavengers, e.g. of superoxide d i s m u t a s e (SOD), of two d i h y d r o c h i n o l i n e s (biG, MDS) and of B - c a r o t e n e on ConA s t i m u l a t e d h u m a n adenoidal m a s t cells, and the latter also in C5a s t i m u l a t e d h u m a n p e r i p h e r a l b l o o d mcnocytes. The SOD h a d an inhibitory effect on the h i s t a m i n e release from mast cells only at very high c o n c e n t r a t i o n s (8 x 10-2). The d i h y d r o c h i n o l i n e s , however, inhibited the release d o s e - d e p e n d e n t l y in the range of i0 -z - i0 -6 M and I0 -6 - 10 -4 M respectively. b - c a r o t e n e showed a d o s e - d e p e n d e n t i n h i b i t i o n of the release from m a s t cells in the range of 10 -6 10 -3 M. It had, however, an inhibitory e f f e c t in the m o n o c y t e s only at v e r y h i g h c o n c e n t r a t i o n (10 -3 M). Our results suggest that radical scavengers might exert some antiallergic and a n t i i n f l a m m a t o r y actions.

(Subsidized by a research grant of the Netherlands Asthma Foundation) Department of Pharmacology, Utrecht Institute for Pharmaceutical Sciences, P.O. Box 80.082, 3508 TB Utrecht, The Netherlands.

K.

Institute for P h a r m a c o l o g y & Toxicology, M e d i c a l Faculty, RWTH, D-52057 A a c h e n

Bolsmann,

Rll

41

43

SUPEROXIDE ANION PRODUCTION IS DEPENDENT ON CALCIUM INFLUX AND IS NOT INHIBITED BY SK&F 96365 INfMLP-STIMULATED DIFFERENTIATED HL-60 CELLS A. Gallois, J.-L. Bueb & E. J. Tschirhart. SK&F 96365, (1-(beta-(3-(4-methoxy-phenyl)propoxyl)-4-methoxyphenetyl)-lH-imidazole hydrochloride) has been shown to inhibit the receptor-mediated calcium entry (RMCE) in various cell types. We have investigated its effect on intracellular calcium concentration ([Ca2+]i) rise and superoxide anion (02") production induced by the chemotactic peptide N-formyl-Methionyi-Leucine-Phenylalanine (fMLP) in a neutrophil model, the DMSO-differentiated HL-60 cell line. HL-60 cells (ATCC CCL240) were grown in Iscove's medium with bovine serum (20%). Differentiation into neutrophil-like cells was induced by DMSO (1.3%) fox 4 days. [Ca2+]i and 02" were measured simultaneously by a double labelling fluorescent assay, using fura-2 and dihychorhodamine-123 respectively. Cells were activated by fMLP (0.01-1000 nM) in presence or absence of extracellular Ca 2+. Alternatively, SK&F 96365 (10 gM or 30 ~M) was added 3 min before activation byfMLP. HL-60 cells responded tofMLP with an ECs0 of 1.1+0.6 nM and an Ernax of 404+72 nM for [Ca2+]i increase, and an ECso of 2.1_+1.1 nM with an Ernax of 11.7+3.6 pmole/min/106 cells for O2" production. Omission of extracellular Ca 2+ reduced the response tofMLP by 80% for both [Ca 2+]i rise and 02" production, without modifying the ECs0s. Readdition of Ca2+ into the medium restored [Ca2+]i levels and partially 02" production. The purported organic RMCE inhibitor was not able, in presence of extracellular Ca 2+, to fully inhibit Ca 2+ entry observed with the Ca 2+free/Ca 2+ addition protocol. Indeed, SK&F 96365, 30 gM but not 10 ~tM, reduced [Ca2+]i rise by 30% and had no effect on 02 ° production. These results indicate that the [Ca2+]i rise can be mainly attributed to a Ca 2+ influx which is necessary for a full biological response, as evidenced by the major reduction of 0 2 ' production in Ca2+-free conditions. The small effect of SK&F 96365 suggests either its lack of potency in fully inhibiting RMCE, hypothesis strengthened by the reported high ICs0 (10 gM), or a pluripotent mechanism of action, resulting in an underlying antagonism of the previous reported effect.

DIFFERENTIAL ROLE OF PROTEIN KINASE A (PKA) AND PROTEIN KINASE C (PKC) IN THE REGULATION OF UPTAKE OF L-ARGININE AND ITS METABOLISM BY ARGINASE IN RABBIT ALVEOLAR MACROPHAGES (AMs). H. Nematollahi, C. Hey, I. Wessler* & K. Rack6 #, AMs are endowed with a high affinity uptake mechanism for L-arginine (L-Arg), and the metabolism of L-Arg by arginase appears to be an important pathway in these cells. At present, little is known about mechanisms controlling the activity of arginase and that of the uptake system. Recently, we reported (this journal (1995) 351:R79) that arginase activity of cultured rabbit AMs was enhanced after stimulation of adenylyl cyclase by forskolin, an effect further characterized in the present experiments. AMs were obtained by broncho-alveolar lavage of isolated rabbit lungs and were resuspended in DMEM-F12 medium containing 5 % FCS. After dissemination adherent cells (mainly AMs) were cultured for 18 h either in the presence of LPS alone or in combination with forskolin and/or KT 5720 (a PKA selective inhibitor) or phorbol-12-myristate-13-acetate (PMA). L-Arg uptake was studied by determining the accumulation of tritium during 5 min incubation with pH]-L-Arg (18 kBq, 1 #mol/1). Arginase activity was measured by determining the formation of rsu[ H]-L-ornithine ([SH]-L-Orn) during 1 h incubation of the intact ceils with [3H]-L-Arg (37 kBq). In incubation media of AMs cultured in the presence of LPS (10 L~g/ml) alone, 57,400+_3,900 DPM/2.5*106 cells (mean+-SEM, n=32) pH]-LOrn accumulated during 1 h of incubation with [SH]-L-Arg. When forskolin (10 /zmol/1) was additionally present in the culture medium [SH]-LOrn accumulation was enhanced by 170 %, and this effect was prevented by KT 5720 (300 ~mol/1). KT 5720 alone caused a reduction in [SH]-LOrn formation by about 65 %. When PMA (3 or 100 nmol/1) was present during the culture period [3H]-L-Orn formation was reduced by 20-30 %. On the other hand, uptake of [SH]-L-Arg was enhanced about 10fold after PMA had been present during the culture period, but tended to be reduced by about 20 % when forskolin had been present. In conclusion, in rabbit AMs L-Arg uptake is markedly enhanced by PKC-, but not by PKA-dependent mechanisms. On the other hand, arginase activity is markedly enhanced by PKA-, but not by PKC-dependent mechanisms.

Neuroimmunologie & Inflammation. Centre de Recherche Public-Sant&

120, route d'Arlon. L-1150 LUXEMBOURG.

42 LYMPHOCYTE CHEMOAI"FRACTANT FACTOR (LCF) IS INVOLVED IN THE INDUCTION OF AIRWAY HYPERRESPONSIVENESS IN A MURINE MODEL FOR ALLERGIC ASTHMA. E.M. Hessell, W.W. Cruikshank 3, A.J.M. Van Oosterhout 1, G. Hofmanl~ H. Van LoverenZ~ D.M. Center ~ & F.P. Niikamp 1. Patients with allergic asthma suffer from airway hyperresponsiveness (AHR) and inflammation which is characterized by eosinophil and T-lymphocyte infiltration. The precise sequence of events leading to AHR and inflammation is still unknown. Lymphocyte chemoattractant factor (LCF) has been described as a potent ehemoattractant for CD4+ cells (T-lymphocytes, monocytes, eosinophils), and is produced by Ag-stimulated T-lymphocytes. We have shown that in ovalbumin (Ok,) sensitized mice AHR and inflammation could be induced after chronic OA inhalation. The objectives of this study were: 1) to investigate whether LCF was produced after chronic OA inhalation and 2) to determine the role of LCF in the development of AHR and inflammation. Bronchoalveolar lavage fluid (BALF) was collected at 3 and 24 hrs after the last OA inhalation in OA-sensitized mice. Using a Boyden chamber the migratory response of human lymphocytas incubated with these BALF samples was measured. At 3 hrs, 4 out of 6 BALF samples caused migration (160%) when compared to medium. At 24 hrs, 2 out of 6 samples caused migration (170%). BALF of saline-challengedcontrol animals, however, did not induce migration and were inhibitory when compared to medium (50%). At both time points the migration could be largely inhibited by in vitro incubation of the BALI= samples with antibodies to LCF, indicating that LCF was present in the BALF samples. In vivo treatment with antibodies to LCF during chronic OA inhalations in OAsensitized mice (2x500 I~g, i.v.) largely inhibited the induction of AHR (50%), but had no effect on the eosinophil infiltration into the BALF (1.6±0.8x104 cells/BAL). In mice treated with control antibodies normal AHR and eosinophilia (2.3±1.3x104 cells/BAL) was observed. In conclusion: LCF could be detected in the BALF of OA-sensitized mice which were exposed to chronic OA inhalations. Interestingly, LCF production followed the same time course as described for humans. Furthermore, LCF appears to be involved in the induction of AHR, but not in the development of eosinophil infiltration. Further studies are warranted to elucidate the mechanism by which LCF is involved in the induction of AHR. 1Department of Pharmacology, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, P.O.Box 80.082, 3508 TB Utrecht, The Netherlands. 2Nat. Inst. for Public Health & Environm. Protection, Bilthoven, The Netherlands Spulmonary Center, Boston University, Boston, MA, USA.

Department of Pharmacology, University Hospital, J.W. Goethe-Uni~rsity Frankfurt; *Department of Pharmacology, University of Mainz and VInstitute of Pharmacology and Toxicology, University of Bonn, Reuterstr. 2b, D-53113 Bonn.

44

ALLERGEN PROVOCATION CAUSES A DEFICIENCY OF VAGALLY INDUCED NITRIC OXIDE AFTER THE EARLY ASTHMATIC REACTION 1N CONSCIOUS, UNRESTRAINED GUINEA PIGS. M. Schuiling, H. Meurs, A.B. Zuidhofand J. Zaagsma Nitric oxide (NO), as a neurotransmitter of the inhibitory nonadrenergic, noncholinergic (iNANC) nerves, is involved in the regulation of the bronchial tone. Since a deficiency of neurally released NO could be involved in allergen-induced bronchial hyperreactivity (BHR), we investigated the role of NO in the regulation of vagally induced bronchoconstriction in a guinea pig model of allergic asthma, characterized by allergen-induced early (EAR) and late (LAR) asthmatic reactions and BHR after these reactions. Ovalbumin (OA)-sensitized guinea pigs were instrumented with a small latex intrapleural balloon and a bipolar stimulation electrode around the right cervical vagus nerve .to measure vagally induced bronchoeonstriction in conscious, unrestrained animals. Before allergen provocation, electrical stimulation of the intact vagus nerve (1-32 Hz, 0.1 ms, 7.5 mA for 10 s) resulted in a frequency-dependent bronchoconstriction, with a maximal 1.99-+0.15-fold increase in pleural pressure (Pp~) at 32 Hz. Aerosol inhalation of the NO synthase inhibitor N%nitro-L-arginine methyl ester (L-NAME; 12 raM, 15 min) by these animals caused significantly enhanced the vagally induced bronchoconstriction at all frequencies to a maximal 2.30_+0.16-fold increase in Pp~ at 32 Hz (p<0.01). At 6 h after OA provocation (between the EAR and LAR), the effect of L-NAME on vagal nerve stimulated bronchoconstriction had completely vanished at all frequencies (1.93 _+0.08 and 1.90_+0.09-fold increase in Ppl at 32 Hz, pre and post L-NAME, respectively; n.s.). At 24 h aRer provocation (after the LAR) the L-NAME-induced potentiation of vagally induced broncboconstriction was restored (2.08_+0.12 and 2.44_+0.17-fold increase in Ppt at 32 Hz, pre and post L-NAME, respectively; p<0.01). At all conditions used, L-N,/~ME alone did not affect baseline Pp~. These results indicate that (1) NO released from iNANC nerves causes inhibition of vagally induced bronchoconstriction in vivo, (2) the release of NO from iNANC nerves is deficient after the EAR, which could contribute to the enhanced vagal reflex activity after this reaction, and (3) the deficiency of neurally released NO is restored after the LAR. This work is was supported by a grant from the Netherlands Asthma Foundation.

Groningen Utrecht Institute for Drug Exploration, Depamnent of Medicinal Chemistry and Molecular Pharmacology, University of Groningen, Ant. Deusinglaan 2, 9713 AW Groningen, The Netherlands.

R12

45 OPPOSITE EFFECT OF NEUTRALIZING ANTIBODIES AGAINST IL4 AND IFNy ON BLOOD PRESSURE. D. van Heuven-Nolsen, T. Muis, I. van Ark, F.P Nijkamp, Evidence is accumulating that a disbalance in T lymphocyte-mediated immune function contributes to hypertension in man, rat and mouse. T lymphocyte-mediated immune functions result from the action of cytokines. T h l and Th2 lymphocytes are the most prominant subpopulations and IFNy and IL4 are considered to be the key cytoldnes they produce, respectively. The NZBxNZW F1 hybrid mouse develops a spontaneous hypertension. In view of the regulatory function of T h l and Th2 cells on the immune system and T cell function, we investigated the effect of calL4 and cdFN,/ on the development of hypertension in F1 hybrid mice and on the mean arterial pressure (MAP) in several normotensive mice strains. MAP in anesthetized, F1 hybrid mice increases from 64 -* 8 mm Hg at 4 to 6 weeks after birth to 102 -+ 4 mm Hg at 10 to 12 weeks of age. Control antibody treatment had no influence on MAP in all the mice strains investigated, ctlFNy treatment had no influence on the increase in MAP in F1 hybrid mice, but the increase in MAP was prevented by a l i a treatment. ~xlIA had no influence on MAP in normotensive NZW mice, however, ctlFNy caused an increase of the MAP of approximately 30 mm Hg. Mice strains differ from one another regarding their Thl/Th2 balance. NZW and C57BI/6 mice are more inclined to a Thl-like response, whereas Balb c mice show a preferential Th2 response. The results showed that cdFNy increased MAP in NZW and C57B1/6 mice, but was without effect in Balb c mice. In conclusion the present results indicate that the balance of IIA and IFNy directs the development of hypertension, alIA is antihypertensive in the hypertensive F1 hybrid mice and conversely ctlFNy induced hypertension in the T h l responser NZW and C57B1/6 mice. Supported by the Netherlands Heart Foundation (grant no. D92.009). Dept. of Pharmacology, Utrecht Instutite of Pharmaceutical Sciences, Utrecht University, P.O. Box 80.082, 3508 TB Utrecht, The Netherlands.

46 A PUTATIVE PHYSIOLOGICAL ROLE OF KININS IN THE PANCREAS. F.Lembeck & T.Gdesbacher. It was the high content of the enzyme kallikrein in the pancreas which lead to the discovery of the kallikrain-kinin system. A pathological role of kinins in pancreatitis has been postulated already in 1958 by Wade. An expedmetal model for acute pancreatitis was established by infusion of the CCK analogue, caerulein. Under pretreatment with captopril (to inhibit the enzymatic cleavage of kinins) a 30-60 min i.v.. infusion of caerulein induced a severe pancreatic oedema, a pronounced loss of plasma and hypovolaemia. Caerulein caused the release of enzymes into the pancreatic tissue, inducing morphological changes. All these changes were suppressed under pretreatment with the long-acting kinin antagonist icatibant (Hoe140), thus indicating a key role of kinins in this model of pancreatitis. I.v.infusion of Monastral blue (MB) (particles of 3 pm diameter which stick in opened endothelial gaps of venules) served as indicatior for plasma leakage, an eady sign of inflammation. Captopril alone already coused an accumulation of MB in the vessels, indicating a continuous release of kinins in the pancreas. Infusion of bradykinin or caerulein in ineffective doses caused a marked accumulation of MB unter captopril. All the accumulations of MB were completely suppressed under preteratment with icatibant. It seems therefore that, under physiological conditions, released kinins induce microcirculatory changes which allow a better accas of plasma to the secretory cells, thus allowing to adjust their function to the requirement. This conclusion is in congruence with observations in salivary glands or the cardioprotective effect of bradykinin in reperfused hearts. This physiological function differs from the pathophysiological situation of hypersecretion of enzymes as induced by a large dose of caerulein, the model for pancreatitis. F.Lembeck, Institut f.exp.u, klin.Pharmakologie, Universit~itsplatz 4, A-8010 Graz, Austria

47 CHARACTERISTICS OF INTERACTION BETWEEN GALANIN AND ITS ANALOGUES W I T H GALANIN R E C E P T O R IN THE RAT'S S T O M A C H FUNDUS. W. SLlWlNSKI, *P. REKOWSKI, A. HALAMA and R. KOROLKIEWICz Galanin is a novel peptide, which is reported to play a significant role in regulation of gastrointestinal motility. This study investigates affinity of galanine to its specific receptor in rat's stomach fundus strip. Rat gastric fundus strips were prepared according to Vane's method. Contraction of the muscle strips caused by different concentration of galanin were recorded using an isotonic transducer. Based on these results we constructed a conventional dose-response curve. Concentration of galanin (raM) % of maximal effect (+_ SE) l 6.5+ 2.65 3 18.7+ 4.42 10 46.6 +_ 2.72 30 73.2 + 2.44 100 94.7 + 2.44 300 99.4 + 0.56 The results present a mean of at least 10 experiments. EDs0 for galanin action was estimated to be 13.7 riM. Hill's coefficient for galanin equals 1.048. The implication of such result is that galanin follows a classical theory: one drug molecule interacts with one receptor molecule. Experiments are being conducted to construct similar dose-response curve for two other galanin receptor agonists: {Gal(1-14)'[AbuS]-SCY-1} and galantide (M-I 5). Preliminary results suggest these peptides might be strong galanin receptor agonists. Department of Pharmacology, Medical University of Gdafisk, Do Studzienld 38 Street, 80-227 Gdafisk, * Faculty of Chemistry, 18 Sobieski Street, 80952 Gdaflsk

48 MODULATION OF ACETYLCHOLINE R E L E A S E BY OPIOID R E C E P T O R S IN H U M A N N E O C O R T E X G. B. Landwehrmeyer*, O. Gleichauf*, R. Jackisch#, D. Peckys*, W. Seeger§ and T.J. Feuerstein* Both cholinergic neurotransmission and opioid peptides in cerebral cortex are thought to influence memory and cognitive functions in man. It is therefore of interest to investigate the regulation of acetylcholine release by opioid peptides as an important aspect of cholinergic neurotransmission. We studied the modulation of acetylcholine (ACh) release by opioid receptors in superfused slices of surgically removed human neocortex.Tissue slices from 27 patients were prelabeled with [3H]choline and stimulated electrically or by K ÷ (20 mmol/1) to evoke [3H]ACh overflow. Electrically evoked [3H]ACh release was tetrodotoxine-(TTX)sensitive and dependent on the presence of extracellular Ca ++. Evoked [3H]ACh release was significantly increased by the 8-opioid receptor antagonist naltrindole (10-7 mol/1) in the presence of peptidase inhibitors suggesting an endogenous, 8-opioid receptor mediated inhibition of AChrelease by enkephalin. The 8-opioid receptor agonist DPDPE and the ~opioid receptor agonist U-50488H in nanomolar concentrations reduced evoked [3H]ACh release in a concentration-dependent fashion; naltrindole (10 -7 mol/1) and norbinaltorphimine (10 -9 mol/1), respectively, antagonized these effects. Application of the g-opioid receptor agonist DAGO also resulted in an inhibition of [3H]ACh release; however, 6- and K-receptor antagonists were able to block the effect of DAGO. The greceptor agonists morphine and (+)-nortilidine had no effect on evoked [3H]ACh release. Together these results indicate that ACh release in human neocortex is inhibited by agonists on 6- and on ~-receptors, but not by agonists on g-receptors. K+-evoked [3H]ACh overflow in the presence of TTX was inhibited by U-50488H (10 -6 mol/l) but not by DPDPE (10-7 mol/1) suggesting the presence of ~-receptors on cholinergic terminals and the localization of &-receptors on cortical interneurons. The potent effect of the 8-receptor agonist on cortical ACh release is therefore likely to be indirect, by modulation of intrinsic cortical neurons. *Divisionof Clinical Neuropharmacology,Departmentof Neurology,#Departmentof Pharmacology,§Departmentof Neurosurgery,D-79106Freiburg,Germany

R13

49

51

EFFECT OF BETA-ENDORPHIN ON BLOOD PRESSURE-BLOOD V O L U M E RELATIONSHIP W. De JONG, P. SANDOR* J. COX-VAN PUT** and V. WIEGANT**

PK/PD MODELLING OF THE ANTICONVULSANT AND EEG EFFECTS OF PHENYTOIN O.E. Della Paschoa J.W. Mandema, R.A. Voskuyl and M. Danhof

We investigated the effect of beta-endorphin (13-E) on cardiovascular regulation in rats. Controlled stepwise induced hemorrhagic hypotension was used as a cardiovascular challenge. The rats were anesthetized with urethane, paralysed and artificially ventilated. In sham-operated and in hypophysectomized (HX) rats, mean arterial pressure (MAP) was decreased to 80, 60 and 40 mm mHg by blood letting. Although in I-IX plasma 13-E concentration amounted less than 10% o f controls with no difference in cerebrospinal fluid I~-E levels, shed blood volumes o f HX did not differ from values in sham-operated rats. Intracerebroventricular (icv) administration of 13-E made both intact and H X rats more sensitive to blood letting. In contrast, intravenous administration o f 13-E had no effect in both groups. Furthermore, icv administration o f a 13-E antiserum as performed in intact rats resulted in elevated shed blood volumes required to decrease MAP to the three pressure levels. This effect was mimicked by icy naloxone. These data point to a role of a brain 13-E system in the regulation of the systemic arterial pressure-blood volume relationship in controlled hemorrhagic hypotension. Institut de Pharmacologic, Universit6 Louis Pasteur, 11, rue Humann, 67000 STRASBOURG, France; *Research Department, 2nd Institute of Physiology, Semmelweis University of Medicine, BUDAPEST, Hungary; **Rudolf Magnus Institute for Neurosciences, University of Utrecht, UTRECHT, The Netherlands.

Phenytuin (DPH) is one of the most prescribed antiepileptic drags (AED). Surprisingly, very limited information concerning the kinetics of its pharmacological response is available. A major limitation to the assessment of the pharmacokineticpharmacodynamic relationship has been the lack of methods which allow accurate measurement of the effect intensity. Two new promising approaches, the direct cortical stimulation model (CSM) and the quantitative BBG monitoring (EBG), have been proposed. The former supplies information on the ability of an AED to suppress the onset and/or propagation of convulsive activity, whereas the latter has allowed the characterization of several CNS active drags. Purpose of this investigation was to characterize the PK/PD relationship of DPH with these two pharmacodynamic endpoints. In parallel, two groups of male Wistar-derived rats (n=9) were administered a single i.v. dose of DPH (40 mg/kg) and monitored during 5 hours. The increase in the beta frequency band, expressed as total number of waves (TNW), was used as BEG-effect measure for group I. An elevation of the threshold for generalized seizure (TGS), simultaneously with a minor effect on the threshold for localized seizure (TLS), was observed in group II. DPH pharmacokinetics was assessed by arterial blood sampling and plasma levels were analysed by a HPLC technique. The pharmacokinetic profile was described by saturation-elimination kinetics and fitted by a model with Michaelis-Menten elimination. The sigmoidal-Eo,~ model was used to describe the relationship between the EEG-effect and the concentration. For the anticonvulsant effect a non-linear relationship between concentration and effect was observed with no clear maximal effect. The time delay between DPH plasma concentrations and effect was estimated by an equilibration kinetics model using a hysteresis minimization routine. Mean I~o values were 0.077 and 0.108 rain" for groups I and II, respectively. Pharmacodynamic estimates for the EEG yielded an EC(50) of 12.5 pg/ml. It can be concluded that the changes in TNW can be used as a dynamic measure in the EEG model. The newly developed PK/PD model with Michaelis-Menten kinetics accurately characterized the time course of the effects of DPH. Moreover, the derived effects were found to occur in the same concentration range as the "therapeutic range" in humans. Leiden/Amsterdam Center for Drug Research, Division of Pharmacology, P.O.Box 9503, 2300 RA Leiden, The Netherlands.

50

52

PHARMACOKINETIC-PHARMACODYNAMIC MODELLING OF THE EEG EFFECT OF ALFENTANIL IN RATS. E.H. Cox, J.A. Kuipers and M. Danhof

INCREASED ACTIVITY OF CARDIAC G. IN END-STAGE HEART FAILURE: IS IT OF FUNCTIONALI]M~ORTANCE?. O.-E. Brodde, A 2 ~ B r o e d e - S i t z ", M. Vogelsan9 J, H.-R. Zerkowski '

Quantitative EEG monitoring, in combination with simultaneous pharmacokinetic-pharmacodynamic modelling, may be a useful tool to characterize the pharmacodynamics of CNS-active dmgs in-vivo. In order to characterize the pharmacodynamics of alfentanil, the pharmacokinetic-pharmacodynamic relationship was studied in three groups of chronically instrumented rats. Each group received two consecutive infusions of alfentanil with different infusion rates and -lengths (20-, 40- and 60 rain). Serial blood samples were collected for determination of the concentration of the opioid in blood in conjunction with continuous recording of the EEG. Increase of the power in the 0.5-4.5 Hz frequency band of the EEG was used as a pharmacodynamic endpoint. During the experiments, rats were artificially ventilated with air when needed and muscle rigidity was managed with repeated administration of vecuronium bromide. The concentration-time profile of alfentanil could be described with a two-compartment pharmacokinetic model. The values of clearance, volume of distribution at steady state and terminal half-life obtained from the first 20-rain infusion data were (mean + se) 53 -+ 6 mL/min.kg, 1.20 _+ 0.20 L/kg and 24 _+ 2.5 rain, respectively, and no significant difference in the pharmacokinetic parameters was observed between the three treatment groups (p>0.05). The EEG effect of alfentaniI could be directly related to its whole blood concentrations on basis of the sigmoidal Em~~ pharmacodynamic model. The values of Eo, Em~, ECs0 and Hill-factor obtained from the first 20-rain infusion data were (mean --. se) 95 _+ 17 ~V, 57 -+ 5 pV, 202 + 25 ng/mL and 1.5 +-. 0.2, respectively, and no significant difference was observed between the three treatment groups (p>0.05). However, ECs0 values obtained after the second infusion showed a 2-fold increase, indicating the development of tolerance. It is concluded that the in-vivo pharmacodynamics of alfentanil in the rat can be studied using an integrated pharmacokinetic-pharmacodynamic approach. Upon repeated administration, tolerance developed to the EEG effect of atfentanil. The results presented in this study suggest that in the estimation of the in-vivo pharmacodynamic parameters of synthetic opioids, functional tolerance development must be taken into consideration. Leiden/Amsterdam Center for Drug Research, Division of Pharmacology, P.O. Box 9503, 2300 RA Leiden, The Netherlands

In e n d - s t a g e h e a r t f a i l u r e (ESHF) c a r d i a c G is i n creased. To f i n d out whether t h i s is of f u n c t i o n a l importance we s t u d i e d the e f f e c t s of s e v e r a l ~ - a d r e n o c e p t o r (AR) a g o n i s t s , h i s t a m i n e ( H I S ) , s e r o t o n i n (5-HT) and c a r b a c h o l (CARB) on a d e n y l y l c y c l a s e (AC) a c t i v i t y in r i g h t (RA) and l e f t a t r i a (LA) and l e f t v e n t r i c l e s (LV) and on LV f o r c e of c o n t r a c t i o n from 6 p o t e n t i a l h e a r t t r a n s p l a n t donors (NFH) and from 20 p a t i e n t s w i t h ESHF (9 w i t h d i l a t e d (DCM) and 11 w i t h ischemic c a r d i o m y o p a t h y (ICM). In NFH in a l l 3 t i s s u e s i s o p r e n a l i n e (ISO), t e r b u t a l i n e (TER), HIS and 5-HT a c t i v a t e d AC ( e x c e p t LV where 5-HT showed o n l y weak and i n c o n s i s t e n t e f f e c t s ) and CARB i n h i b i ted I S O - s t i m u l a t e d AC; maximal e f f e c t s were (ISO = 100%): RA and LA: TER, HIS, 5-HT 50-65%; CARB 60-90%; LV: TER, HIS 60%; CARB 60%. In ESHF in a l l 3 t i s s u e s not o n l y ISO and TER, but a l s o HIS, 5-HT and GTP s t i m u 1 ~ e d AC was s i g n i f i c a n t l y reduced w h i l e NaF and Mn a c t i v a t e d AC and i n h i b i t i o n of I S O - a c t i v a ted AC by CARB was not a l t e r e d ; no d i f f e r e n c e was observed between ICM and DCM. S i m i l a r l y , not o n l y B-AR a g o n i s t s (ISO, TER, n o r a d r e n a l i n e , a d r e n a l i n e , dobutamine, e p i n i n e and dopamine) but a l s o HIS induced i n c r e a s e s in LV f o r c e of c o n t r a c t i o n were reduced in ESHF (no d i f f e r e n c e between ICM and DCM), w h i l e CARB induced decrease in I S O - s t i m u l a t e d LV f o r c e of c o n t r a c t i o n was unchanged. We conclude t h a t in ESHF i n c r e a s e s in c a r d i a c G do not enhance r e s ponsiveness of G i - c o u p l e d r e c e p t o r s but lead t o a r e d u c t i o n of the e f f e c t s of a l l r e c e p t o r system a c t i n g v i a c y c l i c AMP i n c r e a s e s . I n s t i t u t e of Pharmacology and ~! Dept. of T h o r a c i c S u r g e r y , U n i v e r s i t y of H a l l e ; " j Dept. I n t e r n a l M e d i c i n e , U n i v e r s i t y of Esen, Germany.

R14 53

55

THE INFLUENCE OF HYPERTHYROIDISM ON ADR/~OCEPTOR-MEDIATED RESPONSES IN RAT ISOLATED AORTAE

INFLUENCE OF BIBP3226, A SELECTIVE NPY Y~ ANTAGONIST, ON THE NPY-INDUCED INHIBITION OF NORADRENALINE OVERFLOW IN SWIMMING SPONTANEOUSLY HYPERTENSIVE RATS. J. Smit, A.F. Roffel, H.N. Doods# and J. Zaa,qsma. BIBP3226 has been shown to have potent antagonistic properties on neuropeptide

E.A. Winlder Prins, J.Zwaveling, M. Pfaffendorf, P.A, van Zwieten Hyperthyroidism is known to be associated with several cardiovascular alterations. Significant changes in the phammcodynamic behaviour of the cardiovascular system may be expected, although this matter has not been studied in detail. We have investigated the response to various compounds in isolated aortae obtained from chronic hyperthyroid Wistar rats. ttyperthyruldism was induced by feeding with a chow containing 5 mg L-thyroxine (F4) per kg, for 4 weeks, q[he thoracic aortae were excised and suspended in an organ bath with a physiological salt solution at 37°C, and gassed with 95% 02 and 5% COy Isometric force was measured and recorded. After 1 h of equilibration at a resting tension of 10 mN the preparation was subjected to a depolarising potassium salt solution (dpss), phenylephrine (Phe 10-6Ivl),and a further dpss, with intervals of 20 min. Cumulative concentration response curves (CRC) were constructed for: a~ adrenoreceptor agonists, g adrenoreceptor agonists, forskolin, and dibutyryl cAdVl]?.CRC for vascular relaxation were constructed after precontractiun with Phe 10~M. Schild analysis were perforuxed with the g-adrenoceptor antagonists ICI 118.551 (g2) and CGP 20712A (gO, with Phe for precontraction and isoproterenol as the B-agonist. The results are: control T4-treated control T4-treated -log EC60 ~ (mN) phenylephrine 6.7 - 0.1 6.8 -2_ 0.1 8.1 -+ 0.9 6.9 -+ 0.8 methoxamine 4.8 + 0.2 5.2 + 0.0' 8.8 -+ 0.9 5.3 -+ 0.5* cirazoline 6.9 -+ 0.1 7.0 -+ 0.1 2.7 + 0.4 2.1 +- 0.4 -log F~0 E ~ (%) isoproterenol 5.9 -+ 0.1 6.4 _+ 0.1' 81 +- 5 100 -+ 0* salbutamol 5.9 -+ 0.1 6.2 + 0.2 89 +- 4 98 + 2 terbutaline 5.2 -+ 0.1 6.0 _+ 0.1' 87 -+ 2 100 -+ 0 forskolin 7.2 - 0.0 7.3 - 0.I 100 + 0 100 -+ 0 dibutyvyI cAMP 4.3 -+ 0.0 4.4 -+ 0.0 100 + 0 100 -+ 0 * Indicate a significant (p < 0.05) difference from control. In the presence of the B2-adrenoceptor antagonist ICI 118.551 the Scllild plot became biphasic when constructed in aortae from hyperthyroid rats. g:-Adrenoceptor antagonism with CGP 20712A did not influence the Iso CltC of control preparations, whereas in those of T4-treated animals, a CGP concentration-dependentrightward shift occurred. The Schild analysis revealed a pA2 of 8.83. In conclusion, hyperthyroidism was associated with changes at the receptor level, including the appearance of functional intact B~-adrenoceptors. The second messenger systemwas not affected by hyperthyroidism, as reflected by the unchanged Ct/C of forskolin and dbc.~2VIP. Department of Phaxmacotherapy, Academic Medical C~atre, University of Affnsterdam, Meibergdreef 15, 1105 PuZ Amasterdam, The Netherlands.

Y (NPY) Y1 receptors both in vivo and in vitro. Though these receptors primarily have a postjunctional location, we have recently obtained evidence, using Y1- and Y2- selective agonists, that they are also present prejunctionally (Coppes et al.; EJP 1994,261,311). In the present study the possible role of prejunctional Y~ receptors in NPY-induced inhibition of noradrenaline (NA) overflow was studied in spontaneously hypertensive rats (SHR) in rest and during swimming-exercise. Male SHR rats were provided with two cannulae in the left and right jugular vein to infuse drugs and to sample blood, and an artedal cannula in the abdominal aorta to measure mean arterial pressure (MAP) and heart rate (HR). The rats had to swim against a counter current (22 cm/s) for 10 minutes in warm water (32_+1 °C). Before and during .swimming, MAP and HR were registered to estimate postjunctional effects, and blood samples were taken to determine plasma levels of NA and adrenaline (A). Infusion of NPY (2000 ng/kg/min, starting 12 minutes before exercise) significantly increased MAP with 20-25 mmHg under basal conditions as well as during exercise, and had no signigicant effect on HR in both conditions. Basal NA levels were decreased with 100+17 pg/ml (p<0.001); no effect on basal A levels was found. BIBP3226 (1.0 mg/kg/min for 3 minutes followed by 1.0 mg/kg/hr, starting 15 minutes before exercise) did not have any effect on MAP; however, BIBP3226 completely abolished the NPY-induced increase of MAP in rest and during swimming. NPY inhibited exercise-induced NA overflow to 56_+7% (p<0.O01) of control (infusion of PEG 10%); BIBP3226 alone tended to facilitate exercise-induced NA overflow (119-+10% of control; N.S.). When given together with NPY, BIBP3226 completely reversed the inhibitory effect of NPY and NA overflow again rose above control levels (124_+5%; p<0.05). Exercise-induced A overflow tended to be reduced after NPY infusion (75_+10%, N.S.), this effect was also abolished with BIBP3226. These results confirm earlier findings that the NPY-induced increase of MAP is mediated by Y~ receptors. A role for endogenous released NPY was indicated by the facilitating properties of BIBP3226 on NA overflow during exercise. In addition, clear evidence was provided for the involvement of prejunctional Y1 receptors in the NPY-induced inhibition of exercise induced NA overflow. University of Groningen, Dept. of Medicinal Chemistry & Molecular Pharmacology., A. Deusing/aan 2, 9713 AW Groningen, The Netherlands, # Div. of Pharma Research, Dr. Karl Thomae GmbH, Birkendorfer Str. 65, D-863967 Biberach, Germany.

54

56

COMPARISON OF CARDIOVASCULAR IN VlVO EFFECTS OF NORADRENALINE AND TYRAMINE U . P o l l e r , R . F . S c h 6 f e r s * ) , AoDau1*), O.-E.Brodde

AFFINITY AND BIODISTRIBUTION OF IODO-CGP 12177, A N E W RADIOLIGAND FOR THE IMAGING OF [5-ADRENOCEPTORS E.A.Dubois, G.A. Somsen, J.C. van de Bos, A.G.M. Janssen, H.D. Batink, M. Pfaffendorf, E.A. van Royen, P.A. van Zwieten.

The aim o f t h i s randomised, p l a c e b o - c o n t r o l l e d , s i n g l e b l i n d crossover study was t o c h a r a c t e r i z e adrenoceptor (AR) subtypes mediating myocardial and v a s c u l a r e f f e c t s o f exogenously i n fused n o r a d r e n a l i n e (NA, 10-160 ng/kg/min) and endogenously released NA (by tyramine i n f u s i o n (TY), 5-20 ~g/kg/min) in 6 h e a l t h y male v o l u n t e e r s . NA and TY were infused b e f o r e and a f t e r ~ - , g - o r muscarinic r e c e p t o r blockade with doxazosin (DO; 2 mg p . o . ) , yohimbine (YO; 15 mg p . o . ) , b i s o p r o l o l (BI; 15 mg p . o . ) , p r o p r a n o l o l (PR; 5 mg i . v . ) and a t r o p i n e (AT; 0.015 mg/kg BW i . v . loading dose f o l l o w e d by 0.0015 mg/kg/min by i . v . i n f u s i o n ) . Vascular and myocardial e f f e c t s were assessed by measurement o f d i a s t o l i c (DBP) and s y s t o l i c (SBP) blood pressure, h e a r t r a t e (HR) and HR corrected electromechanical s y s t o l e (qSgc). NA and TY increased SBP by 55 mmHg, and t h i s was p o t e n t i a t e d by AT. NA induced SBP r i s e was antagonized by BI and DO, TY induced increase by BI and PR. NA increased DBP (+ 15 mmHg); t h i s was attenuated by DO and p o t e n t i a t e d by PR, BI and AT. TY s l i g h t l y decreased DBP ( - 7 mmHg); t h i s was a m p l i f i e d by YO but antagonized by BI and PR. In c o n t r a s t t o TY (no i n f l u e n c e ) NA decreased HR, PR enhanced and DO and YO abolished these HR changes. In presence o f AT both NA and TY increased HR. Shortening o f QSgc by NA and TY was suppressed by PR and BI. DO and YO enhanced the NA induced e f f e c t . We conc 1 ude t h a t exogenous l y admin i s t e r e d and endogenous l y r e l e a s e d NA produce d i v e r g e n t c a r d i o v a s c u l a r e f f e c t s . The p r e dominant e f f e c t o f NA seems t o be v a s o c o n s t r i c t i o n viaoC1-ARs t i m u l a t i o n . * ~ - A R - b l o c k a d e unmasked a d i r e c t p o s i t i v e i n 6 t r o p i c e f f e c t of NA, t h a t i s m e d i a t e d by s t i m u l a t i o n o f c a r d i a c 3-ARs. In c o n t r a s t , TY produced n e a r l y no v a s o c o n s t r i c t i o n , b u t p o s i t i v e i n o t r o p i c e f f e c t s v i a s t i m u l a t i o n of gt-ARs. M u s c a r i n i c r e c e p t o r b l o c k a d e w i t h AT potentiated most 6f t h e c a r d i o v a s c u l a r e f f e c t s induced by both e x o g e n o u s l y a d m i n i s t e r e d and e n d o g e n o u s l y r e l e a s e d NA. I n s t i t u t e of Pharmacology, U n i v e r s i t y of H a l l e , and*) Dept. of I n t e r n a l M e d i c i n e , U n i v e r s i t y o f Essen, Germany

g-adrenoceptors (f3-AR) are known to be downregulated because of increased catecholamine levels in patients with congestive heart failure. W e intend to develop new radioligands for the imaging of B-AR in vivo. We compared the in vitro affinity o f 4- (3-t-butylamino-2-hydroxypropoxy)-benzimidazoleone ((+)CGP12177), (S)-4-(3-t-butylamino-2-hydroxypropoxy)-5-iodobenzimidazole-2-one (CYBL2BS), 4-(3-t-butylamino-2-hydroxypropoxy)-7iodo-benzimidazole-2-one (CYBL2A), for the [3-adrenoceptor, using [125I]Iodocyanopindolol as the radioligand. The Ki-values (nanomol/1, means +SEM, n=5),were 1.17+0.42, 28786+_9255, 11.06+_2.08, for CGP12177, CYBL2A, CYBL2BS, nadolol and inadolol, respectively. Subsequently, CYBL2BS was tested in vivo in New Zealand White rabbits (2.5-3.5 kg). Rabbits received an intravenous injection of 50 gCi of CYBL2BS (specific activity > 5000 Ci/mmol) and were sacrificed at 5,15,30,50 minutes, 1,2,4 and 24 hours after injection. Organs were removed, w e i g h e d and radioactivity was measured. Radioactivity levels in the left ventricle (LV) and in the lungs, expressed as % I.D./g tissue x kg body weight, are listed in the table (means +S.E.M., n=3 per time point). LV Lung LV Lung 5'

0.28+0.07

1.49_+0.63

lh

0.20+0.01

0.46-+0.03

15'

0.46+-0.04

0.89+-0.03

2h

0.13-+0.002

0.33_+0.02

30'

0.20+-0.03

0.66_+0.11

4h

0.07-+0.009

0.21+0.002

50' 0.21+-0.01 0.50+-0.04 24h 0.02+_0.002 0.08+-0.01 Although uptake of CYBL2BS in the lungs could be blocked significantly (0.63 + 0.09 in the controls versus 0.33 + 0.02, n=5, in the pretreated animals), uptake in the left ventricle could not be blocked by pre-injection of 0.1gmol propranolol i.v. (0.24 +- 0.02 in the controls versus 0.20 + 0.009, n=5, in the pretreated animals), suggesting non-competitve binding to the receptor, which was confirmed by in vitro experiments. From the present data, we conclude that CYBL2BS may be a promising radioligand for the imaging g - A R i n vivo. Depts. of Nucl. Med. and Pharmacotherapy, Academic Medical Centre, Umversity of Amsterdam Meibergdreef 15, 1105 AZ Amsterdam The Netherlands.

R15 57

59

THE INFLUENCE OF HYPERTHYROIDISM ON ISOPROTERENOL INDUCED RESPONSES OF ISOLATED HEARTS L Zwaveling, E.A. Wmlder Prim, M. Pfaffendorf and P.A. van Zwieten Hyperthyroidism is known to be associated with several cardiovascular alterations. The clinical symptoms of hyperthyroidism are suggestive of an altered sympathetic tone, although this is not reflected by elevated plasma catecholamine concentrations. To investigate whether the cardiac sensitivity to g-adrenoceptor stimulation is changed, we measured the responses to isoproterenol in isolated hearts taken from chronic hyperthyroid animals. Hyperthyroidism was induced by feeding male Wistar mrs of approximately 250 g with 5 mg/kg L-thymxine(T4)-containing chow during four weeks. T4 treatment caused significant increases in serum T4-1evels (treated vs. control: 266.3 ± 0.13 and 55.9 ± 2.1 nmol/l; means ± SEM, n=8; P<0.05) and decreases in serum TSH levels (0.33 ± 0.03 vs. 0.87 _+ 0.13 ng/ml;n=8; P<0.05), The inotmpic effects of isopmterenol were assessed in paced (5 Hz) Langendorff hearts (37°C), whereas the chronotropic effects were investigated in spontaneously beating hearts. The influence of the drug on left ventricular pressure (LVP), dP/dTmax and heart rate (HR,anpaced hearts) was established. The basal left ventricular pressure and dP/dTmax of hyperthyroid anpaced rat hearts were higher compared to control rat hearts.

INHIBITION BY N-ACETYLSEROTONIN OF NITRIC OXIDE SYNTHASE EXPRESSION 1N CULTURED CELLS AND IN THE ANAESTHETIZED RAT P Klemm, M Hecker, H Stockhansen, C C Wu and C Thiemermann Induction of the calcium-independent isoform of nitric oxide (NO) synthase (iNOS) in various cell types has been implicated in the circulatory failure in experimental models of septic shock. Tetrahydrobiopterin (BH4) appears to be an essential co-factor for NO formation and therefore an inhibition of its biosynthesis represents a feasible therapeutic target. We have investigated the effects of an inhibitor of BH 4 synthesis, N-acetylserotonin (NAS), on the expression of iNOS in cultured macrophages and smooth muscle cells in vitro, and on the hypotensive response to bacterial lipopolysaccharide (LPS) in the anaesthetised rat in vivo.NAS (0.01-5 mM) caused a concentration-dependent inhibition of the accumulation of nitrite in the conditioned medium of LPS/interferon-~(IFNq,)stimulated RAW 264.7 macrophages and interleukin-1NIL-113)-activated vascular smooth muscle cells (VSMC). This effect was paralleled by a similar decrease in the iNOS protein content of these cells, as determined by immunoblot analysis.Pretreatment of RAW 264.7 macrophages with BH 2 (0.1 raM) did not restore nitrite formation in the presence of NAS (1 raM). Intravenous administration of NAS (1 mg kg -I min -1 for 30 min) in anaethetised rats strongly prevented the fall in mean arterial blood pressure, restored the pressor response to noradrenaline (1 gg kg-1), and ameliorated the increase in plasma nitrite following exposure to LPS (10 mg kg'l). NAS-pretreatment also attenuated iNOS activity in lung homogenates, as determined by the conversion of radiolabelled L-arginine to L-citrulline, and partially restored the constrictor effect of noradrenaline in aortic rings isolated from LPS-treated rats. Moreover, NAS significantly reduced the rise in the plasma concentration of tumor necrosis factom (TNFa) in response to LPS. These findings suggest that NAS inhibits the expression rather than the activity of iNOS in cultured macrophages and smooth muscle cells. This effect of NAS appears to be independent of the availability of BH 4, but may be related to an attenuation of the release of TNFa following LPS administration, as shown in the anaesthetised rat. This mechanism may also account for the beneficial haemodynamic effect of NAS in our experimental model of endotoxaemia.

Table 1: Effects mediated by isoproterenol (pD2-values from LVP, coronary flow(CF) and HR, max.increase in LVP (ram Hg), n=6-10, means ± SEM. *:P<0,05 treated vs control) in isolated heart taken from hyperthyroid mrs. T4-treated control isoproterenol pD2 (LVP) 8.46 ± 0.07* 8.10 ± 0.05 (paced) pD2 (CF) 8.46 ± 0.09* 8.21 _+ 0.07 LVPmax 119 ± 17' 167 ± 10 isoproterenol pD2 (LVP) 8.84 _+ 0.04* 8.28 _+ 0.11 (anpaced) pD2 (CF) 8.55 _+ 0.16 8.26 ± 0.16 pD2 (HR) 8.81 + 0.11 8.85 -t- 0.11 LVPmax 79 + 10" 154 ± 9 In conclusion, the sensitivity of both paced and unpaced hearts taken from hyperthyroid animals to inotropic responses to the l?,-adrenoceptoragodist isoproterenol proved increased, whereas the chronotropic effects remained uninfluenced. However, both in paced and unpaecd hearts maximal inotropic responses (LVPmax)to fi-adrenergic stimdiatinn were lower in hearts taken from hypertbymid animals. Department of Pharmacothempy, Academic Medical Centre, University of Amsterdam, Meibergdreef 15, I105 AZ Amsterdam, the Netherlands.

CASSELLA AG, Hanauer Landstrasse 536, 60386 Frankfurt

58

6O

SUMATRIPTAN CONSTRICTS PORCINE CAROTID A R T E R I O V E N O U S A N A S T O M O S E S (AVAs) VIA 5-HT m R E C E P T O R S . P. de Vries, J.P.C. Heiligers and P.R. Saxena

PEPTIDOGLYCAN AND LIPOTEICHOIC ACID F R O M S T A P H Y L O C O C CUSAUREUS SYNERGISE IN INDUCING NITRIC OXIDE SYNTHASE SJ De Kimpe, M. Kengatharan, C Thiemermann and JR Vane.

The antimigraine drug sumatriptan constricts carotid AVAs by a 5-HTl-like receptor. The lack of antagonism by metergoline suggested that the 5-HT m receptor subtype may not be involved [1]. We have now examined the effects of a selective 5-HT~D receptor antagonist GR127935 [2] on the sumatriptan-induced reduction of AVA blood flow in anaesthetized pigs. In animals treated with saline, sumatriptan (30-300 gg/kg, iv) reduced the total carotid blood flow by exclusively affecting the AVA fraction; treatment with GR127935 (0.25 and 0.5 mg/kg, iv) potently inhibited these responses (Fig. 1). In addition, GR127935 itself reduced Total c a r o t i d AVA the total carotid and AVA flows b (max change: 19_+4% and 29_+8%, l b ~~_ ~ ' D _ respectively) and antagonized the sumatriptan-induced increases in i b -!~i ~ ~-----~.% skin and ear blood flows. The l ~ above results suggest that the AVA constriction as well as arteriolar -4o ~ dilatation in the skin and ears by :" - * o - - , , , -B0--, ~ sumatriptan are mediated by 5-HTID o ~0 ~00 ~00 o 30 ~o~ ~oo receptors. Since GR127935 has SumatriptRn(pg/kg) Sumntriptan(.~/~ been reported to be a 5-HTID~ Fig. L Effect of sumatriptan in pigs treated with saline agonist, but a 5-HT~o~ antagonist in (@) or GR127935 (~, 0.25 and m, 0.5 mg/kg), a, cells with cloned receptors [3], and P
o ~-:,~!.~i

-,0

[1] [2] [3] [4]

Den Boer MO et al. (1992) NS Arch Pharmacol 345:509-515. Clitherow J W e t al. (1994) J Med Chem 37:2253-2257. Panwels P J, Colpaert FC (1995) Neuropharmacology 34:235-237. Saxena PR, Ferrari MD (1989) Trends Pharmacol Sci I0:200-204.

Department of phannacology, Erasmus University Rotterdam, P.O.box 1738, 3000 DR Rotterdam, The Netherlands.

Gram-positive organisms, such as S t a p h y l o c o c c u s aureus, lack an outer-membrane with endotoxin, but contain peptidoglycan (PepG) and lipoteichoic acid (LTA) as major cell wall components. LTA induces iNOS and circulatory failure (1). Here, we investigated the effect of PepG combined with LTA on the induction of iNOS in cultured macrophages and anaesthetised rats. The release of nitrite, indicative of nitric oxide formation, by murine J774.2 macrophages was measured by the Griess method. Male Wistar rats (200-325g) were anaesthetised with thiopentobarbitone sodium (120mg/kg, ip). The carotid artery was cannulated to obtain arterial blood samples for measurement of blood gasses and the jugular vein for administration of compounds. At the end of the experiment, lungs were removed and iNOS activity was determined in the homegenates by the conversion of L-arginine to L-citrulline. Incubation of macrophages with PepG (1-100~tg/ml for 24 h) alone did not result in an increased nitrite formation. However, in the presence of interferon (IFN)- 7 (10u/ml), PepG caused a concentration-depandent accumulation of nitrite. Interestingly, the nitrite formation caused by LTA alone (10gg/ml) was increased fourfold by simultaneous incubation with PepG (30gg/ml). Injection of PepG (I0 mg/kg) and LTA (3 mg/kg) in anaesthetised rats gave a strong increase in iNOS activity in lungs from 0.3+0.1 to 22+4 pmol L-citrulline/min/mg protein after 360 min, compared to 3.2+0.9 for PepG alone and 2.5_+0.4 fur LTA alone. The marked increase in iNOS activity in lungs was associated with a marked decrease in arterial oxygen pressure from 78+2 mmHg for control to 55_+4 mmHg for PepG and LTA, which was prevented by infusion of the iNOS inhibitor aminoguanidine (70_+4 mmHg; P<0.05; 10 mg/kg/h starting 20 min prior to injection of PepG and LTA). Thus, PepG and LTA synergise in inducing iNOS acitivity, which in anaesthetised rats contributes to the respiratory failure elicited by the Gram-positive bacterial wall components. (1) SJ De Kimpe et al (1995) Br. J. PharmacoI. 114:1317-1323. The William Harvey Research Institute, Medical College of St. Bartholomew's Hospital, Charterhouse Square, London EC 1M 6BQ, UK.

R16

61 MYOGENIC RESPONSIVENESS IN RAT SMALL ARTERIES IS AUGMENTED BY NORADRENALINE THROUGH PRESSUREDEPENDENT DEPOLARIZATION J.P.M.WesselmanI, E.VanBavelI, R.Schubert2, H.Nilsson2, M.J.Mulvany2 The myogenic response refers to the ability of blood vessels to constrict with increased transmural pressure and can be augmented by e.g. noradrenaline (NA). In this study we tested how NA affected the relation between pressure and smooth muscle membrane potential (Em). Rat mesenteric small arteries were cannulated using micro-pipettes. The outer diameter (D) was monitored with a video cmnera. Em was measured using micro-electrodes. Pressure was raised stepwise between 10 and 120 mmHg, either without drugs or with 10 laM NA. D is normalized to D at 120 mmHg and full dilation. Without drugs pressure elevation increased both D and Era. 10 p.M NA at low pressure caused both constriction and depolarization, as well as vasomotion and oscillations in Em. Pressure elevation did not change D, but evoked extra depolarization (see table, data are means_+SEM, n= 6-41 vessels, 'min' and 'max' refer to minima and maxima of NA-induced oscillations, *: p<0.( 5 by t-test). P, mmHg

PSS

NAmin

NAmax

PSS

NAmin

NAmax

10

65.4-+0.5

45.6_+0.8

47.3±0.8

-56.0_+0.7

-56.7-+2.3

-40.0_+2.4

22

71.8±0.6*

46.0_+0.7

47.6-+0.6

-55.3_+1.2

-49.6_+1.9"

-40.1±1.9

86.3±0.6*

46.6_+0.8

48.4_+0.9

-54.1_+1.3

-45.3_+1.4"

-37.9±1.7

80

93.2+_1.1"

50.3_+2.8

52.0±2.9

-51.4-+1.3"

-42.7-+1.6"

-37.7±2.7

120

92.4±1.7*

49.3+_1.8

50.7-+2.2

-47.3-+1.8"

-39.0_+1.5"

-34.8±1.9

44 ,

Em, mV

diameter, %

'

Thus the depolarization upon pressure elevation is augmented by 10 gM NA. We conclude that the augmentation of myogenic responsiveness by NA is, at least in part, due to pressure-dependent depolarization. 1Department of Medical Physics, University of Amsterdam, Meibergdreef 15, 1105 AZ, Amsterdam, The Netherlands; 2Department of Pharmacology, University of Aarhus, University Park 240 , DK-8000 Aarhus C, Denmark.

63 HYPOREACTIVITY OF PERIPHERAL RESISTANCE ARTERIES IN EXPERIMENTAL H E A R T FAILURE FRM Stassen, GE Fazzi, PMH Schiffers and JGR De Mey

In heart failure patients, baroreflex control of blood pressure is impaired and peripheral resistance arteries are hyporesponsive to vasoconstrictor substances. An experimental animal model may be useful to determine the underlying mechanisms. We therefore evaluated the structure and reactivity of the thoracic aorta (AO) and mesenteric resistance-sized arteries (MrA) obtained from rats who underwent left coronary artery ligation (MI) 5 weeks earlier and from sham-operated controls (SHAM). Surgery resulted in marked (>40%) infarction of the free left ventricular wall, in elevated plasma levels of various neurohumoral mediators and in significant increases of cardiac and lung wet weight. The isolated vessels were chemically sympathectomised, mounted in a myograph at individual optimal diameter, exposed to vasoconstrictor agents, fixed and processed for histological examination. In AO no differences were found between SHAM and MI rats with respect to (i) pD 2 and Emax for noradrenaline (NA) and phenylephrine (PHE), (ii) active wall tension in response to 125 mM K + and (iii) lumen diameter and media cross sectional area.Yet, in MrA, Emax for NA (1.92±.15 vs 3.35±.24 mN/mm), Emax for PHE (1.57± vs 2.96±.37) and Emax for K ÷ (1.70±.20 vs 2.73±.27) were significantly reduced, while pD 2 for the agonists, media cross sectional area and lumen diameter were not modified. These observations suggest that in rats with experimental heart failure, structure and contractility of the AO is not altered while MrA display a marked reduction of their contractility. This resistance arterial hyporeactivity does not seem to involve availability or activity of membrane receptors and is not due to structural remodeling of the vessel wail. Supported by the Netherlands Scientific Research Organisation (NWO, 518291 PCJC). Department of Pharmacology and Cardiovascular Research Institute Maastricht, POBox 616, 6200 MD Maastricht, The Netherlands

62

64

PHARMACOLOGICAL AND MORPHOLOGICAL CHARACTERISTICS OF CORONARY ARTERIES AND AORTAE IN HYPERTENSION. B.C.P. H0sken, A.C. van der Wal*, P. Teeling*, M.-J. Mathy, A.J. Pijl, M. Pfaffendorf & P.A. van Zwieten. Hypertension is associated with important structural and functional changes in vascular smooth muscle. The question arises whether different vascular beds react similarly or in a heterogeneous manner to the hypertensive state. Accordingly, we evaluated the contractile response of phenylephrine (PhE) and the endothelium-dependent relaxation induced by methacholine (MCh) in isolated thoracic aortic rings obtained from 30 and 52 weeks old SHR and WKY rats, respectively. In coronary arteries taken from 22 weeks old WKY and SHR we investigated the response to serotonin (5-HT) and MCh. In addition, we quantitatively studied the morphology of the coronary arteries and thoracic aortae taken from 4, 30 and 52 weeks old SHR and WKY, respectively. In the coronary arteries the maximal effect (Emax) of 5-HT was significantly enhanced in the SHR vessels (2.8 _+ 0.4 mN/mm) compared to the Emax in the WKY preparations (0.9 -+ 0.2 mN/mm), and the concentration-response curves (CRC) in the SHR-arteries were shifted leftward (PD2= 6.19 + 0.03 and 6.77 _+ 0.06 for WKY and SHR, respectively). The MCh-induced relaxation was not influenced by hypertension of the donor animals. The pD 2- and Emax- values of the CRC of PhE were the same in the aortic rings taken from age-matched WKY (6.35 + 0.06, 4.3 + 0.4 mN) and SHR (6.52 _+ 0.07, 3.8 + 0.4 mN). The MCh-induced responses (pD2 and Emax) were only impaired in 52 weeks old SHR (6.97 + 0.10, 58.3 -+- 6.4%) when compared with the age-matched WKY (7.29 + 0.08, 87.3 + 3.1%). In coronary arteries taken from the SHR the media/lumen ratio values were significantly (p< 0.05) increased compared to those obtained from the age-matched WKY. Both the media- and lumen- area of the aortae taken from the SHR were increased when compared with those from the age-matched WKY rats. Therefore, no difference in media/lumen ratio between the aortae from the SHR and WKY were observed. In conclusion, the responsiveness and morphological structure of different vascular beds during hypertension are clearly heterogeneous.

THE ROLE OF ACE IN THE PROCESS OF NEO-INTIMA FORMATION IN THE RAT P. A. M a r t o r a n a , M. Paul, M. S. Fernandez-Alfonso, B. A. Sch61kens, Frankfurt/M a n d Heidelberg, D.

Depts. of Pharmacotherapy and Cardiovascular Pathology*, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands.

The role o f ACE on neo-intima formation was i n v e s t i g a t e d in a m o d e l of c a r o t i d b a l l o o n injury in the rat (21-day study) using molecular biological, biochemical, and m o r p h o m e t r i c a l m e t h o d o l o g i e s . Total RNA was higher in t h e lesioned t h a n in t h e control artery a n d r e a c h e d a p e a k at 6 days. PDGF-A mRNA was m o d e r a t e l y i n c r e a s e d (+ 30 %) b e t w e e n 6 a n d 16 days w h e r e a s ACE mRNA was m a r k e d l y (+ 80 %) i n c r e a s e d from d a y 2 to d a y 10 a f t e r the b a l l o o n injury. These d a t a suggest a role for ACE in the d e v e l o p m e n t of t h e neo-intima. Thus, the e f f e c t of the ACE-inhibitor ramiprilat (R) was investigated in this model. R was g i v e n at t h e doses of 0.1,0.3, 1.0, a n d 3.0 m g / k g / d a y for 21 days. At t h e e n d of the study in the R-rats t h e r e was a d o s e d e p e n d e n t d e c r e a s e in DNA c o n t e n t in the lesioned artery as c o m p a r e d to the control rats. Furthermore, also the a r e a o f t h e neo-intima a n d t h e ratio n e o - i n t i m a / m e d i a w e r e d e c r e a s e d b y the R-treatment in a d o s e - r e l a t e d fashion. All these d a t a show t h a t ACE plays a role in t h e process o f n e o - i n t i m a f o r m a t i o n in this model. P. A. M a r t o r a n a , Cassella AG, H a n a u e r Landstr. 526, 60386 Frankfurt/M, D.

R17

65

67

ACETYLSALCYLIC ACID PROLONGS EPICARDIAL POTENTIAL DURATION AND ENHANCES ARRHYTHMOG E N E I T Y IN I S C H E M I C I S O L A T E D R A B B I T H E A R T S . S.Dhein, E.Gottwald, M.Schott and W.Klaus

Neuronal nicotine and muscarine receptors (M1, M4) control the evoked release of newly-synthesized [~H]acetylcholine in human isolated bronchi T. Reinheimer, M. Pins, U. Tauber, KD. H6hle*, K. Rack~ *~ and I. Wessler

Isolated saline perfused rabbit hearts were treated with increasing concentrations of acetylsalicylic acid (ASA, 0.05, 0.1, 0.5, 1 gM). Epicardial activation and repolarization process were analysed using an epicardial mapping (256 unipolar leads). For each electrode activation and repolarization time was determined. From these data the "breakthrough-points" (BTP) of epicardial activation were determined. At each electrode an activation vector (VEC) was calculated giving direction and velocity of the local excitation wave. The beat similarity of various heart beats compared to control was evaluated by determination of the percentage of identical BTP and of similar VEC (deviation < 5°). At each electrode the local activation recovery interval (ARI) and its standard deviation (of 256 leads, dispersion, DISP) were determined. ASA led to an increase in ARI (7%), a decrease in BTP (by 10%) and VEC (by 15%). In a second series 30 rain regional ischemia was induced by LAD occlusion followed by 30 rain reperfusion in absence or presence of ASA. In control hearts VEC, BTP, ARI were markedly reduced and DISP and was greatly increased. These changes of VEC and DISP were significantly enhanced by 0.5 p.M ASA, so that in all (7/7) ASA treated hearts sustained ventricular fibrillation occured after 20 rain ischemia, whereas in absence of ASA fibrillation was found in only 2/7 hearts during reperfusion and not during ischemia. Blockade of cyclooxygenase with 1 p.M indomethacine did not cause these changes. However, ASA after pretreatment with indomethacine exhibited the same effect as ASA alone. From these results we conclude, that ASA can induce ventricular fibrillation possibly by reduction in cellular coupling and probably independent from its action on cyclooxygenase. Thus, in acute myocardial ischemia ASA may have -besides the well known mad desired antiaggregatory effects- electrophysiologic side effects which seem to be proarrhythmic in regional ischemia at least in this model. (supported by the DFG)

Prejunctional inhibitor3,muscarine autoreceptors play an important role in the local control of acetylcholine release. For example, release of acetylcholine from pulmonary cholinergic neurones is inhibited by stimulation of neuronal muscarine receptors. In the present experiments we have attempted to characterize these musearine receptors in human and rat airways. The release of newly-sy,nthesized [3H]acetylcholine (ACh) was measured in the absence and presence of the M4-preferring agonist clozapine (Liston et al., Life Sci (1995) 56:1018). In addition, the effects of atropine or subtype preferring antagonists (MI: pirenzepine; M2: AFDX-116:M3: para-fluorhexahydrosiladifenidol(pFHHSiD)) on evoked pH]ACh release were investigated to identify the muscarine receptor subtype involved in the endogenous feed-back. Human bronchi (diameter 3-6 ram) obtained from patients with lung cancer at thomcotomy and mt tracheae were isolated and incubated in an organ bath. Airway mucosa was removed at the start of the experiments and the bath medium contained 3 IxM indomethacin and 0.3 pM propranotol to exclude indirect effects on evoked [3H]ACh release. After labelling with [3H]choline (30 rain) the release of [3H]ACh was elicited by two (or four) subsequent periods of transmural stimulation. [3H]ACh released by the first stimulation was regarded as individual control and the antagonists or clozapine were added 18 rain before the second stimulation. In some experiments tubocurarine (TC; 100 p.M)was used to block nicotine receplors Clozapine inhibited evoked [3H]ACh release in a concentration-dependent manner (EC50: 0.10 pM). Atropine (0.1 !aM)prevented the inhibitory effect of 1 gM clozapine indicating a specific, receptor-mediated effect. Atropine (0.1 pM) alone enhanced (40 %) evoked [3H]ACh release, irrespective of the presence or absence of TC. A subtype-specific concentration of pirenzepine (0.1 gM) inhibited (25%) evoked [3H]ACh release but in the presence of TC, i.e. with blocked nicotinic transmission, this inhibition was converted to a facilitation (30 %). AFDX-116 (0.1 and 1 pM), in the presence or absence of TC, produced a slight enhancing effect, whereas p-FHHSiD did not affect [3H]ACh release. In the rat trachea clozapine was without an inhibitory effect but oxotremorine reduced and atropine facilitated evoked [3H]AChrelease. In human airways a rather complex control by neuronal nicotine and muscarine receptors of ACh release appears to exist; facilitatory and inhibitory M1- as well as inhibitory M4-receptors are involved. The facilitatory Ml-receptors are operating only under the condition of an intact nicotinic transmission. Whether this interaction between nicotine and Ml-receptors occurs at the level of the cell body or more distal at the axon and varicosities remains to be elucitated.

Institute for Pharmacology, 50931 Cologne, Gleuelerstr.24, FRG

Department of Pharmacolgy, Univ. Mainz, Obere Zahlbacher Str. 67, D-55101 Mainz *St. Hildegardis Hospital, Clinic for Surgery, D-55101 Mainz **Department of Pharmacology, Univ. Bonn, D-53113 Bonn, Germany

66

68

PHARMACOKINETICS AND CARDIOVASCULAR PHARMACODYNAMICS IN RATS OF THREE ADENOSINE A1-RECEPTOR AGONISTS W I T H DIFFERING LIFOPH1LICITY E.A. van Schalck, G. Cristalli, A.P. IJzerman and M. Danhof

MODULATION OF NORADRENALINE RELEASE BY ENDOGENOUS ACETYLCHOLINE IN THE GUINEA PIG TRACHEA J.R.A. de Haas, P.G.E. Kockelbergh, J. Zaagsma and A.F. Roffel. Guinea pig tracheal smooth muscle is innervated both by sympathetic and parasympathetic nerves. Colocalization of adrenergic and cholinergic varicosities suggests the possibility of prejunctional communication between these two nervous systems. We studied this putative reciprocal regulation by measuring the evoked release of endogenous noradrenaline (NA) and acetylcholine (ACh) from epithelium-denuded guinea pig tracheal preparations in the absence and presence of neuronal uptake or acetylcholinesterase (ACHE) inhibitors. Experiments were performed in the presence of 10 g M tyrosine. Determination of both neurotransmitters was through HPLC with electrochemical detection. Electrical field stimulation (16 Hz, 150 mA, 0.8 ms) during 5 minutes released 94.6 ± 29.1 pmol.(g tissue) "1 N A and 29.8 ± 15.2 pmol.g -1 ACh (n=6). The c~2-adrenoceptor antagonist yohimbine (1 gM) increased N A overflow to 168% of control. Combining yohimbine with the reuptake inhibitor desipramine (DMI, 1 pM) increased N A overflow to 295%. In both situations the ACh release was unaffected. In contrast, the muscarinic antagonist atropine (I ~tM) increased the overflow of both N A and ACh, to 174% and 156% respectively. Addition of neostigmine (1 gM) to atropine caused a slight but not significant increase in N A release whereas the ACh release increased to 2416%. These results indicate that endogenous acetylcholine inhibits its own release and that of noradrenaline through prejunctional muscarinic auto- and heterureceptors respectively, while the increased N A release after yohimbine administration indicates regulation via az-adrenergic autoreceptors. We conclude that reciprocal prejunctional regulation between the adrenergic and the cholinergic system in the guinea pig trachea may play a role in the regulation of neurotransmitter release.

Selectivity of purinergic drug action in vivo is not only determined by the affinity of the compound for the receptor, but also by its pharmacokinetic characteristics. In this study we investigated the relationship between the pharmacoldnetics and the cardiovascular effects of three adenosine agonists with different physico-chemical properties. Conscious male Wistar rats received an intravenous infusion of either 0.40 mg/kg N6-cyclopentyladenosine (CPA), 0.60 mg/kg N6-(p-sulfophenyi)adenosine (SPA) or 0.60 mg/kg 1-deaza-2-chloro-N6-cyclopentyladenosine (DCCA) in 5 min. After a single dose frequent arterial blood samples were drawn and cardiovascular parameters were monitored continuously. Blood concentrations were determined by HPLC and plasma protein binding by ultrafiltration. For each compound the concentration-time profiles could be described by a bi-exponential function. The compounds had large differences in pharmacokinetic properties. This resulted in terminal half-lives of (mean + SEM) 66 + 10, 24 + 1 and 8.2 -+ 0.4 min for SPA, DCCA and CPA, respectively. SPA had a significant lower blood clearance. Due to its higher lipophilicity DCCA had a larger volume of distribution and protein binding. The pharmacokinetic differences led to different durations of action. The individual concentration-heart rate relationships could be adequately described by the sigmoidal E ~ model. For SPA, DCCA and CPA the ECs0 values based on free drug concentrations were (mean + SEM) 423 _+92, 9.5 -+ 1.1 and 1.8 _+0.4 nM, respectively. These in vivo values correlated closely to the affinity of the compounds for the adenosine A~ receptor in vitro, with corresponding K~ values of (mean -+ SEM) 423 + 71, 34 + 7 and 1.2 -+ 0.4 nM, respectively. This study demonstrated that the adenosine agonists had different pharmacokinetics, which in turn had a large effect on the duration of the cardiovascular response. However, on the basis of an integrated pharmacokinetic-pharmacodynamic model estimates of potency could be obtained, which were independent of dose and pharmacokinetics. Leiden/Amsterdam Center for Drug Research, Divisions of Pharmacology and Medicinal Chemistry, P.O. Box 9503, 2300 RA Leiden, The Netherlands.

Supported by grant 93. 70 from the Netherlands Asthma Foundation. Groningen Utrecht Institute for Drag Exploration. Department of Medicinal Chemistry and Molecular Pharmacology, University of Groningen, Antonius Deusinglaan 2, 9713 AW, Groningen, The Netherlands.

R18 69

71

PREJUNCTIONAL MUSCARINIC M 2 AUTORECEPTORS IN THE AIRWAYS OF CONSCIOUS UNRESTRAINED GUINEA PIGS UNDER CONDITIONS OF BRONCHIAL HYPERREACTIVITY R.E.J. ten Berge, M. Krikke, B.C.H. Teisman, A.F. Roffet and J. Zaagsma Dysfunction of inhibitory M2 autoreceptors on vagal nerve endings in the airways of ovalbumin-sensitized and -challenged guinea pigs has been demonstrated in vitro (Br J Pharmacol 1995, 114: 881), and in rive using anesthetised animals (J Appl Physiol 1991, 71: 2255). We sought to establish the extent and time course of such dysfunction in conscious unrestrained guinea pigs: airway function was monitored as an increase in plenral pressure using an intrapleural fluid-filled cannula, and the cervical vagus nerve was provided with a stimulation electrode. Before antigen challenge, a significant increase of vagally induced (1-32 Hz, 0.1 ms, 7.8 mA for 10 s) bronchoconstriction was found with 0.1 and 1.0 mM of inhaled gallamine, the maximum effect being a two-fold increase; with 10 raM, bronchoconstriction was depressed by 50%. Six hours after antigen challenge (i.e. after the early allergic reaction in this model of allergic asthma - see e.g. J All Clin Immunol 1994, 93: 1021) no increase of nerve stimulation-induced bronchoconstriction by gallamine was found anymore; bronchial responsiveness to inhaled histamine was enhanced 4.5_+0.8-fold. Both after the late allergic response (24 h after challenge; 1.6+0.l-fold histamine hyperresponsiveness) and 4 days after allergen challenge (normal histamine responsiveness) gallamineinduced potentiation of bronchoconstriction was largely restored. The results clearly demonstrate that vagally mediated bronchoconstriction can be repeatedly induced in conscious unrestrained guinea pigs. Furthermore, M2 autoreceptors controlling such bronchoconstriction are completely dysfunctional after the early, but not after the late, allergic reaction. Since it has been shown that histamineinduced bronchoconstriction is partly mediated via activation of the vagus nerve, the dysfunction of autoinhibitory M~ receptors may contribute to the strongly enhanced responsiveness to histamine after the early allergic response. Supported by the Netherlands Asthma Foundation.

cq-ADRENOCEPTOR SUBTYPES AND ADRENERGIC NERVES IN RAT ARTERIES AND KIDNEYS. G.M.J. Janssen, R. Maes, J.G.R. De Mey At least 3 molecular entities display the general characteristics of cqadreneceptors, such as a high binding affinity for prazosin (KD 0.1 nM). Two pharmacological subtypes can be dissociated by their affinity for ligands such as (+) niguldipine (NIG) and by the action of the alkylating agent chloroethylchlonidine (CEC): eqA, resistant to CEC and pK~ for NIG > 8.3; otm, blocked by CEC and pK~ for NIG < 7.5. We investigated the relationship between adrenergic nerves and these subtypes in rat arteries and kidneys. In segments of thoracic aorta and carotid artery (vessels that contain little noradrenaline (NA), <0.2 ng/#g DNA) all specific binding sites for [3H] prazosin could be classified as cqB. In superior mesenteric, renal and femoral arteries (1-3 ng NA//z DNA) a significant population of cqA-AR could be identified, but the majority of the binding sites ( > 75 %) consisted of ~m. On the other hand, in 1st and 4th order mesenteric resistance arteries (15-25 ng NA/I~g DNA) the [~H]-prazosin binding sites consisted primarily (> 70%) of alA and to a lesser extent of cqB. Two weeks after chemical sympathectomy of the rats with 6-hydroxydopamine, the density of cqB in arteries was not modified but that of a~A was drastically reduced. Microsomes of whole kidneys contained a mixed population of prazosin binding sites (60% alA, 40% ~m) which was only marginally modified following sympathectomy (50% alA, 50% re)Thus, in rat arteries a reversible positive relationship seems to exist between the presence of adrenergic nerves that of cq^-adrenergic receptors. In rat kidney, on the other hand, neither c~t adrenergic receptor subtype seems to be related to sympathetic nerves. University of Limburg, Department of Pharmacology, P.O. Box 616, 6200 MD Maastricht, The Netherlands.

Groningen/Utrecht Institute for Drug Exploration, Department of Medicinal Chemistry & Molecular Pharmacology, University of Groningen, Antonius Deusinglaan 2, NL-9713 AW Groningen, The Netherlands

7O INTERACTION OF TRIPITRAMINE WITH MUSCARINIC RECEPTORS IN GUINEA PIG ATRIA AND LUNG A.F. Roffel, J.H. Davids. C.R.S. Elzin~a and J. Zaa~sma Contraction of guinea pig lung strip is not mediated by M3 receptors but by a novel subtype, or by a mixture of M2 and M3 (or M4) receptors (Enr J Pharmacol 250: 267). We further investigated the nature of the muscarinic receptor in this preparation using the new antagonist tripitramine, which clearly discriminates between these subtypes (pKi M2 9.5, M3 6.2, M4 7.9) (J Med Chem 1993, 36: 3734). Guinea pig atria were used as an M2 receptor model, and trachea as M3. Based on recent literature (Life Sci 1995, 56: 837) the influence of equilibration time was investigated as well. It was found with 60 min antagonist incubation in guinea pig electrically paced left atria and spontaneously beating right atria, that tripitramine produced unproportionally larger shifts at higher concentrations, apparent pKB values being 9.55+0.06 and 9.39+0.06, respectively, at 0.1-0.3 /zM, but only 8.50+0.10 and 8.65+0.09 at 3-10 nM (means+ S.E.M. of n=1012). In left atria, rightward shifts of methacholine concentration response curves induced by 30 nM tripitramine increased from 1.33 log units after 60 min to 1.78 log units after 120 rain and 1.94 log units after 180 rain of incubation, yielding apparent pKB values of 8.85+0.08, 9.30+0.10 and 9.46+ 0.21 respectively (n=4-6). Thus, this concentration of tripitramine requires at least 120 min of incubation to approximate equilibrium in guinea pig atria. In guinea pig lung strip, tripitramine also produced unproportionalty larger shifts at higher concentrations, especially at 30 and 60 rain of incubation, but increasing incubation time to 120 min produced a Schild plot with slope not significantly different from unity for concentrations ranging from 10 nM to 0.3 /xM (1.10+_0.11, n=4). The apparent pKB value of 8.76+0.05 (n=16) was lower than that obtained on M2 receptors in guinea pig atria but higher than that found in guinea pig trachea (apparent pKB value 6.07+0.15, n=7 estimated using tripitramine 0.3-3 /zM), and than reported for M4 (7.9). Since the shape of the methacholine concentration response curves was not clearly changed by tripitramine, the present results indicate the involvement of a novel receptor subtype in guinea pig lung strip contraction rather than of (a mixed population containing) M4. Groningen/Utrecht Institute for Drug Exploration, Department of Medicinal Chemistry & Molecular Pharmacology, University of Groningen, Antonius Deusinglaan 2, NL-9713 AW Groningen

72 NOIb~DRENALINE-INDUCEDCONTIL~CTIONOF MOUSEISOLATEDSPLEEN IS MEDIATED BY ALPHAIB-ADRENOCEPTORS M. El tze Several e1-adrenoceptor (el-AR) agonists, adrenaline = noradrenalind >> L-phenylephri'ne > methoxamine > SDZ NVI 085 > cirazoline, evoked contraction of the isolated mouse spleen (MS), whereas indanidine was ineffective. The contra~tions were sensitive to chloroeth~Iclonidine (CEC, 6xi0M), resistant to isradipine (10 M), and were antagonized by low concentrations of spiperone, but relatively high concentrations of tamsulosin, 5-methyl-urapidil, WB 4101 and (+)-niguldipine. Affinities (pA~ values) of subtype-selective antagonists at MS e1-ARs s~gnificantly correlated with those at eIR-ARs in guinea-pig spleen (GPS), rat aorta (RA) and pK~ vaTffes at eIR binding sites in rat l i v e r , but d i f fered from pA9 valudg at ela-ARs in rat vas deferens (RVD) and pK. value~ at rat cortical e . . and rabbit l i v e r e ~ bindin~ sites (Taddei et a l . , 1993, ~ife Sci. 53, PL 177):

Tissue

MS

GPS

RA

RVD

etA

Spiperone Tamsulosin 5-Methyl-urapidil WB 4101 (+)-Niguldipine Phentolamine Flesinoxan

8.3 8.6 7.0 8.3 6.3 7.4 5.6

8.1 8.3 6.9 7.9 6.3 6.9 5.7

7.8 9.6 7.0 8.5

7.6 10.2 9.1 9.6

7.2 5.5

8.5 7.0

8.1 10.3 9.1 9.9 10.4 8.2

aIB 8.7 8.4 6.7 7.6 6.4 7.1

eIC 7.7 9.1 7.8 8.9 8.3 7.5

Thus, from the potencies of agonists, sensitivity of the contractions to CEC but their resistance to calcium channel blockade and differential a f f i n i t i e s of eI-AR subtypeselective antagonists, the el-AR mediating s~lenic smooth muscle contraction in mouse, like that in rat, rabbit and guinea-pig, can best be characterized as B subtype. Department of Pharmacology, Byk Gulden, 78467 Konstanz, FRG.

R19 73 OF THE ACTION OF NORADRENALINE VASCULAR DOPAMINE D1 R E C E P T O R S CHARACTERISATION

75 AT

P.H. Van der G r a a f and P.R. Saxena In order to account for steep Schild plots obtained with compounds classified as cqadranoceptor antagonists, it has been reported that noradrenaline (NA), in addition to its cq-adrenoceptor-mediated contractile effect, may have a relaxatory action in the rat small mesanteric artery (s.m.a.) assay (Van der Graaf et al., 1995). In this study, a relaxatory action of NA has been exposed in the rat isolated, endothelium-denuded, s.m.a. (n = 4-5) precontracted by the thromboxane Az-mimetic, U46619. NA, but not the selective ct2-adrenoceptoragonist, UK14304, produced concentrationdependent contraction of the s.m.a. (pECso- 5.7 _+0.1). After precontraetion with 0. I t~M U46619, 10 nM-30 /aM NA produced a further contraction (pECso - 6.1 _+0.2), while higher concentrations of NA produced small, but significant, retaxatory responses. In the presence of 1 /aM prazosin, 0.1-30 ~M NA produced concentration-dependent relaxation (pICso = 5.9 + 0.1) after precontraction with 0.1 #M U46619. The NA relaxatury curve was completely inhibited by 1 #M of the B,/g2-adrenoceptorantagonist, timolol. However, when the concentration of prazosin was increased by 10-fold (10 taM), NA once again produced concentration-dependent relaxation (plCs0= 4.5 _+0.2). This relaxation curve was not blocked by a 10-fold higher concentration of timolol (10 ~M), nor by the presence of idazoxan (10 /aM), cyanopindolol (10 /aM), N~-nitro-Larginine methyl ester (L-NAME, 100/aM), indometbacin (10 #M) or sulpiride (1 #M). However, baloperidol (10 #M) and (+)SCH-23390 (10 nM) produced significant inhibition of the relaxation, suggesting the involvement of dopamine (DA) D~receptors. Following U46619 precontraction, DA also produced concentration-dependent relaxation (pICs0 = 5.4 _+0.1), which was significantly inhibited by haloperidol and (+)SCH-23390. Pretreatment with 10 /~M phenoxybenzamine for 60 rain produced a significant inhibition of the DA and NA relaxation curves and application of the operational model of agonism (Black & Left, 1983) yielded estimates of the affinity (pKA= 5.3 -+0.2 and 4.4 _+0.2) and efficacy (log 'r = 0.06 + 0.11 and 0.01 _+0.10) for DA and NA, respectively, at D I receptors. HV723 (0.1 and 1 /aM), a ligand which yielded a Schild plot slope parameter of unity as an antagonist of NA in the contractile assay (Van der Graaf et al., 1995), produced concentration-dependent inhibition of the NA-mediated relaxation (pA2 ~8). The results of this study indicate that NA can activate D~ receptors mediating relaxation in the rat s.m.a, at concentrations which were encountered in our previous receptor classification experiments using competitive cq-adrenoceptor antagonists. Black JW, LeffP (1983) Prec. R. Soc. Land. B. 220:141-162 Van der GraafPH, Sbankley NP, Black JW (1995)Br. J. Pharmacol. 114: 425P

P H A R M A C O L O G I C A L CHARACTERIZATION OF THE RECEPTORS INVOLVED IN THE M O D U L A T O R Y EFFECTS OF SEROTONIN ON [3H]-DOPAMINE RELEASE FROM RAT STRIATAL SLICES B.J. van Viler, E.J.M. Montulet, M.A.W. van der Neut & R.R. Terpstra Serotonin (5-HT) and dopamine have been proposed as mediators of several neuropsychiatric diseases, including depression, schizophrenia and obsessive compulsive disorder. Most research about 5-HT and dopamine has focused on these compounds individually, with less attention for the pharmacology of central interactions among serotonergic and dopaminergic systems. Therefore, in the present study, we investigated the receptors involved in the modulatory effects of serotonin on [3H]-dopamine release from rat striatal slices. 5-HT (107-10 ~) caused a dose dependent increase in spontaneous [SH]-dopamine release. The selective 5-HT receptor agonists 8-OH-DPAT, 5-carbexyamidotryptamine (5-CT), DOB, DOI and 2-Methyl-5-HT tested at the same concentration range did not affect [3H]-dopamine release, indicating that 5HT-evoked dopamine release is not mediated by either the 5-HTIA, 5-HTlo, 5-HT 2 or 5-HT 3 receptor. The effects of selective 5-HT receptor ligands, including the 5-HT 4 receptor antagonist GR 113808, as well as of selective dopamine reuptake inkibitors GBR13069 and nomifensine and the highly selective 5-HT reuptake inhibitor fluvoxamine, will be discussed. Solvay Duphar BV, Department of Pharmacology, PO Box 900 1380 D A Weesp, The Netherlands.

Department of Pharmacology, Erasmus University, PO Box 1738, 3000 DR Rotterdam, The Netherlands.

74 IN VIVO REGULATION OF E X T R A C E L L U L A R D O P A M I N E IN THE RAT P R E F R O N T A L CORTEX BY e~2-ADRENOCEPTORS. M.G.P.Feenstra, M.H.A.Botterblom, H . F . M . v a n Uum, Experimental studies in rodents and primates have shown that both dopamine (DA) and noradrenaline (NA) are involved in the cognitive functions (e.g. short term memory, attention) of the prefrontal cortex (PFC). It is therefore of considerable interest that these two transmitters show interactions on various levels. As there are clear differences in the conditions under which activation of either mesocortical DA neurons or coerulocortical NA neurons occurs, the possible interactions might have important functional implications. We have studied the possibility that N A affects in vivo D A release in freely moving rats, using microdialysis. Male rats were implanted with bilateral microdialysis cannulae in the medial PFC or in the nucleus accumbens. One or two days later microdialysis experiments were carried out with on line HPLC detection of D A and/or N A in the dialysate. The ~2-agonists bromoxidine (UK14,304) and moxonidine or the antagonist 2-methoxyidazoxan (RX 821002) were added to the perfusion fluid. As we previously reported (e.g. Brain Res. 1994, 635: 238-248) extracellular NA was strongly and concentration-dependently decreased by 0.1 - 10 /aM moxonidine and 0.01 - 10 /~M bromoxidine (maximally to 10-20% of control). The tissue concentration of these agents will have been 10-15% of the concentration in the perfusion fluid. Both agonists also reduced D A with the same potency (maximal effect 40-60% of control). Perfusion with the antagonist had a small effect on both N A and DA, with a maximal increase of 30-50%. The agonist-induced decrease in D A was not found in the nucleus accumbens. These results show that N A may regulate extracellular DA in the PFC in vivo, that this effect is selective for the PFC and that a small endogenous inhibitory effect is present. Whether the mechanism involves ~xz-adrenoceptors presynaptically located on D A terminals or an indirect effect of the altered N A concentration is subject to further studies. Netherlands Institute for Brain Research, Graduate S c h ~ "~reurosciences Amsterdam, Meibergdreef 33, 1105AZ Amsterdam ZO, "[~lc Netherlands.

76 c~2-AUTORECEPTORS IN PORCINE BRAIN CORTEX ARE e~2A A.U. Trendelenhurg, N. Limberger and K. Starke Presynaptic c~2-autoreceptors are mainly ,X2D in the rat, mouse and guinea pig and ~X2Ain the rabbit. It was suggested that the majority of presynaptic ~2adrenoceptors belong to the ,X2A/D group, across mammalian species (Trendelenburg et al. 1993, Naunyn-Schrnledeberg's Arch Pharmacol 348:35). We now extended these studies to the pig. In order to characterize presynaptic ~x2-autoreceptors in porcine brain cortex, pK d values of 12 ~2-adrenoceptor antagonists and of the partial agonist oxymetazoline against the ~x2-adrenoceptor agonlst UK 14304 were determined. Slices of the brain cortex were preincubated with 3H-noradrenaline and then superfused in the presence of desipramine 1 #M. Four periods of electrical stimulation were applied, each consisting of 6 pulses/ 100 Hz, and cumulative concentration-inhibition curves of UK 14304 were determined. UK 14304 reduced the evoked tritium Compound pK d overflow with an EC50 of 0.84 nM. All compounds tested caused parallel shifts of the UK 14304 concenMK 912 9.5 tration-inhibition curve to the right with pK d values RX 821002 9.1 listed in the Table. A comparison of these PKdS with Rauwolscine 9.0 data from the literature indicates that the a2Oxymetazoline 8.9 autoreceptors in porcine brain cortex are ~2A: there is WB 4101 8.3 an excellent correlation with PKdS at C~2Abinding sites Efaroxan 8.3 in, for example, HT29 cells (r=0.98; P<0.001; Blaxall Phentolamine 8.0 et al. 1991, J Pharmacol Exp Ther 259:323), but no or Spiroxatrine 7.9 only a poor correlation with PKdS at CC2B,a2C and ~X2D Benoxathian 7.6 binding sites. Moreover, the porcine receptors are (-)-Mianserin 6.5 similar to the presynaptic C~2A-autoreceptors in rabbit Corynanthine 6.3 brain, but there is less agreement with the C~2c-autoreARC 239 6.0 ceptors in human kidney and with the C~2D-autoreceptors Prazosin 5.8 in rat, guinea-pig and mouse brain. The classification of the presynaptic c~2-autoreceptors in porcine brain cortex as ~2A is in accord with the hypothesis that the majority of presynaptic o~2-adrenoceptors belong to the ~X2A/D group across mammalian species. This is the first functional subtype determination of ~2-autoreceptors in the pig. Pharmakologisches Institut, Hermann-Herder-Strasse 5, D-79104 Freiburg i. Br.

R 20 77

79

~2-AUTORECEPTORS ARE tX2D IN MOUSE ATRIA C. Wahl and A.U. Trendelenburg

THE INFLUENCE OF DIFFERENT PHARMACOLOGICAL AGENTS ON SMOOTH MUSCLE CONTRACTION E V O K E D B Y

It has been suggested that the majority of presynaptic c~2-adrenoceptors in the rat, mouse and guinea pig are eZ2D. However, the a2-autoreceptors in rat atria were an exception from this rule (Limberger et al. 1992; Br J Pharmacol 107: 246). Do atrial c~2-autoreceptors also deviate from other presynaptic tx2adrenoceptors in the mouse? To answer this question, pK d values of 15 ~adrenoceptor antagonists against the c~2-adrenoceptor agonist UK 14304 and the pK a value of the agonist oxymetazoline were determined. Mouse atrial slices were preincubated with 3H-noradrenaline, then superfused in the presence of desipramine 1/zM and stimulated four times with 2 trains of 20 pulses/50 Hz, train interval 120 s. Cumulative concentration-inhibition curves for UK 14304 and oxymetazoline were determined. Both agonists a-Antagonist pK d reduced the evoked tritium overflow, EC50 values 7.9 nM (UK 14304) and 12.9 nM (oxymetazoline). All MK 912 9.4 antagonists shifted the UK 14304 concentration-inhibition RX 821002 8.8 curve to the right with pK d values listed in the Table. Rauwolscine 8.3 Partial receptor inactivation by pretreatment with Efaroxan 8.4 phenoxybenzamine (0.3/~M) shifted the concentrationIdazoxan 8.0 inhibition curve of oxynaetazoline to the right and Phentolamine 8.0 diminished its maximal effect. From equieffective Spiroxatrine 7.7 concentrations without and with phenoxybenzamine WB 4101 7.5 pretreatment the pK a.value of oxymetazoline (7.7) was SKF 104856 7.4 calculated. A comparison of the antagonist PKdS with data Benoxathian 6.7 from the literature indicates that the a2-autoreceptors in SKF 104078 6.6 mouse atria are most similar to ~2D: the PKdS correlate ARC 239 6.6 excellently with PKdS at C~2D binding sites in, for Prazosin 6.3 example, bovine pineal gland (r=0.95; P < 0 . 0 0 1 ; Tolazoline 6.3 Simonneaux et al. 1991; Mol Pharmacol 40:235); there is Corynanthine 5.8 less agreement with a2A, C~2Band ~2C" Moreover, the PKdS correlate excellently with PKdS at presynaptic a2Dautoreceptors in, for example, mouse brain cortex (r=0.96; P < 0 . 0 0 1 ; Limberger et al. in press; Naunyn-Schmiedeberg's Arch Pharmacol). The high pK a value of oxymetazoline also argues against C~2B and ~2C" It is concluded that the presynaptic ~2-autoreceptors in mouse atria are ~2D in accordance with the initial suggestion.

GALANIN. R. K O R O L K 1 E W l C Z , W. SLIWIIqSKI, * P. R E K O W S K I A N D • A. H A L A M A As we have s h o w n in our previous w o r k galanm causes contraction o f smooth muscle strips isolated from rat's stomach fundus. To a p p r o a c h its mechanism o f action w e investigated the influence o f several pharmacological tool-substances on smooth muscle contraction elicited b y galanin. The dose-response curves o f galanin were constructed in presence o f dibenamme, glibenclamide, diltiazem and papavenne. Dibenamine in concentration o f 10 p.M a n d glibenclamide in concentration o f I and 10 p M seem not to have an effect o n the doge-response curve o f galanin. Diltiazem a n d papaverine both in concentration o f 0.1; 1; 10 g M decrease contraction evoked by galanin, in a dose-dependent manner. Conclusions: 1 / T h e contraction p r o d u c e d by galanin depends neither on cholinergic and adrenergic receptors n o r on the state o f potassium channels 2/Diltiazem and papaverine inhibited the galanin action in a non-competive manner.

Department of Pharmacology, Medical University of Gdafisk, Do Studzienki 38 Street, 80-227 Gdafisk, * Faculty o f Chemistry, University o f Gdafisk, 18 Sobieski Street, 80-952 Gdafisk, Poland

Pharmakologisches Institut, Hetmann-Herder-Strasse 5, D-79104 Freiburg i. Br.

78

8O

DIFFERENCES IN BOTH THE T Y P I C A L AND A T Y P I C A L fl-ADRENOCEPTOR POPULATIONS MEDIATING RELAXATION O F GUINEA P I G AND RAT OESOPHAGEAL SMOOTH MUSCLE

CHARACTERIZATION OF THE SECRETORY RESPONSE TO SEROTONIN (5-HT) IN ISOLATEDTHE GUINEAPIG COLONICEPITHELIUM.

R.E.P. de Boer. P.A. Kroezen. M. Fransen, F. Brouwer and J. Zaagsma In rat oesophagus muscularis mucosae, we have shown that ll-adrenoceptormediated relaxation involved predominantly lls- , but also l~-adrenoceptors (BJP 1993, 110, 442-446.). In the present study, the nature of the ft-adrenoceptorg in guinea pig oesophagus smooth muscle was investigated and compared to those in rat oesophagus. Guinea pig single muscularis mucosae strips (7x2 ram.) were mounted for isotonic recording. Methacholine (1 /zM) was used to raise muscle tone and all experiments were carried out in the presence of corticosterone (10 t~M) to block extraneuronal uptake. (-)-Isoprenaline induced concentration dependent relaxations with a pD2-value of 7.78 + 0.10 (11). Both with the I~l-selective antagonist ICI 89,406 and the B~-seleetive antagonist ICI 118,551, up to high concentrations, antagonism remained purely competitive with pA2-values of 8.84 + 0.07 (23) and 7.06 + 0.05 (19), respectively, indicating a major fi~-adrenoceptor population. Relaxations to Fenoterol and Cc25 however, were biphasic, indicating the presence of another ll-adrenoceptor subtype. To investigate this in more detail, a series of Bs-selective agonists was used. Their potencies decreased in the order CGP 12,177 > BRL 37,344 > SR 58611 > ZD 2079, whereas CI 316,243 was totally inactive. On rat oesophagus, a totally different order was found: CI 316,243=BRL 37,344 > SR 58611 > ZD 2079 > > CGP 12,177. In addition, high concentrations (1-100 /tM) of the non-selective I~-antagonist carazolol antagonized BRL 37,344-induced relaxations in rat oesophagus, but not in guinea pig oesophagus. In conclusion, the results indicate the presence of a minor 1~3-adrenoceptor population in guinea pig oesophagus in addition to a majority of I~aadrenoceptors. The nature of the f~s-adrenoceptors in guinea pig oesophagus may be different from the fls-adrenoceptors mediating relaxation in rat oesophagus. Groningen Utrecht Institute for Drug Exploration, Department of Medicinal Chemistry and Molecular Pharmacology, University of Groningen, Antonius Deusinglaan 2, 9713 AW Groningen, The Netherlands.

M. Gladis-Villanueva.W. Sehmutzler.K.-U.Petersen. Expositions of the guinea pig colon to histamine, PGE2, 5-HT or cAMP are known to induce electroganicanion secretion.We have furtherinvestigatedthe effect of 5-HT using full thicknessand isolated epithelial (scraped)preparations.The tissues were mounted in Ussing-typechambersand continuouslyshort-circuitedfor determinationof tissue conductance(Gt), transepithelialvoltage(Pd) and short-circuitcurrent (Ise). Addition of 0,1 mM 5-HT to the serosal side caused a significant increase in Isc, being more pronounced and longer lasting in full thickness than in scraped preparations(peak 5,8 -~ 1,1 vs. 2,1 :~ 0,6 #Eq/h/cm2, p<0,05). In full thicknesspreparationsbut not in isolatedepithelia,the Isc increasewas abolishedby pretreatmentwith tetrodotoxin(TTX, I gM to the serosal side). In contrast,hexamethonium(10p.Mto the serosalside) entirelyand atropine(10p.M to the serosal side) partially failed to inhibit the response of the full thickness preparations.The cyclooxyganaseinhibitors indomethacinand areclofenamate(0,1 and 0,3 p.M respectivelyto the serosal side) modified the responses neither in full thickness nor in scraped preparations. Metoclopramide, ketanserin and methysergide(each at 0,1 laM to the serosal side) were more efficientin blockingthe responsesof isolatedepitheliathan that of full thicknesspreparations.The 5HT3 antagonistondansetron(0.1 mM to the serosalside) almostcompletelyinhibitedthe responseof scraped preparationsbut only partially reduced the Isc increase in full thickness preparations. In combination, methysergideplus ondansetronabolishedany response to 5-HT in both full thickness and scrapedpreparations.Our results suggestthat the effects of 5-HT in guinea pig colonic mucosa are mediatedboth by 5-HT receptorslocatedon the epithelialcells (probably5-HT1 and 5-HT3)and indirectlythrough acetylcholinereleasefrom the cholinergicneurons. lnstitut far Pharmakologieund Toxikologie,RWTHAachen,52057Aachen,FRG.

R 21 81 BLOCKADE OF P2-PURINOCEPTORS BY ISOTHIOCYANATES Ralph Bfiltmann, Birgit Pause, Gerhart Kurz and Klaus Starke The blockade by small aromatic and aliphatic isothiocyanato-sulphonates of P2x-purinoceptors in rat vas deferens and of P2y-purinoceptors in guinea-pig taenia coli was investigated. The compounds were 2-, 3- and 4-isothiocyanatobenzene-l-sulphonate (olBS, mlBS and pIBS), 4-isothiocyanatonaphthalene-l-sulphonate (pINS), l-isothiocyanatonaphthalene-2-sulphonate (uINS), 2-isothiocyanatonaphthalene-l-sulphonate (BINS), 4-isothiocyanatobenzene-l,3disulphonate (IB1,3dS), 2-isothiocyanatobenzene- 1,4-disulphonate (IB1,4dS) and 2-isothiocyanatoethylene-l-sulphonate 0ES). In rat vas deferens, effects on concentration-contraction curves of a,B-methylene ATP (a,6-MeATP; 0.l to 320 laM) were studied. IES (up to 1 mM) caused no change. The main effect of the other compounds was flattening of the agonist concentration-contraction curve. At concentrations between 32 pM (pINS, aINS) and 320 /aM (IB1,3dS, IB1,4dS) contractions elicited by ct,B-MeATP were totally suppressed. None of the compounds altered contractions elicited by high potassium. The antagonism against a,B-MeATP was only partly reversible: 25 to 66 % of the inhibition of responses to tt,B-MeATP (3.2 pM) persisted after washout of the isothiocyanate compounds. In guinea-pig taenia coli, effects on concentration-relaxation curves of adenosine 5-O-(2-thiodiphosphate) (ADPBS) were studied. All compounds except IES (1 mM) and BINS (100/aM) shifted the curve to the right without changing the maximum; apparent antagonist K B values were 13 (pINS) to 238/aM (olBS). The results indicate blockade of P2-purinoceptors by small aromatic isothiocyanates, pINS and cdNS are relatively potent and selective P2x-purinoceptor antagonists, completely suppressing the responses to a,B-MeATP at a concentration of 32/aM but causing only small rightward shifts of the ADPI3S concentration-relaxation curve at 100 /aM. The different nature of antagonism indicates differences in the ligand binding site of the two P2-purinoceptor subtypes. Pharmakologisches Institut, Hermann-Herder-Strasse 5; Biochemisches Institut, Albert-Strasse 21, D-79104 Freiburg, FRG

82 PRESYNAPTIC P2-PURINOCEPTORS AT SEROTONINERGIC AXONS IN RAT BRAIN CORTEX H. Koch, I. yon KQgelgen and K. Starke Noradrenergic axons terminals in the peripheral and central nervous system of the rat possess P2y-like P2-purinoceptors which, when activated, decrease the release of noradrenaline, In the present study we examined the question whether such receptors also occur at the serotoninergic axons in the rat brain cortex. Slices of the brain cortex were preincubated with [3H]-serotonin and then superfused with medium containing 6-nitroquipazine 1 pM and metitepin 1 pM to block neuronal serotonin uptake systems and serotonin autoreceptors, respectively (see Limberger et al., Naunyn-Schmiedeberg's Arch Pharmacol 332:324-331, 1986). The slices were electrically stimulated by trains of 10 pulses/1 Hz. Of the nucleotides tested, ATP (30 - 300 ,uM), ATP~/S (300/IM), and diadenosine-pentaphosphate (AP5A; 300 /IM) reduced the evoked overflow of tritium by up to 30 %. AMP (30 pM) and cq~-methyleneATP (30/IM) reduced evoked tritium overflow by 15 %, whereas the P2y-agonist 2-methylthio-ATP (3 - 300 pM) caused no change. Of the nucleosides tested, only 2-chloroadenosine (0.3 pM, but not 0.03 and 3 /JM) reduced the evoked overflow of tritium (by about 15 %). The adenosine A 1-receptor agonist cyclopentyladenosine (0.03 - 3 pM), the A2a-receptor agonist 2-p-(2-carbonylethyl)-phenethylamino-5'-Nethylcarboxamido-adenosine (CGS-21680; 0.003 - 3 pM), and the A 3receptor agonist N6-2-(4-aminophenyl)ethyl-adenosine (APNEA; 0.03 3 pM) caused no change. The Al-receptor antagonist 8-cyclopentyl1,3-dipropylxanthine (DPCPX; 3 nM) did not change the effects of ATP, ATlaS and 2-chloroadenosine. The non-subtype selective adenosine receptor antagonist 8-p-sulphophenyl-theophylline (100 pM) abolished the inhibition by 2-chloroadenosine without changing the inhibitory effects of ATP and ATPTS. The results indicate the operation of inhibitory P2-purinoceptors (probably not of the P2y-type) and the absence of adenosine A 1-, A2aand A3-receptors at serotoninergie axons in rat brain cortex. This is the first description of presynaptic P2-purinoceptors at serotoninergic axon terminals. Pharmakologisches Institut, Hermann-Herder-Str. 5, D-79104 Freiburg

83 PEROXYNITRITE INDUCES A I R W A Y EPITHELIAL D A M A G E AND HYPERRESPONSIVENESS IN THE GUINEA PIGS

G Sadeghi Hashjin, G Folkerts, PAJ Henricks, AKCP Verheyen, HJ Van Der Lind& IEM Dik, and FP Nijkamp Peroxynitrite (ONE)O-) is a cytotoxic product of the rapid reaction between nitric oxide and superoxide which may initiate inflammation. Isolated perfused tracheae from guinea pigs were incubated from lumenal side for 15 rain with 10 pM peroxynitrite. Thereafter, concentration-response curves to histamine and methacholine were constructed on the preparations. Peroxynitrite caused a significant hyperresponsiveness; the maximal contractions in response to histamine and methacholine were enhanced by 30% and 40%. respectively (see also figure). In the treated 16O I ~ Trutea group a clear epithelial damage as well as eosinophil destruction were detected after histological examination. Moreover, 3, 5, and 10 days | /6 / /o after intra-tracheal instillation of peroxynitrite (100 nmol), a -7 -6 -5 -4 -3 significant rise in pulmonary log Histamine [M] resistance to histamine of anaesthetized animals was observed. It is suggested that the generation of peroxynitdte from nitric oxide and superoxide radicals during inflammatory processes induces epithelial damage leading to airway hyperresponsiveness. These findings may have clinical implications since airway hyperresponsiveness, inflammation and epithelial damage are characteristic features in patients suffering asthma.

;//o

Department of Pharmacology, Utrecht institute for Pharmaceutical Sciences, Utrecht University, P.O. Box 80.082, 3508 TB Utrecht, The Netherlands

84 ROLE OF SUPEROXIDE ANION AND NITRIC OXIDE IN ALLERGEN - INDUCED BRONCHIAL HYPERREACTIVITY IN GUINEA PIGS - AN EX r I V e STUDY. J. de Boer, H. Meurs, F.M.H. Pouw, J. Zaaqsma. Using a guinea pig model of asthma, characterized by ovalbumin (OA)induced early (EAR) and late (LAR) asthmatic reactions and development of bronchial hyperreactivity after these reactions, we have recently shown that at 6h after OA challenge in vivo (after the EAR), a deficiency of endogenous nitric oxide (NO) may contribute to an increased responsiveness of isolated perfused tracheae to intraluminally (IL) applied methacholine (MeCh). One possible mechanism for the observed deficiency of NO could be its reaction with enhanced levels of inflammation-induced superoxide (02-). In the present study we examined the effect of endogenous O2- on the regulatory role of NO in MeCh-induced airway obstruction. Using the constant flow perfusion model described by Munakata et al. (JAP 1988; 64:466-471 ), we compared the in vitro responsiveness to IL applied MeCh of intact tracheae from OA-sensitized guinea pigs, 6h after OA-challenge, with that of tracheae from nonchal[enged control animals, in the absence or presence of the extraluminally (EL) applied 02- scavanger superoxide dismutase (SOD, 100 U/ml) or the NO synthase inhibitor L-NAME (10.4 M, IL). Agonist-induced differential pressure changes in the lumen were expressed as %Ap of the effect of EL applied 40mM KCI. In control preparations, the maximal response (Ema,) tO MeCh was significantly increased in the presence of LNAME (from 68.1% + 4.5% to 109.0% + 16.8%, P
R 22 85

87

DEFICIENCY OF ENDOGENOUS NITRIC OXIDE 1N ALLERGEN-INDUCED BRONCHIAL HYPERREACTIVITY IN CONSCIOUS, UNRESTRAINED GUINEA PIGS M. Schuiling, A.B. Zuidhof, M.A.A. Bonouvrie, J. Zaagsma and H. Meurs As a physiological mediator of airway smooth muscle relaxation, nitric oxide (NO) is considered to play an important role in regulating bronchial tone. In a guinea pig model of asthma, characterized by allergen-induced early (EAR) and late (LAR) asthmatic reactions, bronchial hyperreactivity (BHR) and airway inflammation (Santing et al., J. Allergy Clin. Immunol. 1994; 93; 1021-1030), we investigated the role of endogenous NO in the regulation of airway responsiveness to inhaled histamine and in allergen-induced BHR to this mediator after the EAR and LAR. Aerosol inhalation of the NO synthase inhibitor N%nitro-L-arginine methyl ester (LNAME; 12 mM, 15 min) by conscious, unrestrained, ovalbumin (OA)-sensitized guinea pigs caused a significant 1.93+0.20-fold (p<0.01) increase in bronchial reactivity towards histamine when measured at 30 min after administration of the inhibitor. The increase in histamine reactivity was abolished in 2.5h (1.33+0.23-fold; n.s.) to 6h (l.06+0.06-fold; n.s.) after administration. A significant increase in bronchial reactivity to histamine (3.73+0.67-fold; p<0.01) was also observed at 5h after OA challenge (between EAR and LAR). Subsequent inhalation of L-NAME had no significant effect on the observed histamine BHR after the EAR (histamine reactivity ratio pre/post L-NAME 1.00_+0.07; n.s.). At 23h after OA challenge (after the LAR) a diminished, but still significant BHR to histamine (2.19_+0.40; p<0.05) was observed, which was, however, significantly enhanced after inhalation of LNAME (histamine reactivity ratio pre/post L-NAME 1.57_+0.19; p<0.01). Inhalation of saline instead of L-NAME did not affect histamine reactivity before and at 5 and 23 h after allergen challenge. These results indicate that (1) inhibition of NO synthesis causes BHR to histamine, (2) a deficiency of NO is involved in allergeninduced BHR after the EAR and (3) restoration of NO synthesis or effectiveness may contribute to the reduced BHR to histamine observed after the LAR. This work was supported by a grant from the Netherlands Asthma Foundation.

INHIBITION OF NITRIC OXIDE-MEDIATED A M I N O - S A L I C Y L I C ACID. B.A. Peskar, D. Pallapies and B.M. Peskar

Groningen Utrecht Institute for Drug Exploration, Department of Medicinal Chemistry and Molecular Pharmacology, University of Groningen, Ant. Deusinglaan 2, 9713 AW Groningen, The Netherlands.

EFFECTS

BY 5-

Rat peritoneal neutrophils (RPN) and rat aortic strips are known to produce continuously nitric oxide (NO). The release of NO can be determined by the antiaggregatory effect of RPN and the tone of the aortic strips, respectively. We have used these experimental systems to investigate the interference of 5aminosalicylic acid (5-ASA) with NO effects. Strips of rat thoracic aorta mounted in organ baths and precontracted by phenylephrine were further contracted by 5-ASA (50-200 p,M) in a concentration-dependent manner. Mechanical removal of the endothelium or addition of methylene blue (10 p,M) or oxyhemoglobin (2 ~M) abolished the effect of 5-ASA. RPN obtained 4 h after i.p. injection of oyster glycogen were added to suspensions of washed human platelets and aggregation was induced by threshold concentrations of thrombin. The decrease of optical density was monitored for 4 min in the absence or presence of 5-ASA and, in some experiments, superoxide dismutase. RPN were found to inhibit thrombin-induced aggregation of the ptatelet suspensions. 5-ASA (50-250 p,M) antagonized the inhibitory action of RPN in a concentration-dependent manner (ICs0:140 }aM). The antagonism of the antiaggregatory effect of RPN by 5-ASA was lost in the presence of superoxide dismutase (60 U/roll. Furthermore, 5-ASA similarly inhibited the antiaggregatory effect of the NO donor 3-morpholinosydnonimine (SIN-1, 0.5 - 4.0 gM), while the antiaggregatory effect of iloprost (0.5 - 3.0 nM) was not affected. The results indicate that 5-ASA inactivates NO resulting in increased tone of rat aortic strips and antagonizes NO-mediated inhibition of platelet aggregation by a SOD-sensitive mechanism. Since NO can act as a cytotoxic mediator, its inactivation by 5-ASA could contribute to the therapeutic activity of the drug, e.g. in inflammatory bowel disease. Department of Experimental and Clinical Pharmacology, University of Graz, Austria, and Department of Experimental Clinical Medicine, Rnhr-University of Bochum, Germany.

86

88

A ROLE FOR THE NITRIC OXIDE/CYCLIC GUANYLATE CYCLASE SIGNAL TRANSDUCTION PATHWAY IN THE HISTAMINERGIC NERVE SYSTEM OF THE RAT BRAIN. I.B.G. Paouav*. H-P. Vess. J. de Vente. H.W.M. Steinbusch. H. Tim_merman,and A. Bast. To investigate a possible role of the nitric oxide/cyclic gu,'mylate mouophosphate signal transduction pathway in the histaminergic nerve system, an immunocymehemical method was used. This method visualizes cyclic guanylate monephosphate production in tissue slices. Upon stimulation with hist,'unine an enhanced cyclic guanyhtte monophosphate production was found in the suprachiasrnaficnucleus ~md the medial preopdc area of the rat br~n. This eftect was completely abolished when the nitric oxide synthase inhibitor L-nitro ,'u'gininemethyl ester was applied to the tissue. The nitric oxide donor sodium nitroprusside gave ,an enh,'mcedcyclic gmmylatemouophosphate production in the entre sectien. These results indicate a role tier the nitric oxide/cyclic guanylate monophosphate transduction pathway in the hist~uninergiu signal tmnsduction in the rat brain. However, extensive research is necessary to quantity these effects and to identify the histaminergic receptor subtypes inw~lvedin this process.

ON THE TOXICITY OF NITRITE AND NITRATE IN RATS W.Vleeming, A van de Kuil, A.B.T.J. Boink, G.J.A.Speijers, J Meulenbelt and D.J. de Wildt

Department of Ph,'wmacochemistry,Vdje llniversiteit Amsterdam, De Boetel~mn1083. 1081 HV Pansterd,'un.The Netherhmds.

High dietary nitrate intake due to the high nitrate content of several drinking water resources or of certain vegetables, cultivated at low temperature and light intensity, has raised concern with respect to the possible health effects. In humans 5-10 % of the ingested dose of nitrate is converted into nitrite by salivary or gastrointestinal reduction. Following administration of low doses of nitrite, but not of nitrate, hypertrophy of the zona glomerulosa of the adrenals of rats has been reported. We studied the effects of nitrate (NAN%; 100 pmoVkg) and nitrite (NaNO2; 0, 10, 30, 100, 300, and 1000 p.mol/kg) on blood pressure (BP) during 75 minutes in anaesthetized rats. Nitrite dose dependently decreased BP. The maximal decrease in systolic BP caused by 1000 IJ.mol/kg was 65.4 %. The decrease in BP by nitrite occurred rapidly (during the 5 min infusion period) and was accompanied by an increase in blood methaemoglobin (MetHb). Addition of methylene blue (2 mg/kg/iv at t= 60 min) reversed MetHB values to control values but did not restore nitrite-induced hypotension. This indicates a minor contribution of NO to the nitrite-ieduced decrease in blood pressure Plasma nitrite and nitrate concentration measurements showed that already after 5 min approximately 2/3 of the nitrite dose had been converted into nitrate. Infusion of nitrate caused a small transient increase in BP and no conversion of nitrate into nitrite was observed. We concluded that hypotension was the primary effect of nitrite which may have been counteracted by MetHb formation. Thus, MetHb formation and concurrent conversion of nitrite into nitrate acted as an efficient detoxifying process to prevent prolonged decrease of BP. Because administration of nitrite, but not of nitrate, decreased BP and induced hypertrophy of the adrenal zona glomerulosa it might be speculated that the nitrite-induced decrease in BP may have a role in the aetiology of the hypertrophy of the adrenal zona gloraerulosa. National Institute of Public Health and Environmental Protection, PO Box 1, 3720 BA Bilthoven, The Netherlands.

R 23 89

91

ENHANCED ENDOGENOUS PRODUCTION OF TETRAHYDROBIOPTERIN AND NITRIC OXIDE DURING SEPTIC SHOCK IN RATS AND RABBITS. J.G.C. van Amsterdam, C. van de Berg, J.D. te Biesebeek, J. Zuidema, W. Vleeming, D.J. de Witdt and H. Rokos',

HYDROLYSIS OF MORPHINE-3-ESTERS: IN-VITRO STUDIES ON PREPARATIONS FROM PLASMA, BRAIN, AND LIVER OF THE RABBIT. C. Mignat, D. Heber* and A. Ziegler

induction of systemic infection triggers the immune system to release a large number of inflammatory mediators. The details of the cascade ultimately leading to the septic shock syndrome is poorly understood despite intensive investigations. Recently it was assumed that the NO synthase enzyme, generating the pleiotropic mediator nitric oxide (NO) was involved in the pathophysiology of septic shock. A large rise in the serum level of total nitric oxide metabolites was observed some hours after the initiation of septicemia in the rat. In vitro studies suggested that the expression of inducible NO synthase preceeds this large increase in NO production, in order to become an active enzyme the newly expressed NO synthase requires tetrahydrobioperin as cofactor. Enhanced tetrahydrobiopterin synthesis can be accomplished by activating/inducing the ratelimiting enzyme GTP-hydmlase. Interestingly, both enzymes (NO-synthase and GTP-hydrolase) are induced in-vitro by the same factors like LPS or combinations of cytokines. The aim of the present study was to determine the time course (from to 0 to 10 hours) of the elicited production of tetrahydrobiopterin and NO in two models of septic shock (rat versus rabbit). In serum, the sum of NO-metabotltes (nitrate+nitrite) was assayed colorimetdcally after reduction with Klebsiella pneumoniae and tetrahydrobiopterin after oxidation to biopterin by radioimmunoassay. In the rat systemic administration of both E. coil or LPS induced a large increase in NO synthase activity (29-fold) while in rabbits the increase was very modest compared to rats (only 37 %). In both species a lag time of 3-4 hours preeeeded the gradual rise in NO-metabolitea (up to 10 hours post treatment). As expected a gradual increase in biopterin level was observed in the same rats (from 224 to 395 I.tM). In rabbits, however, the increase in biopterin serum level was much more pronounced (from 239 to 1227 gM). Present results show that both pathways are activated within 4 hours after the introduction of systemic septicemia. In addition both species appear to differ in the kinetics and extend of the activated routes. Whether these differences are relevant for the pathophysiology of septic shock is to be elucidated.

Recently, low opioid receptor affinity as well as modifiable hydrolytic stability have been demonstrated for morphine-3-esters, suggesting that these compounds display prndrug properties which may be useful for the development of slowrelease morphine formulations (Mignat et al., Naunyn-Schmiedeberg's Arch. Pharmacoh 351:R585, 586, 1995). In order to investigate the hydrolytic capacity of different organs, the degradation of five morphine-3-esters was evaluated in plasma mrd in homogenates frmn brain and liver of the rabbit (standardized on a protein content of 34mg/ml). The morphine-3-esters (3-pivaloylmorphine [A], 3(2-methylbenzoyl)morphine [B], 3-(2-phenylbenzoyl)morphine [C], 3-(2,2-diphenylpropionyl)morphine [D], and 3-(2,6-dimethylbenzoyl)morphine [E]) were incubated at 37°C m~d the hydrolysis yielding morphine was monitored for up to 24h by using a HPLC-method.

National Institute of Public Health and Environmental Protection, PO Box 1, 3720 BA Bilthoven, The Netherlands; "Henning-Berlin GmbH, Berlin, FRG.

Department of Pharmacology and Department of Phannaceutical Chemistry*, Christian-Albrechts-University, Hospitalstra$e 4, D-24105 Kiel, FRG

90

92

BENZODIAZEPINE RECEPTORS ARE INVOLVED IN THE INCREASED STRIATAL DOPAMINE RELEASE CAUSED BY INTRASTRIATAL TAURINE

H I P P O C A M P A L N E T W O R K P R O P E R T I E S O F BETA R H Y T H M I N D U C E D BY M E T A B O T R O P I C G L U T A M A T E RECEPTORS H.W.G.M. Boddeke & P. Boeijinga

M. Ruotsalainen, M. Majasaari, L. Ahtee Intracerebroventricular taurine affects the function of striatal dopaminergic neurons in a similar way to the inhibitory neurotransmitter GABA (1). We recently showed that intrastriatal taurine increases the release of striatal dopamine (DA) in anaesthetised rats (2) as well in freely moving rats by a tetrodotoxin-sensitive manner (3). In the present experiments the mechanism of this effect was further elucidated by the studying the interactions of taurine with GABA-benzodiazepine receptor using microdialysis technique in freely moving rats. Experiments were performed 42-46 h hours after implantation of concentric probe. Extracellular concentrations of DA, 3,4-dihydroxyphanylacetic acid (DOPAC) and homovanillic acid (HVA) were assayed by HPLC with electrochemical detection. Intrastriatal infusion of 150 mM taurine for 2 hours increased the release of DA maximally by 170%. Extracellular level of DOPAC was increased maximally by 100% and that of by HVA 60%, respectively. Diazepam (5 mg/kg i.p.) completely abolished the enhancement of striatal DA release caused by taurine, but did not alter the increase of DOPAC or HVA. The depressing effect of diazepam on the enhancement of DA release caused by taurine was antagonized by fiumazenil (10 mg/kg i.p.). Diazepam or flumazenil alone at the used doses did not affect the striatal DA release. The findings that diazepam abolished the increase of striatal DA release caused by taurine and that diazepam's effect was antagonized by fiumazenil, suggest that the GABAbenzodiazepine receptor complex is involved in the effect of intrastriatal taurine on DA release. 1) 2) 3)

Panula-Lehto E. et al. (1992) Natmyn-Sehmiedeberg'sArch Pharmaeol. 346:57-62 M~JdnenM. et al. (1993) Natmyn-Sehmiedeberg'sArch Pharmacol. 347:R135 M~ikinenM. et al. (1994) Can. J. Physiol. Pharmacol. 72:425

Department of Pharmacy, Division of Pharmacology and Toxicology, BioCenter IB, P.O.Box 56, FIN-00014 University of Helsinki, Finland

Brain Plasma

A

B

C

D

E

0.5

6,0

> 700

> 700

> 700

< 0.1

0.4

16

500

> 700

Liver < 0.1 < 0.1 3,9 330 > 700 As can be seen from the half-lives (h), the range order of the hydrolysis rate by the different organs is similar, irrespective of which morphine-3-ester is considered. The in-vitro results indicate that the liver homngenates show the highest and brain homogenates the lowest enzymatic activity, with plasma revealing an intermediate position. Thus, it may be presumed that when applying lnorphine-3-ester, the major part will be hydrolyzed in the liver. However, the results show that some, albeit significantly smaller, degradation may occur in the brain, which may become important due to the higher availability of morphine-3-esters directly in the brain as the site of action of morphine.

Administration of the metabotropic glutamate receptor (mGluR) agonist 1S,3R-ACPD induces rhythmic activity with a dominant frequency of 20 + / 2 I-Iz in rat hippocampal slices. This beta-rhythmic activity is inhibited by the mGluR antagonist (+)et-MCPG and is mainly determined by GABA-ergic and not by ionotropic glutamatergic transmission. The rankorder of mGluR agonist potency of the rhythmic effect was quisqualate > ACPD = glutamate, suggesting involvement of either mGluR1 or mGluR5 receptors. A phase reversal of the rhythmic activity was observed in the apical dendritic area suggesting that the generator activity is mainly carried by pyramidal neurons. In line with this observation, spike triggered averaging of pyramidal neurons and of oriens/alveus interneurons showed that the firing of both cell types was in phase with the EEG activity, suggesting involvement in the rhythmic activity. The 20 Hz beta oscillations may serve to potentiate synaptic transmission and we propose that metabotropic receptors may play a role in sensory processing and cognitive function. Preclinical Research, Sandoz Pharma Ltd., CH 4002 Basel, Switzerland

R 24 93

95

MECHANISM OF EMETIC EFFECTS OF TRAMADOL AND ITS 0-DESMETHYL METABOLITE IN FERRETS

PK/PD MODELLING OF THE INTERACTION BETWEEN PHENYTOIN AND SODIUM VALPROATE (VPA) O.E. Della Paschoa., J.W. Mandema, R.A. Voskuyl and M. Danhof

E. Morgenstem, E. Fridedchs* and A. Stattmann Tramadol ('rRA), an opiod analgesic drug, differs from morphine by its lower incidenco of side effects, e.g. respiratory depression, constipation and induction of tolerance and dependence. However it can induce nausea and emesis, a common side effect of opioids. In addition to opioid mechanisms TRA has been found to affect seretonergic and noradrenergic (NAergic) mechanisms. Several metabolites have been descdbod for TRA, one of them, o-desmethyitramadol (DES-TRA), is pharmacologically active. Both enantiomeres of TRA and of DESTRA differ in their influence on opioid and NAergic mechanisms. In the present study the emetic effect of beth enantiomeres of TRA and DES-TRA were investigated in ferrets. All drugs were given i.p. Emesis of each animal was mesuared over a period of 60 min. The following parameters were evaluated: latency, number of retches, vomits and emesis episodes, mean duration of single episodes, total duration of emesis, number of ferrets that react with emesis. The following results were obtained: (±)-TRA induced emesis only at the highest dose of 46,41 mg/kg, which is at the toxic level. (-)-TRA did not produce emetic effects up to a toxic dose of 31,6 mg/kg. (+)-TRA induced weak emetic effects in both above mentioned doses (31,6 and 46,41 mg/kg) without toxic effects.There was a tendency of naloxone to inhibit this effect. (-)-DES-TRA induced dose-dependent emesis in doses between 14,68 and 46,41 mg/kg. Naloxon produced a complete inhibition. (+)-DES-TRA produced emesis already at 1 mglkg. It was fully inhibited by naloxon. From previous studies it is known that the 5 substances differ in their activity at p-opioid and NA uptake mechanisms: whereas (-)-TRA produces NA uptake inhibition, (+)-TRA and (-)-DES-TRA act at both mechanisms, (+)-DES-TRA has selective IJ-opioid effects. Therefore, regarding the induction of emesis in the ferret model, it is concluded, that the opioid mechanism of TRA is responsible for this side effect.

Data on the pharmacodynamic interaction of phenytoin (DPH) and VPA in v i v o are scarce. Pharmacokinetic-pharmacodynamic (PK/PD) modelling is a straightforward approach to characterize pharmacodynamic interactions. Recently reported, the direct cortical stimulation model (CSM) permits the application of such approach. Briefly, it consists in quantifying the anti-convulsant effect on the basis of direct cortical stimulation with a ramp-shaped pulse train, which allows the determination of two thresholds, one for localized and another for generalized seizure activity within the same animal. Aim of the present study was to characterize the pharmacokineticpharmacodynamic interaction between DPH and VPA in the CSM. 8 male Wistarderived rats were allocated in 3 groups according to a parallel group design (add-on v e r s u s monotherapy). Group I was administered an i.v. infusion to achieve steadystate concentration of VPA. Subsequently, a dose of 40 mg/kg DPH was infused during 5 rain at a rate of 0.1 ml.min~. Likewise, group II received a continuous saline infusion and the same dose of DPH. Group III received only the continuous infusion of VPA. The time course of the concentration and the anticonvulsant effect were determined up to 6 hours after drug administration. Residual blood was used to assess protein binding. Concentration of DPH in plasma was assayed by a HPLC method. The elevations of the threshold for localized seizure activity (TLS) and of the threshold for generalized seizure activity (TGS) above their baseline represented the anticonvulsant effect. The pharmacokinetics of DPH was described by saturationelimination kinetics and fitted by a model with Michaelis-Menten elimination. The time delay between DPH plasma concentrations and effect was estimated by an equilibration kinetics model using a hysteresis minimization routine. The anticonvulsant effect of DPH was reflected by an elevation of the TGS without alteration of the TLS. DPH pharmacokinetics was not significantly altered in the presence of VPA. A significant shift in the concentration-effect relationship of DPH was observed in the presence of VPA. Taking into account that single administration of VPA resulted in only minor effects and no marked differences in protein-binding were observed, one may conclude that there is a synergistic pharmacodynarnic interaction between VPA and DPH.

PharrnakologischeForschungsgesellschaftBIOPHARM GmbH, Affed-Kowalke8tr. 4, D 10315 Berlin, *Gn3nenthalGmbH, Forschungszentrum, D-52078 Aachen

Leiden/Amsterdam Center for Dmg Research, Division of Pharmacology, P.O.Box 9503, 2300 RA Leiden, The Netherlands.

94 AFFII~ITIES OF THE qWO MAJOR METABOI/TES OF CLOZAPINE AND OF A SERIES OF ATYPICAL NI~JROL~F±'£CS FOR H 3 RECEPIDRS E. Schlicker and I. Marr Clozapine exhibits an intermediate affinity and antagonistic potency at H 3 receptors (PKi/PA2 of 6.2 - 7.1; Psycho~nrmacology 116, 464, 1994; Brit J Pharmacol II__44, 1523, 1995). We investigated whether this property is shared by its two major metabolites as well as by olanzapine (which is chemically related to clozapine) and by 4 atypical neuroleptics of different chemical structure. Binding of 3H-N~-methylhistamine to rat brain cortex membranes was displaced by olanzapine (pKi 5.45), N-desmethylclozapine (5.33), clozapine-N-oxide (4.94), thioridazine (4.78), zotepine (4.75), remoxipride (4.51) and risperidone (4.43). The metabolites of clozapine as well as olanzapine were also studied in a functional H 3 receptor model. In mouse brain cortex slices preincubated with 3H-noradrenaline, the three compounds did not affect the electrically (0.3 Hz) evoked tritium overflow. However, they shifted to the right the concentration-response curve of histamine for its inhiq~itory effect on the evoked tritium overflow; the pA 2 values were: olanzapine - 5.80; Ndesmethylclozapine - 5.84 and clozapine-N-oxide - 4.36. The present results suggest that the affinity and antagonistic potency of the two major metabolites of clozapine and of olanzapine at H 3 reoeptors is lower than that of clozapine. The affinity of the other atypical neuroleptics is very low (pKi < 4.8).

96

Institut f/Jr Pharmakolcgie und Toxikologie, Rheinische Friedrich-wilhelms-Universit~t Bonn, ReuterstraBe 2b, D53113 Bonn, Germany

NITRENDIPINE INHIBITS DIRECT NORADRENALINE- AND NPYINDUCED VASOCONSTRICTION BUT NOT POTENTIATION BY NPY 1N RAT MESENTERIC MICROVESSELS H. Chert u , C. Fetcher 1, R. Schafers 1, G. Wambach 2, M.C. MicheP Using a Mulvany-Halpern myograph chamber we have previously shown that the sympathetic transmitter noradrenaline and its cotransmitter NPY both produce direct vasoconstricting effects in isolated rat mesenteric microvessels via t~a-adrenergic and Yl NPY receptors, respectively; additionally NPY can potentiate the effects of noradrenaline via a Y1 receptor in this preparation which results in a reduced EC50 without major alterations of maximal noradrenaline effects (Nannyn-Schmiedeberg's Arch. Pharmacol. 351 Suppl., R150, 1995). We have now compared the sensitivity of the direct noradrenaline and NPY responses and of the NPY potentiation response to the Ca 2+ entry blocker nitrendipine. Nitrendipine (300 nM) reduced maximal contractile responses to noradrenaline in control and in chloroethylclonidinetreated (10 gM, 30 min, 37 ° C) vessels by approximately 30-40%, and caused a right shift of the concentration-response curve for noradrenaline towards higher concentrations by approximately 0.3 log units. Chloroethylclonidine treatment alone had no statistically significant effects. Nitrendipine reduced the contractile response to 100 nM NPY by approximately 60-70%. In contrast the potentiating response to 100 nM NPY was not significantly affected. Thus, in the presence of nitrendipine addition of NPY fully restored the reduced potency of noradrenaline and almost completely restored its maximal effects. We conclude that in isolated rat mesenteric microvessels a~A-adrenoceptor- and Y~ NPY receptor-mediated direct contractile effects may at least partly depend on influx of extracellular Ca 2+ through dihydropyridine-sensitive channels, whereas potentiation of the noradrenaline response by Y~ NPY receptors does not. We speculate that an enhancement of the availability of intracellular Ca 2+ might underly the potentiation response. tDept, of Medicine, University of Essen, 45122 Essen,2 and Medicine, St. Elisabeth Hospital, 45697 Herten, Germany

Dept. of

R 25

97

99

RADIOLIGAND BINDING DETECTS NPY RECEPTORS 1N RABBIT AND GUINEA PIG BUT NOT IN RAT OR HUMAN KIDNEY A. Bischoff, A. K/3tting, W. Erdbriigger, M. Schimiczek, D. Grandt, M.C. Michel

VASOCONSTRICTOR EFFECTS OF ANGIOTENSIN II AND ITS DEGRADATION PRODUCTS ANGIOTENSIN III AND ANGIOTENSIN IV Q. Li, J. Zhang~ M. Pfaffendorf, P.A. van Zwieten

We have previously shown that diuretic and natriuretic NPY effects in anesthetized rats are likely to be mediated via an extrarenal NPY receptor (Joint Meeting of the Dutch and German Hypertension Leagues, Mtinster 1994). Moreover, it has previously been reported that NPY receptors cannot be detected in rat kidney by autoradiography using [~25I]NPY due to NPYdegrading peptidases which cannot be inhibited without interfering with binding itself (Br. J. Pharmacol. 104:321-326, 1991). We have reinvestigated this issue using [125I]NPY and [125I]PYY as the ligands and compared kidney of rats with those of rabbits, guinea pigs and humans. Incubation of rat renal membranes with [lzSI]NPYresulted in marked ligand degradation as assessed by HPLC analysis of the incubation medium despite the presence of multiple peptidase inhibitors. This was somewhat ameliorated but not fully inhibited when intact rat renal cells were used in the presence of peptidase inhibitors plus colchicine to block cytoskeleton-dependent peptidase secretion. In contrast [~25I]PYY(in the presence of peptidase inhibitors) was not subject to major degradation by rat renal membranes. Nevertheless radioligand binding experiments with rat or human renal membranes and [t25I]PYY as the ligand did not detect quantifiable specific binding. In contrast under the same assay conditions specific [~25I]PYYbinding was detectable in rabbit and guinea pig renal membranes, and binding in both species was competed for with high affinity by unlabeled NPY or PYY. We conclude that the failure of binding studies to detect NPY receptors in rat and human renal membranes is not due to ligand degradation by peptidases or inadequate assay conditions. We speculate that rat and man in contrast to rabbit and guinea pig express only very few renal NPY receptors and/or that their expression is restricted to a small subset of renal cells.

The heptapeptide angiotensin Ill (Ang III) and the hexapeptide angiotensin IV (Ang IV) are the N-terminal degradation products of the octapeptide angiotensin II (Aug II). In the present study, the contractile effects of Ang II, Ang III and Ang IV were determined in rat aorta. Isolated rat aortic rings (endothelium-intact or -denuded) were set up in a 10 ml organ bath with Krebs' solution at 37°C. Isometric force was recorded. A resting tension of 1 g was maintained. After the preparation was challenged with a depolarizing potassium solution, cumulative concentration-response curves (CRC) of Ang II, Aug III and Ang IV were constructed. The following protocols were performed: 1) CRCs for each peptide were constructed 30 rain after addition of the various drugs. 2) The second CRCs of Ang II, Ang III and Ang IV were repeated lh after the first CRCs were constructed. In endothelium-denuded preparations, all three peptides caused concentrationdependent contractions with similar maximal responses. Ang III proved approximately 4 times less potent than Ang II, whereas Ang IV was about 1000 times less active than Ang II. Losartan (10 - 300 nM) caused parallel rightward shifts of the CRCs for all three peptides. The Schild plot slope for the effect of losartan on Aug III curves was significantly lower than unity (0.68 -+ 0.06, p < 0.05). PD123177 (1-[(4-amino-3-methylphenyl) methyl]-5-(diphenylacetyl)4,5,6,7-tetrahydro-lH-imidazole[4,5-C]pyridine-6-carboxylic acid) did not influence the CRCs for Ang II and Ang IV. However, the Ang III curves were moderately shifted leftward in the presence of PD123177 (0.1 gM). Destruction of the endothelium or incubation with the NO-synthesis inhibitor NG-methyl-L-arginine acetate (0.1 mM) significantly enhanced the contractile responses to all three peptides. The presence of endothelium significantly enhanced the development of tachyphylaxis to all three peptides. However, in endothelium-denuded preparations, the Emax value of the second curve for Aug II was about 50%, compared with the first one. In contrast, for Ang III and Ang IV the comparative Emax values were as high as 90% and 100%, respectively. Our results indicate that both Aug III and Ang IV are less potent but similarly efficacious vasoconstrictor agents compared with Ang II. Their contractile effects are also mediated by ATl-receptors and may be modulated by the endothelium. Tachyphylaxis induced by Ang III and Ang IV proved much weaker than that for Ang II. Tachyphylaxis appears to be enhanced by the intact endothelium.

Dept. of Medicine, University of Essen, 45122 Essen, Germany

Department of Pharmacotherapy, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands

98

100

PREJUNCTIONAL MODULATION BY ANGIOTENSINS ON NORADRENALINE RELEASE FROM SYMPATHETIC NEURONS IN RABBIT AORTA Storgaard T. and Nedergaard O.A. The aim of the present work was to examine the modulating effect of angiotensin I, IX and III (AI, AII and AIII) on stimulation-evoked noradrenaline release from postganglionic sympathetic neurons in rabbit isolated aorta. Rings of aorta were preloaded with (-)-3H-noradrenaline and subsequently washed with salt solution. The spontaneous 3H-outflow and field-stimulation-evoked 3H-overflow were collected in fractions. Cumulative addition of AI (10-8-106 M), AII (3x10-~1-10-7 M) and AIII (3x10-1°-10-6 M) enhanced the stimulation-evoked 3H-overflow up to 142, 165 and 188 %, respectively. The order of potency was A I I > AIII > AI. Edetate disodium (NazEDTA; 3x10 -5 M) did not alter the enhancing effect of AI (10-8-10-7 M). Captopril (10-6-10.5 M) and lisinopril (10-6 M) markedly attenuated the facilitating effect of AI (10-9-10-7 M) but did not alter the facilitation seen with All (10-1°-10.8 M). However, 10-s M captopril enhanced the facilitatory effect of AII (10-8 M). The aminopeptidase inhibitor amastatin (3x10 -6 M) did not alter the facilitating effect of AII (10-9-10-8 M). Captopril (10-7-104 M), lisinopril (10-7-104 M) and amastatin (10-9-3x10-6 M) alone did not alter the stimulation-evoked 3H-overflow. The enhancement seen with AII (3xl0-tl-10 -8 M) was independent of stimulation frequency (1-10 Hz). The facilitation induced by single concentrations (10-9-10-7 M) of AI, AII and AIII on repeated stimulations waned with time, i.e. development of tachyphylaxis. Cocaine (3x10 -5 M) plus corticosterone (4x10 -s M) did not alter the enhancing effect of AII. We conclude that the AII-induced enhancement of 3H-overflow is due to an increased release of 3H-noradrenaline rather than inhibition of neuronal and extraneuronal uptake. Furthermore, the enhancing effect of AI is mainly due to its conversion to AII, while the effect of AII is not dependent on a conversion to AIII.

INHIBITORY EFFECTS OF THE ANGIOTENSIN II RECEPTOR ANTAGONISTS BIBS 39 AND BIBR 277 ON [Dzs]-ANGIOTENSIN II BINDING AND ANGIOTENSIN II-INDUCED CA2+ MOBILIZATION IN RAT AORTIC SMOOTH MUSCLE CELLS J. Zhangr M. Pfaffendorf and P.A. van Zwieten We investigated the effects of two novel nonpeptidergic angiotensin II (Ang IX) receptor antagonists, 4'-[(2-n-butyl-6-cyclohexylaminocarbonylaminobenzimidazole-1-yl) - methyl ] biphenyl-2-carboxylic acid (BIBS 39) and 4'[ ( 1,4' -dime thyl-2 '-pro pyl [2, 6'-bi - 1H-b enzimi dazol e ]- 1'-y 1)methyl] - [ 1,1 'biphenyl]-2-carboxylic acid (BIBR 277) on specific [125I]-AnglI binding and Ang U-induced increases of [Ca2+] in cultured rat aortic smooth muscle cells. Radioligand binding experiments were performed by incubating aliquots of cell suspensions (3 × 105 cells) with varying concentrations of Ang II or its antagonists in the presence of 0.2 nM of [125I]-Ang I1 (specific activity 2200 Ci/mM) in 12 x 75 mm polystyrene tubes. Specific binding was defined as the total [125I]-Ang II bound minus the nonspecific binding. The inhibitory concentration (ICs0) of an inhibitor that caused 50% displacement of the specific binding of [125I]-Ang II was calculated. Intracellular [Ca2+] were measured by loading the cells with fura-2. The cell suspension (4 x 106 cells/ml) was incubated with fura-2/AM (4 gM) for 40 min at room temperature with continuous shaking. Fura-2 fluorescence was detected with a Detascan dual-wavelength fluorometer at room temperature (excitation 340 and 380 nm, emission 505 to 510 nm) and intracellular [Ca2+] was calculated. Specific [125I]-Ang II binding was inhibited by Ang 11, losartan, BIBS 39 and BIBR 277 with ICs0 values of 3.4 nM, 55.4 nM, 48.2 nM and 7.9 nM, respectively. Ang II (100 nM) rapidly increased the [Ca2+] from 160 nM up to 450 nM. The three Ang II receptor antagonists studied (losartan, BIBS 39 and BIBR 277) caused a concentration-dependent inhibition of Ang IIinduced increases of the intracellular calcium concentration. Our results indicate that BIBS 39 and BIBR 277, two newly synthesized compounds, are potent nonpeptidergic Ang 11 receptor antagonists in rat aortic smooth muscle cells. Among the three antagonists used, B1BR 277 proved to be the strongest inhibitor in attenuating the Ang II-provoked increase of intracellular calcium concentration.

Department of Pharmacology, Odense University, Winsloewparken 19, DK-5000 Odense C, Denmark.

Department of Pharmacotherapy, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, the Netherlands

R 26 101

103

LOSARTAN INHIBITS ARTERIAL AND VENOUS CONSTRICTOR RESPONSES TO ANGIOTENSIN II IN THE HUMAN FOREARM J. Baan, P,C. Chang, P. Vermeij, M. Pfaffendorf, P.A. van Zwieten

EFFECT OF CHRONIC ANGIOTENSIN II INFUSION ON AORTIC COMPLIANCE IN ANESTHETIZED RATS. D.L. Brouwers, F.R.M. Stassen, D.S. van lngen Schenan, G.E. Fazzi, H.J.M.G. Nelissen-Vrancken, J.G.R. De Mey, and J.F.M. Smits. This study was undertaken to examine the effects of chronic elevation of plasma angiotensin II (AngII) levels on aortic structure and compliance in the rat. Accordingly, Wistar rats were infused s.c. with 250 ng/kg/min AnglI for 14 days (AII; n=7). Both AII and control rats (CON; n=8) were cannulated with an arterial catheter for measurement of mean arterial pressure (MAP). Thoracic aortic diameter, compliance coefficient (CC), and distensibilitycoefficient (DC) were determined non-invasively in anesthetized animals (pentobarbital; 60mg/kg Lp.) using a B-mode imager attached to a vessel wail tracking system. After sacrifice, medial cross sectional area (CSA), media thickness (Mt) and wall-to-lumen ratio (W/L) of the thoracic aorta were determined. AnglI infusion significantly increased MAP in conscious rats. This was normalized when the rats were anesthesized, thus making it possible to determine CC and DC under isobaric conditions (see table). Although two-week infusion of AnglI induced thoracic aorta hypertrophy (increased CSA, Mt, and W/L), this was without effect on compliance parameters.

Angiotensin II causes arterial vasoconstriction, which is mediated by angiotensin II type 1 (AT 1) receptors. Losartan, a selective ATl-receptor antagonist, is known to induce arterial dilation, thus reducing cardiac afterload in heart failure patients. Losartan may also cause venous dilation, leading to preload reduction. The responsible receptor subtype, and the effects of Iosartan on the venous responses to angiotensin II have not been investigated in any detail so far. Accordingly, we studied the arterial and the venous responses to angiotensin II, before and after Iosartan, in the human forearm vascular bed of 6 healthy subjects. Angiotensin II (0.01, 0.1, 1.0 and 10.0 ng/kg/min) was infused into the brachial artery and changes in forearm blood flow (FBF) and maximal venous outflow (MVO) were measured by venous occlusion plethysmography and expressed as ml/100ml/min. Before measurements were performed, the vascular forearm system was predilated by infusion of sodium nitroprusside (5 ng/kg/min). The protocol was repeated in the same subjects after oral treatment with Iosartan (50 mg once daily for 5 days). Angiotensin [I concentration-dependentlydecreased FBF from 4.5 to 1.2 (-71%) and decreased MVO from 176 to 79 (-55%). The pECs0 value for the arterial constriction was 9.1 and that for the venous constriction 8.3. Losartan inhibited the FBF and MVO responses to angiotensin II: FBF decreased from 5.4 to 2.3 (-57%; p < 0 . 0 5 ) and MVO decreased from 197 to 153 (-22%; p<0.05). The pEC50 values were 8.6 and 7.9, respectively. In conclusion, angiotensin 11 induces venous constriction, qualitatively similar to the arterial constriction, in the vascular beds of the human forearm, most likely mediated by ATcreceptors. The efficacy was less in capacitance vessels than in resistance vessels. Losartan counteracts the angiotensin II-induced constriction of both the capacitance and the resistance vessels.

MAP mmHg (conscious)

MAP mmHg (anesth.)

CC, DC, 1 0 ~ m m 2 / k P a 10~/kPa

CON

123±4

117± 11

139.64.9.4

52.54.6.1

AII

153±7'

106~=7

133.3±21.7

50.14.6.2

diameter, ~m

CSA, mm2

Mt, ,am

W/L

969±35

0.485±0.018

77.2+4.1

0.169-±0.014

CON

AII 867±39 0.603±0.043* 103.64-8.7' 0.255±0.029* Data are mean~SEM.; * P<0.05. Thus, aortic wall hypertrophy resulting from chronic elevation of plasma AnglI concentration does not alter the dynamic compliance of the vessel.

Dept. of Pharmacotherapy, Academic Medical Center, Meibergdreef 15, 1105 AZ Amsterdam and Dept. of Nephrology, Academic Hospital of Leiden, The Netherlands.

Dept. of Pharmacology, Cardiovascular Research Institute Maastricht (CARIM), University of Limburg, Maastricht, The Netherlands.

102

104

CHRONIC AMINOOUANTDINE TREATMENT INCREASES ARTERIOLAR REACTIVITY TO ANGIOTENSIN II AND NOREPINEPHRINE IN RATS. F.R.L Crijns, H, van Essea, H. A.J. Struijker Boudier and B.H.R. Wolffenbuttel. Aminoguanidine (AG) treatment has been proven to prevent or decrease vascular dysfunctions in diabetes. AG acts as an inhibitor of advanced glycation endproduct (AGE) formation, but it also inhibits nitric oxide (NO) synthase. The latter could induce abnormal vasoreactivity. We therefore studied the effects of AG treatment on arteriolar constriction induced by norepinephrine (NE) and angiotensin II (Angll). At the age of 7-8 weeks Wistar Rp rats were implanted with a dorsal mierocirculatory chamber that allows reactivity measurements of striated muscle arterioles in conscious rats. A group treated with AG (50 mg.kg-lday "] s.c., AGR, n=8), and an untreated control group (CR, n=10) were used. After six weeks treatment, videorecurdings of At (large), A2 (intermediate) and A3 (small) arterioles were made at base-line, after i.v. infusion of NE (0.06, 0.2, 0.6 and 2 p.g.kg-].min4) and after i.v. infusion ofAnglI (0A and 0.3 Bg.kg-].min% Body weight and mean arterial pressure (MAP) were comparable in both groups (mean:i:SE, 254±7 g and 124±2 mmHg in CR vs. 238±7 g and 124±2 mmHg in AGR). MAP rose dose-dependently in treated and untreated animals following AnglI infusion (+34±5 mmHg and +51±4 in CR vs. +42±4 and +56±8 in AGR) and following NE infusion (+64-2 mmHg, +18±4 and +40±5 in CRvs. +5:t:1, +27±4 and +454-2 in AGR). Base-line diameters of Al, A2 and A3 arterioles were comparable in both groups. Arteriolar responses were markedly enhanced in the AG treated rats after AnglI infusion (change in diameter for 0.3 ~tg.kg4.min'l: -164-2% in A1, -14±3% in A2 and -194-5% in A3 vs. -4±2% in A1, -34-3% and -5:~3% in A3). NE infusion also caused an increased constriction in arterioles of the AGR group (change in diameter after 2/ag.kg-1.min4 NE: -164-4% in A1 arterioles, -20~:5% in A2 and -15±4% in A3 vs. -7±2% in A1, -7±5% in A2 and - 10-x4% in A3). We conclude that aminoguanidine treatment in conscious non-diabetic rats has no influence on mean arterial pressure but markedly enhances vasoconstriction in skeletal muscle arterioles after angiotensin II or norepinephrine infusion.

MOXONIDINE EXERT RENAL ACTIONS IN SPONTANEOUSLY HYPERTENSIVE RATS OF THE MONSTER STRAIN BUT NOT IN tN' SPONTANEOUSLY HYPERTENSIVE RATS H. Hoha,qe, C. Jahl, K. Hess, C. Reinhardt, J. Greven*, E. Schlatter Spontaneously hypertensive rats (SHR) are believed to have an altered receptor/ second messenger coupling. Recently we could recently demonstrate in Sprague Dawley (SD) rats, that moxonidine exerts besides its antihypertensive properties, additional renal effects. The purpose of this study was to investigate the renal effects of imidazoline compounds in two models of genetically determined hypertension, anaesthetised spontaneously hypertensive rats (SHR) and spontaneously hypertensive rats of the MOnster strain (SHRMs).

Dept.s. of Internal Medicine and Pharmacology, University of Limburg, P.O. Box 5800, 6202 AZ Maastricht, The Netherlands.

moxonidine

SHRMs A Na + excretion 5.9# A urine flow 35# A RR 19#

SHR -0.4 -3.4 -30

Tab.1 Effects of moxonidine (0.5 mg/kg b.w.) on A Na ÷ excretion (!Jmol/min*100g b.w., urine flow (IJI/min*100g b.w.) and blood pressure (mmHg) in anaesthetised SHR and SHRMs. #=p<0.05 SHR vs. SHRMs, n=5. Our study provides evidence, that moxonidine exerts different actions on kidney function and blood pressure in these two models of genetically determined hypertension. In SHR, moxonidine failed to increase blood pressure, Na + excretion and urine flow. This may indicate, that SHR have a reduced number of o~2 adrenoceptors /imidazoline receptors or have a defective second messenger coupling. Medical Department D, University MOnster Albert Schweitzer Str.33 D-48129 MOnster

R 27 105

107

DO ~3-BLOCKERS W I T H VASODILATING PROPERTIES O F F E R A BETTER E N D U R A N C E EXERCISE P E R F O R M A N C E THAN CLASSICAL/3-BLOCKERS?

DESENSITIZATION OF THROHBOXAN Ag-HEDIATED CARDIAC EFFECTS IN SPONTANEOUSLY HYPERTENSIVE RATS C. Giessler, I . Hoffmann-Heinroth, K. P6nicke, O.-E. Brodde

L. Van Bortel, J. Wijnen, F. Hartgens, H. Kuipers, H. Stluijker Boudier, and M. van Baak

We have r e c e n t l y shown t h a t , i n the isolated perfused r a t heart, p l a t e l e t a c t i v a t i n g f a c t o r (PAF)induced decreases in coronary flow and v e n t r i c u l a r c o n t r a c t i l i t y are mediated mainly i n d i r e c t l y via the release of thromboxan A9 (TXAg); these e f f e c t s were not changed in hearts gf spoMtaneously hypertensive rats (SHR) despite markedly enhanced TXA9release suggesting t h a t in SHR responsiveness of" TXAg-receptors might be desensitized (Giessler et a l . ~ Naunyn-Schm Arch Pharmacol 351 (Suppl):R 434, 1995). To t e s t t h i s hypothesis in t h i s study we assessed the effects of the TXAg-receptor agonist

Endurance exercise capacity - an important determinant of quality of life in physically active patients - is decreased by/3-blocking drugs. Various mechanisms have been proposed in this respect, i.e., a decease in muscle perfusion, leading to a decrease in oxygen and/or energy substrate transport capacity during exercise. Therefore,/3-blockers with vasodilating properties (flB+VD) might offer a better muscle perfusion and a better endurance performance (E). The present study investigates the effect on E of a 2-week treatment with carvedilol (C;/3B+VD) 25 mg once daily (o.d.) and with propranolol slow release (pSR) 80 mg o.d. Sixteen patients (42_+2 y, 78 + 3 kg, 175 + 3 cm) with hypertension completed the double-blind cross-over study. The 2 cross-over periods were separated by a placebo (PL) period of at least 2 weeks. Measurements were performed 2 h after the last drug intake. Versus baseline, pSR did not decrease resting blood pressure (rBP). The decrease in E time was limited to 15%. C decreased rBP ( p < 0 . 0 0 1 ) from 1 3 6 + 1 4 / 9 5 + 1 1 mmHg ( m e a n + SEN[, PL) to 1 2 5 + 4 / 8 6 + 3 mmHg. Heart rate (rHR) did not change. E time was 6 4 + 3 min during PL and was lower (p < 0.001) during C (41 + 3 min). In conclusion, although regularly advocated as initial dose in antihypertensive treatment, pSR 80 mg o.d. did not provide a full 24-h decrease in rBP. C decreased rBP significantly. In contrast to other fl-blocking drugs, C did not decrease rHR substantially. The decrease in E (36%) was comparable with that during classical flblockade. This study shows that ~-blocking drugs with vasodilating properties do not per se offer a better E than classical fl-blockers.

U 46619 ( ( 1 R - ( 1 ~ , 4 ~ , 5 B ( Z ) , 6 ~ ( 1 E ~ 3 S * ) ) ) - 7 - ( 6 - ( 3 - h y droxy-l-octenyl)-2-oxabicyclo(2.2.1)hept-5-yl)-5h e p t e n o i c a c i d ) on c o r o n a r y f l o w and v e n t r i c u l a r contractility ( d e t e r m i n e d as dp/dtm= v and d p / d t = i . ) in i s o l a t e d p e r f u s e d h e a r t s f r o m 1~=24 weeks o 1 ~ ' " normotensive Wistar-Kyoto (WK~) r a t s ~ a n d a g e - m a t c h e d SHR. In WKY r a t s U 46619 (10 -~ - 10 -~ m o l / 1 ) caused concentration-dependent d e c r e a s e s in c o r o n a r y f l o w and v e n t r i c u ] a r contractility; t h e s e e f f e c t s were a n t a g o n i z e d by t h e T X A g - r e c e p t o r a n t a g o n i s t SQ 29548 ((1S-(l¢,2~(Z),3~,4~))t7-(3-((2((phenylamino)carbo nyl)hydracino)methyl)-7goxabicyclo(2.2.1)hept-2-yl) -5-heptenoic a c i d ) (10 -~ m o l / l ) indicating that they are mediated via TXAg-receptor stimulation. In SHR, h o w e v e r , U 4 6 6 1 9 - i n d b c e d d e c r e a s e s in c o r o n a r y f l o w and v e n t r i c u ] a r contractility were s i g n i f i c a n t l y r e d u c e d . We c o n c l u d e t h a t in SHR c a r d i a c r e c e p t o r s m e d i a t i n g T X A g - i n d u c e d d e c r e a s e s in c o r o n a r y f l o w and v e n t r i c u l ~ r contractility are desensitized.

Depts. of Pharmacology, Human Biology, and Physiology, Cardiovascular Research Institute Maastricht, University of Limburg, Maastricht, The Netherlands

I n s t i t u t e of Pharmacology, U n i v e r s i t y of Halle, Germany

106

108

EFFECT OF THE ~ - A D R E N O R E C E P T O R A G O N I S T ERGOTAMINE ON THE M E C H A N I C A L VESSEL W A L L PROPERTIES OF THE COMMON CAROTID ARTERY

POTENTIATION OF THROMBIN-INDUCED MITOGENESIS IN C O R O N A R Y A R T E R Y S M O O T H M U S C L E C E L L S BY T I t R O M B O X A N E

M. Barenbrock, C. Spieker, St. Evers 1, A.P.G. Hoeks2, W. Zidek, K.-H. Rahn, Depart. of Medicine D and 1Neurologie, Univ. of M0nster, FRG, 2Depart. of Biophysics, Univ. of Limburg, Maastricht, NL The role of vascular smooth muscle activity for the mechanical vessel wall properties of large arteries are not known. The effect of the c~adrenoreceptor agonist ergotamine on the viscoelastic properties of the common carotid artery was investigated in 10 patients with ergotamine abuse (age 40+8 years) and in 10 healthy controls (age 42+9 years). The patients and controls were matched in respect of age, sex and blood pressure. Arterial distension was determined by using a multigate Doppler system, blood pressure was measured by a sphygmomanometer. Blood pressure was 139+10/77+9 mmHg in the ergotamine group and 135+12/75+9 mmHg in the control group (n.s.). The enddiastolic diameter of the common carotid artery was not significantly different between both groups (ergotamine: 8,3+0,4 mm; controls: 6,6+0,6 mm, n.s.). In the ergotamine group, arterial distensibility (DC) was significantly lower than in the control group (DC 16,7+6,0 10-3/kPa in the ergotamine, DC 21,9+4,6 10-3/kPa in the control group; p<0,02). The results show that the c~-adrenoreceptor agonist ergotamine can decrease arterial distensibility. Since arterial compliance is decreased in hypertension independent from blood pressure elevation, vasoconstrictive stimuli may modify arterial compliance in hypertension by changes of large arterial smooth muscle activity. Department of Medicine D, University of Mtinster, Albert-SchweitzerStr. 33, 48149 MOnster, Germany

T.-Ph. Zucker*, D. B6nisch, E. Brasclmeider+, E. Glusa+and K. Schr6r Thrombin is known as a potent mitogen for vascular smooth muscle cells (SMC), however no information is available on thrombin-induced mitogenesis in ogronary atte~ SMC. The role of thromboxane A2 (TXA2)in SMC-proliferation is discussedcontroversial. The ptesunt study investigates the effect of the TXA2 mimetic U46.619 (9,11Mideoxy-ll, 9epoxymethano~ostaglandin F2) on thrombin-indneed DNA synthesis in cultured bovine coromry artery SMC. Confluent cells (passage 4-6) were 404 incubated for 24h with U46.619 (0.3-30 • + thrombin r pM) in the absence or preseme of cto + vehicle A thrombin (0.1-2.0 IUIml) in serum-free 30" medium, supplemented with indo- '-~ methacin (3 HM). DNA synthesis was assessed as [3l-I]thymidineincorporation. Q" 20Thrombin stimulated the [3H]tJaymidine incorporation in a comentration- "~ dependent n~,,,~ (ECs0 = 0.3 IU/ml). ~ 10The thrombin (1.0 IU/ml) -induced DNA synthesis was further increased2fold in the additional ptesenee of U46.619 at concentrations, which wea~ 0

.

ineffective alone, with a maximum at 3

6

/L.I

1'0 3b

taM (n = 3, *) = P < 0.05 for thrombin [U46619] (Old) + U46.619 vs. thrombin alone and vs. U46.619 alone, repecfively (Figure). This potentiating effect was completely ta~vented by a selective TXA2 receptor-antagonist. In a comparable fashion, TRAP-14 (SFLLRNPNDKYEPF) (10 pM) -stimulated [3n]thymidine incorporation was also potentiated by U46.619, indicating that the catalytic site of thrombin is not directly involved in the pomntiation by TXA2. The above results suggest, that vasoconstrictory eicosanoids released from activated platelets might aggrnvatedthe thrombin-inducedgrowth response following coronary injury, e.g. in restenosis subsequent to PTCA. This potentiating i ~ o n should be consideredin experimental pharmacological prevention of corom~ SMC growth. Institute fiat Pharmakologie and *Klinische Anaesthesiologie, Heimich-Heine-Universit~t Diissetdorf, Moorensla,.5, 40225 Diisseldorf,Germany, Forschungszentrum fin"V a s ~ Biologie und Medizin, Friedrich~hiller-Universit~t Jana, NordNiuserStr. 78, 99089 Erfurt, Germany.

R 28

109

111

PREVENTION OF BLOOD SUPPLY TO THE RAT BRAIN IN LIVE ESCHERICHIACOLI SEPTIC SHOCK BY INTRAVENOUSCIPROFLOXACIN C van den Berg, C.M.Kasbergen, J.D te Biesebeek, W.Vleerning and D.J. de Wildt.

NEUROHUMORAL CHANGES IN RATS WITH HEART FAILURE PM.H. Schiffers, J. Bost, J.F.M. Smits, J.G.R. De Mey

In the course of the septic shock syndrome cerebral hypoperfusionmay lead to ischemia and to increased risk of developmentof an altered mental status and neurological disorders. Antibiotic therapy is an important therapeuticalapproach to treat sepsis. Therefore, the present study was focused on the effect of ciprofloxacinon cerebral blood flow (CBF) during sepsis in rat. In anesthetized non-septiccontrol rats, after administrationof phenylephrineand sodium nitmprusside,within the mean arterial pressure (MAP) windowof 34 mm Hg to 118 mm Hg, the CBF was constant. So, autoregulationof CBF in anaesthetized rats was intact. In septic rats, starting at one hour after administration of live E.coli, both CBF and MAP declined with a similar time course. Ciprofioxacin injections ( 12 mg/kg/iv at t= -5 rain and 2, 4, 8 h) 1) preventedreduction of CBF, 2) maintained intracerebral carotid blood flow portly through redistribution of flow from extra- to intracerebralvascular beds, 3) preventedsystemic hypotension4) prevented metabolicacidosis, and 4) enhanced 10 h survival rate from 17 up to 100 %. These results might indicate a loss of autoregulatorycontrol of CBF in sepsis and emphasizethe importanceof early administrationof antibiotics in the therapeutic approachof sepsis. National Institute of Public Health and EnvironmentalProtection,PO Box 1, 3720 BA Biithoven,The Netherlands.

Both in patients and animal models of heart failure an increased resistance is observed in peripheral vascular beds due to vasoconstriction and/or structural changes. Because various neurohumoral factors are potent vasoconstrictors and have mitogenic effects on vascular smooth muscle cells, we evaluated neurohumoral changes after myocardial infarction in rats induced by ligation of the left coronary artery. We measured catecholamines (NA, A), angiotensin II (ANG II), aldosteron (ALDO), vasopressin (VASO), endothelin (ET) and atrial natriuretic peptide (ANP) in plasma sampled at 1 and 5 weeks after infarct induction (MI) or SHAM surgery. Plasma levels of ALDO and NA were not altered after 1 or 5 weeks as compared to controls. After 1 week of ligation of the coronary artery, plasma levels of A (104.0_+30.6pg/ml), ANG II (105.7+_23.1 pg/ml), ET (3.05+0.67 pg/ml), VASO (23.4_+7.7 pg/ml) and ANP (92.6+_21.7 pg/ml) were significantly elevated as compared to SHAM operated animals (55.9+6.8 pg/ml; 38.0+5.4 pg/ml; 2.60+0.31 pg/ml; 6.7+1.5 pg/ml; 42.2.+.2.8 pg/ml, respectively). At 5 weeks after infarction only the significant elevation of plasma ANP levels persisted (ANG II and ET were also elevated but not statistically significant). These results suggest a transient activation of various neurohumoral systems following myocardial infarction in the rat. Initially these include mediators that exert vasoconstriction and hypertrophic actions on the vasculature. At later points in time only the vasodilator and antimitogenic agent ANP remained elevated. Consequences of this for cardiovascular adaptations in heart failure remain to be established. Department of Pharmacology, University of Limburg, Postbox 616, 6200 MD Maastricht, The Netherlands.

110

STUDIES ON THE INFLUENCE OF LIPOPHILICITY OF SOME DOBUTAMINE-ANALOGUES IN PAPILLARY MUSCLES. M. Rauland, R. Shirvani~ W. Klaue~ K. G0ttler

112 ALTERED CALCIUM SENSITIVITY OF RESISTANCE ARTERIAL SMOOTH MUSCLE IN EXPERIMENTAL HEART FAILURE M.J.J.M.F. Willemsen, G.M.J. Janssen, F.R.M. Stassen, LG.R. De Mey.

The present investigations were performed to compare the myocardial actions of dobutamine (DOB) with different new synthetized dobutamine-analogues. At first, homoveratrum acid and a N-substituted secundary amine were condensated to an amide, which could be reduced with lithium aluminum hydride in a second step to the corresponding amine: 4-[2-[[(3-Phenyl)ethyl]amino]ethyl]-l,2-benzenediol (DOB-ANA-1), 4- [2-[[3-(4-Hydroxyphenyl)ethyl]amino]ethyl]- 1,2-benzenediol (DOB-ANA-2), 4- [2-[[(3-Phenyl)propyl]amino]ethyl]-1,2-benzenediol (DOB-ANA-3) and 4-[2-[[3-(4-Hydroxyphenyl)propyl]amino]ethyl]- 1,2- benzenediol (DOB-ANA-4). The lipid solubility of compounds was determined in the following order: DOB-ANA-1 < DOB-ANA-4 < DOB < DOB-ANA-2 < DOB-ANA-3. Contractile force and effective refractory period were investigated in papillary muscles from guinea-pig hearts at a temperature of 37° C. It has been found that all compounds tested increased contractile force in the following order of potency: DOB-ANA-2 (+ 93 + 5 % by 10s M) < DOB-ANA-1 (+ 236 +_9 % by 10,5 M) < DOB-ANA-4 (+ 95 +_14 % by104 M) < DOB-ANA-3 (+127 +_14 % by10 "6M)
In human and experimental heart failure, the reactivity of peripheral resistance arteries to vasoconstrictor agents is impaired. We evaluated whether the responsiveness to calcium is modified in resistance arterial smooth muscle under this condition. We therefore isolated mesenteric resistance arteries from rats in which myocardial infarction had been induced by ligation of the left coronary artery (MI) and from sham operated controls (SHAM). The vessels were denuded of endethelium, sympathectomised and depleted of neuropeptides. They were mounted in myographs for recording of isometric tension development. Calcium concentration-resonse curves were constructed in the continuous presence of 125 mM K + or of 10 taM phenylephrine (PHE) after depletion ofintracellular calcium stores. Calcium sensitivity (Ca-pD 2 ) was also determined in depolarised vessels in the presence of PI-IE and of 10 taM isoproterenol (ISO) which increased and decreased Ca-pD 2 . Three weeks after MI, the Ca-pD2 was not modified under either of the following conditions: K ÷, PHE, K÷+ PHE or K ÷ + ISO. Five weeks after MI, however, the Ca-pD2 was markedly reduced in the presence of PHE alone while not being modified in depolarised preparations with or without PHE or ISO. At this point in time, the density of ~x-adrenergic receptors in mesenteric resistance arteries (Bmax in [3H]-prazosin binding assays) was not reduced. These observations indicate that after MI in the rat, a modification of excitation-contraction coupling progressively develops in pheripheral resistance arterial smooth muscle. This may involve cellular hyperpolarisation and/or less effective coupling of receptors to calcium-influx.

Institut for Pharmakologie der Universit&t zu K61n, Gleuelerstr. 24, D-50931 K61n, FRG

Department of Pharmacology and Cardiovascular Research Institute Maastricht, University of Limburg, PO box 616, 6200 MD Maastricht, The Netherlands.

R 29

113

115

CRATAEGUS-EXTRACT PROLONGS ACTION POTENTIAL DURATION IN

INFLUENCE OF SALBUTAMOL ON HEARTS FROM DIABETIC HYPERTENSIVE RATS. OHM Beenen, M Pfaffendorf, PA van Zwieten. In the present study the effect of the ~,2-adrenoceptor agonist salbutamol was studied on various parameters in isolated Langendorff hearts of hypertensive diabetic rats. Male spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY) of 12 weeks of age were made diabetic by a bolus injection of streptozotocin (55 mg/kg i.v). 8 Weeks later the inotropic effect of salbutamol was assessed in paced (6Hz) Langendorff hearts (37°C), perfused at constant pressure (55 mmHg), and in separate experiments with constant flow pertusion. Left ventricular pressure (LVP), coronary flow (CF) and coronary perfusion pressure (CPP) were measured.

GUINEA PIG PAPILLARY MUSCLE

A. Mt3tler, W. Linke, Y. Zhao, S. Dhein, W. Klaus Crataegus-extract (CRA) has been shown to be effective in the treatment of patients suffering from moderate heart failure (NYHA II). In order to find out which mode of action CRA has on cardiac muscle preparations, we studied its effects on guinea pig papillary muscle using conventional microelectrode techniques. (T=35°C; regular pacing at 1 Hz). Two concentrations of CRA were tested: 3 mg/I and 10 mg/l. While

the

lower concentration

had almost

no effect,

the

higher

concentration lead to an increase in the force of contraction (+12.0_+3.0%; Mean_+SEM; p < 0.05) and a significant prolongation of action potential duration at the levels of 20%, 50% and 90% repolarisation. Action potential duration was prolonged by 8.5+2.3 ms, 12.5-+2.6 ms and 11.7+_2.9 ms, respectively (control APDgo: 172-+4 ms).

In addition, a

tendency towards a reduction of the maximum upstroke velocity of the action potential was observed. Our results confirm the positive inotropic action of CRA seen in clinical studies. Unexpectedly, we found a prolongation of action potential duration, which may explain the increase in refractory period in guinea pig hearts observed by other investigators. The findings suggest, that CRA besides its positive inotropic action might have some class Ill-like antiarrhythmic effect.

Institut for Pharmakologie, Universit~t zu K61n, Gleueler Str. 24, 50931 K01n, Germany

CW DW CS DS

LVP (E~)

log ECho

CF (Em~)

CPP (Em~x)

104 + 7 91 + 3 83+8 101 _+10

-6.32 + 0.06 -6.37 + 0.04 -6.60 _+0.05# -6.51 + 0.08

6.2 + 0.6 9.7 + 1.1" 4.7_+0.6 10.2 _+0.9*

20.4 _+1.8 29.5 _+1.7" 10.3_+1.6# 27.4 -+ 1.2*

Table 1: maximal increase in LVP (mmHg), log ECso (M), max increase CF (ml/g wet heart weight/minute) and maximal decrease in CPP (mmHg). Means + SEM, n=4-7, *: p<0.05 vs non-diabetic control, #: p<0,05 vs non-hypertensive control (CW: control WKY, DW: diabetic WKY, CS: control SHR, DS: diabetic SHR). No difference was found between the increases in LVP in the four groups of rats, although hearts from SHR tended to be more sensitive to salbutamol than hearts from WKY. In hearts from all diabetic animals a stronger salbutamolinduced rise in coronary flow was found. Accordingly, during perfusicn at constant flow a stronger decrease in coronary perfusion pressure was seen in hearts from diabetic animals compared to their non-diabetic controls. Hearts from normoglycemic SHR showed an impaired decrease in coronary perfusion pressure compared to control hearts. Diabetes mellitus appears to increase the efficacy of 132-adrenoceptor-mediated vasodilation in the coronary system, whereas hypertension per se showed the opposite effect. Dept of Pharmacotherapy, Academic Medical Centre, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands.

114

116

CONTINUOUS VERSUS INTERMITTENT OUABAIN TREATMENT AND THE EFFECT ON CARDIAC FUNCTION IN AWAKE RATS WITH MYOCARDIAL INFARCTION H.J.M.G. Nelissen-Vrancken, J-F. Wang, E.A. Raes and J.F.M. Snails Acute and chronic treatment of normal rats with cardiac glycosides are generally believed not to improve cardiac function. To investigate the effects of cardiac glycosides on cardiac function in a rat model for heart failure, we studied central hemodynamics in rats with myocardial infarction (MI) following chronic ouabain treatment. Three weeks before treatment, MI was induced by ligation of the left coronary artery of Wistar rats. The animals received no treatment, or either intermittent (once dally 14.4 mg/kg s.c.) or continuous treatment (14.4 mg/kg.d, osmotic minipump) with ouabain for 2 weeks. The animals were instrumented with an electromagnetic flow probe on the ascending aorta and an arterial catheter for measurement of resp. cardiac output (CO) and mean arterial pressure (MAP), and a venous catheter for injection of a volume load. Intermittent treatment with ouabain resulted in a significant increase in total peripheral resistance (TPR) at rest (untreated: 1.25±0.15; intermittent: 1.52±0.07 mmHg.min/ml; mean±SEM), whereas MAP and CO at rest and after volume loading were not significantly affected. In contrast, continuous treatment of MI animals with ouabaln resulted in an improvement of CO both at rest (untreated: 74±5; continuous: 83±4 ml/min) and after volume loading (untreated 105±7; continuous: 134±8 ml/min). TPR at rest decreased significantly following continuous treatment (1.08±0.08 mmHg.min/ml). In contrast to observations in normal rats, the present experiments demonstrate an improvement of cardiac function in MI rats following continuous treatment of ouabain, which may be due to changes in Na+,K+-ATPase following MI. The differences in effect between the two therapeutic designs in MI animals are likely to depend on differences in variation of plasma concentration of ouabain during the day.

IN VITRO CHARACTERIZATION OF S 1197, A NOVEL OPTICALLY PURE DIASTEREOMERICFIBRINOGEN RECEPTORANTAGONIST. B. Jablonkq, M. Just, W. K6nig, and H. UoStilz. RGD (Arg-Gly-Asp)-peptides are competitive platelet fibrinogen receptor antagonists inhibiting platelet aggregation and thrombus formation. S 1197 ((S)(3-(2-(4-(S)-(4-(Amino-imino-methyl)-phenyl)-4-methyl-2,5-dioxo-imidazolidin -1-yl)-acetylamino))-3-phenyl-propionic acid hydrochloride) is a non-peptidic RGD-mimetic compound synthetized in the optically pure diastereomeric confguration. The mechanism of action of S 1197 was characterized in various in vitro experiments: - S 1197 inhibited platelet aggregation in human gel-filtered platelets (GFP), platelet rich plasma (PRP), and whole blood in a dose-dependent manner, independent from.the agonist used. ICso-values of platelet aggregation varied between 20 and 100 nM in the different systems. In contrast, ristocetin-induced platelet agglutination in GFP was not influenced by S 1197. - The inhibition of platelet aggregation was reversible, as shown by gel-filtration of S 1197-pretreated PRP and subsequent aggregation experiments. - S 1197 inhibited 125I-fibrinogen (Fg) binding to ADP- activated human GFP in a concentration-dependent manner with a K~-value of 9 riM. In comparison to the less potent diastereomere, S 1197 exhibited an 200-fold higher binding affinity, thus demonstrating pronounced stereoselective binding characteristics between both optically pure diastereomeric partners for platelet GP IIb-IIIa. - Beside ]25I-Fgbinding, binding of 125I-vonWillebrand Factor to GP IN-Ilia of ADP-activated GFP was also prevented by comparable S 1197-concentrations (ICso = 8 nM); ristocetin-induced von Willebrand Factor binding to GP Ib-IX remained unchanged in the presence of 100 girl S 1197. - The S 1197-induced inhibitory effect on platelet aggregation results from a competitive inhibition of Fg binding to GP I/b-Ilia as shown by a rightward shift of the fibrinogen saturation isotherm without a change in the number of maximal fibrinogen binding sites in the presence of S 1197. - 125I-Fg binding experiments with the isolated and immobilized human GP IIb-IIIa confirmed the potent, stereoselective, and competitive inhibitory effects of S 1197 found with intact human platelets. In conclusion, S 1197 is an optically pure diastereomeric fibrinogen receptor antagonist, that potently and reversibly prevents aggregation by competitive inhibition of fibrinogen binding to GP IIb-ilIa of activated human platelets.

Dept. of Pharmacology, Cardiovascular Research Institute Maastricht (CARIM), University of Limburg, P.O Box 616, 6200 MD Maastricht, the Netherlands.

Pharma Research, Hoechst AG, 65926 Frankfurt; Germany

R 30 117

119

PHARMACOKINETIC-PHARMACODYNAMIC MODELLING OF T H E ANTI-LIPOLYTIC EFFECTS OF T H E ADENOSINE A r R E C E P T O R AGONIST N6-(P-SULFOPHENYL)ADENOSINE (SPA) IN RATS E.A. van Schaick. H.J.M.M. de Greef, M.W.E. Langemeijer, A.P. IJzerman and M. Danhof

EFFECTS OF ~-ADRENOCEPTOR AGONISTS, DOPAMINE AND GLUCAGON ON PROPRANOLOL INTOXICATION IN RATS. J.D. te Biesebeek, A.E. ToeI, J.Meulenbelt, J.Wemer, W.Vleeming and D.J de Wiidt.

For therapeutic use of adenosine analogues, it is important to develop compounds with selectivity of action. In this study tissue-selectivity of the adenosine A~-receptor agonist SPA was investigated by quantification of the hemodynamic and antilipolytic effects on the basis of an integrated pharmaookinetic-pharmacodynamic (PK/PD) model. Pharmacokinetics and pharmacodynamics were determined after intravenous administration of N6-(p-sulfophenyl)adenosine (SPA) to male Wistar rats. Serial arterial blood samples were drawn for the determination of blood SPA concentrations and plasma non-esterificd fatty acid (NEFA) levels. Blood pressure and heart rate were monitored continuously. For each individual rat the relationship between the SPA concentrations and the NEFA lowering effect could be described by the indirect suppression model. Administration of SPA at different rates and doses (0.06 mg/kg in 5 rain, 0.06 mg/kg in 15 min and, 0.12 mg/kg in 60 min) revealed uniform pharmacodynamic parameter estimates. The averaged parameters (mean _+ SEM, n=19) were Em~: -80 -+ 3% (% change of baseline), ECs0:23 -+ 2 ng/ml and Hill factor: 2.3 _..+0.3. . In addition the model estimated the elimination rate constant of NEFAs (0.097 _+ 0.007 min% In another group, which received 0.40 mg/kg SPA in 15 rain, pharmacodynamic parameters for both heart rate and antilipolytic effect were derived within the same animal. SPA inhibited lipolysis at concentrations lower than those required for an effect on heart rate. The ECs0 values (mean + SEM, n=6) were 131 -+ 31 ng/ml and 20 -+ 2.9 ng/ml for heart rate and NEFA lowering effect, respectively. In conclusion, the relationship between blood concentrations of SPA and anti-lipolytic effect could be described by the indirect suppression model, yielding estimates of potency and activity of the drug in vivo. The results showed a 6-fold difference in potency of SPA between the effects on heart rate and NEFAs. On the basis of preclinical PK/PD modelling selectivity of action of newly developed purinergic drugs can be assessed in vivo.

Severe ]3-blocker intoxication is a life-threatening condition. We studied the efficacy of the hydrophUic 151,2-agonist isoprenaline (ISO), the lipophilic I~cagonist flerobuterol (FLE), the lipophilic [32-agonist elenbuterol (CLEN), dopamine (DOP) and glucagon (GLU) in the treatment of dl-propranolol (PROP) intoxication. Experiments were performed in spontaneously breathing (SB) and artificially ventilated (AV) anaesthetized rats. Treatment was started 0 or 30 min after institution of continuous infusion of a toxic dose of PROP (30 mg/kg/h) In SB and in AV PROP-intoxicated rats, compared to saline neither ISO (10-50 gg/kg/min) nor FLE (1-10 i.tg/kg/min) or CLEN (10-50 p.g/kg/min) had any effect on cardiovascular parameters, blood gas values and suwival times. Death, due to respiratory arrest in SB rats and due to cardiovascular collapse in AV-rats occurred in about 75 and 250 min, respectively. When treatment by FLE was started simultaneously with infusion of PROP (at t=O rain) also no beneficial effects were observed. Both, DOP (25 gg/kg/min) and GLU (initial 100 i.tg/kg followed by 1 gg/kg/min) were ineffective in SB PROP-intoxicated rats but had beneficial effects on mean arterial pressure in AV PROP-intoxicaled rats. However, no increase in survival time was observed. Treatment by the combination of DOP and GLU decreased the survival time beth in SB and AV PROP-intoxicated rats. We concluded that: 1) I~-agonists are ineffective in treatment of propranolof intoxication in rats. Mechanisms other than I~-adrenergic receptor interactions seem to play a more important role in propranolol intoxication in rats. 2) Support of respiration seems to be of mare importance than optimalization of hemodynamic parameters because restoration of hemodynamic parameters by dopamine or glucagon did not increase survival time whereas artificial ventilation did prolong survival time by about factor 3 in propranolol-intoxicated rats. 3) Combination therapy with dopamine and glucagon may be harmful in the treatment of propranolol intoxication. National Institute of Public Health and Environmental Protection, PO Box 1, 3720 BA Bilthoven, The Netherlands.

Leiden/Amsterdam Center for Drug Research, Divisions of Pharmacology and Medicinal Chemistry, P.O, Box 9503, 2300 RA Leiden, The Netherlands.

118

120

CONTRIBUTION OF THE INDIVIDUAL ENANTIOMERS TO RACEMIC PROPRANOLOL INTOXICATION IN RATS. A van de Kuil, A.E. Toet, J.Meulenbelt, J.Wemer, W.Vleeming and D.J de Wildt.

PROPERTIES OF 1lg-HYDROXYSTEROID DEHYDROGENASES IN THE RENAL CELL LINE LLC-PK1.

13-blockers have a wide variety of clinical indications and their wide-spread availability contributes to an increasing incidence of ~-bleoker intoxication. We studied the contribution of the individual enantiomers to racemic propranoiol (dl-pmp) intoxication in spontaneously breathing (SB) and artificially ventilated (AV) anaestbetized rats. In SB rats, infusion of a toxic dose of dl-prop, I-prop or d-prop (30 mg/kg/h) caused a decrease in heart rate, mean arterial blood pressure (MAP), PaOa, pH, an increase in PaCO2 , ultimately resulting in death by respiratory arrest. The survival time of the d-prop group was longer compared to the dl-prop group. The decrease in MAP was initiated at 0.4, 0.5 and 0.7 of the survival time for I-prop, dl-prop and d-prop, respectively. In AV rats infusion of dl-prop, I-prop or d-prop (30 mg/kg/h) caused similar effects on hemodynamic parameters as observed in SB rats, whereas blood gas values remained fairly stable. In all propranol treated rats artificial ventilation increased survival time by a about a factor 3. Death was the result of a progessive bradycardia and decrease of MAP. Survival time (rain) in SB and AV rats after continuous infusion of dl-prop , I-prop, d-prop or saline are shown in the table below. Values are means _+ SEM of 10 experiments. The observation period for SB and AV rats was 150 and 300 minutes, respectively. dl-prop SBrats A V rats

68+

6

276+17

I-prop 79+

7

271_+ 19

d-prop

saline

91+5"

150"*

266+22

300

slgnlncanuy.=nerent versus Ul-prop; -- slgnlrmanuy,alnerenl versus o1-, I-, ano a-prop. Based on the results obtained in SB rats it was concluded that d-prop is the least toxic compound while dl-prop seems to be the most toxic compound in rats. This more toxic effect is assumed to be the result of a potentiation of effects of the individual enantiomers. Respiratory insufficiency seems to be strongly involved in propranclol intoxication. National Institute of Public Health and Environmental Protection, PO Box 1, 3720 BA Bilthoven, The Netherlands.

E. MObus, E. Maser, and J.J.R. Hermans*, llg-Hydroxysteroid dehydmgenases (llB-HSD), by converting lIgglucocorticoids to their ll-ketones, are assumed to regulate cortJcosteroid hormone action, as they determine the concentration of active glucocorticoids at their effector sites (being e.g. glucocorticoid- and mineralocorticoid receptors). This mechanism has been shown to be important in various physiological processes involved in blood pressure regulation, e.g. renal salt-handling. However, up to now, there is relatively few information on whether and bow this enzyme system is influenced by inter- and intracellular signalling mechanisms. In this study we have tried to obtain insight whether the renal cell line LLC-PK1 may represent a valuable tool for studying the effect of hormonal impulses on renal llB-HSD. We found that in this cell line considerable lIg-hydroxysteroid dehydrogenase activity (measured as tile conversion of cortisol to cortisone) is present, which appears unidirectional, since no reduction of cortisone could be demonstrated. The liBoxidation of cortisol occurs at 2-5 times higher rates in the presence of 1 mM NAD than in the presence of 1 n~Vl NADP. Using NAD as a cofactor, llB-HSD displayed a Km for cortisol of 0.35 gM. Western blot analysis with antibodies against 11B-HSD1 revealed no positive signals for this isoform in homogenates of LLC-PK1 cells. The absence of fetal calf serum in cell culture medium, 24 hours before enzyme assay, decreased cortisol oxidation rates 1.5 and 2.8 fold for NAD and NADP respectively, pointing to the presence of an essential (possibly upregulating) factor in serum. Pretreaenent of the cells with 1 FM dexamethasone appeared to induce the NADP dependent liB-oxidation of cortisol 2.5 times. This effect was evident after approximately 2 hours and lasted at least 32 hours; after 72 hours tile enzyme activity appeared to be returned to normal. For the NAD dependent liB-oxidation of cortisol, no effects were appareut after dexamethasone treatment, possibly pointing to the existence of multiple 1IB-HSD isoforms in LLC-PKI cells. Taken together, LLC-PKI cells may provide an interesting instnmlent for the study of the regulation of renal I1B-HSD expression or activity by endogenous or exogenous substances that modulate renal function. Current and future studies will be directed on the further characterization of the llB-HSD isoforms in the LLC-PK1 cells as well as on the effect of various external stimuli on 11B-HSD in LLC-PK1. Dept. Pharmacology and Toxicology, Philipps University, Marburg, Germany Dr Hemlans has been granted a post-doctoral fellowship by the Alexander yon Humboldt Stiftung.

R 31 121

123

NOVEL ALDOSTERONE BINDING-PROTEINS IN PORCINE LIVER MICROSOMES: CHARACTERIZATION AND SOLUBILIZATION C. MEYER, M CHRIST, and M. WEHLING During the past decades specific binding sites have been described in membranes for various steroids. They are discussed in context with rapid non-genomic effects of steroids like the stimulation of the Na+/H+-exchanger by aldosterone, IP~ and flee intracellular calcium. Using [1,2,6,7-3H] aldosterone ([3I-I] aldosterone) as radioligand, specific binding sites for aldosterone were identified and characterized in microsomal preparations from porcine liver. The reversible binding of [3H] aldosterone was saturable and Scatchard analysis revealed two apparent dissociation constants (Ka) , K d l = 11 nM and K0a = 118 nM. Maximum binding capacity was approximately 700 fmol mg -~ microsomal protein and binding showed a distinct pH-optimum at pH 7.2. Binding was also dependent on temperature, and was reduced by more than 70% when membrane vesicles were pretreated with the protease trypsin. The kinetics of radioligand association to microsomes was rapid ( h a = 3-5 min). A range of steroids was tested for their ability to compete with the radioligand. The binding site showed binding selectivity for aldosterone with cortisol being a weak agonist at 1000-fold higher concentrations only. Evidence for their location at plasma membranes is given by the inhibition of binding by GTP-7-S pointing to a Gprotein dependent receptor as binding protein. Among those detergents tested to optimize conditions for solubilization, n-octylglucoside was most favorable and solubilized 25% of the radioligand-binding protein complex in undissociated form. These binding sites exposed unique pharmacological properties which are similar to those found for aldosterone membrane binding in human lymphocytes and pig kidney, and for rapid aldosterone effects on sodium-proton-exchange and second messengers.

COMPARISON OF METABOLISM OF DIFFERENT DIHYDROPYRIDINE CALCIUM CHANNEL BLOCKERS LN RABBIT LIVER MICROSOMES. K. Otter, C. Mignat and A. Ziegler

Medizinische Klinik, Klinikum Innenstadt, Division of Clinical Pharmakology, University of Munich, 80336 Munich, Germany

DHP

in-vivo t~ EL

(h)

Vj0.40(~tM/min)

V=~ (~aM/min)

Km

(gM)

25 gM

50 gM

100 [aM

Nifedipine

3.4

0.56

0.77

1.11

1,7

55,2

Isradipine

8.4

0.80

1.03

1.92

4,95

162,3

Felodipine

10.2

0.61

0.97

1.41

2,53

79,4

Nilvadipine 11.0 0.67 1.02 1.48 2,53 71,8 As shown in the table (in-vivo t~¢~E~adapted from: Kelly & O'Malley, 1992, Clin Pharmacokinet 22:416-433), the results revealed that degradation of the DHPs tested was quite similar with respect to the V,~- and Kin-values. It is suggested, therefore, that hepatic metabolism does not provide the main determinant for the observed differences in in-vivo elimination rates of DHPs. Department of Pharmacology, Christian-Albrechts-Universit/it, Hospitalstrage 4, D-24105 Kiel, FRG

122 PHARMACOKINETIC-PHARMACODYNAMIC MODELLING REVERSIBLE DRUG EFFECTS IN T H E P R E S E N C E DISTRIBUTIONAL ARTERIOVENOUS CONCENTRATION DIFFERENCES B. Tuk, V.M.M. Herbert, J.W. Mandema and M. Danhof.

Despite to the similiarity in chemical structure, dihydropyridine calcium channel blockers (DHPs) are cleared from the body with markedly different elimination rates. This finding may be explained either by differences in the volume of distribution (i.e. due to differences in protein binding) or by differences in the rate of hepatic elimination. The aim of the present study was to evaluate whether differences in the rate of hepatic elimination might contribute to the observed differences in the elimination half-life (tt~., EL) of DHPs. Since the main metabolic pathway of DHPs in the liver is assumed to be the oxidation of the pyridine ring by the cytochrome.P-450 enzyme system (B~3cker & Guengerich, 1986, J Med Chem 29: 1596-1603), we used microsmnes of rabbit liver to evaluate the hepatic degradation of DHPs (25gM-100p.M). After incubation at 37°C in the presence of hnM NADPH, samples were taken at 0, 10, 20, 30, and 40 min and quantified by HPLC-UV. The initial rate of oxidation (V~0.4o)was proved to follow MichaelisMenten kinetics, thus allowing the calculation of V,,,~, and Kin, respectively.

124 OF OF

A new approach is presented for the analysis of non steady-state pharmacokinetic and pharmacodynamic data on basis of venous drug concentrations. In the model, unobserved arterial blood concentrations are linked to the venous compartment by first-order rate constant kvo. In contrast to previously published methods, concentrations at the effect site are not estimated by numerical deconvolution, but on basis of an analytical solution to the mathematical equations. The value of the extended effect-compartment link model was determined by computer simulations and a pharmacokinetic-pharmacodynamic experiment in rats, using either arterial or venous concentrations to drive the pharmacodynamic fitting procedure. The results of the simulations show that, when using a conventional effect compartment model for venous drug concentrations, significant bias in the estimated values of the ECs0 may occur up to -70%. With the extended link model, accurate estimates of the pharmacodynamic parameters are obtained upon any given combination of the values keo and kvo, though precision of the parameter estimates is reduced. Precision could be improved by increasing the number of observations of the pharmacological response intensity. The outcome of the pharmacokinetic-pharmacodynamic modelling in rats, using midazolam as model compound, confirmed the findings of the computer simulations. Leiden/Amsterdam Center for Drug Research, Division of Pharmacology, Sylvius Laboratory, PO Box 9503, 2300 RA Leiden.

B I O A V A I L A B I L I T Y OF T W O A Q U E O U S C A F F E I N E F O R M U L A T I O N S IN H E A L T H Y V O L U N T E E R S Matthey B, Biederbick W, Joseph G, Rump AFE, Biederbick S, Theisohn M, and Klaus W Caffeine is used in premature infants to prevent the apnoea and bradycardia syndrome. Caffeine citrate diluted in pure water has a bitter taste. To improve the taste of the drug a new formulation of caffeine with addition of rasberry syrup was made by the pharmacy department of the university hospital of Cologne. The bioavailabilty of the rasberry syrup formulation of caffeine citrat was compared to the caffeine citrat solution in water. Six healthy adults (4 male, 2 female, age from 22 to 45 years) received 5 mg/kg bodyweight caffeine' citrate of the respective formulations in a randomized cross over study. Multiple venous blood samples were drawn, centrifagated and the plasma stored at -20°C until analysis. Extraction was done with 2.5 ml dichlormethane, organic phase was evaporated in an Hetovac. Caffeine, theophylline and thenbromine concentrations were determined by HPLC (Hypersil ODS 5p,m column 250 mm length, 4 mm diameter, elution 0,5 mmol/l NaH2PO 4 : acetonitrile (920:80 pH=5), UV-detection: 270 nm, detection limit about 50 ~tg/1) with hydroxyethyltheophylline as internal standard. Pharmacokinetic analysis was performed using the TOPFIT-computer program. The bioavailability of the caffeine citrate in rasberry solution was slightly better than those of the caffeine citrate in pure water. Caffeine citrate in water AUC [mg x minx 1-~] 2422 (4- 2220) Cm,~ [rag/l] 4.29 (4-0.71) Tmax [rain] 47.0 (±21.5)

Caffeine citrate in rasberry-syrup 2546 (:51905) 4.96 (:k0.72) 32.5 (4-22.1)

However, change of the dosing regime was not necessary as the small increase of bioavailabilty has no clinical impact. Institute of Pharmacology, University of Cologne, Gleueler Str. 24, D-50931 Krln, Germany

R 32 125

127

COMPARISON OF FREE AND TOTAL PLASMA AND SALIVA CONCENTRATIONS OF CAFFEINE AFTER ORAL ADMINISTRATION TO HEALTHY VOLUNTEERS

7,12-Dimethylbenz[a]anthracene and Tacrolimus Human Intestinal Cells

Joseph G, Biederbiek W, Kahle M, Biederbiek S, Theisohn M, and Klaus W Caffeine binding to plasma proteins varies between 10 and 30% of the total caffeine plasma concentration. Since the free caffeine concentration is responsible for the pharmacological effects the free caffeine plasma concentrations should be used for therapeutic drug monitoring (TDM). Determination of free plasma caffeine is expensive, but saliva concentrations of caffeine may be a good substitute. In the present study we compared total and free plasma with the saliva concentrations of caffeine to prove the usefullness of saliva for the determination of the free plasma concentration of caffeine. Six healthy volunteers (age 22 to 45 years, 2 female, 4 male) received orally 5 mg/kg bodyweight caffeine citrate. Multiple saliva and venous blood samples were taken. Blood samples were centrifuged and plasma und saliva samples were stored at -20°C until analysis. To determine the free caffeine concentrations plasma ultrafiltrate was produced using the Amicon MPS-1 system (YMT membrane). Plasma and ultrafiltrate were extracted with 2.5 ml dichlormethane, the organic phase was evaporated in an Hetovac. Caffeine concentrations were determined by HPLC using hydroxyethyltheophylline as internal standard. The saliva samples were centrifuged for direct HPLC analysis using an external standard procedure. The ratio free caffeine/total caffeine in plasma was 73.5 % ( ± 6.3 %) varying between 67.3 % to 82.5 % intraindividually. Saliva taken later then 180 minutes after caffeine intake had the same caffeine concentration as free caffeine in plasma. Determination of caffeine concentrations in saliva seems to be an easy and cheap way of assessing free caffeine concentrations in serum. Institute of Pharmacology, University of Cologne, Gleueler Str. 24, D-50931 Kfln, Germany

Metabolism in

Bader A 1, Lampen A 1, Bestmann T 1, Streit F 1, Gonschior A.KI., Witte L2, Schiebel H.M. 2, Esselmann HI., Liebsch C 1, Christians UI., Sewing K-Fr 1. lInstitnt ftir Allgemeine Pharmakologie Mediziniscbe Hochschule Hannover, 2Institut for Organische Chemic, Technische Universit~t Braunschweig,Germany The poor oral bioavailability of several drugs has recently drawn the attention to the expression and activity of the cytochrome P450 system (CYP) in the gastrointestinal barrier. In addition to drugs also environmental toxic agents such as the procarcinogen 7,12 dimethylbenz[a]anthracene (DMBA) are potential sobstrates of intestinal CYPs. In vitro models of intestinal biotransformalion including human intestinal cell lines suchas CaCo-2 ceils have previously been used as a tool to study drug transport or expression of cytochrnmes but not for metabolic activity of compounds of pharmacological or toxicological relevance. The aim of this study was to analyze the biotransformalion of the immunosuppressant tacrolimus and the procarcinogen DMBA in intestinal cell lines. The shortage of a fully characterized model required first to screen the presently available cellbased in vitro models of the rat and human intestine for gene and protein expression of CYP. Methods: The cell lines tested included rat duodenal cell line IEC6, rat ileal IEC 18, fetal human HnTu80, fetal human small intestinal FHS, human duodenal HCT8, human colon CaCo-2. CYP 1A1, 1A2, 2C 9/10 and 3A expression was analyzed using Northern and Western blotting. Metabolism of DMBA was assessed by GC-MS and tacrolimos by HPLC/MS. Tacrnlimns metabolites were extracted from human bile and characterized by I-IPLC/MS and NMR. Synthetic DMBA metabolite standards were obtained from NCI Chemical Carcinogen Repository (USA). Results: Only the human intestinal cell line CaCo-2 simultaneously expressed mRNA and protein of CYPIA1 and CYP3A but not CYP 3A4. DMBA metabolites (mass) detected included 7-methylbenz[a]anthracene-12methanol (288), 7,12-dimethylbenz[a]anthracene-dihydrodiol (290,4), 7-methyl-12hydroxymethyl-benz[a]anthracene (272), 7-hydroxymethyl-12-methyl-benz[a]anthracene (272) and possibly the dihydrated form of either of the former two metabolites (274, no standard available). CaCo-2 cells also metabolized tacmlimns. The following metabolites were detected: 13-O-demethyl tacrolimus (789,5) 13,15-O-didemethyl tacrolimns (791.5), 12-hydrnxy tacrolimus (819,5) and demethyl-dihydroxy tacrolimus (821.5). The human intestinal ceil lines FHS, even though CYP 3A was detected, did not metabolize taerolimus. Neither direct incubation with tacrolimus n~ microsomal preparations of the cells were able to generate tacrolimns metabolites. Conclusion: This study demonstrates tacrolimus and DMBA metabolism in human intestinal cells. CYP 1A1 expression in CaCo-2 cells is identical to that in the human duodenum. CaCo-2 ceils, however, are likely to contain a fetal form of CYP3A as CYP 3A was detected by unspecific Westem Blot but not CYP 3A4 by the more specific RNA probe. Nevertheless typical tacrolimus metabolites were generated in CaCo-2 cells. CaCo-2 cells may therefore be a representative tool to study xenobiotic metabolism in the human gut. Supported by SFB 280 project A8.

126

128

C O R R E L A T I O N O F P L A S M A AND S A L I V A C O N C E N T R A T I O N S OF CAFFEINE AFTER ORAL APPLICATION OF CAFFEINE CITRATE SOLUTION

METABOLISM OF THE M A C R O L I D E IMMUNOSUPPRESSANT TACROLIMUS IN THE PIG GUT M U C O S A IN THE USSING CHAMBER

Biederbiek W, Rump AFE, Biederbick S, Kahle M, Joseph G, Theisohn M, and Klaus W . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Therapeutic drug monitoring (TDM) in serum is difficult in premature infants because of the limited blood volume and difficulties in venopuncture. TDM in saliva seems to be an alternative. However, the lack of cooperation in new born infants and the size of the commercially available saliva sampling systems make it difficult to get sufficient amounts of saliva in reproducible quality. Absorbent swaps normally used in ophthalmic surgery were taken to collect saliva in six healthy adults, who received 5 mg/kg bodyweight caffeine-citrate. Saliva and blood samples were taken at 15, 30, 45, 60, 75, 90, 120, 180, 240, 360, 480, and 600 minutes after caffeine-citrate ingestion. The absorbent swaps were centrifuged, the filtrate was collected in vials for direct HPLC analysis using an external standard procedure. Caffeine was not bound to the absorbent swaps. Venous blood samples were centrifuged and the plasma stored at -20°C until analysis. Extraction was done with 2.5 ml dichlormethane, organic phase was evaporated in an Hetovac. Caffeine concentrations in plasma were determined by HPLC using hydroxyethyltheophyllineas internal standard. Saliva samples taken in the first 180 minutes after oral caffeine uptake showed frequently higher caffeine concentrations than plasma. Saliva concentrations taken more than 180 minutes after caffeine uptake showed fixed saliva/plasma ratio. This ratio was constant intraindividually. Saliva samples collected with absorbent swaps from ophthalmic surgery seem to he an excellent device for therapeutic drug monitoring of caffeine. Further studies will focus on the feasibility for TDM in premature infants. Saliva samples should not be taken earlier than 180 minutes after oral administration. Institute of Pharmacology, University of Cologne, Gleueler Str. 24, D-50931 K~31n,Germany

U. Christians i , A. L a m p e n1, A.-K. G o n s c h i o r1, A. Bader 1, I. Hackbarth 1, W. von Engelhardt2, and K.-F. Sewing I The macrolide tacrolimus (FK506), used as immunosuppressant, is a cytochrome P450 (CYP) 3A substrate in the liver. The metabolism of tacrolimus and the transport of its metabolites in the pig gut was studied in the Ussing Chamber. Tacrolimus and its metabolites were quantified using HPLC/mass spectrometry. In the Ussing chamber demethyl, didemethyl, hydroxy and hydroxydemethyl tacrolimus were generated. The concentrations formed were dependent on tacrolimns concentration and incubation time. The metabolite pattern was not different from that after incubation of tacrolimus with human small intestinal or liver microsomes. The metabolite concentrations formed were highest in the duodenum and declined in the order duodenum > jejunum > ileum > colon > stomach. Since tacrolimus metabolism was inhibited by the specific CYP3A inhibitors troleandomycin and ketoconazole, it was concluded that these enzymes are involved in intestinal metabolism of tacrolimus. Tacrolimus metabolites reentered the mucosal chamber ( > 90 % ) and went through the mucosal preparation into the serosal chamber. It is concluded that tacrolimus is metabolized in the intestine, that the metabolites are able to go back into the gut lumen as well as into the portal vein and that small intestinal metabolism and transport is a potential determinant of the oral bioavailability of tacrolimus.

Supported by DFG grant SFB280, project A8. IInstitut fiir All~emeine Pharmakologie, Medizinische Hochschule Hannover, and ~Physiologisches Institut, Tier~irztliche Hochschule Hannover, Hannover, Germany

R 33 129

131

A N O V E L ras-RELATED G T P - B I N D I N G P R O T E I N (rab26) W I T H UNIQUE STRUCTURAL FEATURES AND PREDOMINANT EXPRESSION IN TESTIS A. Brauers, A. Sch0rrnann, S. MafAmann, P. Miihl-Zfirbes a n d H . G . Joost

RELATION BETWEEN BINDING OF IMIDAZOLINE/GUANIDINIUM C O M P O U N D S AND INHIBITION OF KATp-CHANNELS IN INSULIN-SECRETING CELLS. C. Herrmann,P. Ratzka, C. Dickel, I. Rustenbeckand A. Hasselblatt.

A novel rag-related GTP-binding protein with GTPase activity was cloned by a PCR-based approach with degenerate oligonucleotides matching the domains PM1 and PM3 o f the ARF-subfamily. One PCR-product, which was isolated in addition to 6 ARF and 4 ARF-like isotypes, exhibited a sequence unrelated to ARF, and was used as a probe to screen a rat fat cell and brain library. A full length eDNA was isolated comprising an open reading frame of 221 amino acids. A highly homologous human eDNA sequence was obtained by PCR from testis (identity of nucleotide sequence 91.3%, and of deduced amino acid sequence 96.4%). The sequence contains all motifs presumably involved in GTP binding and hydrolysis. Since the highest homology o f the sequence wag with members of the tab-family (sequence identity 31-33%), the gene was designated rab26. However, the homology to other members o f the tab-family is mainly restricted to the six GTP-binding domains. In addition, tab26 exhibited several structural features which are unusual for the tab-family. Northern blots of RNA from various rat tissues revealed a 1.2 kb transcript which was found exclusively in testis, and a 2 kb transcript which was detected in lower levels in heart, sceletal muscle and adipocytes. A recombinant fusion protein was expressed in E. cell, partially purified, and specific binding o f [35S]GTP[S] and GTPase activity were assayed. Specific GTP binding approached an equilibrium within 15 minutes. Furthermore, the protein showed an intrinsic GTPase activity with a kinetic constant of 0.085 rain -1. Our data indicate that tab26 is a novel GTPase with a unique structure, presumably exerting a specific, possibly testis-related function. Institut fi~r Pharmakologie und Toxikologie, Medizinische Fakult~it der RWTH-Aachen, Wendlingweg 2, D-52057 Aachen, Germany

The enhancement of glucose-induced insulin secretion by imidazoline compounds is related to an inhibitory action of these agents on KATP channel activity in pancreatic B-cells. However, it is not clear, which binding site mediates the imidazoline-induced closure of KATP channels. We therefore characterized the effects of four representative imidazoline or guanidinium compounds on KATP channel activity in pancretic B-cells and compared this action with binding to membranes from insulinoma cells (.HIT-cells). Binding experiments were performed by displacing ~[H]-clonidine with imidazoline- or guanidinium-compounds in the presence of 100 I.tM norepinephrine to mask ~-receptors. The competition curves indicated the presence of two imidazoline/guanidinium binding sites. Binding to the high affinity, low capacity site had Kis of 2.9 nM, 17.3 nM and 32.8 nM for guanabenz, idazoxan and clonidine, respectively. Binding to the low affinity, high capacity site displayed a different order of affinity with Ks of 0,6 I~M, 4.9 ~tM and 39.6 IxM for guanabenz, clonidine and idazoxane. Amiloride d splaced c enid ne only from one binding site with an IC50 of 5.2 I.tM. In patch-clamp experiments, using the inside,out configuration, guanabenz inhibited KATP channel activity with an IC50 of around 2 I.tM and caused complete, but reversible closure at 10 ~tM. Clonidine inhibited channel activity down to 32% of control value with an IC50 of 0.9 ~tM. Idazoxan in micromolar concentrations also inhibited KATP channel activity, however, there was no direct relation between concentration and channel closure. Amiloride was ineffective, only at concentrations above 100 ~tM a marginal effect could be seen. These data suggest that binding to the low affinity site rather than to the high affinity site is related to the effect of these compounds on KATP channel activity. Instituteof Pharmacology,Universityof GSttingen,D-37075Germany

130

132

L A C K OF CORRELATION BETWEEN MDR-1 EXPRESSION AND VOLUMEACTIVATION OF CHLORIDE-CURRENTS IN RAT COLON CANCER CELLS.

SPERMINE INHIBITS TRANSITIONAL CA 2+ RELEASE FROM ISOLATED RAT LIVER MITOCHONDRIA. I. Rustenbeck,D. L0ptien, H. Reiter and S. Lenzen*

C. De Greefz, S. van der Heyden~, F. Viana2, Raeyrnaekers2, G. Droogmans2, B. Nilius :.

].

Eggermont~, E. De Bruijn ~, L.

It has been proposed that P-glycoprotein (P-gp), the product of the human MDR-1 gene, may function not only as a drag transporter but also, depending on the conditions, as a volume-activated Cl--channel. However, P-gp expression and amplitude of swelling-activated Cl'--eurrents were not correlated in a number of cell types, whereas in others a close con'elation between Cl-chatmel activity and P-gp expression has been observed. We studied the correlation between expression of the mdr-i genes (a and b) at the mRNA and protein level and volume-activation of chloride-channels in rat colon cancer CC531 cells by means of RT-PCR, Western blotting and patch clamp, respectively. Three phenotypically different MDR cell lines were used: CC531 ~'+/', CC531 '~'++ and CC531 '~'-. At the mRNA level, the parental cell line CC531 '~'+/showed significantly less mdr-la expression compared to CC531 '~'+÷, the drug resistant cell line induced from the parental CC531 cells by growth in the presence of colchicine. CC531 ~ " was a spontaneous revertant of the drug resistant cell line to a drug sensitive one, but with a maintained level of mdr-la mRNA. In none of the three cell lines, mdr-lb could be detected. At the protein level, a difference in mdr-1 expression between CC531 ''a'÷~, CC531 '~'- and CC531 ~'++ was observed. Although the amount of mdr-la mRNA detected in CC531 ~ was comparable to that found in CC531 ~d'++, the amount of mdr-I encoded protein in CC531 ~ " was remarkably reduced. In all three cell types, cell swelling activated chloride-currents which could be blocked by NPPB. Chloride-currents measured at the K + reversal potential of -90mV were not significantly different (~86.1 ± 19.1pA/pF, n = 5 in CC531 ~r+~, -59.5±13.1pA/pF, n=6 in CC531 '~r÷+ and -68.1±15.3pA/pF, n=7 in CC531~). These data show a discrepancy between mdr-1 expression (both at the RNA and at the protein level) and channel activity and are difficult to reconcile with the hypothesis that mdr-1 encoded P-gp, in addition to its established involvement in drug transport, would mediated volume-activated Cl'-currents. These findings make it unlikely that mdr-1 encoded P-gp is a volume-activated Cl--channel or a regulator of this channel. 1Lab Cancer Res & Clin One, Antwerp University, Universiteitsplein, 1 (T3), 2610 Wilrijk, Belgium 2KULeuven, Dep of Physiology, Campus Gasthuisbcrg, 3000 Leuven, Belgium

The membrane permeability transition of the mitochondria is characterized by a sudden release of ions and low molecular weight compounds from the matrix space, followed by colloid-osmotic swelling of the mitochondria. This event is discussed as a critical step in toxic cell death. The poly.amine, spermine, a physiological component of the cytoplasm, inh~bits ca 2+ release associated with the permeability transition and enhances the Ca2+ accumulation capacity of isolated rat liver mitochondria. While an inhibition of transitional Ca2+ release is also exerted by ADP or Cyclosporin A these agents donot enhance the m tochondrial Ca2+ accumulation. On the other hand, several polyamine analogues like poly-L-lysine or gentam c n, enhance mitochondrial Ca2+ accumulation, but do not protect against transitional Ca2+ release. Apparently, both effects of spermine are related to binding of this polycation to the outer face of the inner mitochondrial membrane. The association curve of spermine binding to intact isolated mitochondria is biphasic, indicating a competition between spermine and other cations. By displacing Ca2+ and K+ ions from anionic membrane sites spermine may act as a protective agent to preserve mitochondrial functional integrity under conditions of ischemic or toxic stress. Institutfor Pharmakologieund Toxikologie,Universit&tG0ttingen, D-37075G0ttingen Institutf0r KlinischeBiochemie,MedizinischeHochschuleHannover, D-30623Hannover

R 34

133

135

PROTEIN KINASE C EXERTS A POSITIVE FEEDFORWARD CONTROL OF METHACHOLINE- AND HISTAMINE-INDUCED Ca2+ SIGNALLING IN ISOLATED AIRWAY SMOOTH MUSCLE CELLS. BH. Hoitinz, R. Kuipers, C.R.S. Elzinga, J. Zaagsma, and H. Meurs. Activation of protein kinase C (PKC) by phorbol esters has previously been shown to inhibit agonist-induced changes in intracellular Ca2+ concentration ([Ca2+]i) in various cell types, including airway smooth muscle cells, suggesting that PKC plays a role in the negative feedback regulation of agonist-induced Ca2+ signalling. However, since various isoenzymes of PKC have been identified in mammalian tissue, with distinct sensitivities to Ca2+, lipid activators and phorbol esters, the significance of the latter observation with respect to agonist-induced PKC activation is still uncertain. As a more direct approach to assess the role of PKC in agonist-induced Ca2+ signalling in airway smooth muscle, we investigated the effect of a recently available potent and highly specific, but isoenzyme-unselective, PKC inhibitor, the aminoalkyl bisindolylmaleimJdeGF 109203X, on methacholine (MeCh)- and histamine (His)-induced [Ca2+]ichanges in isolated bovine tracheal smooth muscle cells. Both contractile agonists produced a (concentration-dependent) rapid and transient increase in [Ca2+]i, followed by a steady state [Ca:+]i that was substantially higher than resting level. In the presence of 10 ~tM GF 109203X, the peak-rise in Ca2+ in response to 1, 10, and 100 ~tM MeCh and 100 gM His was slguificantly reduced to 48±3, 75~-6, 644-4, and 604-8 % of control, respectively, while the steady state rise in Ca2+ was reduced to 744-3, 844-2, 934-3, and 524-6 % of control, respectively (n=5; P<0.05 for all observations). In the presence of 5 mM EGTA - which abolishes the steady-state phase of Ca2+ influx - GF 109203X inhibited the agunist-induced Caz+ transients to a similar extent (37:e3, 65~:2, 72:t:3, and 52~-6 % of control, respectively; n=5; P<0.001 for all observations). The results for the first time indicate that PKC activation in smooth muscle cells by contractile agonists exerts a positive, feedforward control of Ca2+mobilization and influx induced by these agonists.

STAUROSPORINE AND THE MORE SPECIFIC PKC INHIBITOR GF109203X INDUCE CA2+ RELEASE FROM INTERNALSTORES AND CA2+ ENTRY. H. Sipma, L. van der Zee, A. den Hertog and A. Nelemans.

Department of Medicinal Chemistry and Molecular Pharmacology, University of Groningen, Ant. Deusinglaan 2, 9713 AW Groningen, The Netherlands.

We investigated the effect of the specific protein kinase C (PKC) inhibitor GF109203X and the more aspeciflc protein kinase inhibitor staurosporine on basal and agonist stimulated Ca 2÷ metabolism in DDT 1 MF-2 smooth muscle cells. Intracellular Ca 2+ concentrations ([Ca2+]) were measured using the fluorescent Ca 2+ probe Indo-1. The maximal increase in [Ca2+]i, but not the sustained elevation of [Ca2+]i after stimulation of cells with histamine (100 /~M), was reduced (60 %) by pretreatment of cells with GF109203X or staurosporine for 45 min. In the absence of extracellular Ca2+ or after blockade of plasmamembrane Ca 2+ channels with La3+ (50/zM), GF109203X and staurosporine caused an inhibition (60 %) of histamine induced Ca2+ release. These protein kinase inhibitors did not affect the histamine induced formation of inositol 1,4,5-trisphosphate, suggesting that activation of the phospholipase C signalling pathway was not reduced. Exposure of cells to GF109203X (5 /~M) gave rise to a slight increase in [Ca2+]~, whereas the effect of staurosporine (100 nM) on [Ca~+]i could not be deducted due to autofluoresence of staurosporine. The passive release of Ca2+ evoked by thapsigargin, an inhibitor of intracellular store Ca2+-ATPase, was likewise strongly inhibited (65 %) by GF109203X and staurosporine, which shows that these compounds depleted these stores. This latter effect was most likely not dependent on PKC, since it was also observed after exposure of cells to phorbol 12-myristate 13-acetate (1 ttM) for 48 h, a treatment known to evoke downregulation of most PKC isozymes. Pretreatment of cells with GF109203X and staurosporine enhanced Ca 2+ entry as measured by accumulation of extracellularly added nSca2+, possibly by the mechanism of store dependent Ca2+ entry. Department of Clinical Pharmacology, State University of Groningen, 9713 BZ, Groningen, The Netherlands.

134

136

LOE 908 (R,S-(3,4-DIHYDRO 6,7-DIMETHOXYISOQUINOLINE-1YL)-2-PH ENYL-N,N-di-[2-(2,3,4-TRIMETI-IOXYPRENYL)ETHYL]ACETAMIDE) INHIBITS Ca 2+ RELEASE-ACTIVATED Ca2+-ENTRY IN VASCULAR ENDOTHELIAL CELLS. A. Eneabo, R. Schmidt, W.R. Kukovetz and K. Groschner

CLONING AND CHARACTERIZATION OF A NOVEL PROTEIN KINASE RELATED TO 5'-AMP ACTIVATED PROTEIN KINASE W. Becket, J. Heukelbach, H. Kentrup, H.G. Joost

Depletion of intracellular Ca 2+ stores leads to activation of Ca 2+ permeable ion channels in the plasma membrane of endothelial cells. We tested LOE 908 a blocker of non-selective cation channels for its ability to affect the Ca 2+ release-activated Ca 2+ entry pathway in endothelial cells cultured from human umbilical vein. Depletion of intracellular Ca 2+ stores was initiated with ionomycin (30-100 nM), and changes in intracellular free Ca 2+ concentration ([Ca2+]i) were monitored with Fura-2. In parallel, ionomycin-induced membrane currents were recorded using the perforated-patch technique. Ca 2+ release-activated Ca 2+ entry was measured as the increase in [Ca2+]i which was observed upon restoration of extracellular Ca 2+ levels following ionomycin-induced depletion of Ca 2+ stores in nominally Ca2+-free solution. LOE 908 at concentrations > l~tM significantly suppressed the increase in [Ca2+]i induced by elevation of extraceliular Ca 2+. Membrane currents were recorded in an extracellular solution containing 10 mM Ca 2+ supplemented with 65 mM TEA and 0.3 mM DIDS to block Ca2+-activated K + and CI- currents. Ionomycin induced a membrane current which reversed at about +10 mV and exhibited sensitivity to inhibition by La 3+ (50 taM). This ionomycin-induced current was reversibly blocked by 10 taM LOE 908. Our results suggest that the isoquinoline derivative LOE 908 blocks Ca 2+ permeable ion channels which are activated by depletion of Ca 2+ stores in vascular endothelial cells. Department of Pharmacology and Toxicology, University of Graz, Universit~_tsplatz 2, A-8010 Graz, Austria. Supported by the Austrian Research Funds (P10185)

In eukaryots, a vast number of different protein kinases is engaged in the regulatory phosphorylation of proteins. The strong sequence conservation of some regions in the catalytic domain of protein kinases allowed us to amplify partial cDNA's of protein kinases by polymerase chain reaction (PCR) with degenerate primers. One of the cloned PCR products was used to isolate a cDNA clone of a novel protein kinase (PSK100) from a rat fat cell cDNA library. The encoded polypeptide (746 amino acids, Mr=81,627 ) comprises a consensus kinase domain of 270 amino acids located at the N-terminus. A recombinant glutathion-S-transferase-PSK100 fusion protein catalysed autophosphorylation as well as phosphorylation of histone, confirming that PSK100 has indeed protein kinase activity. By Northern blot hybridization, a 5 kb mRNA was detected in brain, heart, fat cells, intestine, testis, ovary, adrenal gland and thymus. Sequence comparisons classify PSK100 into the SNF1 family of protein kinases. SNF1 from yeast and its mammalian homologue 5'-AMP activated protein kinase (AMPK) are involved in the regulation of cellular lipid and carbohydrate metabolism. Outside the catalytic domain, PSK100 shows no extended similarity with SNFl-related protein kinases or any other protein, but harbours several characteristic structural features. These include two PEST sequences, a nuclear localization signal that is preceded by a stretch of 10 consecutive acidic residues, and a glycinerich region. Furthermore, we detected a small region of homology (3035 amino acids) C-terminal of the catalytic domain that is also present in other SNFl-related protein kinases, including AMPK. These data suggest that the C-terminal part of PSK100 may represent a new type of regulatory domain of protein kinases. Institut f/ir Pharmakologie und Toxikologie, Medizinische Fakultat der RWTH Aachen, Wendlingweg 2, D-52057 Aachen, Germany

R 35 137

139

IDENTIFICATION OF DOMAINS AND AMINO ACIDS OF THE GLUCOSE TRANSPORTER GLUT4 WHICH ARE INVOLVED IN FUNCTION AND LIGAND BINDING (IAPS-FORSKOLIN AND CYTOCHALASIN B). S. Wandel, A. Sehtirmann, A. Buchs, A. Powers and H.G. Joost Cytochalasin B (KD approximately 10-TMin GLUT4, 10`6or higher in GLUT2) and forskolin (KD 200 nM in GLUT4, not detectable in GLUT2) are considered as specific ligands of mammalian glucose transporters. In order to analyse the domains or amino acids of the GLUT4 that are involved in ligand binding of cytochalasin B and forskolin, chimerae of the human GLUT2 and GLUT4 were constructed by site-directed mutagenesis and PCR. Furthermore, 6 conserved tyrosine residues of GLUT4 were altered to phenylalanine by site-directed mutagenesis. Constructs and wild-type glucose transporters were subcloned into the mammalian expression vector driven by the cytomegalovirus promotor (pCMV), and were transiently expressed in COS-7 cells. Cells were homogenized after 48 h, and plasma membranes, high- and low-density microsomes were isolated by differential centrifugation. Glucose transport activity was assayed after solubilization and reconstitution into lecithin liposomes. Binding of forskolin was assessed by photolysis in the presence of [l:zsI]IAPS-forskolin, electrophoretic separation and autoradiography. Cytochalasin B binding was assayed by incubation of plasma membranes with tritiated cytochalasin B. Exchange of the Cterminal loop in GLUT4 produced a chimera with glucose transport activity, IAPS-forskolin and cytochalasin B binding similar to that of GLUT4. In contrast, exchange of 6 terminal helices reduced the binding of eytochalasin B and IAPSforskolin, but failed to affect transport activity. Exchange of tyrosine 143 reduced both transport activity and cytochalasin B binding but failed to affect IAPSforskolin binding. Exchange of tyrosine 292 produced a large decrease of cytochalasin B binding, but failed to affect IAPS-forskolin binding or glucose transport activity. All other tyrosine residues exhibited normal binding and activity. These data suggest that contact sites for inhibitory ligands are located in the C-terminal half of the transporter. In addition, contact sites for cytochalasin B are located in helix 4 and 7 but differ from those of IAPS-forskolin.

INTERACTION OF GABA, NPY AND SOMATOSTATIN WITH INOSITOLPHOSPHATE FORMATION 1N RAT CEREBRAL CORTEX A. Jussofie I, M.C. Michel 2

Institut fiir Pharmakologie und Toxikologie, Medizinische Fakultftt der RWTH Aachen, Wendlingweg 2, 52057 Aachen, Germany, and Vanderbilt University, Division of Endocrinology, Nashville, TN, USA.

Interneurons in rat cerebral cortex corelease GABA, NPY and somatostatin which typically act through GABAA receptors (an intrinsic ion channel) and GABAB, NPY and somatostatin receptors which couple via G~-like proteins. We have investigated whether GABA, NPY and somatostatin alone or in combination can affect inositol phosphate formation and or potentiate the folTnation via c~-adrenoceptors or muscarinic acetylcholine receptors in rat cerebral cortex slices. Inositol phosphate formation was determined in cortical slices (350 x 350 gm) prelabeled with [3H]-myo-inositol during a 45 rain incubation in the absence or presence of agonists; total [3H]inositol phosphates were separated by Dowex AG I-X8 column chromatography. Neither NPY (1 p-M), somatostatin (1 gM), GABA (100 p-M), the GABAA agonist muscimol (100 p-M) or the GABAB agonist baclofen (100 ~tM) significantly affected inositol phosphate formation, and their combination was also ineffective. In contrast inositol phosphate formation was enhanced by the musearinic agonist carbachol (1 mM) or the ~l-adrenoceptor agonist noradrenaline (10 gM), but this was not affected by NPY or somatostatin. In contrast GABA significantly potentiated carbachol- or noradrenalineinduced inositol phosphate formation but this was not mimicked by muscimol or baclofen. On the other hand, the GABAA antagonist bicucullin (100 p.M) inhibited the potentiating effect of GABA whereas the GABA B antagonist phaelofen (100 p-M) had only little if any inhibiting effects. We conclude that the cotransmitters GABA, NPY and somatostatin do not directly affect inositol phosphate formation. GABA (but not NPY and somatostatin) potentiates inositol phosphate formation in rat cerebral cortex via an GABAA receptor but this effect is not mimicked by muscimol. ~Dept. of Physiological Chemistry, University of Essen ~md Dept. of Medicine, University of Essen, 45122 Essen, Germany

138

140

MUTATIONS OF TRANSCRIPTION FACTOR PIT-1 IMPAIR GENE EXPRESSION AND PROLIFERATION OF PITUITARY GH3 CELLS H. J. Steinfelder, J. Weichert, F. E.Wondisford°

COMPARISON OF THE SIGNALING PATHWAYS STIMULATING GLUCOSE TRANSPORT IN RESPONSE TO INSULIN AND PDGF IN 3T3-L1 PREADIPOCYTES C.Huppertz and H.G. Joost

Pit-t is a cell specific transcription factor found only in lacto-, somato- and thyrntropic cells of the anterior pituitary that is involved in basal and TRH regulated expression ofprolaetin, GH and TSH-subunlt genes. Dwarf mice models revealed that mutations in the pit-1 gene resulted in decreased levels of GI-I, prolactin and TSH as well as in a reduction of lacto-, somato- and thyrotropic cells. In the present study we tried to evaluate the effects of single amino acid mutations of Pit-I on proliferation of laetosomatotropic GH3 cells as well as on prolactin reporter gene expression in these cells. Therefore, mutations from threonine to alanine at position 219 or 220 were performed and respective eDNAs cloned into expression vectors driven by the CMV promoter. These threonines are phosphorylated by protein kinases A and C. In order to study the role of Pit-1 on GH3 cell proliferation we stably transfected cells with Pit-I mutants 219 and 220. These chimeric cells expressed endogenous Pit-1 as well the respective mutant proteins and were tested for their basal growth rate and the proliferative effect of TRH. Both mutant lines grew at slower rates than cells transfeeted with a wild type Pit-I eDNA. On the other hand, TRH stimulated growth in the mutant lines at least as good as in control cells, Transient eotransfection experiments of these mutant plasmids or a wild type Pit-1 expression vector together with a reporter construct coding for luciferase under the control of the prolactin promoter revealed that basal luciferase expression was not significantly impaired by these phosphorylation defect mutations but the response towards TRH stimulation was reduced by 35 - 40 %. Therefore, it can be concluded that the loss of threonines 219 or 220 and thus disabled phosphorylation of Pit-I by protein kinases A and C reduces stimulation of gene expression as well as basal proliferation of GH3 cells. Institute of Pharmacologyand Toxicology, Robert-Koch-Str. 40, D-37075 G~ttingen, *ThyroidUnit, Beth Israel Hospital,Boston,USA

Murine 3T3-L1 fibroblasts differentiate in vitro to an insulin-sensitive, adipocyte-like phenotype. The stimulation of the insulin receptor and its intrinsic tyrosine kinase activity leads to the increase of glucose uptake. The same response of glucose transport is produced by the stimulation of the IGF1 receptor, the closest relative of the insulin receptor. In order to assess the specificity of other tyrosine kinase-controlled pathways, we studied the differential expression of the PDGF receptor and the effects of PDGF on glucose transport in 3T3-L1 ceils before and after differentiation. Levels of mRNA of the PDGF receptor decreased markedly during differentiation. Similarly, the tyrosinephosphorytation of the PDGF receptor detected by immunoprecipitation with a phosphotyrosine antiserum was markedly reduced upon differentiation. In the confluent, undifferentiated cells that exhibit a moderate insulin-sensitivity (6-fold stimulation), PDGF exerted an insulin-like stimulation of 2-deoxyglucose uptake (ECs0 0,5 nm). In contrast, PDGF produced only 17% of the effect of insulin (18-fold) in the differentiated cells. In the immunoprecipitates of the differentiated cells a marked tyrosine-phosphorylation of IRS-1 was detected after stimulation with insulin but not with PDGF. In the fibroblasts a protein of 160 kDa was moderately tyrosine-phosphorylated after stimulation with PDGF but not with insulin. The stimulatory effects of PDGF and insulin on 2-deoxyglucose uptake appeared both mediated by phosphatidylinositol 3-kinase (PI3-kinase), because they were inhibited by the specific inhibitor Wortmannin. In conclusion, the data suggest that the effects of insulin and PDGF in 3T3-L1 cells are mediated by different pathways that both give rise to PI3-kinase activation and glucose transport stimulation. Institut ftir Pharmakologie und Toxikologie, Medizinische Fakult~t der RWTH Aachen, Wendlingweg 2, D-52057 Aachen, Germany.

R 36 141 P L A T E L E T - A C T I V A T I N G - F A C T O R INDUCES [Ca2+]iRISE AND SUPEROXIDE ANION GENERATION IN UMBILICAL-CORD-BLOOD DERIVED EOSINOPHILS MATURING IN VITRO

143 AIRWAY NYPERRESPONSIVENESS INDUCED BY 13-HYDROXY. OCTADECADIENOIC ACID (13-HODE) IS MEDIATED BY SENSORY NEUROPEPTIDES

D.M. Zardini, P. Heuschling, J.-L. Bueb, E.J. Tschirhart.

F. Engels, A.H. van Houwelingen,T.L. Buckley, M.J. van de Velde, P.A.J. Henricks, F.P. Nijkamp.

Eosinophils have been implicated in the pathogenesis of various allergic and non allergic diseases such as bronchial asthma, atopic dermatitis and parasitosis. In these disorders, eosinophils are considered to be important effector cells which can cause local damage to infiltrated tissue via the release of reactive oxygen species (O2") and a number of cytotoxic protein~ stored in their cytoplasmic granules. Platelet-activating factor (PAF), a potent inflammatory mediator, has also been shown to be implicated in the pathogenesis of bronchial asthma. Hence, the aim of the present study was to clarify the possible relationship between PAF and eosinophils in view of their involvement in various allergic reactions. Because eosinophils are a minority constituent of the peripheral blood cells, a method was developed that permitted isolation of large numbers of cells (about 2xl06/ml cord blood). In the presence of interleukin-3 and -5 (10 ng/ml each) eosinophil precursors from human umbilical cord blood mononuclear cells, were regularly differentiated into mature eosinophil-like cells, expressing normal morphology and cyanide-resistant peroxidase. 02" production and [Ca2+]i were measured in these in vitro differentiated cells after PAF stimulation, using 2 fluorescent probes: fura-2AM (2.5 gM) was used to measure [Ca2+] i variations, and the oxidative burst indicator dihydrorhodamine-123 (llxM) was used to assess 02" production. We report here, that PAF (0.1 pM - 1 gM) induced a concentration dependent increase in [Ca2+]i with a maximun of 485 + 50 nM (n=3) at 100 nM. Furthermore, we found that PAF (10 n M - 3 p.M) induced a concentration-dependent production of 02" with maximum effect at 1 IxM (n=4) and an half maximum effect at around 120 nM (n=2). Considering the studied parameters, these results suggest: that human cord-derived eosinopbils demonstrate functional characteristics similar to human peripheral blood eosinophils after activation and thus, may serve as a model for other eosinophil biological activity studies that a sustained elevation in [Ca2+]i (a basic signalling event in many secretory cells) is presumably involved in functional responses, like 02' production, in eosinophils.

The linoleic acid metabolite 13-HODE induces airway hyperresponsiveness in guinea pigs in vitro as well as in vivo (Eur. J. Pharmacol. 1991; 197: 233-234). In the present experiments we studied the possible involvement of sensory neuropeptides in this effect of 13-HODE. Incubation of guinea pig isolated tracheal rings with 13-HODE (1 ~.M) for 30 rain induced a 34% increase of the maximal contraction to histamine. Capsalcin treatment of the tracheal rings, which resulted in initial release of neuropeptides from sensory nerves and subsequently led to total depletion of the sensory neuropepfides, fully inhibited the effect of 13-HODE. Further experiments were performed to try and implicate the tachykinins (e.g. Substance P and neurokinin A) in the 13-HODE-induced hyperresponsiveness. Use of selective tachykinin NK 1 and NK2 receptor antagonists revealed that 13-HODE-induced tracheal hyperresponsiveness was mediated by activation of NK1 receptors. We conclude from these experiments that the lipid mediator 13-HODE may affect sensory nerves, leading to the release of neuropeptides which subsequently activate NK1 receptors. This cascade of events results in airway hyperresponsiveness to histamine. Department of Pharmacology, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, P.O. Box 80082, 3508 TB Utrecht, The Netherlands.

Neuroimmunologie & Inflammation, Centre de Recherche Public-Sant6, 120 Route d'Arlon. L-1150 Luxembourg.

142

144

15-DEOXYSPERGUALIN (DSG) IS EFFECTIVE IN THE TREATMENT OF DEXTRAN SULFATE INDUCED COLITIS. J.K. Lindner, U. Nicolay DSG is a novel immunomodulatory drug derived from the antitumor antibiotic spergualin produced by Bacillus laterosporus. In animal models DSG showed efficacy in the treatment of several autoimmune diseases and transplant rejection. DSG was tested for efficacy in the model of acute dextran sulfate induced colitis (DSS). The colitis was induced by the method of Okayasu et al. (Gastroenterology, 98:964-702, 1990). Female CBA mice were applied 5% DSS in drinking water for 9 days. The severity of the colitis was determined by the measurement of the occurrence of occult blood in stool, shortening and histological examination of the large bowel. DSG was applied i.p. beginning at day 0 of the application of DSS until day 9. All DSG doses -> 7.5 mg/kg showed a significant increase of the length of large bowel. Pooled mean differences ranged between 0.52 and 1.18 cm, and the extend of occult blood in faeces was significantly reduced in these animals, too. With respect to the histological changes the 3 DSGdoses of 10, 12.5 and 15 mg/kg displayed significantly better values than the positive controls pertaining to them.

MUCOSAL MAST CELL ACTIVATION AND EARLY VASCULAR PERMEABILITY CHANGES IN A DTH REACTION IN THE RAT SMALL INTESTINE

Behringwerke AG, Dept. of Immunoregulation, P.O. Box 11 40, 35001 Marburg, Germany

Aletta D. Kraneveld, Dicky van Heuven-Nolsen, Thea Muis, Andries Sj. Koster* and Frans P. Nijkamp In this study, the role of mucosal mast cells (MMC) in the small intestinal DTH (delayed type of hypersensitivity) reaction was investigated. Male rats were skin sensitized with dinitrefluorobenzene (DNFB) and challenged intragastrically with dinitrobenzene sulfonic acid (DNBS). Depletion (short-term dexamethasone treatment) and stabilization (doxantrazole treatment) of MMC before and at time of challenge were very effective in reducing edema formation, 48 hours after the challenge. This suggested that the MMC plays a role in initiating the DTH reaction. MMC activation and vascular leakage during the initiating phase of the DTH reaction (0-60 min after challenge) was investigated by measuring release of rat mast cell protease II (RMCP-II) and Evans Blue tissue accumulation, respectively. MMC stabilization was effected by doxantrazole treatment. In addition, the influence of sensory nerves was studied by means of neonatal capsaicin-induced depletion of sensory neuropeptides. In the initiating phase, a significant increase in vascular permeability was found in DNFB-sensitized rats, associated with RMCPII release. Doxantrazole treatment resulted in a significant reduction of vascular leakage and RMCP-II release. Neonatal capsaicin pretreatment abolished the DTH-induced early vascular response as well as MMC activation. The findings of this study are consistent with an important role of the MMC in the initiating phase of the DNFB-induced DTH reaction in the small intestine of the rat. The results with neuropeptide depletion indicate that MMC activation early after the challenge is under sensory nervous control. ADK was supported by a Glaxo (Ware, UK) PhD. scholarship. *presenting author: A.Sj. Koster, Department of Pharmacology, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, P.O. Box 80082, 3508 TB Utrecht, The Netherlands

R 37 145

147

MAST CELLS CONTRIBUTE TO THE INCREASED VASCULAR PERMEABILITY ASSOCIATED WITH INTESTINAL DELAYED-TYPE HYPERSENSITIVITY RESPONSE IN THE MOUSE A.D. Kraneveld, A. Chandrasekar*, A.Sj. Koster, B.K. Wershil*, F.P. Nijkamp.

CHANGESOF GUINEAPIG TRACHEALSMOOTHMUSCLECONTRACTION H.J.M. van Hoof*t, H-P. Voss*, L. van Breet and A. Bast*

The role of mast ceils (MC.s) in DTH responses has been controversial. Several studies have shown that MCs are not essential for the full expression of cutaneous DTH responses and a recent report did not detect a significant contribution of MCs in TNBS-induced colitis. We examined the role of MCs in intestinal DTH in the mouse using a model which has been shown to produce an increase in vascular permeability and the release of MC mediators in the rat (Gastro. 106:A715). MC-deficient WBB6F1-W/Wv, MC-defieient WCB6F1-SI/SI d mice and the respective normal (+/+) littermate mice received an epicutaneous application of either dinitrofluorobenzene (25 gg) or vehicle (control) on day 0 and 1. On day 5, the mice were challenged orally with dinitrobenzene sulphonic acid (30 p_g). A cutaneous DTH response was elicited in the right ear by application of DNFB (40 ttg) while the left ear received vehicle. Cutaneous DTH reactions were assessed by measurement of ear thickness and changes in vascular permeability in the small intestine were assessed by measurement of Evan's blue extravasation at 24 hrs, as previously described (Br. J. Pharmacol. 114:1483). Normal (+/+) mice developed a significant increase in Evan's blue extravasation at sites of intestinal DTH compared with sites of control reactions (WBB6F1+/+: 0.41±0.05 vs 0.19±0.03; WCB6FI-+/+: 0.49±0.03 vs 0.22±0.01 ml plasma/g dry weight, DTH vs control, respectively, p<0.01). In contrast, no difference was detected between DTH and control reactions in MC-deficient mice (WBB6F11W/V¢~: 0.22±0.03 vs 0.20±0.02; WCB6F1-+/+:0.29±0.01 vs 0.27±0.02 ml plasma/g dry weight, DTH vs control respectively). As previously reported, there was no difference in ear swelling associated with cutaneous DTH in the normal (+/+) mice compared to the respective MC-deficient mice. We next examined the intestinal DTH response in MC-deficient W/Wv mice which had their MCdeficiency corrected by transplantation of bone marrow (BM) derived from the +/+ littermates. BM transplanted W / W v mice exhibited a significant increase in the extravasation of Evan's blue dye during intestinal DTH compared to controls. These data confirm previous observations that MCs do not play a significant role in cutaneous DTH reactions. However, our findings suggest that MCs contribute significantly to changes in vascular permeability associated with small intestinal DTH responses. Dept. of Pharmacology, Utrecht Institute for Pharmaceutical Sci.ences, Utrecht University, PO Box 80.082, 3508 TB Utrecht, The Netherlands. Div. of Exp. Pathology, Beth Israel Heap., Harvard Medical School, Boston, MA, USA.

THE ROLE OF CYCLO-OXYGENASE PRODUCTS IN OZONE-INDUCED

We investigated the role of cyclo-oxygenase products in the ozone-induced changes of guinea pig tracheal smooth muscle contractions after stimulation with methacholine and histamine. Male Dunkin-Hartley guinea-pigs were exposed to 3 ppm ozone for two hours. Tracheas were removed within 30 rain. after exposure and changes in tracheal smooth muscle tension were recorded isometrically. After exposure to ozone an increase in maximal contraction (i.e. hyperreactivity) was observed after stimulation with the muscarinic agonist methacholine (0.632 + 0.075 g vs. 0.900 + 0.088 g). After stimulation with the histaminergic agonist histamine a decrease in maximal contraction (i.e. hyporeactivity) was found after ozone-exposure (0.884 + 0.062 g vs. 0.547 + 0.063 g). To study the role of cyclooxygenase products in these contractions we repeated the experiments after addition of 10 gM indomethacin to the buffer. In the presence of this cyclooxygenase inhibitor no differences were observed in maximal contraction levels between the control and ozone-exposed situation after both methacholine and histamine stimulation (methacholine 1.454 + 0.069 g vs. 1.603 + 0.119 g; histamine 1.098 + 0.078 g vs, 1.303 + 0.098 g). These results suggest the involvement of cyclo-oxygenase products in ozoneinduced changes of guinea-pig tracheal smooth muscle contraction and to confirm this we are currently investigating which cyclo-oxygenase products are formed in control situations and after exposure to 3 ppm ozone for two hours. *Leiden/Amsterdam Center for Drug Research, Department of Pharmacochemistry, Vrije Universiteit, Amsterdam, The Netherlands tDepartment of Inhalation Toxicology, National Institute of Public Health and Environment, Bilthoven, The Netherlands

146

148

MODULATION OF ALLERGEN-INDUCED BRONCHOCONSTRICTION, BRONCHIAL HYPERREACTIVITY AND AIRWAY INFLAMMATION BY INHALATION OF SUBTYPE SELECTIVE PHOSPHODIESTERASE INHIBtTORS IN CONSCIOUS, UNRESTRAINED GUINEA PIGS. R.E. Santing, A.A.B. Rohof, N.M. van der Zee and J. Zaansma. Using a guinea pig model of allergic asthma (Santing et al., JACI 1994, 93, 1021-30) the effects of inhaled bronchodilatory doses of the phosphodiesterase (PDE) inhibitors rolipram (PDE IV selective), ORG 9935 (PDE III selective) and ORG 20241 (dual PDE Ill/IV inhibitor with some selectivity for PDE IV) on allergen-induced early (EAR) and late (LAR) asthmatic reactions, bronchial hyperreactivity (BHR) to histamine and airway inflammation were investigated. Aerosol inhalation (15 min) of solutions of 2.5mM rolipram, 100mM ORG 9935, 10 and 100mM ORG 20241 caused a significant 1.7 to 1.9-fold reduction in the bronchial reactivity to histamine. A significant protection against histamineinduced bronchoconstriction by these inhibitors was observed during 90, 120, 90 and 150 min, respectively. In some of the animals inhaling 100mM ORG 20241, a protection during up to 480 rain was observed. When inhaled 1 h prior to allergen exposure, all inhibitors caused a significant reduction in maximal bronchoconstriction during EAR, while all - except the lower concentration of ORG 20241 - showed a reduction of the total area under the time-response curve (AUC) during the EAR. The AUC of the LAR was significantly inhibited by ORG 9935, while a tendency to a reduced AUC was found for the other conditions. Rolipram, ORG 9935 and ORG 20241 (10 and lOOmM) caused a significant inhibition or even complete protection of the BHR observed at 6h after allergen challenge (after the EAR). Similarly, all inhibitors, except the lower concentration of ORG 20241 (P=0.06), significantly inhibited the BHR at 24h after challenge (after the LAR). Bronchoalveolar lavage studies at this time point indicated a reduced infiltration of eosinophils, lymphocytes and macrophages after inhalation of ORG 9935, while a strongly reduced infiltration of eosinophils was found with 100mM ORG 20241. Neutrophil infiltration tended to be inhibited after rolipram, ORG 9935 and 100mM ORG 20241. The results indicate that inhalation of single bronchodilating doses of PDE III, IV and Ill/IV selective inhibitors may protect against allergen-induced bronchoconstriction and BHR, and may thus be useful in the treatment of allergic asthma. A t least for the PDE Ill and dual PDE Ill/IV selective inhibitors the effects on BHR and LAR may be related to inhibition of inflammatory cell infiltration.

IS AN RGDS-BINDING DOMAIN INVOLVED IN THE TRANSMIGRATION OF POLYMORPHNUCLEAR LEUKOCYTES IN THE EXTRAVASCULAR SPACE OF ISCHEMIC MYOCARD1UM ?

Groningen Utrecht Institute for Drug Exploration, University Centre for Pharmacy, Department of Medicinal Chemistry and Molecular Pharmacology, Antonius Deusinglaan 2, 9713 AW Groningen.

E. Gottwald, M. Schott, S. Dhein, W. IGaus Polymorphnuclear leukocytes (PMN) cause, once migrated into the extravascular space, marked cellular damage during ischemia/reperfusion. By the liberation of multiple mediators such as e.g. oxygen free radicals and leukotrienes they enlarge the infarction zone and enhance transendothelial migration of further PMNs. We therefore tried to prevent transmigration by antiadhesive drugs in an experimental setting where spontaneously beating rabbit hearts were perfused according to the Langendorff-technique with tyrode solution alone (control), tyrode solution plus autologous PMNs, PMNs incubated with RGDS-peptide for integrine blockade or chondroitinsulfate C for selectine blockade. PMN counts, determined microscopically by counting a defined number of fields of view, in the isehemie border and centre zone, in the PMN-perfused hearts were twice as high as compared to control hearts whereas neither RGDS-peptide nor chondroitinsulfate C treated hearts showed elevated PMN tissue levels. PMN counts in the normoxic zone of each of the series differed not significantly. Severity and area of isehemia were determined by ST-segment mapping. Recovery of ST-segment elevation is impaired in the after PMN-perfusion alone which is not the case after seiectine or integrine blockade. Thus we conclude that an RGDS-binding domain is involved in the adhesion of PMNs to the vascular endothelial wall although e.g. gp IIb/IIIa-complex, the RGDS-receptor of platelets, is not yet described on neither cell. (Supported by the DFG). Institute of Pharmacology, University of Cologne, Germany

R 38 149 TOLUENE DIISOCYANATE-INDUCED HYPERREACTIVITY IN THE MOUSE AIRWAYS H. Scheerens~ T.L. Buckley, H. Van Loveren and F.P. Niikamp Toluene diisocyanate (TDI), a low molecular weight compound, is known to cause occupational asthma in man, however, the mechanisms involved in this reaction are unknown. In this study a murine model was developed to investigate TDI-induced asthma. Mice were skin-sensitized twice on day 0 and day 1 with 1% TDI (sensitized group) or with vehicle control (nonsensitized group). On day 8 all mice were challenged intranasally with 1% TDI. Tracheal reactivity to the muscarinie receptor agonist carbachol was measured in vitro 2, 24 and 48 h after the challenge. The sensitized mice exhibited marked tracheal hyperreactivity (increase in Emax) compared to the nonsensitized mice; this hyperreactivity was significant 24 h after the challenge (Emax: nonsensitized mice; 1915 -+ 180 mg, sensitized mice; 2768 _+ 171 rag, n=10-12 mice/group, p<0.01). In further experiments, the relationship between TDI-induced hyperreactivity and the presence of neutrophils was investigated. Myeloperoxidase (MPO), which is an enzyme located in neutrophil granules, was measured in lung tissue taken from sensitized and nonsensitized mice using a standard speetrophotometric method. In direct correlation with hyperreaetivity, MPO was significantly enhanced in TDI-sensitized mice 24 h after the challenge (nonsensitized mice: 4877 _+ 688 optical density/g tissue; sensitized mice: 18006 _+ 10233 optical density/g tissue, n=I0-12 mice/group, p<0.05). In summary, these results demonstrate that TDI is capable of inducing tracheal hyperreaetivity which is coincident with enhanced MPO activity in the mouse airways. Our current research is focussing on whether the neutrophil accumulation/activation is crucial for the induction of airways hyperreactivity in this model. Dept. of Pharmacology, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, PO Box 80.082, 3508 TB Utrecht, The Netherlands. 150 INHIBITION OF HYPERREACTIVITY BY ANTIBODIES DIRECTED AGAINST THE CELL ADHESION MOLECULES LFA-1 AND ICAM-1 IN A MURINE MODEL FOR NON-ALLERGIC ASTHMA P.G.M. Bloemen, P.A.J. Henricks, T.L. Buckley, M.C. van den Tweel, F.A.M. Redegeld, A.Sj. Koster, and F.P. Nijkamp. Airways hyperreactivity and leukocyte accumulation (particularly mononuclear leukocytes and neutrophils) are prominent features of delayed type hypersensitivity (DTH) reactions in the mouse lung. In this study the relationship between these two phenomena was investigated using monoclonal antibodies (mAbs) directed against the adhesion molecules LFA-1, Mac-1 and ICAM-I. The pulmonary DTH reaction was induced by skin-sensitizing mice with the hapten dinitrofluorobenzene (DNFB) or vehicle on two consecutive days. Five days later, mAbs or control Ab (rat IgG) were injected i.v. 2 h before (2001.tg) and 2 h after (200~g) an intranasal challenge with the same hapten. Tracheal reactivity to carbachol was determined 24 h after the challenge. In mice treated with control Ab marked hyperreactivity (increase in Emax) was observed 24 h after the challenge (see Table). Treatment with anti-LFA-1 or anti-ICAM-1 inhibited the tracheal hyperreactivity to control levels, whereas anti-Mac-1 had only a partial effect on this response (see Table). Treatment Vehicle Group (Emax) DNFB Group (Emax) rat IgG 1778=127 mg 2679=174 mg ** anti-LFA-1 2014=201 mg 1943=124 mg anti-Mac-1 1988=162 mg 2367+139 mg * anti-ICAM-1 2129=120 mg 1965=143 mg (n=10-12 mice/group; * p<0.05 ** p<0.01 vehicle vs DNFB groups) Besides tracheal hyperreactivity, a decrease in the T-cells in the blood and increased numbers of neutrophils in the broncho-alveolar lavage fluid and blood were observed 24 h after the challenge. The decrease of T-cells was markedly inhibited by treatment with anti-LFA-1 or antiICAM-t while anti-Mac-1 had little effect on this parameter. The elevation of neutrophils was markedly inhibited by anti-LFA-1 and by anti-Mac-I, whereas treatment with anti-ICAM-1 had no influence. In summary, the adhesion molecules LFA-1 and ICAM-1 play an important role in the development of tracheal hyperreactivity possibly via T cells in the lung tissue. Department of Pharmacology, Utrecht Institute for Pharmaceutical Sciences, P.O. Box 80.082, 3508 TB Utrecht, The Netherlands.

151

FURTHER EVIDENCE THAT INDUCTION OF PHOSPHOLIPASE A2/LIPOXYGENASE PATHWAY IN RAT ALVEOLAR MACROPHAGES (AMs) IS SUPPRESSED BY ENDOGENOUS NITRIC OXIDE. G Brunn, I. Wessler* & K. Rack6#, Lipopolysaccharides (LPS) can induce nitric oxide (NO) synthase in AMs (see Hey et al. 1995, this journal, 354, in press). Arachidonic acid (AA) metabolites, and particularly leukotriene B4 (LTB4), are important proinflammatory mediators released from AMs. Recently, we reported that A 23 187 evoked LTB 4 release from rat isolated, cultured AMs (Brunn et al. 1995, Pharmacol Res, in press) was markedly enhanced when NO synthesis was inhibited by NG-monomethyl-L-arginine (L-NMMA) during exposure to LPS, and this effect is further characterized in the present study. AMs were cultured for 18 h as described (Hey et al. 1995, this journal 354, in press) in the absence or presence of LPS (10 /zg/ml) and/or L-NMMA (100 #mol/l) and/or cycloheximide (10/zmol/1). Cells were then incubated for 2 h in Krebs medium containing 370 kBq 3H-AA. Thereafter the outflow of 3H-compounds was determined during 2 consecutive 50 min periods; A 23 187 (10 #mol/1) was present during the 2rid period. 3H-Compounds were separated by gradient reverse phase HPLC allowing the identification of 3H-LTB4, 3H-AA and several other AA-metabolites. Spontaneous outflow of 3H-LTB4 amounted about 170 DPM/50 min and was not significantly affected by the different culture protocols. In AMs cultured under control conditions, A 23 187 increased the 3H-LTB4 outflow about 3fold (compared to the individual initial outflow), and a similar increase was observed in AMs cultured in the presence of LPS. When in addition to LPS, L-NMMA was present in the culture medium, A 23 187 increased 3H-LTB4 release about 15fold, and this effect was largely reduced when the L-arginine concentration in the culture medium had been increased from 0.7 to 3 mmol/1, whereas addition of 2.3 retool/1 D-arginine had no effect. When the protein synthesis had been inhibited by cycloheximide during the culture period, A 23 187 evoked 3H-LTB4 release was almost abolished, both from control AMs and AMs treated with LPS in combination with L-NMMA. In conclusion, induction of the phospholipase A2/5-1ipoxygenase pathway in rat AMs is suppressed by endogenous NO. Department of Pharmacology, University Hospital, J.W. Goethe-Uni~rsity Frankfurt; g*Department of Pharmacology, University of Mainz and t t lInstitute of Pharmacology and Toxicology, University of Bonn, Reuterstr. 2b, D-53113 Bonn.