ROYAL A C A D E M Y OF MEDICINE IN IRELAND SECTION OF BIOMEDICAL SCIENCES P r o c e e d i n g s o f S u m m e r M e e t i n g h e l d June, 1994. ACTIVATION OF MAXI-CONDUCTANCE K § CHANNELS BY PURINERGIC AGONISTS IN HUMAN NONPIGMENTED CILIARY BODY EPITHELIUM

Thapsigargin (TGN) (0.51,tM) increased CI efflux from both cell types. In the CFPAC-I cells efflux was raised to 51.6 + 1.6% in the presence of TGN (n = 3). CI efflux from TPAC cells was increased to 75.8 + 1.8% of total C1 by TGN (n = 3). Significant increases in CI efflux were observed in both cell types in the presence of ATP (10p.M), efflux from CFPAC-I cells reaching 35.1 + 2.3% of total CI (p < 0.05, n = 6), while in TPAC cells efflux was raised to 56.4 + 1.6% (p < 0.01, n = 7). These results indicate that it is possible to activate CI efflux in cells expressing the CF defect. These cell models are therefore useful in investigating theraputic strategies in CF. Supported by the Cystic Fibrosis Association of Ireland. References 1. Cliff, W. H., Shoumacher, g. A., Frizzell, R. A. cAMP-activated C1 channels in CFTR transfected cystic fibrosis pancreatic epithelial cells. Am. J. Physiol., 1992: v262, pCl154 - Cl160. 2. Rugolo, M., Mastrocola, T., De Luca, M., Romeo, G., Galietta, L. J.V. A volume-sensitive chloride conductance revealed in human keratinocytes by 36C1- efflux and whole cell patch clamp recording. Biochimica et Biophysica Acta, 1992: vl 112, p 39-44.

A. Cullinane*, M. Coca-Prados, B. Harvey: Cellular Physiology Research Unit, Department of Physiology, University College, Cork. *Department of Ophthalmology, Yale University Medical School, New Haven, U.S.A. Using the patch-clamp technique, we have investigated, the effects of purinergic agonists on ion channel activity in an SV40 transformed cell line from human nonpigmented ocular ciliary epithelium (HNPE). The cells were isolated 4 days after attaining confluency and bathed in standard Krebs solution. The patchpipette contained an intracellular-like potassium solution (135 mM KCI, buffered to pCa 6, pH 7.2). Single channel activity was measured as the product of the number of open channels (N) and the single channel open probability (Po)" The predominant ion channel was found to be a calcium-activated potassium channel of high conductance (200pS - Maxi-K). Exposure of adenine nucleotides to the HNPE activated this channel in cell-attached patches by increasing both N and Po" The addition of 100 ItM ATP to the bath caused a thirtyfold increase in Po and a four-fold increase in N within 30s. Activation of maxi-K channels had a potency order of ATP>Adenosine>UTP which would indicate the response is mediated via a type 2 purinergic receptor. Since the membrane patch is isolated from the bath, channel activation by extracellular ATP must involve a receptor-coupled intracellular signal. The increase in Maxi-K~ channel activity is likely to result from purinergic receptor activation of calcium influx and/or the release of calcium from intracellular stores. This study shows that neurotransmitters can activate K § secretion in HNPE and may thus regulate aqueous humour production. Funded by The Health Research Board.

ACTIVATION OF SODIUM-PROTON EXCHANGE BY ALDOSTERONE STIMULATES POTASSIUM RECYCLING IN ISOLATED HUMAN COLON D. Maguire, G. O'Sullivan, B. J. Harvey. Cellular Physiology Research Unit, Department of Physiology, & Department of Surgery, Mercy Hospital, University College, Cork. Normal human colon epithelium was mounted in Ussing chambers and potassium transport across the basolateral membrane was studied in isolation, by increasing the electrical conductance of the luminal cell membranes (perforated with the ionophore nystatin) and establishing a lumen to serosa K§ gradient (substituting K§ for Na § in the mucosal Krebs solution). Under these conditions, the short circuit current (SCC) is a measure of electrogenic K § recycling across the basolateral cell membranes. Aldosterone (10nM) added to the basolateral bath produced an immediate increase in the K+-dependent SCC and this effect was completely inhibited (n=16 p<0.001) by addition of basolateral amiloride at a concentration (100 I.tM) which blocks Na§ § exchange. If aldosterone activation of K § channels is transduced via Na/ H exchange then modulation of the exchanger activity by known cytosolic inhibitors (e.g. cAMP) or activators (phorbol esters) of the exchanger should affect the atdosterone response. Pre-incubation of the colon epithelium with 50 IxM forskolin (to elevate intracellularcAMP) blocked the immediate aldosterone response (n=8 p<0.001). Addition of 10 nM phorbol 12-myristate acetate, PMA, (to elevate intracellular protein kinase C) caused an immediate increase in basolateral membrane K +conductance similar to aldosterone. Stimulation of SCC by PMA was prevented by pre-treatment with amiloride (n=8 p<0.001). Inhibition of protein kinase C by chelerythrine (10 nM) prevented the immediate aldosterone-induced increase in basolateral membrane K§ efflux.

ACTIVATION OF CHLORIDE TRANSPORT IN CYSTIC FIBROSIS PANCREATIC CELL LINES C. O'Reilly. M. P. Ryan. Department of Pharmacology, University College Dublin, Belfield, Dublin 4. Cystic Fibrosis (CF) is a disease of the secretory epithelia, attributed to abnormal regulation of chloride transport across epithelial cells. Pancreatic adenocarcinoma cells derived from a patient with CF (CFPAC-1 cells) and CFPAC-I cells transfected with wild type CFTR (TPAC cells) were used in this study, providing a model to compare chloride (CI) transport in CF and normal epithelia. 1 Efflux was measured using a modification of the method of Rugolo et al. 2 Basal efflux from CFPAC- 1 ceils reached a maximum of 28.2 + 2.3% of total Cl.(n = 6). TPAC cells displayed a significantly higher (p -< 0.01) rate of basal efflux with 46.5 + 2.3% of total C1 (n = 6) transported from the cells over the 10 min period studied. Forskolin (101.tM) significantly (p < 0.05) stimulated CI efflux from the TPAC cells to 69.3 + 1.6% of total C1 (n = 3) but had no effect upon efflux from the CFPAC-1 cells. 79

80 Royal Academy of Medicine in Ireland We conclude that the immediate up-regulation of basolateral K§conductance by aldosterone is dependent on activation of Na§ H§ exchange and transduced via changes to PKC and cAMP. Funded by the Wellcome Trust.

THE EFFECT OF SODIUM CONTAINING AND SODIUM FREE ISOTONIC SOLUTIONS ON URINARY EXCRETORY PA'Iq'ERNS IN MAN AND THE MINIPIG E. M. Gebruers, W. J. Hall, A. M. Harris. Department of Physiology, University College, Cork. Administration of isotonic solutions in man and the minipig leads to alterations in the urinary excretory patterns of salt and water. In the human there is a significant diuresis which is water led ~but in contrast a sodium led diuresis is seen in the minipig2. In both species the control diurnal sodium surge is augmented by sodium containing isotonic solutions. When a sodium free isotonic solution (Mannitol, 20 ml/kg body weight) was given to the minipig, we found that the diurnal pattern of sodium excretion was attenuated. Sodium excretion pre-infusion gave a median value of 47 ~tmol/min (24,139 - upper and lower quartiles), and at the peak of the diuresis was 69 ~tmol/min (11,135.). This observation was confirmed in humans where the pre-infusion median value was 152 ~tmol/min (69,263) and at the peak of the diuresis was 125 ttmol/min (70,173). These results lend credence to the suggestion that sodium monitoring sites in the gut affect sodium excretion. Ethics committee approval was obtained for the human experiments. The minipigs, after sedation with'Stresnil' (azaperone), 5mg/kg, were anaesthetized with 'Saffan' (alphaxolone 9mg/ml and alphadalone acetate 3mg/ml), 0.2-0.3 ml/kg. Grant support from the H.R.B.is gratefully acknowledged. References

1. Crotty, T. B., Gebruers, E. M., Hall, W. J. Signals from the stomach and/or intestines contribute to the diuresis on drinking isotonic fluids. J. Physiol. 406, 57P, 1988. 2. Gebruers, E. M., Hall, W. J., Harris, A.M. A urinary excretory response to intragastric infusion of a bowel lavage solution in the anaesthetized minipig. J. Physiol. 446, 455P, 1991.

EFFECTS OF ACETAZOLAMIDE ON VENTILATORY RESPONSES TO HYPOXIA AND HYPERCAPNIA IN CONSCIOUS RATS K. D. O'Halloran, A. K. Curran, A. Bradford. Department of Physiology, Royal College of Surgeons in Ireland, Dublin 2. Carbonic anhydrase inhibitors such as acetazolamide are used in the treatment of a variety of disorders but their effects on breathing are complex and controversiaP. Ventilation was measured by barometric plethysmography in 16 unrestrained, conscious, tracheostomized (under ether inhalation) rats breathing air containing 0, 3, 6 and 9% COz or 10% 02 in N 2, before and 40 minutes after administeringacetazolamide (50 mg/kg, I.P.; n = 8) or vehicle (n = 8). Values (mean + SD) for minute ventilation (in ml/min/100g body weight) before and during acetazolamide treatment were respectively 32.8 + 11.1 and 70.7 + 30.0 for air, 56.7 + 12.7 and 67.7 _+18.8 for 10% 02, 48.4 _+13.8 and 68.9 +_30.1 for 3% CO 2, 77.7 __28.0 and 90.2 -+ 55.0 for 6% CO 2 and 77.8 _+28.1 and 73.3 + 37.9 for 9% CO 2. Acetazolamide significantly increased

I.J.M.S. January, 1995

(Student's t test, p < 0.05) minute ventilation breathing air without affecting respiratory frequency but abolished ventilatory responses to hypoxia and hypercapnia. Injection of vehicle had no effect on the measured variables. The complete supression of hypoxic sensitivityby acetazolamide has been previously reported in humans and anaesthetized cats ~ but not the abolition of hypercapnic sensitivity which may be related to the absence of a respiratory frequency response in tracheostomized conscious rats. Supported by the Health Research Board and Royal College of Surgeons in Ireland. Reference

1. Teppema, L. J., Rochette, F., Demedts, M. Ventilatory effects of acetazolamide in cats during hypoxemia. J. Appl. Physiol. 1992: 72(5), 1717-1723.

PROPOFOL AND ALFENTANIL FOR INTUBATION IN CHILDREN H. E. Bunting, P. McConaghy, C. McLoughlin. Department of Anaesthesia, Belfast City Hospital, Lisburn Road, Belfast. This study assessed intubating conditions in children having anaesthesia induced with propofol and alfentanil. With ethical committee approval and informed parental consent, 60 ASA I/1I children, aged 3 to 12 years, undergoing elective ENT surgery were studied. Without premeditation, the patients were randomly allocated to anaesthetic induction with alfentanil 5, 10 or 15 ug/kg followed by propofol titrated to loss of consciousness. Three aspects of intubating conditions were assessed on a 4 point scale; (1) laryngoscopy (2) vocal cord movement (3) coughingI. Conditions were considered excellent if all 3 scores were 2 or less. The results were subjected to one way ANOVA and t-test where appropriate. A p value of < 0.05 was considered significant. The 3 groups were of equal size. With increasing doses of alfentanil the induction dose of propofol decreased significantly and the intubating conditions improved significantly. There was a significant difference in intubating conditions between the 5 and 10 ug/kg groups and the 5 and 15 ug/kg groups, but not between the 10 and 15 ug/kg groups. Intubation was successful at the first attempt in 70, 95 and 95% of cases, and conditions were excellent in 20%, 70% and 80% of cases in the 5, 10 and 15 ug/kg groups respectively. This study showed that induction using alfentanil 10 ug/kg with propofol may provide adequate intubatingconditions. Higher doses of alfentanil provided no additional benefit. Reference

1. Helbo-Hansen, S., Ravlo, O., Trap-Andersen, S. The influence of alfentanil on the intubating conditions after priming with vecuronium. Acta Anaesthesiol. Stand. 1988; 32: 41-44.

TRANSMEMBRANE SINGALLING IN NORMAL AND DYSTROPHIN-DEFICIENT MUSCLE D. Sweeney, O. Hardiman. Department of Pharmacology and Department of Human Anatomy & Physiology University College Dublin. Duchenne and Becker muscular dystrophy (DMD/BMD) are allelic disorders characterized by the absence or abnormality of

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the structural protein dystrophin. 3 animal models of DMD/BMD have been described. Although there are significant differences between the human and animal forms of dystrophin deficiency, all are associated with true muscle fibre hypertrophy. The nature and mechanism of this hypertrophy is poorly characterized, however there is evidence to suggest that it may develop as a result of altered transmembrane and/or intracellular signalling processes. Previous investigators have suggested that the activity of the transmembrane signalling enzyme adenylyl eyclase is altered in DMD. The activity of adenylyl cyclase has not been studied in other animal models of DMD. Using 16 age-matched male mdx and C57 control mice we have shown that basal activity is consistently twofold higher in the mdx model compared to controls, and that activated adenylyl cyclase activity is significantly elevated (sodium fluoride activation: mdx: 8.4 + 6 (mean + s.d) pmol cAMP/mg protein/min; control 3.5 + 3 (mean + s.d.) pmol cAMP/mg protein/min, p<0.03). (Statistical analysis performed using ANOVA and Student's t test). We propose that this up-regulation may contribute to the fibre hypertrophy characteristic of dystrophin deficiency.

CYCLIC AMP, CYCLIC GMP AND PROTEIN CONTENT OF HATCHED BOVINE BLASTOCYSTS M. Grealy, J. M. Sreenan. Department of Animal Reproduction, Teagasc, Belclare, Tuam, Co. Galway. There is a paucity of information on the control of growth, differentiation and embryo-maternal signalling in pre-implantation bovine embryos. Bovine blastocysts hatch at Day 9 of pregnancy and then undergo rapid growth and elongation before implantation. We measured total protein content and basal cAMP and cGMP levels in blastocysts during this period. Embryos were flushed from the uteri of superovulated heifers at Days 13, 14, 15 and 16 of pregnar/cy during mid-ventral laparotomy. Heifers were anaesthetized with thiopentone sodium (5g i.v.), followed by closed circuit anaesthesia with halothane and oxygen. The embryos were stored in 0.1 M HCI in liquid nitrogen until assay when they were thawed, sonicated and centrifuged. The resulting supernatants were used to assay cAMP and cGMP (NEN ~ I RIA) and the pellets used for protein determination (Pierce micro BCA). Protein concentration increased 60-fold between Day 13 (7.94 _+ 3.6 Ixg, mean + SEM, n=6) and Day 16 (476 _+ 142 ~tg, n=7; P < 0.005, ANOVA). cAMP and cGMP concentrations were highest at'Day 14 (25.4 _+ 2.9 and 4.5 _+ 1.7 fmol/~tg protein respectively, n=18), had decreased by Day 15 (11.18 _+0.77 and 0.36 _+0.07 fmol/lxg protein, n=23) and remained low at Day 16 (14.5 +_0.78 and 0.31 _+0.07 fmol/~tg protein, n=17; P < 0.0005 ANOVA, for Day 14 vs 15 or 16). Day 14 is the stage at which bovine embryos begin to elongate and this coincides with increased cyclic nucleotide levels. Funded by the European Union

PROLINE SPECIFIC ENZYMES OF GUINEA-PIG BRAIN L. Gilmartin 1, G. O'Cuinn 2. ~Department of Biochemistry, University College, Galway. 2Department of Life Sciences, Regional Technical College, Galway. At least eight different proline specific peptidases have been

described in mammalian cells. To date we have identified five of these enzymes in guineapig brain cells, four in the cytoplasm and one On the synaptosomal membrane. It is not clear why the brain cells require five proline specific peptidases to release proline from peptides. In particular the necessity for both amino peptidase P and soluble dipeptidyl aminopeptidase IV is not immediately obvious as both appear to metabolise X-Pro-Y-Z type substrates, albeit in different ways. We have characterised the substrate specificitiesof the dipeptidyl aminopeptidase IV and aminopeptidase P and found that while the former peptidase can release aminoacylproline residues from the N-termini 9f a wide range of peptides, it was unable to cleave between two adjacent prolines at positions 2 and 3 from the Nterminus. It could however release prolylproline from peptides where this sequence occurs at the N-terminus. In contrast aminopeptidase P hydrolysed the N-terminal amino acid from peptides where proline is present at position 2 from the Nterminus and activity appears not to be impaired if proline is present also at position 3. If we take the model substrate Leu-Pro-Pro-Ser it would appear from our characterisation studies that dipeptidyl aminopeptidase IV is unable to cleave between the two prolines. Aminopeptidase P on the other hand can remove the leucine residue allowing dipeptidyl aminopeptidase IV to subsequently remove the prolylproline dipeptide. Thus the requirement for aminopeptidaseP must in part be due to the restricted specificity of dipeptidyl aminopeptidase when confronted with X-Pro-ProY-Z sequences.

CAN INTRINSIC ACTIVITIES OF D 2 RECEPTOR AGONISTS BE PREDICTED FROM RADIOLIGAND BINDING MEASURES? M. Lawlor, K. M. O'Boyle. Department of Pharmacology, University College Dublin, Belfield, Dublin 4. While several studies have attempted to correlate radioligand binding measures with measures of intrinsic activity, there is little consensus as to which binding measurement should be used 1,2.The present study investigated the binding characteristics of a range of dopamine D2 receptor ag0nists 2 to identify which binding measurement(s) best predict agonist intrinsic activity. D 2 receptors in bovine caudate were labelled with [3H] spiperone and displacement and saturation experiments were carried out. Correlations between intrinsic activity and individual binding measures were investigated using the Spearman rank correlation test. Displacement of 0.3 nM [3H] spiperone by (-)quinpirole, dopamine, R(-) apomorphine or S(-)3-PPP was best described by a two site model (high affinity, K, and low affinity, KL), while displacement by terguride, SDZ208-911 or SDZ208-912 was best described by a one site model. There was no correlation between agonist intrinsic activity and K H, K L o r %R w However significant correlations were found between agonist intrinsic activity and Hill slope (p<0.05), KL/KH rati ~ (p<0.01) and % decrease in Bm~ induced by 2K i (p<0.05) or 10K i (p<0.01) concentrations of agonist. SDZ208-911 and SDZ208-912 were generous gifts of the Sandoz corporation. This work was supported by EOLAS and University College Dublin. References

I. O'Boyle, K. M., Waddington, J. L. Agonist and antagonist interactions with D~ dopamine receptors: Agonist induced masking of

82 Royal Academy of Medicine in Ireland DI receptors depends on intrinsic activity. Neuropharm. 1992: 31,177183. 2. Lahti, R. A., Figur, L. M., Piercey, M. F. et al. Intrinsic activity determinations at the dopamine D2 guanine nucleotide-binding proteincoupled receptor: Utilization of receptor state binding affinities. Mol. Pharmacol. 1992: 42, 432-438.

DOPAMINE D 1 RECEPTORS IN THE R A T FRONTAL CORTEX ARE GUANINE NUCLEOTIDE-INSENSITIVE C. B. Farrell, K. M. O'Boyle. Department of Pharmacology, University College Dublin, Belfield, Dublin 4. It has been reported that two subtypes of dopamine D 1receptors can be distinguished in human brain on the basis of their sensitivity to guanine nucleotides~l~: guanine nucleotide-sensitive D~ receptors, which are coupled to adenylyl cyclase and guanine nucleotide-insensitive D~ receptors, which are not. In this study, dopamine D l receptors located in the rat frontal cortex were assessed for their mode of interaction with dopamine and sensitivity to guanine nucleotides. Dt receptors were labelled with 2 nM [3H]SCH 23390 and displacement curves constructed using increasing concentrations of dopamine. The effects of pretreating membranes with 200 ~tM Gpp(NH)p or heat (60~ for 5 min) and the effect of sodium ions on dopamine displacements were examined. Dopamine displacement of [3H]SCH 23390 binding to frontal cortex D I receptors was best described by amodel assuming one binding site with a K~ value of 7257 + 1573 nM (n=4). Neither pretreatment with Gpp(NH)p nor heat had an effect on the ability of dopamine to displace ligand binding. In contrast, the absence of sodium ions increased the potency of dopamine to displace [3H]SCH 23390 binding (Ki 1022 + 242 nM, n=4, P<0.05, Oneway ANOVA). These findings suggest that, in contrast to the rat striatum t2~, the rat frontal cortex contains only dopamine D~ receptors that are guanine nucleotide-insensitive. This work was supported by Health Research Board and University College Dublin. References

1. De Keyser, J. Subtypes and localization of dopamine receptors in human brain. Neurochem. Int. 1993: 22, 83-93. 2. Farrell, C. B., O'Boyle, K. M. Partial conversion of D 1 dopamine receptors from high to low agonist affinity by Gpp(NH)p, heat and Nethylmaleimide. Br. J. Pharmacol. 1994: 110, 5P.

AN INVESTIGATION OF THE HYPOALGESIC EFFECTS OF COMBINATIONS OF TRANCUTANEOUS ELECTRICAL NERVE STIMULATION (T.E.N.S.) PARAMETERS UPON MECHANICAL PAIN THRESHOLD N. E. L. Foster, D. M. Walsh, G. D. Baxter, J. M. Alien. Biotherapeutics Research Group, Departments of Biological and Biomedical Sciences and Occupational Therapy & Physiotherapy, University of Ulster, Jordanstown, BT37 OQB, N. Ireland. Transcutaneous Electrical Nerve Stimulation (T.E.N.S.) has become a popular non-pharmacological method of pain relief, yet confusion still surrounds the selection of optimal stimulation parameters. This double-blind, placebo-controlled trial investigated the effect of four combinations of frequencies (4Hz &

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January, 1995

110Hz) and pulse durations (50l.ts & 2001.ts) upon experimentally induced mechanical pain. The distribution of the superficial radial nerve was mapped in the dominant forearm and hand to ensure normal innervation in 48 healthy, T.E.N.S.-naive volunteers. Two measurements of Mechanical Pain Threshold (MPT) were taken, using a pressure algometer, at 3 standardized points in the first dorsal web-space. Subjects were randomly allocated to one of 6 experimental groups: Group 1 ( l l 0 H z & 200p.s), Group 2 ( l l 0 H z & 501.ts), Group 3 (4Hz & 501xs), Group 4 (4Hz & 2001.ts), Placebo (nonactive socket) and Control. In Groups 1 to 4, T.E.N.S. was applied using a T.E.N.S. Model 120Z stimulator (ITO, Tokyo, Japan) for 3 five minute periods. Two hydrogel electrodes (3.5 cm x 5 cm) were attached to the skin (1 cm apart) directly over the course of the superficial radial nerve and current intensity was increased to maintain a 'strong but comfortable' sensation. MPT measurements were recorded before and after each five minute period of stimulation. The lowest of the 2 MPT readings from each recording point and the difference from baseline scores were used for analysis. Analysis of variance and post hoc Fisher tests revealed that application of one combination of parameters (110Hz & 2001xs) produced a significant increase in MPT (p<0.01), indicating significant hypoalgesia.

INVESTIGATIONS OF ENDOTHELIUM-DEPENDENT RELAXATIONS IN RAT MESENTERIC ARTERIES T. Cawley, E. Breslin, J. R. Docherty. Department of Physiology, RCSI, Dublin 2. Endothelium-dependent relaxation (EDR) involves at least one factor termed endothelium-derived relaxing factor (EDRF), thought to be nitric oxide (NO). However, controversy exists as to whether a second factor termed endothelium-derived hyperpolarising factor (EDHF) may be involved. Mesenteric arteries of diameter 200-350 I.tm were mounted in small vessel myographs for the investigation of contractions to the thromboxane mimetic U46619 and relaxations to acetylcholine (ACh). The nitric oxide synthase inhibitor N~-monomethyl-L-arginine (LNMMA) (1001xM) caused a significant 6 fold shift in the potency of ACh from 7.37+0.20 (-log ECso) to 6.59_+0.16 (n=7) (P<0.01) and a significant reduction in the maximum relaxation from 59.0+8.4% to 30.1+7.4% (P<0.01), and the guanylate cyclase inhibitor methylene blue (10 I.tM), alone or in combination with L-NMMA, virtually abolished responses. It is concluded that EDR of rat mesenteric artery to ACh involve predominantly nitric oxide. Supported by the Health Research Board and RCSI.

A LOW MOLECULAR WEIGHT FRACTION FROM FOLLICULAR FLUID AFFECTS GRANULOSA CELL PROLIFERATION, FOLLICLE GROWTH AND OVULATION RATE A. C. Hynes 1, J. M. Sreenan 2, M. T. Kane ~. Departments of tPhysiology, University College, Galway and 2Animal Reproduction, Teagasc, Belclare, Tuam, Co. Galway. There is increasing evidence that local ovarian factors modulate the effects of the pituitary gonadotrophins in controlling

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ovulation in monovular species 1. This study reports on the isolation of a factor from bovine follicular fluid and its effects on ovarian function in vivo and in vitro. Results were analysed by analysis of variance. The low molecular weight fraction (<10,000 Mr) of follicular fluid was obtained by membrane filtration, lyophilised and fractionated on a Sephadex G-25 gel filtration column. Granulosa 4 . 9 cells were plated (20 x 10/well) in culture medium and precultured for 24h prior to treatment for 24h with 4 concentrations (0.00, 0.01, 0.10 and 11xg/ml) of each peak. Only one peak (peak 4), inhibited (P<0.001) granulosa cell proliferation as measured both by haemocytometer counting and [3H]thymidine incorporation into acid-precipitable material. Female Sprague-Dawley rats were either injected with saline or two levels of peak 4 (0.006, 0.024 mg/animal/day) on 3 days. Peak 4 decreased uterine (P<0.01) and ovarian (P<0.01) weight and large follicle numbers (P<0.01) but increased small follicle numbers (P<0.01). For immunisation studies rats were given a primary (501xg) and 1 booster injection (201.tg) of a HSA-Peak 4 conjugate. Sheep were given a primary (3mg) and 8 booster injections (lmg). Immunisation increased large follicle numbers and decreased small follicle numbers (P<0.01) in rats, and increased ovulation rate in sheep. Reference

1. Ackland, J. F., Schwartz, N. B., Mayo, K. E., Dodson, g. E. Nonsteroidal signals originating in the gonads. Physiol. Rev. 1992: 72, 731-787.

ROLE OF THE PHOSPHATIDYLINOSITOL SYSTEM IN MOUSE EMBRYO STEM CELLS C. Duffy, M. T. Ka'ne. Department of Physiology, University College, Galway9 Embryonic stem (ES) cells are pludpotenfial cells derived from the inner cell mass of preimplantation embryos. Because the phosphatidylinositol IPtdlns) second messenger system is present in rabbit e m b r y o s , we set out to investigate the role of the Ptdlns system in a mouse ES cell line. ES cells were cultured 9 3 . . . . . with myo-[2- H]mosltol. The phosphomoslttdes were extracted and separated by thin layer chromatography and the inositol phosphates by HPLC anion exchange chromatography. [3I-I]inositol was incorporated into the phosphoinositides Ptdlns, Ptdlns4P and Ptdlns(4,5)P 2 with similar relative proportions to those reported in other cells. [3H]inositol was also incorporated into a fourth phospholipid, tentatively identified as an inositolglycan 9 3 . , by its Rf value and the fact that [ H]-glucosamine was incorporated into a lipid with the same Rf value. [3H]-inositol was also incorporated into a number of inositol phosphates with the greatest incorporation after 24 h being into InsP 5. Following Serum starvation and labelling with [3H]inositol for 24 hr, the addition of 10% serum for 2 and 20 min caused a significant (P<0.05, analysis of variance) increase in incorporation into the InsP, InsP 2, InsP 3 and InsP 4 peaks. The non dialysable fraction of serum (>10,000 Mr) caused a similar degree of activation. This indicates that the Ptdlns system may have a role in mediating the effects of serum growth factors on ES cells. References

1. Fahy, M. M., Kane, M. T. Incorporation of [3H]inositol into phosphoinositides and inositol phosphates by rabbit blastocysts. Mol. Reprod. Dev. 1993: 34, 391-395.

THE EFFECTS OF LOW DENSITY LIPOPROTEIN FROM DIABETIC SUBJECTS ON LIPOPROTEIN LIPASE SECRETION BY CULTURED HUMAN MACROPHAGES A. Mohd. Nor, A. H. Johnson, G. H. Tomkin, P. B. Collins. Department of Biochemistry, Royal College of Surgeons in Ireland, Dublin 2. Macrophages are implicated in atherogenesis and express the enzyme lipoprotein lipase which degrades triglyceride-rich lipoprotein in the body. Diabetic subjects are four to five times more prone to develop atherosclerosis than normal subjects. This study compares the effects of low density lipoprotein (LDL) isolated from 8 Type II diabetic subjects (all male, mean age 55 + 3yr) with that from 8 healthy controls (all male, mean age 51 + 4yr) on the expression of lipoprotein lipase LPL activity by cultured human monocyte-derived macrophages. Lipoprotein lipase activity in culture medium and cells was measured by an isotopic assay method. Preliminary experiments indicated secretion of enzyme activity into the medium at day 7 of culture9 Uttracentrifugallyprepared LDL (20 Ixg/ml final concentration) from control and diabetic subjects was added to cells on day 6 and both medium and cells were assayed for lipoprotein lipase activity on day 7. Increased medium levels of lipoprotein lipase activity were found in six of the diabetic subjects' LDL samples relative to that of controls9 The mean effect of LDL from the diabetic group as a whole was to increase medium LPL activity by 33% (11.55+1.2 vs. 8.62_+0.8 cpm/ng DNA** for diabetic and control LDL respectively; p<0.05). Previous studies suggested altered composition of LDL in the diabetic state which may enhance its uptake by cells. This may have profound implications in a diabetic milieu for the processing of macrophages of triglyceride-rich lipoproteins. **Mean + S.E.M., n=8

REGULATION OF BASOLATERAL K § CHANNELS BY ALDOSTERONE AND CALCIUM IN FROG SKIN EPITHELIUM D. (5 Cr6nfn, B. J. Harvey. Cellular Physiology Research Unit, Department of Physiology, University College, Cork. Frog skin epithelium was mounted in an Ussing chamber and the apical membrane was perforated with 5000 I.U./ml nystatin. In the presence of a transepithelial K § gradient, the short-circuit current (Iscc) was generated by the basolateral membrane K § conductance (GK). At low cystolic Ca 2§ levels (100 nM) the tolbutamide (100 ~tM)-sensitive compomnent of G K (KATP) channel) accounted for 70.3+6.8% (mean + SEM, n=4) of Isc C. The remaining Isc C was blocked by tetrapentyl ammonium (TPA 100 9 . 2+ . txM). At high mtracellular Ca concentrations 9500 nM), the TPA-inhibited component ofG K (Kca channel) was dominant and generated 93.4 + 4 . 8 (n=4) of Isc C. Alaosterone (1o M) added to the basolateral bath, produced an immediate increase in Isc c which after 2 hours had risen to 32.8+5.5% (n=4) above control. When the epithelium was exposed to a high concentration of Ca 2§ aldosterone induced a fall in Isc c to levels less than 50% of control. We conclude that aldosterone has rapid non-genomic effects on basolateral membrane K § conductance by upregulating KATp channels or d6wngrading Kca channels depending on the level of intracellular calcium. Funded by The Health Research Board and Wellcome Trust.

84 Royal Academy of Medicine in Ireland ADRENERGIC AND CHOLINERGIC COMPONENTS OF THE RESPONSES OF THE ISOLATED SHEEP BLADDER NECK MUSCLE TO ELECTRICAL FIELD STIMULATION S. McCloskey, K. D. Thornbury. Department of Physiology, School of Biomedical Science, The Queen's Univeristy of Belfast, 97 Lisburn Road, Belfast, BT9 7BL. The response to electrical field stimulation (EFS) of the sheep bladder neck muscle consists of a mixture of relaxation, contraction and poststimulus contraction. The object of this study was to examine the contractile responses to EFS after they had been unmasked by an NO synthase inhibitor. Sheep bladders were obtained from an abattoir and circulatory-orientated strips were cut from the region just below the trigone. These were mounted in organ baths and stimulated with 1 minute trains of 0.3 ms pulses at constant freequencies of 0.5 - 8Hz. L-NOARG (100 ~tM) blocked relaxations and post stimulus contractions and unmasked further contraction, consisting of a rapid "spike" which declined to a more sustained plateau. Atropine (1 I.tM) reduced the "spike" at all frequencies tested by less than 15% (n=12, P<0.05), but reduced the plateau by approximately 50% (n= 12, P<0.05). In the presence of atropine, prazosin (1 I.tM) abolished both spike and plateau (n=6, P<0.05), although yohimbine (1 IxM) was without effect. These results suggest that the contractions unmasked by NO-synthase blockade are mediated by both noradrenaline and ACh. Noradrenaline, acting via et1 receptors, makes the greater contribution to the phasic contraction, while both transmitters contribute equally to the plateau.

INVESTIGATIONS OF THE VASCULAR ACTIONS OF CHLOROETHYLCLONIDINE (CEC) IN RAT AORTA M. O'Rourke, S. Kearns, J. R. Docherty. Department of Physiology, RCSI, Dublin 2. CEC is an ct-adrenoceptor alkylating agent which shows ctladrenoeeptor subtype selectivity. We have investigated the unusual actions of CEC in rat aorta. CEC (100 IxM) significantly shifted the potency of NA from 7.53!-0.21 (vehicle, n=l 1) (-logEC50) to 5.54_+0.26 (n=5) (P<0.001), producing a 100 fold shift in potency, and produced a significant reduction in the maximum response to NA from 120.5+10.8% of control (vehicle) to 76.7+5.1% of control. The response to NA following CEC was biphasic: an early prazosin sensitive txl-response and a late prazosin insensitive response, which was also insensitive to yohimbine (t~-antagonists). In the absence of CEC, the response to NA was entirely prazosin sensitive. In receptor protection experiments in which receptors were occupied by receptor ligands prior to addition of CEC, the ligands NA or Yombine (beth 10 gM), but not prazosin (10 I.tM), protected against the actions of CEC against NA. It is concluded that CEC acts as an irreversible %-adreneceptor agonist in rat aorta. CEC is an example of a novel class of drugs: irreversible agonist. Supported by RCSI.

DAILY RUMINATION TIME IN SHEEP IS DIRECTLY RELATED TO THE MASS OF "LONG FIBRE" IN THE FORESTOMACH D. P. Campion, B. F. Leek. Department of Veterinary Physiology and Biochemistry, U.C.D., Veterinary College, Dublin 4. For rumination to be evoked, sheep require forestomach sensory

I.J.M.S. January, 1995

epithelial receptor excitation by dietary "long fibre". Four Suffolk-cross sheep fitted with ruminal cannulae were housed in metabolic crates and fed a low fibre pelleted concentrate diet for periods of 9 days to reduce rumination to zero. Jaw movements were recorded for 24h periods to monitor the duration of true rumination. On the ninth day, different masses (5g, 20g, 35g and 50g) of 10mmlong polyethylene fibre were added intraruminally at 12.00 hours. During the next 24h thesee masses evoked rumination totalling 162.5:30, 313+28, 295+17 and 403+50 min.day"1 (mean + S.E.M.) respectively. Thus the time spent ruminating per day was found to be directly related to the mass of 10mm fibre added into the rumen (F=22.9, p<0.001). This result accords with an earlier one (1) in which 2 of 3 concentrate-fed sheep, in response to their "fibre appetite" ingested 42.1 and 42.0g of the same polyethylene fibre and increased rumination from 9 _l zero to 179 and 177 finn.day respectively. We conclude that daily rumination time in sheep is directly related to the mass of "long fibre" in the forestomach and that sheep have a rumination drive expressed through a "fibre appetite". Reference

1. Campion, D. P., Leek, B. F. Investigation of a possible "appetite" for fibre in adult sheep, lr. J. Med. Sci9 1994: 163. 102.

SUPERIOR LARYNGEAL NERVE SECTION ALTERS THE VENTILATORY RESPONSE TO HYPERCAPNIA IN CONSCIOUS NEONATAL GUINEA PIGS A. K. Curran, K. D. O'Halloran, A. Bradford. Department of Physiology, Royal College of Surgeons in Ireland, Dublin 2. Laryngeal stimuli evoke! greater reflex respiratory effects in neonates compared to adults but little is known about the effects of laryngeal denervation on vefitilatory control in neonates. Ventilation was measured by barometric plethysmography in 17 unrestrained, conscious, neonatal ( 10-14 days old~guinea pigs breathing air containing 0, 3, 6 and 9% CO 2 of 10% O in N 2 before (sham operated, n=8) and after (n=9) cutting the superior laryngeal nerves (SLN) under ether inhalation. While breathing air, respiratory frequency was significantly (Student's t test, p<0.05) reduced (63.9-2-_21.1% of control, mean -1-SD) and tidal volume was increased ( 154-Z-68.2%)by SLN section. However, minute ventilation was unaffected (98.1+_55.2%). SLN section had no effect on minute ventilatory responses to hyercapnia or hypoxia except for 6% CO 2 where minute ventilation was reduced (66.6+_37.2% of intact value). However, respiratory frequency was reduced and tidal volume increased for all inspired gases. Hypoxia had no effects on ventilation either before or after SLN section. These results suggest that laryngeal afferent activity is important in the regulation of breathing in neonates, a finding consistent with the extensive and complex sensory function of the larynx. Supported by the Health Research Board, Royal College of Surgeons in Ireland and Wellcome Trust. Reference

1. Lucier, G. E., Storey, A. T., Sessle, B. J. Effects of upper respiratory tract stimuli on neonatal respiration: Reflex and single neuron analysis in the kitten. Biol. Neo. 1979: 325, 81-89.

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EFFECTS OF RECURRENT LARYNGEAL NERVE SECTION ON THE FALL IN LARYNGEAL RESISTANCE CAUSED BY COOL AIR IN ANAESTHETISED RATS A. Bradford l, K. D. O'Halloran l, A. K. Curran 1, R. G. O'Regan 2. Department of 1Physiology, Royal College of Surgeons in Ireland, Dublin 2, Department-of 2Human Anatomy and Physiology, University College, Dublin 2. 9

1

We have previously demonstrated that cooling of the isolated larynx reduces laryngeal resistance in anaesthetized rats with sectioned superior laryngeal nerves. We proposed that the mechanism is a direct effect of temperature on laryngeal muscles or on mucosal blood flow. However, part of the effect could be due to reflexes mediated by recurrent laryngeal nerve afferents. In 7 anaesthetized (chloralose, 100 mg/kg and urethane, 1000 mg/kg I.P.), Wistar rats, breathing spontaneously through a lowcervical tracheostomy, warm (36~ or cool (30~ air was applied at constant flow (20 ml/s/kg body weight) to the isolated larynx with sectioned superior laryngeal nerves. Laryngeal airflow, subglottic temperature and pressure, spontaneous tracheal airflow and arterial blood pressure were recorded. Cutting the recurrent laryngeal nerves caused a significant increase (Student's t test, p<0.05) in laryngeal resistance (+28.6_+8.7%, mean _+ SE) and abolished resistance oscillations in phase with breathing. However, the fall in laryngeal resistance caused by cool air was unaffected (-3.8_+6.9% before and -27.6+_3.6% after section). Therefore, laryngeal cooling causes a large fall in laryngeal resistance which is not of reflex origin. We propose that cooling reduces mucosal blood flow, thereby increasing laryngeal crosssectional area. Supported by the Health Research Board and Royal College of Surgeons in Ireland. Reference

I. O'Halloran, K. D., Curran, A. K., Brad~'ord,A. The influence of cold air, 1-menthol and CO 2 on ventilation and laryngeal resistance in the anaesthetized rat. Ir. J. Med. Sci. 1994: 163., 96-97.

MODULATION OF ADENYLYL CYCLASE ACTIVITY IN CULTURED HUMAN MUSCLE O. Hardiman, N. McLaughlin, D. Sweeney, Department of Human Anatomy and Physiology and Department of Pharmacology, University College Dublin. In vitro myogenesis recapitulates the programme of myogenesis in vivo. During the process of muscle differentiation, cAMP plays an Important role in the control of gene expression and in the integration of metabolic functions, cAMP generation may be affected by drugs or hormones that interact with the membrane-bound enzyme adenylyl cyclase, including adrenergic agents and glucocorticoids. In this Study, clonally derived cultured human muscle cells were exposed to a beta-agonist (isoprenaline) or glucorticoids during differentiation, and adenylyl cyclase activity was evaluated in prepared membranes. In control cultures, there was considerable interclonal variation in basal, sodium-fluoride and forskolila-stimutated adenylyl cyclase activity (Range: 3pmol cAMPmg protein/rain - 32 pM01 cAMP protein/ min). A distinct differentiation-associated decline in activity was noted in basal activity with a more variable effect on sodium flu0ride-stimulated and forskolin-stimulated activity following differentiation. No significant desentization was observed following long-term stimulation with isoprenaline. Cultures dif-

feted in their response to steroids: adenylyl cyclase activity was enhanced up to 5-fold in some clones, and was significantly inhibited by up to 10-fold in other clones. Pre-treatment of steroid-treated cultures with pertussis toxin (PTX) resulted in a two-fold increase in sodium fluoride-stimulated activity (without PTX: 9.5_+6.1 pMol cAMP/mg protein/min, with PTX: 26_+4 pMol cAMP/mg protein/min, p<0.05) indicating that the effects of steroids may be mediated in part by modulation of G-protein activity. These findings indicate a substantial heterogeneity among myoblast clones with respect to the modulating effect of steroids and beta-adrenergic agents on adenylyl cyclase activity.

EFFECTS OF ANTHRACENE-9-CARBOXYL ACID ON GENIOHYOID AND DIAPHRAGM MUSCLE ACTIVITY IN ANAESTHETIZED RATS K. D. O'Halloran, A. K. Curran, A. Bradford. Department of Physiology, Royal College of Surgeons in Ireland, Dublin 2. Anthracene-9-carboxyl acid (9-AC) induces a model of 1 myotonia which has been extensively investigated in vitro. However, very little is known about its in vivo effects and respiratory responses have not been investigated. Geniohyoid and diaphragm EMG activity was recorded in 8 anaesthetized (chloralose and urethane, 100 & 1000 mg/kg respectively, I.P.), spontaneously breathing, Wistar rats before, . . . . -3 during and after direct apphcatlon of 10 M 9-AC or vehicle to the muscles. In 18 trials, 9-AC significantly increased (student's t test, p<0.05) peak integrated diaphragm activity (+101.2_+2.0%). Diaphragmatic stimulation was almost immediate and lasted for several respiratory cycles. Unexpectedly, direct application of 9-AC to the diaphragm greatly increased geniohyoid muscle activity (+409.7_+237.2%) and this effect persisted following bilateral vagotomy. Application of vehicle had no effects. In conclusion, 9-AC directly excites diaphragm but not geniophyoid muscle activity in vivo. This difference between the two muscles may be due to differences in fibre composition. Geniohyoid activation following application of the compound to the diaphragm may be due to stimulation of phrenic afferents. Supported by the Health Research Board and Royal College of Surgeons in Ireland. References

1. Palade, P. T., Barachi, R. L. On the inhibition of muscle membrane chloride conductance by aromatic carb0xylic acids. J. Gen. Physiol. 1977: 69, 879-896.

FIBRIN POLYMERISATION. EVIDENCE FOR A SECONDARY POLYMERISATION SITE ON THE CARBOXY TERMINAL END OF THE Aa CHAIN USING A HUMAN FIBRIN SPECIFIC MURINE MONOCLONAL ANTIBODY J. Gaffney, T. A. Edgell, *J. M. Walker. National Institute for Biological Standards and Control, Hertfordshire, EN6 3QG and *Division of Biosciences, University of Hertfordshire, Hertfordshire, A L l 0 9AB. Since fibrin is involved in a wide variety of pathological conditions, control of its deposition and lysis is important. A fibrin specific monoclonal antibody (mab A1 l) was found to affect fibrin aggregation both in plasma and purified systems.

86 Royal Academy of Medicine in Ireland affect fibrin aggregation both in plasma and purified systems. The purified IgG and its F(ab)2 shortened the lag phase, enhanced the fibrin aggregation rate and yielded thicker fibrin fibres by scanning electronmicroscopy (sem), while the Fab fragment seemed to have the reverse effect. This suggested that IgG and F(ab) 2 from mab A 11, both being dimeric molecules, acted as chelating agents during fibrin polymerisation enhancing the optical density of the resultant large fibrin fibres. In order to define the site on the fibrin to which mab A11 is targeted various domainal structures of fibrinogen (denoted X, Y, D and E) andits polypeptide chains were investigated by immunoblotting and ELISA techniques. It was concluded that the epitope to which A 11 is targeted is in the 40 KD carboxy terminal region of the Aa chain of fibrinogen. It was concluded that the carboxy terminal of the Aa chain contains a secondary polymerisation lregion. Since interference with this latter site using the Fab from Mab A11 yields an optically (~, = 340 nm) inactive fibrin it can be concluded that the carboxy Aa-located polymerisation site is related to bundling or fibrin fibre thickening. Since A11 binds strongly to fully polymerised fibrin (K D - lO'l~ the antibody could not be directed to the polymerisation site itself but rather to an epitope which is at least conformationally adjacent to the site.

I.J.M.S. January, 1995 Prolonged TET exposure is known to impair myelinogenesis and may lead to demyelination of the axons within white matter of brain and spinal cord. Mitochondria and microtubule formation are directly affected by TET, causing inhibition of energy . 1 metabolism and lntraneuronal t r a n s p o r t . Although the pathogenesis of TET has not been fully elucidated, much of its neurotoxicity may be attributed to its lipophilic nature and subsequent membrane affinity. In the present study, the effects of TET (1, 2 and 4 mg/kg) administered intraperitoneally, on neurotransmitter concentration, locomotor activity and specific organ histology were examined in the male Sprague-Dawley rat. TET caused a significant decrease in the concentration of noradrenaline, serotonin, doamin and 7-aminobutyric acid with an increase in 5-HIAA concentration also observed (P<0.05). Locomotor activity 30 mins after a singe injection of TET showed no significant changes. Examination of body organs following TET administration reveals no histological damage in the liver, spleen or kidney. Triethyltin has been shown to effect a number ofneurotransmitter systems in the central nervous system having no acute effect on locomotor activity and no histological damage in the liver, spleen or kidney of treated animals Reference

AN ASSESSMENT OF THE HYPOTHERMIC RESPONSE TO RECEPTOR AGONISTS FOLLOWING STREPTOZOTOCIN ADMINISTRATION IN THE RAT C. P. MacSweeney, C. Faherty, J. P. Kelly, B. E. Leonard. Department of Pharmacology, University College, Galway. Streptozotocin (STZ) is an antibiotic which is selectively toxic to the pancreas, thereby rendering the animal diabetic. Previous studies have shown that neurochemical changes occur in the rat brain as a result of STZ-induced diabetes I. The aim of this experiment was to assess the hypothermic response to the selective receptor agonists apomorphine (DA-1 receptor; 1 mg/kg), clonidine (a-2 adrenergic receptor; 0.2 mg/ kg) and 8-OH-DPAT (5-HT1A receptor agonist; 0.15 mg/kg). This was carried out at intervals following a single administration of STZ (60 mg/kg, s.c.) in male Sprague-Dawley rats. T h e r e was no difference in the hypothermic response to apomorphine or 8-OH-DPAT at any of the time points. At all time points (days 4, 8, 11 and 15), the hypothermic response to clonidine was increased in the STZ group. It can be concluded that there is an alteration in the a - 2 responsiveness in the STZinduced diabetes. Reference

1. McCall, A. L. The impact of Diabetes on the CNS. Diabetes 1992: 41,557-570.

THE EFFECTS OF TRIETHYLTIN ON BIOCHEMICAL, BEHAVIOURAL AND HISTOLOGICAL PARAMETERS IN THE RAT C. Faherty, B. Farley, B. E. Leonard. Department of Pharmacology, University College, Galway. Triethyltin (TET) is a neurotoxic organometal that causes a severe but reversible cerebral oedema throughout the white matter of both brain and spinal cord. Vacuoplation of the myelin sheath at the interperiod line is a prominent feature of TET exposure.

1. Aschner, M., Aschner, J. L. Cellular and molecular effects of trimethyltin and triethyltin: Relevance to organotinneurotoxieity. Neurosci. Biobehav. Rev. 1992: 16, 427-435.

PROTECTIVE EFFECTS OF LISURIDE AND PIRIBEDIL AGAINST ISCHAEMIA-INDUCED HYPERACTIVITY AND HIPPOCAMPAL NEURODEGENERATION IN THE GERBIL M. O'Neill 1, J. M. Reymann 2, H. Allain2, B. E. Leonard 1. 1Pharmacology Department, University College Galway. 2Laboratoire de Pharmacologie Experimentale et Clinique, Rennes, France. In Mongolian gerbils, bilateral carotid occlusion (BCO) followed by reperfusion causes uniform destruction of the CA1 pyramidal neurons in the hippocampus. Recently, the role of dapamine in cerebral ischaemia has been investigated. Dopamine and serotinin have vasoactive actions and microdialysis studies have shown that there is a large release of these neurotransmitters during ischaemia In the present series of experiments the effects of lisuride (0.5 mg/kg i.p.) and piribedil (10 mg/kg i.p.) administered 1 hour before 5 min BCO on locomotor activity 1, 2, 7 and 8 days after surgery and histological paramaters 9 days after surgery in the gerbil was examined. Sham operated animals were used as controls and animals were anaesthetised with 2% halothane delivered with 0 2 at 1 litre/min via a face mask (n=10 per group). Results indicated that 5 min BCO caused a large increase in locomotor activity: 1 (P<0.03 and 2 (P<0.05) days after surgery and extensive damage in the CA1 region of the hippocampus as shown using histological methods. Both lisuride and piribedil attenuated the ischaemia-indueed hyperactivity (P<0.05) and were neuroprotective as shown by histological methods. These results suggest that the dopamine released during ischaemia contributes to the neuronal damage. Drugs acting on the dopaminergic system may have possible neuroprotective effects in cerebral ischaemi

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SIGMA LIGANDS ON BIOCHEMICAL PARAMETERS IN A MODEL OF FOCAL CEREBRAL ISCHAEMIA IN THE MOUSE M. Caldwell, M. Jennings, B. Earley, B. E. Leonard. Department of Pharmacology, University College, Galway. Cerebral ischaemia caused by eitller cardiovascular or brain trauma causes a selective pattern o f neurodegeneration. The use of animal models is crucial to the study of ischaemic brain injury. The middle cerebral artery occlusion (MCAO) mouse model is considered to be particularly useful in the study of focal ischaemia. The exact mechanism of cell death during ischaemia has not been fully elucidated, but glutamate levels increase. This increased glutamate, acting on N-methyl-d-aspartate (NMDA) receptors . . . . . . 2.1inmates a cascade of excitatory amino acids, Ca and free radical mediated toxicity and cell death. In the present study, the effects of the NMDA antagonist MK801, the mixed NMDA antagonist and sigma receptor ligand ifenprodil and putative sigma receptor ligands on the levels of biogenic amines and nitric oxide synthase (NOS) levels I 72 and 96 hours post MCAO were examined. Sham operated and unoperated animals were used as controls. Treatment drugs were administered 5, 15, 45 min, 3, 6, 24, 30, 48 and 54 hr post surgery. Results indicated MCAO caused a significant increase in the levels of biogenic amines L-DOPA, dopamine, HVA and 5-HIAA and a significant decrease in noradrenaline and serotonin 96 hr post surgery (P<0.05). NOS activity is also increased 72 and 96 hr post surgery (P<0.05) in the MCAO control when compared to unoperated and sham controls. All drugs administered caused a significant decrease in NOS activity in all brain regions measured (P<0.05). In conclusion, this study demonstrates altered neurochemical function in this model of ischaemia, mainly affecting the dopaminergic, noradrenergic and serotonergic systems and nitric oxide production. The NMDA/sigma receptor ligands used seem to reduce the ischaemia induced NOS activity by producing free radical production. Reference

be displayed in renal, hepatic, pulmonary and ocular epithelia. Using appropriate computer software and excitation wavelengths it is possible to construct a three-dimensional image of single cells showing the spatial distribution of Ca 2§ within a cell. Images of single cells can be rotated in space and viewed from the perspective of luminal or basolateral membranes. Funded by The Wellcome Trust.

ACTIVATION OF POTASSIUM CHANNELS BY MEMBRANE STRETCH IN NON-PIGMENTED EPITHELIUM FROM HUMAN OCULAR CILIARY BODY A. Cullinane, B. Harvey. Cellular Physiology Research Unit, Department of Physiology, University College, Cork. We have used the cell-attached and excised inside-out membrane configurations of the patch-clamp technique to demonstrate the effects of membrane stretch and air exposure on calciumdependent K § channels (Kca) in an immortalised cell line from human ocular ciliary body non-pigmented epithelium. Normally, Kca channels are quiesent at the resting membrane potential and at low intracellular [Ca2§ Their activity can be increased by (i) depolarisation 50 mV, (ii) raising intracellular [Ca 2§ above 100 nM or (iii) membrane stretch. Activation of Kca channels by voltage or Ca 2§ is instantaneous whereas the effect of membrane stretch is latent, requiring up to 60 sec of sustained stretch for full activation. When membranes containing stretch-activated Kca channels are excised inside-out from the cell, the channel openings rapidly inactivate. Brief air exposure of the quiescent membrane patch rapidly reactivates Kca channels to levels comparable with the cell-attached configuration. The effects of membrane stretch may depend on channel regulation by cytoskeleton attachments. Funded by The Health Research Board.

1. Bredt, D. S., Snyder, S. H. Isolation ofnitdc oxide synthase, a ealmodulin requiring enzyme. Proc. Natl. Acad. Sci. USA, 1990: 87, 682-685.

CELLULAR REGULATION OF BASOLATERAL POTASSIUM ION CHANNELS IN HUMAN COLON

THREE-DIMENSIONAL IMAGING OF INTRACELLULAR FREE CALCIUM ION ACTIVITY IN EPITHELIAL CELLS E. Prosser, A. Cullinane, V. Urbach, E. Horwitz, B. Harvey. Cellular Physiology Research Unit, Department of Physiology, University College, Cork. Many cellular processes are dependent on changes in the level of intracellular free calcium activity (Ca2§ Cell volume regulation, proliferation, fluid secretion, exocytosis, for example, are 2+ all initiated by increases in Ca r In epithelial ceils, the changes in Ca2+i triggered by hormones and neutrotransmitters may exhibit oscillations and have a spatial distribution, localised to either apical or basolateral membranes. The real-time detection and quantification of the spatial pattern of the dynamic Ca2"1"i signal in living cells, can provide unambiguous answers as to the identification and localisation of membrane Ca 2§ channels, intracellular Ca 2+ pools and membrane targets. Subcellular digital-imaging of the fluorescence pattern of the Ca 2+ fluoroprobe FURA-2 using STARWISE FLUO system (IMSTAR, Pads) will be demonstrated at the meeting and some illustrative results of Ca2+ i signalling and cell-cell coupling will

B. J. Harvey, G. O'Sullivan, D. Maguire. Cellular Physiology Research Unit, Department of Physiology and Department of Surgery, Mercy Hospital, University College, Cork. We present evidence for two different types of K + channels on their pharmacology and sensitivity to intracellular pH, Ca 2+ and ATP in basolateral membranes of isolated human colon epithelium mounted in Ussing chambers. Basolateral membrane K § current was measured by voltage clamp using nystatin to permeabilise the luminal membrane. Tolbutamide (100 gM), inhibited 90% of the basolateral K § current (n=32, p<0.001), and the remaining K § current was inhibited by tetrapentylammonium (TPA= 100 gM). The tolbutamide-sensitive current was increased by raising intracellular pH or lowering intracellular ATP or Ca 2§ The TPA-sensitive K § 2.1. current was upregulated by intracellular Ca and displayed a bell-shaped sensitivity to pH with maximum current at pH 7.5. We conclude that steady-state K § recycling occurs via ATPsensitive channels which are metabolically coupled to sodium absorption rates via the Na/K pump activity. A second type of + 9 9 . 2.1. K channel is activated by cystosohc Ca and is possibly involved in cell volume regulation. Funded by the Wellcome Trust. ION CHANNELS IN HUMAN AND PORCINE

88 Royal Academy of Medicine in Ireland

I.J.M.S. January,

ION CHANNELS IN HUMAN AND PORCINE CHONDROCYTES E. R. Horwitz, B. J. Harvey. Cellular Physiology Research Unit, Department of Physiology, University College, Cork. Synthesis and breakdown of cartilage matrix are dependent upon chondrocyte function9 Mechanical changes affect chondrocyte function. This effect is mediated via changes in ionic fluxes across the cell membrane I. The role of ion channels in this process is not fully understood and, therefore, we investigated ion Channels in both porcine and human chondrocytes. Articular chondrocytes were obtained from porcine metaphalangeal joints and human chondrocytes from femoral heads using a standard enzymatic dissociation. Cell electrophysiology was then investigated using a patch-clamp technique. The extracellular solution contained mM: 138 NaC1, 5.6 KCL, 5 CaCI2, 10 HEPES, pH 7.4 with NaOH. Electrode filling solution contained (Mm): 107 KCI, 1.033 MgC12, 1.292 CaC12, 5 EGTA, 10 HEPES, pH 7.2 with KOH. In human cells a K § channel of small conductance (=10 pS) and a rectifying CI" channel (55 pS) were found9 In porcine cells a rectifying K§ channel (98 pS upon depolarisation and 18 pS upon hyperpolarisation) and a C1 channel (20-30 pS) were found. K§ channel activity was present at the spontaneous membrane potential, but little CI~ channel activity. 9 2+ + . . . TheexlstenceofCa -dependentK actlvltymchondrocyteshas 9 2 ~ . been previously demonstrated. This is the first demonstration of chloride channelsand other potassium channels in human chondrocytes. The authors thank the H.R.B. for supporting this project. References

1. Urban,J. P. G., Hall, A. C. Physicalmodifiersof cartilage metabolism. In: Kuettner,K. etal. (ed). Articularcartilageand osteoarthritis.RavenPress,

New York, 1992: 393-405. 2. Wright,M. O., Stockwell,R. A., Nuki,G. Responseof plasmamembrane to appliedhydrostaticpressure in chondrocytesand fibroblasts.Connective Tissue Research, 1992: 28, 49-70.

ACTIVATION OF A CALCIUM-DEPENDENT CHLORIDE CHANNEL BY EXTRACELLULAR URIDINE TRIPHOSPHATE IN HUMAN LUNG EPITHELIUM V. Urbach, E9 Prosser, J-P. Raffin, S. Thomas, B. J. Harvey9 Cellular Physiology Research Unit, Physiology Department, University College, Cork. In cystic fibrosis, mutations in the CF gene product, CFTR, cause a defect in chloride secretion and possible therapy could involve pharmacological activation of alternative chloride secretion pathways. We have studied the effect of extracellular uridine triphosphate (UTia) on C1- channel activity in the apical membrane, using patch-clamp techniques in human lung primary cell cultures. When UTP (10"aM) was added on the luminal side of the epithelium, an outward-rectifying chloride channel (Clot : 30-40

1995

pS) was activated in previously quiescent cell-attached patches. -4 . . . 2+ UTP (10 M) also increased lntracellular calcmm (Ca i ) 5-fold above resting levels in cells loaded with Fura-2, measured by spectofluorimetry. The half-maximal increase in Ca2+i was obtained at [UTP] = 10-6 M. The activation of a chloride channel by extracellular UTP could thus result from a rise in cystosolic calcium via purinergic receptor activation9 This hypothesis is strengthened by the observation in excised inside-out patches that the Clor channel is inactive at Ca2§i = 10nM and is activated (increased open channel probability) for Ca2+i > 100riM. Supported by the Wellcome Trust and the Cystic Fibrosis Association of Ireland9

LOCALISATION OF THE CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR IN HUMAN PLACENTAL BRUSH BORDER MEMBRANE VESICLES AND ESTABLISHED CELL LINES D. A. Egan, A. O'Farrell, C. O'Reilly, M. P. Ryan. Department of Pharmacology, University College Dublin, Belfield, Dublin 4. The aims of this study included localisation of the cystic fibrosis transmembrane conductance regulator (CFTR) to human placental brush border vesicles (BBMV) and established cell lines. The CFTR is an integral membrane glycoprotein with a 9 1 . predicted molecular mass of approximately 170 KD . Previous studies from this laboratory, demonstrated that BBMV possess a chloride channel2, which may be CFTR. BBMV were isolated from human term placentae by differential centrifugation and characterised. T84 and rat foetal lung fibroblasts (RFLF) were used as positive and negative controls, 9 2 respectively. Membrane protein preparations were separated using electrophoresis. Results indicated the presence of a protein band at approximately 190 KD. BBMV membrane proteins were also assessed by Dot Blot and found to possess a protein which acted as an antigen for an anti-CFTR antibody. Western Blot analysis confirmed that this protein was CFTR CFTR was localised in two human pancreatic cell lines. CFPAC cells derived from a cystic fibrosis (CF) patient stained weakly for CFTR, while TPAC cells corrected for the CF defect stained strongly at the surface membrane9 In conclusion, CFTR was successfully localised in both placental BBMV and established cell lines. The authors acknowledge grant support from The Cystic Fibrosis Association of Ireland. References

1. Fuller, C. M., Howard, M. B., Bedwell, D. M., Frizzel, R. A. Antibodies against the cystic fibrosis transmembrane regulator. Am. J. Physiol. 1992: 262(31), C396-C404. 2. Failer, D., Ryan, M. P. Factors affecting chloride conductance in apical membrane vesicles from human placentae. J. Membrane Biol. 1992: 130, 227-239.