1P
ALTERED INOSITOL PHOSPHATE METABOLISM IN TAIL ARTERIES IN SPONTANEOUSLY HYPERTENSIVE RATS
S.B. Guild, S. Jenkinson & T.C. Muir, Department of Pharmacology, University of Glasgow, Glasgow G12 8QQ A change in polyphosphoinositide (PPI) metabolism, the signal-transduction mechanism mediating the contractile responses to noradrenaline (NA, Abdel-Latif, 1986), manifest as an enhanced inositol triphosphate (IP3) accumulation, occurs in spontaneously-hypertensive rats (SHR) to NA (Jenkinson et al, 1990). The site of this alteration has now been investigated.
NA-stimulated PPI metabolism in tail artery segments (1cm, 5mg wet wt.) from age-matched Wistar Kyoto (WKY) and spontaneously-hypertensive rats (SHR), was estimated by measuring the accumulation of inositol monophosphate (IP1), inositol bisphosphate (IP2) and IP3 (Akhtar and Abdel-Latif, 1984). The rates of formation and decay, after adding prazosin (10-6M) to terminate NA-stimulated PPI hydrolysis of these IP's were measured. IP3 accumulation increased significantly more in tissues from SHR than in those from WKY at each effective concentration of NA. This increase was parallelled by an equivalent reduction in IP1, consistent with the lack of any significant difference in total IP accumulation between the two populations. Time course studies suggest that the enhanced NA-evoked IP3 accumulation, in arteries from SHR rats, is due to a reduced rate of metabolism of both IP3 and IP2 rather than to a greater formation of IP3 from PPI's. This enhanced IP3 accumulation may result in an increased calcium mobilisation and so account for the increased contractility to NA of such arteries. We acknowledge the support of the Nuffield Foundation, the Wellcome Trust and SmithKline Beecham.
Abdel-Latif, A.A. (1986) Pharmacol. Rev. 38, 227-272. Akhtar, R.A. & Abdel-Latif, A.A. (1984) Biochem. J. 263, 291-300. Jenkinson, S., Guild, S.B. & Muir, T.C. (1990) Brit. J. Pharmacol. 101, 534P.
2P
INVOLVEMENT OF NITRIC OXIDE IN INHIBITORY JUNCTION POTENTIALS IN CANINE PROXIMAL COLON
H.H. Dalziel , K.D. Thornbury+, S.M. Ward, A. Carl, D.P. Westfall & KM. Sanders. Depts of Physiology & Pharmacology, University of Nevade-Reno, School of Medicine, Reno, NV 89557, U.S.A. and +Dept of Physiology, The Queen's University of Belfast, Belfast BT9 7BL, N. Ireland (Introduced by M.A. Khoyi).
Nitric oxide has been implicated in non-adrenergic, non-cholinergic (NANC) relaxation in a variety of visceral muscles. The underlying mechanism of NANC relaxation is hyperpolarization of smooth muscle membrane (i.e. inhibitory junction potentials; IJPs). Experiments were performed to study the basis of NANC IJPs in circular muscle cells of the canine proximal colon. Cells near the myenteric surface of the circular smooth muscle layer were specifically studied using intracellular microelectrodes. The effects of an inhibitor of NO-synthase,J.-NG-nitroarginine methyl ester (L-NAME, 1M ), on the nerve-mediated UPs, and the ability of authentic NO and the NO carrier, nitrosocysteine (cys-NO; synthesized according to method of Field et al.; 1978) to mimic NANC hyperpolarizations were evaluated. Circular muscle cells in this region had average resting potentials of -43 ± 2 mV (n= 1). NANC nerve stimulation (0.1 Hz) produced UPs averaging 24.4 ± 4 mV in magnitude and 2.6 ± 0.2 s (n=7) in duration. The NO-synthase inhibitor, L-NG-nitroarginine methIyl ester (L-NAME, 100gLM), reduced the UP size to 3 ± 1 mV (P carbachol (4.51) > pilocarpine (4.23) = McN-A-343 (4.23) > methacholine (3.25) > bethanechol (3.16). There was no change in the potency of carbachol in two consecutive curves, constructed at 60 min intervals (curve 1 -log EC50 = 4.21; curve 2 -log EC50 = 4.23) suggesting that the tissue did not display desensitisation under the conditions employed. The pA2 values (mean ± SEM, n=4), with Schild slopes in parenthesis, for atropine, pirenzepine, methoctramine and (±)para-fluoro-hexahydro-siladifenidol were 9.09 ± 0.08 (0.92 ± 0.11), 8.26 ± 0.11 (1.07 ± 0.10), 6.05 ± 0.09 (1.01 ± 0.07) and 7.11 ± 0.11 (1.08 ± 0.07), respectively. The similarity between agonist potency (-log EC50) and apparent affinity (-log KA), suggests an absence of a substantial receptor reserve associated with the contractile response. The pA2 values indicate that stimulation of the M1 muscarinic receptor subtype mediates contraction. In this respect, the canine femoral vein resembles the canine saphenous vein in terms of the muscarinic receptor population (Eglen et al., 1990) and provides a further example of postjunctional, smooth muscle M1 muscarinic receptors. Eglen, R.M., Michel, A.D., Montgomery, W.W., Kunysz, E.A., Machado, C.A. & Whiting, R.L. (1990) Br. J. Pharmacol., 99, 637-642. Furchgott, R.F. & Bursztyn, P. (1967) Ann. N.Y. Acad. Sci., 144, 882-899. Hulme, E.C., Birdsall, N.J.M. & Buckley, N.J. (1990) Ann. Rev. Pharmacol. Toxicol., 30, 663-674. Rubanyi, G.M. & Vanhoutte, P.M. (1988) Blood Vessels, 23, 75-81.
49P
SOURCES OF CALCIUM MOBILISED BY a-ADRENOCEPTORS IN HUMAN SUBCUTANEOUS RESISTANCE ARTERIES
N.A. Parkinson, A.D. Hughes, P.S. Sever. Dept Clinical Pharmacology, St Mary's Hospital Medical School, London W2 1NY. The a2-adrenoceptor has been located postjunctionally in human subcutaneous resistance artcries (Niclsen el al 1989). Little is known of the mechanism of action of the postjunctional ct2-adrenoceptor or the relative importance of either the extracellular or intracellular pool of calcium ions. To investigate which source of calcium was of more importance, in vitro studies were undertaken on human subcutaneous resistance arteries (internal diameter 126-412ptm). 13 resistance arteries were obtained from 10 patients undergoing surgery and studied using a myograph (Mulvany & Halpern, 1977). Maximum responses to 118mM potassium depolarising solution (KDS), noradrenaline (NA) (1 OiM) and azepcxole (BHT) (litLM) an (12-adrenoceptor agonist, were ascertained in physiological saline solution (PSS) containing 2.5mM calcium, or calcium-free PSS containing 1mM BAPTA. In other experiments, the maximum response to caffeine (20mM), NA (1 0tM) and BHT (1 was determined in the absence and presence of ryanodine (1I01±M, incubated for 30 min). Table 1. Table 2.
OtiM)
BHT (n=5)
PSS 41.4±6.0 Ca-free PSS 3.43±1.31 piperoxan (+5.6 + 1.7 'C) > idazoxan (+4.6 ± 1.1 'C) > imiloxan (+4.1 + 1.3 'C) > SKF 104078 (+2.0 ± 1.9 "C) > BDF-6143 (+ 1.3 + 0.8 "C). Prazosin (0.3 mg/kg, s.c.) did not increase TS; propranolol (10 mg/kg, s.c.) evoked a small increase in Ts (+2.9 ± 1.0 'C). Pentolinium (210 mg/kg, s.c.) elicited a dose-related increase in Ts, which was elevated by 4.4 ± 1.3 'C after a dose of 10 mg/kg. Tc was reduced in a dose -related manner; a dose of 10 mg/kg reduced Tc by 3.8 + 0.8'C. This effect, together with a lowering of blood pressure, could explain why ganglionic blockade failed to elevate Ts to the same extent as RS-15385-197. Drug vehicles (1 ml/kg, s.c.) had no effects on Ts or Tc.
These data indicate that q2-adrenoceptor antagonists elevate tail skin temperature in the rat. This phenomenon has previously been noted for yohimbine (Wilson & Fregly, 1985). It is not known whether this is a central or peripheral effect, or whether this is due to an increase in total tail blood flow, redistribution of blood flow from deep vessels (either arterial or venous) to the tail surface, or opening of arteriovenous anastomoses. In view of the positive results with pentolinium, and the negative results with prazosin, our working hypothesis is that vasoconstriction within the tail, which prevents heat loss at an ambient temperature of around 19 'C, involves post-junctional vascular a2-adrenoceptors. This suggestion is supported by indirect evidence from in vitro studies of this vascular bed (Templeton et al., 1989). Brown, C.M., Clague, R.U., Kilpatrick, A.T., MacKinnon, A., Martin, A.B. & Spedding, M. (1990) Br. J. Pharmacol. 99, 272P. Clark, R.D., Repke, D.B., Kilpatrick, A.T., Brown, C.M., MacKinnon, A.C., Clague, R.U. & Spedding, M. (1989) J. Med. Chem. 32, 2034-2036. Wilson, K.M. & Fregly, M.J. (1985) Peptides 6, 695-701. Templeton, A.G.B., Macmillan, J., McGrath, J.C., Storey, N.D. & Wilson, V.G. (1989) Br. J. Pharmacol. 97, 563-57 1.
51 P
DRUG-INDUCED ENHANCEMENT OF CONTRACTIONS TO CLONIDINE IN RAT TAIL ARTERY RINGS IN VITRO
D.M. Aidulis , A. Fugisang, S.A.H. Savage & D. Pollock, Department of Pharmacology, University of Glasgow, Glasgow, G12 8QQ
Alpha2-adrenoceptors occur on smooth muscle in the rat tail artery (MacLean & McGrath, 1990; Rajanayagam et al., 1990). These receptors are elusive and various techniques have been used to reveal them. This study investigated some of the conditions necessary to demonstrate enhanced postsynaptic alpha -adrenoceptor mediated responses to clonidine (CLON). Segments (3-4 mm) of proximal tail artery from male Wistar rats (150-200 g) were suspended between pairs of stainless steel hooks, inserted through the lumen, in Krebs buffer (370C), gassed with 95% 0 /5% C02. The resting tension on each artery ring was set at 1 g. After 2 h equilibration, responses to irugs were recorded isometrically. Responses to a standard submaximal concentration (10 nM) of CLON were examined in the absence and presence of submaximal concentrations of phenylephrine (PE, 0.5 uM), potassium chloride (KCl, 0.03 M) or vasopressin (VP, 0.4 miu m-1 . The standard res onse to CLON was also examined in rings precontracted with PE (0.5,uM), KCl (0.03 M) or VP (0.4 miu ml ) and then relaxed with isobutylmethylxanthine (IBMX, 10 um). The response to CLON (10 nM) was enhanced in the presence of PE (0.5 ,uM) but not in the presence ofKCl (0.03 M) or VP (0.4 miu ml-1). When the artery ring was precontracted with PE, KCl or VP and relaxed with IBMX (10uM), the superimposed response to CLON (10 nM) was enhanced respectively by 115 ± 17% (mean ± s.e. mean, 0.05>P>0.01, n=7), 933 + 15%, (PP>0.01, n=7). In each case the enhanced response to CLON was similar to that produced by the precontracting agonist alone (PE/CLON, R=0.97, P
ALTERED INOSITOL PHOSPHATE METABOLISM IN TAIL ARTERIES IN SPONTANEOUSLY HYPERTENSIVE RATS
S.B. Guild, S. Jenkinson & T.C. Muir, Department of Pharmacology, University of Glasgow, Glasgow G12 8QQ A change in polyphosphoinositide (PPI) metabolism, the signal-transduction mechanism mediating the contractile responses to noradrenaline (NA, Abdel-Latif, 1986), manifest as an enhanced inositol triphosphate (IP3) accumulation, occurs in spontaneously-hypertensive rats (SHR) to NA (Jenkinson et al, 1990). The site of this alteration has now been investigated.
NA-stimulated PPI metabolism in tail artery segments (1cm, 5mg wet wt.) from age-matched Wistar Kyoto (WKY) and spontaneously-hypertensive rats (SHR), was estimated by measuring the accumulation of inositol monophosphate (IP1), inositol bisphosphate (IP2) and IP3 (Akhtar and Abdel-Latif, 1984). The rates of formation and decay, after adding prazosin (10-6M) to terminate NA-stimulated PPI hydrolysis of these IP's were measured. IP3 accumulation increased significantly more in tissues from SHR than in those from WKY at each effective concentration of NA. This increase was parallelled by an equivalent reduction in IP1, consistent with the lack of any significant difference in total IP accumulation between the two populations. Time course studies suggest that the enhanced NA-evoked IP3 accumulation, in arteries from SHR rats, is due to a reduced rate of metabolism of both IP3 and IP2 rather than to a greater formation of IP3 from PPI's. This enhanced IP3 accumulation may result in an increased calcium mobilisation and so account for the increased contractility to NA of such arteries. We acknowledge the support of the Nuffield Foundation, the Wellcome Trust and SmithKline Beecham.
Abdel-Latif, A.A. (1986) Pharmacol. Rev. 38, 227-272. Akhtar, R.A. & Abdel-Latif, A.A. (1984) Biochem. J. 263, 291-300. Jenkinson, S., Guild, S.B. & Muir, T.C. (1990) Brit. J. Pharmacol. 101, 534P.
2P
INVOLVEMENT OF NITRIC OXIDE IN INHIBITORY JUNCTION POTENTIALS IN CANINE PROXIMAL COLON
H.H. Dalziel , K.D. Thornbury+, S.M. Ward, A. Carl, D.P. Westfall & KM. Sanders. Depts of Physiology & Pharmacology, University of Nevade-Reno, School of Medicine, Reno, NV 89557, U.S.A. and +Dept of Physiology, The Queen's University of Belfast, Belfast BT9 7BL, N. Ireland (Introduced by M.A. Khoyi).
Nitric oxide has been implicated in non-adrenergic, non-cholinergic (NANC) relaxation in a variety of visceral muscles. The underlying mechanism of NANC relaxation is hyperpolarization of smooth muscle membrane (i.e. inhibitory junction potentials; IJPs). Experiments were performed to study the basis of NANC IJPs in circular muscle cells of the canine proximal colon. Cells near the myenteric surface of the circular smooth muscle layer were specifically studied using intracellular microelectrodes. The effects of an inhibitor of NO-synthase,J.-NG-nitroarginine methyl ester (L-NAME, 1M ), on the nerve-mediated UPs, and the ability of authentic NO and the NO carrier, nitrosocysteine (cys-NO; synthesized according to method of Field et al.; 1978) to mimic NANC hyperpolarizations were evaluated. Circular muscle cells in this region had average resting potentials of -43 ± 2 mV (n= 1). NANC nerve stimulation (0.1 Hz) produced UPs averaging 24.4 ± 4 mV in magnitude and 2.6 ± 0.2 s (n=7) in duration. The NO-synthase inhibitor, L-NG-nitroarginine methIyl ester (L-NAME, 100gLM), reduced the UP size to 3 ± 1 mV (P carbachol (4.51) > pilocarpine (4.23) = McN-A-343 (4.23) > methacholine (3.25) > bethanechol (3.16). There was no change in the potency of carbachol in two consecutive curves, constructed at 60 min intervals (curve 1 -log EC50 = 4.21; curve 2 -log EC50 = 4.23) suggesting that the tissue did not display desensitisation under the conditions employed. The pA2 values (mean ± SEM, n=4), with Schild slopes in parenthesis, for atropine, pirenzepine, methoctramine and (±)para-fluoro-hexahydro-siladifenidol were 9.09 ± 0.08 (0.92 ± 0.11), 8.26 ± 0.11 (1.07 ± 0.10), 6.05 ± 0.09 (1.01 ± 0.07) and 7.11 ± 0.11 (1.08 ± 0.07), respectively. The similarity between agonist potency (-log EC50) and apparent affinity (-log KA), suggests an absence of a substantial receptor reserve associated with the contractile response. The pA2 values indicate that stimulation of the M1 muscarinic receptor subtype mediates contraction. In this respect, the canine femoral vein resembles the canine saphenous vein in terms of the muscarinic receptor population (Eglen et al., 1990) and provides a further example of postjunctional, smooth muscle M1 muscarinic receptors. Eglen, R.M., Michel, A.D., Montgomery, W.W., Kunysz, E.A., Machado, C.A. & Whiting, R.L. (1990) Br. J. Pharmacol., 99, 637-642. Furchgott, R.F. & Bursztyn, P. (1967) Ann. N.Y. Acad. Sci., 144, 882-899. Hulme, E.C., Birdsall, N.J.M. & Buckley, N.J. (1990) Ann. Rev. Pharmacol. Toxicol., 30, 663-674. Rubanyi, G.M. & Vanhoutte, P.M. (1988) Blood Vessels, 23, 75-81.
49P
SOURCES OF CALCIUM MOBILISED BY a-ADRENOCEPTORS IN HUMAN SUBCUTANEOUS RESISTANCE ARTERIES
N.A. Parkinson, A.D. Hughes, P.S. Sever. Dept Clinical Pharmacology, St Mary's Hospital Medical School, London W2 1NY. The a2-adrenoceptor has been located postjunctionally in human subcutaneous resistance artcries (Niclsen el al 1989). Little is known of the mechanism of action of the postjunctional ct2-adrenoceptor or the relative importance of either the extracellular or intracellular pool of calcium ions. To investigate which source of calcium was of more importance, in vitro studies were undertaken on human subcutaneous resistance arteries (internal diameter 126-412ptm). 13 resistance arteries were obtained from 10 patients undergoing surgery and studied using a myograph (Mulvany & Halpern, 1977). Maximum responses to 118mM potassium depolarising solution (KDS), noradrenaline (NA) (1 OiM) and azepcxole (BHT) (litLM) an (12-adrenoceptor agonist, were ascertained in physiological saline solution (PSS) containing 2.5mM calcium, or calcium-free PSS containing 1mM BAPTA. In other experiments, the maximum response to caffeine (20mM), NA (1 0tM) and BHT (1 was determined in the absence and presence of ryanodine (1I01±M, incubated for 30 min). Table 1. Table 2.
OtiM)
BHT (n=5)
PSS 41.4±6.0 Ca-free PSS 3.43±1.31 piperoxan (+5.6 + 1.7 'C) > idazoxan (+4.6 ± 1.1 'C) > imiloxan (+4.1 + 1.3 'C) > SKF 104078 (+2.0 ± 1.9 "C) > BDF-6143 (+ 1.3 + 0.8 "C). Prazosin (0.3 mg/kg, s.c.) did not increase TS; propranolol (10 mg/kg, s.c.) evoked a small increase in Ts (+2.9 ± 1.0 'C). Pentolinium (210 mg/kg, s.c.) elicited a dose-related increase in Ts, which was elevated by 4.4 ± 1.3 'C after a dose of 10 mg/kg. Tc was reduced in a dose -related manner; a dose of 10 mg/kg reduced Tc by 3.8 + 0.8'C. This effect, together with a lowering of blood pressure, could explain why ganglionic blockade failed to elevate Ts to the same extent as RS-15385-197. Drug vehicles (1 ml/kg, s.c.) had no effects on Ts or Tc.
These data indicate that q2-adrenoceptor antagonists elevate tail skin temperature in the rat. This phenomenon has previously been noted for yohimbine (Wilson & Fregly, 1985). It is not known whether this is a central or peripheral effect, or whether this is due to an increase in total tail blood flow, redistribution of blood flow from deep vessels (either arterial or venous) to the tail surface, or opening of arteriovenous anastomoses. In view of the positive results with pentolinium, and the negative results with prazosin, our working hypothesis is that vasoconstriction within the tail, which prevents heat loss at an ambient temperature of around 19 'C, involves post-junctional vascular a2-adrenoceptors. This suggestion is supported by indirect evidence from in vitro studies of this vascular bed (Templeton et al., 1989). Brown, C.M., Clague, R.U., Kilpatrick, A.T., MacKinnon, A., Martin, A.B. & Spedding, M. (1990) Br. J. Pharmacol. 99, 272P. Clark, R.D., Repke, D.B., Kilpatrick, A.T., Brown, C.M., MacKinnon, A.C., Clague, R.U. & Spedding, M. (1989) J. Med. Chem. 32, 2034-2036. Wilson, K.M. & Fregly, M.J. (1985) Peptides 6, 695-701. Templeton, A.G.B., Macmillan, J., McGrath, J.C., Storey, N.D. & Wilson, V.G. (1989) Br. J. Pharmacol. 97, 563-57 1.
51 P
DRUG-INDUCED ENHANCEMENT OF CONTRACTIONS TO CLONIDINE IN RAT TAIL ARTERY RINGS IN VITRO
D.M. Aidulis , A. Fugisang, S.A.H. Savage & D. Pollock, Department of Pharmacology, University of Glasgow, Glasgow, G12 8QQ
Alpha2-adrenoceptors occur on smooth muscle in the rat tail artery (MacLean & McGrath, 1990; Rajanayagam et al., 1990). These receptors are elusive and various techniques have been used to reveal them. This study investigated some of the conditions necessary to demonstrate enhanced postsynaptic alpha -adrenoceptor mediated responses to clonidine (CLON). Segments (3-4 mm) of proximal tail artery from male Wistar rats (150-200 g) were suspended between pairs of stainless steel hooks, inserted through the lumen, in Krebs buffer (370C), gassed with 95% 0 /5% C02. The resting tension on each artery ring was set at 1 g. After 2 h equilibration, responses to irugs were recorded isometrically. Responses to a standard submaximal concentration (10 nM) of CLON were examined in the absence and presence of submaximal concentrations of phenylephrine (PE, 0.5 uM), potassium chloride (KCl, 0.03 M) or vasopressin (VP, 0.4 miu m-1 . The standard res onse to CLON was also examined in rings precontracted with PE (0.5,uM), KCl (0.03 M) or VP (0.4 miu ml ) and then relaxed with isobutylmethylxanthine (IBMX, 10 um). The response to CLON (10 nM) was enhanced in the presence of PE (0.5 ,uM) but not in the presence ofKCl (0.03 M) or VP (0.4 miu ml-1). When the artery ring was precontracted with PE, KCl or VP and relaxed with IBMX (10uM), the superimposed response to CLON (10 nM) was enhanced respectively by 115 ± 17% (mean ± s.e. mean, 0.05>P>0.01, n=7), 933 + 15%, (PP>0.01, n=7). In each case the enhanced response to CLON was similar to that produced by the precontracting agonist alone (PE/CLON, R=0.97, P
