1P

K+ CHANNEL OPENERS DILATE BOTH LARGE AND SMALL CORONARY ARTERIES IN CONSCIOUS DOGS

C. Drieu La Rochelle, B. Riou, A. BerdeaurF & J.F. Giudicelli, Department of Pharmacology, Faculte de M&decine Paris-Sud, 63, rue Gabriel Peri, 94276 - Le Kremlin-Bicetre Cidex, France. Cromakalim (C) and pinacidil (P) are two K channel openers which induce coronary vasodilation through both a direct (vasorelaxation) and an indirect (increase in myocardial oxygen demand) mechanisms (Kawashima and Liang, 1985; Cook and Hof, 1988). However, if this vasodilatory effect is well documented for coronary arterioles, it has not yet been studied in large epicardial conductance vessels which are of major importance in the pathophysiology of coronary vasospasm. The present study was thus designed to investigate the effects of C and P on large and small coronary arteries and to compare them to those of nitroglycerin (N) in conscious dogs. Large coronary artery diameter (CD) and coronary blood flow (CBF) were measured in 6 conscious dogs previously instrumented with ultrasonic crystals (sonomicrometry) and electromagnetic flow probes implanted on the circumflex coronary artery. Mean arterial pressure (MAP, intra-aortic catheter) and hear: rate (HR, electrocardiogram) were monitored. Coronary vascular resistance (CVR) was calculated as the MAP/CBF ratio. Experiments were conducted at least three weeks after initial surgery and each dog received (i.v. bolus) ir, random order and on separate days N (0.03 to 10 pg/kg), C (1 to 10 pg/kg) and P (3 to 30 pg/kg). One day at least elapsed between two successive injections. At doses up to 0.3 pg/kg, N selectively increased CD without simultaneously altering either hemodynamic or other coronary parameters. In contrast, C and P at all doses and N at doses higher than 0.3 pg/kg always simultaneously and dose-dependently increased CD, CBF and HR whereas MAP and CVR were decreased (all p 24 337±43 8-12 CK 10pM -78.1±1.6 17.0±2.7 156±13 >24 233±32 16-20 11.2±3.1 97.0±6.9 CK 1pM -80.9±0.6 7.0±0.8 190±28 16-20 212±17 >24 7.4±1.1 Diazoxide 300pM 100.7±5.2 -76!0±0.7 14.5±2.8 154±20 1§-20 173±24 >24 Diazoxide 100pM 97.9±12.5 12.7±0.8 denotes change in Mean ± s.e.mean(n=3-7). tmax denotes time taken to reach maximum response (min); parameter For CK the rate of rise of K efflux was greater than that for Rb and this difference was more marked at the higher concentration level. For 1 and 10pM CK, K efflux reached its maximum between 8-12 min and 16-20 min respectively whereas Rb efflux was still rising at the end of the 24 min exposure period to the drug. The maximum changes in K efflux were greater than those for Rb. In contrast, diazoxide (300pM and 100pM) caused similar rises in both K and Rb efflux and changes in K efflux paralleled those of Rb. For CK the tmax values for the changes in K efflux, membrane potential and tension were similar. These results may indicate that the effects of CK at a Rb-impermeable K channel are largely responsible for the observed electrical and In contrast, the effects of diazoxide may involve a K channel permeable to Rb. mechanical changes. J. Longmore is supported by a grant from Rhone Poulenc. Bray, K.M. & Weston, A.H. (1989) Br. J. Pharmacol., (Proc. Manchester Meeting). Edwards, G. & Weston, A.H. (1989) Br. J. Pharmacol., (Proc Manchester Meeting).

4P A.

DIFFERENTIAL EFFECTS OF OUABAIN ON K+-INDUCED CONTRACTIONS OF GUINEA-PIG TRACHEAL AND AORTIC SMOOTH MUSCLE Lawson,

Laboratoires Jouveinal -

1,

rue

des Moissons,

B.P.

100,

94265

Fresnes

Cedex, France

(introduced by I. Cavero).

NaXK4-ATPase

may be partially responsible for the maintenance of basal tone of guinea-pig tracheal smooth muscle (Chideckel et al., 1987). In addition, this enzyme may have an important role in allergic diseases as indicated by the increase in Na+-K+ exchange after sensitization of the guinea-pig with antigen (Souhrada and Souhrada, 1982). The aim of this study was to compare the effects of the Na+,K+-ATPase inhibitor, ouabain, on K+-induced contractions of tracheal and aortic smooth muscle preparations of the guinea-pig.

Tracheal and aortic rings (devoid of epithelium and endothelium, Dunkin-Hartley guinea-pigs were suspended in modified Krebs solution, C02 and 02 (5:95). A load of 2g was applied to each ring. After viability was assessed by a single exposure to K (56mM). A single (10-100mM) was constructed per preparation after a 20 min incubation Results are expressed as mean ± s.e.mean.

respectively), obtained from male maintained at 370C and gassed with a 90 min equilibration period ring concentration-response curve to K+ with vehicle, ouabain or diltiazem.

a small initial The concentration-response curve to K+ on the trachea, was multiphasic consisting of contraction (10-12mM; Emaxi: 0.39 ± 0.07g, n = 12), followed by a return to basal tone (18mM) and then a second contractile phase (25-100mM; EAX2z: 2.14 ± 0.lOg). Ouabain (0.l-lOO1M), whilst not modifying the first phase, inhibited the contractions to 25-100mM KP (ICso: 0.64 ± 0.1M, n = 4). In contrast, diltiazem (0.1-10pM) inhibited both contractile phases to K+ (IC5o of EmaX2: 1.58 ± 0.1iM, n = 4). The K curve on the aorta was monophasic. Ouabain displaced the K+ curve to the left (ECWo : control 23.4 ± 1.5mM, n = 7; 3pM ouabain 8.2 ± 0.1mM, P< 0.05,n = 4) with no modification of maximum. Diltiazem induced a rightward displacement of the curve (response to 80mM K+: control 3.17 + 0.33g, n = 7; lOpM diltiazem 0.87 ± 0.19g, P< 0.05, n = 4). In conclusion, ouabain inhibited the contractile responses to K of guinea-pig tracheal smooth muscle but enhanced those of the aorta. Contractions of smooth muscle to Ke are the consequence of voltage operated calcium channel activation as indicated by the results with diltiazem. Further studies, however, are required to determine the role, if any, of VOCs in the observed effects of ouabain.

Chideckel E.W., Frost J.L., Mike P. and Fedan J.S.(1987) Br. J. Pharmacol., 92, 609-614 Souhrada M. and Souhrada J.F. (1982) Respir. Physiol., 47, 69-81

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2-DEOXY-D-GLUCOSE, AN INHIBITOR OF GLYCOLYSIS, INHIBITS CROMAKALIM BUT NOT SODIUM NITROPRUSSIDE INDUCED VASORELAXATION

A.D.Wickendenm

and T.L.Grant.

I.C.I.

Pharmaceuticals, Bioscience Dept.II, Alderley Park, Macclesfield,

Cheshire, SK10 4TG. Cromakalim is a novel vasodilator thought to act by opening K*-channels in vascular smooth muscle (Hamilton et al.,1986). Although the site(s) of action of cromakalim remains to be established recent findings have implicated the involvement of ATP-dependent K*-channels similar to those expressed in pancreatic B-cells (Buckingham et al., 1989, Davies et al., 1989). The aim of the present study was to compare the effect of 2deoxy-D-glucose (2-DG), an inhibitor of glycolysis, with cromakalim. In addition the vasorelaxant effects of cromakalim and sodium nitroprusside were compared following inhibition of glycolysis. Rings of rat thoracic aorta were suspended in Krebs' bicarbonate buffer at 370C and bubbled with 95Z02/5%C02. Changes in tension were recorded isometrically under a resting tension of 1g. The effects of 2-DG were assessed following incubation in Krebs' buffer in which glucose was replaced with an equimolar amount of 2-DG and supplemented with sodium acetate (5iM) to maintain oxidative phosphorylation.

Pre-incubation (30mins.) with cromakalim (lOuM) inhibited the response to noradrenaline (NA, luM) by 55.3 ± 2.6% in standard Krebs' buffer. This effect was reversed by glibenclamide ( 10.3 ± 6.1% inhibition of NA in the presence of glibenclamide, lOuM). In contrast, pre-incubation with 2-DG Krebs' markedly enhanced the response to NA (94.9 ± 24.0%). Enhanced responses in the presence of 2-DG were unaffected by glibenclamide(lOuM) or by pre-treatment with nitrendipine (luM). In additional experiments, cumulative concentration response curves were constructed to either cromakalim or sodium nitroprusside on NA(luM) precontracted tissues before and after incubation in 2-DG Krebs'. Following 2hr. incubation with 2-DG Krebs' the relaxant effect of cromakalim was markedly reduced ( 24.2 ± 7.3% of control activity). Under identical conditions the relaxant effect of sodium nitroprusside was unchanged.

The results clearly show that 2-DG does not mimic the effects of cromakalim. The mechanism by which 2-DG enhances the NA response is not clear but the since it was not prevented by nitrendipine it is unlikely to involve enhanced calcium uptake through voltage operated calcium channels. The finding that 2-DG inhibits cromakalim induced vasorelaxation is consistent with the hypothesis that cromakalim is acting via glycolytically regulated ATP-dependent K*-channels. However, the involvement of other ATP-dependent processes cannot be ruled out.

Buckingham,R.E., et al. (1989). Br.J.Pharmacol. 97,57-64. Davies,N.W., et al. (1989). J. Physiol., in press. Hamilton,T.C., et al. (1986). Br.J.Pharmacol.88,103-111.

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THE CARDIOVASCULAR EFFECTS OF SDZ PCO 400, A NOVEL K+ CHANNEL OPENER

J.R. Fozard*, K. Menninger, N.S. Cook, S. Blarer & U. Quast, Preclinical Research, Sandoz Ltd., CH-4002 Basel, Switzerland SDZ PCO 400 ((-)-(3S,4R)-3,4-dihydro-3-hydroxy-2,2-dimethyl-4-(3-oxo-cyclopent-1-enyloxy)-2H-1-benzopyran-6carbonitrile) induces relaxation of vascular smooth muscle by a mechanism consistent with opening of K channels (Quast et al., 1990). The present studies were carried out to define the cardiovascular profile of SDZ PCO 400 in vivo and to compare its effects with those of the prototype K channel opener, cromakalim. Male spontaneous hypertensive (SH) rats (320 - 380 g) were implanted with indwelling femoral arterial cannulae and used for experiments in the conscious state after a minimum I week recovery period. Other male SH rats (340 - 370 g) were anaesthetized with thiobutabarbital (Inaktin*), 150 mg/kg, and set up for recording blood pressure (BP), heart rate (HR) and, using miniaturized Doppler probes, renal, mesenteric, hindquarters and carotid blood flows (Wright & Fozard, 1988). Male and female rhesus monkeys (6 - 12 kg) were trained to sit quietly in restraining chairs and BP and HR were measured plethysmographically from the arm. Blood samples were withdrawn from a cannulated leg vein for determination of plasma renin. In conscious SH rats acute administration of SDZ PCO 400, 0.1 - 0.3 mg/kg p.o., induced dose-dependent decreases in BP which were rapid in onset (< 15 min) and of long duration (> 5 h). Increases in HR were also seen which were, however, neither dose-dependent nor well sustained. Cromakalim, 0.3 - 1 mg/kg p.o., induced similar maximum falls in BP to those seen with SDZ PCO 400. The effects were, however, less well sustained than those of SDZ PCO 400 unlike the accompanying tachycardia which was, at the higher dose, greater and of longer duration than that induced by SDZ PCO 400. The antihypertensive effect of SDZ PCO 400, 0.1 mg/kg p.o., was well maintained on repeated daily dosing for 4 days. In anaesthetized SH rats, both SDZ PCO 400, 0.1 - 0.3 mg/kg p.o., and cromakalim, 0.3 - 1 mg/kg p.o., produced similar, sustained falls in BP associated with vasodilatation in the renal, mesenteric, hindquarters and carotid vascular beds. In conscious rhesus monkeys, SDZ PCO 400, 0.1 - 0.3 mg/kg i.v. or p.o., gave dose-related and substantial falls in BP accompanied by tachycardia and an increase in plasma renin activity. SDZ PCO 400 was some 3-fold more active than prazosin by the oral route in this model although for any given fall in BP the changes in renin activity were similar. Cromakalim was only weakly active in the rhesus monkey: 0.1 - 1 mg/kg i.v. led to only shortlived falls in BP which were nevertheless associated with tachycardia and an increase in plasma renin activity; by the oral route, cromakalim was inactive up to 3 mg/kg. In conclusion, SDZ PC0 400 is a potent and long-lasting BP lowering agent when given orally to conscious SH rats and rhesus monkeys. Its haemodynamic profile is that of a peripheral vasodilator, consistent with an action primarily to open K+ channels in vascular smooth muscle. Quast, U. et al. (1990) This meeting Wright, C.E. & Fozard, J.R. (1988) Eur. J. Pharmacol. 155, 201 - 203

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THE CARDIOVASCULAR EFFECTS OF SDZ PCO 400 IN VIVO. COMPARISON WITH CROMAKALIM

U. Quast*, S. Blarer, P.W. Manley, N.S. Cook, C. Pally, & J.R. Fozard, Preclinical Research, Sandoz Ltd., CH-4002 Basel, Switzerland Potassium channel openers such as cromakalim (BRL 34915) comerise a new class of smooth muscle relaxants thought to act by increasing the membrane permeability for K . In this report, we describe the in vitro and in vivo vasorelaxant effects of a newly synthesized compound, SDZ PCO 400 ((-)-(3S,4R)-3,4-dihydro-3-hydroxy2,2-dimethyl-4-(3-oxo-cyclopent-1-enyloxy)-2H-1-benzopyran-6-carbonitrile). Comparisons have been made with cromakalim and/or its active (-) enantiomer, BRL 38227. Rat portal veins were loaded with 86Rb (as a marker for K+) 8gd mounted in a superfusion chamber for simulRb+ efflux (Quast, 1987). Both SDZ PCO 400 and taneous measurement of spontaneous contractile activity and BRL 382278 nhibited spontaneous activity with similar potency (pIC50 ca. 7.9) and increased the rate constant of Rb efflux concentration-dependently up to a maximum of 60% (pED30 ca. 6). The respective (+) enantiomers were 100 to 200 fold weaker in eliciting these effects. Potassium channel openers lose their vasodilator activity in depolarizing medium (see e.g. Quast & Cook, 1989). The vasorelaxant potency of SDZ PCO 400 and BRL 38227 (and their (+) enantiomers) were therefore compared on rat aortic rings precontracted with 0.1 pM noradrenaline in Krebs solution containing either 5 or 55 mM KCl (the latter in the presence of 0.1 pM isradipine). In 5 mM KCl, the (-) enantiomers relaxed the noradrenaline contraction up to 90% with a pIC45 value of 7.5. Under depolarizing conditions, both compounds were about 500 times less active in inhibiting the noradrenaline contraction, consistent with the dominant mechanism of vasorelaxation being K+ channel opening. The inhibition was stereospecific under either condition. In the rat isolated perfused mesenteric artery bed, SDZ PCO 400 and cromakalim inhibited contractions elicited by 1 s pulses of noradrenaline (100 pM) with pIC40 values of 7 2 and 6.7 respectively, indicating that they may interfere with the ability of noradrenaline to release Ca + from intracellular stores in resistance vessels (Quast, 1989). In normotensive anaesthetized thiobutabarbital (Inakting, 150 mg/kg i.p.) rats both SDZ PCO 400 and cromakalim, 0.1 mg/kg i.v., decreased blood pressure by ca. 35 mmHg and increased heart rate by ca. 30 beats/min. The effects on blood pressure were markedly reduced after pretreatment (15 min) with glibenclamide, 20 mg/kg i.v., a known inhibitor of the effects of K channel openers (Quast & Cook, 1989). In conclusion, the data show that SDZ PCO 400 induces vasorelaxation in vivo and in vitro by opening of K+ channels in the smooth muscle membrane. Quast, U. (1987) Br. J. Pharmacol. 91, 569 - 578 Quast, U. (1989) Br. J. Pharmacol. 97, Suppl. 114P Quast, U. & Cook, N. (1989) J. Pharmacol. Exp. Therap. 250, 261 - 271

8P

INTERACTIONS BETWEEN K+ CHANNEL OPENERS AND THE SYMPATHETIC NERVOUS SYSTEM IN PITHED SPONTANEOUSLY HYPERTENSIVE RATS

C. Richer*, P. Mulder & J.F. Giudicelli, Dtpartement de Pharmacologie, Faculte de Me&decine Paris-Sud, 94276 Le Kremlin-Bicetre Cedex, France. In vitro (Hamilton et al., 1986) and in vivo studies (Buckingham, 1988) have previously shown that cromakalim attenuates adrenergically-mediated contractile and pressor responses. The aims of the present study were (1) to assess whether the previously described systemic sympathoinhibitory effect of cromakalim also develops at the regional vascular levels and if so, whether it affects homogeneously or not the different vascular beds, and (2) to investigate if this sympathoinhibitory effect is a specific property of cromakalim or if it is also shared by other K+ channel openers, e.g. SR 44866, a newly developed drug of this group (Richer et al., 1989). Male SHRs were anesthetized with pentobarbital (50 mg/kg i.p.), artificially ventilated and pithed. Blood pressure and

heart rate were measured from the cannulated carotid artery. Cardiac output and regional (renal, mesenteric and hindlimb) flows were measured from Doppler velocity probes placed on the upper aorta, the renal and mesenteric arteries and the terminal aorta respectively. Corresponding vascular resistances were calculated. Cromakalim (I pg/kg/min, 15 min), SR 44866 (0.25 pg/kg/min, 15 min) or their solvent (0.09 ml/kg/min, 15 min) were then infused and their effects on the systemic and regional vascular responses (a) to increasing i.v. doses of either cirazoline, a selective al-adrenoceptor agonist (0.3 to 10 pg/kg) or UK-14,304, a selective 0(2-adrenoceptor agonist (3 to 100 pg/kg), and (b) to increasing frequencies of electrical stimulation of the spinal cord (0.25 to 4 Hz) were investigated and compared. Finally, the effects of the K+ channel openers vs spinal cord stimulation were also assessed after restoration of blood pressure to its pre-drug level by an infusion of either PGF20( (30 pg/kg/min) or vasopressin (4 mU/kg/min). The two K+ channel openers did not affect postsynaptic o 1- but slightly reduced postsynaptic o(2-adrenoceptor-mediated systemic pressor and regional vasoconstrictor responses. Both drugs significantly decreased the systemic pressor and regional vasoconstrictor responses elicited by spinal cord stimulation. These sympathoinhibitory effects were not homogeneously distributed among the-different vascular beds, the decreasing rank order being: mesentery>kidney>hindlimb. Simultaneously, the spinal cord stimulation-induced tachycardia remained unaffected. Restoration of blood pressure and vascular tone to pre-K + channel openers infusion levels by PGF2oe and vasopressin respectively did not affect and abolished the sympathoinhibitory effects of cromakalim and SR 44866. We conclude that in SHRs the two investigated K+ channel openers exert similar vascular syrrmpathoinhibitory effects which might thus be a common property to this group of drugs. These sympathoinhibitory effects which do not affect homogeneously the different vascular beds are not dependent upon the arterial blood pressure level and are most likely presynaptically located. Buckingham, R.E. (1988) Br. J. Pharmacol. 93, 541-552. Hamilton, J.R., Weir, S.W. & Weston, A.H. (1986) Br. J. Pharmacol. 88, 103-111. Richer, C., Mulder, P., Doussau, M.P. & Giudicelli, J.F. (1989) Arch. Mal. Coeur 82, 1333-1337.

THE EFFECT OF CROMAKALIM IN GUINEA-PIG STOMACH CANNOT BE ASCRIBED SOLELY TO OPENING K+ CHANNELS

9P

T.Tomital & AF. Brading', 'Department of Physiology, Faculty of Medicine, Nagoya University, Nagoya 466 Japan and University Department of Pharmacology, South Parks Road, Oxford OXI 3QT

Cromakalim will relax spontaneous tone in all smooth muscles so far studied. A common underlying chanism appears to be the opening of K chaels normally causinghyperpolarization ofthe smooth muscle plasma mmbrane, a decrease in membrane resistance and an Increase in K or Rb fluxes (Cook, 1988). The spontaneous mechanical actvty stomach muscle consists of rhythmic phasic contractions which are associated with regular slow depolarng pottials or slow waves (romita, 1981). These slow waves do not invarlablygeneratetensLon butiftheyarelarge enough eithertotrlggerCabasedactionpotentlals. ortheopeningofslowvoltage sensitive Ca channels, then contraction normally results. To investigate the actions of cakalim on stomach muscle, circular muscle strips were dissected from the antral region, mounted as previously described (Ohba et al., 1975) and perfused with Hepes-Tris buffered ungassed physiological saline either warmed to 32-35°C or at room temperature (25°C). Mechanical activity was recorded using a home-mountedAkers transducer, and membrane potentials were recorded with conventional KCI filled microelectrodes. imin

~mN |l mV

=U

f

FIgure 1. The effects of 3 x 1O-Mcomakalim on the mechanical (upper trace) and electrical Qower trace) activity of a circular muscle strip from guinea-pig stomach at 35°C. The drug was applied for the period indicated by the bar.

Figure 1 shows the effects of3pM cromakalim at 35°C. Note the membIrane hyperpolarization. and the cessaton of th mechanical activity, but that the slow waves persist with a slight increase in frequency, and an enhanced early spike component he hyperpolarization is assocated with a small increase in membrane conductance, is enhanced in K-free solution and reduced as extracellularKis elevated, consistent with an increase in K permeability. The increase In slow wave frequency is more pronounced at room temperature, and not obviously related to the increased K permeability. The most obvious and unexpected finding is the clear dissociation between the mechanical and electrical events, suggesting that crmakalm has a profound effect on electro-mechanical coupling in the stomach. Cook, N.S. (1988) Trends in Pharmacol. Sc., 9, 21-28

lop

ANTAGONISM OF RELAXIN BY GLIBENCLAMIDE IN THE UTERUS OF THE RAT IN VIVO

S.J. Downing* & M. Hollingsworth, Snmoth Muscle Research Group, Department of Physiological Sciences, University of Manchester, Oxford Road, Manchester M13 9PT. hanim of action my 1hlaxin is an inhibitor of utArine cmatractinm in vivo (Parter et al., 1979) %*hse be via insed ymetrial cAW amicentratios, altough these were not well corrlated trally with inhibition of contractions in vitro (Sartorn et al., 1980) . K-annel epuurs are also uterine relaxants (Hslll nawrth et al., 1987) ard are adi in vivo by giblamide (Sadraei et al., 1989) 7[e aim of this study was to determine w&bther re in was ized by glib lmide ocqxaedwith slbitaml, a uterine relaxant which acts via stimulation of adenylate cyclase. Fteale rats, 200-250g, were nsthetized with tri czethariol (240mg kg-1 i.p.), bilaterally ovariectanized to bolus and uipped with an intrauterine balloon and jugular vein c ila. After 24 h uterini res i.v. doses of porcine relaxin (2, 5, 20Og kg-1), or salmo1 (10, 50, 200jg kg-1) ware determined. i for 60 min followim relaxnt bolus was expressed as % of the intal for Interral of uterine c 60 mun prior to eac dose. FPzr bours after the last relant dose, glibenclamide (2Qmg kg-1) or vehicle (0.02N NaC in 4% glucose w/v, 5ml kg-) was infsed i.v. over 5 min followad 10 min later by a single bolus dose of relaxant (rreladn; 2-200pg kg-1, sa hbtamol; 10, 50 or 20Cpg kg-1, 20 rats per dose) -Ie % inhibition of uterine contractions was det-eined over 60 min as before. Pre-infusion log MD50 values for each animal and post-infusion log ID50 values from mean data were estimated using probit analysis. Uterine sensitivity to relaxin was unchanged in vehicle-infused rats (log ID50(Mg kg-i): pre-infusion, 1.03+0.04; post-infusion, 0.99). Glibenclamide infusion significantly reduced uterine responses to all s Of relaxin (p