Dual Mechanism of Action of Nicorandil on Rabbit Corpus Cavernosal Smooth Muscle Tone

Dual mechanism of action of nicorandil on rabbit corpus cavernosal smooth muscle tone

GC Hsieh1*, T Kolasa1, JP Sullivan1 and JD Brioni1

1Neurological and Urological Diseases Research, Abbott Laboratories, Illinois, USA

The potential of ATP-sensitive potassium channel openers (KCOs) for the treatment of male erectile dysfunction has recently been suggested based on positive clinical outcomes following intra-cavernosal administration of pinacidil. Agents that increase the levels of cGMP via elevation of nitric oxide (NO) nitroglycerin, for example, are also effective in improving erectile function preclinically and clinically. The aim of the present study was to determine the effects and mechanism of the action of nicorandil on rabbit corpus cavernosum. The in vitro regulation of smooth muscle tone was assessed in isolated cavernosal tissues pre-contracted with phenylephrine. Nicorandil, but not its major metabolite, relaxed phenylephrine-precontracted cavernosum smooth muscle with an EC50 of 15 mM. The effects of nicorandil were only partially reversed by the KATP channel blocker glyburide (10 mM) or by a soluble guanylate cyclase (sGC) inhibitor 1H-[1,2,4] oxadiazole [4,3-a] quinoxalin-1-one (ODQ, 3 mM). However, a combination of ODQ and glyburide completely blocked the relaxant effects of nicorandil. The results of the present study indicate that nicorandil can relax rabbit cavernosal tissue in vitro via a mechanism that involves activation of KATP channels and stimulation of soluble guanylate cyclase. International Journal of Impotence Research (2001) 13, 240±246.

Keywords: nicorandil; erectile dysfunction; KATP channel; guanylate cyclase

Introduction and NO donors) have shown limited ef®cacy in clinical trials for male erectile dysfunction.4 How- ever, since these agents relax the peripheral vascu- Corpus cavernosal smooth muscle tone is controlled lature via a similar mechanism and are prone to by a number of discrete signaling pathways co- systemic side-effects,5 they must be administered ordinated at the level of the peripheral and central locally by intracavernosal injection or topically. nervous system. This smooth muscle tone ulti- ATP-sensitive potassium channels (KATP) are an mately regulates penile ¯accidity and erection.1 important family of potassium channels that couple Contraction is primarily mediated by alpha- cellular energy metabolism to membrane electrical adrenoceptor stimulation1,2 while relaxation is 6,7 activity. At physiologic ATP levels, KATP chan- mediated by the interaction of several types of nels remain mostly in their closed state while at low neurotransmission, including nonadrenergic ± non- ATP levels, the channels open. Potassium channel cholinergic (NANC) neural input and the release of openers (KCOs) such as L-cromakalim, diazoxide, nitric oxide,1,3 Nitric oxide, released from NANC pinacidil and its analogue P1075 activate KATP nerve terminals and from cholinergically-activated channels producing hyperpolarization of vascular endothelial cells, diffuses into adjacent smooth and non-vascular smooth muscle membranes which muscle cells where it activates soluble guanylate reduce Ca2 in¯ux through voltage-activated L-type cyclase. This increases the intracellular levels of Ca2 channels, thus resulting in smooth muscle cyclic guanosine monophosphate (cGMP) that leads relaxation.6 Recently, the prototypic potassium to the relaxation of cavernosal smooth muscle. The channel openers such as L-cromakalim and pinaci- increased blood ¯ow and engorgement of the dil, when administered as intracavernosal injec- trabecular spaces results in veno-occlusion and tions, have been found effective in the initiation penile erection. Nitrovasodilators (organic nitrates and maintenance of penile erection in experi- mental animal models8±10 and in clinical study in humans.11 *Correspondence: G Hsieh, Neurological and Urological Nicorandil (N-(2-hydroxyethyl)nicotinamide ni- Diseases Research, D-4ND, AP-9, Abbott Laboratories, 100 Abbott Park Road, Abbott Park, IL 60064-6119, USA. trate ester) is an orally ef®cacious anti-anginal E-mail: [email protected] drug.12 While this agent has been shown to relax Received 21 November 2000; accepted 16 April 2001 human corpus cavernosum,13 the mechanism under- KATP and guanylate cyclase activation by nicorandil GC Hsieh et al 241 lying this effect remains to be fully elucidated. The dissected free of the tunica albuginea and surround- present study was designed to investigate the ing connective tissues). Each tissue strip was mechanism of action of nicorandil in rabbit corpus longitudinally cut into three strip preparations cavernosum. Speci®cally, the role of the KCO and measuring approximately 26267 mm (unstretched nitrate activities associated with this compound was length, mean weight 30 mg). investigated. Additionally, the possibility that the relaxant effects of nicorandil are mediated via its major metabolite was also assessed. Measurement of isometric tension

Materials and methods The strips were transferred and mounted to organ baths (10 ml) containing Kreb ± Henseleit buffer solution (pH 7.4) maintained at 37C by a thermo- Drugs regulated water circuit. The buffer solution con- tained D-glucose 2.0 g=l, MgSO4 0.141 g=l, KH2PO4 Nicorandil (N-(2-hydroxyethyl) nicotinamide nitrate 0.16 g=l, KCl 0.35 g=l, NaCl 6.9 g=l (Sigma Chemical ester) and its metabolite N-(2-hydroxyethyl) nicotin- Co) plus CaCl2.2H2O 0.373 g=l and NaHCO3 2.1 g=l. The buffer was continuously aerated with a mixture amide (Figure 1), KATP channels openers L-croma- of 95% O =5% CO . The tissue strips were loaded kalim, P1075, and KATP channel blocker glyburide 2 2 were synthesized at Abbott Laboratories (Abbott with a resting tension of 2 g and equilibrated for Park, IL). Guanylate cyclase inhibitor ODQ (1H- 90 min. During equilibration, the bath solution was [1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one), diazoxide, replaced every 10 ± 15 min. Changes in isometric sodium nitroprusside, and phenylephrine were tension of muscle tissues were measured using a purchased from Sigma Chemical Co. (St Louis, force-displacement transducer (Grass FT03 model, MO). Nicorandil was dissolved in dimethyl sulf- Grass Instruments Co., Quincy, MA) and recorded oxide (DMSO) to make a 100 mM stock solution. All on a Grass 7D model polygraph. Repeated adjust- drugs, unless indicated otherwise, were further ment of tension was performed if necessary. No diluted with ethanol before adding into the buffer changes in tension were made after the experiment solutions in tissue bath chamber so that the ®nal was started. total solvent (DMSO plus ethanol) concentrations in the assays was less than 0.5% (v=v) which itself did not alter the smooth muscle tone. Stock solutions of Drug treatment phenylephrine prepared in saline included 0.1% ascorbic acid as an antioxidant. The relaxation effects of nicorandil, nicorandil metabolite, L-cromakalim, P1075, diazoxide, and Tissue preparation sodium nitroprusside were assessed in rabbit corpus cavernosum preparations precontracted with phenylephrine (1.5 mM). At stable tension, compounds The in vitro regulation of smooth muscle tone was to be tested were added cumulatively at half log unit assessed using isolated rabbit cavernosum smooth increments, and a new concentration was not added muscle strips mounted in organ bath chamber and until the response to the previous one had attained a pre-contracted with phenylephrine. Corpus caver- steady state tension. nosum tissues were prepared from adult male New In another set of experiments, tissue preparations Zealand white rabbits (Covance Research Produc- were contracted with phenylephrine 1.5 mM and tion, Kalamazoo, MI), weighing 3.0 ± 3.5 kg, after subsequently treated with 3 ± 10 mM ODQ (a guany- euthanasia by intravenous pentobarbital sodium late cyclase inhibitor), 10 mM glyburide (for compe- injection. The two corpus cavernosum strips were titive blockade of ATP-dependent K channel), carefully dissected under a illuminated magni®er (a or combination of ODQ (3 mM) and glyburide slit was made in the proximal end of the tunica and (10 mM) for 15 min. The inhibitors were added after extended distally and the corpus cavernosum was the phenylephrine-induced contraction reached

O O O OH N N NO2 H H N N

Figure 1 Structure of nicorandil and its metabolite N-(2-hydroxyethyl) nicotinamide.

International Journal of Impotence Research KATP and guanylate cyclase activation by nicorandil GC Hsieh et al 242 steady-state. A cumulative concentration ± response Effects of KATP channel openers P1075, curve was then constructed for the relaxant agents L-cromakalim, and diazoxide on phenylephrine above described. contracted corpus cavernosal strips

In order to determine the effect of other KCOs Data analysis lacking nitrate activity on rabbit corpus cavernosal smooth muscle tone, three reference openers, P1075, Sigmoid curves were ®tted to concentration- L-cromakalim, and diazoxide, were evaluated. All response data by nonlinear regression analysis three KCOs relaxed the tissue preparation in a concentration-dependent manner. The EC values (GraphPAD, San Diego, CA) to obtain EC50 values 50 as appropriate. Data are expressed as the mean for P1075, L-cromakalim and diazoxide were 0.064, Æ s.e.m. 0.75, and 85 mM, respectively (Figure 3). The rank order of potency of these compounds in this tissue is similar to that reported previously for other smooth muscle tissues.14 L-cromakalim-induced relaxa- Results tion was completely inhibited by the pretreatment with the selective KATP channel blocker, glyburide Effects of nicorandil and its metabolite on (Figure 4). phenylephrine contracted corpus cavernosal strips

Phenylephrine contracted isolated rabbit corpus cavernosal tissue strips in a concentration-depen- Effects of guanylate cyclase activator sodium dent manner with an EC50 of 1.5 mM. To investigate nitroprusside on phenylephrine contracted the relaxant effects of nicorandil, corpus caver- corpus cavernosal strips nosum strips were precontracted with phenylephrine (1.5 mM) and then treated with increasing concentrations of compound (1078 ±1074 M). Nico- The NO-donor sodium nitroprusside (1079 ± randil elicited a concentration-dependent relaxation 1075M) produced a concentration-dependent relax- of smooth muscle strips (Figure 2); the potency ation of the cavernosal tissue strips (Figure 5) with (EC50) determined from cumulative concentration ± an EC50 value of 0.37 mM. Pretreatment with the response curve was 15 mM. In contrast, the major guanylate cyclase inhibitor ODQ (3 and 10 mM) metabolite of nicorandil (N-(2-hydroxyethyl) nicotin- completely blocked the relaxant effects of sodium amide) did not relax phenylephrine precontracted nitroprusside. In contrast, glyburide (10 mM) had no cavernosum tissue at concentrations up to 100 mM effect on the relaxant properties of sodium nitro- (Figure 2). prusside (data not shown).

-20 -20 0 0 20 20 40 40 60 60

%Relaxation 80 Nicorandil P1075

%Relaxation 80 100 Nicorandil metabolite L-Cromakalim 100 Diazoxide 120 -9 -8 -7 -6 -5 -4 -3 120 -12 -11 -10 -9 -8 -7 -6 -5 -4 -3 -2 Log [Nicorandil, M] Log [Compounds, M]

Figure 2 Effects of nicorandil and its metabolite on phenylephrine (1.5 mM) precontracted rabbit corpus cavernosum strips. Figure 3 Relaxant effects of K-ATP channel openers P1075, L- Data are expressed as means s.e.m. (n 4 ± 5). Phenylephrine at cromakalim and diazoxide on contractions induced by phenyl- 1.5 mM elicited a stable contractile response in tissue strips with ephrine 1.5 mM in rabbit corpus cavernosal strips. Data are an increased tension response of 2.25 g. expressed as means Æ s.e.m. (n 5).

International Journal of Impotence Research KATP and guanylate cyclase activation by nicorandil GC Hsieh et al 243 -20 -20 0 0 20 20 40 40 L-Cromakalim 60 60 Nicorandil L-Cromakalim %Relaxation 80 %Relaxation 80 + Glyburide 10 µM +10µM Glyburide 100 100 + ODQ 3 µM 120 120 -8 -7 -6 -5 -8 -7 -6 -5 -4 -3 Log [L-Cromakalim, M] Log [Nicorandil, M]

Figure 4 Relaxant effects of K-ATP channel opener L-cromaka- Figure 6 Cumulative concentration response for nicorandil in lim on phenylephrine (1.5 mM) precontracted rabbit corpus relaxing the phenylephrine (1.5 mM) precontracted rabbit corpus cavernosum in the absence and presence of K-ATP channel cavernosal tissue strips in the absence and presence of K-ATP blocker glyburide (10 mM). Data are expressed as means Æ s.e.m. channel blocker glyburide (10 mM) or guanylate cyclase inhibitor (n 4). ODQ (3 mM). Data are expressed as mean Æ s.e.m. (n 4).

Effects of glyburide and ODQ on the relaxant only partially ( 40%) reversed. Similarily, the response to nicorandil effects of nicorandil (100 mM) were only partially reversed by ODQ (3 mM). However, in the presence of the combination of glyburide (10 mM) and ODQ The studies with KCOs and sodium nitroprusside (3 mM), the relaxant effects of nicorandil (100 mM) described above indicated that both KATP channel were completely antagonized (Figure 7). activation and guanylate cyclase activation can relax corpus cavernosum. To determine if the relaxant effects of nicorandil are mediated via one or both of these pathways, the effects of glyburide and ODQ Discussion alone or in combination on the relaxant effects of nicorandil were investigated. As shown in Figure 6, the relaxant effects of nicorandil at concentra- In this report we have found that nicorandil tions <30 mM were completely blocked by glybur- effectively relaxes rabbit corpus cavernosum smooth ide. However, higher concentrations (100 mM) were muscle strips pre-contracted with phenylephrine

-20 -20 0 0 20 20 40 40 Nicorandil 60 60 SNP Nicorandil

%Relaxation 80 %Relaxation 80 SNP + ODQ 3 µM + ODQ 3 µM 100 SNP + ODQ 10 µM 100 + Glyburide 10 µM 120 120 -10 -9 -8 -7 -6 -5 -4 -9 -8 -7 -6 -5 -4 -3 Log [Nicorandil, M] Log [SNP, M]

Figure 7 Cumulative concentration response for nicorandil in Figure 5 Relaxant effects of guanylate cyclase activator sodium relaxing the phenylephrine (1.5 mM) precontracted rabbit corpus nitroprusside (SNP) on phenylephrine (1.5 mM) precontracted cavernosal tissue strips in the absence and presence of the rabbit corpus cavernosal tissue strips in the absence and presence mixture of K-ATP channel blocker glyburide (10 mM) and of guanylate cyclase inhibitor ODQ (3 and 10 mM). Data are guanylate cyclase inhibitor ODQ (3 mM). Data are expressed as expressed as mean Æ s.e.m. (n 4). means Æ s.e.m. (n 4).

International Journal of Impotence Research KATP and guanylate cyclase activation by nicorandil GC Hsieh et al 244 in vitro. This relaxation can be partially blocked by nicorandil-induced relaxation, indicating that solu- glyburide, suggesting activation of ATP-sensitive K ble guanylate cyclase activation plays an important channels; or partially inhibited by ODQ, suggesting role on the relaxation effects of nicorandil on soluble guanylate cyclase activation. At concentra- phenylephrine precontracted cavernosum smooth tions less than 30 mM, nicorandil appears to act muscle. ODQ completely inhibits the effects of NO through the opening of potassium channels. At donor sodium nitroprusside induced relaxation higher concentrations, nicorandil appears to act (Figure 5). Guanylate cyclase catalyzes the biosyn- through the NO-activated guanylate cyclase. While thesis of cGMP from GTP and exists in two isozyme both pathways contribute to these effects, they are forms, the soluble form and the membrane-bound independent. Further, our studies demonstrate form21 By formation of cGMP as a second messen- that N-(2-hydroxyethyl) nicotinamide, a main deni- ger, guanylate cyclase plays a critical role in trated metabolite of nicorandil, is pharmacologically different physiological processes, eg smooth muscle inactive. relaxation, platelet aggregation, and neuronal trans- Potassium channels regulate membrane potential mission.1,2,21 The most important physiological and modulate smooth muscle tone.6 Recent studies activator of soluble guanylate cyclase is the endo- both in vitro and in vivo have documented the thelium-derived relaxing factor, which is con- potential physiological=pathophysiological impor- sidered to be NO or an NO-related compound. tance of potassium channels to the modulation of Activation of soluble guanylate cyclase by NO is corpus cavernosal smooth muscle tone.15 ± 18 The heme-dependent.22 ± 24 Therapeutically, vaso- KATP and maxi-K potassium channel subtypes are dilatory agents such as nitroprusside, linsidomine thought to be the two most physiologically relevant (SIN-1), and nitroglycerin (glyceryl trinitrate) which potassium channels expressed in corpus cavernosal activate soluble guanylate cyclase after conversion smooth muscle.16,17 Signi®cant reduction in the to NO have shown ef®cacy in limited clinical trials sensitivity of precontracted corpus cavernosum in humans for the treatment of erectile dysfunction smooth muscle tissue strips excised from diabetic by intracavernous injection.4 However, since these patients to relaxation by structurally diverse KATP agents relax the peripheral vasculature via a similar channel modulators pinacidil and P1075, when mechanism and are prone to systemic side-effects,5 compared to the tissues obtained from non-diabetic they must be administered locally by intracavern- patients, suggests that a decrease in expression or an osal injection or topical application. impairment of function in these channels may occur Recently advances in the physiology of male in erectile dysfunction.18 Transfection of the human sexual function have established that corpus caver- maxi-K hSlo cDNA into aged rats in vivo has shown nosum smooth muscle relaxation is the primary that increased expression of the human maxi-K event of penile erection.1 Clinically, intracavernous channel produces the physiologically relevant im- injection therapy is the most effective pharmaco- provements in penile function.15 Thus, gene therapy logical treatment for erectile dysfunction. However, or therapeutic approaches to enhance existing the treatment discontinuation rate for the intraca- channel activity may be effective treatments for vernous injection therapy of alprostadil programs is erectile dysfunction. 50% in many studies because of the method of Agents that decrease cell excitability, due to administration and side effects of penile pain and hyperpolarization of cell membranes following K burning sensations.4 Our results suggest that nicor- channel opening, may offer a novel therapeutic andil, a hybrid molecule with KATP channel opening approach.6,20 Recently, in limited clinical studies in and nitrate-like properties, appears to hold promise humans, the prototypic potassium channel openers as direct effector of corpus cavernosum smooth such as pinacidil11 and PNU-8358719 when injected muscle relaxation (Figure 8). NO released from intracavernosally, were found to be effective in nicorandil rapidly diffuses into the smooth muscle the initiation and maintenance of penile erection. cells to activate guanylate cyclase, which is respon- Unlike intracavernosal injection of PGE1 there were sible for the conversion of GTP to cGMP. The no complaints of penile pain often associated with increase of cGMP initiates a series of reactions that injection. ultimately reduce intracellular Ca2, leading to the In contrast to relaxation by the potassium channel smooth muscle relaxation. The opening of KATP opener L-cromakalim which was completely channels causes cellular membrane hyperpolariza- blocked by glyburide (Figure 4), nicorandil-induced tion that reduces calcium ion in¯ux through a relaxation on the smooth muscle tone was only decreased activity of voltage-dependent calcium partially inhibited through blocking the glyburide- channels. This in turn results in corpus cavernosum sensitive KATP channel (Figures 6 and 7). These data smooth muscle relaxation. The ®ndings of the mode suggest that other pathways besides potassium and mechanism of action of nicorandil suggest channel opening activity are responsible for the that compounds like nicorandil with a dual mech- effects of nicorandil. The present studies also anism may be of clinical importance for male demonstrate that ODQ, a selective inhibitor of erectile dysfunction because ef®cacy is expected soluble guanylate cyclase, signi®cantly inhibits to be increased by employing two independent

International Journal of Impotence Research KATP and guanylate cyclase activation by nicorandil GC Hsieh et al 245

Figure 8 Dual mechanism of nicorandil in relaxing rabbit corpus cavernosum smooth muscle. mechanisms, when drug is administered locally to 3 Burnett A. Nitric oxide in the penis: physiology and achieve high enough doses that both K channel pathology. J Urol 1997; 157: 320 ± 324. ATP 4 Meinhardt W, Kropman RF, Vermeij P. Comparative toler- opening and guanylate cyclase activation are in- ability and ef®cacy of treatment for impotence. Drug Safety volved. Nicorandil is a potent vasodilating agent for 1999; 20: 133 ± 146. the treatment of myocardial hypoxia and two 5 Leopold JA, Loscalzo J. New developments in nitrosovasodi- postulated mechanisms of action on different parts lator therapy. Vasc Med 1997; 2: 190 ± 202. 12,25 6 Lawson K. Potassium channel openers as potential therapeutic of the vascular systems have also been reported, weapons in ion channel disease. Kidney Int 2000; 57: 838 ± the nitrate-like effect of nicorandil inducing the 845. vasodilation of large coronary arteries, and its 7 Lee SW, Wang HZ, Christ GJ. Characterization of ATP- sensitive potassium channels in human corporal smooth opening action on KATP channels dilating the coronary resistance vessels. muscle cells. Int J Impot Res 1999; 11: 179 ± 188. 8 Trigo-Rocha F et al. The effect of intracavernous injection of In conclusion, nicorandil, but not its non-NO potassium channel openers in monkeys and dogs. Int J Impot containing metabolite, induced corpus cavernosum Res 1995; 7: 41 ± 48. smooth muscle relaxation, suggesting the impor- 9 Giraldi A, Wagner G. Effects of pinacidil upon penile erectile tance of NO in the mechanism. Consistent with this tissue, in vitro and in vivo. Pharmacol Toxicol 1990; 67: 235 ± hypothesis, ODQ, an inhibitor of soluble guanylate 238. 10 Holmquist F, Andersson KE, Fovaeus M, Hedlund H. K ( )- cyclase, inhibited nicorandil-mediated smooth channel openers for relaxation of isolated penile erectile tissue muscle relaxation. Furthermore, glyburide, an from rabbit. J Urol 1990; 144: 146 ± 151. ATP-sensitive K channel blocker, also inhibited 11 Moon DG, Byun HS, Kim JJ. A K-ATP channel opener as a the relaxatory effects of nicorandil. Taken together, potential treatment modality for erectile dysfunction. Br J Urol Int 1999; 83: 837 ± 841. these data suggest that a dual mechanism involving 12 Goldschmidt M, Landzberg BR, Frishman WH. Nicorandil: a both NO-mediated activation of soluble guanylate potassium channel opening drug for treatment of ischemic cyclase and the opening of potassium channels is heart disease. J Clin Pharmacol 1996; 36: 559 ± 572. responsible for the effects of nicorandil on corpus 13 Hedlund P, Holmquist F, Hedlund H, Andersson KE. Effects of nicorandil on human isolated corpus cavernosum and caver- cavernosum smooth muscle. nous artery. J Urol 1994; 151: 1107 ± 1113. 14 Gopalakrishnan M et al. Characterization of the ATP-sensitive potassium channels (K-ATP) expressed in guinea pig bladder Acknowledgements smooth muscle cells. J Pharmacol Exp Ther 1999; 289: 551 ± 558. 15 Christ GJ et al. Intracorporal injection of hSIo cDNA in rats The authors are grateful to Dr Robert B Moreland for produces physiologically relevent alterations in penile func- his valuable comments on the manuscript. tion. Am J Physiol 1998; 275: H600 ± H608. 16 Lee SW, Wang HZ, Christ GJ. Characterization of ATP-senstive potassium channels in human corporal smooth muscle cells. Int J Impot Res 1999; 11: 179 ± 198. References 17 Fan SF, Brink PR, Melman A, Christ GJ. An analysis of the maxi-K (KCa) channel in cultured human corporal smooth 1 Moreland RB, Nakane M, Hsieh GC, Brioni JD. Prospectives for muscle cells. J Urol 1995; 153: 818 ± 825. pharmacotherapy of male erectile dysfunction. Current Opi- 18 Giraldi A et al. Differential relaxation of human corpus nions CPNS Drugs 2000; 2: 283 ± 302. cavernosum smooth muscle by potassium openers: evidence 2 Traish AM, Kim NN, Goldstein I, Moreland, RB. Alpha that relaxation is both agonist dependent and altered by adrenergic receptors in the penis. J Androl 1999; 20: 671 ± 682. diabetes melitus. Int J Impot Res 1995; 7: 1 ± 10.

International Journal of Impotence Research KATP and guanylate cyclase activation by nicorandil GC Hsieh et al 246 19 Vick RN, Patel M, Benevides M, Carson CC. The ef®cacy, 23 Zhao Y, Brandish PE, Ballou DP, Marletta MA. A molecular safety, and tolerability of intracavernosal PNU-83587 in the basis for nitric oxide sensing by soluble guanylate cyclase. treatment of erectile dysfunction. J Urol 2000; 163: 20. Proc Natl Acad Sci USA 1999; 96: 14753 ± 14758. 20 Quast U. Potassium channel openers: pharmacological and 24 Denninger JW et al. Interaction of soluble guanylate cyclase clinical aspects. Fundam Clin Pharmacol 1992; 6: 279 ± 293. with YC-1: kinetic and resonance Raman studies. Biochemistry 21 Garthwaite J et al. Potent and selective inhibition of nitric 2000; 39: 4191 ± 4198. oxide-sensitive guanylyl cyclase by 1H-[1,2,4]oxadiazolo[4, 3- 25 Berdeaux A et al. Differential effects of nitrovasolidators, K a]quinoxalin-1-one. Mol Pharmacol 1995; 48: 184 ± 188. channel openers and nicorandil on large and small coronary 22 Denninger JW, Marletta MA. Guanylate cyclase and the arteries in conscious dogs. J Cardiovasc Pharmacol 1992; NO=cGMP signaling pathway. Biochim Biophys Acta 1999; 20(Suppl 3): S17 ± S21. 1411: 334 ± 350.

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