Br. J. Pharmacol. (1992), 105, 715-719
C) Macmillan Press Ltd, 1992
Effects of pinacidil on guinea-pig isolated perfused heart with particular reference to the proarrhythmic effect 'Roberto Padrini, Sergio Bova, Gabriella Cargnelli, *Donatella Piovan & *Mariano Ferrari Department of Pharmacology and *Institute of Internal Medicine, University of Padova, Padova, Italy 1 The effects of pinacidil (10, 30, 50,M) on contractility (+dP/dt,,), coronary perfusion pressure (cP), and ECG intervals (PR, QRS, QT) have been studied on constant-flow perfused guinea-pig hearts, driven at four frequencies (2.5, 3, 3.5, 4 Hz). 2 Pinacidil decreased +dP/dt,,,, cP and the QT interval in a dose-dependent manner, whereas the PR interval was increased. QRS duration was not modified. All these effects were independent of driving frequency. Pinacidil decreased the interval from Q-wave to T-wave peak (QTpeak) to a greater extent than the QT interval, thus decreasing the QTpeak/QT ratio. This effect, unlike that on QT interval, was more evident at the highest frequency of stimulation. 3 In 4 out of 20 hearts treated with pinacidil sustained ventricular fibrillation (VF) occurred following a short run of premature ventricular beats (R on T phenomenon). 4 In separate experiments, an attempt to induce VF electrically was made at drug concentrations ranging from 1OpM to 1OM (8 experiments for each concentration). In control conditions and at the lowest concentration of pinacidil tested (1OpM) VF could never be induced; in the presence of 30pMm pinacidil VF was induced in 5 out of 8 experiments. Drug concentrations higher that 50 gM permitted the induction of VF in every case. 5 Although the concentrations of pinacidil producing ventricular fibrillation are 30-40 times higher than those found in patients under long term treatment with this agent, it is suggested that caution should be used in prescribing this drug, at least in patients suffering from myocardial ischaemia. Keywords: Pinacidil; heart contractility; coronary tone; ECG; ventricular fibrillation
Introduction As a typical 'K channel opener' (KCO), pinacidil has been shown to relax various types of smooth muscle by increasing K conductance and hyperpolarizing the cell membrane (Weston, 1989). These properties account for the successful clinical application of pinacidil in antihypertensive therapy (Goldberg, 1988) and for the therapeutic potential of KCOs in the treatment of bronchial asthma, irritable bladder syndrome and angina pectoris (Cook, 1988). At a cardiac level, pinacidil shortens the action potential (AP) duration of ventricular and Purkinje cells (Smallwood & Steinberg, 1988); decreases the automaticity of Purkinje fibres (Steinberg et al., 1988); decreases contractility and sinus rate (at least in isolated preparations) (Longman et al., 1988; Steinberg et al., 1988; Nielsen et al., 1989); and increases coronary flow (Kawashima & Liang, 1985). Increasing amounts of experimental data have become available on the influence of pinacidil on heart rhythmicity. This aspect of the action of the drug, while of great clinical importance, is somewhat puzzling in that pinacidil, on the one hand, can favour re-entrant type arrhythmias (acute ischaemia models; Wollenberg et al., 1989; Chi et al., 1990) and, on the other, it exhibits a definite antiarrhythmic action on arrhythmias produced by increased automaticity (subacute ischaemia, barium-induced arrhythmias, Kerr et al., Steinberg et al., 1988). The present work describes the pharmacodynamic profile of pinacidil in Langendorff-perfused guinea-pig hearts driven at different stimulation rates and, specifically, focuses on the influence of the drug on heart rhythmicity in normoxic conditions.
Methods The preparation used was the constant flow-perfused guineapig heart (Langendorff preparation). An extensive description of the procedure followed can be found elsewhere (Bova et al., I Author for correspondence at: Department of Pharmacology, Largo Egidio Meneghetti 2,1-35131 Padova, Italy.
1989). In brief, guinea-pigs of either sex (weight: 250-350 g) were killed by a blow to the neck and the heart was rapidly removed and retrogradely perfused within 45s of removal. The perfusion medium was a modified Krebs-Henseleit solution (mM: NaCl 118, KCI 4.7, CaCl2 2.5, MgSO4 1.2, NaCO3 25, KH2PO4 1.2, glucose 11.1 and pyruvate 2) bubbled with an 02:CO2 gas mixture (95%:5%) and maintained at 370C (pH 7.4 + 0.01). The flow of perfusate was kept constant at about 10mlg-' tissue min-' with a Gilson peristaltic micropump. After excising the sinus node the heart was driven at various frequencies (see below) with a S88 Grass stimulator through platinum electrodes placed on the left atrium. Intraventricular pressure was measured by means of a rubber balloon inserted into the left ventricle and connected to a mechano-electrical transducer (MARB P82). The maximal positive derivative of the intraventricular pressure (+ dP/dt,,,j,,) was taken as an index of contractility. Surface ECG was recorded by means of two electrodes, one placed on the crux cordis and the other on the left ventricle free wall. The signal, amplified with a P16 Grass amplifier, was monitored with a Tektronics oscilloscope and simultaneously recorded on magnetic tape. PR, QRS and QT intervals were measured on magnified photographs of the tracings 'frozen' on the oscilloscope screen. In addition to QT duration the interval from Q-wave to T-wave peak was also determined (QTpeak) and the QT/QTpeak ratio was calculated. Coronary perfusion pressure (cP) was also monitored to assess the change in coronary bed resistance. A solution of pinacidil was infused, with a peristaltic pump, through a T-junction at very low flow rate (28 times lower than the total flow); the very small increase in the total flow rate induced when infusion was started did not elicit any detectable change of the parameters measured. The drug concentrations in the infusing solutions were calculated in order to obtain final pinacidil concentrations of 10, 30, 50 M in the aortic cannula.
Study protocol The effects of pinacidil on contractility (+dP/dt,,.jX), coronary resistance (cP) and ECG intervals were evaluated at four
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driving frequencies: 2.5, 3, 3.5 and 4Hz. The choice of such a narrow range of frequencies was justified for two technical reasons: (1) in some preparations a frequency lower than 2.5 Hz allows a spontaneous junctional rhythm to arise; (2) at frequencies higher than 4Hz the stimulus artifact on ECG tracings can fall close to the end of preceding T wave, making it impossible to measure the QT interval. Each preparation was first allowed to recover for 30-45 min with stimulation at 2.5 Hz and then frequency was increased in 0.5 Hz steps up to 4Hz, leaving 5min for stabilization at each frequency step. After 15 min exposure of the heart to a single drug concentration the same stimulation procedure was repeated. Four to six experiments were performed at each concentration. Repetition of the stimulation protocol per se was found to have no influence on the cardiac parameters measured.
Determination of ventricularfibrillation threshold In a separate series of experiments an attempt was made to induce ventricular fibrillation (VF) by electrical stimulation in basal conditions and after 15min exposure to 10, 30, 50 and 100pUM pinacidil (n = 8 at each concentration). After removing the atrial free wall and septum, stimulating electrodes were fitted over the upper edge of the intraventricular septum and a stimulation protocol similar to that described by Almotrefi & Baker (1980) was applied. Driving frequency was suddenly increased from 2.5 to 20 Hz and current intensity was progressively increased either until VF occurred or an intensity 10 fold greater then the basal value was reached.
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Figure 1 Frequency-dependence of the cardiac parameters of the guinea-pig isolated perfused heart measured under control conditions (n = 16). Changes are expressed as a percentage of the value observed at 2.5 Hz. Symbols: (0) +dP/dtm,,; (A), PR interval; (0) coronary pressure; (A), QT interval. Significant change from baseline: * P < 0.05.
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At the stimulation rate of 2.5 Hz the mean control values (±s.e.) of Cp, dP/dt,,aX, PR interval, QRS interval, QT interval duration and QTpeak/QT ratio were 64.0 + 2.2 mmHg, 1405 + 86mmHgrs-, 48.6 + 2.2ms, 22.5 + 0.4ms, 180.1 + 3.4 ms and 0.900 + 0.004, respectively. As expected, when the driving frequency was increased in steps, + dP/dtmax and PR increased while QT and cP decreased (Figure 1). QRS duration and QTpeak/QT ratio were rate-independent. In the presence of pinacidil (10, 30 or 5OpM) coronary pressure and cardiac contractility decreased, QT shortened and PR lengthened in a dose-dependent way, while QRS duration was not modified. The effects of pinacidil were virtually identical at any frequency applied, hence only the effects observed as 2.5 Hz are shown in Figure 2. Although the narrow range
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concentration (50M) were -74.0% + 2.2 for +dPdt x -51.3% + 3.6 for cP, -34.3% + 4.2 for QT and + 16.2 + 2.6 for PR. As well as shortening the QT interval duration, pinacidil changed the morphology of the T-wave (Figure 3) in that it shortened the QTpeak interval to a greater extent than the QT interval, thus decreasing the QTpeak/QT ratio. The effect on QTpeak/QT ratio was dose-dependent and, unlike that on QT interval, also rate-dependent at least at the highest concentration tested (50M). Figure 4 shows the effect of pinacidil
Figure 3 Pinacidil-induced ECG changes in guinea-pig isolated perfused heart. The drug lengthens- PR interval, shortens QT interval and modifies QT-wave shape. (a) Control (2.5 Hz); (b) pinacidil 50gM.
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5
to the shortening of repolarization time (Smallvwood & Steinberg, 1988), we analyzed the relationship betwee n QT changes and +dP/dt,,..0X changes. Figure 5 shows that t]he decrease in +dP/dt,,,, paralleled QT shortening only up to a 15-20% QT change, whereas greater QT variation was not aAssociated with further appreciable + dP/dtx depression. Taking into account the biological variabilityy of QT duration in untreated hearts (160-215 ms at 2.5 Hz, 155-205ms at 3Hz, 145-19Oms at 3.5 Hz, 140-180ms at 4 Hz) a further correlation was sought at each frequency step between pretreatment QT value and drug-induced QT sihortening. The results indicate that QT shortening did not correlate with nnt elknwrnl otwn). pretreatment QT duration at any dose level (dat a,a
Proarrhythmic effects In 4 of 20 experiments, sustained ventricular fitlbrillation (VF) occurred after 16-28 min exposure to pinacidil I(in one case at 30gM and in three cases at 50 p.M) and lasted unktil KCl (0.1 ml of a 0.54 M solution) was infused as a bolus into the aortic cannula. No clear relationship was noted betweten the onset of VF and the frequency applied (2.5-4 Hz). Furtlher analysis of the ECG tracings revealed that VF was always preceded by a short run of premature ventricular beats, the Ilast one rising from the peak of the T wave (R on T phenomen4on) (Figure 6). In order to study this proarrhythmic effect bietter, we tried to induce VF electrically by increasing, first tihe driving frequency (20 Hz), and then the stimulus current I(see Methods). Under control conditions and after exposure 1to 10pM pinacidil VF could never be induced. At a drug cooncentration of 30uM, VF occurred in 5 out of 8 experiments at stimulation currents 2 to 5 times higher than the basal va lue. In 3 of 5 cases VF stopped spontaneously after 3-5 s (non-sustained
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Figure 6 Ventricular fibrillation occurring after exposure to 5OPM pinacidil in guinea-pig isolated perfused heart. Numbers represent: (1) last normal beat; (2) first premature ventricular beat; (3) second premature ventricular beat rising from the T-wave; (4) ventricular fibrillation.
VF), while in the other 2 cases the arrhythmia was still present after 5 min (sustained VF). At higher drug concentrations (50 and 100pM) VF was always inducible and in every case the arrhythmia was of the sustained type. Apart from in one experiment at 50 UM concentration in which current intensity had to be increased 1.5 fold, in all the others it was sufficient to increase the driving frequency to 20Hz in order to induce VF.
Discussion In agreement with the findings of other investigators (Kawashima & Liang, 1985; Longman et al., 1988; Smallwood & Steinberg, 1988; Steinberg et al., 1988; Nielsen et al., 1989), our results show that in the range of concentrations between 10 and 50piM, pinacidil decreases coronary tone and heart contractility, shortens ventricular repolarization time time (PR) without modify(QT) and prolongs AV conduction ing intraventricular conduction time (QRS). While coronary vessels appear to be the tissue most sensitive to the actions of pinacidil, the parameter which undergoes the greatest percentage variation at the maximal dose tested is +dP/dtmax (- 74%). Such a marked negative inotropic effect is not generally seen in vivo, probably due to the lower doses employed and to the sympathetic activation induced by vasodilatation (Kawashima & Liang, 1985; Nichols et al., 1986). The mechanism by which pinacidil depresses heart contractility has not yet been clarified. It is generally accepted that drugs which shorten AP duration can reduce Ca-influx during the plateau phase (Sheu & Lederer, 1985). However, our data show that QT shortening greater than 15-20% is not accompanied by further substantial +dP dt.. depression, suggesting that the decrease of Ca-entry induced by AP shortening reaches a ceiling. The magnitude of all the effects observed, except that on QTpeak/QT ratio (see below), was completely independent of driving frequency in the 2.5-4 Hz range. This behaviour is different from that of other drugs interacting with ionic channels, such as Na and Ca entry blockers (Hondeghem & Katzung, 1984) or some K channel blockers (Hondeghem & Snyders, 1990), which exhibit various patterns of use-dependence. A possible explanation of the rate-independent effects of pinacidil may be that it binds to a voltage activated K channel in the heart with an affinity which is independent of the activation state of the channel. Alternatively, pinacidil could bind to a K channel which is not gated by the membrane potential of cardiac cells. Recent experimental evidence (Fan et al., 1990; Tzeng & Hoffman, 1990; Arena & Kass, 1989a,b) strongly suggests that the main site of action of pinacidil at the myocardial level is the ATP-sensitive K channel. The gating kinetics of such a
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channel seem to be almost independent of membrane potential (Kakei et al., 1985) and this property is thus consistent with the use-independent effects of pinacidil. Furthermore, in normoxic conditions this K current is completely inhibited by high intracellular ATP levels (Noma, 1983) so that it does not normally contribute to cell repolarization. This feature could explain why the action of pinacidil on QT duration is independent of QT duration in the pretreatment period. In fact, if pinacidil were to act on a K current normally contributing to cell repolarization (e.g. the delayed rectifier) its effect should be less marked in preparations where K current is already high, namely where QT interval is shorter. Another feature worth considering regarding the action of pinacidil is the change of T-wave morphology (Figure 3), as reflected by the decrease of QTpeak/QT ratio. It is difficult to understand why the effect of pinacidil on QTpeak interval is more marked than that on QT interval and why only the former is rate-dependent. A simple explanation may be that these two effects are the outcome of different ionic mechanisms. It is generally acknowledged that T wave normally results from the asynchronous repolarization of the cells within the ventricular myocardium (Burgess, 1979). The first to repolarize are the epicardial cells, the last are the endocardial. Since the maximum voltage of T wave (T peak) occurs when most of the cells with short action potential duration (APD) are fully repolarized, a decrease in QTpeak interval should reflect a quicker repolarization of the epicardial cells. It has recently been shown that the epicardium (unlike the endocardium) has a large transient outward K current ('to) (Litovsky & Antzelevitch, 1988; Wang et al., 1991) and that pinacidil can augment the Ca-dependent component of this current (4t2) (Tzeng & Hoffmann, 1990). Thus, a reduction in the QTpeak/QT ratio could reflect a non-homogeneous drug-induced shortening of AP in the myocardial cell population. Given that the dispersion of APD is a well-known arrhythmogenic condition (Kuo et al., 1983), it should be taken into account in explaining the occurrence of VF in some of our experiments. Finding this type of proarrhythmic effect in our model was worthy of note for at least two reasons: (1) To our knowledge it is the first time that pinacidil has been shown to promote VF under non ischaemic conditions. Wollenben et al. (1989) found that pinacidil reduces the time required for the Langendorff-perfused rat heart to develop VF following acute global ischaemia. Chi et al. (1990) were able to demonstrate that in infarcted dogs exposed to a second episode of acute ischaemia, pinacidil increases the likelihood of electrically inducing VF. In contrast, other authors (Nielsen et al., 1989)
who tested the drug (0.15-100pM) on perfused rabbit hearts in normoxic conditions did not observe any proarrhythmic effects. The reason for the discrepancy between these findings and our data is not clear but species-specific factors may be involved. (2) The guinea-pig heart is intrinsically resistant to electrically-induced VF because of its small size (short re-entry circuit length) and its relatively long QT interval (long refractory period) compared to that of other species with similar heart sizes (rats, for example). It is well recognized (Fozzard & Arnsdorf, 1986) that in the presence of a functional or anatomical circuit, re-entrant arrhythmias can occur only when: RP < L/V, where: RP = refractory period of the re-excited area; L = circuit length and V = slowest conduction velocity. Pinacidil is able to create the functional substrate for reentry simply by shortening refractoriness (QT interval), without delaying intraventricular conduction time (QRS). It is also conceivable that pinacidil may be responsible for the short runs of premature ventricular beats preceding the spontaneous VF episodes. In this respect it is noteworthy that Steinberg et al. (1988), studying the electrophysiological effects of pinacidil on canine Purkinje fibres, detected, as we did, the appearance of arrhythmias at concentrations > 30pM in about 20% of preparations. The true clinical relevance of these experimental observations cannot be inferred with certainty. The highest concentration found in serum of hypertensive patients treated with pinacidil is about 30-40 times lower than that producing VF in our experiments (Carsen et al., 1983) and, so far, no proarrhythmic effects have been reported in man. Nevertheless, in 30% of patients treated with pinacidil, T-wave changes were observed (Goldberg, 1988) and little is known about drug tolerability in the subgroup of hypertensive patients with ischaemic heart disease. Recent evidence indicates that a new KCO (SR 44866) increases long term mortality in rats with healed myocardial infarction (Mulder et al., 1990). It therefore seems reasonable to recommend careful surveillance, at least in patients suffering from myocardial ischaemia. Finally, on mere experimental grounds, the combined exposure of guinea-pig heart to pinacidil, plus electrical stimulation, may represent a new, valuable model to induce sustained VF and study the effectiveness of antiarrhythmic drugs. This work was supported by grants from the Consiglio Nazionale delle Ricerche and from the Ministero della Universita e della Ricerca Scientifica e Tecnologica, Roma. Pinacidil was kindly supplied by Leo Pharmaceuticals, Denmark.
References ALMOTREFI, A.A. & BAKER, J.B.E. (1980). The antifibrillatory potency of aprindine, mexiletine, tocainide and lignocaine compared on Langendorff-perfused hearts of rabbits and guinea pigs. J. Pharm. Pharmacol., 32, 746-750. ARENA, J.P. & KASS, R.S. (1989a). Activation of ATP-sensitive K channels in heart cells by pinacidil: dependence on ATP. Am. J. Physiol., 257, H2092-H2096. ARENA, J.P. & KASS, R.S. (1989b). Enhancement of potassium-sensitive current in heart cells by pinacidil. Evidence for modulation of the ATP-sensitive potassium channel. Circ. Res., 65, 436-445. BOVA, S., PADRINI, R., CARGNELLI, G., PIOVAN, D. & FERRARI, M.
(1989). The influence of acidosis on myocardial uptake and electrocardiographic effects of disopyramide. Eur. J. Pharmacol., 168, 179-185. BURGESS, MJ. (1979). Relation of ventricular repolarization to electrocardiographic T wave-form and arrhythmia vulnerability. Am. J. Physiol., 236, H391-H402. CARSEN, L.E., KARDEL, T., JENSEN, H.E., TANGO, M., & TRAPJENSEN, J. (1983). Pinacidil, a new vasodilator: pharmacokinetics and pharmacodynamics of a new retarded release tablet in essential hypertension. Eur. J. Clin. Pharmacol., 25, 557-561. CHI, L., UPRICHARD, A.C.G. & LUCCHESI, B.R. (1990). Profibrillatory actions of pinacidil in a conscious canine model of sudden coronary death. J. Cardiovasc. Pharmacol., 15, 452-464.
COOK, N.S. (1988). The pharmacology of potassium channels and their therapeutic potential. Trends Pharmacol. Sci., 9, 21-28. FAN, Z., NAKAYAMA, K. & HIRAOKA, M. (1990). Pinacidil activates the ATP-sensitive K channel in inside-out and cell-attached patch membranes of guinea-pig ventricular myocytes. Pflugers Arch.; 415, 387-394. FOZZARD, H.A. & ARNSDORF, M.F. (1986). Cardiac electrophysiology. In The Heart and Cardiovascular System. ed. Fozzard, H.A., Haber, E., Jennings, R.B., Katz, A.M. & Morgan, H.E. pp. 1-30. New York: Raven Press. GOLDBERG, M.R. (1988). Clinical pharmacology of pinacidil, a prototype for drug which affect potassium channels. J. Cardiovasc. Pharmacol., 12 (suppl. 2), S41-S47. HONDEGHEM, L.M. & KATZUNG, B.G. (1984). Antiarrhythmic agents: the modulated receptor mechanism of. action of sodium and calcium channel-blocking drugs. Annu. Rev. Pharmacol. Toxicol., 24, 387-423. HONDEGHEM, L.M. & SNYDERS, D.J. (1990). Class III antiarrhythmic agents have a lot of potential but a long way to go. Reduced effectiveness and dangers of reverse use dependence. Circulation, 81, 686-690. KAKEI, M., NOMA, A. & SHIBASAKI, T. (1985). Properties of adenosine-triphosphate-regulated potassium channels in guineapig ventricular cells. J. Physiol., 263, 441-426.
PROARRHYTHMIC EFFECT OF PINACIDIL KAWASHIMA, S. & LIANG, C.S. (1985). Systemic and coronary hemodynamic effects of pinacidil, a new antihypertensive agent, a awake dogs: comparison with hydralazine. J. Pharmacol. Exp. Ther., 232, 369-375. KERR, M.J., WILSON, R. & SHANKS, R.G. (1985). Suppression of ventricular arrhythmias after coronary artery ligation by pinacidil, a vasodilator drug. J. Cardiovasc. Pharmacol., 7, 875-883. KUO, C.S., MUNAKATA, K., REDDY, C.P. & SURAWICZ, B. (1983). Characteristics and possible mechanism of ventricular arrhythmia dependent on dispersion of action potential durations. Circulation, 67, 1356-1367. LITOVSKY, S.H. & ANTZELEVITCH, C. (1988). Transient outward current prominent in canine ventricular epicardium but not endocardium. Circ. Res., 62, 116-126. LONGMAN, S.G., CLAPHAM, J.C., WILSON, C. & HAMILTON, T.C.
(1988). Cromakalim, a potassium channel activator: a comparison of its cardiovascular haemodynamic profile and tissue specificity with those of pinacidil and nicorandil. J. Cardiovasc. Pharmacol., 12, 535-542. MULDER, P., FARNES, P., RICHER, C., CAMILLERI, J.-P. & GUID-
ICELLI, J.-F. (1990). K' channel openers decrease long term survival in rats with healed myocardial infarction. Eur. J. Pharmacol., 183, 658 (abstract). NICHOLS, D.P., MURTAGH, J.G., SCOTT, M.E., MORTON, P. &
SHANKS, R.G. (1986). Acute haemodynamic effects of pinacidil in man. Br. J. Clin. Pharmacol., 22, 287-292. NIELSEN, C.B., MELLEMKJAER, S. & NIELSEN-KUDSK, F. (1989). Pinacidil uptake and effects in the isolated rabbit heart. Pharmacol. Toxicol., 64, 14-19.
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NOMA, A. (1983). ATP-regulated K+ channels in cardiac muscle. Nature, 305, 147-148. SHEU, S.S. & LEDERER, W.J. (1985). Lidocaine's negative inotropic and antiarrhythmic actions. Dependence on shortening of action potential duration and reduction of intracellular sodium activity. Circ. Res., 57, 578-590. STEINBERG, M.I., ERTEL, P., SMALLWOOD, J.K., WYSS, V. & ZIMMER-
MAN, K. (1988). The relation between vascular relaxant and cardiac electrophysiological effects of pinacidil. J. Cardiovasc. Pharmacol., 12 (suppl. 2), S30-S40. SMALLWOOD, J.K. & STEINBERG, M.I. (1988). Cardiac electrophysiological effects of pinacidil and related pyridylcyanoguanidines: relationship to antihypertensive activity. J. Cardiovasc. Pharmacol., 12, 102-109. TZENG, G.N. & HOFFMAN, B.F. (1990). Actions of pinacidil on membrane currents in canine ventricular myocytes and their modulation by intracellular ATP and cAMP. Pfliigers Arch., 415, 414-424. WANG, Z., FERMINI, B. & NATTEL, S. (1991). Repolarization differences between guinea pig atrial endocardium and epicardium: evidence for a role of Ito' Am. J. Physiol., 260, H1501-H1506. WESTON, A.H. (1989). Smooth muscle K channel openers; their pharmacological and clinical potential. Pflugers Arch., 414 (suppl. 1), S99-S105. WOLLENBERG, C.D., SANGUINETTI, M.C. & SIEGEL, P.K.S. (1989). Influence of ATP-potassium channel modulators on ischemiainduced fibrillation in isolated rat hearts. J. Mol. Cell. Cardiol., 21, 783-788.
(Received November 13, 1990 Revised October 21, 1991 Accepted November 6, 1991)
C) Macmillan Press Ltd, 1992
Effects of pinacidil on guinea-pig isolated perfused heart with particular reference to the proarrhythmic effect 'Roberto Padrini, Sergio Bova, Gabriella Cargnelli, *Donatella Piovan & *Mariano Ferrari Department of Pharmacology and *Institute of Internal Medicine, University of Padova, Padova, Italy 1 The effects of pinacidil (10, 30, 50,M) on contractility (+dP/dt,,), coronary perfusion pressure (cP), and ECG intervals (PR, QRS, QT) have been studied on constant-flow perfused guinea-pig hearts, driven at four frequencies (2.5, 3, 3.5, 4 Hz). 2 Pinacidil decreased +dP/dt,,,, cP and the QT interval in a dose-dependent manner, whereas the PR interval was increased. QRS duration was not modified. All these effects were independent of driving frequency. Pinacidil decreased the interval from Q-wave to T-wave peak (QTpeak) to a greater extent than the QT interval, thus decreasing the QTpeak/QT ratio. This effect, unlike that on QT interval, was more evident at the highest frequency of stimulation. 3 In 4 out of 20 hearts treated with pinacidil sustained ventricular fibrillation (VF) occurred following a short run of premature ventricular beats (R on T phenomenon). 4 In separate experiments, an attempt to induce VF electrically was made at drug concentrations ranging from 1OpM to 1OM (8 experiments for each concentration). In control conditions and at the lowest concentration of pinacidil tested (1OpM) VF could never be induced; in the presence of 30pMm pinacidil VF was induced in 5 out of 8 experiments. Drug concentrations higher that 50 gM permitted the induction of VF in every case. 5 Although the concentrations of pinacidil producing ventricular fibrillation are 30-40 times higher than those found in patients under long term treatment with this agent, it is suggested that caution should be used in prescribing this drug, at least in patients suffering from myocardial ischaemia. Keywords: Pinacidil; heart contractility; coronary tone; ECG; ventricular fibrillation
Introduction As a typical 'K channel opener' (KCO), pinacidil has been shown to relax various types of smooth muscle by increasing K conductance and hyperpolarizing the cell membrane (Weston, 1989). These properties account for the successful clinical application of pinacidil in antihypertensive therapy (Goldberg, 1988) and for the therapeutic potential of KCOs in the treatment of bronchial asthma, irritable bladder syndrome and angina pectoris (Cook, 1988). At a cardiac level, pinacidil shortens the action potential (AP) duration of ventricular and Purkinje cells (Smallwood & Steinberg, 1988); decreases the automaticity of Purkinje fibres (Steinberg et al., 1988); decreases contractility and sinus rate (at least in isolated preparations) (Longman et al., 1988; Steinberg et al., 1988; Nielsen et al., 1989); and increases coronary flow (Kawashima & Liang, 1985). Increasing amounts of experimental data have become available on the influence of pinacidil on heart rhythmicity. This aspect of the action of the drug, while of great clinical importance, is somewhat puzzling in that pinacidil, on the one hand, can favour re-entrant type arrhythmias (acute ischaemia models; Wollenberg et al., 1989; Chi et al., 1990) and, on the other, it exhibits a definite antiarrhythmic action on arrhythmias produced by increased automaticity (subacute ischaemia, barium-induced arrhythmias, Kerr et al., Steinberg et al., 1988). The present work describes the pharmacodynamic profile of pinacidil in Langendorff-perfused guinea-pig hearts driven at different stimulation rates and, specifically, focuses on the influence of the drug on heart rhythmicity in normoxic conditions.
Methods The preparation used was the constant flow-perfused guineapig heart (Langendorff preparation). An extensive description of the procedure followed can be found elsewhere (Bova et al., I Author for correspondence at: Department of Pharmacology, Largo Egidio Meneghetti 2,1-35131 Padova, Italy.
1989). In brief, guinea-pigs of either sex (weight: 250-350 g) were killed by a blow to the neck and the heart was rapidly removed and retrogradely perfused within 45s of removal. The perfusion medium was a modified Krebs-Henseleit solution (mM: NaCl 118, KCI 4.7, CaCl2 2.5, MgSO4 1.2, NaCO3 25, KH2PO4 1.2, glucose 11.1 and pyruvate 2) bubbled with an 02:CO2 gas mixture (95%:5%) and maintained at 370C (pH 7.4 + 0.01). The flow of perfusate was kept constant at about 10mlg-' tissue min-' with a Gilson peristaltic micropump. After excising the sinus node the heart was driven at various frequencies (see below) with a S88 Grass stimulator through platinum electrodes placed on the left atrium. Intraventricular pressure was measured by means of a rubber balloon inserted into the left ventricle and connected to a mechano-electrical transducer (MARB P82). The maximal positive derivative of the intraventricular pressure (+ dP/dt,,,j,,) was taken as an index of contractility. Surface ECG was recorded by means of two electrodes, one placed on the crux cordis and the other on the left ventricle free wall. The signal, amplified with a P16 Grass amplifier, was monitored with a Tektronics oscilloscope and simultaneously recorded on magnetic tape. PR, QRS and QT intervals were measured on magnified photographs of the tracings 'frozen' on the oscilloscope screen. In addition to QT duration the interval from Q-wave to T-wave peak was also determined (QTpeak) and the QT/QTpeak ratio was calculated. Coronary perfusion pressure (cP) was also monitored to assess the change in coronary bed resistance. A solution of pinacidil was infused, with a peristaltic pump, through a T-junction at very low flow rate (28 times lower than the total flow); the very small increase in the total flow rate induced when infusion was started did not elicit any detectable change of the parameters measured. The drug concentrations in the infusing solutions were calculated in order to obtain final pinacidil concentrations of 10, 30, 50 M in the aortic cannula.
Study protocol The effects of pinacidil on contractility (+dP/dt,,.jX), coronary resistance (cP) and ECG intervals were evaluated at four
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driving frequencies: 2.5, 3, 3.5 and 4Hz. The choice of such a narrow range of frequencies was justified for two technical reasons: (1) in some preparations a frequency lower than 2.5 Hz allows a spontaneous junctional rhythm to arise; (2) at frequencies higher than 4Hz the stimulus artifact on ECG tracings can fall close to the end of preceding T wave, making it impossible to measure the QT interval. Each preparation was first allowed to recover for 30-45 min with stimulation at 2.5 Hz and then frequency was increased in 0.5 Hz steps up to 4Hz, leaving 5min for stabilization at each frequency step. After 15 min exposure of the heart to a single drug concentration the same stimulation procedure was repeated. Four to six experiments were performed at each concentration. Repetition of the stimulation protocol per se was found to have no influence on the cardiac parameters measured.
Determination of ventricularfibrillation threshold In a separate series of experiments an attempt was made to induce ventricular fibrillation (VF) by electrical stimulation in basal conditions and after 15min exposure to 10, 30, 50 and 100pUM pinacidil (n = 8 at each concentration). After removing the atrial free wall and septum, stimulating electrodes were fitted over the upper edge of the intraventricular septum and a stimulation protocol similar to that described by Almotrefi & Baker (1980) was applied. Driving frequency was suddenly increased from 2.5 to 20 Hz and current intensity was progressively increased either until VF occurred or an intensity 10 fold greater then the basal value was reached.
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Figure 1 Frequency-dependence of the cardiac parameters of the guinea-pig isolated perfused heart measured under control conditions (n = 16). Changes are expressed as a percentage of the value observed at 2.5 Hz. Symbols: (0) +dP/dtm,,; (A), PR interval; (0) coronary pressure; (A), QT interval. Significant change from baseline: * P < 0.05.
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At the stimulation rate of 2.5 Hz the mean control values (±s.e.) of Cp, dP/dt,,aX, PR interval, QRS interval, QT interval duration and QTpeak/QT ratio were 64.0 + 2.2 mmHg, 1405 + 86mmHgrs-, 48.6 + 2.2ms, 22.5 + 0.4ms, 180.1 + 3.4 ms and 0.900 + 0.004, respectively. As expected, when the driving frequency was increased in steps, + dP/dtmax and PR increased while QT and cP decreased (Figure 1). QRS duration and QTpeak/QT ratio were rate-independent. In the presence of pinacidil (10, 30 or 5OpM) coronary pressure and cardiac contractility decreased, QT shortened and PR lengthened in a dose-dependent way, while QRS duration was not modified. The effects of pinacidil were virtually identical at any frequency applied, hence only the effects observed as 2.5 Hz are shown in Figure 2. Although the narrow range
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concentration (50M) were -74.0% + 2.2 for +dPdt x -51.3% + 3.6 for cP, -34.3% + 4.2 for QT and + 16.2 + 2.6 for PR. As well as shortening the QT interval duration, pinacidil changed the morphology of the T-wave (Figure 3) in that it shortened the QTpeak interval to a greater extent than the QT interval, thus decreasing the QTpeak/QT ratio. The effect on QTpeak/QT ratio was dose-dependent and, unlike that on QT interval, also rate-dependent at least at the highest concentration tested (50M). Figure 4 shows the effect of pinacidil
Figure 3 Pinacidil-induced ECG changes in guinea-pig isolated perfused heart. The drug lengthens- PR interval, shortens QT interval and modifies QT-wave shape. (a) Control (2.5 Hz); (b) pinacidil 50gM.
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5
to the shortening of repolarization time (Smallvwood & Steinberg, 1988), we analyzed the relationship betwee n QT changes and +dP/dt,,..0X changes. Figure 5 shows that t]he decrease in +dP/dt,,,, paralleled QT shortening only up to a 15-20% QT change, whereas greater QT variation was not aAssociated with further appreciable + dP/dtx depression. Taking into account the biological variabilityy of QT duration in untreated hearts (160-215 ms at 2.5 Hz, 155-205ms at 3Hz, 145-19Oms at 3.5 Hz, 140-180ms at 4 Hz) a further correlation was sought at each frequency step between pretreatment QT value and drug-induced QT sihortening. The results indicate that QT shortening did not correlate with nnt elknwrnl otwn). pretreatment QT duration at any dose level (dat a,a
Proarrhythmic effects In 4 of 20 experiments, sustained ventricular fitlbrillation (VF) occurred after 16-28 min exposure to pinacidil I(in one case at 30gM and in three cases at 50 p.M) and lasted unktil KCl (0.1 ml of a 0.54 M solution) was infused as a bolus into the aortic cannula. No clear relationship was noted betweten the onset of VF and the frequency applied (2.5-4 Hz). Furtlher analysis of the ECG tracings revealed that VF was always preceded by a short run of premature ventricular beats, the Ilast one rising from the peak of the T wave (R on T phenomen4on) (Figure 6). In order to study this proarrhythmic effect bietter, we tried to induce VF electrically by increasing, first tihe driving frequency (20 Hz), and then the stimulus current I(see Methods). Under control conditions and after exposure 1to 10pM pinacidil VF could never be induced. At a drug cooncentration of 30uM, VF occurred in 5 out of 8 experiments at stimulation currents 2 to 5 times higher than the basal va lue. In 3 of 5 cases VF stopped spontaneously after 3-5 s (non-sustained
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Figure 6 Ventricular fibrillation occurring after exposure to 5OPM pinacidil in guinea-pig isolated perfused heart. Numbers represent: (1) last normal beat; (2) first premature ventricular beat; (3) second premature ventricular beat rising from the T-wave; (4) ventricular fibrillation.
VF), while in the other 2 cases the arrhythmia was still present after 5 min (sustained VF). At higher drug concentrations (50 and 100pM) VF was always inducible and in every case the arrhythmia was of the sustained type. Apart from in one experiment at 50 UM concentration in which current intensity had to be increased 1.5 fold, in all the others it was sufficient to increase the driving frequency to 20Hz in order to induce VF.
Discussion In agreement with the findings of other investigators (Kawashima & Liang, 1985; Longman et al., 1988; Smallwood & Steinberg, 1988; Steinberg et al., 1988; Nielsen et al., 1989), our results show that in the range of concentrations between 10 and 50piM, pinacidil decreases coronary tone and heart contractility, shortens ventricular repolarization time time (PR) without modify(QT) and prolongs AV conduction ing intraventricular conduction time (QRS). While coronary vessels appear to be the tissue most sensitive to the actions of pinacidil, the parameter which undergoes the greatest percentage variation at the maximal dose tested is +dP/dtmax (- 74%). Such a marked negative inotropic effect is not generally seen in vivo, probably due to the lower doses employed and to the sympathetic activation induced by vasodilatation (Kawashima & Liang, 1985; Nichols et al., 1986). The mechanism by which pinacidil depresses heart contractility has not yet been clarified. It is generally accepted that drugs which shorten AP duration can reduce Ca-influx during the plateau phase (Sheu & Lederer, 1985). However, our data show that QT shortening greater than 15-20% is not accompanied by further substantial +dP dt.. depression, suggesting that the decrease of Ca-entry induced by AP shortening reaches a ceiling. The magnitude of all the effects observed, except that on QTpeak/QT ratio (see below), was completely independent of driving frequency in the 2.5-4 Hz range. This behaviour is different from that of other drugs interacting with ionic channels, such as Na and Ca entry blockers (Hondeghem & Katzung, 1984) or some K channel blockers (Hondeghem & Snyders, 1990), which exhibit various patterns of use-dependence. A possible explanation of the rate-independent effects of pinacidil may be that it binds to a voltage activated K channel in the heart with an affinity which is independent of the activation state of the channel. Alternatively, pinacidil could bind to a K channel which is not gated by the membrane potential of cardiac cells. Recent experimental evidence (Fan et al., 1990; Tzeng & Hoffman, 1990; Arena & Kass, 1989a,b) strongly suggests that the main site of action of pinacidil at the myocardial level is the ATP-sensitive K channel. The gating kinetics of such a
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channel seem to be almost independent of membrane potential (Kakei et al., 1985) and this property is thus consistent with the use-independent effects of pinacidil. Furthermore, in normoxic conditions this K current is completely inhibited by high intracellular ATP levels (Noma, 1983) so that it does not normally contribute to cell repolarization. This feature could explain why the action of pinacidil on QT duration is independent of QT duration in the pretreatment period. In fact, if pinacidil were to act on a K current normally contributing to cell repolarization (e.g. the delayed rectifier) its effect should be less marked in preparations where K current is already high, namely where QT interval is shorter. Another feature worth considering regarding the action of pinacidil is the change of T-wave morphology (Figure 3), as reflected by the decrease of QTpeak/QT ratio. It is difficult to understand why the effect of pinacidil on QTpeak interval is more marked than that on QT interval and why only the former is rate-dependent. A simple explanation may be that these two effects are the outcome of different ionic mechanisms. It is generally acknowledged that T wave normally results from the asynchronous repolarization of the cells within the ventricular myocardium (Burgess, 1979). The first to repolarize are the epicardial cells, the last are the endocardial. Since the maximum voltage of T wave (T peak) occurs when most of the cells with short action potential duration (APD) are fully repolarized, a decrease in QTpeak interval should reflect a quicker repolarization of the epicardial cells. It has recently been shown that the epicardium (unlike the endocardium) has a large transient outward K current ('to) (Litovsky & Antzelevitch, 1988; Wang et al., 1991) and that pinacidil can augment the Ca-dependent component of this current (4t2) (Tzeng & Hoffmann, 1990). Thus, a reduction in the QTpeak/QT ratio could reflect a non-homogeneous drug-induced shortening of AP in the myocardial cell population. Given that the dispersion of APD is a well-known arrhythmogenic condition (Kuo et al., 1983), it should be taken into account in explaining the occurrence of VF in some of our experiments. Finding this type of proarrhythmic effect in our model was worthy of note for at least two reasons: (1) To our knowledge it is the first time that pinacidil has been shown to promote VF under non ischaemic conditions. Wollenben et al. (1989) found that pinacidil reduces the time required for the Langendorff-perfused rat heart to develop VF following acute global ischaemia. Chi et al. (1990) were able to demonstrate that in infarcted dogs exposed to a second episode of acute ischaemia, pinacidil increases the likelihood of electrically inducing VF. In contrast, other authors (Nielsen et al., 1989)
who tested the drug (0.15-100pM) on perfused rabbit hearts in normoxic conditions did not observe any proarrhythmic effects. The reason for the discrepancy between these findings and our data is not clear but species-specific factors may be involved. (2) The guinea-pig heart is intrinsically resistant to electrically-induced VF because of its small size (short re-entry circuit length) and its relatively long QT interval (long refractory period) compared to that of other species with similar heart sizes (rats, for example). It is well recognized (Fozzard & Arnsdorf, 1986) that in the presence of a functional or anatomical circuit, re-entrant arrhythmias can occur only when: RP < L/V, where: RP = refractory period of the re-excited area; L = circuit length and V = slowest conduction velocity. Pinacidil is able to create the functional substrate for reentry simply by shortening refractoriness (QT interval), without delaying intraventricular conduction time (QRS). It is also conceivable that pinacidil may be responsible for the short runs of premature ventricular beats preceding the spontaneous VF episodes. In this respect it is noteworthy that Steinberg et al. (1988), studying the electrophysiological effects of pinacidil on canine Purkinje fibres, detected, as we did, the appearance of arrhythmias at concentrations > 30pM in about 20% of preparations. The true clinical relevance of these experimental observations cannot be inferred with certainty. The highest concentration found in serum of hypertensive patients treated with pinacidil is about 30-40 times lower than that producing VF in our experiments (Carsen et al., 1983) and, so far, no proarrhythmic effects have been reported in man. Nevertheless, in 30% of patients treated with pinacidil, T-wave changes were observed (Goldberg, 1988) and little is known about drug tolerability in the subgroup of hypertensive patients with ischaemic heart disease. Recent evidence indicates that a new KCO (SR 44866) increases long term mortality in rats with healed myocardial infarction (Mulder et al., 1990). It therefore seems reasonable to recommend careful surveillance, at least in patients suffering from myocardial ischaemia. Finally, on mere experimental grounds, the combined exposure of guinea-pig heart to pinacidil, plus electrical stimulation, may represent a new, valuable model to induce sustained VF and study the effectiveness of antiarrhythmic drugs. This work was supported by grants from the Consiglio Nazionale delle Ricerche and from the Ministero della Universita e della Ricerca Scientifica e Tecnologica, Roma. Pinacidil was kindly supplied by Leo Pharmaceuticals, Denmark.
References ALMOTREFI, A.A. & BAKER, J.B.E. (1980). The antifibrillatory potency of aprindine, mexiletine, tocainide and lignocaine compared on Langendorff-perfused hearts of rabbits and guinea pigs. J. Pharm. Pharmacol., 32, 746-750. ARENA, J.P. & KASS, R.S. (1989a). Activation of ATP-sensitive K channels in heart cells by pinacidil: dependence on ATP. Am. J. Physiol., 257, H2092-H2096. ARENA, J.P. & KASS, R.S. (1989b). Enhancement of potassium-sensitive current in heart cells by pinacidil. Evidence for modulation of the ATP-sensitive potassium channel. Circ. Res., 65, 436-445. BOVA, S., PADRINI, R., CARGNELLI, G., PIOVAN, D. & FERRARI, M.
(1989). The influence of acidosis on myocardial uptake and electrocardiographic effects of disopyramide. Eur. J. Pharmacol., 168, 179-185. BURGESS, MJ. (1979). Relation of ventricular repolarization to electrocardiographic T wave-form and arrhythmia vulnerability. Am. J. Physiol., 236, H391-H402. CARSEN, L.E., KARDEL, T., JENSEN, H.E., TANGO, M., & TRAPJENSEN, J. (1983). Pinacidil, a new vasodilator: pharmacokinetics and pharmacodynamics of a new retarded release tablet in essential hypertension. Eur. J. Clin. Pharmacol., 25, 557-561. CHI, L., UPRICHARD, A.C.G. & LUCCHESI, B.R. (1990). Profibrillatory actions of pinacidil in a conscious canine model of sudden coronary death. J. Cardiovasc. Pharmacol., 15, 452-464.
COOK, N.S. (1988). The pharmacology of potassium channels and their therapeutic potential. Trends Pharmacol. Sci., 9, 21-28. FAN, Z., NAKAYAMA, K. & HIRAOKA, M. (1990). Pinacidil activates the ATP-sensitive K channel in inside-out and cell-attached patch membranes of guinea-pig ventricular myocytes. Pflugers Arch.; 415, 387-394. FOZZARD, H.A. & ARNSDORF, M.F. (1986). Cardiac electrophysiology. In The Heart and Cardiovascular System. ed. Fozzard, H.A., Haber, E., Jennings, R.B., Katz, A.M. & Morgan, H.E. pp. 1-30. New York: Raven Press. GOLDBERG, M.R. (1988). Clinical pharmacology of pinacidil, a prototype for drug which affect potassium channels. J. Cardiovasc. Pharmacol., 12 (suppl. 2), S41-S47. HONDEGHEM, L.M. & KATZUNG, B.G. (1984). Antiarrhythmic agents: the modulated receptor mechanism of. action of sodium and calcium channel-blocking drugs. Annu. Rev. Pharmacol. Toxicol., 24, 387-423. HONDEGHEM, L.M. & SNYDERS, D.J. (1990). Class III antiarrhythmic agents have a lot of potential but a long way to go. Reduced effectiveness and dangers of reverse use dependence. Circulation, 81, 686-690. KAKEI, M., NOMA, A. & SHIBASAKI, T. (1985). Properties of adenosine-triphosphate-regulated potassium channels in guineapig ventricular cells. J. Physiol., 263, 441-426.
PROARRHYTHMIC EFFECT OF PINACIDIL KAWASHIMA, S. & LIANG, C.S. (1985). Systemic and coronary hemodynamic effects of pinacidil, a new antihypertensive agent, a awake dogs: comparison with hydralazine. J. Pharmacol. Exp. Ther., 232, 369-375. KERR, M.J., WILSON, R. & SHANKS, R.G. (1985). Suppression of ventricular arrhythmias after coronary artery ligation by pinacidil, a vasodilator drug. J. Cardiovasc. Pharmacol., 7, 875-883. KUO, C.S., MUNAKATA, K., REDDY, C.P. & SURAWICZ, B. (1983). Characteristics and possible mechanism of ventricular arrhythmia dependent on dispersion of action potential durations. Circulation, 67, 1356-1367. LITOVSKY, S.H. & ANTZELEVITCH, C. (1988). Transient outward current prominent in canine ventricular epicardium but not endocardium. Circ. Res., 62, 116-126. LONGMAN, S.G., CLAPHAM, J.C., WILSON, C. & HAMILTON, T.C.
(1988). Cromakalim, a potassium channel activator: a comparison of its cardiovascular haemodynamic profile and tissue specificity with those of pinacidil and nicorandil. J. Cardiovasc. Pharmacol., 12, 535-542. MULDER, P., FARNES, P., RICHER, C., CAMILLERI, J.-P. & GUID-
ICELLI, J.-F. (1990). K' channel openers decrease long term survival in rats with healed myocardial infarction. Eur. J. Pharmacol., 183, 658 (abstract). NICHOLS, D.P., MURTAGH, J.G., SCOTT, M.E., MORTON, P. &
SHANKS, R.G. (1986). Acute haemodynamic effects of pinacidil in man. Br. J. Clin. Pharmacol., 22, 287-292. NIELSEN, C.B., MELLEMKJAER, S. & NIELSEN-KUDSK, F. (1989). Pinacidil uptake and effects in the isolated rabbit heart. Pharmacol. Toxicol., 64, 14-19.
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NOMA, A. (1983). ATP-regulated K+ channels in cardiac muscle. Nature, 305, 147-148. SHEU, S.S. & LEDERER, W.J. (1985). Lidocaine's negative inotropic and antiarrhythmic actions. Dependence on shortening of action potential duration and reduction of intracellular sodium activity. Circ. Res., 57, 578-590. STEINBERG, M.I., ERTEL, P., SMALLWOOD, J.K., WYSS, V. & ZIMMER-
MAN, K. (1988). The relation between vascular relaxant and cardiac electrophysiological effects of pinacidil. J. Cardiovasc. Pharmacol., 12 (suppl. 2), S30-S40. SMALLWOOD, J.K. & STEINBERG, M.I. (1988). Cardiac electrophysiological effects of pinacidil and related pyridylcyanoguanidines: relationship to antihypertensive activity. J. Cardiovasc. Pharmacol., 12, 102-109. TZENG, G.N. & HOFFMAN, B.F. (1990). Actions of pinacidil on membrane currents in canine ventricular myocytes and their modulation by intracellular ATP and cAMP. Pfliigers Arch., 415, 414-424. WANG, Z., FERMINI, B. & NATTEL, S. (1991). Repolarization differences between guinea pig atrial endocardium and epicardium: evidence for a role of Ito' Am. J. Physiol., 260, H1501-H1506. WESTON, A.H. (1989). Smooth muscle K channel openers; their pharmacological and clinical potential. Pflugers Arch., 414 (suppl. 1), S99-S105. WOLLENBERG, C.D., SANGUINETTI, M.C. & SIEGEL, P.K.S. (1989). Influence of ATP-potassium channel modulators on ischemiainduced fibrillation in isolated rat hearts. J. Mol. Cell. Cardiol., 21, 783-788.
(Received November 13, 1990 Revised October 21, 1991 Accepted November 6, 1991)
