Electrophysiological analysis of the negative chronotropic effect of endothelin-1 in rabbit sinoatrial node cells.

1. Electrophysiological effects of endothelin-1 (ET-1) were studied in rabbit sinoatrial node (SAN) using conventional microelectrode and whole-cell voltage and current recordings. 2. In rabbit SAN, RT-PCR detected ET(A) endothelin receptor mRNA. ET-1 (100 nM) increased the cycle length of action potentials (APs) from 305 +/- 15 to 388 +/- 25 ms; this effect was antagonised by the ET(A) receptor-selective antagonist BQ-123 (1 microM). ET-1 increased AP duration (APD50) by 22%, depolarised the maximum diastolic potential (MDP) from -59 +/- 1 to -53 +/- 2 mV, shifted the take-off potential by +5 mV and decreased the pacemaker potential (PMP) slope by 15%. Under exactly the same experimental conditions, ET-1 caused a positive chronotropic effect in guinea-pig SAN with a decrease of 13% in APD50, a shift of -4 mV in the take-off potential and an increase of 8% in the PMP slope. 3. Rabbit SAN exhibited two major cell types, distinguished both by their appearances and by their electrophysiological responses to ET-1. Whereas the spontaneous pacing rate and the PMP slope were similarly decreased by ET-1 (10 nM) in both cell types, ET-1 depolarised MDP from -67 +/- 1 to -62 +/- 4 mV in spindle-shaped cells but hyperpolarised it from -73 +/- 1 to -81 +/- 3 mV in rod-shaped cells. ET-1 decreased APD50 by 8 and 52% and shifted the take-off potential by +5 and -9 mV in spindle- and rod-shaped cells, respectively. 4. ET-1 decreased the high-threshold calcium current (I(CaL)) by about 50% in both cell types, without affecting its voltage dependence, and decreased the delayed rectifier K+ current (I(K)) with significant shifts (of +4.7 and +14.0 mV in spindle- and rod-shaped cells, respectively) in its voltage dependence. It was exclusively in rod-shaped cells that ET-1 activated a sizeable amount of time-independent inward-rectifying current. 5. The hyperpolarisation-activated current (I(f)), observed exclusively in spindle-shaped cells, was significantly increased by ET-1 at membrane potentials between -74.7 and -84.7 mV whereas it was significantly decreased at more negative potentials. ET-1 significantly decreased the slope of the current-voltage (I-V) relation of the I(f) tail without changing its half-maximum voltage. 6. The overall negative chronotropic influence of ET-1 on the whole rabbit SAN is interpreted as resulting from the integration of its different actions on spindle- and rod-shaped SAN cells through electrotonic interaction.

Ryanodine sensitizes the Ca(2+) release channel (ryanodine receptor) to Ca(2+) activation.

Ryanodine, a plant alkaloid, is one of the most widely used pharmacological probes for intracellular Ca(2+) signaling in a variety of muscle and non-muscle cells. Upon binding to the Ca(2+) release channel (ryanodine receptor), ryanodine causes two major changes in the channel: a reduction in single-channel conductance and a marked increase in open probability. The molecular mechanisms underlying these alterations are not well understood. In the present study, we investigated the gating behavior and Ca(2+) dependence of the wild type (wt) and a mutant cardiac ryanodine receptor (RyR2) after being modified by ryanodine. Single-channel studies revealed that the ryanodine-modified wt RyR2 channel was sensitive to inhibition by Mg(2+) and to activation by caffeine and ATP. In the presence of Mg(2+), the ryanodine-modified single wt RyR2 channel displayed a sigmoidal Ca(2+) dependence with an EC(50) value of 110 nm, whereas the ryanodine-unmodified single wt channel exhibited an EC(50) of 120 microm for Ca(2+) activation, indicating that ryanodine is able to increase the sensitivity of the wt RyR2 channel to Ca(2+) activation by approximately 1,000-fold. Furthermore, ryanodine is able to restore Ca(2+) activation and ligand response of the E3987A mutant RyR2 channel that has been shown to exhibit approximately 1,000-fold reduction in Ca(2+) sensitivity to activation. The E3987A mutation, however, affects neither [(3)H]ryanodine binding to, nor the stimulatory and inhibitory effects of ryanodine on, the RyR2 channel. These results demonstrate that ryanodine does not "lock" the RyR channel into an open state as generally believed; rather, it sensitizes dramatically the channel to activation by Ca(2+).

Inhibition by a novel anti-arrhythmic agent, NIP-142, of cloned human cardiac K+ channel Kv1.5 current.

NIP-142 was shown to prolong atrial effective refractory period and to terminate atrial fibrillation and flutter in in vivo canine models. To obtain information on its antiarrhythmic action, we examined the effect of NIP-142 on cloned human cardiac K+ channel Kv1.5 (hKv1.5) currents stably expressed in a human cell line using whole-cell voltage clamp methods. NIP-142 inhibited the hKv1.5 current in a concentration-dependent and voltage-independent manner. The inhibition was larger at the end of depolarizing pulse than at the outward current peak. The IC50 for inhibition of the steady-state phase was 4.75 microM. A cross-over phenomenon was observed when current traces in the absence and presence of NIP-142 were superimposed. Inhibition of hKv1.5 current by NIP-142 was frequency-independent; changing the depolarizing pulse frequencies (0.1, 0.2, 1 Hz) and little effect on the degree of inhibition. NIP-142 decreased the maximal peak amplitude of kHv1.5 current at the first command pulse after 3 min rest in the presence of the drug. These results suggest that NIP-142 has inhibitory effects on the hKv 1.5 current through interaction with both open and closed states of the channel, which may underlie its antiarrhythmic activity in the atria.

Involvement of Ca2+ waves in excitation-contraction coupling of rat atrial cardiomyocytes.

Two-dimensional and line-scan analyses of the early phase Ca2+ transients in rat cardiomyocytes were performed with a rapid-scanning laser confocal microscope and fluo-3 to elucidate the mechanism of activation of Ca2+ release from the sarcoplasmic reticulum in atrial myocytes which lack a well developed T-tubular network. On electrical stimulation of ventricular myocytes, Ca2+ concentration began to rise earliest at the Z-line level and became uniform throughout the cytoplasm within about 10 msec. In contrast, on stimulation of atrial myocytes, the earliest rise in Ca2+ occurred at the cell periphery and then spread to the cell interior; cytoplasmic Ca2+ became uniform after more than 30msec. The velocity of the propagation of rise in Ca2+ was 112 +/- 5.1 microm/sec (n = 10), which was similar to that of spontaneous Ca2+ waves observed in atrial and ventricular myocytes. No difference in frequency, amplitude and kinetics of spontaneous Ca2+ sparks was observed between the subsarcolemmal and central regions of atrial myocytes. Ryanodine concentration-dependently decreased the contractile force of isolated rat atrial and ventricular tissue preparations; the sensitivity was higher in atrial myocytes. The present study visualized the involvement of a propagated Ca2+-induced-Ca+ release mechanism in atrial but not ventricular myocytes. This difference may underlie some of the atrioventricular difference in response to physiological and pharmacological stimuli.

Inhibition of T-type and L-type Ca(2+) currents by aranidipine, a novel dihydropyridine Ca(2+) antagonist.

The effects of aranidipine, a novel dihydropyridine Ca(2+) channel antagonist, on membrane currents in guinea pig ventricular myocytes and on action potentials in rabbit sinoatrial node tissue were examined. In myocytes, aranidipine (10 nmol/l to 1 micromol/l) concentration-dependently decreased T-type and L-type Ca(2+) currents. Aranidipine (1 micromol/l) had little effect on K(+) currents. In the sinoatrial node, 0.1 micromol/l aranidipine increased cycle length, and decreased +V(max) and the slope of the phase 4 depolarization. Thus, inhibition of both T-type and L-type Ca(2+) currents by aranidipine may partly explain its potent negative chronotropic activity.

Possible requirement of phosphonate moiety for efonidipine effects on the sino-atrial node action potential.

The effects of efonidipine, a 1,4-dihydropyridine phosphonate, and structurally related compounds on rabbit sino-atrial node action potential were examined with microelectrodes. 3NIC5NZ has a phosphonate moiety identical to that of efonidipine at the C5 position of the dihydropyridine ring and a side chain identical to nicardipine at C3, while 3NZ5NIC has C5 and C3 side chains identical to nicardipine and efonidipine, respectively. All four compounds decreased the slope and prolonged the early and late phases of pacemaker depolarization. The selectivity for the late phase against the early phase was in the order of efonidipine > 3NIC5NZ >> nicardipine > 3NZ5NIC. Thus, the phosphonate moiety at C5 position of the may be important for the characteristic prolongation of the late phase pacemaker depolarization by efonidipine.

Frequency-dependent blockade of T-type Ca2+ current by efonidipine in cardiomyocytes.

Efonidipine is a dihydropyridine Ca2+ antagonist with inhibitory effects on both L-type and T-type Ca2+ channels and potent bradycardiac activity especially in patients with high heart rate. In the present study, we examined the frequency dependence of efonidipine action on the T-type Ca2+ channel in isolated guinea-pig ventricular myocytes. The potency of efonidipine to inhibit the T-type Ca2+ current was higher under higher stimulation frequencies. The IC50 values were 1.3 x 10(-8), 2.0 x 10(-6) and 6.3 x 10(-6) M under stimulation frequencies of 1, 0.2 and 0.05 Hz, respectively. The reduction of T-type Ca2+ current amplitude was not accompanied by change in the time course of current decay. Efonidipine (10 microM) inhibited T-type Ca2+ current elicited by depolarization from holding potentials ranging from -90 to -30 mV by about 30%; the voltage-dependence of steady-state inactivation was not changed by the drug. Efonidipine slowed the recovery from inactivation following an inactivating prepulse. In conclusion, efonidipine was shown to have frequency-dependent inhibitory effects on the T-type Ca2+ channel, which could be explained by slow dissociation of the drug from the inactivated state of the channel.

Calcium channel antagonistic effects of AH-1058, a novel antiarrhythmic drug, on guinea-pig myocardium.

Effects of AH-1058, a novel cyproheptadine derivative with high antiarrhythmic activity in in vivo arrhythmia models, were studied in guinea-pig myocardium. In coronary-perfused right ventricular tissue preparations, AH-1058 (10(-5) M) shortened the action potential duration with little effect on the resting membrane potential, maximum rate of rise and overshoot. AH-1058, 10(-7) M to 10(-5) M, concentration-dependently decreased the contractile force. The increase in contractile force by Ca2+ was markedly inhibited by 3 x 10(-6) M AH-1058 while that by isoproterenol was only slightly affected. In isolated ventricular myocytes, AH-1058 concentration-dependently decreased the nicardipine sensitive transient inward current with no effect on steady state currents, and decreased the amplitude of the evoked Ca2+ transient. These results suggest that AH-1058 has Ca2+ channel antagonistic effects which may contribute to its antiarrhythmic activity.

Effects of hirsutine and dihydrocorynantheine on the action potentials of sino-atrial node, atrium and ventricle.

The effects of hirsutine, an indole alkaloid from Uncaria rhynchophylla MIQ. JACKSON with antihypertensive, negative chronotropic and antiarrhythmic activity, and its C3 structural epimer, dihydrocorynantheine, on membrane potentials of rabbit sino-atrial node and guinea-pig right ventricle and left atrium were studied with microelectrode techniques. In sino-atrial node preparations, hirsutine and dihydrocorynantheine (0.1 microM to 10 microM) concentration-dependently increased cycle length, decreased slope of the pacemaker depolarization (phase 4 depolarization), decreased maximum rate of rise and prolonged action potential duration. In atrial and ventricular preparations, both compounds (0.1 microM to 30 microM) concentration-dependently decreased maximum rate of rise and prolonged action potential duration. These results indicate that hirsutine and dihydrocorynantheine have direct effects on the action potential of cardiac muscle through inhibition of multiple ion channels, which may explain their negative chronotropic and antiarrhythmic activity.

Effects of mibefradil, a selective T-type Ca2+ channel antagonist, on sino-atrial node and ventricular myocardia.

The effects of mibefradil, a non-dihydropyridine Ca2+ channel antagonist, on the action potential configuration of isolated rabbit sino-atrial node preparations, membrane currents of guinea-pig ventricular myocytes and the contractile force of isolated ventricular papillary muscles were examined. In sino-atrial node preparations, 10 microM mibefradil decreased the slope of the pacemaker depolarization (phase 4 depolarization) and maximum rate of rise, and shifted the threshold potential to the positive direction with no effect on action potential duration. In ventricular myocytes, 1 microM mibefradil inhibited the T-type Ca2+ current by about 40% while it had no effect on the L-type Ca2+ current. At 10 microM, mibefradil inhibited the L-type and T-type Ca2+ currents by about 40% and 90%, respectively. Mibefradil had no effect on contractile force at concentrations up to 1 microM. Thus, mibefradil was shown to produce potent prolongation of the pacemaker depolarization, mainly through inhibition of the T-type Ca2+ current. It is suggested that the T-type Ca2+ current may not be involved in ventricular contraction.

Inhibition of myocardial L- and T-type Ca2+ currents by efonidipine: possible mechanism for its chronotropic effect.

Effects of efonidipine, a dihydropyridine phosphonate Ca2+ channel antagonist, on the guinea-pig heart were compared with those of nifedipine. In the sino-atrial node, 1 microM efonidipine produced increase in cycle length accompanied by prolongation of the phase 4 depolarization which was not prominent with 0.1 microM nifedipine. In ventricular myocytes, both efonidipine and nifedipine produced inhibition of the L-type Ca2+ current, nifedipine being tenfold more potent than efonidipine. Efonidipine also inhibited the T-type Ca2+ current at higher concentrations but nifedipine did not. Both Ca2+ channel antagonists had no or only a weak effect on K+ currents. In addition, 40 microM Ni2+, which selectively inhibited the T-type Ca2+ current, had no effect on myocardial Ca2+ transients and contractile force. In conclusion, efonidipine was shown to have inhibitory effects on both L- and T-type Ca2+ currents, which may contribute to its high negative chronotropic potency.

Effects of Ca2+ channel antagonists on sinus node: prolongation of late phase 4 depolarization by efonidipine.

Effects of various Ca2+ channel antagonists on the action potential configuration of rabbit sino-atrial node tissue were examined with standard microelectrode techniques. All Ca2+ channel antagonists decreased the maximum rate of phase 0 depolarization (Vmax) and increased the cycle length. The potency order to increase the cycle length was nisoldipine = verapamil > nifedipine = clentiazem > efonidipine > diltiazem. The potency order to decrease Vmax and to shift the threshold potential to a positive direction was the same as that to increase the cycle length, indicating that the major mechanism of negative chronotropism was inhibition of the L-type Ca2+ current. All Ca2+ channel antagonists except efonidipine shifted the maximum diastolic potential to the positive direction, decreased the action potential amplitude and prolonged the action potential duration. The effects of nifedipine were slightly weaker than those of other drugs when compared at equally bradycardiac concentrations. These differences may reflect differences in drug effects on currents other than the L-type Ca2+ current. A characteristic feature of efonidipine was selective suppression of the later phase of pacemaker depolarization with no effect on action potential amplitude and duration. Similar suppression of the later phase was observed with 50 microM Ni2+, which is reported to inhibit the T-type, but not L-type, Ca2+ current. Thus, efonidipine appears to suppress selectively the later phase of pacemaker depolarization through inhibition of both L- and T-type Ca2+ currents, which may be the underlying mechanism for its reported potent negative chronotropic but weak inotropic activity.

Comparative effects of gallopamil and verapamil on the mechanical and electrophysiological parameters of isolated guinea-pig myocardium.

The effects of gallopamil, D600, a methoxy derivative of verapamil, on the mechanical and electrophysiological parameters of isolated guinea-pig myocardial preparations were compared with those of verapamil. Both gallopamil and verapamil produced concentration-dependent negative chronotropic and negative inotropic effects in isolated right atrial and right ventricular papillary muscles, respectively. The negative chronotropic and negative inotropic potencies of gallopamil were 7.2 and 4.3 times higher than those of verapamil, respectively. Gallopamil decreased the action potential duration of isolated papillary muscles without substantially affecting other action potential parameters, while verapamil decreased not only the action potential duration but also the maximum rate of rise and amplitude. In voltage-clamped single ventricular myocytes, gallopamil as well as verapamil decreased the L-type Ca2+ current amplitude. The potency orders for the shortening of the action potential duration and inhibitory effects of the L-type Ca2+ current amplitudes were verapamil > gallopamil. These results indicate that gallopamil has higher negative chronotropic and inotropic potency than verapamil as a result of factors other than L-type Ca2+ current inhibition.

Developmental changes in action potential and membrane currents in fetal, neonatal and adult guinea-pig ventricular myocytes.

Developmental changes in electrophysiological properties were investigated in enzymatically isolated ventricular cardiomyocytes from fetal (45-55 days after conception), neonatal (1-5 days after birth) and adult (45-60 days after birth) guinea-pigs. Action potentials were elicited at 1 Hz in current-clamp mode, and membrane currents were measured using whole cell voltage clamp method. Action potential durations at 50% and 90% repolarization decreased between fetal and neonatal periods and increased between neonatal and adult periods, while there was no substantial age-related change in resting membrane potential and action potential overshoot. Because cell membrane capacitance increased with age, indicating a developmental increase in cell size, current density was normalized to membrane capacitance for each cell. The L-type calcium current (lCaL) density at 0 and + 10 mV was significantly smaller in fetal and neonatal cells than in adult ones, although the voltage dependence and inactivation kinetics were similar among the three age groups. The delayed rectifier K+ current (lK) density at 0 and + 30 mV was significantly smaller in fetal cells than in neonatal and adult ones. No significant difference in the inward rectifier K+ current (lK1) density was observed among the three age groups. Thus, the electrophysiological properties of the guinea-pig ventricular myocytes were demonstrated to change during pre- and postnatal development. The observed changes in action potential duration could be explained by changes in the balance between lK and lCaL.

Relative selectivity for negative chronotropic and inotropic effects of a novel dihydropyridine derivative CD-832.

The effects of CD-832 [(4R)-(-)-2-(nicotinoyl-amino) ethyl 3-nitroxypropyl 1,4-dihydro-2,6-dimethyl-4,3-nitrophenyl, 3,5-pyridine dicarboxylate], a novel dihydropyridine derivative, on various guinea-pig myocardial preparations were compared with those of nifedipine, verapamil and diltiazem. CD-832 decreased the action potential duration of isolated papillary muscles without substantially affecting other parameters. In voltage-clamped single ventricular myocytes, CD-832 decreased the L-type Ca2+ current amplitude while having little effect on outward currents. CD-832 and other Ca2+ channel antagonists produced negative chronotropic effects in isolated right atrial preparations and negative inotropic effects in right ventricular papillary muscles, respectively, in a concentration-dependent manner. The potency order for the negative chronotropic effect was CD-832 > nifedipine > verapamil > diltiazem, while that for the negative inotropic effect was nifedipine > verapamil > or = CD-832 > diltiazem. The ratio, EC20 for negative inotropic effect divided by EC20 for negative chronotropic effect, which was considered to be an index of selectivity for negative chronotropic effect was highest for CD-832, the ratio for CD-832, nifedipine, verapamil and diltiazem being 5.4, 0.11, 0.25 and 0.37, respectively. These results indicate that CD-832 is an L-type Ca2+ channel antagonist with relative selectivity for a negative chronotropic effect rather than for a negative inotropic effect. This 'chrono-selective' cardiosuppressive effect of CD-832 could be of value in the treatment of cardiovascular diseases such as angina pectoris.

Myocardial and vascular effects of efonidipine in vitro as compared with nifedipine, verapamil and diltiazem.

1. Effects of efonidipine on isolated myocardial and aortic preparations were compared with those of nifedipine, verapamil and diltiazem. 2. All drugs produced concentration-dependent negative chronotropic effects on isolated guinea-pig atrial preparations. The potency order was efonidipine > or = nifedipine > diltiazem > or = verapamil, EC30 values being 3.08 x 10(-8)M, 3.48 x 10(-8)M, 1.27 x 10(-7)M and 1.47 x 10(-7)M, respectively. 3. Nifedipine, verapamil and diltiazem produced concentration-dependent negative inotropic effects on isolated guinea-pig left atrial preparations. The potency order was nifedipine > verapamil > diltiazem, EC30 values being 4.94 x 10(-8)M, 1.49 x 10(-7)M and 8.03 x 10(-7)M, respectively. Efonidipine, even at 1 microM produced no inotropic effect: 10 microM efonidipine decreased the contractile force by about 20%. 4. All drugs concentration-dependently attenuated the KCl-induced contraction of isolated rat aortic ring preparation. The potency order was nifedipine > efonidipine > verapamil > diltiazem, EC30 values being 2.98 x 10(-9)M, 1.24 x 10(-8)M, 3.96 x 10(-8)M and 2.13 x 10(-7)M, respectively. 5. Thus, efonidipine was demonstrated to be a potent vasodilator with negative chronotropic but minimal negative inotropic activity, which may be of benefit in the treatment of cardiovascular disorders.

Inhibitory effects of terfenadine on the rising phase of action potentials and sinus rates in isolated guinea-pig myocardium.

1. Effects of terfenadine on the ventricular action potential and sinus rate of isolated guinea-pig myocardial preparations were examined to elucidate whether the drug has any cardioinhibitory effect. 2. Terfenadine significantly and concentration dependently decreased the maximum rate of rise (Vmax) of action potentials, the decrease of which was 60% of the control value at 2 x 10(-5) M. 3. Terfenadine decreased the sinus rate concentration dependently at 10(-6) M and higher concentrations; in some preparations, spontaneous beating was abolished at 2 x 10(-5) M (sinus arrest). 4. On the other hand, KW-4679 ((Z)-11-[3-(dimethylamino)propylidene]-6-11-dihydrodibenz[b, e]oxepin- 2-acetic acid hydrochloride), a new antiallergic drug having higher antiallergic potency than terfenadine, exerted a weaker inhibitory effect on the Vmax without affecting the sinus rate. 5. In conclusion, the antiallergic drug terfenadine has inhibitory effects on isolated myocardia. The cardioinhibitory effects of antiallergic drugs seem to be independent of their antiallergic potency.