Esmolol inhibits Na+ current in rat ventricular myocytes

Esmolol is a unique cardioselective, intravenous, ultra-short acting, beta1-adrenergic blocking agent. It has been widely applied in treating ventricular and supraventricular arrhythmias, especially in emergency situations. In this study the effects of esmolol on sodium current (I(Na)) were investigated by the whole cell patch-clamp recording technique in isolated adult rat ventricular myocytes. The results indicated that esmolol reversibly inhibited I(Na) in a concentration-dependent manner, with an IC50 of 74.2 +/- 0.60 micromol l(-1) with a Hill coefficient of 1.02 +/- 0.04. This inhibition was voltage- and frequency-dependent. Esmolol decreased the peak of the I-V relationship curve at -35 mV from 16.97 +/- 1.68 pA/pF to 6.96 +/- 0.51 pA/pF. The steady-state inactivation curve of I(Na) was shifted to more negative potentials, the voltage at half-inactivation changing from -78.75 +/- 2.3 mV in control to -85.94 +/- 3.2 mV in the presence of esmolol. The development of resting inactivation from closed states was accelerated by esmolol, the time constant was shortened from 62.75 +/- 3.21 ms to 24.93 +/- 2.43 ms, whereas the activation curve was not altered. I(Na) from inactivation could not be recovered completely in the presence of esmolol. These results suggest that esmolol inhibits I(Na) through sodium channel in rat ventricular myocytes by mechanisms involving preferential interaction with the inactivated state and acceleration of the development of inactivation directly from resting state. Therefore, the effect of inhibitory sodium of esmolol may play a vital role in its antiarrhythmic efficacy.     

V102862 (Co 102862): a potent, broad-spectrum state-dependent blocker of mammalian voltage-gated sodium channels

1. 4-(4-Fluorophenoxy)benzaldehyde semicarbazone (V102862) was initially described as an orally active anticonvulsant with robust activity in a variety of rodent models of epilepsy. The mechanism of action was not known. We used whole-cell patch-clamp techniques to study the effects of V102862 on native and recombinant mammalian voltage-gated Na+ channels. 2. V102862 blocked Na+ currents (I(Na)) in acutely dissociated cultured rat hippocampal neurons. Potency increased with membrane depolarization, suggesting a state-dependent mechanism of inhibition. There was no significant effect on the voltage dependence of activation of I(Na). 3. The dissociation constant for the inactivated state (K(I)) was approximately 0.6 microM, whereas the dissociation constant for the resting state (K(R)) was >15 microM. 4. The binding to inactivated channels was slow, requiring a few seconds to reach steady state at -80 mV. 5. The mechanism of inhibition was characterized in more detail using human embryonic kidney-293 cells stably expressing rat brain type IIA Na+ (rNa(v)1.2) channels, a major Na+ channel alpha subunit in rat hippocampal neurons. Similar to hippocampal neurons, V102862 was a potent state-dependent blocker of rNa(v)1.2 channels with a K(I) of approximately 0.4 microM and K(R) approximately 30 microM. V102862 binding to inactivated channels was relatively slow (k(+) approximately = 1.7 microM(-1) s(-1)). V102862 shifted the steady-state availability curve in the hyperpolarizing direction and significantly retarded recovery of Na+ channels from inactivation. 6. These results suggest that inhibition of voltage-gated Na+ channels is a major mechanism underlying the anticonvulsant properties of V102862. Moreover, understanding the biophysics of the interaction may prove to be useful in designing a new generation of potent Na+ channel blocker therapeutics.     

Biphasic effects of haloperidol on sodium currents in guinea pig ventricular myocytes

Aim： To study the effects of haloperidol on sodium currents （INa） in guinea pig ventricular myocytes. Method： Whole-cell patch clamp technique was employed to evaluate the effects of haloperidol on INa in individual ventricular myocytes. Results： Haloperidol （0.1-3 wnol/L） inhibited INa in a concentration-dependent manner with an IC50 of 0.253±0.015 larnol/L. The inhibition rate of haloperidol （0.3 μmol/L） on INa was 22.14%±0.02%, and the maximum conductance was reduced. Haloperidol significantly reduced the midpoints for the activation and inactivation of INa by 2.09 and 4.09 mV, respectively. The time constant of recovery was increased. The increase in time intervals could only recover by 90.14%±1.4% （n=6）; however, haloperidol at 0.03 μmol/L enhanced INa conductance. The midpoints for the activation and inactivation Of INa were shifted by 1.38 and 5.69 mV, respectively, at this concentration of haloperidol. Conclusion： Haloperidol displayed a biphasic effect on INa in guinea pig cardiac myocytes. High concentrations of haloperidol inhibited INa, while lower concentrations of haloperidol shifted the activation and inactivation curve to the left. Full recovery of recovery curve was not achieved after 0.3 μmol/L haloperidol administration, indicating that the drug affects the inactivated state of sodium channels.     

A multifunctional aromatic residue in the external pore vestibule of Na+ channels contributes to the local anesthetic receptor

Voltage-gated Na(+) (Na(v)) channels are responsible for initiating action potentials in excitable cells and are the targets of local anesthetics (LA). The LA receptor is localized to the cytoplasmic pore mouth formed by the S6 segments from all four domains (DI-DIV) but several outer pore-lining residues have also been shown to influence LA block (albeit somewhat modestly). Many of the reported amino acid substitutions, however, also disrupt the inactivated conformations that favor LA binding, complicating the interpretation of their specific effects on drug block. In this article, we report that an externally accessible aromatic residue in the Na(v) channel pore, DIV-Trp1531, when substituted with cysteine, completely abolished LA block (e.g., 300 microM mexiletine induced a use-dependent block with 65.0 +/- 2.9% remaining current and -11.0 +/- 0.6 mV of steady-state inactivation shift of wild-type (WT) channels versus 97.4 +/- 0.7% and -2.4 +/- 2.1 mV of W1531C, respectively; p < 0.05) without destabilizing fast inactivation (complete inactivation at 20 ms at -20 mV; V(1/2) = -70.0 +/- 1.6 mV versus -48.6 +/- 0.5 mV of WT). W1531C also abolished internal QX-222 block (200 microM; 98.4 +/- 3.4% versus 54.0 +/- 3.2% of WT) without altering drug access. It is interesting that W1531Y restored WT blocking behavior, whereas W1531A channels exhibited an intermediate phenotype. Together, our results provide novel insights into the mechanism of drug action, and the structural relationship between the LA receptor and the outer pore vestibule.     

芍药苷对小鼠海马CA1区神经元钠电流的抑制作用

AIM: To study the blockade of paeoniflorin (Pae) on I(Na) in the acutely isolated hippocampus neurons of mice. METHODS: The whole-cell patch clamp technique was used. RESULTS: Pae inhibited I(Na) in frequency-dependent and concentration-dependent manners, with an IC50 of 271 micromol/L. Pae 0.3 mmol/L shifted the activation potential of the maximal I(Na) from -40 mV to -30 mV, shifted the steady-state activation and inactivation curves toward more positive and negative potentials by 10.8 mV, and 18.2 mV, respectively, and postponed the recovery of I(Na) inactivation state from (4.2+/-0.7) ms to (9.8+/-1.2) ms. CONCLUSION: Pae inhibited I(Na) in mouse hippocampus neurons.     

双苯氟嗪对豚鼠心室肌细胞L-钙电流的影响

目的:观察双苯氟嗪(Dip)对豚鼠心室肌细胞L-型钙电流(I_(Ca-L))的影响。方法:酶解法制备单个心室肌细胞。应用全细胞膜片箝技术记录豚鼠单个心室肌细胞钙电流。结果:在0.3-30μmol/L范围内,Dip可浓度依赖性地降低电压依赖性激活I_(Ca-L)峰值,被Dip 3μmol/L所抑制的I_(Ca-L)在冲洗5min后可得到部份恢复。但Dip对I_(Ca-L)的电压依赖特征,最大激活电压,以及I_(Ca-L)稳态激活无明显影响。在Dip3μmol/L存在下,半数激活电压(V_(0.5))和斜率参数(к)与对照组相比,差异均无显著性。V_(0.5)分别为(-12.8±1.7)mV和(-13.2±2.4)mV,к分别为(7.1±0.4)mV和(7.5±0.5)mV(P>0.05)。Dip3μmol/L可明显使钙电流稳态失活曲线左移,加速钙通道电压依赖性稳态失活。V_(0.5)分别为(-19.7±2.4)mV和(-31±6)mV,к分别为(3.6±0.3)mV和(1.8±0.2)mV(P<0.05).Dip 3μmol/L还使I_(Ca-L)从失活状态下的恢复明显减慢。结论:Dip主要作用于L-型钙通道的失活状态,加速钙通道失活,并使其从失活状态下恢复减慢,从而抑制I_(Ca-L)。     

葛根素抑制大鼠心室肌细胞的钠电流

AIM: To study the effect of puerarin (Pue) on Na+ channel in rat ventricular myocytes. METHODS: Whole-cell patch-clamp technique was applied on isolated cardiomyocytes from rats. RESULTS: Pue inhibited cardiac INa in a positive rate-dependent and dose-dependent manner, with an IC(50) of 349 micromol/L. The kinetics of blockage of cardiac sodium channel by Pue resembled the ClassIa/Ic of antiarrhythmic agents. Pue 300 micromol/L did not alter the shape of the I-V curve of INa, but markedly shifted the steady-state inactivation curve of INa towards more negative potential by 15.9 mV, and postponed the recovery of INa inactivation state from (21.9+/-1.6) ms to (54.4+/-3.4) ms (P<0.01). It demonstrated that the steady state of inactivation was affected by Pue significantly. CONCLUSION: Pue protected ventricular myocytes against cardiac damage and arrhythmias by inhibiting recovery from inactivation of cardiac Na+ channels.     

Effect of resveratrol on L-type calcium current in rat ventricular myocytes

AIM: To study the effect of resveratrol on L-type calcium current (I(Ca-L)) in isolated rat ventricular myocytes and the mechanisms underlying these effects. METHODS: I(Ca-L) was examined in isolated single rat ventricular myocytes by using the whole cell patch-clamp recording technique. RESULTS: Resveratrol (10-40 micromol/L) reduced the peak amplitude of I(Ca-L) and shifted the current-voltage (I-V) curve upwards in a concentration-dependent manner. Resveratrol (10, 20, 40 micromol/L) decreased the peak amplitude of I(Ca-L) from -14.2+/-1.5 pA/pF to -10.5+/-1.5 pA/pF (P<0.05), -7.5+/-2.4 pA/pF (P<0.01), and -5.2+/-1.2 pA/pF (P<0.01), respectively. Resveratrol (40 micromol/L) shifted the steady-state activation curve of I(Ca-L) to the right and changed the half-activation potential (V0.5) from -19.4+/-0.4 mV to -15.4+/-1.9 mV (P<0.05). Resveratrol at a concentration of 40 micromol/L did not affect the steady-state inactivation curve of I(Ca-L), but did markedly shift the time-dependent recovery curve of I(Ca-L) to the right, and slow down the recovery of I(Ca-L) from inactivation. Sodium orthovanadate (Na(3)VO(4); 1 mmol/L), a potent inhibitor of tyrosine phosphatase, significantly inhibited the effects of resveratrol (P<0.01). CONCLUSION: Resveratrol inhibited I(Ca-L) mainly by inhibiting the activation of L-type calcium channels and slowing down the recovery of L-type calcium channels from inactivation. This inhibitory effect of resveratrol was mediated by the inhibition of protein tyrosine kinase in rat ventricular myocytes.     

Mechanism of inhibition of the sodium current by bepridil in guinea-pig isolated ventricular cells.

1. Effects of bepridil, a sodium-, calcium-, and potassium-antagonistic agent, on the Na+ current were studied by the whole cell voltage clamp technique (tip resistance = 0.5 MOhm, [Na]i and [Na]o 10 mmol l-1 at 20 degrees C). 2. Bepridil produced tonic block (Kdrest = 295.44 mumol l-1, Kdi = 1.41 mumol l-1; n = 4). 3. Bepridil (100 mumol l-1) shifted the inactivation curve in the hyperpolarization direction by 13.4 +/- 2.7 mV (n = 4) without change in the slope factor. 4. In the presence of 50 mumol l-1 bepridil, bepridil showed use-dependent block at 2 Hz, whereas changes in pulse duration did not significantly effect this use-dependent block (81% +/- 2% at 10 ms, 84% +/- 3% at 30 ms, 86% +/- 3% at 100 ms; n = 4). 5. After removal of fast inactivation of the Na+ current by 3 mmol l-1 tosylchloramide sodium, bepridil (50 mumol l-1) still showed use-dependent block which was independent of the holding potential. 6. The recovery time constant from the bepridil-induced use-dependent block was 0.48 s at holding potential of -100 mV and 0.51 s at holding potential of -140 mV. 7. These results indicate that bepridil could bind to the receptor in the sodium channel through the hydrophobic and the hydrophilic pathway and leave the receptor through the hydrophobic pathway in the lipid bilayer. The binding and dissociation kinetics of this drug were shown to be fast, and the accumulation of the drug in the sodium channel appeared to be small.(ABSTRACT TRUNCATED AT 250 WORDS)     

双苯氟嗪对哇巴因诱发豚鼠乳头肌迟后除极和触发活动的影响

目的:研究双苯氟嗪(DiP)对哇巴因和高钙诱发豚鼠乳头肌迟后除极(DADs)和触发活动(TA)的影响.方法:应用哇巴因(1 μmol/L)和高钙(5.4 mmol/L)诱发稳定且可重复的迟后除极和触发活动.用细胞内玻璃微电极技术记录DADs和TA诸参数.结果:(1)预先应用DiP(10,30 μmol/L)对DADs和TA有明显的抑制作用.在DiP(30 μmol/L)作用下,DADs的幅度由(10.5±2.2)降到(3.6±0.3)mV,DADs时程由(230±19)缩短到(152±14)ms,引起DADs的时间从(21±5)延长至(66±11)min,TA未见发生.(2)诱发DADs后再给予Dip(10,30μmol/L),DADs和TA被明显抑制.在Dip(30μmol/L)作用下,DADS的幅度由(10.4±1.2)降至(3.3±0.6)mV,DADs的时程由(218±22)缩短至(159±26)ms,TA未见发生.结论:Dip对哇巴因和高钙诱发的豚鼠乳头肌迟后除极和触发活动有抑制作用,这可能与其抑制L-型钙通道和/或肌浆网钙释放从而减轻细胞内钙超载有关,并由此产生抗心律失常作用.     

Critical molecular determinants of voltage-gated sodium channel sensitivity to mu-conotoxins GIIIA/B

GIIIA/B mu-conotoxins block the rat skeletal muscle sodium channel (rNa(v)1.4) with high affinity by binding to specific residues in the pore. However, human Na(v)1.4 (hNa(v)1.4) channels, which are resistant to block by GIIIA/B, have these same pore residues. We used chimera constructs, site-directed mutagenesis, and electrophysiological techniques to investigate which residues determine GIIIA/B selectivity. Exchange of serine 729 in the D2/S5-S6 linker of rat Na(v)1.4 with leucine (S729L), the corresponding residue in hNa(v)1.4, reduces the sensitivity of rNa(v)1.4 by approximately 20-fold and largely accounts for the differential sensitivity of rNa(v)1.4 and hNa(v)1.4 to both GIIIA and GIIIB. To determine whether D2/S5-S6 linker residues might contribute to the resistance of neuronal channels to GIIIA/B, we exchanged residues in this linker that differed between rNa(v)1.4 and neuronal channels. Substitution of aspargine 732 with lysine (N732K), the corresponding residue in rNa(v)1.1a and rNa(v)1.7, reduced the GIIIB sensitivity of rNa(v)1.4 by approximately 20-fold. The N732K substitution, however, only reduced GIIIA sensitivity of rNa(v)1.4 by approximately 4-fold, demonstrating that GIIIA and GIIIB have distinct interactions with the D2/S5-S6 linker. Our data indicate that naturally occurring variants in the extra-pore region of the D2/S5-S6 linker contribute to the isoform-specific sensitivity of sodium channels to GIIIA/B. Because S729 and N732 are not part of the high-affinity binding site for mu-conotoxins, these extra-pore residues probably influence the accessibility of the toxin to the binding site within the pore and/or the stability of the toxin-channel complex. Our results should aid the development of toxins that block specific neuronal sodium channel isoforms.     

Demonstration of functional M3-muscarinic receptors in ventricular cardiomyocytes of adult rats

1 Muscarinic receptors (M-receptors) in the mammalian heart are predominantly of the M(2)-subtype. The aim of this study was to find out whether there might exist an additional myocardial non-M(2)-receptor. 2 For this purpose, we assessed, in adult rat isolated ventricular cardiomyocytes, carbachol-induced [(3)H]-inositol phosphate (IP) formation, and its inhibition by M-receptor antagonists. 3 Carbachol (10(-7)-10(-3) mol l(-1)) increased IP-formation (maximal increase: 14+/-3% above basal, n=6). This increase was significantly enhanced by pretreatment with pertussis toxin (PTX, 250 ng ml(-1) for 20 h): maximal increase was 31+/-5%, pEC(50)-value was 5.08+/-0.33 (n=6). 4 In PTX-pretreated cardiomyocytes 100 micromol l(-1) carbachol-induced IP-formation was inhibited by atropine (pK(i)-value: 8.89+/-0.10) and by the M(3)-receptor antagonist darifenacin (pK(i)-value: 8.67+/-0.23) but was not significantly affected by the M(1)-receptor antagonist pirenzepine (1 micromol l(-1)) or the M(2)-receptor antagonists AF-DX 116 and himbacine (1 micromol l(-1)). 5 In conclusion, in adult rat cardiomyocytes there exists an additional, non-M(2)-receptor, that is coupled to activation of the phospholipase C/IP(3)-pathway; this receptor is very likely of the M(3)-subtype.     

Effects of moxonidine and clonidine on potassium excretion in Sprague-Dawley rats.

Recently we demonstrated that clonidine and moxonidine exert specific action on fractional fluid and Na+ excretion in anaesthetised Sprague-Dawley rats. Classically, most of the diuretics used induce an increased K+ excretion, at least in part due to Na+ load in the distal tubule and exchange of Na+ by K+. Therefore, we studied the effects of moxonidine and clonidine on K+ excretion in anaesthetised Sprague-Dawley rats. Moxonidine (0.25 and 0.5 mg kg-1 body wt. i.v.) increased transiently K+ (1.0 +/- 0.3 -1.9 +/- 0.4 and 0.9 +/- 0.2 -2.9 +0.7 micromol min-1 100 g body wt.) and Na+ (1.4 +/- 1.0 -6. 9 +/- 3.1 and 0.8 +/- 0.36 -6.6 +/- 1.5 micromol min-1100 g body wt.) excretion. Clonidine (0.25 mg kg-1) caused a more pronounced increase in K+ (1.0 +/- 0.1 -2.7 +/- 0.4 micromol min-1 100 g body wt.) and Na+ (0.6 +/- 0.3 -9.5 +/- 0.4 micromol min-1 100 g body wt.) excretion, whereas the higher dose of 0.5 mg kg-1 body wt. had less effect as compared to moxonidine (K+: 0.8 +/- 0.1 -1.7 +/- 0.2 micromol min-1 100 g body wt., Na+: 0.3 +/- 0.1 -3.4 +/- 1.0 micromol min-1 100 g body wt.). The increased electrolyte excretion returned (similar to moxonidine) to baseline levels within 20 min after injection of the drugs. Antagonists such as idazoxan and yohimbine did not change K+ and Na+ excretion by their own. Both, the non-selective imidazoline/alpha2-adrenoceptor antagonist idazoxan and the pure alpha2-adrenoceptor antagonist yohimbine attenuated the moxonidin-induced effects on K+ and Na+ excretion. This could be also observed with clonidine and simultaneous injection of these two antagonists. Our results demonstrate that moxonidine and clonidine also increase renal K+ excretion in this animal model. K+and Na+ excretion show a parallel behaviour, indicating that the increased K+ excretion is mainly due to Na+ load in the tubular system.     

Lysophosphatidylcholine augments Ca(v)3.2 but not Ca(v)3.1 T-type Ca(2+) channel current expressed in HEK-293 cells

Lysophosphatidylcholine (LPC) has been shown to induce electrophysiological disturbances to arrhythmogenesis. However, the effects of LPC on the low-voltage-activated T-type Ca(2+) channels in the heart are not understood yet. We found that LPC increases the T-type Ca(2+) channel current (I(Ca.T)) in neonatal rat cardiomyocytes. To further investigate the underlying modulatory mechanism of LPC on T-type Ca(2+) channels, we utilized HEK-293 cells stably expressing alpha1G and alpha1H subunits (HEK-293/alpha1G and HEK-293/alpha1H), by use of patch-clamp techniques. A low concentration of LPC (10 micromol/l) significantly increased Ca(v)3.2 I(Ca.T) (alpha1H) that were similar to those observed in neonatal rat cardiomyocytes. Activation and steady-state inactivation curves were shifted in the hyperpolarized direction by 5.1 +/- 0.2 and 4.6 +/- 0.4 mV, respectively, by application of 10 micromol/l LPC. The pretreatment of cells with a protein kinase C inhibitor (chelerythrine) attenuated the effects of LPC on I(Ca.T) (alpha1H). However, the application of LPC failed to modify Ca(v)3.1 (alpha1G) I(Ca.T) at concentrations of 10-50 micromol/l. In conclusion, these data demonstrate that extracellularly applied LPC augments Ca(v)3.2 I(Ca.T) (alpha1H) but not Ca(v)3.1 I(Ca.T) (alpha1G) in a heterologous expression system, possibly by modulating protein kinase C signaling.     

海葵毒素anthopleurin—Q对豚鼠心室肌细胞钠电流的作用

目的:研究从海葵(Anthopleura xanthogrammica)提取的毒素anthopleurin-Q(AP-Q)对豚鼠心室肌钠电流(I_(Na))的作用。方法:用酶消化法分离豚鼠单个心室肌细胞,用全细胞膜片箝技术记录心室肌细胞钠电流。结果:AP-Q 3-30nmol/L浓度依赖性地增大I_(Na),EC_(50)、为104nmol/L(95％可信范围:78-130nmol/L)。AP-Q 300nmol/L使I-V曲线左移,使半数激活电压从(-36.3±2.3)mV变为(-43±23)mV(n=6,P<0.01),半数失活电压从(-75±6)mV变为(-59±5)mV(n=6,P<0.01)。AP-Q 300nmol/L使I_(Na)半数恢复时间从(114±36)ms缩短为(17±2)ms(n=6,P<0.01),并明显减慢I_(Na)的快速失活时间常数(τ_f)。结论:AP-Q对I_(Na)有促进作用并减慢其失活过程。     

Electrophysiological effects of fluoxetine in mammalian cardiac tissues

Fluoxetine is a widely used antidepressant compound having selective serotonin reuptake inhibitor properties. In this study, the actions of fluoxetine were analyzed in guinea pig, rat, rabbit and canine ventricular myocardiac preparations using conventional microelectrode and whole cell voltage clamp techniques. Low concentrations of fluoxetine (1-10 micromol/l) caused significant shortening of action potential duration (APD) and depression of the plateau potential in guinea pig and rabbit papillary muscles and single canine ventricular myocytes. In rat papillary muscle, APD was not affected by fluoxetine (up to 100 micromol/l), however, the drug decreased the force of contraction with EC50 of 10 micromol/l. Fluoxetine (10 micromol/l) also decreased the maximum velocity of depolarization and action potential overshoot in each species studied. At this concentration no effect was observed on the resting membrane potential; high concentration (100 micromol/l), however, caused depolarization. In voltage clamped canine ventricular myocytes, fluoxetine caused concentration-dependent block of the peak Ca2+ current at 0 mV with EC50 of 5.4+/-0.94 micromol/l and Hill coefficient of 1.1+/-0.14 (n=6). In addition, 10 micromol/l fluoxetine shifted the midpoint of the steady-state inactivation curve of the Ca2+ current from -20.7+/-0.65 to -26.7+/-1 mV (P<0.001, n=8) without changing its slope factor. These effects of fluoxetine developed rapidly and were fully reversible. Fluoxetine did not alter voltage-dependence of activation or time constant for inactivation of I(Ca). Fluoxetine had no effect on the amplitude of K+ currents (I(K1) and I(to)). The inhibition of cardiac Ca2+ and Na+ channels by fluoxetine may explain most cardiac side effects observed occasionally with the drug. Our results suggest that fluoxetine may have antiarrhythmic (class I + IV type), as well as proarrhythmic properties (due to impairment of atrioventricular or intraventricular conduction and shortening of repolarization). Therefore, in depressed patients with cardiac disorders, ECG control may be suggested during fluoxetine therapy.     

Voltage-dependent and frequency-independent inhibition of recombinant Cav3.2 T-type Ca2+ channel by bepridil

Effects of bepridil on the low voltage-activated T-type Ca2+ channel (CaV3.2) current stably expressed in human embryonic kidney (HEK)-293 cells were examined using patch-clamp techniques. Bepridil potently inhibited ICa,T with a markedly voltage-dependent manner; the IC50 of bepridil was 0.4 micromol/l at the holding potential of -70 mV, which was 26 times as potent as that at -100 mV (10.6 micromol/l). Steady-state inactivation curve (8.4 +/- 1.7 mV) and conductance curve (5.9 +/- 1.9 mV) were shifted to the hyperpolarized potential by 10 micromol/l bepridil. Bepridil exerted the tonic blocking action but not the use-dependent block. Bepridil had no effect on the recovery from inactivation of T-type Ca2+ channels. Thus, high efficacy of bepridil for terminating atrial fibrillation and atrial flutter may be considered to be attributed, at least in a part, to the T-type Ca2+ channel-blocking actions.     

Mechanisms underlying the contractile response to endothelin-1 in the rat renal artery

We assessed the functional response and the mechanisms following receptor stimulation of endothelin-1 (ET-1) in the rat renal artery. In this study, isometric tension was recorded in renal artery rings without endothelium. Cumulative application of ET-1 from 0.1 to 100 nmol/l induced a sustained concentration-dependent contraction in the renal artery. Submaximal contraction induced by 10 nmol/l ET-1 in 2.5 mmol/l Ca(2+) and in the absence of inhibitors was used as control response (100%). The relative contribution of different sources of Ca(2+) in ET-1-induced contraction was evaluated. The contractile response to 10 nmol/l ET-1 in 2.5 mmol/l Ca(2+ )(1.2 +/- 0.2 g) was significantly inhibited either in Ca(2+)-free solution containing 100 micromol/l ethylene glycol bis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (0.6 +/- 0.1 g) or after depletion of intracellular Ca(2+) stores (0.62 +/- 0.05 g). The contribution of phospholipase C and protein kinase C was evaluated by using their inhibitors 2-nitro-4-carboxyphenyl N,N-diphenylcarbamate (NCDC) and [1-(5-isoquinolinesulfonyl)-2-methylpiperazine] (H-7), respectively. The contractile response to 10 nmol/l ET-1 was inhibited by 10 micromol/l NCDC (to 80 +/- 6%) and 30 micromol/l H-7 (to 76.6 +/- 6.5%). We found that 1 micromol/l nifedipine inhibited the ET-1-induced contraction (to 48.7 +/- 6.9%), indicating the contribution of Ca(2+) influx through voltage-gated L-type Ca(2+) channels to this response. Further, the inhibitory effect of nifedipine was to a greater extent as compared with NCDC or H-7. Additive inhibition of ET-1-induced contraction was not observed in the presence of both nifedipine and NCDC. We also evaluated the role of the ionic transport system in the ET-1-induced response by using 20 nmol/l 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), an inhibitor of Na(+)-H(+) exchange, or 100 micromol/l ouabain, an inhibitor of Na(+)-K(+)-ATPase. The response to ET-1 was decreased by both EIPA (to 61.6 +/- 8.4%) and ouabain (to 62.1 +/- 8.6%). The contribution of Na(+)-Ca(2+) exchange to ouabain action was tested using the inhibitor dimethyl amiloride HCl (10 micromol/l). The decrease in ET-1-induced contraction by the combination of ouabain and dimethyl amiloride HCl was similar to that observed with ouabain alone. In view of these observations, both extra- and intracellular sources of Ca(2+) contribute to the contractile response induced by ET-1 in the renal artery. Our findings also revealed the importance of Ca(2+) influx through voltage-gated L-type Ca(2+) channels in mediating contraction to ET-1 in the renal artery, whereas a minor role of phospholipase C and protein kinase C was observed. Na(+)-H(+) exchange and Na(+)-K(+)-ATPase also play a role in the ET-1-induced contraction in renal artery. Moreover, the contribution of Na(+)-K(+)-ATPase in ET-1 contraction is not an Na(+)-Ca(2+) exchange-related process.     

Properties of voltage-gated Na+ channels in the human rhabdomyosarcoma cell-line SJ-RH30: conventional and automated patch clamp analysis.

Conventional and automated patch clamp electrophysiology were used to characterise the Na+ current of the SJ-RH30 human rhabdomyosarcoma. In conventional recordings SJ-RH30 cells exhibited a fast activating, fast inactivating Na+ current at potentials positive to -40 mV; in full current-voltage curves maximum current occurred between -20 and -10 mV. Inactivation kinetics at 0 mV were biexponential with time constants of 0.5 and 3.7 ms. Deinactivation at -90 mV also exhibited two kinetic components. Tetrodotoxin (TTX) blocked the Na+ current completely at 1 microM. The NaV 1.4 selective toxin mu-CTx-GIIIB reversibly blocked the Na+ current approximately 60% at 10 microM. Very similar biophysical behaviour was observed in automated patch clamp and conventional recordings. For example, inactivation mid-point was -72+/-2 mV (slope factor 7.2+/-0.2) in automated patch clamp and -74+/-2 mV (slope factor 7.4+/-0.4) with conventional recording. The corresponding values for activation mid-point were -33.2+/-2.4 and -30.3+/-2.7 mV (slope 5.8+/-0.3 and 6.4+/-0.3, respectively). The throughput of the automated method was used to generate additional pharmacological data on inhibition of the Na+ current. TTX inhibited with an IC50 of 23 nM. Mu-CTx-GIIIB also inhibited the channel in a concentration-dependent manner. Inhibition produced by both tetracaine and amitriptyline were shown to be frequency-dependent. Our experiments indicate that the Na+ current of SJ-RH30 cells arises mainly from channels with a phenotype like recombinant NaV 1.4 channels. The suitability of these cells for automated patch clamp suggests they may be useful for higher throughput studies of the interaction of drugs with human skeletal muscle Na+ channels.     

Pharmacological profile of SL 59.1227, a novel inhibitor of the sodium/hydrogen exchanger

1. The NHE1 isoform of the Na(+)/H(+) exchanger plays an important role in the regulation of intracellular pH and in cardiac cell injury caused by ischaemia and reperfusion. SL 59.1227 is a novel imidazolypiperidine Na(+)/H(+) antiport inhibitor which is structurally unrelated to previously described acylguanidine inhibitors such as cariporide. 2. Recovery of pH(i) following an intracellular acid load was measured in CCL39-derived PS120 variant cells, selectively expressing either NHE1 or NHE2 isoforms of the Na(+)/H(+) exchanger. pH(i) recovery was potently and selectively slowed by SL 59.1227 in NHE1-expressing cells (IC(50) 3.3+/-1.3 nM) versus NHE2-expressing cells (2.3+/-1.0 microM). The respective IC(50) values for cariporide were 103+/-28 nM (NHE1) and 73+/-46 microM (NHE2). 3. In anaesthetized rats following left coronary artery occlusion (7 min) and reperfusion (10 min) SL 59.1227 (10 - 100 microg kg(-1) min(-1) i.v.) inhibited ischaemia-mediated ventricular tachycardia (71 - 100%) and reperfusion-induced ventricular fibrillation (75 - 87%) and prevented mortality. Bolus i.v. administration of SL 59.1227 (1 mg kg(-1)) produced anti-arrhythmic effects when administered either before or during ischaemia. 4. Cardiac infarct size was determined in anaesthetized rabbits following left coronary artery occlusion (30 min) and reperfusion (120 min). Infarct size measured as a percentage of the area at risk was 36.2+/-3.4% (control group) versus 15.3+/-3.9% (SL 59.1227 0.6 mg kg(-1) i.v.). 5. SL 59.1227 is the first example of a potent and NHE1-selective non-acylguanidine Na(+)/H(+) exchanger inhibitor. It possesses marked cardioprotective properties.