Caffeine actions on currents induced by calcium-overload in Purkinje fibers

The ionic events underlying the changes induced by caffeine in calcium-overloaded Purkinje fibers were studied by means of a voltage-clamp technique. The following results were obtained. In fibers exposed to strophanthidin (5 X 10(-7) M), a depolarizing clamp of suitable magnitude or duration is followed by an oscillatory current (Ios) often superimposed on a decaying inward tail current (the "tail current"). Caffeine (9 mM) abolishes Ios and increases the tail current. Caffeine has similar actions when calcium overload is induced by increasing [Ca]0 or decreasing [Na]0. The magnitude of the tail current is reduced by decreasing [Ca]0. The tail current appears with repolarizations to -40 mV or more negative values as Ios appears in the absence of caffeine. As with Ios the tail current can be triggered twice (during and after a test clamp of suitable characteristics), becomes more inward with repeated clamps and becomes larger with larger or longer conditioning clamps. During the recovery from caffeine exposure, the tail current decreases gradually as Ios returns progressively. It is concluded that both Ios and tail current are caused by calcium overload but are affected in opposite direction by caffeine, apparently because caffeine decreases the calcium overload in the sarcoplasmic reticulum (abolition of Ios) and increases the calcium to be extruded from the sarcoplasm (increase in the tail current).     

Effects of Buthus martensii (Karsch) scorpion venom on sodium currents in rat anterior pituitary (GH3/B6) cells.

The effects of two fractions (II, containing anti-insect toxins, and III, containing eight anti-mammal toxins) isolated from the venom of the Old World scorpion Buthus martensii (Karsch) on Na+ currents of rat anterior pituitary cells (GH3/B6 cells) were investigated using the whole-cell configuration of the patch clamp technique. Fraction II induced a temporary, and fraction III a permanent increase of the Na+ current amplitude. Application of each of the venom fractions resulted in a flattening of the curve relating steady state Na+ inactivation to membrane potential. In addition, the two fractions had specific effects. Fraction II shifted the voltage dependence of Na+ current activation by -42 mV, and the voltage dependence of Na+ inactivation by -25 mV in the absence of a conditioning depolarizing pre-pulse. Slowing of Na+ inactivation was most prominent at negative membrane potentials, resulting in a steady Na+ inward current at the holding potential of -80 mV. Fraction III induced a pronounced slowing of Na+ inactivation leading to an increase of peak Na+ currents and to incomplete steady state Na+ inactivation even at positive membrane potentials.     

Action of the 1,4-dihydropyridine derivative, KW-3049, on the smooth muscle membrane of the rabbit mesenteric artery.

1. The effect of 1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridine-dicarboxylic acid methyl 1-(phenylmethyl)-3-piperidinyl ester hydrochloride (KW-3049) on the smooth muscle membrane of the rabbit mesenteric artery was investigated using microelectrode and single electrode voltage clamp methods. 2. In whole tissue preparations KW-3049 did not alter the resting membrane potential of the artery, but did inhibit the action potential evoked by a depolarizing current in the presence of 10 mM tetraethylammonium (TEA). 3. Using the voltage clamp technique, the effects of KW-3049 on the inward current evoked in solution containing 100 mM BaCl2 were observed. When the membrane was held at -60 mV, KW-3049 inhibited the inward current in a concentration-dependent manner. The inward current evoked by a larger depolarizing pulse was inhibited to a larger extent than that evoked by a smaller one. 4. When the membrane was held at -80 mV, the inward current evoked at test potentials of -10 and 0 mV was enhanced by low concentrations of KW-3049 (below 100 nM). 5. KW-3049 accelerated the rate of inactivation of the inward current and shifted the voltage-dependent inactivation curve to the left. 6. KW-3049 has a long-lasting inhibitory action on smooth muscle cells, since the inhibition of the inward current persisted for over 1 h after the removal of KW-3049. 7. Our results suggest that KW-3049 has a selective and long-lasting action on the Ca channels of the smooth muscle cell membrane of the rabbit mesenteric artery. This agent has both facilitatory and inhibitory actions on the Ca channel, depending on the values of the holding and command potentials.     

Effects of ciguatoxin on current and voltage clamped frog myelinated nerve fibre

The effects of 0.25 X 10(-9) and 1.25 X 10(-9) g/ml of purified ciguatoxin (CgTX) upon the node of Ranvier of frog isolated nerve fibres were investigated under current and voltage clamp conditions. When added to the external solution, CgTX induced spontaneous action potentials at a frequency of about 100 Hz, which were reversible upon removal of the toxin. Under voltage clamp conditions, CgTX modified neither linear leakage and capacity currents nor K current, but reversibly induced a maintained (late) inward current (IL) during long lasting depolarizations. IL, as well as the peak Na current, was suppressed by tetrodotoxin (300 nM). The steady-state inactivation curve of the Na current showed that a fraction of the current (corresponding to IL) did not inactivate. IL activated and reversed at voltages about 30 mV more negative than the peak Na current (recorded under control conditions or in the presence of CgTX). During a given depolarizing pulse, the amplitude of IL depended on the holding potential. IL was about three times greater when the holding potential was -70 mV rather than -120 mV. We conclude that CgTX specifically interacts with and modifies Na channels. We also conclude that the effects of CgTX depend on membrane potential.     

Noradrenaline activates a calcium-activated chloride conductance and increases the voltage-dependent calcium current in cultured single cells of rat portal vein.

1. Membrane responses were recorded by a patch pipette technique in cultured cells isolated from rat portal vein. Using the whole-cell mode, pressure ejections of noradrenaline evoked depolarization (current clamp) and inward current (voltage clamp) at membrane potentials of -60 to -70 mV. The noradrenaline-induced response was reversibly blocked by prazosin indicating that the response was mediated by alpha 1-adrenoceptors. 2. The ionic mechanism of the noradrenaline-induced inward current was investigated in potassium-free caesium-containing solutions. Alteration of the chloride equilibrium potential produced similar changes in the reversal potential of the noradrenaline-induced current, indicating that noradrenaline opened chloride-selective channels. There was no evidence implicating sodium or calcium as the charge-carrying ion. 3. Caffeine applied in the bathing solution also induced a transient increase in chloride conductance but the noradrenaline-induced response was lost after application of caffeine. This is interpreted to mean that the increase in chloride conductance induced by noradrenaline and caffeine can occur as a consequence of a rise in intracellular calcium concentration depending on release of calcium from the same intracellular stores. 4. In the presence of caffeine, noradrenaline increased both the voltage-dependent calcium and chloride membrane conductances during application of repetitive depolarizing pulses. It is concluded that in isolated cells of the rat portal vein the depolarization in response to noradrenaline is mediated by an increase in chloride conductance depending on both the calcium release from intracellular stores and the increase of the voltage-dependent calcium current.     

Actions of the digitalis analogue strophanthidin on action potentials and L-type calcium current in single cells isolated from the rabbit atrioventricular node.

1. The atrioventricular node (AVN) of the heart is vital to normal cardiac function and is a major site of antiarrhythmic drug action. This study describes the effects of the cardiac glycoside analogue strophanthidin on spontaneous action potentials and L-type calcium current recorded from single AVN cells isolated from the rabbit heart. 2. With a standard KCl-based internal dialysis solution, exposure to 50 microM strophanthidin produced a progressive depolarization of the maximum diastolic potential and a reduction in action potential amplitude and upstroke velocity. Sustained application resulted in the loss of action potentials and occurrence of spontaneous 'bell-shaped' depolarizations. 3. Cells were whole-cell voltage clamped at -40 mV and depolarizing voltage clamps applied. With a standard KCl-based internal dialysis solution, exposure to 50 microM strophanthidin caused a large reduction of ICa,L at all potentials between -30 and +40 mV (n = 4). At + 10 mV, the mean ICa,L amplitude was reduced from -232 +/- 65 pA to -48 +/- 26 pA (P < 0.05; 1 test; n = 5 cells). 4. To record ICa,L more selectively, cells were dialysed with a Cs-based pipette solution. A short strophanthidin exposure reduced ICa,L amplitude from -250 +/- 31 pA to -88 +/- 19 pA (P < 0.001; n = 8 cells). For both KCl and CsCl-based solutions it was observed that sustained exposure to strophanthidin for several minutes caused spontaneous inward fluctuations in the membrane current record similar to the 'ITI' (arrhythmogenic oscillatory transient inward) current shown for other cardiac cells. 5. When the calcium chelator BAPTA was added to the pipette solution (10 mM), the reduction in ICa,L by strophanthidin was largely eliminated (P > 0.1), and no spontaneous inward current fluctuations were observed after sustained exposure to strophanthidin (n = 8 cells). 6. When external Ca in the perfusate was replaced with Ba, strophanthidin did not significantly reduce the Ba current through L-type calcium channels (n = 5 cells). 7. We conclude that strophanthidin reduces ICa,L by an indirect action, mediated by the rise in intracellular calcium (Cai) which follows inhibition of the Na/K pump caused by cardiac glycosides. The appearance of spontaneous ITI with strophanthidin would also seem to be mediated by a rise in Cai, and may contribute to the spontaneous oscillations in membrane potential observed after prolonged strophanthidin exposure.     

Actions of the novel neuroprotective agent, lifarizine (RS-87476), on voltage-dependent sodium currents in the neuroblastoma cell line, N1E-115.

1. The actions of the neuroprotective agent, lifarizine (RS-87476-190), on voltage-dependent Na+ currents have been examined in the neuroblastoma cell line, N1E-115, using the whole-cell variant of the patch clamp technique. 2. At a holding potential of -80 mV, lifarizine reduced the peak Na+ current evoked by a 10 ms depolarizing step with an IC50 of 1.3 microM. At holding potentials of -100 and -60 mV the IC50 concentrations of lifarizine were 7.3 microM and 0.3 microM, respectively. 3. At a holding potential of -100 mV, most channels were in the resting state and the IC50 value for inhibition of Na+ current should correspond to the dissociation constant of lifarizine for resting channels (KR). KR was therefore estimated to be 7.3 microM. 4. In the absence of lifarizine, recovery from inactivation following a 20 s depolarization from -100 mV to 0 mV was complete within 2 s. However, in the presence of 3 microM lifarizine recovery took place in a biexponential fashion with time constants of 7 s and 79 s. 5. Lifarizine (1 microM) had no effect on steady-state inactivation curves when conditioning pre-pulses of 1 s duration were used. However, when pre-pulse durations of 1 min were used the curves were shifted to the left by lifarizine by about 10 mV. Analysis of the shifts induced by a range of lifarizine concentrations revealed that the apparent affinity of lifarizine for the inactivated state of the channel (K1) was 0.19 microM. 6. Lifarizine (1 microM) had no effect on chloramine-T-modified Na+ currents, suggesting no significant open channel interaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Caffeine-induced inhibition of calcium channel current in cultured smooth cells from pregnant rat myometrium.

1. The inhibitory effect of caffeine on the calcium channel current was investigated in cultured myometrial cells isolated from pregnant rats. 2. Caffeine inhibited the calcium channel current elicited from a holding potential of -70 mV in a concentration-dependent manner. The IC50 was estimated to be 35 mM. 3. The caffeine inhibition was not enhanced when calcium channels were opened by a conditioning depolarizing pulse sequence or when the number of inactivated calcium channels was increased at depolarized holding potentials. 4. Caffeine antagonized the specific binding of (+)-[3H]-isradipine to myometrial membranes. The IC50 value found in binding experiments was similar to the IC50 value for half-maximal inhibition of calcium channel current. Caffeine decreased the maximal binding capacity of (+)-[3H]-isradipine to myometrial membranes without any significant change in the dissociation constant. 5. The results indicate that caffeine interacts with a site closely associated with the voltage-dependent calcium channels in myometrial cells and, in turn, inhibits calcium influx.     

Use- and voltage-dependent depression by ethmozine (moricizine) of the rapid inward sodium current in single rat ventricular muscle cells

Effects of ethmozine (moricizine) on the rapid inward sodium current (INa) were studied in freshly isolated single cells of rat ventricular myocardium. INa was measured by means of a patch clamp method for observing integral ionic currents. Ethmozine was applied extracellularly to a small cell membrane patch at concentrations of 10, 20, and 40 microM. At a stimulation frequency of 0.1 Hz the drug decreased the peak INa without producing a shift of the current-voltage curve, but shifted the V0.5 of the steady-state inactivation curve by -6 mV. At frequencies of 2-5 Hz the ethmozine-induced block exhibited a prominent use dependence, with trains of depolarizing clamp pulses 5-50 ms in duration eliciting maximal INa from holding potentials at which the steady-state inactivation variable h infinity was close to 1. The use-dependent inhibition of INa became more pronounced with an increase in both stimulation rate and pulse duration. In contrast to what has been observed in the node of Ranvier of the frog, the present results indicate that ethmozine binds to both inactivated and open Na+ channels, but that the contribution of the open channel block to the overall block at depolarizing clamp step durations of several hundred milliseconds is small in comparison with the contribution of the block of inactivated channels.     

The activation gate of the sodium channel controls blockade and deblockade by disopyramide in rabbit Purkinje fibres.

1. The effect of disopyramide on the maximum upstroke velocity (Vmax) and the sodium current of rabbit cardiac Purkinje fibres was studied with the two-microelectrode voltage-clamp technique. 2. In the absence of stimulation the drug did not cause block at membrane potentials ranging from -100 to -65 mV. Use-dependent block of Vmax was most pronounced at -75 mV. At hyperpolarized membrane potentials development of use-dependent block was faster than at depolarized membrane potentials. The time course of development of use-dependent block was not significantly influenced by the duration of the depolarizing pulse. These results strongly suggest that disopyramide predominantly blocks activated sodium channels. 3. The relative decrease of the sodium current at the beginning of a 2 s depolarizing clamp to -45 mV was almost the same as at the end, implying a rapid blockade of activated sodium channels. The Hill plots were linear with slopes ranging from 0.978 to 1.08 indicating a first order reaction; the dissociation constant for activated channels was 70 microM. 4. Recovery of Vmax from use-dependent block during rest was strongly voltage-dependent, the time constant of recovery increasing upon hyperpolarization. When the fraction of charged molecules was reduced by changing the pH of the external solution, the voltage-dependence of recovery was decreased. In contrast, recovery of Vmax for a change in holding potential from -80 to -95 mV was very fast during repetitive stimulation. 5. It is concluded that disopyramide blocks the sodium channel during activation and is trapped in the channel when the activation gate closes.     

Regulation by GTP and its stable thiol derivatives of calcium current components in rat nodose ganglion neurons

The calcium current components of acutely dissociated nodose ganglion neurons were characterized using the whole-cell variation of the patch-clamp technique. Many neurotransmitters regulate neuronal calcium currents via GTP binding (G) proteins and in some cases affect calcium current components selectively. To determine whether G proteins regulated these current components in the absence of ligand binding, recording pipettes contained 0.1 mM GTP, guanosine 5'-O-(thiodiphosphate) (GDP-beta-S), or guanosine 5'-O-(thiotriphosphate) (GTP-gamma-S). Nodose ganglion neurons had three calcium current components, similar to T, N, and L current components found in other sensory neurons. Isolated T currents did not diminish in magnitude during a 20-min recording, but there was a progressive loss of currents containing the N and L current components. The reduction of current magnitude was primarily dependent on the extent of intracellular dialysis and not on the holding potential (Vh) or stimulus frequency. When GDP-beta-S was substituted for GTP in the pipette solution, there was no change in the T current or in the rate of run-down of N and L current components. Substitution of GTP-gamma-S for GTP in the pipette solution resulted in a moderate (approximately 40%) loss of isolated T current. This effect was most evident on T currents evoked at relatively positive clamp potentials (Vc, -30 to -15 mV) and occurred relatively late (approximately 10 min) in the recording. In the presence of GTP-gamma-S, currents evoked from Vh = -80 mV, containing the N and L current components, were reduced 40-60%, with a lesser effect on those currents evoked from Vh = -40 mV, containing primarily the L current component. The average time to peak current (Ip) was increased 3-4-fold in the presence of GTP-gamma-S, and the Vc at which the maximal peak current was evoked was shifted +10 to 20 mV. These effects were evident within 2-5 min after initiation of the whole-cell recording. Pretreatment of neurons with pertussis toxin attenuated or blocked the effects of GTP-gamma-S. We conclude that nodose ganglion neurons have T-, N-, and L-type calcium current components, which had different stability during whole-cell recording. Activation of G proteins with GTP-gamma-S reduced N greater than T much greater than L currents, effects reduced in the presence of pertussis toxin. Thus, the calcium current components of nodose ganglion neurons were regulated by cytosolic constituents and by Gi- or Go- type G proteins.     

Induction of the oscillatory current by low concentrations of caffeine in sheep cardiac Purkinje fibres

Summary The actions of low concentrations of caffeine (0.5–2 mmol/1) on the transient inward oscillatory current (I_os) and the inward tail current (I_ex) were studied in sheep cardiac Purkinje fibres by means of a two microelectrode voltage clamp method. The following results were obtained. Caffeine: 1. induced an I_os when this current was not already present; 2. increased the amplitude (within limits) and consistently decreased the time to peak of an already present I_os; 3. increased I_ex upon which I_os may be superimposed; 4. shifted the depolarizing threshold for the appearance of I_os to more negative and the repolarizing threshold to less negative values; 5. increased the effects of strophanthidin of I_os and I_ex; 6. exaggerated the effects of high [Ca]_o which by itself mimicked some of the actions of caffeine; 7. had a small effect of I_os and I_ex in low [Ca]_o; 8. reversed the effect of norepinephrine on I_ex; 9. enhanced the effects of trains of clamps and of longer clamp steps on I_os and I_ex. It is concluded that low concentrations of caffeine facilitate the manifestations of calcium overload thereby inducing or exaggerating the oscillatory and tail currents and that these effects are modulated by the cellular calcium load but are not mediated through adrenergic mechanisms. Send offprint requests to M. Vassalle at the above address     

Strontium, nifedipine and 4-aminopyridine modify the time course of the action potential in cells from rat ventricular muscle

Action potentials, initiated by brief depolarizing pulses, were recorded from single cells isolated from rat ventricular muscle. These action potentials showed a rapid upstroke to about +30 mV, followed by two phases of repolarization referred to as the early and late phases of the action potential. Nifedipine (1 microM), which blocks the second inward current (Isi) carried by Ca in these cells, shortened the early phase. Substitution of strontium for calcium in the solution bathing the cells, a procedure which prolongs Isi, prolonged the early phase. 4-Aminopyridine (1 mM), which inhibits transient outward current, prolonged the early phase with either calcium or strontium in the external solution. It is concluded that both Isi and transient outward current contribute to the early phase of the action potential in rat ventricular muscle. It is also suggested that Isi does not directly contribute to the late phase, since the characteristics of the late phase are not compatible with such a role, and the possibility of additional inward current is investigated in the accompanying paper (Mitchell et al., 1984).     

Block of sodium channels by psychotropic drugs in single guinea-pig cardiac myocytes.

1. Effects of imipramine and haloperidol on voltage-gated sodium channels were investigated in guinea-pig isolated ventricular myocytes by the whole-cell patch clamp technique. Some additional experiments were also performed with chlorpromazine for the purpose of comparison. 2. All test drugs in micromolar concentrations suppressed the amplitude of peak sodium current associated with step depolarization from a holding potential of -140 mV in a reversible manner. The order of potency was chlorpromazine greater than imipramine greater than haloperidol. 3. Dose-response curves obtained with a holding potential of -140 mV were best fitted by 2:1 stoichiometry in all three drugs and were shifted in the direction of lower concentrations when a holding potential of -90 mV was used. 4. The drug-induced block was not associated with any change in the time courses of sodium current activation and inactivation. 5. Steady-state sodium channel inactivation curve was shifted in the direction of more negative potentials by the drugs. 6. All three drugs also produced marked use-dependent block as demonstrated by a cumulative increase in the block during a train of depolarizing pulses. 7. The use dependence was due to a higher affinity of the drugs for the inactivated state of sodium channels than the resting state and to a very slow repriming of the drug-bound sodium channels from inactivation. 8. The steady-state and use-dependent block of voltage-gated sodium channels by psychotropic drugs may contribute to their cardiotoxic and perhaps antiarrhythmic effect.     

Effects of captopril on membrane current and contraction in single ventricular myocytes from guinea-pig.

1. Single guinea-pig ventricular myocytes were voltage-clamped and cell length was measured with a photodiode array. 2. Captopril (1 x 10(-5) M) reduced both peak early current and active shortening in response to a depolarizing clamp pulse along a similar time course. 3. From a holding potential of around -45 mV peak early inward current was reduced by 37 +/- 9% (P less than 0.001) on exposure to captopril. The early current-voltage relationship was shifted outwards by captopril indicating a reduction in membrane conductance through the L-type calcium channel (ICa). 4. The amplitude of cell shortening in response to depolarizing voltage steps was reduced but the voltage-dependence of contraction after captopril was unchanged. 5. A small negative shift of the potential at which ICa was half-activated was observed after captopril. There was no change in the voltage-dependence of the inactivation variable or in the time-dependence of repriming for ICa. 6. The actions of captopril on ICa and developed shortening were dose-dependent and took place in the same proportion when Ica was increased by isoprenaline. 7. These results are discussed in relation to the effects of captopril on Ica and contraction and to its clinical usage.     

阿霉素对豚鼠单一心肌细胞钙电流(I_(Ca-L))的影响

目的　探讨阿霉素 (adriamycin ,ADR)诱发心肌病的发病机制。方法　采用膜片钳全细胞记录方法 ,研究ADR对豚鼠单一心肌细胞L型钙通道电流 (ICa L)的影响。结果　用 0 1mmol·L-1ADR灌流前后豚鼠心肌细胞ICa L电流 电压 (I U)关系曲线近似倒钟形 ,其峰值电压在 + 10mV ;0 1mmol·L-1ADR可使ICa L明显增大 ,由灌流前的 ( -0 93± 0 0 5 )nA(n =8)增大到 ( - 1 3 1± 0 0 8) (n =8) (P <0 0 1) ,其增大百分率为灌流前的 ( 4 1 6± 12 3 ) % (n =8)。结论　ADR激活电压依赖性钙通道促进钙内流很可能是ADR造成心肌细胞内钙超载 ,从而诱发心肌病的机制之一。     

Electrophysiological effects of SD-3212, a new antiarrhythmic agent with vasodilator action, on guinea-pig ventricular cells.

1. The effects of SD-3212 on transmembrane action potentials were examined in right ventricular papillary muscles and in single ventricular myocytes isolated from guinea-pig hearts. 2. In papillary muscles, SD-3212 > or = 3 microM caused a significant decrease in the maximum upstroke velocity (Vmax) of action potential without affecting resting membrane potential. The inhibition of Vmax was enhanced at higher stimulation frequencies. 3. In the presence of SD-3212, trains of stimuli at rates > or = 0.5 Hz led to a use-dependent inhibition of Vmax. The time constant for the recovery of Vmax from the use-dependent block was 1.3 s. 4. Voltage-dependence of Vmax inhibition by SD-3212 was investigated in single myocytes. The curves relating membrane potential and Vmax were shifted by SD-3212 (10 microM) in a hyperpolarizing direction by 6.2 mV. 5. In myocytes treated with SD-3212 (10 microM), the Vmax of test action potentials preceded by conditioning clamp to 0 mV was decreased progressively as the clamp pulse duration was prolonged. Vmax of test action potentials following a long (1 s) 0 mV clamp recovered at a time constant ranging from 1.01 to 1.22 s, being shorter at the more negative potential within a range from -70 to -90 mV. 6. These findings suggest that the primary electrophysiological effect of SD-3212 is a use- and voltage-dependent inhibition of sodium channels. From the onset and offset kinetics of the use-dependent block, SD-3212 is located between fast and intermediate kinetic Class-I drugs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative effects of clarithromycin on action potential and ionic currents from rabbit isolated atrial and ventricular myocytes

Prolongation of QT interval by several antibacterial drugs is an unwanted side effect that may be associated with development of ventricular arrhythmias. The macrolide antibacterial agent clarithromycin has been shown to cause QT prolongation. To determine the electrophysiologic basis for this arrhythmogenic potential, we investigated clarithromycin effects on (i). action potentials recorded from rabbit Purkinje fibers and atrial and ventricular myocardium using conventional microelectrodes and (ii). potassium and calcium currents recorded from rabbit atrial and ventricular isolated myocytes using whole-cell patch clamp recordings. We found that (i). clarithromycin (3-100 microM) exerted concentration-dependent lengthening effects on action potential duration in all tissues, with higher efficacy and reverse frequency-dependence in Purkinje fibers. However, clarithromycin did not cause development of early afterdepolarizations, and the parameters other than action potential duration were almost unaffected; (ii). clarithromycin (10-100 microM) reduced the delayed rectifier current. Significant blockade (approximately 30%) was found at the concentration of 30 microM. At 100 microM, it decreased significantly the maximum peak of the calcium current amplitude but failed to alter the transient outward and inwardly rectifier currents. It was concluded that these effects might be an explanation for the QT prolongation observed in some patients treated with clarithromycin.     

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.     

Action of angiotensin II, 5-hydroxytryptamine and adenosine triphosphate on ionic currents in single ear artery cells of the rabbit.

1. Angiotensin II, 5-hydroxytryptamine (5-HT) and adenosine triphosphate (ATP) evoked a transient inward current in isolated single car artery cells of rabbit held at -60 mV by whole cell voltage clamp in physiological saline using a KCL-containing pipette solution. Under these conditions agonist did not activate a calcium-dependent potassium current. 2. Responses to each agonist were transient and desensitized rapidly. Inward current at -60 mV holding potential was not abolished by blockade of voltage-dependent calcium channels or by buffering intracellular calcium with BAPTA, a calcium chelator, or following depletion of intracellular calcium stores with ryanodine. 3. The shape of the current-voltage relationships and the reversal potentials of the current induced by angiotensin II, 5-HT and ATP were similar under a variety of ionic conditions. Agonist-induced current was unaffected by replacing intracellular chloride with citrate ions or by replacing intracellular sodium with caesium or extracellular sodium with barium or calcium. Replacement of extracellular sodium with Tris shifted the reversal potential in all cases by around 30 mV negatively. 4. These data suggest that angiotensin II, 5-HT and ATP activate similar cationic conductances which are relatively non-selective allowing mono- and divalent cations to cross the smooth muscle cell membrane. These channels may allow the influx of calcium under physiological conditions.