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 labedipinedilol‐A,third‐generation dihydropyridine‐type calcium blocker,on ouabain‐induced arrhythmia

Labedipinedilol‐A, a novel dihydropyridine‐type calcium antagonist with α/β‐adrenoceptor blocking properties, has been reported to produce a cardioprotective effect against ischemia reperfusion injury in rats. We investigated the protective effects of labedipinedilol‐A on ouabain‐induced tonotropy and arrhythmias in isolated whole atria, and using patch‐clamp techniques to study the underlying mechanism of its antiarrhythmic activity on isolated cardiac myocytes. Labedipinedilol‐A (10 µM) suppressed the tonotropic effect of ouabain significantly and prolonged the onset time of extra‐systole (arrhythmia) induced by ouabain in isolate atria. In the voltage‐clamp study, labedipinedilol‐A (1–100 µM) reduced the peak amplitude of sodium inward current (I_Na) and L‐type calcium current (I_Ca‐L), and shifted the current‐voltage (I‐V) curve upward in a concentration‐dependent manner. In contrast, the addition of labedipinedilol‐A increased transient outward potassium current (I_to) and inward rectifier potassium current (I_K1) significantly. Labedipinedilol‐A (10 µM) also effectively depressed the isoproterenol‐induced increase in the Ca²⁺ current. These results show that labedipinedilol‐A blocks I_Ca‐L and I_Na, and increases I_to and I_K1. These findings indicate that labedipinedilol‐A produces direct cardiac action, probably due to the inhibition of cardiac Na⁺ and Ca²⁺ channels. Our results suggest that labedipinedilol‐A may reduce the membrane conduction through inhibition of ionic channels which decrease ouabain‐induced arrhythmia. Drug Dev Res 69:26–33, 2008 © 2008 Wiley‐Liss, Inc.     

The effects of ryanodine and caffeine on Ca-activated current in guinea-pig ventricular myocytes.

1. Action potentials from guinea-pig single ventricular myocytes were interrupted by application of a 300 ms voltage clamp to -40 mV in order to evoke the Ca-activated tail current which is thought to be carried by Na:Ca exchange. Stimulation frequency was 1 Hz and temperature 36 degrees C. 2. The actions of ryanodine (1 microM and 10 microM) and caffeine (1 mM and 10 mM) on Ca-activated tail currents were investigated. 3. Exposure to 10 mM caffeine and ryanodine reduced tail currents associated with very abbreviated (12 ms duration) action potentials and greatly reduced the difference between first and steady-state tail currents at this action potential duration. These observations were interpreted in terms of suppression of Ca release from the sarcoplasmic reticulum (SR) stores. 4. Tail current decay during the voltage clamp is thought to reflect the fall in [Ca]i which accompanies muscle relaxation. Current decay is dependent on Ca extrusion via Na:Ca exchange and on Ca accumulation by the SR stores. Time constants of tail current decay were seen to decrease with increasing action potential duration. This relationship was not affected by 1 mM caffeine or 1 microM ryanodine. Ryanodine at 10 microM and 10 mM caffeine abolished this relationship and increased the time constants of current decay. An increase in the time constant of tail current decay was thought to reflect a reduction in the rate of Ca accumulation by the sarcoplasmic reticulum. 5. The actions of caffeine and ryanodine on the Ca-activated tail currents are consistent with a dose-dependent leakage of Ca from the SR Ca stores.(ABSTRACT TRUNCATED AT 250 WORDS)     

3,6-[二甲氨基]-二苯骈碘因甲酸盐对豚鼠单—心室肌细胞动作电位和慢内向钙电流的影响

采用全细胞电压钳技术,观察了3,6-[二甲氨基]-二苯骈碘因甲酸盐(IHC-64)对豚鼠单一心室肌细胞动作电位和慢内向钙电流(I_si)的影响。结果表明,IHC-64 20和200 μmol/L可明显缩短APD₂₀和APD₉₀。予先给予钙通道阻滞剂尼索地平可取消IHC-64缩短APD₂₀的作用。在电压钳条件下,上述浓度的IHC-64对I_si有明显的抑制作用,I_si由给药前的1.92±0.41 nA分别降低至给药后1.73±0.50 nA(P<0.05)和0.62±0.38 nA(P<0.01).提示IHC-64可能对心肌细胞钙通道有阻滞作用。     

Caffeine-induced decreases in the inward rectifier potassium and the inward calcium currents in rat ventricular myocytes.

The effects of high (20 mM) concentrations of caffeine were studied on the transmembrane voltage and currents in rat single ventricular myocytes by the whole cell configuration of the patch clamp technique. Rapid application of caffeine released Ca2+ from the sarcoplasmic reticulum and induced a Ni(2+)-sensitive transient inward current with concomitant change of the transmembrane voltage from -72.6 +/- 0.4 to -68.0 +/- 0.6 mV (n = 4). Maintained application of caffeine lengthened the action potential duration (APD90) from 66.7 +/- 16.9 to 135.1 +/- 34.1 ms (n = 4) and depressed the amplitude of both the inward rectifier potassium and the inward calcium currents. It is concluded that these effects of caffeine should be recognized when it is used as a tool to study electromechanical coupling.     

Voltage-dependent inhibition of the delayed outward potassium current by OPC-8490, a novel positive inotropic agent,in isolated atrial myocytes of guinea-pig heart

Summary To elucidate the ionic mechanisms underlying prolongation of the action potential by OPC-8490, a novel positive inotropic agent, we examined effects of the drug on the membrane currents in isolated atrial myocytes of guinea-pig heart. The tight-seal whole cell clamp technique was used. In the current clamp condition, OPC-8490 prolonged the atrial action potential in a concentration-dependent fashion. In the voltage clamp condition, OPC-8490 affected neither the conventional L type Ca current nor the outward I _K1 current significantly. The drug blocked the delayed outward K current (I _K) in a concentration-dependent fashion. At a constant concentration of the drug, inhibition of _{I K} became more marked as the membrane potential became more negative. The tail current of I _K could be fitted by a sum of two exponentials (fast and slow components). OPC-8490 blocked the fast component more prominently than the slow one. Time constants of both components were not affected significantly by the drug. In conclusion, OPC-8490 prolongs the cardiac action potential mainly by blocking I _K in a voltage-dependent and time-independent fashion. Send offprint requests to Y. Kurachi at the above address     

The actions of ryanodine on Ca2+-activated conductances in rat cultured DRG neurones; evidence for Ca2+-induced Ca2+ release

The whole-cell recording technique was used to investigate the actions of a calcium release channel ligand, ryanodine, on calcium-activated chloride conductances, and to evaluate ryanodine-sensitive Ca²⁺-induced Ca²⁺ release from intracellular stores in cultured neonatal rat DRG neurones. The aim of the project was to use ryanodine as a pharmacological tool to evaluate calcium-induced calcium release in the cell bodies of cultured DRG neurones. Action potential after-depolarizations were attenuated by extracellular application of the chloride channel blocker, niflumic acid (10 μM), and by ryanodine (10 μM); these actions occurred without concurrent changes in evoked action potentials. Ryanodine and caffeine (10 mM) activated calcium-dependent conductances and the responses to ryanodine were attenuated by depletion of caffeine-sensitive Ca²⁺ stores. The current clamp data were complicated by changes in potassium conductances so studies were carried out under voltage clamp and voltage-activated calcium currents and calcium-activated chloride and non-selective cation currents were isolated pharmacologically. Ryanodine (10 μM) evoked delayed, inward, calcium-activated non-selective cation and chloride currents which reversed close to 0 mV and were attenuated by N-methyl-d-glucamine, niflumic acid and dantrolene. Consistent with actions on action potential after-depolarizations, niflumic acid (10 μM) and ryanodine (10 μM) attenuated calcium-activated chloride currents evoked by calcium entry through voltage-activated calcium channels. Niflumic acid and ryanodine had no effects on voltage-activated calcium currents evoked from a holding potential of –90 mV by voltage step commands to 0 mV. In conclusion calcium-activated chloride conductances appear to be activated in part by calcium released from ryanodine-sensitive stores, and significant calcium-induced calcium release may occur locally in cell bodies of DRG neurones as a result of calcium entry through voltage-activated channels during an action potential. Received: 6 July 1998 / Accepted: 30 November 1998     

粉防己碱对犬心浦氏纤维慢内向电流的作用

应用双微电极电压钳技术,观察粉防己碱(Tet) 对犬心脏浦氏纤维细胞的慢内向电流I_si的影响,发现Tet对I_si的峰值有明显减弱作用。Tet的这种效应呈剂量及时间依赖性。当Tet浓度为30μmol/L和100μmol/L,给药后10min时,I_si的峰值分别由给药前的47.9±5.6 nA和38.4±21.5nA降至21±7.8nA和6.3±7.7nA,P值均小于0.01。Tet100μmol/L对由二价阳离子Sr²⁺所介导的I_si亦有明显的抑制作用。结果提示Tet是一慢通道阻滞剂。     

Effects of Goniopora toxin on the membrane currents of bullfrog atrial muscle

Summary The effects of Goniopora toxin (GPT), isolated from the Goniopora species, on the action potential and membrane currents of bullfrog atrial muscle were studied using the double sucrose-gap method. GPT at concentrations above 10 nmol/l prolonged the duration of the action potential and sometimes induced arrhythmias. The prolongation was also induced in the presence of Ca channel blockers (Mn or verapamil). These effects were not reversed by continuous superfusion of GPT-free solution, but were rapidly antagonized by tetrodotoxin (1 mol/l). The resting potential was not affected by GPT. Voltage clamp experiments revealed that sustained inward current flows following the fast sodium current upon depolarization, in the presence of GPT. The current was elicited by the toxin in Mn-treated fibers and abolished in the presence of TTX. The delayed outward current (I _x) was slightly reduced; the background K current (I _K1), inward background current (I _b) and slow inward current (I _slow) were not altered by GPT. These results suggest that GPT acts on sodium channels to give rise to a prolonged sodium current which is in turn responsible for the prolongation of the action potential.This work was supported in part by a Grant-in Aid for Special Project Research (No. 58110007) from the Ministry of Education, Science and Culture, Japan (M.F.)     

ON THE ANTIARRHYTHMIC ACTIONS OF MAGNESIUM IN SINGLE GUINEA-PIG VENTRICULAR MYOCYTES

1. The hypotheses that magnesium quickly abolishes arrhythmias by acting as a calcium antagonist or by increasing outward potassium currents were tested in guinea-pig isolated ventricular myocytes by recording membrane potentials and currents by means of a single microelectrode discontinuous voltage clamp method. 2. High [Mg²⁺]₀ (4-16 mmol/L) slightly increased the amplitude and duration of the action potential (AP) in some myocytes, but overall the changes were not significant. 3. High [Mg²⁺]₀ did not decrease the slow inward current (Ic_a) and had little effect on voltage- and time-dependent outward potassium currents whether or not Ic_a was allowed to flow. 4. Zero [Mg²⁺]₀ decreased the duration, but not amplitude, of the AP. Zero [Mg²⁺]₀ had little effect on Ic_a and on outward currents except for a small increase in outward current in the region of the negative slope of the inward rectifier currentvoltage relationship. 5. In our myocytes, in contrast to [Mg²⁺]₀ high [Ca²⁺]₀ significantly increased the amplitude and decreased the duration of the AP; at the same time, high [Ca²⁺]₀ increases Ic₂. and the outward potassium current. 6. High [Mg²⁺]₀ decreased the amplitude of the oscillatory potentials (V_os) induced by various Ca₂₊-over1oading procedures (high [Ca²⁺]₀, noradrenaline, strophanthidin and barium). 7. It is concluded that the mechanisms by which high [Mg²⁺]₀ quickly suppresses cardiac arrhythmias are related to an extracellular action of Mg²⁺ and do not include a block of I_Ca, or an increase in outward current. Mg²⁺ can be antiarrhythmic by decreasing V_os amplitude and possibly by screening the fixed negative charges at the external surface of the sarcolemma.     

Effects of taurine on the transmembrane ionic currents of rat and guinea pig ventricular fibres.

The effects of taurine on electrical properties of isolated myocardial ventricular fibres were studied in rat and guinea pig. The action potential amplitude was decreased in these species while the plateau and the repolarization phase were not altered. This suggests that simple inspection of the plateau phase is not sensitive enough to pick up changes in outward current since voltage clamp experiments showed that taurine decreased both potassium current and fast inward current. However, the slow inward calcium current remained unchanged with taurine in each species. These results suggest that the inotropic effects of taurine on rat and guinea pig heart contraction already described, were not due to modifications of the cell membrane permeability for calcium. It seems more reasonable to assume that the cardiac actions of this compound result from intracellular changes in calcium availability.     

Effect of methamphetamine on potassium and L-type calcium currents in rat ventricular myocytes

The mechanisms of cardiac toxicity caused by methamphetamine (MA) are poorly understood at present. This study was designed to investigate the effects of MA on ionic currents in myocardial cells. The effects of MA on transient outward potassium current (I_to), inward rectifying potassium current (I_K1), and L-type calcium current (I_Ca-L) in isolated rat ventricular myocytes were studied using the whole-cell patch clamp technique. It was demonstrated that MA inhibited the I_to, I_K1, and I_Ca-L in the rat ventricular myocytes concentration-dependently. MA shifted left the I_to steady-state inactivation curve and shifted down the recovery curve, but had no influence on the steady-state activation curve. MA did not affect the I_Ca-L steady-state activation curve or steady-state inactivation curve, but shifted down the recovery curve. We concluded that MA had inhibitory effects on the I_to, I_K1, and I_Ca-L in ventricular myocytes, which might be one of the possible electrophysiological mechanisms of cardiac damage caused by MA.     

The actions of nickel on membrane currents activated by hyperpolarisation in single cells from the rabbit atrioventricular node

• 1.1. The atrioventricular node (AVN) is vital for cardiac function as it normally provides the only conduction route for the cardiac impulse from atria to ventricles and can act as a pacemaker for the ventricles if the sinoatrial node (SAN) fails. We have shown previously that whilst 80–90% of AVN myocytes do not possess I_f (we have termed these type I cells), a small proportion (10–20%) of AVN cells (type 2) do exhibit I_f.
• 2.2. The present study describes the effects of the divalent cation nickel (Ni) on membrane currents activated by hyperpolarising voltage clamps from −40/mV in type 1 and type 2 cells at 35°C, using the whole cell patch clamp technique. In type 2 cells 5 mM Ni enhanced the amplitude of I_f. At −120 mV the mean Ni-activated I_f was −1.85 ± 0.28 pA/pF (mean ± SEM; n = 5). Ni significantly enhanced I_f at −70 mV and at all potentials negative to this (p < 0.05 at −70, −80, −90 and −110 mV; 0.05 < p < 0.1 at −100 mV; p < 0.005 at −120 mV).
• 3.3. In type 1 cells, which exhibit a small time-independent inward current on hyperpolarisation there was no activation of If by Ni (p > 0.1 at all potentials between −40 mV and −120 mV).
• 4.4. In type 1 cells 5 mM Ni significantly reduced the time-independent inward current activated by a hyperpolarising pulse to −120 mV (p < 0.02) and had a smaller effect at −110 and −100 mV (0.05 < p < 0.1 at these potentials). With pulses to less negative potentials there was no significant alteration of the time-independent current.
• 5.5. An additional observation was that the fast sodium current activated on repolarisation of the membrane potential to −40 mV after a hyperpolarising voltage clamp appeared to be blocked by Ni. However, this apparent blockade reflected a positive shift in the activation threshold for I_Na, since a repolarising step to −30 mV could still elicit I_Na.
• 6.6. Ni is known to block sarcolemmal Na/Ca exchange in cardiac cells, and one possible mechanism for the enhancement of I_f by Ni in type 2 cells is increased intracellular Ca via Na/Ca exchange blockade increasing I_f. The reduction in end pulse current in type 1 cells is also consistent with Na/Ca exchange current blockade. A second possibility of the enhanced I_f in type 2 cells with Ni is a positive shift of the activation curve for I_f in the presence of an increased concentration of external divalent cations.

     

牛磺酸对低钙环境下豚鼠心室肌细胞ICa的影响

目的　观察在低钙环境下 ,牛磺酸对豚鼠单一心室肌细胞钙离子电流 (ICa)的影响。方法　应用全细胞膜片钳制技术。结果　低钙状态下 ,豚鼠心室肌细胞ICa减小 ,此时牛磺酸可促进Ca2 +内流 ,使ICa增加 (P 0 .0 1) ,此作用具有电压依赖性 ,对反转电位无明显影响。此作用随细胞外Ca2 +浓度的降低而有所增强 ,并且是可逆的。结论　牛磺酸能增加低钙状态下的心室肌细胞Ca2 +内流。     

Cardiac mechanical and electrophysiologic modulations of guinea-pig by caffeine and thapsigargin

• 1.1. The effects of caffeine and thapsigargin on the contractile force and the action potential in guinea-pig papillary muscles were examined.
• 2.2. Caffeine (1 to 10 mM) initially increased contractile force in a concentration-dependent manner. Subsequently, 1 mM caffeine decreased it as compared with precaffeine level (but not significantly). At 5 mM or 10 mM, caffeine also decreased contractile force, but the decrease was still positive as compared with control level.
• 3.3. Exchange to low [Ca]_o(0.9 mM) or high[k]_o(8 mM) decreased steady-state value during exposure to 1 mM caffeine. Addition of 1 μM thapsigargin (TG) decreased the steady-state value during exposure to 1 mM caffeine, but enhanced it with 5 mM and 10 mM caffeine. TG (1 μM) alone increased the force.
• 4.4. In electrophysiologic studies, caffeine shortened the action potential duration (APD) in a concentration-dependent manner. In the presence of caffeine (1 mM), high[K]_o shortened APD and decreased the action potential amplitude and resting potential.
• 5.5. These results suggest that in the presence of caffeine and/or thapsigargin calcium overload might not occur in the left ventricular papillary muscles of the guinea-pig heart.

     

Cellular mechanisms underlying carbachol-induced oscillations of calcium-dependent membrane current in smooth muscle cells from mouse anococcygeus

1. At a holding potential of −40 mV, carbachol (50 μM) produced a complex pattern of inward currents in single smooth muscle cells freshly isolated from the mouse anococcygeus. Membrane currents were monitored by the whole-cell configuration of the patch-clamp technique. Previous work has identified the first, transient component as a calcium-activated chloride current (I_Cl(Ca)) and the second sustained component as a store depletion-operated non-selective cation current (I_DOC). The object of the present study was to examine the cellular mechanisms underlying the third component, a series of inward current oscillations (I_oscil) superimposed on I_DOC.
2. Carbachol-induced I_oscil (amplitude 97±11 pA; frequency 0.26±0.02 Hz) was inhibited by the chloride channel blocker anthracene-9-carboxylic acid (A-9-C; 1 mM), and by inclusion of 1 mM EGTA in the patch-pipette filling solution.
3. In calcium-free extracellular medium (plus 1 mM EGTA), carbachol produced an initial burst of oscillatory current which lasted 94 s before decaying to zero; I_oscil could be restored by re-admission of calcium. The frequency, but not the amplitude, of I_oscil increased with increasing concentrations of extracellular calcium (0.5–10 mM).
4. Inclusion of the inositol triphosphate (IP₃) receptor antagonist heparin (5 mg ml⁻¹) in the patch-pipette filling solution, or pretreatment of cells with the sarcoplasmic reticulum (SR) calcium ATPase inhibitor cyclopiazonic acid (CPA; 10 μM), prevented the activation of I_oscil by carbachol. Caffeine (10 mM) activated both I_Cl(Ca) and I_DOC and prevented the induction of I_oscil by carbachol. Caffeine and CPA also abolished I_oscil in the presence of carbachol, as did both a low (3 μM) and a high (30 μM) concentration of ryanodine.
5. Carbachol-induced I_oscil was abolished by the general calcium entry blocker SKF 96365 (10 μM) and by Cd²⁺ (100 μM), but was unaffected by La³⁺ (400 μM). As found previously, I_DOC was also blocked by SKF 96365 and Cd²⁺, but not La³⁺; the inhibition of I_DOC preceded the abolition of I_oscil by 27 s with SKF 96365 and by 30 s with Cd²⁺. Nifedipine (1 μM) produced a partial inhibition of the carbachol-induced I_oscil frequency at holding potentials of −20 mV and −60 mV and, in addition, reduced I_DOC at −60 mV by 18%.
6. It is concluded that carbachol-induced inward current oscillations in mouse anococcygeus cells are due to a calcium-activated chloride current, and reflect oscillatory changes in cytoplasmic calcium ion concentration. These calcium oscillations are derived primarily from the SR stores, but entry of calcium into the cell is necessary for store replenishment and maintenance of the oscillations. Capacitative calcium entry (via I_DOC) appears to be important not only for sustained contraction of this tissue, but also as a route for re-filling of the SR and, therefore, represents an important target for the development of novel and selective drugs.

     

Magnesium isoglycyrrhizinate inhibits L-type Ca<Superscript>2+</Superscript> channels,Ca<Superscript>2+</Superscript> transients,and contractility but not hERG K<Superscript>+</Superscript> channels

To explore the cardiovascular protective effects of Magnesium isoglycyrrhizinate (MI), especially the underlying cellular mechanisms related to L-type calcium channels and myocardial contractility, and to examine the effects of MI on hERG K⁺ current expressed in HEK293 cells. We used the whole-cell patch clamp technique, video-based edge detection and dual excitation fluorescence photomultiplier systems to explore the effect of MI on L-type Ca²⁺ currents (I_Ca-L) and cell contraction in rat cardiomyocytes. We also examined the rapidly activating delayed rectifier potassium current (I_Kr) expressed in HEK293 cells using a perforated patch clamp. MI inhibited I_Ca-L in a dose-dependent manner, with a half-maximal inhibitory concentration (IC₅₀) of 0.22 mg/ml, and the maximal inhibitory effect was 61.10 ± 0.59%. MI at a concentration of 0.3 mg/ml reduced cell shortening by 24.12 ± 3.97% and the peak value of the Ca²⁺ transient by 36.54 ± 4.96%. MI had no significant influence on hERG K⁺ channels expressed in HEK293 cells at all test potentials. MI exerts protective effects on the heart via the inhibition of I_Ca-L and cell shortening in rat cardiomyocytes. However, MI had no significant influence on I_Kr; thus, MI may exert cardioprotective effects without causing drug-induced long QT syndrome.     

Theophylline Effects on Phasic Tension and Slow Inward Current of Isolated Frog Atrial Fibres: Influence on the Action of Isoprenaline

Abstract: The action of theophylline on phasic tension and slow inward current (I_si) was investigated on isolated voltaged-clamped frog atrial fibres. The action of theophylline was both dose- and time-dependent; the most effective dose was 2.10^?3M. Theophylline induced a rapid positive inotropic effect which cannot be attributed to a β-adrenoceptor stimulation or to an adenosine antagonism; this effect was closely related to an increase in slow inward current (I_si), probably due to an inhibition of phosphodiesterase activity and a subsequent increase in cAMP content. The decline of the positive inotropic effect of theophylline was discussed in terms of additional effects of the drug. Theophylline, consistently with its phosphodiesterase inhibitory action, was shown to potentiate the effects of isoprenaline on phasic tension and I_si; however no potentiation was observed after reserpine-induced depletion of endogenous catecholamines. It might be thought that non-specific effects of reserpine on some steps of cardiac excitation-contraction coupling couteract the effects of theophylline.     

Effects of an extract of Ginkgo biloba On the action potential and associated transmembrane ionic currents in mammalian cardiac myocytes: Inhibition of isoproterenol-induced chloride current

Ventricular myocytes of guinea pig heart were used to examine the effects of an extract of Ginkgo biolba (EGb 761) on the action potential and individual transmembrane ionic currents. Elecrophysiological events were recorded using the “whole-cell” configuration of the patch-clamp technique. A systematic analysis of the data revealed that EGb 761 (5-50 μg/ml) did not affect the normal action potential or the various ionic currents involved in its generation; i.e., fast inward sodium current (I_Na), inward calcium currents (I_CaT and I_CaL), delayed outward potassium current (I_K), inward rectifying potassium current (I_K1), and ATP-senstive potassium current (I_K-ATP) evoked by 2,4-dinitrophenol (2,4-DNP). However, EGb 761 (≥5 μg/ml) elicited a pronouced concentration-dependent and reversible inhibiton of isoproternol-induced Cl^? current (I_Cl), the maximal effect being observed at 50 μg/ml. This current may be significantly involved in sympathetic hyperactivity, hypoxia, and ischemia, pathophysiological conditions for which EGb 761 offers therapeutic benefit. The basic mechanism(s) underlying the inhibitory effect of EGb 761 on I_Cl and the constituent(s) of EGb 761 responsible for this action remain to be identified, but it seems clear, from results which showed that neither ginkgolide B (50–500 ng/ml) nor biolobalide (150–1,500 ng/ml) influenced this current, that a terpenoid constituent of EGb 761 is probably not involved.     

Pharmacological modulation of transmitter release by inhibition of pressure-dependent potassium currents in vestibular hair cells

Vestibular vertigo may be induced by increases in the endolymphatic pressure that activate pressure-dependent K⁺ currents (I_K,p) in vestibular hair cells. I_K,p have been demonstrated to modulate transmitter release and are inhibited by low concentrations of cinnarizine. Beneficial effects against vestibular vertigo of cinnarizine have been attributed to its inhibition of calcium currents. Our aim was to determine the extent by which the inhibition of I_K,p by cinnarizine may alter the voltage response to stimulating currents and to analyze whether such alterations may be sufficient to modulate the activation of Ca²⁺ currents and transmitter release. Vestibular type II hair cells from guinea pigs were studied using the whole-cell patch-clamp technique. In current clamp, voltage responses to trains of stimulating currents were recorded. In voltage clamp, transmitter release was assessed from changes in the cell capacitance, as calculated from the phase shift during application of sine waves. Cinnarizine (0.05–3?µM) concentration dependently reversed the depressing effects of increases in the hydrostatic pressure (from 0.2 to 0.5 cm H₂O) on the voltage responses to stimulating currents. Voltage protocols that simulated these responses were applied in voltage clamp and revealed a significantly enhanced transmitter release in conditions mimicking an inhibition of I_K,p. Cinnarizine (≤0.5?µM) did not inhibit calcium currents. We conclude that cinnarizine, in pharmacologically relevant concentrations, enhances transmitter release in the presence of elevated hydrostatic pressure by an indirect mechanism, involving inhibition of I_K,p, enhancing depolarization, and increasing the voltage-dependent activation of Ca²⁺ currents, without directly affecting Ca²⁺ current.