Effect of nonachlazine on transmembrane ionic currents in the frog atrial trabeculae

The effect of the antianginal drug nonachlazine displaying antiarrhythmic properties on transmembrane ionic currents in the frog atrial fibers was studied in experiments on isolated trabeculae of the frog atria. The transmembrane ionic currents were measured by a voltage clamp technique based on a double sucrose gap arrangement. Nonachlazine (1.03 X 10(-5) mol/l) decreased the amplitude of the fast inward current whatever the magnitude of membrane potential. The drug inhibited the slow inward current and prevented the adrenaline-increased permeability of the slow sodium-calcium channel if external sodium ions were replaced by choline chloride. Nonachlazine (1.03 X 10(-5) mol/l) diminished the amplitude of the inward ionic current in a calcium-free medium as well. The stimulatory effect of prostacycline (2 X 10(-7) mol/l) on the fast inward ionic current was inhibited by nonachlazine. The data obtained suggest that the antiarrhythmic effect of nonachlazine might be linked with the inhibition of the fast sodium inward current and the slow calcium inward current.     

Possible existence of transient TTX-sensitive chloride current in the membrane of isolated cardiomyocytes

Cardiomyocytes enzymatically isolated from rat and guinea pig ventricular tissue were investigated under conditions of intracellular perfusion and voltage clamp at 18-20 degrees C. Perfusion with 135 mmol/l Tris(HF), pH 7.2 was used to eliminate outward potassium currents. The dependence of inward current (elicited by depolarizing pulses from a holding potential level of--120 mV) on low external TTX concentrations (from 10(-13) to 10(-10) mol/l) was studied. Similar TTX concentrations increased the amplitude of the inward current and changed its kinetics in a large number of cells tested. The effect was fully reversible. The effect could be evaluated in a net form by digital subtraction of the current obtained after the application of a low external TTX concentration from the initial current in a TTX-free solution. The TTX concentration dependence of the difference current could be fitted by one-to-one binding curve with Kd = (1.0 +/= 0.4) x 10(-12) mol/l. TTX-induced current changes were absent in low sodium or chloride-free external solutions. The outward current (a block of which by TTX produced the inward current changes observed) showed a reversal potential consistent with the chloride nature of such a current. The existence of a transient TTX-sensitive Na-dependent potential gated chloride current in the membrane of isolated cardiomyocytes is postulated.     

A new mechanism of action of vasopressin on the neuronal membrane]

It is shown in this work that vasopressin at the concentrations of 1 x 10(-16) to 1 x 10(-6) mol/l decreased statistically significant the amplitude of the electrosensitive sodium and calcium ionic currents of the mollusc's Lymnaea stagnalis neuronal membrane. This peptide increased the amplitude of the fast potassium current at the concentration of 1 x 10(-16) and 1 x 10(-15) mol/l. It decreased the fast potassium current and did not change the slow potassium current at the concentrations more than 1 x 10(-9) mol/l.     

Ca2+-antagonistic properties of phospholipase A2 inhibitors, mepacrine and chloroquine

The effects of putative phospholipase A2 inhibitors mepacrine and chloroquine on membrane ionic currents were studied in intact frog atrial trabeculae. Both agents decreased slow calcium channel current Isi and fast sodium channel current If. Isi was affected twice at least in comparison to If. Half-block of Isi was observed at approximately 10(-6) mol/l mepacrine and at approximately 10(-5) mol/l chloroquine. These effects on transmembrane ionic transport should be considered when using the above agents as phospholipase inhibitors or antiarrhythmic drugs.     

Is inward calcium current in crayfish muscle membrane constituted of one or two components?

Two inward currents were observed in crayfish muscle membrane during depolarization steps by the method described by Adrian et al. (1970). Under voltage clamp conditions, hyperpolarization steps elicited a large current (leak current If), associated with an inward voltage dependent current. This inward current was inhibited by niflumic acid (NA), a drug known to block Cl---HCO-3 exchange (Cousin et Motais 1982; Br?lè et al. 1983b). Dynamic outward currents triggered by depolarizing steps were inhibited to a great extent by TEA, the not inhibited portion disappearing when procaine (2 mmol/l) was added to external solution. In the presence of TEA, procaine and NA, it was thus possible to dissect the regenerative calcium current (ICa) into two components: a "fast component" (ICa1) and a "slow component" (ICa2). The reversal potential of ICa was 65 mV (for [Ca]0 = 2.8 mmol/l), and [Ca]i could be calculated to be 1.6 X 10(-5) mol/l. This value of [Ca]i is the same as calculated from values reported by Hencek and Zachar (1977). ICa1 was triggered at a threshold membrane potential of -45 mV and ICa2 at -30 mV. Moreover, the inactivation kinetics for ICa1 was faster than that for ICa2. Our results are in perfect agreement with those obtained by Zahradník and Zachar (1982) who postulated two populations of calcium channels.     

TTX-sensitive Na(+) and nifedipine-sensitive Ca(2+) channels in rat vas deferens smooth muscle cells.

The inward currents in single smooth muscle cells (SMC) isolated from epididymal part of rat vas deferens have been studied using whole-cell patch-clamp method. Depolarising steps from holding potential -90 mV evoked inward current with fast and slow components. The component with slow activation possessed voltage-dependent and pharmacological properties characteristic for Ca(2+) current carried through L-type calcium channels (I(Ca)). The fast component of inward current was activated at around -40 mV, reached its peak at 0 mV, and disappeared upon removal of Na ions from bath solution. This current was blocked in dose-dependent manner by tetrodotoxin (TTX) with an apparent dissociation constant of 6.7 nM. On the basis of voltage-dependent characteristics, TTX sensitivity of fast component of inward current and its disappearance in Na-free solution it is suggested that this current is TTX-sensitive depolarisation activated sodium current (I(Na)). Cell dialysis with a pipette solution containing no macroergic compounds resulted in significant inhibition of I(Ca) (depression of peak I(Ca) by about 81% was observed by 13 min of dialysis), while I(Na) remained unaffected during 50 min of dialysis. These data draw first evidence for the existence of TTX-sensitive Na(+) current in single SMC isolated from rat vas deferens. These Na(+) channels do not appear to be regulated by a phosphorylation process under resting conditions.     

Role of transmembrane ion currents in the mechanism of action of beta-adrenoblockers

The effects of propranolol, oxprenolol, pindolol, labetalol, and atenolol on the isoproterenol-stimulated slow calcium current as well as on spontaneous slow calcium and fast sodium currents were studied in frog trabeculae by the double sucrose bridge technique. Administration of beta-adrenoblockers in low doses (10(-7)-10(-6)M) antagonized the stimulatory effect of isoproterenol on the slow calcium current (pindolol greater than oxprenolol greater than or equal to propranolol = labetalol greater than atenolol). When administered in high doses (2 X 10(-6)-10(-4)M) the beta-adrenoblockers reduced the slow calcium current. Meanwhile, some of them (propranolol, oxprenolol, labetalol) suppressed the fast sodium current.     

Ammonia and proton gated channel populations in trigeminal ganglion neurons.

The existence of three distinct types of proton-gated sodium currents classified in accordance with their kinetics of desensitization as fast, medium and slow, has been confirmed in the present study on isolated rat trigeminal ganglion neurons. The emphasis was put on the investigation of a peculiar medium type of a chemically gated channel population, using the concentration jump method. The features of the medium-type mechanism allow to distinguish it from the other already known types: it was insensitive to the Ca antagonist verapamil (in contrast to proton-gated sodium current found in dorsal root ganglion neurons), displayed a strong dependence of the kinetics of desensitization on the membrane potential, and (besides the apparent proton-gating) was activated and desensitized by the application of ammonia containing solution at normal pH values. The effect of ammonia itself appeared to be a good tool for the separation of fast and slow proton-gated responses. The results obtained allow to postulate a nonspecific proton-activation of medium-type receptor-channel complexes and their specificity to ammonia (Kd = 10(-4) mol/l) as an agonist.     

Ionic currents in dispersed chemoreceptor cells of the mammalian carotid body

Ionic currents of enzymatically dispersed type I and type II cells of the carotid body have been studied using the whole cell variant of the patch-clamp technique. Type II cells only have a tiny, slowly activating outward potassium current. By contrast, in every type I chemoreceptor cell studied we found (a) sodium, (b) calcium, and (c) potassium currents. (a) The sodium current has a fast activation time course and an activation threshold at approximately -40 mV. At all voltages inactivation follows a single exponential time course. The time constant of inactivation is 0.67 ms at 0 mV. Half steady state inactivation occurs at a membrane potential of approximately -50 mV. (b) The calcium current is almost totally abolished when most of the external calcium is replaced by magnesium. The activation threshold of this current is at approximately -40 mV and at 0 mV it reaches a peak amplitude in 6-8 ms. The calcium current inactivates very slowly and only decreases to 27% of the maximal value at the end of 300-ms pulses to 40 mV. The calcium current was about two times larger when barium ions were used as charge carriers instead of calcium ions. Barium ions also shifted 15-20 mV toward negative voltages the conductance vs. voltage curve. Deactivation kinetics of the calcium current follows a biphasic time course well fitted by the sum of two exponentials. At -80 mV the slow component has a time constant of 1.3 +/- 0.4 ms whereas the fast component, with an amplitude about 20 times larger than the slow component, has a time constant of 0.16 +/- 0.03 ms. These results suggest that type I cells have predominantly fast deactivating calcium channels. The slow component of the tails may represent the activity of a small population of slowly deactivating calcium channels, although other possibilities are considered. (c) Potassium current seems to be mainly due to the activity of voltage-dependent potassium channels, but a small percentage of calcium-activated channels may also exist. This current activates slowly, reaches a peak amplitude in 5-10 ms, and thereafter slowly inactivates. Inactivation is almost complete in 250-300 ms. The potassium current is reversibly blocked by tetraethylammonium. Under current-clamp conditions type I cells can spontaneously fire large action potentials. These results indicate that type I cells are excitable and have a variety of ionic conductances. We suggest a possible participation of these conductances in chemoreception.     

Ionic requirements for bursting activity in lobster stomatogastric neurons

Summary The ionic requirements for bursting activity have been investigated in the electrically coupled PD-AB cells group of the Stomatogastric ganglion in the lobster.The passive electrical properties and coupling parameters have been determined in either current or voltage clamp conditions. In voltage clamped cells, the current displayed slow inward transients with superimposed fast transients corresponding respectively to the slow waves and spikes of the coupled undamped cells. The amplitude and frequency of the slow transients were reduced upon hyperpolarization.Cyclic conductance changes were observed with short current pulses, the coupling ratio also changes cyclically being lower during the bursts and slowly increasing during the interburst interval.The slow wave amplitude increased in low K-saline. The post-burst hyperpolarization but not the top level of the wave behaved like a potassium electrode for [K]_o higher than 10 mM/l.TEA at low concentration (1 to 5 mM/l) increased the slow wave amplitude by lifting its top level by 10 to 20 mV. The post-burst hyperpolarization remained almost unchanged and its K-dependence was not altered by TEA.Low Na-saline reduced the slow wave amplitude (6 to 11 mV per decade). The Na-dependence increased in the presence of TEA. Slow waves devoid of spikes persisted in 10% Na saline containing TEA. 10^–9 M/1 TTX blocked the spikes. 10^–7 M/1 TTX blocked the slow waves.Mg-free saline had no effect on the slow wave. In Ca-free saline the cells depolarized and the bursting activity tended to vanish. Repolarization with current led to long lasting slow waves deprived of post-burst hyperpolarization. The bursting ceased when EDTA was added to the Ca-free saline.Cobalt (up to 10 mM/l) was similar to Ca-free saline in its effects; lengthening of the wave and blockage of the repolarization. Replacing Ba for Ca produced large (up to 70 mV) slow waves which were reduced by Co and Ca.Bistable states were observed in various experimental conditions. It is concluded that the slow waves are produced by activation of sodium and calcium currents. The amplitude of the slow wave is modulated by the simultaneous activation of a TEA-sensitive K current. The repolarization is caused by increased K current activated by the inward calcium current. The slow pacemaker potential in the interburst interval corresponds to the slow disappearance of the K current.This work was supported by N.I.H. grant no. 09322, NSF grant no. 00250, and a Guggenheim Foundation Fellowship to A.D.S. and by the CNRS and a DGRST grant no. 16501891 to M. Gola. We are grateful to Stuart Thompson and Felix Strumwasser for helpful comments and to Barbara McLean for technical assistance.     

Voltage clamp analysis of embryonic heart cell aggregates

The double-microelectrode voltage clamp technique was applied to small spheroidal aggregates of heart cells from 7-d chick embryos. A third intracellular electrode was sometimes used to monitor spatial homogeneity. On average, aggregates were found to deviate from isopotentiality by 12% during the first 3--5 ms of large depolarizing voltage steps, when inward current was maximal, and by less than 3% thereafter. Two components of inward current were recorded: (a) a fast, transient current associated with the rapid upstroke of the action potential, which was abolished by tetrodotoxin (TTX); and (b) a slower inward current related to the plateau, which was not affected by TTX but was blocked by D600. The magnitudes, kinetics, and voltage dependence of these two inward currents and a delayed outward current were similar to those reported for adult cardiac preparations. From a holding potential of -60 mV, the peak fast component at the point of maximal activation (-20 mV) was -185 microA/cm2. This value was about seven times greater than the maximal slow component which peaked at 0 mV. The ratio of rate constants for the decay of the two currents was between 10:1 and 30:1.     

The relationship among intracellular sodium activity, calcium, and strophanthidin inotropy in canine cardiac Purkinje fibers

The role of sodium and calcium ions in strophanthidin inotropy was studied by measuring simultaneously the electrical, mechanical, and intracellular sodium ion activities in electrically driven cardiac Purkinje fibers under conditions that change the intracellular sodium or calcium level (tetrodotoxin, strophanthidin, high calcium, and norepinephrine). Tetrodotoxin (TTX; 1-5 X 10(-6)M) shifted the action potential plateau to more negative values, shortened the action potential duration, and decreased the contractile tension and the intracellular sodium ion activity (aiNa). The changes in tension and in aiNa caused by TTX appear to be related since they had similar time courses. Strophanthidin (2-5 X 10(-7)M) increased tension and aiNa less in the presence of TTX, and, for any given value of aiNa, tension was less than in the absence of TTX. Increasing extracellular calcium (from 1.8 to 3.3-3.6 mM) or adding norepinephrine (0.5-1 X 10(-6)M) increased tension and decreased aiNa less in the presence than in the absence of TTX. When two of the above procedures were combined, the results were different. Thus, during the increase in aiNa and tension caused by strophanthidin in the presence of TTX, increasing calcium or adding norepinephrine increased tension markedly but did not increase aiNa further. In a TTX-high calcium or TTX-norepinephrine solution, adding strophanthidin increased both tension and aiNa, and the increase in tension was far greater than in the presence of TTX alone. The results indicate that: (a) the contractile force in Purkinje fibers is affected by a change in aiNa; (b) a decrease in aiNa by TTX markedly reduces the inotropic effect of strophanthidin, possibly as a consequence of depletion of intracellular calcium; (c) increasing calcium influx with norepinephrine or high calcium in the TTX-strophanthidin solution produces a potentiation of tension development, even if aiNa does not increase further; and (d) when the calcium influx is already increased by high calcium or norepinephrine, strophanthidin has its usual inotropic effect even in the presence of TTX. In conclusion, the positive inotropic effect of strophanthidin requires that an increase in aiNa be associated with suitable calcium availability.     

Omega-conotoxin blockade of calcium currents in cultured neonatal rat cardiomyocytes: different action on EGTA-modified calcium channels

Calcium currents from neonatal rat ventricular heart muscle cells grown in primary culture were examined using the "whole-cell" voltage clamp technique. An inward current characterized by large amplitude and slow inactivation decay was induced when the extracellular Ca2+ concentration was reduced by EGTA. This current was suppressed by extracellular Na+ removal, or by calcium antagonists, and increased by epinephrine and BAY K 8644. These findings suggest that this current is carried by sodium ions through Ca channels. Both Ca and Na currents through calcium channels were irreversibly blocked by omega-conotoxin. Complete blockade developed 10-15 minutes after the toxin introduction in the extracellular solution. Blockade of Na currents through calcium channels was characterized by a transient increase of current amplitude without any changes in its kinetics and voltage-dependent properties. Structural differences between calcium channels in rat and guinea-pig and frog cardiomyocytes were suggested.     

Properties of an early outward current in single cells of the mouse ventricle

Single ventricular myocytes of adult mice were prepared by enzymatic dissociation for voltage clamp experiments with the one suction pipette dialysis method. After blocking the Na current by 10(-4) mol/l TTX early outward currents (IEO) with incomplete inactivation could be elicited by clamping from -50 mV to test potentials (VT) positive to -30 mV. Interfering Ca currents were very small (less than 0.6 nA at VT = 0 mV). The approximation of IEO by the q4r-model showed a pronounced decrease in the time constant of activation (tau q) to more positive potentials. At 50 ms test pulses the time course of the incomplete inactivation could be described by two exponentials and a constant. The time constant of the fast exponential (tau r1) showed a slight decline towards more positive test potentials (8.1 +/- 1.0 ms at -10 mV; 5.8 +/- 1.2 ms at +50 mV, mean +/- SD, n = 5) whereas the time constant of the slow exponential (tau r2) was voltage independent (41.1 +/- 7.9 ms, mean +/- SD, n = 5). The contributions of the fast exponential and the pedestal increased towards positive test potentials. The Q10 value for the time constants of activation and fast inactivation was 2.36 +/- 0.19 and 2.51 +/- 0.09 (mean +/- SD, n = 3), respectively. After an initial delay the recovery of IEO at a recovery potential of -50 mV could be fitted monoexponentially with a time constant of 16.3 +/- 2.9 ms (mean +/- SD, n = 3). The time course of the onset of inactivation determined with the double pulse protocol was slower than the decay at the same potential, and could be described as sum of a fast (tau = 18.4 +/- 6.0 ms) and a slow (tau = 62.1 +/- 19.9ms, mean +/- SD, n = 3) exponential. IEO could be blocked completely by 1 mmol/l 4-aminopyridine at potentials up to +20 mV. Stronger depolarizations had an unblocking effect.     

Cadmium-induced blockade of the cardiac fast Na channels in calf Purkinje fibres

The fast transient inward current elicited by depolarizations above about -60 mV in calf Purkinje fibres was found to be depressed by Cd in concentrations less than 1 mM. The Cd-sensitive current, which strongly depended on external Na, was recorded in the presence of 2 mM MnCl2 and was blocked by TTX, indicating that a contamination from slow Ca-dependent currents could be discounted. The current reduction caused by Cd was also observed in nominally Ca-free solutions. The Cd-induced depression of the fast Na current was not accompanied by changes in the current kinetic parameters, as revealed by comparing inactivation curves and peak current voltage relations at different Cd concentrations, and could be attributed to a voltage-independent channel blocking action. Half-blockade occurred at 0.182 +/- 0.06 mM (n = 4). Plots of peak current amplitude as a function of the Cd concentration showed that the cooperation of two Cd ions was required to block a single channel.     

Membrane currents induced by inflow of sodium ions into mollusk giant neurons

Membrane currents induced by inflow of sodium ions were investigated in giant neurons of the molluskHelix pomatia during tetanic stimulation or prolonged membrane depolarization under voltage clamp conditions. The membrane current thus produced consists of two components, a fast component with a reversal potential close to the potassium equilibrium potential, and a slow component only slightly dependent on membrane potential in the region from −50 to −90 mV. Addition of strophanthin K to the external solution, or replacement of sodium in the external solution by lithium or calcium abolished the slow component of the membrane current and reduced the fast component. It is concluded that the slow component appears as the result of activation of the sodium pump under electrogenic conditions, where-as the fast component arises as the result of an increase in potassium permeability, possibly coupled with intensive activity of this pump.     

Comparison of binding affinities and calcium current inhibitory effects of a 1,4-dihydropyridine derivative (PN 200-110) in vascular smooth muscle

The binding of (+) (3H) PN 200-110 to high and low affinity sites in mammalian portal vein smooth muscle membranes was characterized. Binding affinities were 0.09 and 30 nM for the high and low affinity sites, respectively, and binding site densities were 45 and 400 fmoles/mg of protein for the respective sites. (+) PN 200-110 blocked both fast and slow calcium currents in isolated cells from portal vein smooth muscle. The blockade of slow calcium current was voltage-dependent as PN 200-110 bound with higher affinity to inactivated slow calcium channels (IC50 = 0.03 nM) than to resting channels (IC50 = 0.15 nM). The blockade of fast calcium current was voltage-independent (IC50 = 45 nM). The IC50 values found from electrophysiological experiments for the binding to inactivated slow and fast calcium channels are similar to the Kd values determined by radioligand binding.     

Electrical characteristics of frog atrial trabeculae in the double sucrose gap.

The electrical behavior of small single frog atrial trabeculae in the double sucrose gap has been investigated. The currents injected during voltage clamp experiments did not behave as predicted from the assumption of spatial uniformity of the voltage across a Hodgkin-Huxley membrane. Much of the difference is due to the geometrical complexities of this tissue. Nonetheless, two transient inward currents have been identified, the faster of which is blocked by tetrodotoxin (TTX). The magnitude of the slower transient varies markedly between preparations but always increases in a given preparation with increase of external calcium. The fast transient current traces, at small to intermediate depolarizations, are often marred by the presence of notches and secondary peaks due most probably to the loss of space clamp conditions. In many preparations these could be removed by reducing the current magnitude through application of a partially-blocking dose of TTX. Conversely, in the preparations whose fast transient was fully blocked by TTX, notches and secondary peaks in the slow transient could by induced through increasing calcium concentration and thereby the slow current magnitude. Previously used techniques for the measurement of the reversal potential of the fast inward transient have been shown to be invalid. In so far as they can be measured, the reversal potentials of the fast and slow inward transient are in the same neighborhood, i.e. around 120 mV from rest. The true values may be quite a bit apart. The total charge flow in the capacitive transient was measured for different sized nodes and preparations. From these data and estimates of plasma membrane area per unit trabecular volume, specific membrane capacitances of around 3 muF/cm2 were calculated for small bundles. The apparent ion current densities on this basis are approximately 1/10 of those measured in axons. The capacitive current occurring in small bundles decayed as the sum of at least three exponential functions of time. On the basis of these data and the anomalously large stable node widths, we suggest a coaxial core model of the preparation with the inner elements in series with an additional large extracellular resistance.     

Oxytocin actions on voltage-dependent ionic channels in pregnant rat uterine smooth muscle cells.

The effects of oxytocin, a uterotonic polypeptide hormone, on the voltage-dependent slow calcium, fast sodium, and potassium channel currents were studied using whole-cell voltage clamp of freshly isolated cells from late pregnant (18-21 day) rat myometrium. The calcium current was rapidly inhibited by oxytocin (about 25% inhibition at 20 nM) in a dose-dependent manner, and this inhibitory effect was completely reversible by washout. However, inhibition was not observed when barium was used as the charge carrier. Sodium current and potassium current were not modified by oxytocin, thus sodium and potassium currents may not play important roles in oxytocin-induced augmentation of uterine contraction. It is concluded that oxytocin stimulates uterine contraction by mechanisms other than augmentation of the voltage-dependent calcium current, e.g., by release of Ca from sarcoplasmic reticulum (by inositol triphosphate) or by activation of a receptor-operated Ca channel. The inhibition of the slow calcium current may be induced by the elevation of [Ca]i.     

The ionic mechanism of the pentylenetetrazol convulsions

The ionic dependence and the nature of conductance was examined at slowly inactivating inward current in metacerebral giant cells of Helix pomatia, induced by 50 mM pentylenetetrazol. Ramp and square wave depolarizations in voltage clamp mode revealed, that withdrawal of sodium ions prevented this current to flow. While TTX was ineffective, Mn, Co and Ni-ions and verapamil blocked the current. It is concluded that PTZ, especially in presence of TEA impairs calcium channels, which loose their specificity and transmit sodium ions, with very slow kinetics.