Jacques Lenfant

Mode of action of bradycardic agent, S 16257, on ionic currents of rabbit sinoatrial node cells

1 The effect of the bradycardic agent S 16257 on the main ionic mechanisms of diastolic depolarization in sinoatrial node cells isolated from rabbit heart, was investigated by the patch‐clamp technique in whole‐cell and macro‐patch recordings. 2 In whole‐cell conditions, S 16257 induced a marked exponential use‐dependent blockade of the hyperpolarization‐activated If current, without shift of the voltage range of its activation curve. The rate of block increased with the drug concentration. The IC50 for the block of If was 2.8 × 10−6 m. 3 A similar use‐dependent decline of If was obtained with 3 μm S 16257, in cell‐attached and in inside‐out macro‐patch configurations, suggesting that the bradycardic agent interacts with If channels from the inside of the cell. 4 A high concentration of S 16257 (10 μm) had no detectable effect on T‐type calcium current and slightly decreased L‐type calcium current (−18.12±0.66%), without significant use‐dependent blockade. 5 S 16257 had no effect on the delayed outward potassium current IK at 3 μm and slightly decreased it only at high concentrations, −16.3±1.2% at 10 μm. In contrast, zatebradine, another bradycardic agent, reduced IK by 20.3±2.5% at 3 μm. 6 In conclusion, S 16257 may lower heart rate without significant negative inotropic action. In comparison with zatebradine, S 16257 had less effect on IK suggesting less prolongation of repolarization time.

The effects of amiodarone on the sinus node activity of the rabbit heart

The action of amiodarone (1.5 X 10(-5) M) on sinus node activity of spontaneously beating isolated right atria of rabbit at 30 degrees C was investigated using a microelectrode technique. The drug significantly increased the action potential duration and decreased the slope of diastolic depolarization, both effects leading to a reduction of the sinus rate. In contrast to beta-blocking agents, amiodarone reduced but did not completely abolish the adrenergic effects on the sinus node activity. It is concluded that the amiodarone-induced bradycarida observed in clinical trials might be due to a direct effect of the drug on the sinus node.

Mechanism of muscarinic control of the high-threshold calcium current in rabbit sino-atrial node myocytes

The mechanism of the action of acetylcholine (ACh) on the L-type calcium current (ICa,L) was examined using a whole-cell voltage-clamp technique in single sino-atrial myocytes from the rabbit heart. ACh depressed basal ICa,L at concentrations in the range 0.05–10 μM, without previous β-adrenergic stimulation. The ACh-induced reduction of ICa,L was reversed by addition of atropine, indicating that muscarinic receptors mediate it. Incubation of cells with a solution containing pertussis toxin led to abolition of the ACh effect, suggesting that this effect is mediated by G proteins activated by muscarinic receptors. Dialysis of cells with protein kinase inhibitor or 5′-adenylyl imidodiphosphate, inhibitors of the cAMP-dependent protein kinase, decreased basal ICa,L by about 85% and suppressed the effect of ACh. The ACh effect was also absent in cells dialysed with a non-hydrolysable analogue of cAMP, 8-bromo-cAMP. The results suggest that, in basal conditions, a large part of the L-type calcium channels should be phosphorylated by protein kinase A stimulated by a high cAMP level correlated with a high adenylate cyclase activity. The depressing effect of ACh on ICa,L may occur via inhibition of the high basal adenylate cyclase activity leading to a decrease of cAMP-dependent protein kinase stimulation and thus to a dephosphorylation of calcium channels.

Characterization of a Ca2+-activated Nonselective Cation Channel during Dedifferentiation of Cultured Rat Ventricular Cardiomyocytes

Cardiac hypertrophy is associated with electrical activity modifications, including sustained depolarization, that lead to a propensity for arrhythmias. The ionic currents underlying the sustained depolarization are not well defined. Similar modifications were reported on adult rat cardiomyocytes in primary culture undergoing dedifferentiation. Using the single-channel measurements on these cells, we identified the appearance of a Ca2+-activated nonselective cation channel (NSCCa) during the dedifferentiation process. In excised inside-out patches the channel presented a linear I/V relationship with a conductance of 26.5 pS. It was equally selective for Na+ and K+ and impermeable to Cl? and Ca2+ ions. The open probability increased with depolarization and with rise in intracellular calcium concentration. The channel activity was reduced by intracellular ATP and suppressed by flufenamic acid. Channel detection increased after incubation with a purinergic receptor agonist (ATPgS) or a PKC activator (PMA). Furthermore, occurrence of the channel developed during the culture. Absent at one day in vitro (d.i.v.), channel activity was present in 5, 46, 27 and 19% of patches after 4, 7, 14 and 21 d.i.v., respectively. We suggest that the channel may be associated with pro-arrhythmic signaling, in particular during the release of transmitters from autonomic nerve endings in the hypertrophied hearts.

Characterization of a hyperpolarization-activated current in dedifferentiated adult rat ventricular cells in primary culture

1 The presence of a hyperpolarization‐activated pacemaker (If)‐like current was tested in dedifferentiated adult rat ventricular myocytes up to 12 days in primary culture with the whole‐cell patch clamp technique. 2 An If‐like current was found and characterized on freshly isolated and cultured ventricular cells. Both activation and density of the current varied in relation to the stage of dedifferentiation. The current was activated from ‐92.0 ± 2.5 and ‐63.0 ± 1.0 mV at the beginning (4‐day‐cultured cells) and end of the dedifferentiation process (12 days), respectively. Its density measured at ‐170 mV progressively increased from ‐2.34 ± 0.36 to ‐6.12 ± 0.64 pA pF−1 between the two farthest stages of cellular remodelling. In freshly isolated cells the current was activated at ‐108.0 ± 1.5 mV and its current density measured at ‐170 mV was ‐1.97 ± 0.56 pA pF−1. 3 The current was blocked by extracellular CsCl (3 mM) in a voltage‐dependent manner. Modification of reversal potentials obtained at various values of [K+]o (5.4 or 25 mM) and [Na+]o (140 or 30 mM) suggests that the current was carried by both K+ and Na+ ions. 4 It is concluded that the hyperpolarization‐activated inward current, recorded in freshly isolated and in cultured ventricular cells has characteristics similar to those of If. In adult rat ventricular cells it is activated in a non‐physiological potential range, but can be elicited in a more physiological range when the cells are remodelled through a dedifferentiated way. It is suggested that such a current could be implicated in ventricular arrhythmias developed in pathological events.

Activation of the Ca2+-Activated Nonselective Cation Channel by Diacylglycerol Analogues in Rat Cardiomyocytes

Introduction: Cardiac hypertrophy is associated with changes in electrophysiologic properties due to ionic channel modifications and increases in protein kinase C (PKC) activity and diacylglycerol (DAG) content. These changes may contribute to an increased propensity for arrhythmia. Similar electrophysiologic modifications have been reported in adult rat cardiomyocytes undergoing dedifferentiation in primary culture.

Evidence for two types of calcium currents in frog cardiac sinus venosus cells

Two types of calcium currents were recorded in single sinus venosus cells of the frog heart, using the whole-cell patch-clamp technique. The threshold potentials were approximately − 65 mV for T-type current and − 40 mV for L-type current. The amplitude and time course of T-type current were unaffected by exchanging calcium for barium, while the amplitude of L-type current was increased and its decay slowed. T-type current was neither modified by 10−6 M nifedipine nor by 10−7 M isoprenaline in contrast with the effects of these agents on L-type current. T-type current began to inactivate at −90 mV and was fully inactivated at −45 mV. Its steady-state inactivation curve was approximately 35 mV negative to that of L-type current. Overlap of activation and inactivation relationships was present for both T-and L-type currents and was maximal at − 57 and − 30 mV, respectively. It was concluded that T-and L-type currents can easily be separated by their voltage, kinetic and pharmacological differences. The presence of a high density of T-type current may be correlated to its contribution to the pacemaking function of the sinus venosus cells.

Characterization of an angiotensin-II-activated chloride current in rabbit sino-atrial cells

Single sino-atrial cells from rabbit heart were voltage-clamped using the whole-cell configuration of the patch clamp technique under conditions in which most of the ionic and exchange currents known in pacemaker cardiac cells were minimized. Extracellular angiotensin II (AII) activated a time-independent background current. The current-voltage relation of this current showed an outward rectification. The reversal potential was −20 mV with 156 mm Cl− external solution and 54 mm Cl− internal solution. This reversal potential shifted with changes in the transmembrane Cl− gradient in the fashion expected for a chloride current. Anthracene-9-carboxylic acid and diphenylamine 2-carboxilic acid (chloride channels blockers) were found to be effective in blocking the AII-sensitive current. The linear segment of the current-voltage relation can be totally inhibited by the competitive AII-receptor (AT1) antagonist losartan and by the presence of intracellular protein kinase C inhibitor, whereas the outward rectification is only slightly changed. It is concluded that sino-atrial cells should contain protein-kinase-C-sensitive chloride channels which may be activated by angiotensin II via the stimulation of the AT1 receptors.

Isolated cells of the frog sinus venosus: properties of the inward current activated during hyperpolarization

Single sinus venosus cells from frog, Rana esculenta, were isolated using an enzymic dispersion procedure, involving applications of collagenase and protease. About 40%–60% of the cells showed spontaneous contractions. Isolated cells were studied in the whole-cell configuration. Regenerative action potentials were tetrodotoxin-insensitive and similar to those recorded in multicellular preparations. Hyperpolarizing pulses in the voltage range negative to −50 mV induced the activation of a time-dependent inward current, which was blocked by 4 mM caesium but less affected by barium ions. A lower concentration of caesium (1 mM) exerted a voltage-dependent reduction of the current and decreased the spontaneous pacing rate. The activation range of the hyperpolarization-activated current approximately extended from −50 mV to −110 mV, but varied from cell to cell. A high variability was observed in the behaviour of the activation kinetics. The current had a reversal potential near −20 mV that was shifted positively by increasing the external potassium concentration (from 3 mM to 30 mM) and negatively by reducing the external sodium concentration (from 115 mM to 30 mM). The hyperpolarization-activated inward current of the frog sinus venosus cell appears to be carried by both sodium and potassium ions. It shows electrophysiological properties similar to those of the If current of the mammalian heart. The role of the current in the spontaneous activity is discussed.

Effects of the replacement of chloride by methylsulphate on the membrane currents in frog atrial trabeculae

SummaryThe effect of the replacement of extracellular chloride by methylsulphate was investigated on isolated frog atrial trabeculae using the voltage clamp technique. In methylsulphate solution, the following results were obtained:1.After a transient depolarization of the resting potential, the action potential was lengthened and the diastolic depolarization rate of the repetitive activity was reduced.2.In voltage clamp conditions, the current-voltage relationships and the steady-state inactivation curves of both early and secondary inward currents were unaffected3.The study of the evolution of the instantaneous background currents with membrane potential showed a decrease of the current values.4.The first component of the time-dependent outward currents was unaffected but the second component was reduced without change of its timeconstant value. Assuming the absolute value of the chloride equilibrium potential at about −30 mV, it was suggested that the lengthening of the action potential in methylsulphate solution might be explained by the decrease of an influx of chloride ions which should participate in the repolarization. The reduction of the diastolic depolarization rate might be due to the decrease of an outflux of chloride ions which should be involved in the inward background currents unmasked by the deactivation of the time-dependent outward currents.