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. 4 A high concentration of S 16257 (10 yM) 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 pM and slightly decreased it only at high concentrations, -16.3+ 1.2% at 10 gM. In contrast, zatebradine, another bradycardic agent, reduced IK by 20.3+2.5% at 3 gM. 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 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 microM, without previous beta-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.

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.