The dihydropyridine derivatives CGP 28392 and BAY K 8644 exert a strong positive inotropic effect in mammalian cardiac muscle, presumably by increasing Ca influx during the action potential. Analysis of the drug effects at the level of single Ca channels by means of the patch-clamp method revealed complicated changes in the kinetics of channel gating. The prevailing effect is a prolongation of the mean open time of Ca channels. In addition, the intervals between channel openings can be slightly prolonged. Ensemble averages of single-channel traces showed a concentration-dependent increase in the mean current amplitude by the drugs. In contrast to -adrenoceptor agonists, the increase in Ca current by the dihydropyridine derivatives is not associated with an increase in the intracellular cyclic AMP level.As a relatively new class of drugs, dihydropyridine derivatives have gained considerable interest both therapeutically and experimentally. Nifedipine and nitrendipine are Cachannel blockers (1, 2) widely used in antihypertensive therapy. These drugs were also the first to be used as radioligands in an attempt to label and characterize Ca channels (3-5). Recently, however, two new drugs of this class have been synthesized that have been proposed to increase rather than to decrease the Ca permeability of excitable cells (6, 7). We have investigated the effects of these drugs [CGP 28392 (6) and BAY K 8644 (7); see structures I and II, respectively] on single Ca channels in cultured cardiac cells by means of the patch-clamp method (8). We have found that both drugs modify the voltage-dependent gating of Ca channels and thereby prolong the open state of these channels. METHODSPrimary cultures of cardiac cells were prepared from hearts of neonatal rats as described elsewhere (9, 10). Myocardial cells were seeded on coverslips at a low density (105 cells per ml). Well-differentiated spindle-like cardiac cells showing cross-striations were used 1-3 days after seeding for the electrophysiological measurements.Single Ca-channel recordings were made with the patchclamp method (8, 11). The holding potential of the patch was identical to resting potential, which is around -70 mV in these cells as estimated from occasional break-through of the patch. The recording pipette contained 96 mM BaCl2 solution with 20 ,uM tetrodotoxin, buffered to pH 7.4 with 10 mM Hepes. Single-channel currents, with Ba + as charge camers, were measured from cell-attached membrane patches with seal resistances between 10 and 100 GQ. The cells were superfused with saline of the following composition in mM: NaCl, 137; KCl, 5.4 4824The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Modulation of ion channels is of increasing interest as it is an important step in the regulation of cellular functions. We have analysed the effect of 8-bromocyclic AMP on Ca2+ channels in cultured cardiac cells by the patch-clamp method and report here that there was a large increase in the probability of opening of the channels. On the basis of a recently proposed kinetic reaction scheme we suggest that cyclic AMP-dependent phosphorylation of Ca2+ channels primarily promotes the forward rate constants which lead to the open state of a Ca2+ channel during depolarization.

SUMMARY1. Primary cardiac cell cultures were prepared from the hearts of neonatal rats. The patch-clamp method (Hamill, Marty, Neher, Sakmann & Sigworth, 1981) was applied for studying whole-cell Na+ currents and single-channel Na+ currents, respectively.2. Whole-cell recordings yielded voltage-and time-dependent Na+ currents which could be blocked by tetrodotoxin.3. Single-channel Na+ currents were directly compared in cell-attached patches and in inside-out patches.4. In cell-attached patches the elementary current was about -1 pA at -10 mV and the slope conductance over a 50 mV voltage range was 15.1 + 1-6 pS (mean + S.D.). Inactivation during depolarization and after conditioning clamp steps, in the steady state, resulted from a reduced opening probability of Na+ channels.5. In inside-out patches, with identical solutions at both membrane surfaces, there was a large (40-50 mV) shift of channel opening and inactivation kinetics towards more negative potentials. However, for levels of comparable opening probabilities, mean open times ofNa+ channels were similar in cell-attached and inside-out patches.Tetrodotoxin (10-20 fSM) had no effect on Na+ channels when applied from the inside, but blocked them completely after application to the outside membrane surface.

Single cells or cell clusters composed of 3-10 cells were isolated from the S-A and A-V nodes of the rabbit heart by coronary perfusion of collagenase dissolved in Ca-free Tyrode solution (0.04 %, for 1 hr). For comparison, atrial and ventricular cells were also isolated from the same heart. Shapes of the isolated nodal cell were either rod or round and nodal cells were slightly smaller than ventricular cells. Spontaneous activity was observed in both rod and round nodal cells. The action potentials had the configurations similar to those recorded from larger conventional preparations. The membrane current recorded from the small nodal cell clusters (5-10 cells) by the two-microelectrode voltage clamp technique showed a time course similar to that of previous recordings from conventional preparations, but the amplitude of the currents was 5-10 times smaller. The isolated cells showed normal sensitivities to both acetylcholine and epinephrine. Findings given in this study indicate that the isolated cells maintain the typical membrane characteristics of the nodal cells and that they are suitable for electrophysiological studies of the cardiac pacemaker cell.