R56865 INHIBITS CATECHOLAMINE RELEASE FROM BOVINE CHROMAFFIN CELLS BY BLOCKING CALCIUM CHANNELS

1 The effects of R56865 (a new class of cardioprotective agent which prevents Na+ and Ca2+ overload in cardiac myocytes) on catecholamine release, whole-cell current through Ca2+ channels (I(Ba)) and cytosolic Ca2+ concentrations, [Ca2+]i, have been studied in bovine chromaffin cells. 2 R56865 caused a time- and concentration-dependent blockade of catecholamine release from superfused cells stimulated intermittently with 5 s pulses of 59 mM K+. After 5 min superfusion, a 3 muM concentration inhibited secretion by 20%; the blockade increased gradually with perfusion time, to reach 85 % after 40 min. The IC50 to block secretion after 5 min periods of exposure to increasing concentrations of R56865 was around 3.1 muM. The blocking effects of R56865 were reversible after 5-15 min wash out. In high Ca2+ solution (10 mM Ca2+), the degree of blockade of secretion diminished by 20% in comparison with 1 mM Ca2+. 3 In electroporated cells, R56865 (10 muM) did not modify the secretory response induced by the application of 10 muM free Ca2+. 4 R56865 blocked the peak I(Ba) current in a concentration- and time-dependent manner; its IC50 was very similar to that obtained for secretion (3 muM). The compound not only reduced the size of the peak current but also promoted its inactivation; when the effects of R56865 were measured at the most inactivated part of the current, its IC50 was 1 muM. Both the inactivation and the reduction of the peak currents were reversible upon washing out the drug. 5 In fura-2-loaded single chromaffin cells the basal [Ca2+]i of around 100 nm was elevated to a peak of 1.5 muM by the application of a 5 s pulse of 59 mM K+. R56865 (10 muM) did not affect the basal [Ca2+]i but blocked by 90% the K+-evoked increase. This effect was fully reversible after 5 10 min of wash out. 6 The results are compatible with the idea that R56865 blocks Ca2+ entry into K+-depolarized chromaffin cells by promoting the inactivation of voltage-dependent Ca2+ channels; this would lead to the limitation of the rise in [Ca2+]i and of the release of catecholamines. The restriction of catecholamine release may favour indirectly the known direct beneficial cardioprotective actions of R56865.