R56865 INHIBITS CATECHOLAMINE RELEASE FROM BOVINE CHROMAFFIN CELLS BYBLOCKING CALCIUM CHANNELS

1 The effects of R56865 (a new class of cardioprotective agent which p revents Na+ and Ca2+ overload in cardiac myocytes) on catecholamine re lease, whole-cell current through Ca2+ channels (I(Ba)) and cytosolic Ca2+ concentrations, [Ca2+]i, have been studied in bovine chromaffin c ells. 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 concentr ation inhibited secretion by 20%; the blockade increased gradually wit h perfusion time, to reach 85 % after 40 min. The IC50 to block secret ion after 5 min periods of exposure to increasing concentrations of R5 6865 was around 3.1 muM. The blocking effects of R56865 were reversibl e after 5-15 min wash out. In high Ca2+ solution (10 mM Ca2+), the deg ree of blockade of secretion diminished by 20% in comparison with 1 mM Ca2+. 3 In electroporated cells, R56865 (10 muM) did not modify the s ecretory response induced by the application of 10 muM free Ca2+. 4 R5 6865 blocked the peak I(Ba) current in a concentration- and time-depen dent manner; its IC50 was very similar to that obtained for secretion (3 muM). The compound not only reduced the size of the peak current bu t also promoted its inactivation; when the effects of R56865 were meas ured at the most inactivated part of the current, its IC50 was 1 muM. Both the inactivation and the reduction of the peak currents were reve rsible 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) d id not affect the basal [Ca2+]i but blocked by 90% the K+-evoked incre ase. This effect was fully reversible after 5 10 min of wash out. 6 Th e results are compatible with the idea that R56865 blocks Ca2+ entry i nto K+-depolarized chromaffin cells by promoting the inactivation of v oltage-dependent Ca2+ channels; this would lead to the limitation of t he rise in [Ca2+]i and of the release of catecholamines. The restricti on of catecholamine release may favour indirectly the known direct ben eficial cardioprotective actions of R56865.