EFFECT OF DIHYDROPYRIDINES ON CALCIUM CHANNELS IN ISOLATED SMOOTH-MUSCLE CELLS FROM RAT VENA-CAVA

1 Whole-cell patch-clamp method was applied to single smooth muscle cells freshly isolated from the rat inferior vena cava. 2 Depolarizing pulses, applied from a holding potential of -90 mV, activated both Na+ and Ca2+ channels. The fast Na+ current was inhibited by nanomolar concentrations of tetrodotoxin (TTX). The slow Ba2+ current (measured in 5 mM Ba2+ solution) was inhibited by Cd2+ and modulated by dihydropyridine derivatives. When the cells were held at a holding potential of -80 mV, racemic Bay K 8644 increased the Ba2+ current (ED50 = 10 nM) while racemic isradipine inhibited the current (IC50 = 21 nM). 3 The voltage-dependency of isradipine blockade was assessed by determining the steady-state availability of the Ca2+ channels. From the shifts of the inactivation curve in the presence of isradipine, we calculated a dissociation constant of 1.11 nM for inactivated Ca2+ channels. Scatchard plots of the specific binding of (+)-[H-3]-isradipine obtained in intact strips incubated in 5.6 mM or 135 mM K+ solutions confirmed the voltage-dependency of isradipine binding. 4 Specific binding of (+)-[H-3]-isradipine was completely displaced by unlabelled (+/-)-isradipine, with an IC50 of 15.1 nM. This value is similar to the IC50 for inhibition of the Ba2+ current (21 nM) in cells maintained at a holding potential of -80 mV. 5 Bay K 8644 had no effects on the Ba2+ current kinetics during a depolarizing test pulse. The steady-state inactivation-activation curves of Ba2+ current were not significantly shifted along the voltage axis. 6 The present data suggest that existence of two distinct dihydropyridine binding sites which can be bound preferentially by agonist or antagonist derivatives.