Effects of calcium channel blockers on spontaneous electrical activity of freshly isolated three-day-old embryonic chick ventricle

Abstract

The effects of four major types of organic Ca2+ channel blockers, verapamil, nifedipine, diltiazem and fendiline and of tetrodotoxin (TXX), a fast Na+ channel blocker, on the action potential (AP) of freshly isolated 3-day-old embryonic chick ventricle (3d ECV) were investigated to resolve the controversy about the ionic basis of upstroke. The APs were characterized by a maximum diastolic potential (MDP) of -60 mV, an overshoot (Eov) of 16 mV and a maximum upstroke velocity (+Vmax) of 42 V s-1. All four Ca2+ channel blockers (0.1-40 microM) and TTX (0.1-80 nM) produced a dose-dependent reduction in +Vmax and Eov. MDP was also reduced by Ca2+ channel blockers in a dose-dependent manner but was unaffected by TTX. A significant linear correlation between MDP and +Vmax was observed for verapamil (r = 0.99), nifedipine (r = 0.99), diltiazem (r = 0.96) and fendiline (r = 0.98). Surprisingly, all Ca2+ channel blockers produced a dose-dependent positive chronotropic effect leading to cessation of firing at high doses (20-40 microM). In preparations becoming quiescent with high doses of verapamil (20-40 microM), elevated extracellular concentrations of Ca2+ (up to 9.6 nM) and isoproterenol (0.5-40 microM) failed to restore spontaneous APs. Electrical stimulation also failed to elicit APs in preparations inhibited by verapamil, diltiazem and fendiline. The inhibition of +Vmax by TTX demonstrates that fast Na+ channels were involved in the upstroke of AP in 3d ECV. Voltage-dependent inactivation of fast Na+ channels during depolarization (reduction in MDP) by the Ca2+ channel blockers explains their inhibitory effect on +Vmax and indicates that L-type Ca2+ channels had no significant role in the upstroke. A positive chronotropic effect of the Ca2+ channel blockers further suggests that slow Ca2+ channels are not involved in automaticity in freshly isolated 3d ECV.