Differential effects of an L-type Ca2+ channel antagonist on activity- and phosphorylation-enhanced release of acetylcholine at the neuromuscular junction of the frog in vitro

Abstract

The effects of the selective L-type Ca(2+)-channel antagonist nimodipine on changes in the electrophysiological correlates of acetylcholine release induced by activity in the motor nerve or by inhibition of protein dephosphorylation were studied in the isolated sartorius muscle of the frog. Nimodipine (1 microM) had no effect on basal miniature endplate potential (mEPP) frequency or on the quantal content of the endplate potential (EPP) evoked at 0.33 Hz. Stimulation of the motor nerve at frequencies of 20, 40 and 50 Hz progressively increased the quantal content and at 50 Hz caused an increase in mEPP frequency measured at the end of the train. Nimodipine (1 microM) had no effect on the change in either parameter caused by activity in the motor nerve. The L-channel agonist BAYK 8644 had inconsistent effects, causing at 0.33 Hz an increase in quantal content at 55% of the neuromuscular junctions examined. Application of the phosphatase inhibitor okadaic acid (1 microM) caused an increase in miniature endplate potential and/or current [mEPP(C)] frequency but not in quantal content. The increase in spontaneous frequency was reduced by the Ca(2+)-channel blockers nimodipine (1 microM) and cadmium (75 microM). The amplitude of mEPP(C)s was increased by okadaic acid (1 microM), but neither the decay time constant of miniature endplate currents (mEPC) nor the amplitude of endplate currents evoked by iontophoretic application of carbachol was so altered. The activity of electric eel acetylcholinesterase was unchanged by okadaic acid. The present data do not support the concept that the recruitment of normally silent L-type Ca(2+)-channels contributes to activity-dependent increases in acetylcholine release. The results obtained with okadaic acid suggest that protein phosphorylation and dephosphorylation may regulate the activity of L-type channels and the packaging of acetylcholine.