Facilitation of spontaneous acetylcholine release induced by activation of cAMP in rat neuromuscular junctions

Abstract

Regulation of neurotransmitter release is thought to involve modulation of therelease probability by protein phosphorylation. Activation of the CAMP-proteinkinase A (PKA) pathway has been shown to facilitate synaptic transmission inmammalian neuromuscular synapses, although the relevant phosphorylation targetsare mostly unknown. We found that the inhibitor of the phosphodiesteraseaminophylline (1 m.M AMIN), the membrane-permeable analog of CAMP, 8-Br-CAMP (5 mM) and, the diect adenylate cyclase activator, forskolin (20 PM),induced an increase of miniature end-plate potentials (MEPPs) frequency in ratneuromuscular junctions. We investigated the possible involvement of the voltagedependentcalcium channels (VDCC), since these proteins are known to bephosphorylated by PKA. But this possibility was ruled out, since the increase inMEPPs frequency was not attenuated by the VDCC blocker Cd” (100 pM) and itwas observed when AMIN was studied on hyperosmotic response, which isindependent of [Ca2’10 and of Ca2’ infhrx through the VDCC. The lack of action ofAMIN on MEPPs frequency when [Ca’+]i was diminished by exposing thepreparations to zero Ca2’-EGTA solution (isotonic condition) or when nerveterminals were loaded with a permeant Ca2’ chelator (BAPTA-AM) (hypertoniccondition), indicate that cAMPmediated presynaptic facilitation is a function ofnerve terminal Ca2” concentration We also found that AMIN exerted a comparableincrease in MEPPs f%equency in control and high K” (10 and 15 mM), suggesting asingle mechanism of action for spontaneous and K+-induced secretion.