Spontaneous acetylcholine release in mammalian neuromuscular junctions

Spontaneous secretion of the neurotransmitter acetylcholine inmammalian neuromuscular synapsis depends on theCa²⁺ content of nerve terminals.The Ca²⁺ electrochemical gradientfavors the entry of this cation. We investigated the possibleinvolvement of three voltage-dependent Ca²⁺ channels (VDCC) (L-, N-, andP/Q-types) on spontaneous transmitter release at the rat neuromuscularjunction. Miniature end-plate potential (MEPP) frequency was clearlyreduced by 5 µM nifedipine, a blocker of the L-type VDCC, and to alesser extent by the N-type VDCC blocker, -conotoxin GVIA (-CgTx,5 µM). On the other hand, nifedipine and -CgTx had no effect onK⁺-induced transmitter secretion.-Agatoxin IVA (100 nM), a P/Q-type VDCC blocker, preventsacetylcholine release induced byK⁺ depolarization but failed toaffect MEPP frequency in basal conditions. These results suggest thatin the mammalian neuromuscular junction Ca²⁺ enters nerve terminalsthrough at least three different channels, two of them (L- and N-types)mainly related to spontaneous acetylcholine release and the other(P/Q-type) mostly involved in depolarization-induced neurotransmitterrelease. Ca²⁺-bindingmolecule-related spontaneous release apparently binds Ca²⁺ very rapidly and wouldprobably be located very close toCa²⁺ channels, since the fastCa²⁺ chelator (BAPTA-AM)significantly reduced MEPP frequency, whereas EGTA-AM, exhibitingslower kinetics, had a lower effect. The increase in MEPP frequencyinduced by exposing the preparation to hypertonic solutions wasaffected by neither external Ca²⁺concentration nor L-, N-, and P/Q-type VDCC blockers, indicating thatextracellular Ca²⁺ is notnecessary to produce hyperosmotic neurosecretion. On the other hand,MEPP frequency was diminished by BAPTA-AM and EGTA-AM to the sameextent, supporting the view that hypertonic response is promoted by"bulk" intracellular Ca²⁺concentration increases.