Increased non-quantal release of acetylcholine after inhibition of endocytosis by methyl-β-cyclodextrin: the role of vesicular acetylcholine transporter

We investigated the role of the vesicular acetylcholine transporter in the mechanism of non-quantal (non-vesicular) secretion of neurotransmitter in the neuromuscular synapse of the rat diaphragm muscle. Non-quantal secretion was estimated electrophysiologically by the amplitude of end-plate hyperpolarization after inhibition of cholinesterase and nicotinic receptors (H-effect) or measured by the optical detection of acetylcholine in the bathing solution. It was shown that 1 mM methyl-β-cyclodextrin (MCD) reduced both endocytosis and, to much lesser extent, exocytosis of synaptic vesicles (SV) thereby increasing non-quantal secretion of acetylcholine with a concurrent decrease in axoplasm pH. During high-frequency stimulation of the motor nerve, that substantially increases vesicles exocytosis, the non-quantal secretion was further enhanced if the endocytosis of SV was blocked by MCD. In contrast, non-quantal secretion of acetylcholine did not increase when the MCD-treated neuromuscular preparations were superfused with either vesamicol, an inhibitor of vesicular transporter of acetylcholine, or sodium propionate, which decreases intracellular pH. These results suggest that the proton-dependent, vesamicol-sensitive vesicular transporters of acetylcholine, which become inserted into the presynaptic membrane during SV exocytosis and removed during endocytotic recycling of SV, play the major role in the process of non-quantal secretion of neurotransmitter.

▶Methyl-β-cyclodextrin (MCD) decreases endocytosis in motor nerve terminals (NT). ▶MCD increases non-quantal secretion of acetylcholine (NQSACh) & acidifies cytoplasm. ▶Stimulation of MCD-treated NT further increases NQSACh and decreases cytosolic pH. ▶Inhibition of vesicular ACh transporters (VAChT) prevents the effect of stimulation. ▶VAChT are responsible for non-vesicular secretion of ACh in neuromuscular synapse.