Is an acetylcholine transport system responsible for nonquantal release of acetylcholine at the rodent myoneural junction?

Abstract

Experiments were performed to investigate the effects on the spontaneous, nonquantal release of acetylcholine (AcCho) from motor nerve terminals of substances known to inhibit the AcCho transport system present in cholinergic synaptic vesicles. In mouse diaphragms, the hyperpolarization normally produced by d-tubocurarine in the end. plate area of muscle fibers that had been treated by an anticholinesterase was partly or completely blocked by 2-(4-phenylpiperidino)cyclohexanol (AH5183, 0.1-1 ,sM), quinacrine (0.1 1AM), and tetraphenylborate (1 IAM). Since the sensitivity of the subsynaptic area to AcCho was not changed, the block of the hyperpolarizing action of d-tubocurarine indicated an inhibition of the spontaneous, nonquantal release of AcCho. This was confirmed in experiments on rat diaphragm using direct radioenzymatic measurement of the AcCho released into the incubation medium. The release of AcCho from the innervated diaphragm was decreased by about 50% in the presence of AH5183 (0.01-1 FAM) and by 42% in the presence of quinacrine (0.1-1 FM). The AcCbo released was presumably neural, since the release of AcCho from 4-day denervated diaphragms was not diminished by either AH5183 or quinacrine. The results indicate that the spontaneous release of AcCho from the motor nerve terminals is highly sensitive to low concentrations of specific inhibitors and is probably mediated by a carrier. It is proposed that spontaneous release is due to the incorporation into the membrane of the nerve terminal during exocytosis of the vesicular transport system responsible for moving AcCho into the vesicle.Acetylcholine (AcCho) is released from the nerve terminal of the neuromuscular junction in several ways. There is electrophysiological evidence for the random release of quanta of AcCho, which gives rise to small, transient postsynaptic depolarizations (miniature endplate potentials or MEPPs), and for the stimulation evoked multiquantal release, which gives rise to endplate potentials (1).In The mechanism underlying the nonquantal release of AcCho is unknown. Synaptic vesicles isolated from the electric organ of Torpedo have been shown to take up AcCho by an active process (9). If the transport system responsible for this is present in vesicles in the nerve terminal of the neuromuscular junction, and if it retains its orientation following the incorporation of the vesicle membrane into the axon membrane during exocytosis, then it would move AcCho from the axoplasm into the extracellular space. We have investigated the effects on nonquantal release of several agents that block the transport of AcCho by vesicles (10, 11) to determine whether the release may be due to this transport system. Nonquantal release has been measured electrophysiologically and by direct analysis of bath AcCho. METHODSElectrophysiological Experiments. The experiments were performed on hemidiaphragms dissected from decapitated female white specific-pathogen-free mice. The thoracic side of the diaphragm was carefully cleaned of pleura, which ...

Direct measurement of ACh release from exposed frog nerve terminals: constraints on interpretation of non‐quantal release.

“…Based on the frequency and size of miniature endplate potentials (MEPPs) from these terminals, they also conclude that spontaneous quantal release comprises only about 1 % of total spontaneous ACh release. Subsequent studies of non-quantal ACh release from amphibian and mammalian nerve-muscle preparations have further expanded these observations (Vyskocil & Illes, 1977;Miledi, Molenaar & Polak, 1980;Vyskocil, Nikolsky & Edwards, 1983;Edwards, Dolezal, Tucek, Zemkova & Vyskocil, 1985;Sun & Poo, 1985; Oen, Polak & van der Laaken 1987; Edwards, Dolezal, Tucek, Zemkova & Vyskocil, 1988). Based on these measurements, most of the spontaneous ACh release from a nerve-muscle preparation is believed to be non-quantal.…”

Effect of alpha‐latrotoxin on the frog neuromuscular junction at low temperature.

“…ACh is released from a terminal both in a steady 'leak' (Katz & Miledi, 1977;Vyscocil & Illes, 1979;Edwards, Dolezal, Tucek, Zemkova & Vyskocil, 1985), and in a pulsatile manner as multimolecular packets, or quanta (del Castillo & Katz, 1954). The total release, i.e.…”

“…The 'leak', or non-quantal component of release, is difficult to measure directly since its major known effect is to produce a small, steady depolarization at an end-plate when acetylcholinesterase has been thoroughly inhibited. Therefore, neither its function or origin within the terminal are known (Edwards et al 1985). Quantal release, on the other hand, is easily detected by electrophysiological means, and its function is well known since it generates the transient end-plate potentials (EPPs) and miniature end-plate potentials (MEPPs) that mediate neuromuscular transmission (Fatt & Katz, 1952;del Castillo & Katz, 1954;Boyd & Martin, 1956).…”

The nature and origin of calcium‐insensitive miniature end‐plate potentials at rodent neuromuscular junctions.

“…Since AH-5183 has been shown not only to block the uptake of ACh into synaptic vesicles but also the leakage of ACh from the nerve terminal cytoplasm (Edwards et al 1985, Vyskocil, 1985, the question arose which of these effects was responsible for the reduction in slow m.e.p.p. amplitude and frequency.…”

“…Slow m.e.p.p.s were reduced in frequency and amplitude by the drug AH-5183, which blocks the active uptake of ACh from the cytoplasm into synaptic vesicles as well as the molecular leakage of ACh from the nerve terminal (Anderson et al 1983 a;Edwards et al 1985;Vyskocil, 1985 Kloot, 1986). This is presumably because of the large number of synaptic vesicles present in the nerve terminal.…”