The effect of curare on the release of acetylcholine from mammalian motor nerve terminals and an estimate of quantum content.

Abstract

SUMMARY1. Curarized and non-curarized rat hemidiaphragm muscles were indirectly stimulated in vitro.2. The fluid bathing the active curarized muscles was eluted through a dextran gel (Sephadex G-10), effecting a complete separation of ACh from curare. The acetylcholine fraction was then assayed on an isometric leech muscle preparation.3. Prostaglandin (PGE1) in a concentration fifteen times that estimated to be released from the skeletal muscle preparation did not affect the response of leech muscle to ACh.4. The amount of ACh released by curarized muscles (4.9 x 10-18 mole/ impulse. junction) was not significantly different from that released by non-curarized muscles (46 x 10-18 mole/impulse. junction). These quantities are similar to those obtained by previous workers. It is concluded that curare in a paralytic dose does not affect the output of ACh from motor nerve terminals stimulated at low frequencies.5. Spontaneous release of ACh from non-curarized muscles was estimated at 0-45-0'65 p-mole/min. hemidiaphragm. It is calculated that only 2 % of this amount could give rise to post-synaptic electrical events, the remainder having a non-synaptic source. 6. The number of molecules of 'quantal' ACh released by stimulated muscle is calculated as 2-5 x 106/impulse . junction, taking account of the non-synaptic release. The number of ACh molecules in one quantum was estimated to be 6250, an amount that could be easily accommodated in one synaptic vesicle.

Changes in total and quantal release of acetylcholine in the mouse diaphragm during activation and inhibition of membrane ATPase.

“…Mitchell & Silver (1963) considered the amount 9 E. S. VIZI AND F. VYSKOCIL of ACh involved in m.e.p.p. production to be only 1-3 % and similarly, Fletcher & Forrester (1975) estimated the quantal spontaneous release in the rat diaphragm to be only 2 % of the total release. According to our data the resting quantal release represents in the mouse preparation not more than 10 % of the total amount of ACh, this value being close to previous estimates.…”

“…Similarly, Fletcher & Forrester (1975) calculated that no more than 2 % of the resting release of ACh could be responsible for the production of m.e.p.p.s.…”

Acetylcholine compartments in mouse diaphragm. Comparison of the effects of black widow spider venom, electrical stimulation, and high concentrations of potassium.

“…This indicates that there is an appreciable secretion of ACh in a nonquantized form, particularly at rest (22,32). Recent estimates of the number of ACh molecules in a quantum range from 6 to 10 x 103 (12,25). If we assume a value of 8 x 10 a, then the rate of secretion of ACh due to quanta is 0.4 pmol/15 min for a resting hemidiaphragm (7,000 junctions, mean mepp frequency of 5/s).…”

“…If we assume that all of the extra ACh released by K is secreted as quanta, then we calculate the number of ACh molecules in a quantum to be 13 x 10 a, a figure only 30% greater than the upper limit estimated by Kuffler and Yoshikami (25). 1 When 25 mM K is applied in the presence of HC-3, the rate of release of ACh increases, but the increase is not sustained and the rate even-1 Fletcher and Forrester (12) have estimated the quantal size in rat diaphragms to be only 6 • 103 molecules. Their calculation assumed an epp to be composed of 400 quanta.…”

Prejunctional modulation of acetylcholine release from the skeletal neuromuscular junction: link between positive (nicotinic)− and negative (muscarinic)‐feedback modulation

“…They concluded that curare had principally a postjunctional action. Since then several attempts have been made to study the prejunctional effect of (+)-tubocurarine (Chang et al, 1967;Fletcher & Forrester, 1975), but until now attempts to demonstrate the inhibitory effect of (+)-tubocurarine on the evoked release of ACh by neurochemical methods (i.e. assay of the released ACh) have been unsuccessful.…”