Nicotinic Agonists Antagonize Quantal Size Increases and Evoked Release at Frog Neuromuscular Junction

1. Previous studies at the frog neuromuscular junction showed that quantal size can be increased two- to fourfold by a variety of treatments, including prior exposure to hypertonic solution (which activates protein kinase A) and insulin (which acts via an unknown pathway). Size increases largely because quanta contain more acetylcholine (ACh). 2. Now the effects of cholinergic agonists on the increases in quantal size have been studied. One muscle from a frog was kept for 2 h in hypertonic sodium gluconate solution. The miniature endplate potential (MEPP) sizes were measured in saline: they had increased about fourfold. The paired muscle went through the same experimental sequence, except that an agonist was added to the hypertonic gluconate solution. Again MEPP sizes were measured in saline. The increase in quantal size was significantly depressed by 0.2 microM 1,1-dimethyl-4-phenyl- piperazinium (DMPP). The sequence of effectiveness of agonists was: DMPP > carbachol > or = ACh = cytisine > oxytremorine. This sequence suggests that the receptor belongs in the nicotinic class. 3. Quantal size is doubled after 1 h in insulin. One micromolar carbachol largely blocked the size increase. 4. The effects of cholinergic antagonists were tested by keeping the experimental muscle in hypertonic gluconate solution containing 1 microM carbachol plus an antagonist. The controls were paired muscles kept in hypertonic gluconate solution (without carbachol or antagonist). MEPP sizes were measured in saline. The depressing action of carbachol on the increase in MEPP size was blocked by 0.2 microM neuronal-bungarotoxin (nBTX). The sequence of effectiveness of antagonists was: nBTX > trimethaphan > d-tubocurarine (dTC). Ten micromolar atropine (without carbachol) depressed the increase in quantal size. Therefore, the antagonist potency of atropine could not be adequately tested. Carbachol action was not blocked by 10 microM hexamethonium or 10 microM mecamylamine. 5. Once quanta are made large they can be converted back to normal size by cholinergic agonists. Muscles in which quantal size had been enlarged were exposed to hypertonic solutions containing the agonist. Quantal size was reduced to a fraction of its former value when the hypertonic solution contained 1 microM carbachol- or 1 microM DMPP. One micromolar oxytremorine had no effect. Carbachol still reduced quantal size when applied in low-Ca2+ solutions, so it does not appear to act by elevating intracellular [Ca2+]. 6. Previous work suggested that the treatments produce a subpopulation of large quanta that are positioned for release.(ABSTRACT TRUNCATED AT 400 WORDS)