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Experiments on isolated phrenic-diaphragmatic preparations from rats showed that low acetylcholine concentrations increase the work capacity of exhausted muscle by increasing the level of evoked quantum transmitter release; acetylcholine also induced hyperpolarization of muscle fiber membranes. The effects of acetylcholine persisted for long periods of time. The modulating actions of acetylcholine are mediated by structures with different pharmacological characteristics from those of typical n- and m-cholinoreceptors; these mechanisms involved ouabain-sensitive isoforms of Na+,K+-ATPase and, perhaps, membrane K+ channels. The data obtained here support the possible existence of long-term neuronal regulation of the efficiency of neuromuscular transmission involving non-quantal acetylcholine, these mechanisms presumably developing differently in muscle fibers with different functional characteristics and abilities to adapt to physiological loading.