Evidence for the role of non-quantal acetylcholine in the maintenance of the membrane potential of rat skeletal muscle.

1. Resting membrane potentials of rat diaphragm muscles cultured in Trowell T8 medium were measured in vitro. After 3 hr in culture the resting membrane potential of muscle fibres within 2.5 mm of nerve section (;near') was -68.3 +/- 0.4 mV (nineteen preparations). This was significantly lower (P < 0.001) than the resting potential (-74.0 +/- 0.4 mV) measured in muscle fibres 8-10 mm from the site of nerve section (;far') in the same preparations. A difference between the ;near' and the ;far' fibres was maintained in muscles cultured for 6 and 12 hr. Miniature end-plate potentials were present in both ;near' and ;far' fibres cultured for 3 and 6 hr and ceased after 12-15 hr.2. The presence of carbamylcholine (10(-7) or 10(-8) M) maintained the resting membrane potential of ;near' fibres close to that of ;far' fibres at 3, 6 and 12 hr. For example, at 3 hr in the presence of 10(-8) M-carbamylcholine the mean resting potential was 75.6 +/- 0.5 mV in ;near' fibres and 76.1 +/- 0.4 mV in ;far' fibres (four preparations). A similar effect was produced in preparations exposed to anticholinesterases: diisopropylphosphorofluoridate (DFP) (10(-7) M), neostigmine (10(-7) M) or physostigmine (10(-5) M).3. Agents that blocked acetylcholine receptors had the reverse effect. In the presence of alpha-bungarotoxin (1 mug/ml.) or d-tubocurarine (10(-5) M) the resting membrane potential of ;far' fibres was reduced to the level of ;near' fibres over the 24 hr period of observation. For example, at 3 hr in the presence of alpha-bungarotoxin the mean resting potential was 67.2 +/- 0.5 mV in ;near' fibres and 68.5 +/- 0.6 mV in ;far' fibres (six preparations). The effect of d-tubocurarine was reversible.4. When muscles were cultured in Ca(2+)-free medium containing 1 mM-EGTA and 10 mM-Mg(2+), there was no difference in membrane potential between ;near' and ;far' fibres and physostigmine (10(-5) M) was ineffective in raising the membrane potential of ;near' fibres.5. It is suggested that non-quantal acetylcholine released from nerve terminals maintains the membrane potential of muscle fibres through a Ca(2+)-dependent mechanism.