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 α-bungarotoxin (1 μg/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 α-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.