Properties of the Cell Membrane of Developing Skeletal Muscle Fibres in Culture and Its Sensitivity to Acetylcholine

Abstract

S U M M A R Y 1. The relationship between determinants of membrane potential has been studied during myogenesis in culture.2. Myoblasts obtained from 1 1-day chick embryo musculature were cultured and intracellular records were made from myoblasts and developing unstriated and striated myotubes.3. Resting membrane potentials increased with time of culture until values equivalent to those obtained from fully developed striated muscle fibres were reached after about 20 days in culture.4. High concentrations of potassium added to the medium failed to affect significantly the resting membrane potentials in myoblasts and myotubes up to 10 days in culture. Changes in external sodium had little effect on values of potentials from myotubes and cells up to 4 days in culture, but removal of sodium after 5 and 6 days in culture caused depolarization. 5. Electrolyte distributions determined for myoblasts and myotubes 4 days after fusion showed no significant change in potassium content, whereas the sodium concentration fell significantly. 6. Acetylcholine applied by microinjection caused two types of response from myoblasts depending upon the level of the resting membrane potential : when the potential was less than -7 mV, hyperpolarization occurred; when it was greater than this value, depolarization occurred. 7. Depolarizing responses to acetylcholine application were regularly obtained from myotubes with resting membrane potentials greater than -7 mV.8. The responses to acetylcholine could be blocked by d-tubocurarine in the medium ; tetramethylammonium (a nicotinic agonist) acted similarly to acetylcholine, but bethanecol (a muscarinic agonist) had no effect.

Electrophysiological and Pharmacological Properties of Skeletal Muscle in Culture

“…Intracellular potamium remained constant at about 140 mM throughout muscle development in culture. In chick muscle, intracellular sodium fell from 56 to 22 mM over a period when the resting membrane potential rose from -10 to -21 mv (14). Catterall(29) estimated the internal sodium concentration of 5-9-day chick myotubes to be 13 mM; Ritchie and Fambrough (20) reported the same value for rat myotubes, although no change during development was observed.…”

“…The increase in membrane potential was correlated with numbers of nuclei in the fiber (4), with fiber thickness (14), and with fiber length (6,181. Fibers of 1000 hm in length usually have attained their maximum resting membrane potential, although the morphology of multiply branched myotubes in culture presents formidable difficulties in measuring fiber length.…”

“…In muscle fibers developed from the fusion of disaggregated primary myoblasts obtained from chick embryos or neonatal rodents, the resting membrane potential changed from a value around -10 mv before fusion and immediately afterward, rising steadily as fiber length and diameter increased to about -50 or -60 mv (4,6,14,(17)(18)(19)(20). Thereafter, little change in the resting membrane potential was observed.…”

“…As the resting membrane potential increases, however, the sensitivity to changes in [K]o increases until there is a virtual linear relationship between log [K]o and membrane potential (14,20,30). The curves obtained with rat myotubes closely followed the theoretical line derived from the Goldman equation arid derived values for the internal ion concentrations (20).…”

Acetylcholine receptors of skeletal muscle cells in cultures of rat thymus glands.

“…However, the results of functional assays based on acetylcholineinduced depolarizations are not so clear because they do not take into account the change in resting membrane potential with age (e.g. Powell & Fambrough, 1973;Dryden, Erulkar & de la Haba, 1974). In the present work sensitivity of developing muscle cells to ionophoretically applied acetylcholine has been expressed in terms of conductance sensitivity, Sg, a measure which is independent of resting membrane 356 THYMUS CHOLINOCEPTORS potential and input resistance and which also corrects for the non-linearity of the dose-response curve (Land et al 1977).…”