Effects of carbamylcholine on membrane potential and Na-K pump activity of cultured rat skeletal myotubes

1. We measured changes in resting membrane potential (E_m) and Na-K pump activity, assayed by ouabain-sensitive⁸⁶Rb uptake, in response to carbamycholine (CCh) and its continued presence in single rat skeletal myotubes in culture. 2. CCh caused immediate depolarization from control E_m (-80 to -85 mV) to near 0 followed by repolarization of varying degrees depending on the age of the culture and temperature of the recording medium; repolarization of E_m was most apparent by culture age 8-9 days in vitro (DIV), E_m reaching values as high as -60 mV by 5-10 min after peak depolarization at 37°C. 3. Input resistance, which decreased during CCh depolarization, increased only slightly during the initial phase of repolarization and then remained essentially unchanged during the major component of membrane repolarization in the presence of CCh. 4. Ouabain, given before CCh, prevented repolarization of E_m and, when given after repolarization had begun, reversed it and caused E_m to return to about -7 mV. 5. Na-K pump activity was decreased in myotubes in which E_m did not repolarize or did so only slightly, and was increased by over 40-50% in myotubes whose E_m repolarized by 40-60 mV, even though CCh was still present in the medium. Inhibition of pump activity in non repolarizing myotubes was related to Na influx, inhibition being reversed to stimulation when CCh was administered to myotubes in Na-free medium. 6. Repeated (three or four times) or prolonged (up to 60-min) administration of CCh to myotubes in which repolarization was hardly expressed (age 6-7 DIV) caused increases both in the amount of repolarization and in⁸⁶Rb uptake, both being related to the number or duration of CCh exposures. 7. We conclude that repolarization of E_m following CCh-induced depolarization of cultured rat skeletal myotubes depends to a large extent on an increase in activity of the electrogenic Na-K pump.