ELECTROGENIC NA-K PUMP CURRENT IN RAT SKELETAL MYOBALLS

Catecholamines and insulin have been reported to hyperpolarize skeletal muscle fibers via stimulation of the electrogenic Na-K pump (Flatman and Clausen, 1979, Nature, 281:580-581). Therefore, the electrogenic Na-K pump current was investigated in cultured colcemid-treated rat skeletal myoballs using whole-cell voltage clamp. Skeletal muscles were taken from newborn rat hindlegs, trypsin digested, and cultured. By day 7, all myoblast cells fused into myotubes. After treatment with the microtubule disrupter colcemid (10(-7) M) for 2 days, some of the myotubes became transformed into spherical myoballs, having an average diameter of 41.2 +/- 1.5 mum (n = 21). The resting membrane potential averaged -56.8 +/- 1.7 mV (n = 40). Ouabain (1 mM) quickly depolarized the myoballs to -51.1 +/- 1.1 mV (n = 27), showing the existence of an electrogenic Na-K pump in the skeletal myoball preparation. The values for the specific membrane resistance and capacitance were 5.5 +/- 1.0 KOMEGA-cm2 (n = 21) and 3.7 +/- 0.3 muF/cm2 (n = 21), respectively. The pump current averaged 0.28 +/- 0.03 pA/pF (n = 10), with the membrane potential at -60 mV and 10 mM intrapipette Na+. The Na-K pump contribution to resting membrane potential was calculated to be 5.7 mV, matching the ouabain-induced rapid depolarization. When the Na-K pump was stimulated with 50 mM intrapipette Na+, the pump current was about doubled (0.52 +/- 0.08 pA/pF; n = 10). Isoproterenol (1 muM) and 8-Br-cAMP (1 mM) also significantly increased pump current by 50% (0.42 +/- 0.04 pA/pF; n = 9) and 64% (0.46 +/- 0.09 pA/pF; n = 7), respectively. In contrast, although insulin and phorbol ester also increased pump current, this increase was not statistically significant. The ineffectiveness of insulin and phorbol ester may be due to colcemid interfering with Na-K pump translocation from internal vesicles to the sarcolemma. (C) 1994 Wiley-Liss, Inc.