INSULIN STIMULATION OF NA H ANTIPORT IN L-6 CELLS - A DIFFERENT MECHANISM IN MYOBLASTS AND MYOTUBES/

Insulin modulation of the Na/H antiport of L-6 cells, from rat skeleta l muscle was studied in both myoblasts and myotubes using the fluoresc ent pH sensitive, intracellular probe 2',7' bis (carboxyethyl)-5(6)-ca rboxyfluorescein. insulin stimulated the Na/H antiport activity in L-6 cells, showing a bell-shaped dose response typical of other insulin r esponses: a maximum at 10 nM (Delta pH of 0.132 +/- 0.007 and 0.160 +/ - 0.040 over basal value, for myoblasts and myotubes, respectively; me ans +/- SD, n = 6-8) and smaller effects at higher and lower concentra tions. Phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, also stimulated the antiport in myoblasts but not in myotube s. Surprisingly the rapid increase in intracellular pH was not observe d when insulin and PMA were added simultaneously to myoblasts; apparen tly these two activators mutually excluded each other. Downregulation of protein kinase C, obtained by preincubation of cells with PMA for 2 0 hr, totally abolished both hormone and PMA effects in myoblasts, whe reas in myotubes insulin stimulation was not affected. Inhibitors of t yrosine kinase activity, such as erbstatin analog and genistein abolis hed insulin effect on the Na/H antiport, both in myoblasts and in myot ubes. Different sensitivity to pertussis toxin in the two cell types s uggests that the differentiation process leads to a change in the sign al pathways involved in the physiological response to insulin. (C) 199 7 Wiley-Liss, Inc.