MODULATION OF THE NA-H ANTIPORT BY INSULIN - INTERPLAY BETWEEN PROTEIN-KINASE-C, TYROSINE KINASE, AND PROTEIN PHOSPHATASES

The insulin modulation of Na-H antiport in rat hepatocytes was studied using the fluorescent, pH-sensitive intracellular probe, 2',7' bis (c arboxyethyl)-5(6)-carboxyfluorescein (BCECF). Our data show that insul in stimulates the Na-H antiport. The dose-response of insulin effect s hows a behavior typical of other insulin responses: a maximum in the p hysiological range (1 nM) and smaller effects at higher and lower horm one concentrations. The time-course of activation is very fast at high hormone concentrations and slow, but reaching a higher value, for the physiological concentrations (0.26 +/- 0.05 and 0.18 +/- 0.022 pH uni ts for 1 nM and 1 mu M insulin respectively). The use of phorbol, 12-m yristate, 13-acetate (PMA), a potent activator of protein kinase C and its inhibitor staurosporine, and the inhibitor of tyrosine kinase erb statin analog, suggests that both protein kinase C and tyrosine kinase could be involved in the mechanism leading to Na-H antiport activatio n by insulin. We suggest that the activation of the antiport involves the two pathways depending on the hormone concentration. In particular , protein kinase C would mediate the effects of high hormone concentra tions, acting as a growth factor, since staurosporine fully inhibited insulin 1 mu M, but only partially 1 nM effects, and tyrosine kinase w ould mediate the effect of insulin 1 nM and only partially 1 mu M. Oka daic acid 1 mu M, a potent inhibitor of protein phosphatases, mimicked the hormone effects on the antiport and abolished the different time- course due to hormone concentration, suggesting a role of kinases and phosphatases in the signal transduction. The effect of all activators was abolished by amiloride analog, 5-(N-ethyl-N-isopropyl) amiloride ( EIPA), confirming the specificity of these effects. (C) 1994 Wiley-Lis s, Inc.