Insulin stimulates the Na+,K+-ATPase and the Na+/K+/Cl- cotransporter of human fibroblasts

Insulin regulation of K+ (Rb+) transport was investigated in cultured human fibroblasts using a non-radioactive method which allows the simultaneous determination of the intracellular concentration of other monovalent cations. Insulin stimulated Rb+ influx through the Na+,K+-ATPase and the Na+/K+/Cl- cotransporter in human fibroblasts. Insulin stimulation was very rapid and maximal effect was observed within 10 min. Insulin stimulation of Rb+ uptake via the Na+,K+-ATPase and the Na+/K+/Cl- cotransporter was dose-dependent, with half-maximal stimulation at 2-3 nM of hormone. Insulin increased the V-max of both transporters involved, affecting only minimally their K-m. In other cells, insulin stimulates the Na+,K+-pump by increasing Naf availability through the Na+/H+ exchanger. In human fibroblasts, insulin stimulation of Na+,K+-ATPase occurred in the presence of ethyl-isopropyl amiloride, an inhibitor of the Na+/H+ exchanger, and without sustained changes in intracellular [Na+]. By contrast, insulin action on Na+,K+-ATPase was impaired by the protein kinase inhibitors staurosporine and genistein. These results indicate that, in human fibroblasts, insulin stimulates both the Na+,K+-ATPase and the Na+/K+/Cl- cotransporter, that stimulation of the Na+,K+-ATPase occurs in the absence of changes in intracellular [Na+], and that protein kinase activity is essential for this insulin action.