Hydrogen peroxide and epidermal growth factor activate phosphatidylinositol 3-kinase and increase sodium transport in A6 cell monolayers

Abstract

Activation of phosphatidylinositol 3-kinase (PI 3-kinase) is required for insulin stimulation of sodium transport in A6 cell monolayers. In this study, we investigate whether stimulation of the PI 3-kinase by other agents also provoked an increase in sodium transport. Both epidermal growth factor (EGF) and H2O2provoked a rise in sodium transport that was inhibited by LY-294002, an inhibitor of PI 3-kinase activity. PI 3-kinase activity was estimated in extracts from A6 cell monolayers directly by performance of a PI 3-kinase assay. We also estimated the relative importance of the PI 3-kinase pathway by two different methods: 1) coprecipitation of the p85 regulatory subunit with anti-phosphotyrosine antibodies and 2) phosphorylation of PKB on both Ser 473 and Thr 308 residues observed by Western blotting. Since the mitogen-activated protein kinase (MAPK) pathway has also been implicated in the regulation of sodium transport, we also investigated whether this pathway is turned on by insulin, H2O2, or EGF. Phosphorylation of ERK1/2 was increased only transiently by insulin and H2O2but quite sustainedly by EGF. Inhibitors of this pathway (U-0126 and PD-98059) failed to affect the insulin and H2O2stimulation of sodium transport but increased substantially the stimulation induced by EGF. The latter effect was associated with an increase in PKB phosphorylation, thus suggesting that the stimulation of the MAPK pathway prevents, in part, the stimulation of the PI 3-kinase pathway in the transport of sodium stimulated by EGF.