Hydrogen peroxide and epidermal growth factor activate phosphatidylinositol 3-kinase and increase sodium transport in A6 cell monolayers.
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 H2O2 provoked 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 H2O2 but quite sustainedly by EGF. Inhibitors of this pathway (U-0126 and PD-98059) failed to affect the insulin and H2O2 stimulation 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.
Markadieu
Nicolas
N
Laboratory of Cell and Molecular Physiology, Campus Erasme CP 611, Université Libre de Bruxelles, BAt E1, niv 6, local 214, Route de Lennik 808, 1070 Bruxelles, Belgium.
Crutzen
Raphaël
R
Blero
Daniel
D
Erneux
Christophe
C
Beauwens
Renaud
R
D000818:Animals; D002460:Cell Line; D002867:Chromones / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D066246:ErbB Receptors / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D007004:Hypoglycemic Agents / Q000494:pharmacology; D007328:Insulin / Q000494:pharmacology; D007668:Kidney / Q000166:cytology*; D020935:MAP Kinase Signaling System / Q000187:drug effects; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D009025:Morpholines / Q000494:pharmacology; D016877:Oxidants / Q000494:pharmacology*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D019000:Phosphotyrosine / Q000378:metabolism; D012964:Sodium / Q000378:metabolism*; D014982:Xenopus laevis; D018909:src Homology Domains
D002867:Chromones; D004791:Enzyme Inhibitors; D007004:Hypoglycemic Agents; D007328:Insulin; D009025:Morpholines; D016877:Oxidants; D019000:Phosphotyrosine; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D004815:Epidermal Growth Factor; D012964:Sodium; D006861:Hydrogen Peroxide; D019869:Phosphatidylinositol 3-Kinases; D066246:ErbB Receptors; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3