Inhibition of insulin-stimulated hydrogen peroxide production prevents stimulation of sodium transport in A6 cell monolayers.

Insulin-stimulated sodium transport across A6 cell (derived from amphibian distal nephron) monolayers involves the activation of a phosphatidylinositol (PI) 3-kinase. We previously demonstrated that exogenous addition of H2O2 to the incubation medium of A6 cell monolayers provokes an increase in PI 3-kinase activity and a subsequent rise in sodium transport (Markadieu N, Crutzen R, Blero D, Erneux C, Beauwens R. Am J Physiol Renal Physiol 288: F1201-F1212, 2005). We therefore questioned whether insulin would produce an intracellular burst of H2O2 leading to PI 3-kinase activation and subsequent increase in sodium transport. An acute production of reactive oxygen species (ROS) in A6 cells incubated with the oxidation-sensitive fluorescent probe 5,6-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate was already detected after 2 min of insulin stimulation. This fluorescent signal and the increase in sodium transport were completely inhibited in monolayers incubated with peggylated catalase, indicating that H2O2 is the main intracellular ROS produced upon insulin stimulation. Similarly, preincubation of monolayers with different chelators of either superoxide (O2(*-); nitro blue tetrazolium, 100 microM) or H2O2 (50 microM ebselen), or blockers of NADPH oxidase (Nox) enzymes (diphenyleneiodonium, 5 microM; phenylarsine oxide, 1 microM and plumbagin, 30 microM) prevented both insulin-stimulated H2O2 production and insulin-stimulated sodium transport. Furthermore, diphenyleneiodonium pretreatment inhibited the recruitment of the p85 PI 3-kinase regulatory subunit in an anti-phosphotyrosine immunoprecipitate in insulin-stimulated cells. In contrast, PI-103, an inhibitor of class IA PI 3-kinase, inhibited insulin-stimulated sodium transport but did not significantly reduce insulin-stimulated H2O2 production. Taken together, our data suggest that insulin induces an acute burst of H2O2production which participates in an increase in phosphatidylinositol 3,4,5-trisphosphate production and subsequently stimulation of sodium transport.