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 [H.sub.2][O.sub.2] 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 [H.sub.2][O.sub.2] 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 [H.sub.2][O.sub.2] is the main intracellular ROS produced upon insulin stimulation. Similarly, preincubation of monolayers with different chelators of either superoxide ([O.sup.*.sub.2]; nitro blue tetrazolium, 100 [micro]M) or[H.sub.2][O.sub.2] (50 [micro]M ebselen), or blockers of NADPH oxidase (Nox) enzymes (diphenyleneiodonium, 5 [micro]M; phenylarsine oxide, 1 [micro]M and plumbagin, 30 [micro]M) prevented both insulin-stimulated [H.sub.2][O.sub.2] 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 [H.sub.2][O.sub.2] production. Taken together, our data suggest that insulin induces an acute burst of [H.sub.2][O.sub.2] production which participates in an increase in phosphatidylinositol 3,4,5-trisphosphate production and subsequently stimulation of sodium transport. ENaC;[H.sub.2][O.sub.2] ; ROS; dichlorodihydrofluorescein; PI 3-kinase