In addtion, the mRNA and protein expression levels of a, b and g ENaC in ATII cells with co administra tion of Akt inhibitor and SGK1inhibitor showed the simi lar changes kinase inhibitor Rapamycin compared with those by LY294002 treatment, and were significantly decreased compared with those by Akt Inhibitors,Modulators,Libraries inhibitor treatment. The bands were absent when proteins were blotted with the a ENaC, b ENaC and g ENaC antibodies in the presence of the blocking peptide both in vivo and in vitro. These results indicated that insulin induced expression of ENaC by Akt phosphorylation via activating PI3K pathway. Exogenous insulin activated the P13K/Akt pathway and inhibited Nedd4 2 in vivo and in vitro To further investigate whether regulation of ENaC Inhibitors,Modulators,Libraries by insulin via PI3K/Akt pathway, the level of Ser473 phos phorylated Akt, a reliable residue to read out of PI3K activity, and Nedd4 2, a binding site for regulation of ENaC function, were measured by western Inhibitors,Modulators,Libraries blot ting and immunoprecipitation.
The protein level of phosphorylated Akt was markedly increased in rat lung by insulin treatment 8 hours after LPS induced ALI. Wortmannin abolished the insulin induced increase in the protein level of phosphorylated Akt. However, the protein level of Nedd4 2 was significantly decreased by insulin treat ment and Inhibitors,Modulators,Libraries was significantly increased by co administra tion of wortmannin and insulin. In ATII cells pretreated with LY 294002 and Akt inhibitor respectively, insulin induced increase in the protein levels of phosphorylated Akt were markedly decreased.
The level of phosphorylated Akt in ATII cells was also significantly blocked by co adminis tration of Akt inhibitor and SGK1inhibitor compared that in cells Inhibitors,Modulators,Libraries treated with insulin. In a contrast, the protein levels of Nedd4 2 were markedly higher in cells pretreated with LY 294002, Akt inhibitor and Akt inhibitor plus SGK1inhibitor compared with those in cells treated with insulin respectively. Western blot analysis of a, b and g ENaC immunocomplexes with anti Nedd4 2 antibody identi fied a band that was the same size as the one observed with ATII cells lysate and no such band was observed with control IgG, which showed Nedd4 2 interacted with a, b and g ENaC in cells under basal conditions. The inhibitory effect of insulin on the levels of Nedd4 2 immunoprecipitated in a, b and g ENaC were significantly abolished by LY 294002 and Akt inhibitor repectively. These findings strongly indicated that the down regulation of Nedd4 2 that interacted with ENaC by insulin via P13K/Akt pathway. Exogenous insulin decreased mortality of rats in LPS induced actue lung injury Insulin treatment significantly improved the survival of rats with ALI, but wortmannin sig nificantly inhibited the survival of rats treated selleck U0126 with insu lin in LPS induced ALI.