tivity may be due to a direct chemical reaction of H2S with H2O2; and that this direct chemical reaction dramatically diminishes the ability of H2O2 to oxidize PTEN and subsequently enhances the apical distribution of PIP3. Such direct chemical reaction has been reported by Geng et al., where H2S directly scavenges superoxide anions and H2O2, and consequently eliminates ROS-induced malondialdehyde generation. Manna and Jain reported that H2S can increase cellular level of PIP3 and can enhance glucose utilization in high concentration of glucose treated adipocytes by activating PI3K and inhibiting PTEN. These authors also demonstrated that there is a decreased cellular PIP3 level and impaired glucose homeostasis in the liver of both type 1 and type 2 diabetic rats. In contrast, our data suggest that NaHS did not affect total expression level of PTEN, but significantly prevented H2O2induced PF-562271 inactivation of PTEN, thereby reversed H2O2-induced accumulation of PIP3 near the apical compartment of A6 cells. The difference between the results obtained by Manna et al. and ours might be explained by cell model-dependence. Nevertheless, diabetes is associated with lower circulating level of H2S 7 H2S Prevents H2O2-Induced Activation of ENaC compared to normal population and diabetes exerts a higher incidence of hypertension. Our results along with the findings by Manna and Jain suggest that H2S may be used for treatment of diabetes associated hypertension by affecting cellular level and distribution of PIP3. Oxidation of PTEN by ROS/H2O2 has been reported in HEK293 cells stimulated with insulin, HeLa cells stimulated with epidermal growth factor, and fibroblasts stimulated with platelet-derived growth factor. It has been 10753475 shown that PIP3 can laterally diffuse in the inner leaflet of epithelial cell membranes from the basolateral membrane domain, where it is generated, to the apical membrane domain. Theoretically, the apical PIP3 would be rapidly degraded because the lipid phosphatase PTEN is mainly located in the apical 
membrane in epithelial cells. H2O2 not only inactivates PTEN but also activates PI3K which phosphorylates PIP2 to produce PIP3. NaHS prevents H2O2 induced inactivation of PTEN, but may not affect the PI3K activity. Therefore, it is not surprising that NaHS prevents accumulation of H2O2-induced PIP3 specifically near the apical compartment but not in the lateral and basal membranes. Since only the levels of PIP3 in the apical membrane are important to ENaC function, we have not quantified the fluorescent intensity of basal and lateral membranes. They may vary from cell to cell. Our data show that NaHS may not prevent accumulation of H2O2-induced PIP3 in the basal and lateral membranes. Leslie and co-workers demonstrated that oxidative inactivation of PTEN in the cells exposed to H2O2 is accompanied by an increase in cellular PIP3. Furthermore, oxidation of PTEN with H2O2 leads to the formation of a disulfide bond between Cys124 residue and a nearby Cys71 residue in the active site, thus inactivating its phosphatase function of PTEN. Oxidized PTEN has two less 26496642 cysteine residues available for alkylation, which should lead to a lower molecular weight form of the protein. Consistent with this notion, we found that the abundance reduced form of PTEN was dramatically reduced in A6 cells incubated with H2O2, whereas the magnitude of oxidized PTEN was significantly augmented in H2O2-treated A6 cells. However, there was no significant change
