So, we hypothesized that LPA-induced VEGF-C expression is mediated through the LEDGF

nts Unless otherwise noted, all chemicals and reagents were purchased from Sigma Aldrich. H2O2 was purchased from LGX818 site Fisher Scientific and was diluted with NaCl solution prior to assessing experiments. Data Analysis Data are presented as mean 6 S.E. Statistical analysis was performed with SigmaPlot and SigmaStat Software. Paired t test or student t test was used for comparisons between pre- and post-treatment activities. Analysis of variance was used for multiple comparisons among various treatment groups. Differences were considered statistically when P,0.05. Results H2O2-induced Enhancement of ENaC Activity is Reversed by H2S in A6 Cells To investigate the effect of H2S on H2O2-induced enhancement of ENaC activity, cell-attached patch-clamp technique was employed. Single-channel current of ENaC was recorded for about 90 min in each experiment. Since the responses had a long latency, in the figures we omitted a period of record and only showed the representative recordings for about six min before and after application of either H2O2 or NaHS to the cells. Consistent with our previous findings, addition of 3 mM H2O2 to the basolateral bath led to a significant increase both in ENaC Po from 0.2860.04 to 0.5760.10 and in the single-channel amplitude of ENaC . In the presence of H2O2, application of 0.1 mM NaHS to the basolateral bath led to a significant decrease in ENaC Po from 0.5760.10 to 0.2960.07 . In contrast, application of 0.1 mM NaHS to the basolateral bath slightly decreased ENaC Po from 0.3260.04 to 0.2660.05; interestingly, H2O2 failed to increase ENaC Po in the cells pretreated with 0.1 mM NaHS . These results suggest that H2S exerts a strong protective effect on H2O2-induced enhancement of ENaC activity 10753475 in A6 cells. intracellular reactive oxygen species were examined with a fluorescent probe, DCF in the presence of H2O2, H2O2+ NaHS, or NaHS, respectively. We performed experiments in the presence of an iron chelator, 50 mM 2,29-dipyridyl, to confirm whether oxidation is due to Fenton chemistry, where we treated the cells with 2,29-dipyridyl for three min followed by exposing to H2O2 for 30 min. Our results show that exposure of A6 cells to H2O2, in the presence of 2,29dipyridyl, induced significant accumulation of intracellular ROS levels. These results suggest that exogenous H2O2-induced oxidation is not due to Fenton chemistry. Furthermore, in the cells pretreated with 0.1 mM NaHS for 30 min, addition of 3 mM H2O2 failed to increase intercellular ROS. We have carried out the MTT experiments to detect whether H2S affects cell viability. The MTT assay showed that H2S at 0.05, 0.1 and 0.3 mM had no effect on cell viability. These results suggest that H2S can reverse H2O2-induced accumulation of intracellular ROS. H2S Diminishes H2O2-induced Elevation of PIP3 Near the Apical Compartment of A6 Cells We have recently shown that H2O2 stimulates ENaC via elevation of PIP3 near the apical compartment of the cell membrane. Therefore, we reasoned that NaHS may diminish exogenous H2O2-induced enhancement of ENaC activity via reducing accumulation of PIP3 near the apical compartment of A6 cells. To test this hypothesis, the cells were 26976569 stably transfected with the EGFP-PH-Akt construct containing the PH domain of Akt which selectively binds to PIP3. Consistent with our previous studies, under control conditions PIP3 was detected mainly in the lateral and basal membranes; treatment of the cells with 3 mM H2O2 for 30 min H2S Attenuates Exogeno