Fig. 2.

H₂S and NO signaling converge on the cGMP/PKG pathway. (A) bEnd3 cells were pretreated with l-NAME (4 mM, 40 min) or ODQ (10 μM, 2 h). Following pretreatments, cells were washed twice with Hanks’ balanced salt solution and then exposed for 3 min to NaHS at the indicated concentrations. cGMP was extracted in 0.1 M HCl and measured by enzyme immunoassay. **P < 0.01 vs. control; ^#P < 0.05 and ^##P < 0.01 vs. corresponding control. (B) sGC enzymatic activity was assayed using [α-³²P]GTP to [³²P]cGMP conversion assay in response to the indicated concentrations of NaHS or DEA/NO. To test whether H₂S affects NO-induced sGC activation, the effect of increasing concentrations of DEA/NO on sGC activity was assessed in the presence or absence of 10 μM NaHS. (C) PDE5A activity was measured in the presence of the indicated concentrations of NaHS or 1 μM sildenafil, as a positive control. *P < 0.05 or **P < 0.01 vs. control. bEnd3 cells were exposed to NaHS at the indicated concentrations and time. Cell lysates were analyzed by SDS/PAGE. PVDF membranes were blotted by using rabbit polyclonal antibodies against phosphorylated (Ser473) or total Akt (D); phosphorylated (Ser1177) or total eNOS (E); phosphorylated (Thr495) or total eNOS (F); and phosphorylated (Ser239) or total VASP (G). Densitometric analysis was performed on three blots from three different experiments using image analysis software. *P < 0.05 and **P < 0.01 NaHS (30 min) vs. control.