FIGURE 6.

S-Nitrosylation inhibits Panx1 currents and ATP release from mouse aortic endothelial cells. A, biotin switch assay on primary mAECs treated with 100 μm GSNO ± 1 mm DTT, 50 μm DEA NONOate ± 1 mm DTT, or 100 μm GSH. Ascorbate (Asc−) was omitted from the assay as a negative control. B, time series showing peak Panx1 whole cell current amplitude from mAECs during application of GSNO, DTT, and CBX. C, current-voltage curves of Panx1 currents from primary mAECs under control conditions (black trace) and following application of 100 μm GSNO (red trace), 1 mm DTT (green trace), and 50 μm CBX (blue trace). C, summary data showing the percentage of Panx1 current inhibition by GSNO or GSH that was reversible by DTT. D, ATP release assay from mAECs stimulated with 1 unit/ml mouse thrombin. Cells were pretreated with 100 μm GSNO, 50 μm DEA NONOate, 100 μm GSH, or 50 μm CBX. A subset of cells was transfected with siRNA against mouse Panx1 to knock down the endogenous protein. All data are presented as percent change in ATP release compared with control (−thrombin). E, inset, Panx1 Western blot of untransfected and Panx1 siRNA-transfected mAECs. Endothelial NOS (eNOS) and GAPDH were used as loading controls. n values are indicated in parentheses. **, p < 0.001; ***, p < 0.0001.