Fig. 5.

Endothelial iNOS/NOX1-generated PN impairs TRPV4_EC channel activity in PH. (A, Left) Representative nonconfocal images for coumarin boronic acid (CBA, PN indicator) fluorescence in ECs from PAs of mice exposed to N, CH (3 wk; 10% O₂), or Su+CH. (Right) Scatterplot of CBA fluorescence intensity in en face preparations of fourth-order PAs from mice exposed to N, CH, or Su+CH (n = 5; ***P < 0.001 versus N; one-way ANOVA). Experiments were performed in the presence of PEG-catalase (H₂O₂ metabolizing enzyme; 500 U/mL) and taurine (hypochlorous acid-lowering agent; 1 mmol/L). (B) TRPV4_EC sparklet traces in one EC from fluo-4–loaded en face PAs (Left) and sparklet activity per site (Right; expressed as NP_O per site) in the absence and presence of the 1400W (iNOS inhibitor; 1 μmol/L) in PAs from C57BL6/J mice exposed to Su+CH (n = 5; **P < 0.01; t test). (C) iNOS mRNA levels in ECs from PAs of N and Su+CH mice, expressed relative to N mice (n = 5; *P < 0.05; t test). (D) Averaged resting RVSP (millimeters of mercury) values in WT and iNOS^−/− mice after exposure to Su+CH (n = 5 to 6; *P < 0.05; t test). (E) TRPV4_EC sparklet activity in PAs from WT and iNOS^−/− mice exposed to Su+CH and iNOS^−/− mice exposed to N (n = 5; * P < 0.05; two-way ANOVA). (F) GSK101 (3–30 nmol/L)-induced vasodilation of PAs from WT and iNOS^−/− mice exposed to Su+CH (n = 5; ***P < 0.001 versus WT [10 nmol/L]; two-way ANOVA). (G) Effect of the NOXA1ds (NOX1 inhibitor, 1 μmol/L) on GSK101 (10 nmol/L)-induced TRPV4_EC sparklet activity in PAs from PAH patients (n = 3; *P < 0.05; t test). (H) Effect of the 1400W (1 μmol/L) on TRPV4_EC sparklet activity in PAs from PAH patients (n = 3; *P < 0.05; t test).