Fig. 2.

Cav-1_EC–TRPV4_EC signaling in PAs lowers resting PAP. (A) Cav-1_EC immunofluorescence in en face preparations of fourth-order PAs. (B) Cav-1_EC mRNA levels in PAs relative to those in Cav-1^fl/fl mice (n = 4; ***P < 0.001; t test). (C) Averaged resting RVSP (Left; n = 5 to 9; *P < 0.05; t test) and PAP values (Right; n = 8 to 11; *P < 0.05; t test). (D) TRPV4_EC sparklet traces in one EC from a fluo-4–loaded en face PAs in response to TRPV4 channel activator GSK101 (10 nmol/L) in Cav-1^fl/fl and Cav-1_EC^−/− mice. (E, Left) Baseline or GSK101-induced (10 to 30 nmol/L) TRPV4_EC sparklet activity, expressed as NP_O per site (n = 5; *P < 0.05 versus Cav-1^fl/fl [Baseline]; ***P < 0.001 versus Cav-1^fl/fl [10 nmol/L]; ns indicates no significance; two-way ANOVA). Experiments were performed in fluo-4–loaded fourth-order PAs in the presence of cyclopiazonic acid (CPA; 20 μmol/L) to eliminate Ca²⁺ release from intracellular stores. (Right) TRPV4_EC sparklet sites per cell in PAs from Cav-1^fl/fl or Cav-1_EC^−/− mice (n = 5; *P < 0.05 versus Cav-1^fl/fl [Baseline]; ***P < 0.001 versus Cav-1^fl/fl [10 nmol/L]; two-way ANOVA). (F) Representative GSK101 (10 nmol/L)-induced outward TRPV4_EC currents in freshly isolated ECs from Cav-1^fl/fl or Cav-1_EC^−/− mice and effect of GSK2193874 (GSK219; 100 nmol/L) in the presence of GSK101 (10 nmol/L). (G) Scatterplot showing TRPV4_EC currents in the presence of GSK101 (10 and 100 nmol/L; n = 5; ***P < 0.001 versus Cav-1^fl/fl [10 nmol/L]; two-way ANOVA). (H, Left) Representative traces showing TRPV4 currents in the absence or presence of Gö-6976 (PKC-α/β inhibitor; 1 μmol/L) in HEK293 cells transfected with TRPV4 only or cotransfected with TRPV4 plus WT Cav-1, recorded in the whole-cell patch-clamp configuration. (Right) Current density plot of TRPV4 currents at +100 mV in the absence or presence of Gö-6976 (1 μmol/L) in HEK293 cells transfected with TRPV4 or TRPV4 + Cav-1 (n = 5; **P < 0.01 versus TRPV4 [−Gö-6976] and TRPV4 + Cav-1 [−Gö-6976]; two-way ANOVA). (I) Effect of Gö-6976 (1 μmol/L) on TRPV4_EC sparklet activity in en face preparations of PAs from Cav-1^fl/fl and Cav-1_EC^−/− mice, expressed as NP_O per site (n = 5; **P < 0.01 versus Cav-1^fl/fl [−Gö-6976]; two-way ANOVA). (J) Percent dilation of PAs in response to GSK101 (3 to 30 nmol/L; n = 6; *** P < 0.001 versus Cav-1^fl/fl [10 nmol/L]; two-way ANOVA). (K) Percent constriction of PAs in response to the thromboxane A2 receptor agonist U46619 (1 to 300 nmol/L; n = 5; *P < 0.05 versus Cav-1^fl/fl [10 and 30 nmol/L]; **P < 0.01 versus Cav-1^fl/fl [100 and 300 nmol/L]; two-way ANOVA).