Regulation of Pulmonary Arterial Pressure by Endothelial Pannexin1–TRPV4 Channel Signaling

Abstract

Endothelial cells (EC) dilate small pulmonary arteries (PAs) and lower the resting pulmonary arterial pressure (PAP). Moreover, the loss of endothelial function is a key contributor to elevated PAP in pulmonary hypertension. Therefore, an in-depth understanding of endothelial mechanisms controlling PAP is necessary for designing novel therapeutic strategies against pulmonary hypertension. Pannexin 1 (Panx1), an ATP efflux pathway on the EC membranes, has emerged as a crucial controller of endothelial function. However, the impact of endothelial Panx1 (Panx1EC) on PA contractility and PAP is not known. Recent studies support the idea that endothelial transient receptor potential vanilloid 4 (TRPV4EC) channels dilate small PAs, and are activated by extracellular ATP (eATP). Since Panx1EC is an ATP efflux pathway, we hypothesized that Panx1EC-TRPV4EC channel signaling dilates PAs and lowers resting PAP. Right ventricular systolic pressure (RVSP), an indirect indicator of PAP, was higher in inducible, EC-specific Panx1 (Panx1EC-/-) and TRPV4 channel knockout (TRPV4EC-/-) mice compared to the respective control mice. The activity of TRPV4EC channels was recorded as individual Ca2+ influx signals (TRPV4EC sparklets) in fluo 4-loaded en face PAs using spinning disk confocal imaging. The TRPV4EC sparklet activity was lower in PAs from Panx1EC-/- mice than the control mice, identifying Panx1EC as a novel promoter of TRPV4EC channel activity. Apyrase (10 U/mL), an ATP hydrolyzing enzyme, also reduced the activity of TRPV4EC sparklets in PAs from control mice but not Panx1EC-/- mice. Pressurized PAs (15 mm Hg) from Panx1EC-/- and TRPV4EC-/- mice showed higher constriction to thromboxane A2 receptor agonist U46619 (1-300 nM) compared to PAs from the respective control mice. These data indicated that Panx1EC-eATP-TRPV4EC signaling lowers PA contractility and PAP. Consistent with previous findings that eATP dilates PAs via purinergic P2Y2 receptor (P2Y2R) signaling, ATP activation of TRPV4EC channels was lost in PAs from inducible P2Y2EC-/- mice. Together, these data identify a novel Panx1EC-eATP-P2Y2REC signaling pathway that promotes TRPV4EC channel activity in PAs. Panx1, P2Y2R, and TRPV4 channels have been shown to localize with the scaffolding protein caveolin 1 (Cav-1), raising the possibility that Cav-1EC may provide a signaling scaffold for Panx1EC-P2Y2REC-TRPV4EC signaling in PAs. Accordingly, P2Y2REC-activation of TRPV4EC channels was lost in PAs from inducible Cav-1EC-/- mice, and these mice showed higher resting RVSPs compared to the control mice. Additionally, in situ proximity ligation assay confirmed the nanometer proximity of Cav-1EC with Panx1EC, P2Y2REC, and TRPV4EC channel in PAs. Overall, our findings identify Cav-1EC-dependent Panx1EC-P2Y2REC-TRPV4EC channel pathway that dilates PAs and lowers PAP. Targeting individual elements of this pathway may provide novel therapeutic options in pulmonary vascular disorders.