Endothelial Pannexin 1–TRPV4 channel signaling lowers pulmonary arterial pressure

Pannexin 1 (Panx1) is an ATP-efflux channel that controls endothelial function in the systemic circulation. However, the roles of endothelial Panx1 in resistance-sized pulmonary arteries (PAs) are unknown. Extracellular ATP dilates PAs through activation of endothelial TRPV4 (transient receptor potential vanilloid 4) ion channels. We hypothesized that endothelial Panx1–ATP– TRPV4 channel signaling promotes vasodilation and lowers pulmonary arterial pressure (PAP). Endothelial, but not smooth muscle, knockout of Panx1 or TRPV4 increased PA contractility and raised PAP. Panx1-effluxed extracellular ATP signaled through purinergic P2Y2 receptor (P2Y2R) to activate protein kinase Cα (PKCα), which in turn activated endothelial TRPV4 channels. Finally, caveolin-1 provided a signaling scaffold for endothelial Panx1, P2Y2R, PKCα, and TRPV4 channels in PAs, promoting their spatial proximity and enabling signaling interactions. These results indicate that endothelial Panx1–P2Y2R–TRPV4 channel signaling, facilitated by caveolin-1, reduces PA contractility and lowers PAP.


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Panx1EC-generated eATP acts through purinergic P2Y2REC stimulation to activate 134 TRPV4EC channels. 135 Bioluminescence measurements confirmed lower baseline eATP levels in PAs from 136 Panx1EC -/mice compared with PAs from Panx1 fl/fl mice ( Fig. 2A), supporting an essential role for 137 Panx1EC channels as an eATP-release mechanism in PAs. PAs from TRPV4EC -/mice, however, 138 exhibited unaltered basal eATP levels. eATP was recently identified as a novel endogenous 139 activator of TRPV4EC channels in the pulmonary circulation 9 . Therefore, we tested whether 140 Panx1EC activates TRPV4EC channels via eATP release. Addition of the eATP-hydrolyzing 141 enzyme apyrase (100 U/mL) reduced the activity of TRPV4EC sparklets in PAs from control mice 142 but not those from Panx1EC -/mice (Fig. 2B), confirming the role of Panx1EC-mediated eATP in 143 TRPV4EC channel activation.

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Similar to TRPV4EC -/and Panx1EC -/mice, P2Y2REC -/mice also showed elevated RVSP 159 and an unaltered Fulton Index (Fig. 2G). Taken together, these findings demonstrate that P2Y2REC  Cav-1EC provides a scaffold for Panx1EC-P2Y2REC-TRPV4EC signaling. 163 Cav-1EC, an essential structural protein in the pulmonary circulation 21,22,30 , has been shown 164 to co-localize with Panx1, P2Y2R, and TRPV4 channels in multiple cell types [17][18][19]31 . Therefore, 165 we hypothesized that Cav-1EC provides a signaling scaffold that supports and maintains the spatial 166 proximity among the individual elements in the Panx1EC-P2Y2REC-TRPV4EC pathway. To clearly 167 delineate the role of Cav-1EC in Panx1EC-dependent signaling, we utilized inducible, endothelium-168 specific Cav-1-knockout mice (Cav-1EC -/-; Fig. 3A and B). The loss of Cav-1EC resulted in elevated 169 RVSP in the absence of right ventricular hypertrophy (Fig. 3C), indicating a crucial role of Cav-1EC 170 in maintaining a low resting PAP. Baseline TRPV4EC sparklet activity and activity induced by a 171 low level of GSK101 (1 nmol/L) were reduced in PAs from Cav-1EC -/mice (Fig. 3D). However,  Cav-1EC provides a signaling scaffold that ensures spatial proximity among the elements of the 225 Panx1EC-P2Y2REC-PKCα-TRPV4EC pathway. Our findings reveal a novel signaling axis that can 226 be engaged by physiological stimuli to lower PAP and could also be therapeutically targeted in 227 pulmonary vascular disorders. Moreover, the conclusions in this study may assist in future 228 investigations of the mechanisms underlying pulmonary endothelial dysfunction.

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Both ECs and SMCs control vascular contractility and arterial pressure. The expression of 230 Panx1 and TRPV4 channels in both ECs and SMCs 8, 18, 35-37 makes it challenging to decipher the 231 cell type-specific roles of Panx1 and TRPV4 channels using global knockouts or pharmacological 232 strategies. Therefore, studies utilizing EC-or SMC-specific knockout mice are necessary for a 233 definitive assessment of the control of PAP by EC and SMC Panx1 and TRPV4 channels.

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Although SMC TRPV4 channels have been shown to contribute to hypoxia-induced pulmonary 235 vasoconstriction, resting PAP is not altered in global TRPV4 -/mice 38, 39 . Further, our studies 236 indicate that SMC Panx1 and TRPV4 channels do not influence resting PAP. Taken together with 237 findings from EC-knockout mice, these results provide strong evidence that endothelial, but not 238 SMC, Panx1 and TRPV4 channels maintain low PA contractility and PAP under basal conditions.

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Recent studies in pulmonary fibroblasts and other cell types suggest that TRPV4 channel-240 mediated increases in cytosolic Ca 2+ can induce eATP release through Panx1 40, 41 . However, the 241 reverse interaction, in which Panx1-mediated eATP release activates TRPV4 channels, has not 242 been explored in any cell type. Since Panx1 is activated by cytosolic Ca 2+ 42 and eATP has been 243 previously shown to activate TRPV4EC channels 9 , bidirectional signaling between Panx1 and 244 TRPV4 channels is conceivable. However, our demonstration that baseline eATP levels are 245 unchanged in PAs from TRPV4EC -/mice rules out a role for TRPV4EC channels in controlling 246 eATP release under baseline conditions. Nevertheless, these data from pulmonary ECs do not rule 247 out potential TRPV4-Ca 2+ -Panx1 signaling in other cell types. Panx1EC-TRPV4EC channel signaling in PAs, but whether such signaling operates in the capillary 256 endothelium and is essential for its physiological function is unclear.

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Purinergic signaling and the endogenous purinergic receptor agonist eATP are essential 258 controllers of pulmonary vascular function 13,26,28,47 . Our discovery of the Panx1EC-P2Y2REC-259 TRPV4EC pathway establishes a signaling axis in ECs that regulates pulmonary vascular function.

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The pulmonary vasculature is a high-flow circulation, and pulmonary ECs have been shown to 261 release eATP in response to flow/shear stress 12 . Therefore, flow/shear stress could be a potential    In conclusion, Panx1EC-TRPV4EC signaling reduces PA contractility and maintains a low 297 resting PAP. This mechanism is facilitated by eATP released through Panx1EC and subsequent 298 activation of P2Y2REC-PKCα signaling. Cav-1EC ensures the spatial proximity among Panx1EC, 299 P2Y2REC, and TRPV4EC channels and also anchors PKCα close to TRPV4EC channels. These 300 findings identify a novel endothelial Ca 2+ signaling mechanism that reduces PA contractility.   MgCl2 hexahydrate, 2.5 CaCl2 dihydrate, 7 dextrose, and 24 NaHCO3) at 37°C and bubbled with 399 20% O2/5% CO2 to maintain the pH at 7.4. All drug treatments were added to the superfusing PSS.  PAs).

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Total number of sparklet sites in a field was divided by the number of cells in that field to obtain 458 sparklet sites per cell.

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Louis, MO, USA) was followed for the detection of co-localized proteins. Lastly, PAs were  Plasmid generation and transfection into HEK293 cells.

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The TRPV4 coding sequence without stop codons was amplified from mouse heart cDNA. The  The authors have no conflicts to disclose.