Endothelial pannexin 1-TRPV4 channel signaling lowers pulmonary arterial pressure in mice

Abstract

Pannexin 1 (Panx1), an ATP-efflux pathway, has been linked with inflammation in pulmonary capillaries. However, the physiological roles of endothelial Panx1 in the pulmonary vasculature are unknown. Endothelial transient receptor potential vanilloid 4 (TRPV4) channels lower pulmonary artery (PA) contractility and exogenous ATP activates endothelial TRPV4 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 increased PA contractility and raised PAP in mice. Flow/shear stress increased ATP efflux through endothelial Panx1 in PAs. 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 in mice.

Keywords: Caveolin 1; Pannexin 1; TRP channel; biochemistry; cell biology; chemical biology; mouse; pulmonary vasculature; purinergic signaling.

Figure 1.. ATP efflux through Panx1_EC ATP activates TRPV4_EC channels in pulmonary arteries (PAs) and lowers pulmonary arterial pressure (PAP).

(A) Left: immunofluorescence images of en face fourth-order PAs from Panx1^fl/fl and Panx1 cKO-EC mice. CD31 immunofluorescence indicates ECs. Center: representative traces showing TRPV4_EC sparklet activity in en face preparations of PAs from Panx1^fl/fl mice in the absence or presence of apyrase (10 U/mL). Dotted lines are quantal levels. Experiments were performed in Fluo-4-loaded PAs in the presence of cyclopiazonic acid (CPA; 20 μmol/L CPA, included to eliminate Ca²⁺ release from intracellular stores). Right: TRPV4_EC sparklet activity (NP_o) per site in en face preparations of PAs from Panx1^fl/fl and Panx1 cKO-EC mice in the presence or absence of apyrase (10 U/mL; n = 5; ***p<0.001 vs. Panx1^fl/fl [-apyrase, 10 U/mL]; ns indicates no statistical significance; t-test). ‘N’ is the number of channels per site and ‘P_O’ is the open state probability of the channel. (B), measurements of ATP (nmol/L) levels in PAs from Panx1^fl/fl, Panx1 cKO-EC, Panx1 cKO-SMC, Trpv4^fl/fl, and Trpv4 cKO-EC mice, and endothelium-denuded PAs from Panx1^fl/fl and Panx1 cKO-SMC mice (n = 5–6; *p<0.05 vs. Panx1 cKO-EC; *p<0.05 vs. Panx1^fl/fl [denuded]; ***p<0.001 vs. Panx1^fl/fl; ***p<0.001 vs. Panx1 cKO-SMC; ns indicates no statistical significance; one-way ANOVA). (C) Average resting right ventricular systolic pressure (RVSP) values in Panx1^fl/fl, Panx1 cKO-EC, and Panx1 cKO-SMC mice (n = 6; ***p<0.001 vs. Panx1^fl/fl; ns indicates no statistical significance; one-way ANOVA). (D) Left grayscale image of a field of view in an en face preparation of Fluo-4-loaded PAs from Panx1^fl/fl and Panx1 cKO-EC mice showing approximately 20 ECs. Dotted outlines indicate an EC (20 μmol/L CPA included to eliminate Ca²⁺ release from intracellular stores). Right: representative traces showing TRPV4_EC sparklet activity in en face preparations of PAs from Panx1^fl/fl and Panx1 cKO-EC mice in response to GSK1016790A (GSK101; 1 nmol/L). Experiments were performed in Fluo-4-loaded PAs in the presence of CPA (20 μmol/L). (E) TRPV4_EC sparklet activity (NP_O) per site and sites per cell in en face preparations of PAs from Panx1^fl/fl and Panx1 cKO-EC mice under baseline conditions (i.e., 20 μmol/L CPA) and in response to 1 nmol/L GSK101 (n = 6; *p<0.05, **p<0.01 vs. Panx1^fl/fl; *p<0.05 vs. Panx1^fl/fl; ns indicates no statistical significance; two-way ANOVA). (F) Left: representative GSK101 (10 nmol/L)-induced outward TRPV4_EC currents in freshly isolated ECs from Panx1^fl/fl and Panx1 cKO-EC mice and effect of GSK2193874 (GSK219, TRPV4 inhibitor, 100 nmol/L) in the presence of GSK101. Currents were elicited by a 200 ms voltage step from –50 mV to +100 mV. Center: scatterplot showing outward currents at +100 mV under baseline conditions, after the addition of GSK101 (10 nmol/L), and after the addition of GSK219 (100 nmol/L; n = 5–6 cells, *p<0.05 vs. Panx1 cKO-EC [+GSK101]; **p<0.01 vs. Panx1 cKO-EC [baseline]; ***p<0.001 vs. Panx1^fl/fl [+baseline]; vs. Panx1^fl/fl [+GSK101]; and Panx1 cKO-EC [+GSK101] vs. Panx1^fl/fl [+GSK101]; two-way ANOVA). Right: scatterplot showing GSK219-sensitive TRPV4_EC currents in response to GSK101 (100 nmol/L; ns indicates no statistical significance; n = 5).

Figure 1—figure supplement 1.. Panx1_SMC mRNA levels in mesenteric arteries from Panx1^fl/fl and Panx1 cKO-SMC mice.

Data presented as a fold change from Panx1^fl/fl (n = 5; ***p<0.001 vs. Panx1^fl/fl; t-test).

Figure 1—figure supplement 2.. TRPV4_EC sparklet activity (NP_O) per site and TRPV4 sparklet sites per cell in en face preparations of pulmonary arteries (PAs) from Panx1^fl/fl and Panx1 cKO-EC mice in response to 30 nmol/L GSK101.

Experiments were performed in Fluo-4-loaded PAs in the presence of cyclopiazonic acid (CPA; 20 μmol/L), included to eliminate Ca²⁺ release from intracellular stores. ‘N’ is the number of channels per site and ‘P_O’ is the open state probability of the channel (n = 6; ns indicates no statistical significance).

Figure 2.. Endothelial Panx1–TRPV4 signaling lowers myogenic and agonist-induced constriction of pulmonary arteries (PAs).

(A) Top: an image showing the left lung and the order system used to isolate fourth-order PAs in this study; bottom: an image of a fourth-order PA cannulated and pressurized at 15 mm Hg. (B) Percentage myogenic constriction of PAs from Trpv4^fl/fl and Trpv4 cKO-EC mice (n = 6; *p<0.05; t-test). (C) Percent constriction of PAs from Trpv4^fl/fl and Trpv4 cKO-EC mice in response to thromboxane A2 receptor agonist U46619 (U466, 1–300 nmol/L; n = 5; *p<0.05 vs. Trpv4^fl/fl [10 nmol/L], **p<0.01 vs. Trpv4^fl/fl [30, 100, and 300 nmol/L]; ##p<0.01 vs. Trpv4^fl/fl; two-way ANOVA). (D) Percentage myogenic constriction of PAs from Panx1^fl/fl and Panx1 cKO-EC mice (n = 6; *p<0.05; t-test). (E) U46619 (U466, 1–300 nmol/L)-induced constriction of PAs from Panx1^fl/fl, Panx1 cKO-EC, and Panx1 cKO-EC mice in the absence or presence of GSK101 (3 nmol/L) (n = 5; **p<0.01 vs. Panx1 cKO-EC, ***p<0.01 vs. Panx1^fl/fl; two-way ANOVA, between groups). (F) Schematic of flow-induced ATP release from isolated and cannulated fourth-order PAs. Shear stress was calculated using the following equation: ${\displaystyle \tau =4(\mu \dot{Q})/(\pi r^3)}$, where μ is viscosity, ${\displaystyle \stackrel{.}{Q}}$ is volumetric flow, and r is internal radius of the vessel. Outflow was collected every 10 min and ATP was measured using Luciferin-Luciferase ATP Bioluminescence Assay. (G) Release of ATP (nmol/L) from PAs of Panx1^fl/fl and Panx1 cKO-EC mice in response to flow/shear stress in the presence of ARL-67156 (ARL; ecto-ATPase inhibitor; 300 μmol/L; 4, 7, and 14 dynes/cm²; n = 6; *p<0.05 vs. Panx1^fl/fl [4 dynes/cm²]; **p<0.01 vs. Panx1^fl/fl [7 dynes/cm²]; ###p<0.001 vs. Panx1 cKO-EC; two-way ANOVA). (H) Release of ATP (nmol/L) from PAs of Trpv4^fl/fl and Trpv4 cKO-EC mice in response to flow/shear stress in the presence of ARL (300 μmol/L; 4, 7, and 14 dynes/cm²; n = 6; *p<0.05 vs. Trpv4^fl/fl [4 dynes/cm²]; #p<0.05 vs. Trpv4 cKO-EC [4 dynes/cm²]; two-way ANOVA).

Figure 2—figure supplement 1.. Percent myogenic constriction in small pulmonary arteries (PAs; 50–100 μm internal diameter) and large PAs (>200 μm internal diameter; n = 6–10; ***p<0.001).

Figure 2—figure supplement 2.. Percent constriction of pulmonary arteries (PAs) from Panx1^fl/fl and Panx1^fl/fl plus apyrase (10 U/mL) mice in response to U46619 (U466; 1–100 nmol/L; n = 5; **p<0.01 vs. Panx1^fl/fl; two-way ANOVA).

Figure 2—figure supplement 3.. Left: representative diameter traces showing ATP (1 μmol/L)-induced dilation of pulmonary arteries (PAs) from Trpv4^fl/fl and Trpv4 cKO-EC mice, pre-constricted with the thromboxane A2 receptor analog U46619 (50 nmol/L).

Fourth-order PAs were pressurized to 15 mm Hg. Center: percent dilation of PAs from Trpv4^fl/fl and Trpv4 cKO-EC mice in response to ATP (1 μmol/L; n = 5–10; ***p<0.001 vs. Trpv4^fl/fl [ATP 1 μmol/L]; t-test). Right: percent dilation of PAs from Panx1^fl/fl and Panx1 cKO-EC mice in response to ATP (1 μmol/L; n = 5–10; ns indicates no statistical significance).

Figure 3.. Endothelial P2Y2R-TRPV4 channel signaling lowers pulmonary artery (PA) contractility and pulmonary arterial pressure (PAP).

(A) Left: immunofluorescence images of en face fourth-order PAs from P2ry2^fl/fl and P2ry2 cKO-EC mice. CD31 immunofluorescence indicates ECs. Right: effects of ATP (1 μmol/L) on TRPV4_EC sparklet activity in the absence or presence of the P2Y1R inhibitor MRS2179 (MRS; 10 μmol/L) or P2Y2R inhibitor AR-C 118925XX (AR-C; 10 μmol/L) in PAs from P2ry2^fl/fl and P2ry2 cKO-EC mice, expressed as NP_O per site (n = 5; ***p<0.001 vs. Control [- ATP]; **p<0.01 vs.+ MRS [- ATP]; ns indicates no statistical significance; two-way ANOVA). ‘N’ is the number of channels per site and ‘P_O’ is the open state probability of the channel. (B) Effects of ATP (1 μmol/L) on TRPV4_EC sparklet activity in the presence of the general P2X1-5/7R inhibitor PPADS (10 μmol/L) and P2X7R inhibitor JNJ-47965567 (JNJ; 1 μmol/L) in PAs of C57BL6/J mice (n = 5; *p<0.05 vs. [-ATP]; one-way ANOVA). (C) Top: representative ATP (10 μmol/L)-induced outward TRPV4 currents in freshly isolated ECs from C57BL6/J mice and the effect of GSK2193874 (GSK219; TRPV4 inhibitor; 100 nmol/L) in the presence of ATP. Currents were elicited by a 200 ms voltage step from –50 mV to +100 mV. Bottom: scatterplot showing outward currents at +100 mV under baseline conditions, after the addition of ATP, and after the addition of GSK219 (100 nmol/L; n = 6 cells; ***p<0.001 vs. baseline; **p<0.01 vs.+ ATP [10 μmol/L]; one-way ANOVA). (D) Left: representative traces showing TRPV4_EC sparklet activity in en face preparations of PAs from P2ry2^fl/fl mice. Dotted lines are quantal levels. Right: TRPV4_EC sparklet activity per site (NP_O) in en face preparations of PAs from P2ry2^fl/fl and P2ry2 cKO-EC mice under baseline conditions (i.e., 20 μmol/L cyclopiazonic acid [CPA]) and in response to 2-thio UTP (P2Y2R agonist, 0.5 μmol/L; n = 5; *p<0.05 vs. P2ry2^fl/fl [-2-thio UTP]; ns indicates no statistical significance; t-test). (E) Left: average resting right ventricular systolic pressure (RVSP) values in P2ry2^fl/fl and P2ry2 cKO-EC mice (n = 6; **p<0.01; t-test). Right: average Fulton index values in P2ry2^fl/fl and P2ry2 cKO-EC mice (n = 5–6; ns indicates no statistical significance). (F) Right: representative diameter traces showing ATP (1 μmol/L)-induced dilation of PAs from P2ry2^fl/fl and P2ry2 cKO-EC mice, pre-constricted with the thromboxane A2 receptor agonist U46619 (U466, 50 nmol/L). Fourth-order PAs were pressurized to 15 mm Hg. Right: percent dilation of PAs from P2ry2^fl/fl and P2ry2 cKO-EC mice in response to ATP (1 μmol/L; n = 5–10; ***p<0.01 vs. P2ry2^fl/fl [ATP 1 μmol/L]; t-test). (G) Percentage myogenic constriction of PAs from P2ry2^fl/fl and P2ry2 cKO-EC mice (n = 5–7; ***p<0.001; t-test). (H) U46619 (U466, 1–300 nmol/L)-induced constriction of PAs from P2ry2^fl/fl, P2ry2 cKO-EC, and P2ry2 cKO-EC mice in the absence or presence of GSK101 (3 nmol/L) (n = 5; ***p<0.001 vs. P2ry2 cKO-EC, ***p<0.001 vs. P2ry2^fl/fl; two-way ANOVA).

Figure 4.. Cav-1_EC provides a signaling scaffold for Panx1_EC–P2Y2R_EC–TRPV4_EC signaling in pulmonary arteries (PAs).

(A) Left: representative traces showing TRPV4_EC sparklets in en face preparations of PAs from Cav1^fl/fl and Cav1 cKO-EC mice in the absence or presence of ATP (1 μmol/L). Dotted lines are quantal levels. Right: TRPV4_EC sparklet activity (NP_O) per site in en face preparations of PAs from Cav1^fl/fl and Cav1 cKO-EC mice in the absence or presence of 1 μmol/L ATP (n = 5; *p<0.05 vs. Cav1^fl/fl [- ATP]; **p<0.01 vs. Cav1^fl/fl [- ATP]; ns indicates no statistical significance; two-way ANOVA). Experiments were performed in Fluo-4-loaded fourth-order PAs in the presence of cyclopiazonic acid (CPA; 20 μmol/L), included to eliminate Ca²⁺ release from intracellular stores. ‘N’ is the number of channels per site and ‘P_O’ is the open state probability of the channel. (B) Percentage dilation of PAs from Cav1^fl/fl and Cav1 cKO-EC mice in response to ATP (1 μmol/L). PAs were pre-constricted with the thromboxane A2 receptor analog U46619 (50 nmol/L; n = 5; ***p<0.01 vs. Cav1^fl/fl; t-test). (C) Top: representative merged images of proximity ligation assays (PLAs) signal, showing EC nuclei and Cav-1_EC:Panx1_EC, Cav-1_EC:P2Y2R_EC, and Cav-1_EC:TRPV4_EC co-localization (white puncta) in fourth-order PAs from Cav1^fl/fl and Cav1 cKO-EC mice. Bottom: quantification of Cav-1_EC:Panx1_EC, Cav-1_EC:P2Y2R_EC, and Cav-1_EC:TRPV4_EC co-localization in PAs from Cav1^fl/fl and Cav1 cKO-EC mice (n = 5; ***p<0.001 vs. Cav1^fl/fl; t-test). (D) Representative PLA images showing EC nuclei, TRPV4_EC:P2Y2R_EC and Panx1_EC:P2Y2R_EC co-localization (white puncta) in fourth-order PAs from Cav1^fl/fl and Cav1 cKO-EC mice. Bottom: quantification of TRPV4_EC:P2Y2R_EC and Panx1_EC:P2Y2R_EC co-localization in PAs from Cav1^fl/fl and Cav1 cKO-EC mice (n = 5; ***p<0.001 vs. Cav1^fl/fl; t-test).

Figure 4—figure supplement 1.. Representative proximity ligation assay (PLA) images showing EC nuclei, TRPV4_EC:P2Y2R_EC and Panx1_EC:P2Y2R_EC co-localization in fourth-order pulmonary arteries (PAs) from P2ry2 cKO-EC mice.

Figure 4—figure supplement 2.. Left: representative proximity ligation assay (PLA) images showing EC nuclei and Cav-1_EC:P2Y1_EC co-localization in fourth-order pulmonary arteries (PAs) from Cav1^fl/fl mice.

Right: quantification of Cav-1_EC:P2Y1_EC co-localization in PAs from Cav1^fl/fl mice (n = 5).

Figure 5.. ATP activates TRPV4_EC channels via phospholipase C–diacylglycerol–protein kinase C (PLC–DAG–PKC) signaling in pulmonary arteries (PAs).

(A) Left: representative traces showing TRPV4_EC sparklet activity in en face preparations of PAs from C57BL6/J mice before and after treatment with ATP (1 μmol/L). Right: effects of U73122 (PLC inhibitor; 3 μmol/L) or Gö-6976 (PKCα/β inhibitor; 1 μmol/L) on TRPV4_EC sparklet activity in en face preparations of PAs from C57BL6/J mice before and after treatment with ATP (1 μmol/L), expressed as NP_O per site. Experiments were performed in Fluo-4-loaded fourth-order PAs in the presence of cyclopiazonic acid (CPA; 20 μmol/L), included to eliminate Ca²⁺ release from intracellular stores (n = 5; *p<0.05 vs. Control [-ATP]; ns indicates no statistical significance; one-way ANOVA). ‘N’ is the number of channels per site and ‘P_O’ is the open state probability of the channel. Dotted lines indicate quantal levels. (B) Left: representative traces showing TRPV4_EC sparklet activity in en face preparations of PAs from C57BL6/J mice in the absence or presence of OAG (DAG analog; 1 μmol/L). Right: effects of U73122 (3 μmol/L) or Gö-6976 (1 μmol/L) on TRPV4_EC sparklet activity in en face preparations of PAs from C57BL6/J mice before and after treatment with OAG (1 μmol/L, n = 6; ^**p<0.01 vs. Control [-OAG]; ^**p<0.01 vs. U73122 [-OAG]; ns indicates no statistical significance; one-way ANOVA). (C) Left: representative traces showing TRPV4_EC sparklets in en face preparations of PAs from C57BL6/J mice in the absence or presence of phorbol myristate acetate (PMA) (PKC activator; 10 nmol/L). Right: effects of U73122 (3 μmol/L) or Gö-6976 (1 μmol/L) on TRPV4_EC sparklet activity in en face preparations of PAs from C57BL6/J mice before and after treatment with PMA (n = 6; *p<0.05 vs. Control [-PMA]; *p<0.05 vs. U73122 [-PMA]; ns indicates no statistical significance; one-way ANOVA). (D) Top: representative traces showing TRPV4_EC sparklet activity in en face preparations of PAs from Cdh5-optoα1AR (adrenergic receptor) mouse before and after light activation (470 nm). Center: scatterplot showing TRPV4 sparklet activity before and after light activation in the absence or presence of PKCα/β inhibitor Gö-6976 (1 μmol/L, n = 4, ***p<0.01 vs. –Gö-6976 [before]; ns indicates no statistical significance; one-way ANOVA). Bottom: scatterplot showing TRPV4 sparklet activity, expressed as sparklet sites per cell, before and after light activation, in the absence or presence of PKCα/β inhibitor Gö-6976 (1 μmol/L; n = 4; ***p<0.001 vs. –Gö-6976 [before]; ns indicates no statistical significance; one-way ANOVA).

Figure 5—figure supplement 1.. A multi-Gaussian to all-points histogram obtained using sparklet traces from X-Rhod-1-loaded pulmonary arteries (PAs), showing quantal (evenly spaced) ΔF/F₀ levels of 0.21.

Figure 5—figure supplement 2.. Left: scatterplot showing TRPV4 sparklet activity, expressed as NP_O per site, before and after light activation, in the presence of TRPV4 inhibitor GSK2193874 (GSK219; 100 nmol/L, n = 4).

‘N’ is the number of channels per site and ‘P_O’ is the open state probability of the channel. Right: scatterplot showing TRPV4 sparklet activity, expressed as sparklet sites per cell, before and after light activation, in the presence of TRPV4 inhibitor GSK219 (100 nmol/L, n = 5; ns indicates no statistical significance).

Figure 6.. Localization of PKCα with Cav-1_EC increases the activity of TRPV4_EC channels in pulmonary arteries (PAs).

(A) Top: representative merged images of proximity ligation assays (PLAs) showing endothelial cell (EC) nuclei and Cav-1_EC:PKC co-localization (white puncta) in fourth-order PAs from Cav1^fl/fl and Cav1 cKO-EC mice. Bottom: quantification of Cav-1_EC:PKC co-localization in PAs from Cav1^fl/fl and Cav1 cKO-EC mice (n = 5; ***p<0.001 vs. Cav1^fl/fl; t-test). (B) Representative traces showing TRPV4 currents in the absence or presence of Gö-6976 (PKC inhibitor; 1 μmol/L) in HEK293 cells transfected with TRPV4 alone or co-transfected with TRPV4 plus wild-type Cav-1, recorded in the whole-cell patch-clamp configuration. (C) Current density scatterplot of TRPV4 currents at +100 mV in the absence or presence of Gö-6976 (1 μmol/L) and after the addition of GSK2193874 (GSK219; TRPV4 inhibitor; 100 nmol/L) in HEK293 cells transfected with TRPV4 alone or TRPV4 plus wild-type Cav-1 (n = 5; **p<0.01 vs. Control [TRPV4]; **p<0.01 vs. Control [TRPV4+ Cav-1]; ns indicates no statistical significance; one-way ANOVA). (D) Current density plot of TRPV4 currents at +100 mV in HEK293 cells transfected with TRPV4+ PKCα or TRPV4+ PKCβ and in the presence of GSK219 (100 nmol/L; n = 5; ***p<0.001 vs. TRPV4+ PKCα; t-test). (E) Schematic depiction of the Panx1_EC–P2Y2R_EC–TRPV4_EC signaling pathway that promotes vasodilation and lowers pulmonary arterial pressure (PAP) in PAs. ATP released from Panx1_EC activates P2Y2R_EC purinergic receptors on the EC membrane. Stimulation of P2Y2R_EC recruits PKCα, which anchors to the scaffolding protein Cav-1_EC in close proximity to TRPV4_EC channels. TRPV4_EC channel-dependent vasodilation lowers PAP.