Interaction Between Pannexin 1 and Caveolin-1 in Smooth Muscle Can Regulate Blood Pressure

Abstract

Objective- Sympathetic nerve innervation of vascular smooth muscle cells (VSMCs) is a major regulator of arteriolar vasoconstriction, vascular resistance, and blood pressure. Importantly, α-adrenergic receptor stimulation, which uniquely couples with Panx1 (pannexin 1) channel-mediated ATP release in resistance arteries, also requires localization to membrane caveolae. Here, we test whether localization of Panx1 to Cav1 (caveolin-1) promotes channel function (stimulus-dependent ATP release and adrenergic vasoconstriction) and is important for blood pressure homeostasis. Approach and Results- We use in vitro VSMC culture models, ex vivo resistance arteries, and a novel inducible VSMC-specific Cav1 knockout mouse to probe interactions between Panx1 and Cav1. We report that Panx1 and Cav1 colocalized on the VSMC plasma membrane of resistance arteries near sympathetic nerves in an adrenergic stimulus-dependent manner. Genetic deletion of Cav1 significantly blunts adrenergic-stimulated ATP release and vasoconstriction, with no direct influence on endothelium-dependent vasodilation or cardiac function. A significant reduction in mean arterial pressure (total=4 mm Hg; night=7 mm Hg) occurred in mice deficient for VSMC Cav1. These animals were resistant to further blood pressure lowering using a Panx1 peptide inhibitor Px1IL2P, which targets an intracellular loop region necessary for channel function. Conclusions- Translocalization of Panx1 to Cav1-enriched caveolae in VSMCs augments the release of purinergic stimuli necessary for proper adrenergic-mediated vasoconstriction and blood pressure homeostasis.

Keywords: Pannexin 1; adrenergic agents; blood pressure; caveolae; muscle, smooth.

Figure 1. Pannexin 1 and caveolin-1 only associate after phenylephrine stimulation

(A) Confocal images and line scan analysis of cultured human VSMCs expressing Panx1-RFP and caveolin-1 GFP following phenylephrine stimulation. Scale bar; 50 μm (low-magnification) and 10 μm (high-magnification) (B) Co-localization analysis of continuous time lapse confocal imaging in human VSMCs throughout acute stimulation (total time= 3 min) with vehicle control (black line; n=11), 500 μmol/L ATP control (red line; n=5), or 100 μmol/L phenylephrine (green line; n=10). *p < 0.05 compared to vehicle control using two-way ANOVA with Dunnett’s posthoc. (C) Heat map representation of percent fluorescence co-distribution using Mander’s coefficient analysis and normalized to baseline (green=positive association; red=negative association). (D) Membrane fractionation and western analysis of VSMCs showing endogenous distribution of Panx1 and caveolin-1 in membrane fractions. (E) Subcellular distribution of caveolin-1 enriched membrane domains in sodium carbonate-based detergent-free cellular fractionation using a discontinuous sucrose gradient (5%–40%), analyzed by immunoblot. Panx1 co-fractionates with caveolin-1 in lipid light rafts at the plasma membrane. (F) Co-immunoprecipitation and quantification of Panx1 and caveolin-1 from subcellular plasma membrane domains was measured following phenylephrine stimulation, n=5. Data analyzed by student’s t-test and presented as mean ± SEM. * p < 0.05.

Figure 2. Pannexin1 and caveolin-1 localize to the plasma membrane near sympathetic nerves

En face immunofluorescence detection of Panx1 and caveolin-1 using proximity ligation assay (PLA) on intact TDA. Sympathetic nerves were labeled with tyrosine hydroxylase (green) and nuclei were labeled with DAPI (blue). (A) Control IgG (rabbit) staining. (B) PLA secondary probe control. (C) Lack of positive PLA between Panx1 and caveolin-1 in VSMCs at baseline conditions. (D–E) Panx1 and caveolin-1 cluster at areas of sympathetic innervation following acute (1 min) phenylephrine treatment (20 μmol/L). Positive PLA amplification is visualized as red punctate spots (white arrows). (E) Quantification of PLA signal between Panx1 and caveolin-1 before and after treatment with phenylephrine. N=3 animals per treatment group. Scale bar; 50 μm.

Figure 3. Inducible deletion of caveolin-1 from smooth muscle cells functionally mimics blunted adrenergic-mediated ATP release in Pannexin 1 deletion

(A) Inducible SMMHC-CreERT2+/Cav1fl/fl (SMC-Cav1fl/fl) mice were injected with tamoxifen (1 mg/kg) to delete caveolin-1 (SMC-Cav1Δ/Δ). (B) Agarose gel from genomic DNA showing Cre-mediated recombination at loxP site in tamoxifen treated mice. (C) Immunostaining of transverse sections of TDAs with anti-caveolin-1 (red), internal elastic lamina (gray), α-SMactin (Acta2), or CD-31 (Pecam1) (green). Nuclei are stained with DAPI (blue). *indicates vessel lumen. Scale bar; 20 μm. Arrows in high magnification indicate endothelial cells. (D) Quantification of caveolin-1 deletion from VSMCs normalized to α-SMactin positive area; n=6 mice. Students t-test was performed, significance indicated by asterisk ***p < 0.001. (E) ATP release from intact TDAs in response to adrenergic vasoconstrictors: phenylephrine (PE; 20 μmol/L) and norepinephrine (NE; 20 μmol/L), or non-adrenergic vasoconstrictors: serotonin (5-HT; 40 nmol/L) and endothelin-1 (ET-1; 40 nmol/L). n = 4 mice. Data displayed as groups and represented as mean ± SEM. Two-way ANOVA and Tukey’s posthoc test was performed for significance; *p < 0.05.

Figure 4. Effects of vascular smooth muscle cell caveolin-1 deletion on vasoconstriction and vasodilation responses in resistance arteries

(A) Contractile responses to increasing concentrations of phenylephrine in TDAs from SMC-Cav1fl/fl control mice (black line; N=4 mice (7 arteries)) and SMC-Cav1Δ/Δ tamoxifen-treated mice (red line; N=6 mice (8 arteries). (B) Effects of VSMC caveolin-1 deletion on endothelial-dependent vasodilation to increasing concentrations of acetylcholine. Concentration-effect curves were fitted to the data using four-parameter, non-linear regression curve. Data assessed by two-way ANOVA with Bonferroni post-hoc test for multiple comparisons. **p < 0.01 ***p < 0.001.

Figure 5. VSMC Caveolin-1 deletion reduces blood pressure

(A) 24 hour mean arterial blood pressure (MAP) of mice across the indicated genotypes. (B) Differences in 24 hour MAP (ΔMAP) across the indicated groups of mice after treatment with tamoxifen or vehicle control. (C) MAP during the nocturnal active period (12 hr dark: 6:00PM–5:59AM), and (D) MAP during the daytime inactive period (12 hr light: 6:00AM–5:59PM). Baseline measurements were made for each individual animal before injections and compared to BP responses 2 weeks after tamoxifen or vehicle control injection. N=4 mice for each treatment group. *p < 0.05, **p < 0.01, ***p<0.001 compared to baseline response using one-way ANOVA (B) or two-way ANOVA (A, C, D), with Tukey’s posthoc test.

Figure 6. VSMC caveolin-1 deletion does not influence cardiac function

Representative transverse and sagittal MRI images of SMC-Cav1^fl/fl control and SMC-Cav1^Δ/Δ hearts. Six short-axis slices were acquired from base to apex, with slice thickness equal to 1mm, in-plane spatial resolution of 0.2 × 0.2 mm², and temporal resolution of 8–12 ms. No differences in size, morphology, or function were detected as summarized in Table 1.

Figure 7. Caveolin-1 deletion prevents the blood pressure-lowering effects of the Panx1 inhibitory peptide (PxIL2P)

Differences in MAP (ΔMAP) measured at baseline and 2 hrs after treatment with the Panx1 inhibitory peptide PxIL2P (20 mg/kg) or scramble control (20 mg/kg) in SMC-Cav1^fl/fl, SMC-Cav1^Δ/Δ, or C57BL/6 mice. Changes in MAP were calculated using each animal’s individual baseline pretreatment. n=4 mice for each treatment group; *p< 0.05 and **p< 0.01 compared to individual baseline response using two-way ANOVA and Tukey’s test.