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. 2023 Feb;80(2):416-425.
doi: 10.1161/HYPERTENSIONAHA.122.20280. Epub 2022 Nov 30.

Amount of Pannexin 1 in Smooth Muscle Cells Regulates Sympathetic Nerve-Induced Vasoconstriction

Luke S Dunaway  1 , Marie Billaud  2 , Edgar Macal  1 , Miranda E Good  3 , Christopher B Medina  4   5 , Ulrike Lorenz  5   6 , Kodi Ravichandran  6   7 , Michael Koval  8   9 , Brant E Isakson  1   10
Affiliations

Amount of Pannexin 1 in Smooth Muscle Cells Regulates Sympathetic Nerve-Induced Vasoconstriction

Luke S Dunaway et al. Hypertension. 2023 Feb.

Abstract

Background: Panx1 (pannexin 1) forms high conductance channels that secrete ATP upon stimulation. The role of Panx1 in mediating constriction in response to direct sympathetic nerve stimulation is not known. Additionally, it is unknown how the expression level of Panx1 in smooth muscle cells (SMCs) influences α-adrenergic responses. We hypothesized that the amount of Panx1 in SMCs dictates the levels of sympathetic constriction and blood pressure.

Methods: To test this hypothesis, we used genetically modified mouse models enabling expression of Panx1 in vascular cells to be varied. Electrical field stimulation on isolated arteries and blood pressure were assessed.

Results: Genetic deletion of SMC Panx1 prevented constriction by electric field stimulation of sympathetic nerves. Conversely, overexpression of Panx1 in SMCs using a ROSA26 transgenic model increased sympathetic nerve-mediated constriction. Connexin 43 hemichannel inhibitors did not alter constriction. Next, we evaluated the effects of altered SMC Panx1 expression on blood pressure. To do this, we created mice combining a global Panx1 deletion, with ROSA26-Panx1 under the control of an inducible SMC specific Cre (Myh11). This resulted in mice that could express only human Panx1, only in SMCs. After tamoxifen, these mice had increased blood pressure that was acutely decreased by the Panx1 inhibitor spironolactone. Control mice genetically devoid of Panx1 did not respond to spironolactone.

Conclusions: These data suggest Panx1 in SMCs could regulate the extent of sympathetic nerve constriction and blood pressure. The results also show the feasibility humanized Panx1-mouse models to test pharmacological candidates.

Keywords: blood pressure; pannexin; smooth muscle; spironolactone; sympathetic nerve.

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Figures

Figure 1:
Figure 1:. Over-expression of Panx1 in smooth muscle cells increases vasoconstriction after sympathetic nerve stimulation.
(A-B) Western blot and quantification of Panx1 abundance in mesenteric arteries of iSMC Panx1 OE and TG Tamox Control mice * p<0.05. (C) EFS-induced constriction of third order mesenteric arteries from Panx1fl/fl, iEC Panx1 KO, and iSMC Panx1 KO mice. **p<0.01, ***p<0.001, **** p<0.0001 Panx1fl/fl vs iSMC Panx1 KO. Data were analyzed by repeated measures two-way ANOVA and Sidak post hoc test.
Figure 2:
Figure 2:. Increased ATP release from mice over-expressing Panx1 in smooth muscle cells.
(A) ATP release from third order mesenteric arteries after stimulation with 10 μM phenylephrine. Data were analyzed by one-way ANOVA and Holm-Sidak post hoc test. ** p<0.01; *** p<0.0001 (B-E) EFS-induced constriction of third order mesenteric arteries with or without apyrase (50 U/mL). Data were analyzed by repeated measures two-way ANOVA and Sidak post hoc test. *p<0.05, **p<0.01, ***p<0.001, **** p<0.0001
Figure 3:
Figure 3:. Pharmacological inhibition of Panx1 inhibits sympathetic nerve induced vasoconstriction.
(A-C) EFS-induced vasoconstriction with or without probenecid (2 mM) (D-F) EFS-induced vasoconstriction with or without spironolactone (20 μM). Data were analyzed by repeated measures two-way ANOVA and Sidak post hoc test. *p<0.05, **p<0.01, ***p<0.001, **** p<0.0001
Figure 4:
Figure 4:. Peptide inhibition of Panx1, but not Cx43, inhibits sympathetic nerve vasoconstriction.
(A-C) EFS-induced constriction with or without the Panx1 inhibitor, PxIL2P (20 μM) or (D-F) EFS-induced constriction with or without the Cx43 inhibitor, Gap19 (20 μM). Data were analyzed by repeated measures two-way ANOVA and Sidak post hoc test. **p<0.01, ***p<0.001, **** p<0.0001
Figure 5:
Figure 5:. Overexpression of human Panx1 in SMC alone increases blood pressure and is rescued by acute injection of spironolactone.
Blood pressure was measured in conscious, unrestrained mice by radiotelemetry. Shown are blood pressures recorded at baseline, after treatment with tamoxifen or vehicle (induction), or 30 minutes after acute treatment with 40 mg/kg spironolactone via intraperitoneal injection. (A) Mice with baseline level of Panx1 (yellow, TG Geno Control; brown, TG Tamox Control) and after induction, had over-expression of Panx1 (red, iSMC Panx1 OE). (B) Mice were baseline lacking Panx1 globally (white, Panx1KO; yellow Panx1KO-TG Geno Control; brown, Panx1KO-TG Tamox Control) and after induction, only had Panx1 in SMC (red, Panx1KO-iSMC OE). n = 5-6, Data were analyzed by two-way ANOVA and Holm-Sidak post hoc test. * p<0.05 vs respective baseline; # p<0.05 vs respective induction.
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