Introduction: Hypoxic pulmonary vasoconstriction (HPV) is an active physiological response to alveolar hypoxia by which pulmonary blood flow is redirected from poorly ventilated areas to lung regions with better oxygen supply. Sustained HPV, however, and subsequent remodeling of the vasculature play a crucial role in the development of chronic hypoxic pulmonary hypertension (PH). Yet, the exact signaling pathway underlying HPV still remains incompletely understood. Recently,  Pannexin 1 (Panx1) mediated ATP-release was identified as an important activator of the TRPV4/TRPC6 signaling cascade which had been previously identified by us and others as integral part of the  hypoxia-induced Ca2+ influx and consequently, contraction of pulmonary artery smooth muscle cells (PASMCs). Earlier work had shown that the antihypertensive mineralocorticoid receptor antagonist Spironolactone (Spiro) prevents the formation of acute pulmonary edema and dyspnea during mountain trekking, i.e. of symptoms commonly attributed to an excessive HPV response at high altitude. Recently, Spiro was identified as a potent inhibitor of Panx1 channels. Therefore, we aimed to investigate the inhibitory effects of Spiro on Panx1 channels as a potential mechanism for an attenuated HPV response.

Methods: To investigate the role of Panx1, HPV was tested in lungs of endothelial cell – and SMC specific Panx1-KO mice as increase in mean pulmonary artery pressure (mPAP) in response to hypoxia.  Lungs of C57Bl6/J and Panx1 KO mice were ventilated with normoxic (20% O2; 5% CO2, 75% N2) or hypoxic gas (1% O2; 5% CO2, 94% N2). mPAP was recorded via pulmonary artery catheter. To test whether Spiro attenuates HPV, C57Bl6/J mice were perfused with either Spiro or vehicle (DMSO). In order to elucidate the role of Panx1 in the development of PH, SMC specific Panx1-KO mice were kept under hypoxic conditions (10% O2) for 5 weeks followed by measurements of right ventricular systolic pressure (RVSP) and cardiac echocardiographic parameters.

Results: Perfusion of isolated mouse lungs with ATP (bolus: 1.79 µmol) caused marked vasoconstriction in line with a potential physiological role for ATP signaling in HPV (ΔmPAP:  Vehicle 1.4±0.4 cmH2O vs.  ATP 4.3±0.3 cmH2O; p<0.05) in WT-mice. Panx1 was expressed in human pulmonary artery endothelial cells (hPAECs) as well as in hPASMCs. The maximal increase in mPAP in response to hypoxia was significantly lower in lungs of mice deficient in SMC-Panx1 as compared to mice lacking Panx1 in ECs, suggesting physiological relevance for SMC-Panx1 in HPV (ΔmPAP: ECPanx1 ko 4.6±0.7 cmH2O vs. SMCPanx1 ko 2.1±0.3 cmH2O; p<0.05). In isolated perfused lungs of WT-mice, Spiro effectively attenuated HPV in a dose dependent manner (ΔmPAP: Vehicle 3.3±0.3 cmH2O vs.  Spiro (20µM) 2.1±0.1 cmH2O; p<0.05) in that change of mPAP in response to acute hypoxia correlated inversely with Spiro dose (rs = 0.958). Pretreatment of isolated mouse lungs using purinergic receptor antagonist Suramin did not decrease HPV response in WT-mice suggesting a role for Panx1 but not for ATP-mediated purinergic signaling. Furthermore, our data revealed that SMC-Panx1-KO did not prevent neither elevated RVSP nor the development of right heart hypertrophy as characteristics of chronic hypoxic PH.

Conclusion: Spiro attenuated the pulmonary vascular response to acute hypoxia, presumably via inhibition of Panx1, but on the other hand, SMC-Panx1-KO might not have an influence on the development of PH.