Introduction: Hypoxic pulmonary vasoconstriction (HPV) redirects pulmonary blood flow from poorly ventilated areas to lung regions with better oxygen supply in order to optimize ventilation/perfusion ratio. Sustained HPV and subsequent remodeling of the vasculature were shown to play a crucial role in the development of pulmonary hypertension (PH). However, the exact signaling pathway underlying HPV remains still incompletely understood. Recently, pannexin 1 (Panx1) has been linked to transient receptor potential vanlloid 4 channel mediated Ca²⁺ signaling in systemic vasoconstriction. Second, the mineralocorticoid receptor (MR) antagonist spironolactone was demonstrated to prevent 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. Since spironolactone was recently identified as a potent inhibitor of Panx1 channels, we aimed to investigate the inhibitory effects of spironolactone on Panx1 channels and its role in HPV-related outcomes.

Methods: To test the role of Panx1, we measured HPV as increase in pulmonary artery pressure (PAP) in response to hypoxia in isolated perfused lungs of endothelial cell (Cdh5-CreERT2/Panx1^fl/fl) and smooth muscle cell specific (SMMHC-CreERT2/Panx1^fl/fl) Panx1-KO mice. C57Bl/6J and Panx1-deficient mice were ventilated with normoxic (20% O₂; 5% CO₂, 75% N₂) or hypoxic gas (1% O₂; 5% CO₂, 94% N₂) and PAP was recorded via a pulmonary artery catheter. To elucidate the role of Panx1 in the development of PH, C57Bl/6J as well as Panx1-KO mice were kept under hypoxic conditions (10% O₂) for 5 weeks followed by measurement of right ventricular systolic pressure, echocardiography and histology.

Results: Panx1 was expressed in human pulmonary artery endothelial cells (hPAECs) as well as in human pulmonary artery smooth muscle cells (hPASMCs). The maximal increase in mPAP in response to hypoxia was significantly lower in lungs of mice deficient in smooth muscle cell Panx1 as compared to mice lacking Panx1 in endothelial cells, suggesting physiological relevance for SMC-Panx1 in HPV. In isolated perfused mouse lungs of C57Bl/6J mice, pharmacological inhibition of Panx1 with spironolactone and the Panx1 specific inhibitory peptide (¹⁰Panx1) effectively attenuated the hypoxia-induced pulmonary vascular response. In addition, our data revealed a positive correlation between increasing spironolactone concentration and decreasing change of mPAP during acute hypoxia. Yet, genetic deletion of Panx1 in either endothelial or smooth muscle cells did not prevent the development of PH in mice. Interestingly, hypoxia did not induce ATP release from hPASMCs, and ATP degradation as well as blockade of purinergic receptors failed to affect HPV. Rather, Panx1 inhibition with spironolactone as well as siRNA-induced knockdown of Panx1 inhibited the hypoxia-induced increase of intracellular Ca²⁺ concentration ([Ca²⁺]_i) in hPASMC suggesting that Panx1 directly or indirectly regulates [Ca²⁺]_i response to hypoxia. In line with this notion, Panx1 overexpression increased [Ca²⁺]_I under hypoxic conditions in HeLa cells

Conclusion: This study demonstrates that spironolactone decreases pulmonary vascular response to acute hypoxia, presumably via inhibition of Panx1. The role of Panx1 in HPV was not attributable to ATP release and purinergic signaling.