Chronic hypoxia-induced upregulation of Ca ²⁺-activated Cl ^- channel in pulmonary arterial myocytes: A mechanism contributing to enhanced vasoreactivity

• A prolonged reduced oxygen level in the lungs, as occurs in patients of many chronic lung diseases and in residents living at high altitude, causes pulmonary hypertension characterized by profound structural and functional changes in pulmonary vasculature. • Many of these changes are ascribed to alterations in Ca ²⁺ homeostasis related to cation channels of pulmonary arterial smooth muscle cells. • Here we report the increase of an anion conductance called calcium-activated chloride channel and the expression of the channel gene TMEM16A in pulmonary arterial smooth muscle cells isolated from rats exposed to 10% oxygen for 3-4 weeks. • The upregulation of the chloride channel contributes to the hyper-responsiveness of pulmonary arteries to serotonin associated with pulmonary hypertension. • These results help us to appreciate the importance of anion channels in the pathophysiology of pulmonary hypertension, and may lead to alternative strategies for the treatment of the disease. Chronic hypoxic pulmonary hypertension (CHPH) is associated with altered expression and function of cation channels in pulmonary arterial smooth muscle cells (PASMCs), but little is known for anion channels. The Ca ²⁺-activated Cl ^- channel (CaCC), recently identified as TMEM16A, plays important roles in pulmonary vascular function. The present study sought to determine the effects of chronic hypoxia (CH) on the expression and function of CaCCs in PASMCs, and their contributions to the vascular hyperreactivity in CHPH. Male Wistar rats were exposed to room air or 10% O ₂ for 3-4 weeks to generate CHPH. CaCC current (I _Cl.Ca) elicited by caffeine-induced Ca ²⁺ release or by depolarization at a constant high [Ca ²⁺] _i (500 or 750 nm) was significantly larger in PASMCs of CH rats compared to controls. The enhanced I _Cl.Ca density in CH PASMCs was unrelated to changes in amplitude of Ca ²⁺ release, Ca ²⁺-dependent activation, voltage-dependent properties or calcineurin-dependent modulation of CaCCs, but was associated with increased TMEM16A mRNA and protein expression. Maximal contraction induced by serotonin, an important mediator of CHPH, was potentiated in endothelium-denuded pulmonary arteries of CH rats. The enhanced contractile response was prevented by the CaCC blockers niflumic acid and T16A _inh-A01, or by the L-type Ca ²⁺ channel antagonist nifedipine. The effects of niflumic acid and nifedipine were non-additive. Our results demonstrate for the first time that CH increases I _Cl.Ca density, which is attributable to an upregulation of TMEM16A expression in PASMCs. The augmented CaCC activity in PASMCs may potentiate membrane depolarization and L-type channel activation in response to vasoconstrictors and enhance pulmonary vasoreactivity in CHPH.