By John F Lilly III1, Fiona Cunningham2, and Normand Leblanc3
Ca2+-activated Cl- channels (CaCCs) encoded by the gene Tmem16a or Anoctamin1 (ANO1) produce membrane depolarization and contraction of vascular smooth muscle cells (VSMCs) when stimulated by endogenous vasoconstrictors acting on Gq-protein coupled receptors. Treprostinil (TPL) is one of several prostacyclin analogs clinically used to treat patients diagnosed with pulmonary arterial hypertension (PAH). TPL reduces pulmonary arterial tone in PAH patients by binding to the IP prostanoid receptor. Activation of this Gs-coupled receptor leads to elevation in cAMP levels causing vasorelaxation by reducing intracellular [Ca2+]i. In this study, we examined whether TPL could exert vasorelaxation of mouse pulmonary arteries (mPA) by inhibiting, at least in part, ANO1-encoded CaCCs. TPL dose-dependently inhibited the contraction of mPA elicited by 10 µM 5-HT (IC50 ≈ 500 nM). The specific ANO1 inhibitor T16Ainh-A01 also dose-dependently inhibited the 5-HT-induced contraction of mPA but had no effect on the KCl-induced contraction (85.4 mM) at concentrations ≤ 10 µM. 200 nM TPL produced a rightward shift of the dose-response curve to T16AInh-A01, suggesting that TPL may inhibit CaCCs. Consistent with this hypothesis, Ca2+-activated Cl- currents (elicited by 1 µM free Ca2+ in the pipette solution) recorded in HEK-293 cells transfected with mouse ANO1 were significantly reduced by TPL (1 µM), or the adenylate cyclase activator Forskolin (10 µM). These preliminary results suggest that elevation of cAMP levels (perhaps through a phosphorylation step involving Protein Kinase A) may down-regulate ANO1-encoded CaCCs and attenuate the depolarization and vasoconstriction triggered by 5-HT. Our findings also suggest that part of the beneficial effects of TPL in PAH patients might be attributable to an interaction with ANO1 channels.
1University of Nevada School of Medicine, Reno, NV 89557-0330 USA
2,3Department of Pharmacology, University of Nevada, Reno, NV
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