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Chronic Hypoxia–Induced Upregulation of Store-Operated and Receptor-Operated Ca²⁺ Channels in Pulmonary Arterial Smooth Muscle Cells

A Novel Mechanism of Hypoxic Pulmonary Hypertension

From the Division of Pulmonary and Critical Care Medicine (M.-J.L., W.-M.Z., X.-R.Y., J.S.K.S.), Division of Gastroenterology (G.P.H.L., C.-M.T.), Johns Hopkins School of Medicine, Baltimore, Md; the Department of Physiology and Biophysics (K.-P.Y.), College of Medicine, University of South Florida, Tampa; and the Department of Physiology and Pathophysiology (M.-J.L.), Fujian Medical University, Fuzhou, Fujian, People’s Republic of China.

Correspondence to James S.K. Sham, PhD, Division of Pulmonary and Critical Care Medicine, Johns Hopkins Asthma and Allergy Center, 5501 Hopkins Bayview Circle, Baltimore, MD 21224. E-mail jsks{at}welchlink.welch.jhu.edu

Chronic hypoxic pulmonary hypertension is associated with profound vascular remodeling and alterations in Ca²⁺ homeostasis in pulmonary arterial smooth muscle cells (PASMCs). Recent studies show that transient receptor potential (TRPC) genes, which encode store-operated and receptor-operated cation channels, play important roles in Ca²⁺ regulation and cell proliferation. However, the influence of chronic hypoxia on TRPC channels has not been determined. Here we compared TRPC expression, and store- and receptor-operated Ca²⁺ entries in PASMCs of normoxic and chronic hypoxic rats. Reverse-transcription polymerase chain reaction (RT-PCR), Western blot, and immunostaining showed consistently that TRPC1, TRPC3, and TRPC6 were expressed in intralobar pulmonary arteries (PAs) and PASMCs. Application of 1-oleoyl-2-acetyl-sn-glycerol (OAG) to directly activate receptor-operated channels, or thapsigargin to deplete Ca²⁺ stores, caused dramatic increase in cation entry measured by Mn²⁺ quenching of fura-2 and by Ca²⁺ transients. OAG-induced responses were 700-fold more resistant to La³⁺ inhibition than thapsigargin-induced responses. siRNA knockdown of TRPC1 and TRPC6 specifically attenuated thapsigargin- and OAG-induced cation entries, respectively, indicating that TRPC1 mediates store-operated entry and TRPC6 mediates receptor-operated entry. In hypoxic PAs, there were 2- to 3-fold increases in TRPC1 and TRPC6 expression. They were accompanied by significant increases in basal, OAG-induced, and thapsigargin-induced cation entries in hypoxic PASMCs. Moreover, removal of Ca²⁺ or inhibition of store-operated Ca²⁺ entry with La³⁺ and SK&F-96365 reversed the elevated basal [Ca²⁺]_i in PASMCs and vascular tone in PAs of chronic hypoxic animals, but nifedipine had minimal effects. Our results for the first time to our knowledge show that both store- and receptor-operated channels of PASMCs are upregulated by chronic hypoxia and contribute to the enhanced vascular tone in hypoxic pulmonary hypertension.

Key Words: pulmonary hypertension • transient receptor potential channels • store-operated Ca²⁺ channels • receptor-operated Ca²⁺ channels

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