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

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 [Ca ²⁺]_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.