A role for receptor-operated Ca(2+) entry in human pulmonary artery smooth muscle cells in response to hypoxia.

Hypoxic pulmonary vasoconstriction (HPV) is an important homeostatic mechanism in which increases of [Ca(2+)](i) are primary events. In this study, primary cultured, human pulmonary artery smooth muscle cells (hPASMC) were used to examine the role of TRPC channels in mediating [Ca(2+)](i) elevations during hypoxia. Hypoxia (PO(2) about 20 mm Hg) evoked a transient [Ca(2+)](i) elevation that was reduced by removal of extracellular calcium. Nifedipine and verapamil, blockers of voltage-gated calcium channels (VGCCs), attenuated the hypoxia-induced [Ca(2+)](i) elevation by about 30 %, suggesting the presence of alternate Ca(2+) entry pathways. Expression of TRPC1 and TRPC6 in hPASMC were found by RT-PCR and confirmed by Western blot analysis. Antagonists for TRPC, 2APB and SKF96365, significantly reduced hypoxia-induced [Ca(2+)](i) elevation by almost 60 %. Both TRPC6 and TRPC1 were knocked down by siRNA, the loss of TRPC6 decreased hypoxic response down to 21 % of control, whereas the knockdown of TRPC1 reduced the hypoxia response to 85 %, suggesting that TRPC6 might play a central role in mediating hypoxia response in hPASMC. However, blockade of PLC pathway caused only small inhibition of the hypoxia response. In contrast, AICAR, the agonist of AMP-activated kinase (AMPK), induced a gradual [Ca(2+)](i) elevation, whereas compound C, an antagonist of AMPK, almost abolished the hypoxia response. However, co-immunoprecipitation revealed that AMPKalpha was not colocalized with TRPC6. Our data supports a role for TRPC6 in mediation of the [Ca(2+)](i) elevation in response to hypoxia in hPASMC and suggests that this response may be linked to cellular energy status via an activation of AMPK.