A role for receptor-operated Ca2+ 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 [Ca2+]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 [Ca2+]i elevations during hypoxia. Hypoxia (PO2) about 20 mm Hg) evoked a transient [Ca2+]i elevation that was reduced by removal of extracellular calcium. Nifedipine and verapamil, blockers of voltage-gated calcium channels (VGCCs), attenuated the hypoxia-induced [Ca2+)]i elevation by about 30%, suggesting the presence of alternate Ca2+ 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 [Ca2+]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 [Ca2+]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 [Ca2+]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.
Department of Cardiology, The First Affiliated Hospital of Southeast University, Nanjing, China.
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D055372:AMP-Activated Protein Kinases / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism; D015687:Cell Hypoxia; D002478:Cells, Cultured; D006801:Humans; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000378:metabolism*; D011651:Pulmonary Artery / Q000166:cytology / Q000502:physiology*; D034741:RNA, Small Interfering / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D050052:TRPC Cation Channels / Q000235:genetics / Q000378:metabolism*; D000074287:TRPC6 Cation Channel
D015220:Calcium Channels; D034741:RNA, Small Interfering; D050052:TRPC Cation Channels; D000074287:TRPC6 Cation Channel; C117032:TRPC6 protein, human; C096351:transient receptor potential cation channel, subfamily C, member 1; D055372:AMP-Activated Protein Kinases; D002118:Calcium