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.
Tang
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Department of Cardiology, The First Affiliated Hospital of Southeast University, Nanjing, China.
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Meng
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Wang
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Gu
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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