Role of calpain in hypoxic inhibition of nitric oxide synthase activity in pulmonary endothelial cells.
Pulmonary artery endothelial cells (PAEC) were exposed to normoxia or hypoxia (0% O(2)-95% N(2)-5% CO(2)) in the presence and absence of calpain inhibitor I or calpeptin, after which endothelial nitric oxide synthase (eNOS) activity and protein content were assayed. Exposure to hypoxia decreased eNOS activity but not eNOS protein content. Both calpain inhibitor I and calpeptin prevented the hypoxic decrease of eNOS activity. Incubation of calpain with total membrane preparations of PAEC caused dose-dependent decreases in eNOS activity independent of changes in eNOS protein content. Exposure of PAEC to hypoxia also caused time-dependent decreases of heat shock protein 90 (HSP90) that were prevented by calpain inhibitor I and calpeptin. Moreover, the HSP90 content in anti-eNOS antibody-induced immunoprecipitates from hypoxic PAEC lysates was reduced, and repletion of HSP90 reversed the decrease of eNOS activity in these immunoprecipitates. Incubation of PAEC with a specific inhibitor of HSP90 (geldanamycin) mimicked the hypoxic decrease of eNOS activity. These results indicate that the hypoxia-induced reduction in eNOS activity in PAEC is due to a decrease in HSP90 caused by calpain activation.
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Department of Medicine, University of Florida College of Medicine, Gainesville 32608-1197, USA.
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D000818:Animals; D002154:Calpain / Q000037:antagonists & inhibitors / Q000502:physiology*; D051242:Caveolin 1; D022461:Caveolins; D002478:Cells, Cultured; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004151:Dipeptides / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000201:enzymology* / Q000473:pathology; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism; D000860:Hypoxia / Q000201:enzymology*; D008565:Membrane Proteins / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors*; D052250:Nitric Oxide Synthase Type III; D010766:Phosphorylation; D011651:Pulmonary Artery / Q000201:enzymology* / Q000473:pathology; D012694:Serine / Q000378:metabolism; D013552:Swine; D014443:Tyrosine / Q000378:metabolism
D051242:Caveolin 1; D022461:Caveolins; D015853:Cysteine Proteinase Inhibitors; D004151:Dipeptides; D018841:HSP90 Heat-Shock Proteins; D008565:Membrane Proteins; C071482:calpeptin; D014443:Tyrosine; D012694:Serine; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D002154:Calpain