Role of calpain in hypoxic inhibition of nitric oxide synthase activity in pulmonary endothelial cells

Yunchao Su, Edward R. Block

Research output: Contribution to journalArticle

63 Citations (Scopus)

Abstract

Pulmonary artery endothelial cells (PAEC) were exposed to normoxia or hypoxia (0% O2-95% N2-5% CO2) 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.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Lung Cellular and Molecular Physiology
Volume278
Issue number6 22-6
StatePublished - Jun 1 2000

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Calpain
Nitric Oxide Synthase Type III
Nitric Oxide Synthase
Endothelial Cells
HSP90 Heat-Shock Proteins
Lung
Pulmonary Artery
Cell Hypoxia
Proteins

Keywords

  • Heat shock protein 90
  • Hypoxia
  • Pulmonary artery

ASJC Scopus subject areas

  • Physiology
  • Pulmonary and Respiratory Medicine
  • Physiology (medical)
  • Cell Biology

Cite this

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title = "Role of calpain in hypoxic inhibition of nitric oxide synthase activity in pulmonary endothelial cells",
abstract = "Pulmonary artery endothelial cells (PAEC) were exposed to normoxia or hypoxia (0{\%} O2-95{\%} N2-5{\%} CO2) 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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AU - Block, Edward R.

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N2 - Pulmonary artery endothelial cells (PAEC) were exposed to normoxia or hypoxia (0% O2-95% N2-5% CO2) 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.

AB - Pulmonary artery endothelial cells (PAEC) were exposed to normoxia or hypoxia (0% O2-95% N2-5% CO2) 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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