Modulation of inducible nitric oxide synthase by hypoxia in pulmonary artery endothelial cells

J. J. Zulueta, R. Sawhney, U. Kayyali, M. Fogel, C. Donaldson, H. Huang, J. J. Lanzillo, P. M. Hassoun

Research output: Contribution to journalArticlepeer-review


The effects of hypoxia on the regulation of inducible nitric oxide synthase (NOS) 2 expression were examined in cultured rat pulmonary microvascular endothelial cells (EC). EC did not express NOS 2 mRNA or protein when exposed to normoxia or hypoxia unless they were pretreated with interleukin (IL)-1β and/or tumor necrosis factor (TNF)-α for 24 h. Induction of NOS 2 by IL-1β+TNF-α was significantly attenuated by concomitant exposure of EC to hypoxia or treatment of EC with antioxidants such as tiron, diphenyliodonium, and catalase, suggesting that NOS 2 expression is dependent on the production of reactive oxygen species. Degradation of IκB and activation of NF-κB, which were both induced by treatment of EC with cytokines, were not altered when the cells were exposed to hypoxia, suggesting that the modulation of NOS 2 expression by hypoxia is unrelated to NF-κB activation. Following stimulation with IL-1β+TNF-α for 24 h, incubation of EC in normoxia resulted in a progressive decline in NOS 2 expression and a calculated half-life of approximately 6 h for NOS 2 mRNA. Hypoxia significantly prolonged the half-life of NOS 2 mRNA (17 h, P < 0.05 versus normoxic EC). The half-life of NOS 2 mRNA was also prolonged by actinomycin D treatment (19.5 and 29.5 h for normoxic and hypoxic EC, respectively), suggesting that transcription of an RNA destabilizing factor or RNAse contributes to NOS 2 mRNA degradation. In EC transiently transfected with the rat NOS 2 promoter, hypoxia and the combination of IL-1β+TNF-α independently increased promoter activity 2.2- and 3-fold, respectively. As opposed to the attenuating effect that hypoxia had on IL-1β+TNF-α-dependent induction of NOS 2 gene expression, the concomitant treatment with IL-1β+TNF-α and hypoxia synergistically increased NOS 2 promoter activity 17.6-fold. Taken together, these results suggest that hypoxia alone does not induce NOS 2 expression in cultured pulmonary microvascular EC, but may modulate cytokine induction of this enzyme at pretranscriptional, transcriptional, and posttranscriptional levels.

Original languageEnglish (US)
Pages (from-to)22-30
Number of pages9
JournalAmerican journal of respiratory cell and molecular biology
Issue number1
StatePublished - 2002
Externally publishedYes

ASJC Scopus subject areas

  • Molecular Biology
  • Pulmonary and Respiratory Medicine
  • Clinical Biochemistry
  • Cell Biology


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