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

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)-1beta and/or tumor necrosis factor (TNF)-alpha for 24 h. Induction of NOS 2 by IL-1beta+TNF-alpha 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 IkappaB and activation of NF-kappaB, 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-kappaB activation. Following stimulation with IL-1beta+TNF-alpha 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-1beta+TNF-alpha independently increased promoter activity 2.2- and 3-fold, respectively. As opposed to the attenuating effect that hypoxia had on IL-1beta+TNF-alpha- dependent induction of NOS 2 gene expression, the concomitant treatment with IL-1beta+TNF-alpha 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.