The mechanism by which nitric-oxide (NO) production increases during hypoxia is unknown. To explore the effect of hypoxia upon endothelial nitric- oxide synthase (ecNOS) activity and expression, we exposed bovine aortic endothelial cells (BAEC) to hypoxia (1% O2) for 0-24 h and measured levels of ecNOS mRNA, protein, and activity. The amount of ecNOS mRNA increases to more than twice the basal level after 6 h of hypoxia. Incubation of BAEC with actinomycin D during hypoxia prevents this increase, demonstrating that higher levels of mRNA observed during hypoxia are due to increased synthesis, not to increased stability of ecNOS mRNA. Levels of ecNOS protein increase throughout 24 h of hypoxia to more than twice normoxic levels. Although ecNOS expression increases within 2 h of hypoxia, total activity remains unchanged. To explore the transcriptional regulation of ecNOS, we constructed a reporter plasmid containing the ecNOS promoter region upstream of the luc gene and transfected this reporter plasmid into BAEC. In this system, hypoxia induces a linear increase over time in the expression of luciferase driven by the ecNOS promoter. It is concluded that hypoxia induces an increase in transcription of ecNOS in endothelial cells, activating the regulatory region of ecNOS by undefined transcription factors.
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