Tyrosine-kinase-dependent regulation of the nitric oxide synthase gene by endothelin-1 in human endothelial cells.

In vascular endothelium, endothelium-derived relaxing factor, predominantly nitric oxide (NO), is synthesized by endothelial NO synthase (eNOS). While regulatory influences on eNOS enzyme activity are widely clarified, little is known about the regulation of the eNOS gene. We investigated the regulatory signaling mechanisms of eNOS mRNA expression and accumulated NO production in human endothelial cells. Northern blot analysis and NO assays demonstrate that the vasoconstrictor peptide endothelin-1 (ET-1) induces the eNOS gene and leads to accumulated NO production. Induction occurs via ETA receptor activation and depends on improved transcript stability. It is maintained for incubation periods of 30-90 min and tapers thereafter. Regulatory signaling mechanisms depend on de novo protein synthesis to control eNOS mRNA fate. Selectively blocking protein tyrosine kinases (PTK) and inhibiting protein kinase C (PKC) inhibit eNOS mRNA expression and accumulated NO secretion. These observations indicate that regulation of eNOS at the genomic level occurs via post-transcriptional mechanisms. Two protein-bound intracellular kinase pathways, PTK and PKC, regulate eNOS mRNA expression and accumulated NO production.