In vascular endothelial cells, tetrahydrobiopterin serves as an essential cofactor required for enzymatic activity of nitric oxide synthase. GTP cyclohydrolase I is the rate-limiting enzyme in the biosynthesis of tetrahydrobiopterin. Previous studies have demonstrated that proinflammatory cytokines stimulate production of tetrahydrobiopterin in endothelial cells. Long-term regulation of GTP cyclohydrolase I gene expression in endothelium has not been studied. The present study was designed to

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... termine whether the cytokines tumor necrosis factor-␣ (TNF-␣), interferon-␥ (INF-␥), and interleukin-1␤ (IL-1␤) stimulate tetrahydrobiopterin synthesis by increasing expression of GTP cyclohydrolase I mRNA in endothelial cells. The relative reverse transcription polymerase chain reaction was used to quantify expression of GTP cyclohydrolase I mRNA in cultured human umbilical vein endothelial cells. Nuclear run-on assay was performed to determine the transcription rate of GTP cyclohydrolase I gene. GTP cyclohydrolase I enzymatic activity and production of tetrahydrobiopterin were measured in cell extracts. After incubation with TNF-␣ (2 g/mL), INF-␥ (200 U/mL), and IL-1␤ (5 U/mL) for 24 hours, significantly increased expression of GTP cyclohydrolase I mRNA was detected. Cytokines increased the transcription rate of GTP cyclohydrolase I 3.6-fold. This increase was associated with increased GTP cyclohydrolase I enzymatic activity and elevation of intracellular levels of tetrahydrobiopterin. An RNA synthesis inhibitor, actinomycin D (2 g/mL), inhibited cytokine-induced expression of GTP cyclohydrolase I gene. A protein synthesis inhibitor, cycloheximide (0.5 g/mL), did not affect expression of GTP cyclohydrolase I mRNA but blocked the increase in enzyme activity, as well as production of tetrahydrobiopterin. Incubation of endothelial cells for 24 hours in the presence of 8-bromoadenosine 3Ј:5Ј-cyclic monophosphate (10 Ϫ3 mol/L) did not affect expression of GTP cyclohydrolase I mRNA. These results demonstrate that in vascular endothelial cells, cytokines increase production of tetrahydrobiopterin by stimulating expression of GTP cyclohydrolase I gene. This effect is apparently due to increased transcription rather than stabilization of mRNA. Regulation of GTP cyclohydrolase I gene expression by cytokines may play an important role in control of endothelial nitric oxide synthesis. (Arterioscler Thromb Vasc Biol. 1998;18:27-32.) Key Words: nitric oxide Ⅲ tetrahydrobiopterin Ⅲ tumor necrosis factor-␣ Ⅲ interferon-␥ Ⅲ interleukin-1␤