Regulation of Nitric Oxide Synthesis by Proinflammatory Cytokines

We have examined cytokine regulation of nitric oxide synthase (NOS) in human umbilical vein endothelial cells (HUVEC). 24-h treatment with IFN-'y (200 U/ml) plus TNF (200 U/ml) or IL-1,8 (5 U /ml) increased NOS activity in HUVEC lysates, measured as conversion of I '4CIL-arginine to [ '4Cj L-citrulline. Essentially, all NOS activity in these cells was calcium dependent and membrane associated. Histamine-induced nitric oxide release, measured by chemiluminescence, was greater in cytokine-treated cells than in control cells. Paradoxically, steadystate mRNA levels of endothelial NOS fell by 94±2.0% after cytokine treatment. Supplementation of HUVEC lysates with exogenous tetrahydrobiopterin (3 ,M) greatly increased total NOS activity, and under these assay conditions, cytokine treatment decreased maximal NOS activity. IFN-y plus TNF or IL-1,8 increased endogenous tetrahydrobiopterin levels and GTP cyclohydrolase I activity, the rate-limiting enzyme oftetrahydrobiopterin synthesis. Intracellular tetrahydrobiopterin levels were higher in freshly isolated HUVEC than in cultured cells, but were still limiting. We conclude that inflammatory cytokines increase NOS activity in cultured human endothelial cells by increasing tetrahydrobiopterin levels in the face of falling total enzyme; similar regulation appears possible in vivo. (J. Clin. Invest. 1994. 93:2236-2243.)