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 '4C IL-arginine to 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.

The onset of neurologic symptoms in a child who had markedly elevated blood phenylalanine levels during the first two weeks of life and who was promptly treated with a low phenylalanine diet, with excellent control of serum phenylalanine levels, suggested that this child had an unusual form of phenylketonuria. In assays of the components of the phenylalanine hydroxylating system (open liver biopsy at 14 months), the activity of phenylalanine hydroxylase was 20 per cent of the average normal adult value. By contrast, no dihydropteridine reductase activity was detected in the patient's liver, brain or cultured skin fibroblasts. Since dihydropteridine reductase is also essential for the biosynthesis of dopamine, norepinephrine, and serotonin, disturbed neurotransmitter function may be responsible for the patient's neurologic deterioration. On the basis of these results, assay of reductase in cultured skin fibroblasts may be advisable in the initial diagnosis of phenylketonuria.

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We have examined the interaction of phenylalanine hydroxylase with phenylalanine, tetrahydropterin cofactors, and an activating phospholipid, lysophosphatidylcholine. Incubation of native phenylalanine hydroxylase with phenylalanine or lysophosphatidylcholine results in an increase in the fluorescence emission of the enzyme at 360 nm, which closely parallels the increase in tetrahydrobiopterin-dependent activity observed under these conditions. The presence of tetrahydrobiopterin in the absence of phenylalanine results in quenching of the enzyme fluorescence emission; this quenching exhibits a sharp end point at about 1 mol of tetrahydrobiopterin bound/mol of enzyme subunit. The binding of tetrahydrobiopterin under these conditions is unexpectedly tight, with an estimated KD of 10-20 nM, while in the presence of lysophosphatidylcholine, the KD is increased to about 25 microM. Quenching experiments with sodium iodide indicate greater exposure of tryptophan residues in the phenylalanine-activated enzyme. The ultraviolet difference spectrum of phenylalanine hydroxylase in the presence of phenylalanine exhibits a peak at 238 nm, which correlates with the fluorescence increase and activation, as well as additional changes in the aromatic region, which do not correlate well with activation. Phenylalanine does not alter the far-ultraviolet circular dichroism spectrum of phenylalanine hydroxylase. In contrast, lysophosphatidylcholine appears to induce a dramatic change in enzyme secondary structure upon activation. These results suggest that activation of phenylalanine hydroxylase results in a conformation change and the exposure of buried tryptophan(s) and possibly a cysteine residue.