Preferential inhibition of inducible nitric oxide synthase in intact cells by the 4-amino analogue of tetrahydrobiopterin

In the present study we demonstrate that the 4-amino analogue of tetrahydrobiopterin, 2,4-diamino-5,6,7,8-tetrahydro-6-(L-erythro-1,2-dihydroxyprol)pteridine (4-amino-H(4)biopterin) binds with high affinity to recombinant endothelial NO synthase and concomitantly inhibits enzyme activity [IC50 = 14.8 +/- 7.5 mu M in the presence of added 5,6,7,8-tetrahydro-L-erythrobiopterin (H(4)biopterin) 10 mu M] as efficiently as previously shown for inducible NO synthase [Mayer, B., Wu, C.Q., Gorren, A.C.F., Pfeiffer, S., Schmidt, K., Clark, P., Stuehr, D.J. & Werner, E.R. (1997) Biochemistry 36, 8422-8427]. In cultured porcine endothelial cells, however, Camino-H(4)biopterin was less effective in inhibiting NO formation (IC50 = 420 +/- 36 mu M) as compared with inhibition of the inducible isoform in murine fibroblasts (IC50 = 15 +/- 4.9 mu M) and in human DLD-1 adenocarcinoma cells (IC50 = 55 +/- 10.3 mu M) In all cells investigated, the inhibitory effect of 4-amino-H(4)biopterin was markedly enhanced by depletion of intracellular H(4)biopterin and could be overcome by increasing intracellular H(4)biopterin concentrations. Endothelial cells contained lower amounts of H(4)biopterin [5.2 +/- 0.3 pmol(mg protein)(-1)] than fibroblasts [19.4 +/- 2.7 pmol (mg protein)(-1)] and DLD-1 cells [8.3 +/- 1.1 pmol(mg protein)(-1)], so that the selectivity of 4-amino-H(4)biopterin towards inducible NO synthase was not explained by differences in the H(4)biopterin levels. Because 4-aminoH(4)biopterin did not suppress expression of NO synthase in cytokine-treated cells, we suggest that high-affinity binding of the inhibitor during protein expression may be responsible for the preferential inhibition of the inducible isozyme in intact cells.