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

In the present study we demonstrate that the 4-amino analogue of tetrahydro biopterin, 2,4-diamino-5,6,7,8-tetrahydro-6-(L-erythro-1,2-dihydroxyprol)pt eridine (4-amino-H(4)biopterin) binds with high affinity to recombinant end othelial 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-erythrobiopter in (H(4)biopterin) 10 mu M] as efficiently as previously shown for inducibl e 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, 8422-842 7]. In cultured porcine endothelial cells, however, Camino-H(4)biopterin wa s less effective in inhibiting NO formation (IC50 = 420 +/- 36 mu M) as com pared with inhibition of the inducible isoform in murine fibroblasts (IC50 = 15 +/- 4.9 mu M) and in human DLD-1 adenocarcinoma cells (IC50 = 55 +/- 1 0.3 mu M) In all cells investigated, the inhibitory effect of 4-amino-H(4)b iopterin was markedly enhanced by depletion of intracellular H(4)biopterin and could be overcome by increasing intracellular H(4)biopterin concentrati ons. 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 selectiv ity of 4-amino-H(4)biopterin towards inducible NO synthase was not explaine d 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 su ggest that high-affinity binding of the inhibitor during protein expression may be responsible for the preferential inhibition of the inducible isozym e in intact cells.