Tetrahydrobiopterin, a redox-active cofactor, is essential for
nitric oxide (NO) biosynthesis. Previous work showed that intracellular
tetrahydrobiopterin levels modulate activity of
nitric oxide synthases (NOSs). The 4-amino analog of
tetrahydrobiopterin is an effective inhibitor of all three purified NOS
isoforms that, in intact cells, preferentially targets the inducible
isoenzyme. In vivo,
4-amino-tetrahydrobiopterin prolonged allograft survival and rescued rats from
septic shock. Here we investigated the effects of
tetrahydrobiopterin and its 4-amino analog on RAW264.7 murine macrophages activated with
lipopolysaccharide. Surprisingly, both tetrahydropteridines inhibited NO formation. This was caused by downregulation of inducible NOS expression rather than by affecting
enzyme activity. In addition, expression of
tumor necrosis factor-alpha was impaired, and apoptosis, as characterized by quantifying
DNA content and
caspase-3 activation and being associated with the formation of a 33 kDa fragment of
nuclear factor-kappaB p65, was induced. The effects of tetrahydropteridines were scavenged by
catalase or
glutathione but not by
superoxide dismutase. Like tetrahydropteridines,
hydrogen peroxide at concentrations comparable to those found in
tetrahydropteridine-treated cultures affected gene expression and cell survival, whereas increasing intracellular
tetrahydrobiopterin levels by
sepiapterin did not. Thus, extracellular tetrahydropteridines suppress gene expression and induce apoptosis in RAW264.7 cells via
hydrogen peroxide formed in the culture medium during autoxidation.