TETRAHYDROBIOPTERIN IS A LIMITING FACTOR OF NITRIC-OXIDE GENERATION IN INTERLEUKIN 1-BETA-STIMULATED RAT GLOMERULAR MESANGIAL CELLS

Treatment of mesangial cells with recombinant human interleukin 1 beta (IL-1 beta) triggers the expression of a macrophage-type of nitric oxide (NO) synthase and the subsequent increase of cellular concentration of cGMP and nitrite production. Tetrahydrobiopterin (BH4) is an essential cofactor for NO synthase, and in the present study we investigated its impact on inducible NO synthesis in mesangial cells. Inhibition of GTP-cyclohydrolase I, the rate-limiting enzyme for BH4 synthesis, with 2,4-diamino-6-hydroxy-pyrimidine (DAHP) potently suppresses IL-1 beta-induced nitrite production and elevation of cellular cGMP levels. This inhibitory effect of DAHP is reversed by sepiapterin, which provides BH4 via the pterin salvage pathway. Most importantly, sepiapterin dose-dependently augments IL-1 beta-stimulated NO synthesis, indicating that the availability of BH4 limits the production of NO in cytokine-induced mesangial cells. N-acetylserotonin, an inhibitor of the BH4 synthetic enzyme sepiapterin reductase, completely abolishes IL-1 beta-stimulated nitrite production, whereas methotrexate, which inhibits the pterin salvage pathway, displays only a moderate inhibitory effect, thus suggesting that mesangial cells predominantly synthesize BH4 by de novo synthesis from GTP. In conclusion, these data demonstrate that BH4 synthesis is an absolute requirement for, and limits IL-1 beta induction of NO synthesis in mesangial cells. Inhibition of BH4 synthesis may provide new therapeutic approaches to the treatment of pathological conditions involving increased NO formation.