INDUCTION OF TETRAHYDROBIOPTERIN SYNTHESIS IN RAT CARDIAC MYOCYTES - IMPACT ON CYTOKINE-INDUCED NO GENERATION

Because tetrahydrobiopterin (BH4) is an essential cofactor for nitric oxide (NO) formation, we investigated whether BH4 synthesis is require d for cytokine-induced NO production in cultured rat cardiac myocytes. The total biopterin content of untreated cardiac myocytes was below o ur limit of detection. However, treatment with interleukin-1 alpha (IL -1 alpha) + interferon-gamma (IFN-gamma) caused a significant rise in biopterin levels and induced NO synthesis. 2,4-Diamino-6-hydroxypyrimi dine (DAHP), a selective inhibitor of GTP cyclohydrolase I (the rate-l imiting enzyme for de novo BH4 synthesis), completely abolished the el evation in biopterin levels induced by IL-1 alpha + IFN-gamma. DAHP al so caused a concentration-dependent inhibition of (IL-1 alpha + IFN-ga mma)-induced NO synthesis. Similarly, N-acetylserotonin, an inhibitor of the BH4 synthetic enzyme sepiapterin reductase, blocked increases i n biopterin levels as well as NO synthesis induced by IL-1 alpha + IFN -gamma. Sepiapterin, substrate for BH4 synthesis via the pterin salvag e pathway, prevented this inhibition by DAHP or N-acetylserotonin, and this effect was blocked by methotrexate. Sepiapterin and, to a lesser extent, BH4 dose dependently enhanced (IL-1 alpha + IFN-gamma)-induce d NO synthesis, suggesting that the concentration of BH4 limits the ra te of NO production. Inducible NO synthase mRNA and GTP cyclohydrolase I mRNA were induced by IL-1 alpha + IFN-gamma in parallel. We thus de monstrate that BH4 synthesis is an absolute requirement for induction of NO synthesis by cytokines in cardiac myocytes.