Induction of tetrahydrobiopterin synthesis in rat cardiac myocytes: impact on cytokine-induced NO generation.
Because tetra-hydrobiopterin (BH4) is an essential cofactor for nitric oxide (NO) formation, we investigated whether BH4 synthesis is required for cytokine-induced NO production in cultured rat cardiac myocytes. The total biopterin content of untreated cardiac myocytes was below our 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-hydroxypyrimidine (DAHP), a selective inhibitor of GTP cyclohydrolase I (the rate-limiting enzyme for de novo BH4 synthesis), completely abolished the elevation in biopterin levels induced by IL-1 alpha + IFN-gamma. DAHP also caused a concentration-dependent inhibition of (IL-1 alpha + IFN-gamma)-induced NO synthesis. Similarly, N-acetylserotonin, an inhibitor of the BH4 synthetic enzyme sepiapterin reductase, blocked increases in biopterin levels as well as NO synthesis induced by IL-1 alpha + IFN-gamma. Sepiapterin, substrate for BH4 synthesis via the pterin salvage 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)-induced NO synthesis, suggesting that the concentration of BH4 limits the rate of NO production. Inducible NO synthase mRNA and GTP cyclohydrolase I mRNA were induced by IL-1 alpha + IFN-gamma in parallel. We thus demonstrate that BH4 synthesis is an absolute requirement for induction of NO synthesis by cytokines in cardiac myocytes.
Kasai
K
K
Department of Endocrinology, Dokkyo University School of Medicine, Tochigi, Japan.
Hattori
Y
Y
Banba
N
N
Hattori
S
S
Motohashi
S
S
Shimoda
S
S
Nakanishi
N
N
Gross
S S
SS
D000818:Animals; D001708:Biopterins / Q000031:analogs & derivatives* / Q000096:biosynthesis; D016207:Cytokines / Q000494:pharmacology*; D004338:Drug Combinations; D004790:Enzyme Induction; D006136:GTP Cyclohydrolase / Q000235:genetics; D007371:Interferon-gamma / Q000494:pharmacology; D007375:Interleukin-1 / Q000494:pharmacology; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D009569:Nitric Oxide / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar
D016207:Cytokines; D004338:Drug Combinations; D007375:Interleukin-1; D012333:RNA, Messenger; D001708:Biopterins; D009569:Nitric Oxide; D007371:Interferon-gamma; D019001:Nitric Oxide Synthase; D006136:GTP Cyclohydrolase; C003402:sapropterin