title: Tetrahydrobiopterin synthesis and inducible nitric oxide production in pulmonary artery smooth muscle.
abstract: We recently reported (Am. J. Respir. Cell Mol. Biol. 7: 471-476, 1992) that a mixture of lipopolysaccharide (LPS) and cytokines produced a time-dependent increase in mRNA and protein expression of inducible nitric oxide synthase (iNOS) in cultured rat pulmonary artery smooth muscle cells (RPASM). In the current study we extend observations on regulation of iNOS in RPASM by showing that de novo synthesis of tetrahydrobiopterin (BH4) is critical for LPS and cytokine-induced NO production. A mixture of LPS and the cytokines gamma-interferon, interleukin-1 beta, and tumor necrosis factor-alpha increased steady-state levels of mRNA of GTP-cyclohydrolase-I (GTP-CH), the rate-limiting enzyme in BH4 biosynthesis. Levels of mRNA to GTP-CH became detectable by 4 h, with further increases at 24 h by Northern blot analysis and reverse-transcriptase polymerase chain reaction. Total intracellular biopterin levels, undetectable under basal conditions, increased after 24 h exposure to LPS and cytokines (to 32.3 +/- 0.8 pmol/mg protein). LPS and cytokine-induced NO production, determined by nitrite concentrations in the medium, was decreased in a concentration-dependent manner by the GTP-CH inhibitor, 2,4-diamino-6-hydroxypyrimidine (DAHP) at 24 h. DAHP also inhibited completely the LPS- and cytokine-induced accumulation of intracellular biopterins. Sepiapterin, which supplies BH4 through a salvage pathway independent of GTP-CH, reversed the effect of DAHP on LPS and cytokine-induced NO production.(ABSTRACT TRUNCATED AT 250 WORDS)
authors: Department of Surgery, University of North Carolina at Chapel Hill 27599-7210.
authors: D K
authors: DK
authors: Nakayama
authors: D A
authors: DA
authors: Geller
authors: M
authors: M
authors: Di Silvio
authors: G
authors: G
authors: Bloomgarden
authors: P
authors: P
authors: Davies
authors: B R
authors: BR
authors: Pitt
authors: K
authors: K
authors: Hatakeyama
authors: H
authors: H
authors: Kagamiyama
authors: R L
authors: RL
authors: Simmons
authors: T R
authors: TR
authors: Billiar
mesh_terms: D000818:Animals; D001708:Biopterins / Q000031:analogs & derivatives* / Q000378:metabolism; D002478:Cells, Cultured; D016207:Cytokines / Q000494:pharmacology; D006136:GTP Cyclohydrolase / Q000235:genetics; D007042:Hypoxanthines / Q000494:pharmacology; D007425:Intracellular Membranes / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism*; D009569:Nitric Oxide / Q000096:biosynthesis*; D009573:Nitrites / Q000037:antagonists & inhibitors; D011651:Pulmonary Artery / Q000166:cytology / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D016207:Cytokines; D007042:Hypoxanthines; D008070:Lipopolysaccharides; D009573:Nitrites; D012333:RNA, Messenger; D001708:Biopterins; D009569:Nitric Oxide; D006136:GTP Cyclohydrolase; C017202:2,4-diaminohypoxanthine; C003402:sapropterin