Werner-FelmayerzvutsronmlkihgfedcbaUSPMIFCBA et al.: Tetrahydrobiopterin-dependent nitric oxide formation in vitro 53 Pleridines Vol. 3, pp. 5 3 - 5 4 Short Communication Tetrahydrobiopterin-Dependent Conversion of L-Arginine to L-Citrulline and Nitric Oxide in Murine and Human Cells 1 ) G. Werner-Felmayer, E. R. Werner, G. Weiss and H. Wächter 2 ' Institut für Medizinische Chemie und Biochemie, Universität Innsbruck, Fritz-Pregl-Str. 3, A-6020 Innsbruck, Austria (Received March 1992) Introduction Nitric oxide (NO) synthase converts L-arginine to NO and L-citrulline. Recent work characterizing this enzyme shows that there exist at least two forms in mammalian cells: a constitutive and Ca 2+ -dependent form which is typically found in endothelium and brain and a cytokine-induciblc, Ca 2+ -independent form which can be found in macrophages but also a variety of other cell types. NO stimulates guanylate cyclase and thus mediates vasodilation and its effects in neurotransmission. Via formation of iron nitrosyl complexes NO inhibits aconitase and ribonucleotide reductase, leading to cytotoxicity [for review on NO synthase see (1)]. Both types of NO synthase require NADPH, flavins and tetrahydrobiopterin as cofactors and it was shown recently that FAD, FMN and tetrahydrobiopterin copurify with the enzyme from porcine cerebellum (2, see also Werner, E. R. et al., this volume). Here we compare the effect of influencing intracellular tetrahydrobiopterin levels on cytokine-induced NO synthase from murine fibroblasts and on the consti- tutive NO synthase from human endothelial cells. lf Support by the Austrian "Bundesministerium für Wissen- schaft und Forschung, Sektion Forschung" is gratefully acknowledged. 2) Author to whom correspondence should be addressed. Pteridines / Vol.3 / No. 1/2 Copyright © 1992 Walter de Gruyter • Berlin • New York Material and Methods Murine fibroblasts isolated from ear dermis of Balb/c mice were cultured as described (3). Human endoth- elial cells from umbilical vein were grown on gelatine- coated surfaces in medium 199 supplemented with 20% (v/v) of heat-inactivated fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin, 0.1 mg/ml strepto- mycin, 50yxwvutsrponmlkihgfedcbaUTRPONMKIHGFDCBA g/ml endothelial cell growth supplement (Boehringer, FRG), 50 g/ml insulin, 0.1 mg/ml trans- ferrin and 0.5% (w/v) of bovine serum albumine. Cells used for experiments were between passage 3 and 12. In murine fibroblasts formation of NO was deter- mined as the amount of nitrite accumulated in super- natants after 48 to 72 hours of cytokine treatment (50 U/ml murine interferon-gamma in combination with 500 U/ml murine tumour necrosis factor-alpha). In endothelial cells NO was measured via formation of cGMP upon activation of constitutive NO synthase by the Ca-ionophore A23187. For this purpose en- dothelial cells grown in 6-well plates were treated for 15 min with 1 mM 3-isobutyl-l-methyl-xanthine in 10 mM Hepes, pH = 7.5, containing 145 mM NaCl, 5 mM KCl, 1 mM MgCl 2 and 2.5 mM CaCl 2 . The re- action was started by addition of 1 Μ A23187. After 4 min the reaction mixture was removed and cGMP levels of individual wells were determined after cell lysis with 0.01 Ν HCl by radioimmunoassay (Amer- sham, UK). Tetrahydrobiopterin synthesis was inhibited by treat- ment of cells with 5 mM 2,4-diamino-6-hydroxy-py- rimidine (DAHP) for 24 hours in endothelial cells and for the period of cytokine induction in murine fibro- Unauthenticated Download Date | 2/25/20 1:32 AM