Differences in nitric oxide synthase activity in a macrophage-like cell line, RAW264.7 cells, treated with lipopolysaccharide (LPS) in the presence or absence of interferon-gamma (IFN-gamma): possible heterogeneity of iNOS activity.
Nitric oxide synthase (NOS) activities were compared in a macrophage-like cell line, RAW 264.7 cells, treated with bacterial lipopolysaccharide (LPS) alone or with LPS and interferon-gamma (IFN-gamma). An about 5-6-fold higher amount of NO2-originating from the nitric oxide radical (.NO) was produced in the culture supernatant of macrophages treated with LPS + IFN-gamma than with LPS alone, depending on both the time of incubation and the dose of LPS. However, the difference in the amounts of iNOS protein in these macrophage extracts was much greater than that in the amount of released NO2-. To estimate the NOS activity after induction of NOS at 37 degrees C for 8 h, we examined both intact cells and cell extracts. The cells were washed and re-incubated in Hank's balanced salt solution (HBSS) containing 1 mM L-arginine and 100 microM carboxy-PTIO, which enabled us to detect trace amounts of .NO by means of rapid reaction of carboxy PTIO with .NO to form .NO2, which was finally converted to NO2-. The results showed an about 7-fold difference between the macrophages pretreated with LPS alone and those with LPS + IFN-gamma. Using the extracts, the NOS activity was assayed with L-[U-14C]arginine as a substrate for NOS in vitro, and the results again revealed an about 7-fold difference between the two types of cell extracts. A kinetic study of the NOS activity by means of in vitro assay suggested that there was little difference in Km value for L-arginine between these two iNOSs. However, it revealed two apparent Kms for beta-NADPH, a co-factor of NOS; one was about 0.4 microM, which was common to these two iNOSs, and the other was about 1.5 and 25 microM for LPS-induced NOS and LPS + IFN-gamma-induced NOS, respectively. The cellular concentration of beta-NADPH was around 14 microM in both LPS- and LPS + IFN-gamma-treated macrophages. These results suggest that there is some heterogeneity of iNOSs induced in macrophages with LPS alone and with LPS + IFN-gamma, and the heterogeneity seems to be due at least in part to the requirement for beta-NADPH.
Noda
T
T
Department of Biochemistry and Cell Biology, National Institute of Health, Tokyo.
Amano
F
F
D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D001565:Benzoates / Q000737:chemistry / Q000378:metabolism; D002250:Carbon Radioisotopes; D002460:Cell Line; D002855:Chromatography, Thin Layer / Q000379:methods; D002956:Citrulline / Q000737:chemistry / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D007093:Imidazoles / Q000737:chemistry / Q000378:metabolism; D007371:Interferon-gamma / Q000494:pharmacology*; D008070:Lipopolysaccharides / Q000633:toxicity*; D008264:Macrophages / Q000187:drug effects / Q000201:enzymology*; D051379:Mice; D009249:NADP / Q000737:chemistry / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000187:drug effects / Q000378:metabolism*; D013379:Substrate Specificity; D013997:Time Factors
D001565:Benzoates; D002250:Carbon Radioisotopes; D007093:Imidazoles; D008070:Lipopolysaccharides; C079393:1,3-dihydroxy-4,4,5,5-tetramethyl-2-(4-carboxyphenyl)tetrahydroimidazole; D002956:Citrulline; D009249:NADP; C068309:N(omega)-hydroxyarginine; D007371:Interferon-gamma; D001120:Arginine; D019001:Nitric Oxide Synthase