Premium
A perfusion system for the long term study of macrophage activation
Author(s) -
Assreuy Jamil,
Moncada Salvador
Publication year - 1992
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1111/j.1476-5381.1992.tb12744.x
Subject(s) - nitrite , perfusion , lipopolysaccharide , nitric oxide , stimulation , chemistry , arginine , biochemistry , nitric oxide synthase , biology , medicine , endocrinology , nitrate , enzyme , amino acid , organic chemistry
1 A closed system was developed for perfusing J774 macrophages in columns. The cells were perfused for up to 100 h, at which time they were still viable. 2 Stimulation with increasing concentrations (0.01–10 μg ml −1 ) of bacterial lipopolysaccharide (LPS) caused the cells to produce increasing amounts of nitrite in the perfusion medium. This production was time‐dependent, reaching a plateau by 48–50 h. 3 The nitrite accumulation caused by 0.1 μg ml −1 of LPS was augmented by priming the cells for 2 h with increasing amounts of interferon‐γ. The nitrite accumulation also reached a plateau under these conditions. 4 N‐iminoethyl‐ l ‐ornithine ( l ‐NIO, 30 μ m ) completely inhibited the accumulation of nitrite whereas dexamethasone (0.3 μ m ) caused 60–70% inhibition. 5 Perfusion of the cells without l ‐arginine prevented the nitrite accumulation. Replacement of this amino acid after 20 or 50 h of perfusion led to a rapid generation of nitrite, the levels of which continued to increase for the duration of the experiment. 6 Thus, the perfusion system can be used to study the kinetics of the activation of the NO synthase and most likely other parameters in J774 cells and probably other cells in culture. An observation already of interest is that the ‘disappearance’ of the NO synthase after its activation can be prevented or reduced by removal of l ‐arginine from the medium.