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Induction of Nitric Oxide Synthase in Rat C6 Glioma Cells
Author(s) -
Feinstein Douglas L.,
Galea Elena,
Roberts Steven,
Berquist Henrik,
Wang Hong,
Reis Donald J.
Publication year - 1994
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.1994.62010315.x
Subject(s) - astrocyte , nitric oxide , lipopolysaccharide , nitric oxide synthase , tumor necrosis factor alpha , citrulline , cytokine , arginine , cell culture , microbiology and biotechnology , omega n methylarginine , nitroarginine , glioma , chemistry , interferon , biology , biochemistry , endocrinology , immunology , amino acid , cancer research , genetics , central nervous system
We have examined the induction of nitric oxide syhthase (NOS) activity in the rat astrocyte‐derived C6 glioma cell line. In contrast to the previous results with primary astrocyte cultures, incubation of C6 cells with bacterial endotoxin lipopolysaccharide (LPS; 1 μg/ml for 24 h) did not stimulate NO 2 production. However, addition of either tumor necrosis factor‐a (TNF‐α) or interferon‐γ (IFN‐γ), cytokines that by themselves had no effect on NOS activity, imparted LPS responsiveness onto these cells in a dose‐dependent manner (EC 50 values of 39 ng/ml of TNF‐α and 9.4 U/ml of IFN‐γ), and the effect of TNF‐α could be further potentiated (twofold) by the presence of interleukin‐1β. The simultaneous presence of TNF‐α and IFN‐γ yielded a greater response than either cytokine alone; however, the respective EC 50 values were not affected. A cytoplasmic extract from induced C6 cells catalyzed the Ca 2+ ‐independent conversion of l ‐arginine to l ‐ citrulline, with an apparent K m of 51.2 n M , and this activity could be blocked by l ‐arginine analogues in the potency order amino > methyl > nitroarginine. Immunoblot analysis revealed an apparent molecular mass of 125 kDa for the NOS protein induced in C6 cells. These results indicate that the combination of LPS plus cytokines can induce NOS activity in C6 glioma cells with properties similar to those of the enzyme expressed in primary astrocyte cultures.