Induction of nitric oxide synthase in rat C6 glioma cells

Abstract

We have examined the induction of nitric oxide synthase (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 microgram/ml for 24 h) did not stimulate NO2 production. However, addition of either tumor necrosis factor-alpha (TNF-alpha) or interferon-gamma (IFN-gamma), cytokines that by themselves had no effect on NOS activity, imparted LPS responsiveness onto these cells in a dose-dependent manner (EC50 values of 39 ng/ml of TNF-alpha and 9.4 U/ml of IFN-gamma), and the effect of TNF-alpha could be further potentiated (twofold) by the presence of interleukin-1 beta. The simultaneous presence of TNF-alpha and IFN-gamma yielded a greater response than either cytokine alone; however, the respective EC50 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 Km of 51.2 microM, 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.