Stimulation of rat C6 glioma ecto-5'-nucleotidase by chronic ethanol treatment

Abstract

Treatment of rat C6 glioma cells in culture with 100 mM ethanol led to increased ecto-5'-nucleotidase activity. Studies were performed to obtain information on the mechanism(s) of action for this effect. Growth of C6 glioma cells in 100 mM ethanol for 6 days increased ecto-5'-nucleotidase activity but had no effect on the pH optima or substrate specificity of the ecto-enzyme. A comparison of enzyme activity of intact cells with that of disrupted cells revealed a selective stimulation of the ecto-enzyme, i.e. the enzyme on the outer plasma membrane. Kinetic studies of cells continuously treated with 100 mM ethanol for 6 days showed an apparent increase in affinity of the ecto-enzyme for 5'-AMP, with no appreciable change in the maximum velocity ( V max). The concentration-dependent inhibition of ecto-5'-nucleotidase activity by Concanavalin A (Con A) was antagonized by ethanol treatment. Double reciprocal plot analysis showed that ethanol acted in an apparently noncompetitive manner with respect to Con A inhibition. It appears that the availability of the catalytic site of the ecto-5'-nucleotidase to exogenous substrates was increased. The data are consistent with rearrangement of the plasma membrane resulting from continuous exposure to ethanol.