CHARACTERIZATION OF THE ACTIVATION OF GLUTAMINASE INDUCED BY N-METHYL-D-ASPARTATE AND POTASSIUM IN CEREBELLAR GRANULE CELLS

Chronic stimulation of cerebellar granule cells with N-methyl-D-aspart ate (NMDA) or KCl induces a specific activation of the enzymes directl y involved in glutamate neurotransmitter synthesis, Phosphate-activate d glutaminase (FAG) activity is enhanced in cultured granule neurons i ncubated with 150 mu M NMDA or 25 mM KCl, Other enzymes are not affect ed by this treatment like lactate dehydrogenase (LDH) and glutamate de hydrogenase (GLDH), which is also a mitochondrial enzyme but not direc tly involved in neurotransmitter synthesis, This effect is dependent o n protein synthesis and is induced after 12 hr of NMDA or KCl stimulat ion, Kinetics of FAG activity showed that K-m values were unaffected, in contrast to V-max values that were increased approximately 70% and 215% over control by NMDA and KCl treatment, respectively, For GLDH, w e found two isoforms that were affected differentially by the experime ntal conditions, Western blot analysis clearly evidenced an increase o f approximately 120-180% in the amount of FAG in NMDA- and KCl-treated cells, whereas GLDH was not significantly modified, These results dem onstrate that the NMDA- and KCl-induced activation of FAG are not due to the modification of the preexisting enzyme, but to an increase in t he synthesis of this enzyme, This suggests that NMDA receptor stimulat ion during critical periods of the cerebellar granule cell development leads to the activation of gene expression involved in the process of cell differentiation. (C) 1996 Wiley-Liss, Inc.