ACTIVATION OF P42 MITOGEN-ACTIVATED PROTEIN-KINASE BY GLUTAMATE-RECEPTOR STIMULATION IN RAT PRIMARY CORTICAL CULTURES

Recent studies have identified at least two homologous mitogen-activat ed protein (MAP) kinases that are activated by phosphorylation of both tyrosine and threonine residues by an activator kinase. To help defin e the role of these MAP kinases in neuronal signalling, we have used p rimary cultures derived from fetal rat cortex to assess the regulation of their activity by agonist stimulation of glutamate receptors and b y synaptic activity. Regulation was assayed by monitoring changes in b oth tyrosine phosphorylation on western blots and in vitro kinase acti vity toward a selective MAP kinase substrate peptide. In initial studi es, we found that phorbol ester treatment increased tyrosine phosphory lation of p42 MAP kinase and stimulated MAP kinase activity. A similar response was elicited by three agonists of metabotropic glutamate rec eptors, i.e., trans-(+/-)-1-amino-1,3-cyclopentane dicarboxylic acid, quisqualate, and (2S,3S,4S)-alpha-(carboxycyclopropyl)glycine. MAP kin ase activity and p42 MAP kinase tyrosine phosphorylation were also sti mulated by the ionotropic glutamate receptor agonist, kainate, but not by N-methyl-D-aspartate. To examine regulation of MAP kinase by synap tic activity, cultures were treated with picrotoxin, an inhibitor of G ABA(A) receptor-mediated inhibition that enhances spontaneous excitato ry synaptic activity. Treatment of cultures with picrotoxin elicited a ctivation of MAP kinase. This response was blocked by tetrodotoxin, wh ich suppresses synaptic activity. These results demonstrate that p42 M AP kinase is activated by glutamate receptor agonist stimulation and b y endogenous synaptic activity.