AN EARLY LOSS IN MEMBRANE-PROTEIN KINASE-C ACTIVITY PRECEDES THE EXCITATORY AMINO ACID-INDUCED DEATH OF PRIMARY CORTICAL-NEURONS

Several lines of evidence indicate that a rapid loss of protein kinase C (PKC) activity may be important in the delayed death of neurons fol lowing cerebral ischemia. However, in primary neuronal cultures, cytot oxic levels of glutamate have been reported not to cause a loss in PKC as measured by immunoblot and conventional activity methods. This app arent contradiction has not been adequately addressed. In this study, the effects of cytotoxic levels of glutamate, NMDA, and lpha-amino-3-h ydroxy-5-methylisoxazole-4-propionic acid (AMPA) on membrane PKC activ ity was determined in cortical neurons using an assay that measures on ly PKC that is active in isolated membranes, which can be used to diff erentiate active enzyme from that associated with membranes in an inac tive state. A 15-min exposure of day 14-18 cortical neurons to 100 mu M glutamate, AMPA, or NMDA caused a rapid and persistent lass in membr ane PKC activity, which by 4 h fell to 30-50% of that in control cultu res. However, the amount of enzyme present in these membranes remained unchanged during this period despite the loss in enzyme activity. The inactivation of PKC activity was confirmed by the fact that phosphory lation of the MARCKS protein, a PKC-selective substrate, was reduced i n intact neurons following transient glutamate treatment. By contrast, activation of metabotropic glutamate receptors by trans-(1 S,3 R)-1-a mino-1,3-cyclopentanedicarboxylic acid was not neurotoxic and induced a robust and prolonged activation of PKC activity in neurons. PKC inac tivation by NMDA and AMPA was dependent on extracellular Ca2+, but les s so on Na+, although cell death induced by these agents was dependent on both ions. The loss of PKC activity was likely effected by Ca2+ en try through specific routes because the bulk increase in intracellular free [Ca2+] effected by the Ca2+ ionophore ionomycin did not cause th e inactivation of PKC. The results indicate that the pattern of PKC ac tivity in neurons killed by glutamate, NMDA, and AMPA in vitro is cons istent with that observed in neurons injured by cerebral ischemia in v ivo.