Modulation of NMDA-mediated excitotoxicity by protein kinase C

Excessive activation of N-methyl-D-aspartate (NMDA) receptors leads to cell death in human embryonic kidney-293 (HEK) cells which have been transfecte d with recombinant NMDA receptors. To evaluate the role of protein kinase C (PKC) activation in NMDA-mediated toxicity, we have analyzed the survival of transfected HEK cells using trypan blue exclusion. We found that NMDA-me diated death of HEK cells transfected with NR1/NR2A subunits was increased by exposure to phorbol esters and reduced by inhibitors of PKC activation, or PKC down-regulation. The region of NR2A that provides the PKC-induced en hancement of cell death was localized to a discrete segment of the C-termin us. Use of isoform-specific PKC inhibitors showed that Ca2+- and lipid-depe ndent PKC isoforms (cPKCs), specifically PKC beta1, was responsible for the increase in cell death when phorbol esters were applied prior to NMDA in t hese cells. PKC activity measured by an in vitro kinase assay was also incr eased in NR1A/NR2A-transfected HEK cells following NMDA stimulation. These results suggest that PKC acts on the C-terminus of NR2A to accentuate cell death in NR1/NR2A-transfected cells and demonstrate that this effect is med iated by cPKC isoforms. These data indicate that elevation of cellular PKC activity can increase neurotoxicity mediated by NMDA receptor activation.