Isoform-specific translocation of protein kinase C following glutamate administration in primary hippocampal neurons

Abstract

High concentrations of glutamate, the major excitatory neurotransmitter in the mammalian brain, lead to intracellular calcium overload resulting in excitotoxic damage and death of neurons. Since protein kinase C (PKC) is involved in neuronal degeneration resulting from cerebral ischemia and from glutamate excitotoxicity, we investigated the effect of glutamate on changes in the cellular distribution of various PKC isoforms in cultured hippocampal neurons in comparison with the effects elicited by the PKC activator phorbol ester. Out of the expressed PKC isoforms α,γ,ε,ζ and λ only the conventional isoforms PKC α and γ responded to glutamate. Using subcellular fractionation and Western blotting with isoform-specific antibodies and immunocytochemical localization with confocal laser scanning microscopy, we observed that phorbol ester and glutamate have different effects on PKC isoform redistribution: Whereas phorbol ester resulted in translocation of PKC α and PKC γ toward a membrane fraction, the glutamate-mediated rise in intracellular calcium concentration induced a translocation mainly toward a detergent-insoluble, cytoskeletal fraction. Immunocytochemical analysis revealed an isoform-specific translocation following glutamate treatment: PKC γ was translocated mainly to cytoplasmic, organelle-like structures, whereas PKC α redistributed to the plasma membrane and into the cell nucleus. The latter result is of special interest, as it indicates that nuclear PKC may play a role in processes of excitotoxic cell damage.