Activation of metabotropic glutamate receptors increases endogenous protein kinase C substrate phosphorylation in adult hippocampal slices

We previously reported (Staak, S., Behnisch, T. and Angenstein, F., Hippocampal long-term potentiation: transient increase but no persistent translocation of protein kinase C (PKC) isoenzymes alpha and beta, Brain Res., 682 (1995) 55-62) that Ca2+-dependent PKC isoenzymes alpha/beta and gamma are not translocated between subcellular compartments after stimulation of glutamate receptor subtypes in hippocampal slices. Extending our previous work in this study in situ phosphorylation of endogenous PKC substrates and the translocation of novel PKC isoenzymes delta and epsilon was analysed to detect PKC activation. Two proteins of approximately 94 kDa and 18 kDa were first characterised to be specific PKC substrates. As control of the technique carbachol was shown to increase in situ phosphorylation of the two substrates without any measurable translocation of PKC protein. Activation of metabotropic glutamate receptors by 50 mu M DHPG also increased the in situ-phosphorylation by 43.9% (94 kDa) and 32.8% (18 kDa) compared to controls but did not induce a measurable subcellular redistribution of conventional and novel PKC isoenzymes. Stimulation by 50 mu M trans-ACPD or 0.1 mM quisqualate enhanced the in situ phosphorylation in the same range, whereas 0.1 mM NMDA was ineffective. To our knowledge this is the first report showing a direct link between metabotropic glutamate receptor activation and increased endogenous PKC substrate phosphorylation in adult hippocampal slices. This PKC activation was not detectable by a redistribution of enzyme protein between subcellular compartments. We, therefore, conclude, that the failure to detect PKC translocation in physiological experiments are not an indicator for unchanged enzyme activity.