Phosphorylation of protein kinase C substrate proteins in rat hippocampal slices - Effect of calpain inhibition

Abstract

Incubation of the acutely dissected rat hippocampal slices in calcium- containing media resulted in spontaneous activation-translocation of classical PKC isoforms and their subsequent (especially γ-type) proteolytic degradation. These changes were blocked by calpain inhibitor MDL 28 170 in 100 μM concentration. Rat hippocampal slices were metabolically prelabelled with Pi and stimulated with NMDA/glycine, depolarization or phorbol dibutyrate (PDBu) treatment. The basal phosphorylation of specific PKC substrates (MARCKS, neuromodulin and neurogranin) was significantly reduced in non-stimulated slices by MDL pretreatment. In contrast, only the slices where calpain activity was inhibited responded to further NMDA or phorbol dibutyrate stimulation by a substantial increase of PKC-dependent protein phosphorylation. It is concluded that the PKC phosphorylation system is severely affected by non-specific activation and a subsequent, calpain- dependent proteolysis in the acutely prepared hippocampal slices. Calpain inhibition by 100 μM MDL partially prevented these changes and increased stimulus-dependent phosphorylation of PKC-specific protein substrates.