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

Incubation of the acutely dissected rat hippocampal slices in calcium-conta ining media resulted in spontaneous activation-translocation of classical P KC isoforms and their subsequent (especially gamma- type) proteolytic degra dation. These changes were blocked by calpain inhibitor MDL 28 170 in 100 m u M concentration. Rat hippocampal slices were metabolically prelabelled wi th (32)Pi and stimulated with NMDA/glycine, depolarization or phorbol dibut yrate (PDBu) treatment. The basal phosphorylation of specific PKC substrate s (MARCKS, neuromodulin and neurogranin) was significantly reduced in non-s timulated slices by MDL pretreatment. In contrast, only the slices where ca lpain activity was inhibited responded to further NMDA or phorbol dibutyrat e stimulation by a substantial increase of PKC-dependent protein phosphoryl ation. It is concluded that the PKC phosphorylation system is severely affe cted by non-specific activation and a subsequent, calpain-dependent proteol ysis in the acutely prepared hippocampal slices. Calpain inhibition by 100 mu M MDL partially prevented these changes and increased stimulus-dependent phosphorylation of PKC-specific protein substrates.