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Phosphorylation of Protein Kinase C substrate proteins in rat hippocampal slices - effect of calpain inhibition

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Domanska-Janik K., Zablocka B., Zalewska T., Zajac H.

vol. 58

issue 4

247-252

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 g- type) proteolytic degradation. These changes were blocked by calpain inhibitor MDL 28 170 in 100 mM concentration. Rat hippocampal slices were metabolically prelabelled with 32Pi 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 mM MDL partially prevented these changes and increased stimulus-dependent phosphorylation of PKC-specific protein substrates.

Interrelations between nuclear-factor kappa B activation, glial response and neuronal apoptosis in gerbil hippocampus after ischemia

100%

Domanska-Janik K., Bronisz-Kowalczyk A., Zajac H., Zablocka B.

Acta Neurobiologiae Experimentalis

2001

vol. 61

issue 1

45-51

Spatial and temporal relations between transcriptional factor NFkB activation and glia reaction in gerbil hippocampus after transient cerebral ischemia has been studied. Activation of protein binding to NFkB consensus oligonucleotide was determined by electrophoretic mobility gel shift assay (EMSA) in homogenates from dorsal (DP- an equivalent of CA1 sector) and abdominal (AbP- containing CA2-4 and gyrus dentatus) parts of hippocampus. A significant activation of NFkB binding was observed exclusively in DP as early as 3 h after ischemia and at this time that response preceded any other morphological signs of postischemic tissue injury. This early enhancement of NFkB binding was followed by microglia activation visualized in CA1 pyramidal region at 24 h of recovery by histochemical staining with lectin from Ricinus communis (RCA-120). Simultaneously, only a moderate increase of immunostaining against glial fibrillary acidic protein (GFAP) was observed homogeneously in all parts of hippocampus. This uniform pattern of astrogliosis was preserved until postischemic day 3-4, when apoptotic DNA fragmentation in CA1 pyramidal neurons had been clearly documented by TUNEL staining. At this period however, continuous elevation of NFkB binding in DP corresponded with similar response manifested also in AbP of the hippocampus. These results evidence a preferential NFkB involvement in an early microglia activation in the apoptogenic CA1 sector, although its role in a later astrocytic response to ischemia could not be neglected too.

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2 Acta Neurobiologiae Experimentalis

1 2001

1 1998

2 Domanska-Janik K.

2 Zablocka B.

2 Zajac H.

1 Bronisz-Kowalczyk A.

1 Zalewska T.

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Phosphorylation of Protein Kinase C substrate proteins in rat hippocampal slices - effect of calpain inhibition

Interrelations between nuclear-factor kappa B activation, glial response and neuronal apoptosis in gerbil hippocampus after ischemia