Differential activation of protein kinase C isoforms following chemical ischemia in rat cerebral cortex slices

Abstract

The aim of the current study was to characterize the effects of chemical ischemia and reperfusion at the transductional level in the brain. Protein kinase C isoforms (alpha, beta(1), beta(2), gamma, delta and epsilon) total levels and their distribution in the particulate and cytosolic compartments were investigated in superfused rat cerebral cortex slices: (i) under control conditions; (ii) immediately after a 5-min treatment with 10mM NaN(3), combined with 2mM 2-deoxyglucose (chemical ischemia); (iii) 1h after chemical ischemia (reperfusion). In control samples, all the PKC isoforms were detected; immediately after chemical ischemia, PKC beta(1), delta and epsilon isoforms total levels (cytosol+particulate) were increased by 2.9, 2.7 and 9.9 times, respectively, while alpha isoform was slightly reduced and gamma isoform was no longer detectable. After reperfusion, the changes displayed by alpha, beta(1), gamma, delta and epsilon were maintained and even potentiated, moreover, an increase in beta(2) (by 41+/-12%) total levels became significant. Chemical ischemia-induced a significant translocation to the particulate compartment of PKC alpha isoform, which following reperfusion was found only in the cytosol. PKC beta(1) and delta isoforms particulate levels were significantly higher both in ischemic and in reperfused samples than in the controls. Conversely, following reperfusion, PKC beta(2) and epsilon isoforms displayed a reduction in their particulate to total level ratios. The intracellular calcium chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, 1mM, but not the N-methyl-d-asparate receptor antagonist, MK-801, 1muM, prevented the translocation of beta(1) isoform observed during ischemia. Both drugs were effective in counteracting reperfusion-induced changes in beta(2) and epsilon isoforms, suggesting the involvement of glutamate-induced calcium overload. These findings demonstrate that: (i) PKC isoforms participate differently in neurotoxicity/neuroprotection events; (ii) the changes observed following chemical ischemia are pharmacologically modulable; (iii) the protocol of in vitro chemical ischemia is suitable for drug screening.