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 (α, β 1 , β 2 , γ, δ and ɛ) 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 10 mM NaN 3 , combined with 2 mM 2-deoxyglucose (chemical ischemia); (iii) 1 h after chemical ischemia (reperfusion). In control samples, all the PKC isoforms were detected; immediately after chemical ischemia, PKC β 1 , δ and ɛ isoforms total levels (cytosol + particulate) were increased by 2.9, 2.7 and 9.9 times, respectively, while α isoform was slightly reduced and γ isoform was no longer detectable. After reperfusion, the changes displayed by α, β 1 , γ, δ and ɛ were maintained and even potentiated, moreover, an increase in β 2 (by 41 ± 12%) total levels became significant. Chemical ischemia-induced a significant translocation to the particulate compartment of PKC α isoform, which following reperfusion was found only in the cytosol. PKC β 1 and δ isoforms particulate levels were significantly higher both in ischemic and in reperfused samples than in the controls. Conversely, following reperfusion, PKC β 2 and ɛ 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, 1 mM, but not the N -methyl- d -asparate receptor antagonist, MK-801, 1 μM, prevented the translocation of β 1 isoform observed during ischemia. Both drugs were effective in counteracting reperfusion-induced changes in β 2 and ɛ 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.