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.