Inhibition of PKCgamma membrane translocation mediated morphine preconditioning-induced neuroprotection against oxygen-glucose deprivation in the hippocampus slices of mice

We previously reported that novel protein kinase C (nPKC)epsilon and N-methyl-D-aspartic acid (NMDA) receptors participated in morphine preconditioning (MP)-induced neuroprotection. In this study, we used Western blot analysis, 2,3,5-triphenyltetrazolium chloride (TTC) staining and lactate dehydrogenase (LDH) leakage assay to determine the involvement of conventional PKC isoforms (cPKC) in MP-induced neuroprotection against oxygen-glucose deprivation (OGD). Hippocampus slices (400-mu m thickness) from healthy male BALB/c mice exposed to OGD for 5-45 min to mimic mild, moderate and severe ischemia in the presence of MP pretreatment. We found that OGD-induced damage in neuronal cell survival rate and LDH leakage could be improved by MP pretreatment (3 mu M) within 20 min of OGD, which was abolished by concomitant incubation with non-selective opioid receptor antagonist naloxone (Nal, 50 mu M). The results of Western blot analysis showed that only cPKC gamma membrane translocation, not alpha, beta I and beta II, increased under the condition of OGD 10 min and 2 h reperfusion (OGD/2 h), and this increment of cPKC gamma membrane translocation was inhibited by MP pretreatment. To further elucidate the role of cPKC gamma in MP-induced neuroprotection, we found that cPKC gamma membrane translocation inhibitor, Go6983 (6 nM) did not affect MP-induced neuroprotection while Go6983 alone exhibited a significant inhibition on OGD-induced increment in LDH leakage and decrease in cell survival rate. These phenomena were defined by the results that Go6983 could restore OGD-induced cPKC gamma membrane translocation, but had no further effect on MP-induced inhibition of cPKC gamma membrane translocation. These results demonstrated that MP can reduce OGD-induced neuronal injuries, and the down-regulation of cPKC gamma membrane translocation might be involved in the neuroprotection. (C) 2008 Elsevier Ireland Ltd. All rights reserved.