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-microm 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 microM) within 20 min of OGD, which was abolished by concomitant incubation with non-selective opioid receptor antagonist naloxone (Nal, 50 microM). The results of Western blot analysis showed that only cPKCgamma membrane translocation, not alpha, betaI and betaII, increased under the condition of OGD 10 min and 2h reperfusion (OGD/2h), and this increment of cPKCgamma membrane translocation was inhibited by MP pretreatment. To further elucidate the role of cPKCgamma in MP-induced neuroprotection, we found that cPKCgamma 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 cPKCgamma membrane translocation, but had no further effect on MP-induced inhibition of cPKCgamma membrane translocation. These results demonstrated that MP can reduce OGD-induced neuronal injuries, and the down-regulation of cPKCgamma membrane translocation might be involved in the neuroprotection.
Liu
Ya
Y
Department of Anesthesiology, Capital Medical University Affiliated Beijing Tongren Hospital, Beijing 100730, China.
Li
Junfa
J
Yang
Jing
J
Ji
Fang
F
Bu
Xiangning
X
Zhang
Nan
N
Zhang
Bingxi
B
D000818:Animals; D002470:Cell Survival / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005947:Glucose / Q000172:deficiency; D006624:Hippocampus / Q000187:drug effects*; D000860:Hypoxia / Q000201:enzymology / Q000473:pathology; D066298:In Vitro Techniques; D019194:Ischemic Preconditioning; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D009020:Morphine / Q000008:administration & dosage*; D018696:Neuroprotective Agents / Q000008:administration & dosage*; D011493:Protein Kinase C / Q000378:metabolism*; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D013997:Time Factors
D004791:Enzyme Inhibitors; D018696:Neuroprotective Agents; D009020:Morphine; C087857:protein kinase C gamma; D011493:Protein Kinase C; D005947:Glucose