Effect of morphine preconditioning on the changes of CaMKIIand NMDA receptors in mice hippocampal slices induced by oxygen-glucose deprivation and recovery

Objective To investigate the effects of morphine preconditioning on the changes of membrane translocation of calcium/calmodulin-dependent kinaseⅡ(CaMKⅡ) and phosphorylation of NMDA receptors in hippocampal slices induced by oxygen-glucose deprivation and recovery (OGDR) .Methods Hippocampal slices (400μm thick) were prepared from adult BALB/C mice of both sexes and incubated in normal artificial cerebrospinal fluid (nACSF) aerated with 95% O2 and 5% CO2 at 37℃for 30 min. The slices were then divided into 5 groups:groupⅠcontrol (C); groupⅡOGDR; groupⅢmorphine preconditioning + OGDR (M); groupⅣnaloxone + morphine preconditioning + OGDR (N + M) and groupⅤnaloxone preconditioning + OGDR (N). In groupⅡ,Ⅲ,ⅣandⅤthe hippocampal slices were deprived of O2 and glucose for 20 min followed by 2 h reoxygenation and glucose supply. The slices were treated with morphine 3μmol·L-1×30 min (groupⅢ) or naloxone 50μmol·L-1×30 min followed by naloxone 50μmol·L-1 + morphine 3μmol·L-1×30 min (groupⅣ) or naloxone 50μmol·L-1×1 h (groupⅤ) before OGDR. The slices were homogenized, supersonicated and centrifuged to separate particulate, cytosolic and total protein immediately before OGDR (T0), at the end of 20 min OGDR (T1) and at 1 h (T2) and 2 h (T3) of reoxygenation and glucose supply for determination of the changes of membrane translocation and total protein of CaMKⅡαand the level of phosphorylation of NRI subtype of NMDA receptors in hippocampal slices (Western blot) . Results The levels of CaMKⅡαwere significantly increased in the paniculate fraction but decreased in the cytosolic fraction at T1 , T2, and T3 as compared with the basaeline values at T0 , while the levels of phosphorylation of NR1 subtype of NMDA receptors were significantly increased at T2 and T3 as compared with the baseline values at T0 in OGDR group. Morphine preconditioning significantly inhibited the changes of the membrane translocation of CaMKⅡαand phosphorylation of NR1 subtype of NMDA receptors induced by OGDR. The inhibitory effects of morphine preconditioning were completely blocked by naloxone. There was no significant change in protein expression of CaMKⅡα.Conclusion Inhibition of membrane translocation of CaMKⅡαand phosphorylation of NMDA receptors by morphine preconditioning plays an important role in the neuroprotection against ischemia-reperfusion injury.