[Effects of cPKCβII/γ membrane translocation ischemic/hypoxic tolerance induced by morphine postconditioning in hippocampal slices].

OBJECTIVE: To determine whether or not morphine postconditioning can induce ischemic/hypoxic tolerance in neurons subjected to reperfusion injury after oxygen-glucose deprivation (OGD).

METHODS: Hippocampal slices of 400 µm thickness were prepared from healthy adult male BALB/c mice. The slices were incubated in oxygen-saturated ACSF without or with calcium, then were subjected to OGD for 20 min. After recovery, the samples were immersed in oxygenated artificial fluid for 2 hours in the presence or absence of morphine postconditioning at 3 µmol/L during the first 5 - 60 min. The assessment of slices injury was performed by a determination of the intensity of slice stain incubated with TTC (2% 2, 3, 5-triphenyltetrazolium chloride) and the leakage rate of LDH also evaluated. At the designated periods during incubation, some slices were immersed into liquid nitrogen for a later analysis of Western blot. The frozen slices were homogenized, sonicated and centrifuged to separate soluble and particulate proteins. 10% SDS-PAGE Western blot was used to identify the changes of membrane-specific translocation of cPKCβII/γ.

RESULTS: After reperfusion, the cell survival significantly decreased with the elongation of OGD (51.4%). The release rate of LDH (184.05%) significantly increased simultaneously. In hippocampal slices postconditioned with morphine for 20 - 60 min, the release rate of LDH (136%, 142%, 144%) significantly decreased as compared with the group OGD. In the hippocampal slices postconditioned with morphine for 10 - 30 min, the cell survival rate (64.9%, 69.9%, 63.5%) significantly increased as compared with reperfusion alone. cPKCγ of particulate fraction increased versus the control. And there was a corresponding decrease of cytosolic fraction. Morphine postconditioning significantly inhibited the cPKCγ isoform-specific membrane translocation. It declined from 136% in the group OGD to 123%, 118%, 114% in the group morphine 20 - 60 min. cPKCβII membrane translocation had no change.

CONCLUSION: Morphine postconditioning can induce ischemic tolerance in nerons. The protective mechanism may be through inhibiting the cPKCγ isoform-specific membrane translocation.