Increased isoform-specific membrane translocation of conventional and novel protein kinase C in human neuroblastoma SH-SY5Y cells following prolonged hypoxia.

Several studies have suggested that protein kinase C (PKC) plays a key role in the mechanism of cerebral ischemic/hypoxic preconditioning (I/HPC). However, detailed information regarding PKC isoforms in response to brain ischemia/hypoxia and their potential role in neuroprotection is unclear. Previous studies in our laboratory have demonstrated that the levels in membrane translocation of conventional PKC (cPKC) betaII, gamma, and novel PKCepsilon (nPKC), but not cPKCalpha, betaI, nPKCdelta, eta, mu, theta, and atypical PKC (aPKC) zeta and iota/lambda, were increased significantly in the hippocampus and cortex of intact mice with hypoxic preconditioning. To further detect cPKC and nPKC isoforms activation following prolonged hypoxia in vitro, we tested the membrane translocation (an indicator of PKC activation) of cPKCalpha, betaI, betaII, and gamma, and nPKCdelta, epsilon, eta, mu, and theta in a human neuroblastoma SH-SY5Y cell line following sustained hypoxic exposure (1% O(2)/5% CO(2)/94% N(2)). Using Western blot and immunocytochemistry methods, we found that the levels of cPKCalpha, betaI, betaII, and nPKCepsilon, but not nPKCdelta, eta, mu, and theta, membrane translocation were increased significantly (P < 0.05, n = 8) in a time-dependent manner (from 0.5 to 24 h) following sustained hypoxic exposure. Similarly, the immunostaining experiment also showed a noticeable translocation of cPKCalpha, betaI, betaII, and nPKCepsilon from the cytosol to the perinuclear or membrane-related areas after 6 h posthypoxic exposure. In addition, no cPKCgamma was detected in this cell line under either a normoxic or hypoxic condition. These results suggested that prolonged hypoxia may induce the activation of cPKCalpha, betaI, betaII, and nPKCepsilon by triggering their membrane translocation in SH-SY5Y cells.