Role of membrane lipids in regulation of vulnerability to oxidative stress in PC12 cells: implication for aging

Previously, we reported that PC12 cells showed increased vulnerability to oxidative stress (OS) induced by H2O2 (as assessed by decrements in calcium recovery, i.e., the ability of cells to buffer Ca2+ after a depolarization event) when the membrane levels of cholesterol (CHL) and sphingomyelin (SPH) were modified to approximate those seen in the neuronal membranes of old animals. The present study was designed to examine whether the enrichment of the membranes with SPH-CHL and increased cellular vulnerability to OS are mediated by neutral SPH-specific phospholipase C (N-Sase) and the intracellular antioxidant GSH. The results showed a significant up-regulation of N-Sase activity by both low (5 μM) and high (300 μM) doses of H2O2. However, under high doses of H2O2 the up-regulation of N-Sase is accompanied by a significant increase in reactive oxygen species and by a decrease in intracellular GSH. The enrichment of membranes with SPH-CHL significantly potentiated the effects of high doses of H2O2, by further reducing the intracellular GSH and further up-regulating the N-Sase activity. Furthermore, repleting intracellular GSH with 20 mM N-acetylcysteine treatment was sufficient to attenuate the effect of a low dose of H2O2 on Ca2+ recovery in SPH-CHL-treated cells. Thus, these results suggested that age-related alterations in the membrane SPH-CHL levels could be important determinants of the susceptibility of neuronal cells to OS.