Vitamin E enhances Ca2+-mediated vulnerability of immature cerebellar granule cells to ischemia

The effects of vitamin E on lipid peroxidation, intracellular free Ca2+ concentration ([Ca2+]i), and cell death were investigated in the postischemic immature cerebellum. Deprivation of oxygen and glucose for 10-min in a suspension of freshly dissociated granule cells from the cerebellum of 9-day-old male rat pups resulted in a recovery-induced consumption of cell nonenzymatic antioxidants (ascorbic acid, glutathione, and α-tocopherol) and development of membrane lipid peroxidation as measured by the thiobarbituric acid method. The rate of lipid peroxidation of the postischemic cells was stimulated, not reduced, by treatment of the cells with vitamin E (5–30 μM α-tocopherol phosphate). In flow-cytometric studies a 10-min period of ischemia resulted in a small increase in intracellular calcium concentration, lipid peroxidation products and cell death, but in the presence of α-tocopherol the same treatment caused a dramatic increase in cell death, accompanied by a large increase in [Ca2+]i and lipid peroxidation products. Pretreatment of the cells with a mixture of three antioxidants (vitamin C/rutin/ubiquinol-10, 10/5/1) or nickel (Ni2+) reduced the α-tocopherol-induced increases in [Ca2+]i, and cell death. Hydrogen peroxide (1 mM) and the water-soluble analogue of vitamin E, trolox (50 μM), mimicked the effect of vitamin E on lipid peroxidation in the postischemic cells. Pretreatment of the cells with the intracellular Ca2+ chelator, BAPTA-AM, reduced both the α-tocopherol-induced increase in [Ca2+]i and cell death. The effect of vitamin E on [Ca2+]i was age dependent and decreased abruptly during maturation of the cerebellum between the first and second weeks of life. Results of in vitro treatment of the immature cerebellar cells with the water-soluble form of vitamin E (α-tocopherol phosphate) suggest that, after consumption of cellular co-antioxidants, vitamin E may be converted to an α-tocopheroxyl radical, which acts as a toxic prooxidant as cellular bioenergetics deteriorate.