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

https://doi.org/10.1016/S0891-5849(98)00157-9Get rights and content

Abstract

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.

Introduction

Brain damage in ischemia is one of the leading causes of death and chronic disability. Clinical and experimental data have suggested that ischemic/reperfusion neuronal injury is at least partly due to oxidative damage caused by lipid peroxidation [1]. Several authors have demonstrated that α-tocopherol, the most active form of vitamin E in vivo, protects the vulnerable hippocampal and other cerebral neurons from ischemic damage via free-radical and active-oxygen scavenging [2], [3], while vitamin E deficiency promotes neuronal damage [4], [5].

Our laboratory has been investigating the mechanisms of ischemia-induced neuronal cell death [6], [7]. In the process of study of the effects of antioxidants, we have made the surprising observation that in the immature cerebellum, which is particularly active in the metabolism and utilization of vitamin E [8], vitamin E shows exactly the opposite of the expected action against ischemia. Using acutely isolated cerebellar granule cells, we find that the water-soluble form of vitamin E, α-tocopherol phosphate, increases cell death resulting from an ischemic challenge. This finding has particular importance, because low, physiological concentrations of vitamin E were sufficient for potentiation of ischemic and reperfusion neuronal injury.

The purpose of this study was to determine the mechanism of α-tocopherol mediated enhancement of the negative consequences of transient ischemia in the immature cerebellum. The effect of the transient ischemia on lipid peroxidation and the concentration of cell antioxidants, as well as the effect of different concentrations of α-tocopherol on lipid peroxidation in control and postischemic cells was investigated, because the prooxidant/antioxidant action of vitamin E may depend on its concentration and the concentration of co-antioxidants [9]. Because vitamin E can affect ion channels of neuronal membranes [10], and because elevations of intracellular calcium are known to be related to neuronal cell death in ischemia [11], the role of intracellular Ca2+ in α-tocopherol-induced mortality of postischemic cerebellar granule cells also was examined.

Section snippets

Cell preparation and transient ischemia

Adult Wistar rats from a breeding colony maintained by the Wadsworth Center were fed a commercial diet from Agway (Syracuse, NY, USA) containing vitamin E at 55 IU/kg and vitamin C at 1045 IU/kg diet. Because postischemic neuronal necrosis can depend on sex [12], only male Wistar rat pups were used for experiments at 9 days of age. In addition, the age dependence of the effect of vitamin E on the mean concentration of intracellular free calcium was measured in rat pups of different ages (from

Results

Vitamin E (10 μM α-tocopherol phosphate) did not affect viability (Fig. 1 , curves A and B), [Ca2+]i, or lipid peroxidation of nonischemic cerebellar granule cells. The vast majority of the cells (90%) were not vulnerable to a 3-min ischemic episode followed by a 1-h reoxygenation (recovery), but a 10-min oxygen and glucose deprivation followed by a 1-h reoxygenation caused an increase in cell death (Fig. 1, curve C). The cell concentrations of ascorbic acid, glutathione, and α-tocopherol were

Discussion

Under normal metabolic conditions, oxygen radicals formed by mitochondria and other cell structures are constantly scavenged by endogenous antioxidants (vitamin E, vitamin C, beta-carotene, glutathione, etc.) and antioxidative enzymes (superoxide dismutase, glutathione peroxidase, catalase, etc.). Oxygen deprivation, glycolytic inhibition, and reoxygenation following ischemia of immature cerebellar granule cells in Tyrode’s solution caused a decrease in the concentration of antioxidants, an

Acknowledgements

This work was supported in part by National Institutes of Health Grants NS 23807 and ES04913 (D.O.C.), and supported in part by the Boehringer Ingleheim Funds (R.L.). We thank Ms. Cythnia Stoner for assistance with early experiments.

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