Decrease of Na(+)-Ca2+ exchange activity by ascorbate in rat brain membrane vesicles.

Na(+)-dependent Ca2+ uptake in rat brain microsomal membrane vesicles was inhibited by preincubating the vesicles with ascorbic acid at 0.1-10 mM. The inhibitory effect of ascorbate was blocked by simultaneous addition of ascorbate oxidase. The decrease in activity was not reversed upon removing the ascorbate. The kinetic study showed that the treatment with ascorbate decreased Bmax without a change in Km for Ca2+. The inhibitory effect by ascorbate was also observed in membrane vesicles derived from osmotically shocked synaptosomes and in reconstituted membrane vesicles. The effect by ascorbate was specific: it did not affect either ATP-dependent Ca2+ uptake in the presence of o-phenanthroline, an inhibitor of lipid peroxidation, or Na(+)-dependent glutamate uptake in the membrane vesicles. The activity of Na(+)-Ca2+ exchange was also decreased by isoascorbic acid, but not by ascorbate 2-sulfate at 1 mM. The treatment with glutathione or 2-mercaptoethanol did not affect the Na(+)-Ca2+ exchange activity, while 1 mM dithiothreitol caused the inhibition which was completely blocked by o-phenanthroline. The effect of ascorbate on Na(+)-dependent Ca2+ uptake was observed even under the conditions which suppress peroxidation of membrane phospholipids.