Glutamate mobilizes [Zn2+]i through Ca2+-dependent reactive oxygen species accumulation

Liberation of zinc from intracellular stores contributes to oxidant-induced neuronal injury. However, little is known regarding how endogenous oxidant systems regulate intracellular free zinc ([Zn2+](i)). Here we simultaneously imaged [Ca2+](i) and [Zn2+](i) to study acute [Zn2+](i) changes in cultured rat forebrain neurons after glutamate receptor activation. Neurons were loaded with fura-2FF and FluoZin-3 to follow [Ca2+](i) and [Zn2+](i), respectively. Neurons treated with glutamate (100 mu M) for 10 min gave large Ca2+ responses that did not recover after termination of the glutamate stimulus. Glutamate also increased [Zn2+](i), however glutamate-induced [Zn2+](i) changes were completely dependent on Ca2+ entry, appeared to arise entirely from internal stores, and were substantially reduced by co-application of the membrane-permeant chelator TPEN during the glutamate treatment. Pharmacological maneuvers revealed that a number of endogenous oxidant producing systems, including nitric oxide synthase, phospholipase A(2), and mitochondria all contributed to glutamate-induced [Zn2+](i) changes. We found no evidence that mitochondria buffered [Zn2+](i) during acute glutamate receptor activation. We conclude that glutamate-induced [Zn2+](i) transients are caused in part by [Ca2+](i)-induced reactive oxygen species that arises from both cytosolic and mitochondrial sources.