Mitochondrial Sequestration and Ca2+-Dependent Release of Cytosolic Zn2+ Loads in Cortical Neurons

Abstract

The endogenous divalent cations, Ca2+ and Zn2+, are both highly toxic upon excessive glutamate triggered intracellular accumulation. Given apparent parallels in their neurotoxic mechanisms, the present study aimed to explore interactions between these cations, by examining effects of moderate intracellular Zn2+ loading on responses to subsequent Ca2+ influx. Cortical cultures were briefly exposed to high-K+ buffer in the presence or absence of Zn2+ (50–100 μM), to activate and permit a modestly toxic amount of Zn2+ to enter through VSCC. After 1 h, the cultures were loaded with fluorescent probes, and 2 h after the Zn2+ exposure, imaged before and after induction of Ca2+ entry or addition of other drugs. In Zn2+ preexposed cultures loaded with the Zn2+ probe, Newport Green, induction of Ca2+ entry through either VSCC or NMDA channels induced cytoplasmic release of sequestered Zn2+. The source of this Ca2+ dependent intracellular Zn2+ release appears largely to be mitochondria, as indicated by the ability of the mitochondrial protonophore, FCCP, the mitochondrial uncoupler, dinitrophenol with the K+ ionophore, valinomycin, or the inducer of mitochondrial permeability transition (mPT), phenylarsine oxide (PAO), to substitute for NMDA in triggering Zn2+ release. Suggesting functional consequences of mitochondrial Zn2+ uptake, Zn2+ preexposures resulted in long-lasting mitochondrial depolarization (assessed with rhodamine 123), and reduced mitochondrial reactive oxygen species generation (assessed with hydroethidine) in response to subsequent NMDA triggered Ca2+ influx.