Under conditions of oxidative stress and disrupted Zn²⁺ buffering, lower levels of Zn²⁺ influx result in mitochondrial Zn²⁺ entry and ROS production.

<div><p><b>A</b>: Effect of DTDP on Zn²⁺-triggered ROS generation. HEt-loaded cultures were exposed to 50 μM Zn²⁺/pyrithione alone (red), in the presence of DTDP (100 µM, blue), or with both DTDP and RR (10 µM, black) as indicated. Note that the Zn²⁺/pyrithione exposure only induced ROS generation in the presence of DTDP, and that the ROS production was eliminated by RR. Traces show mean ± SD values from 4 experiments.</p> <p><b>B</b>: Quantification of HEt fluorescence changes. Values show HEt fluorescence changes (F_x/F₀) at the times indicated on the traces shown in A. Values represent means (± SEM) of the 4 experiments; * indicates difference from control condition (p< 0.01) by 2-tailed t test.</p> <p><b>C</b>, <b>D</b>: Disruption of cytosolic Zn²⁺ buffering by DTDP markedly increases cytosolic Zn²⁺ rises and uptake into mitochondria. Fluo-Zin3-loaded cultures were exposed to 1 μM Zn²⁺ with 90 mM K⁺ (“Zn²⁺/high-K⁺”, to trigger a low level of Zn²⁺ influx), to FCCP (1 µM), or to DTDP (100 µM) as indicated. In C, when the cultures were first exposed to Zn²⁺/high-K⁺ there was a very small FluoZin-3 ∆F, and subsequent FCCP exposure, to depolarize the mitochondria and release mitochondrially-sequestered Zn²⁺ into the cytosol, caused only a slight further increase. However, adding DTDP after the FCCP produced a large FluoZin-3 ∆F, suggesting that the Zn²⁺ entering during the Zn²⁺/high-K⁺ exposure had been largely buffered in the cytosol with little entering the mitochondria. In contrast (<b>D</b>), when the cultures were first exposed to DTDP (100 µM), there was a minimal FluoZin-3 ∆F, but when the DTDP exposure was followed by Zn²⁺/high-K⁺, the cytosolic ∆F was dramatically increased, and subsequent FCCP exposure resulted in a marked further ∆F, indicative of Zn²⁺ having accumulated within the mitochondria. Traces show mean ± SD values from 120 neurons from 4 experiments.</p> <p><b>E</b>: 50 μM Zn²⁺/high-K⁺ exposure cause mitochondrial ROS production only in the presence of DTDP. HEt-loaded cultures were exposed to 50 μM Zn²⁺/90 mM K⁺ alone (red) or after pre-treatment with and in the presence of DTDP alone (100 µM, blue) or with RR (10 µM, black) as indicated. Traces represent time course of HEt ∆F, normalized to baseline values (F_x/F₀) and show mean ± SD values from 4 experiments. </p> <p><b>F</b>: Quantification of HEt fluorescence changes. Values show HEt fluorescence changes (F_x/F₀) at the times indicated on the traces shown in E. Values represent means (± SEM) of the 4 experiments; * indicates difference from control condition (p< 0.01) by 2-tailed t test.</p>