Blockade of the MCU increases cytosolic Zn²⁺ rises while attenuating consequent ROS production.

<div><p>Newport Green or HEt-loaded cultures were exposed to 300 μM Zn²⁺/pyrithione for 5 min alone (red) or after pre-treatment with and in the presence of RR (10 µM) (blue) as described (see Materials and Methods). </p> <p><b>A</b>: Time course of Zn²⁺ rises (assessed as Newport Green F_x/F₀). Traces show mean ± SD values from 4 experiments. </p> <p><b>B</b>: Quantification of Newport Green fluorescence changes. Values show Newport Green fluorescence changes ( F_x/F₀) at the times indicated on the traces shown in A, above. 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>: Time course of ROS generation (assessed as HEt F_x/F₀). Traces show mean ± SD values from 4 experiments. </p> <p><b>D</b>: Quantification of HEt fluorescence changes. Values show HEt fluorescence changes ( F_x/F₀) at the times indicated on the traces shown in C, above. Values represent means (± SEM) of the 4 experiments; * indicates difference from control condition (p< 0.01) by 2-tailed t test.</p></div

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></div

NADPH oxidase (NOX) inhibition attenuates acute Ca²⁺ - but not Zn²⁺-induced ROS production.

<div><p>HEt-loaded cultures were exposed to 100 μM NMDA (30 min) or 300 μM Zn²⁺/pyrithione (5 min) alone (red) or after pre-treatment with and in the presence of apocynin (500 µM), or in glucose-free media supplemented with pyruvate (15 mM) (blue) as described (see Materials and Methods). </p> <p><b>A</b>: Representative images of selected fields of neuronal cultures (“Brightfield”), and pseudocolor images (400x) of HEt fluorescence from these neurons before and 30 min following onset of exposure to NMDA (left) or Zn²⁺/pyrithione (right). The pseudocolor bar shows the 12-bit fluorescence intensity range.</p> <p><b>B</b>: Traces show time course of HEt ∆F, normalized to baseline values (F_x/F₀). Dashed lines show linear extrapolation of baseline. Traces show mean ± SD values from 4 experiments. </p> <p><b>C</b>: Quantification of HEt ∆F changes. Values show F_x/F₀ increases after subtraction of the extrapolated baseline value, 30 min after onset of the exposure. ∆F values each represent means (± SEM) of the 4 experiments; * indicates difference from NMDA alone (p< 0.001) by 2-tailed t test. </p></div