Figure 5. Ca²⁺ attenuates mitochondrial Zn²⁺ accumulation despite exacerbating the consequent dysfunction.

Cultures were loaded with Newport Green, Rhod123 or HEt in 0 or 1.8 Ca²⁺ HSS, and exposed to high K⁺ with 300, 50,10 or 0 μM Zn²⁺ (as indicated, along with 10 μM MK-801, to inhibit Ca²⁺-entry via NMDA receptor activation); DTDP (60 μM), FCCP (1 μM) and/or apocynin (500 μM) were added as indicated. Traces represent mean ± SEM F_x/F₀ values for each dye and represents ≥ 5 experiments consisting of ≥ 120 neurons. Grey bars indicate time points of comparison (NS indicates No Significance, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, by two-tailed t-test [A, C] or by one-way ANOVA with Tukey post hoc [B]).

A). Presence of Ca²⁺ decreases neuronal and mitochondrial Zn²⁺ uptake: Note that presence of Ca²⁺ attenuated both cytosolic Zn²⁺ rise during the exposure and FCCP-induced mitochondrial Zn²⁺ release.

B). Ca²⁺ and Zn²⁺ synergistically induce mitochondrial dysfunction: Neurons loaded with Rhod123 (left) or HEt (right) were exposed to high K⁺/DTDP/MK-801 with 10 μM Zn²⁺ (blue), 1.8 mM Ca²⁺ (red) or with both Zn²⁺ and Ca²⁺ (purple) for 5 min, then washed as indicated. Apocynin was added to HEt-loaded neurons (right) to inhibit contributions from Ca²⁺-dependent NOX activation. Note that despite relatively little effects from Ca²⁺ and Zn²⁺ individually, together they induced significant mitochondrial dysfunction.

C). Overwhelming mitochondrial Zn²⁺ loading induces rapid mitochondrial depolarization: Neurons loaded with Rhod123 (left) or HEt (right) in 1.8 Ca²⁺ HSS were exposed to high K⁺/DTDP/MK-801/Ca²⁺, with 50 (blue) or 300 μM (red) Zn²⁺ for 5 min, followed by wash as indicated. Note that 300 μM Zn²⁺ induced greater loss of ΔΨ_m than 50 μM Zn²⁺, despite both inducing similar levels of ROS generation.