Cytosolic acidification and intracellular zinc release in hippocampal neurons

In neurons exposed to glutamate, Ca²⁺ influx triggers intracellular Zn²⁺ release via an as yet unclear mechanism. As glutamate induces a Ca²⁺-dependent cytosolic acidification, the present work tested the relationships among intracellular Ca²⁺ concentration ([Ca²⁺](i)), intracellular pH (pH(i) ), and [Zn²⁺](i). Cultured hippocampal neurons were exposed to glutamate and glycine (Glu/Gly), while [Zn²⁺](i), [Ca²⁺](i) and pH(i) were monitored using FluoZin-3, Fura2-FF, and 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein, respectively. Glu/Gly applications decreased pH(i) to 6.1 and induced intracellular Zn²⁺ release in a Ca²⁺-dependent manner, as expected. The pH(i) drop reduced the affinity of FluoZin-3 and Fura-2-FF for Zn²⁺. The rate of Glu/Gly-induced [Zn²⁺](i) increase was not correlated with the rate of [Ca²⁺](i) increase. Instead, the extent of [Zn²⁺](i) elevations corresponded well to the rate of pH(i) drop. Namely, [Zn²⁺](i) increased more in more highly acidified neurons. Inhibiting the mechanisms responsible for the Ca²⁺-dependent pH(i) drop (plasmalemmal Ca²⁺ pump and mitochondria) counteracted the Glu/Gly-induced intracellular Zn²⁺ release. Alkaline pH (8.5) suppressed Glu/Gly-induced intracellular Zn²⁺ release whereas acidic pH (6.0) enhanced it. A pH(i) drop to 6.0 (without any Ca²⁺ influx or glutamate receptor activation) led to intracellular Zn²⁺ release; the released Zn²⁺ (free Zn²⁺ plus Zn²⁺) bound to Fura-2FF and FluoZin-3) reached 1 microM.