Cytosolic acidification and intracellular zinc release in hippocampal neurons

Abstract

In neurons exposed to glutamate, Ca2+ influx triggers intracellular Zn2+ release via an as yet unclear mechanism. Since glutamate induces a Ca2+-dependent cytosolic acidification, the present work tested the relationships among intracellular Ca2+ concentration ([Ca2+]i), intracellular pH (pHi), and [Zn2+]i. Cultured hippocampal neurons were exposed to glutamate and glycine (Glu/Gly), while [Zn2+]i, [Ca2+]i and pHi were monitored using FluoZin-3, Fura2-FF, and 2′,7′-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein, respectively. Glu/Gly applications decreased pHi to 6.1 and induced intracellular Zn2+ release in a Ca2+-dependent manner, as expected. The pHi 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 pHi drop. Namely, [Zn2+]i increased more in more highly acidified neurons. Inhibiting the mechanisms responsible for the Ca2+-dependent pHi 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 pHi 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 μM.

Zinc efflux through lysosomal exocytosis prevents zinc-induced toxicity

“…The degree of pH change necessary to cause an effect on Zn 2+ handling, by contrast, significantly exceeds that reported to be caused by Baf. A pH drop below 6.7 is required to trigger an increase in intracellular Zn 2+ according to one set of studies (Kiedrowski, 2012), whereas another set has shown that metallothioneins release Zn 2+ only after cytoplasmic pH drops below 5.0 (Jiang et al, 2000). Thus, the increase in cytoplasmic Zn 2+ caused by Baf likely correlates with the loss of lysosomal function, rather than cytosolic pH changes.…”

Neuronal acid‐induced [Zn2+]i elevations calibrated using the low‐affinity ratiometric probe FuraZin‐1

“…4a, the cells were loaded by an application of 0.1 μM Fura-2FF AM and 0.1 μM FluoZin-3 AM for 5 min only. Earlier data showed that following such loading, the intracellular concentrations of the probes are about 1 μM (Kiedrowski 2012). Since the NEM application caused [Zn 2+ ] i elevations that saturated Fura-2FF and almost saturated FluoZin-3 (Fig.…”

“…Milimolar concentration of Ca 2+ may also affect the FuraZin-1 signal (Kiedrowski 2012). This possibility was clarified in cortical neurons loaded with FuraZin-1 alone.…”

Zinc-Permeable Ion Channels: Effects on Intracellular Zinc Dynamics and Potential Physiological/Pathophysiological Significance

“…Of special note, FluoZin-3 is insensitive to Ca 2+ , which has a significant advantage over previous indicators that cannot differentiate between Zn 2+ and Ca 2+ signals. With the emergence of new indicators, examinations of Zn 2+ and Ca 2+ , simultaneously, using different fluorescent indicators have been reported [191, 192]. These advancements are likely to provide a better understanding of Zn 2+ regulation, conductance and functional importance.…”