Cytosolic zinc release and clearance in hippocampal neurons exposed to glutamate - the role of pH and sodium

P>Although Zn2+ homeostasis in neurons is tightly regulated and its destabilization has been linked to a number of pathologies including Alzheimer's disease and ischemic neuronal death, the primary mechanisms affecting intracellular Zn2+ concentration ([Zn2+](i)) in neurons exposed to excitotoxic stimuli remain poorly understood. The present work addressed these mechanisms in cultured hippocampal neurons exposed to glutamate and glycine (Glu/Gly). [Zn2+](i) and intracellular Ca2+ concentration were monitored simultaneously using FluoZin-3 and Fura-2FF, and intracellular pH (pH(i)) was studied in parallel experiments using 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein. Glu/Gly applications under Na+-free conditions (Na+ substituted with N-methyl-d-glucamine+) caused Ca2+ influx, pH(i) drop, and Zn2+ release from intracellular stores. Experimental maneuvers resulting in a pH(i) increase during Glu/Gly applications, such as stimulation of Na+-dependent pathways of H+ efflux, forcing H+ efflux via gramicidin-formed channels, or increasing extracellular pH counteracted [Zn2+](i) elevations. In the absence of Na+, the rate of [Zn2+](i) decrease could be correlated with the rate of pH(i) increase. In the presence of Na+, the rate of [Zn2+](i) decrease was about twice as fast as expected from the rate of pH(i) elevation. The data suggest that Glu/Gly-induced cytosolic acidification promotes [Zn2+](i) elevations and that Na+ counteracts the latter by promoting pH(i)-dependent and pH(i)-independent mechanisms of cytosolic Zn2+ clearance.