Elevated intracellular zinc and altered proton homeostasis in forebrain neurons

• Published in: Neuroscience 2002-01, Vol.114 (2), p.439-449
• Main Authors: Dineley, K.E, Brocard, J.B, Reynolds, I.J
• Format: Article
• Language: English
• Subjects: acidosis; Animals; Antifungal Agents - pharmacology; BCECF; bicarbonate exchange; Biochemistry and metabolism; Biological and medical sciences; Brain Ischemia - metabolism; Brain Ischemia - physiopathology; Calcium - metabolism; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone - pharmacology; Cells, Cultured; Central nervous system; Female; Fetus; Fundamental and applied biological sciences. Psychology; Homeostasis - drug effects; Homeostasis - physiology; Hydrogen-Ion Concentration - drug effects; Intracellular Fluid - drug effects; Intracellular Fluid - metabolism; Ionophores - pharmacology; microfluorimetry; Neurons - drug effects; Neurons - metabolism; Pregnancy; Prosencephalon - metabolism; Prosencephalon - physiopathology; Protons; Pyridines - pharmacology; Rats; sodium–calcium exchange; Thiones; Up-Regulation - drug effects; Up-Regulation - physiology; Vertebrates: nervous system and sense organs; Zinc - metabolism; Zinc - toxicity
• ISSN: 0306-4522; 1873-7544
• DOI: 10.1016/S0306-4522(02)00294-4

Using the H +-sensitive fluorophore 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) and microfluorimetry, we investigated how elevated intracellular free zinc ([Zn 2+] i) altered intracellular proton concentration (pH i) in dissociated cultures of rat forebrain neurons. Neurons exposed to extracellular zinc (3 μM) in the presence of the Zn 2+-selective ionophore pyrithione (20 μM) underwent intracellular acidification that was not reversed upon washout of the stimulus. Application of a membrane-permeant Zn 2+ chelator, but not an impermeant chelator, partially restored pH i. Removal of extracellular Ca 2+ greatly inhibited [Zn 2+] i-induced acidification, suggesting that acidification was a secondary consequence of Ca 2+ entry. Additional experiments suggested that Ca 2+ entered through the plasma membrane sodium/calcium exchanger (NCE), because a specific inhibitor of reverse mode NCE operation, KB-R7943 (1 μM), significantly inhibited Zn 2+-induced acidification. In addition to the phenomenon of [Zn 2+] i-induced acidification, we found that elevated [Zn 2+] i inhibited neuronal recovery from low pH i. Neurons exposed to a protonophore underwent robust acidification, and pH i recovery ensued upon protonophore washout. In contrast, neurons acidified by the protonophore in the presence of Zn 2+ (3 μM) and pyrithione (20 μM) showed no ability to recover from low pH i. Application of a membrane-permeant Zn 2+ chelator partially restored pH i to pre-stimulus values. Experiments designed to elucidate mechanisms responsible for pH i regulation revealed that neurons relied primarily on bicarbonate exchange for proton export, suggesting that elevated [Zn 2+] i might impede pH i by inhibiting proton efflux via bicarbonate exchange. These results provide novel insights into the physiological effects of raising [Zn 2+] i, and may help illuminate the mechanisms by which Zn 2+ injures neurons.