Elevated intracellular zinc and altered proton homeostasis in forebrain neurons.
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 microM) in the presence of the Zn(2+)-selective ionophore pyrithione (20 microM) 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 microM), 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 microM) and pyrithione (20 microM) 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.
Department of Pharmacology, University of Pittsburgh School of Medicine, W1351 BST, Pittsburgh, PA 15261, USA.
K E
KE
Dineley
J B
JB
Brocard
I J
IJ
Reynolds
D000818:Animals; D000935:Antifungal Agents / Q000494:pharmacology; D002545:Brain Ischemia / Q000378:metabolism* / Q000503:physiopathology; D002118:Calcium / Q000378:metabolism; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D002478:Cells, Cultured; D005260:Female; D005333:Fetus; D006706:Homeostasis / Q000187:drug effects / Q000502:physiology; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D007424:Intracellular Fluid / Q000187:drug effects / Q000378:metabolism*; D007476:Ionophores / Q000494:pharmacology; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D011247:Pregnancy; D016548:Prosencephalon / Q000378:metabolism* / Q000503:physiopathology; D011522:Protons; D011725:Pyridines / Q000494:pharmacology; D051381:Rats; D013871:Thiones; D015854:Up-Regulation / Q000187:drug effects / Q000502:physiology; D015032:Zinc / Q000378:metabolism* / Q000633:toxicity
D000935:Antifungal Agents; D007476:Ionophores; D011522:Protons; D011725:Pyridines; D013871:Thiones; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; C008704:pyrithione; D015032:Zinc; D002118:Calcium