Calcium-sensitive fluorescent dyes can report increases in intracellular free zinc concentration in cultured forebrain neurons.
High concentrations of Zn2+ are found in presynaptic terminals of excitatory neurons in the CNS. Zn2+ can be released during synaptic activity and modulate postsynaptic receptors, but little is known about the possibility that Zn2+ may enter postsynaptic cells and produce dynamic changes in the intracellular Zn2+ concentration ([Zn2+]i). We used fura-2 and magfura-2 to detect the consequences of Zn2+ influx in cultured neurons under conditions that restrict changes in intracellular Ca2+ and Mg2+ concentrations. The resulting ratio changes for both dyes were reversed completely by the Zn2+ chelator, N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine, indicating that these dyes are measuring changes in [Zn2+]i. We found that fura-2 was useful in measuring small increases in [Zn2+]i associated with exposure to Zn2+ alone that may be mediated by a Na+/Ca2+ exchanger. Magfura-2, which has a lower affinity for Zn2+, was more useful in measuring larger agonist-stimulated increases in [Zn2+]i. The coapplication of 300 microM Zn2+ and 100 microM glutamate/10 microM glycine resulted in a [Zn2+]i increase that was approximately 40-100 nM in magnitude and could be inhibited by the NMDA receptor antagonist, MK-801 (30 microM), or extracellular Na+. This suggests that Zn2+ influx can occur through at least two different pathways, leading to varying increases in [Zn2+]i. These findings demonstrate the feasibility of measuring changes in [Zn2+]i in neurons.
Cheng
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Department of Pharmacology, University of Pittsburgh School of Medicine, Pennsylvania 15261, USA.
Reynolds
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D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D004338:Drug Combinations; D005456:Fluorescent Dyes; D016257:Fura-2 / Q000031:analogs & derivatives*; D018698:Glutamic Acid / Q000494:pharmacology; D005998:Glycine / Q000494:pharmacology; D007425:Intracellular Membranes / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism*; D009994:Osmolar Concentration; D016548:Prosencephalon / Q000166:cytology / Q000378:metabolism*; D051381:Rats / Q000196:embryology; D017207:Rats, Sprague-Dawley; D015032:Zinc / Q000378:metabolism* / Q000494:pharmacology
D004338:Drug Combinations; D005456:Fluorescent Dyes; C058776:2-(2-(5-carboxy)oxazole)-5-hydroxy-6-aminobenzofuran-N,N,O-triacetic acid; D018698:Glutamic Acid; D015032:Zinc; D002118:Calcium; D005998:Glycine; D016257:Fura-2