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Volume 17, Number 24, Issue of December 15, 1997 pp. 9554-9564
Measurement of Intracellular Free Zinc in Living Cortical Neurons: Routes of Entry
Received May 15, 1997; revised Sept. 26, 1997; accepted Sept. 30, 1997.
Stefano L. Sensi, Lorella M. T. Canzoniero, Shan Ping Yu, Howard S. Ying, Jae-Young Koh, Geoffrey A. Kerchner, and Dennis W. Choi Center for the Study of Nervous System Injury and Department of Neurology, Washington University School of Medicine, St. Louis, Missouri 63110
We used the ratioable fluorescent dye mag-fura-5 to measure intracellular free Zn2+ ([Zn2+]i) in cultured neocortical neurons exposed to neurotoxic concentrations of Zn2+ in concert with depolarization or glutamate receptor activation and identified four routes of Zn2+ entry. Neurons exposed to extracellular Zn2+ plus high K+ responded with a peak cell body signal corresponding to a [Zn2+]i of 35-45 nM. This increase in [Zn2+]i was attenuated by concurrent addition of Gd3+, verapamil,
-conotoxin GVIA, or nimodipine, consistent with Zn2+ entry through voltage-gated Ca2+channels. Furthermore, under conditions favoring reverse operation of the Na+-Ca2+ exchanger, Zn2+ application induced a slow increase in [Zn2+]i and outward whole-cell current sensitive to benzamil-amiloride. Thus, a second route of Zn2+ entry into neurons may be via transporter-mediated exchange with intracellular Na+. Both NMDA and kainate also induced rapid increases in neuronal [Zn2+]i. The NMDA-induced increase was only partly sensitive to Gd3+ or to removal of extracellular Na+, consistent with a third route of entry directly through NMDA receptor-gated channels. The kainate-induced increase was highly sensitive to Gd3+ or Na+ removal in most neurons but insensitive in a minority subpopulation ("cobalt-positive cells"), suggesting that a fourth route of neuronal Zn2+ entry is through the Ca2+-permeable channels gated by certain subtypes of AMPA or kainate receptors.
Key words: mag-fura-5; voltage-gated calcium channels; sodium-calcium exchanger; calcium; sodium; glutamate; AMPA; NMDA; neurotoxicity; global ischemia; hypoxia
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