- Email: [email protected]
Measurement of Intracellular Free Zinc in Living Neurons
Neurobiology of Disease 4, 275–279 (1997) Article No. NB970160
MINIREVIEW Measurement of Intracellular Free Zinc in Living Neurons L. M. T. Canzoniero, S. L. Sensi, and D. W. Choi1 Center for the Study of Nervous System Injury and Department of Neurology, Washington University School of Medicine, St. Louis, Missouri 63110 Received August 21, 1997, accepted for publication August 21, 1997
Excessive Zn21 influx has been implicated in the pathogenesis of neuronal death after global ischemia or prolonged seizures, but little is presently known about cellular regulation of intracellular free Zn21 ([Zn21]i). In large part, this is because the tools currently available for measuring [Zn21]i are limited in comparison to those available for measuring [Ca21]i or other ions. We outline here approaches to this task that have been taken in the past, and summarize our recent experience using mag-fura-5 to measure [Zn21]i in living cortical neurons exposed to toxic levels of extracellular Zn21. r 1997 Academic Press
Key Words: fura; magfura; fluorescence; calcium channel; glutamate; neurotoxicity; ischemia.
Zn21 AS AN IMPORTANT REGULATOR OF BIOLOGICAL FUNCTIONS Zn21 is an essential component of many metalloproteins, including metalloenzymes and gene transcription factors (Vallee and Falchuk, 1993; Berg and Shi, 1996). Zn21 is also likely a mediator of central neural signaling at excitatory synapses (Frederickson, 1989; Frederickson and Moncrieff, 1994; Smart et al., 1994). Large amounts of chelatable Zn21 are present throughout the mammalian brain, predominantly stored in vesicles within excitatory nerve terminals (Danscher, 1984; Perez-Clausell and Danscher, 1985). This synaptic Zn21 can be released in a Ca21-dependent fashion with neuronal activity (Howell et al., 1984) and may achieve hundred micromolar concentrations in the extracellular space (Assaf and Chung, 1984). Once released, Zn21 may have functionally important effects on the behavior of multiple voltage- or ligand-gated channels (Smart et al., 1994; Harrison and Gibbons, 1994). Given the systematic colocalization of 1
To whom correspondence should be addressed at Department of Neurology, Box 8111, 660 S. Euclid, St. Louis, MO 63110. Fax: 314-362-9462. E-mail: [email protected]. 0969-9961/97 $25.00 Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.
Zn21 to glutamatergic terminals, its powerful ability to downmodulate N-methyl-D-aspartate (NMDA) receptors may be of particular significance (Peters et al., 1987; Westbrook and Mayer, 1987; Christine and Choi, 1990). Furthermore, Zn21 is also a potent blocker of certain forms of GABAA receptors lacking a g subunit (Westbrook and Mayer, 1987; Smart et al., 1991; Draguhn et al., 1990), TTX-insensitive voltage-gated Na1 channels (White et al., 1993), and T type Ca21 channels (Takahashi and Akaike, 1991) and can modulate the gating of certain K1 channels, especially hippocampal A currents (Harrison et al., 1993) and ATP-regulated K1 channels (Kwok and Kass, 1993). Excessive exposure to high concentration of Zn21 can kill cultured cortical neurons (Yokoyama et al., 1986). Previous studies suggest that this neurotoxicity is mediated by Zn21 influx, in large part through voltage-gated Ca21 channels (Weiss et al., 1993), as well as through NMDA receptor-mediated channels (Koh and Choi, 1994) and Ca21-permeable AMPA/kainate receptor-gated channels (Yin and Weiss, 1995). In vivo experiments have suggested that Zn21 can translocate from presynaptic terminals into the postsynaptic cell bodies of neurons injured after prolonged seizures (Sloviter, 1985; Friedrickson, 1989; Suh et al., 1996)
275
276 or transient global ischemia (Tonder et al., 1990; Koh et al., 1996). Blockade of ischemia-induced (Koh et al., 1996) or seizure-induced (Suh et al., 1996) Zn21 translocation by administration of the membrane-impermeable chelator CaEDTA markedly reduces resultant neuronal death.
Canzoniero, Sensi, and Choi
Zinquin fluorescence, even when the neurons were challenged with high concentrations of extracellular Zn21 in the presence of the Zn21-specific ionophore Na1 pyrithione. In contrast, we found Zinquin to work as an indicator of [Zn21]i in HEK-293 cells. Possibly, the failure of Zinquin to work as an indicator within cortical neurons reflects a failure of ethyl ester cleavage and subsequent dye trapping.
MEASUREMENT OF [Zn21]i It would be of interest to know what levels of intracellular cytosolic free Zn21 concentration ([Zn21]i ) are associated with toxic Zn21 influx. Unfortunately, the tools presently available for measuring dynamic changes in [Zn21]i are limited in comparison to those available for measuring intracellular free Ca21 or other ions. Most measurements of brain Zn21 to date have employed techniques requiring tissue fixation, such as Timm’s sulfide staining (Sloviter, 1985) or selenium (Danscher, 1984). In several studies a fluorescentspecific Zn21 dye (TS-Q), 6-methoxy-8-p-toluene sulfonamide quinoline, has been used to visualize Zn21 accumulation in cortical neurons and brain slices. While this Zn21-selective nonratioable fluorescent dye has provided valuable qualitative information about the location of chelatable Zn21 in cells and tissues (Frederickson et al., 1987; Weiss et al., 1993; Yin and Weiss, 1995; Koh et al., 1996), limitations of solubility (requiring very alkaline loading buffers) as well as possible complex interactions with Zn21 bound to membranes have precluded quantitative detection of changes of [Zn21]i in individual living cells (Andrews et al., 1995). Recently, other quinoline derivatives have been developed such as: (1) N-(6-methoxy-8-quinoylyl-carboxybenzoylsulfonamide) (TFLZn) and (2) D-methyl8-p-toluenesulphonamido-6-quinolyloxyacetic acid (Zinquin). Budde et al. (1997) used the water-soluble dye TFLZn to measure changes in [Zn21]i in mossy fiber boutons following electric stimulation in slices. However, in cultured neurons, TFLZn’s low affinity for Zn21 (Kd , 20 µM ) and poorly retention within cells represent major limitations. Zinquin has been successfully employed to examine [Zn21]i in thymocytes, human CLL cells, pancreatic islet cells, and hepatocytes (Zalewski et al., 1993, 1994; Brand and Kleineke, 1996). This new quinoline derivative is available as an ethyl ester and it is highly specific for Zn21. However, its fluorescence is not evenly distributed within cells and its loading capacity is poor. Working with cultured cortical neurons, we were unable to detect changes in Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.
FURA-2 AS Zn21 INDICATOR? Another approach to detecting [Zn21]i is to utilize fura-2-based Ca21-sensitive fluorescent dyes. Fura-2 binds transition metals such as Zn21 with much higher affinity than Ca21, producing a similar (although not identical) shift in emitted fluorescence (Grynkiewicz et al., 1985). Fura-2 as Zn21 indicator has been employed to study D[Zn21]i in response to electrical stimulation in cardiac myocytes (Atar et al., 1995), to characterize depolarization-induced Zn21 entry in chromaffin cells (Vega et al., 1994), to monitor nuclear and cytosolic [Zn21]i changes in bovine liver cells (Hechtenberg and Beyersmann, 1993), and to study the effect of methyl mercury on [Zn21]i in synaptosomal preparation (Denny and Atchison, 1994). However, the main drawback of fura-2 as an indicator of [Zn21]i is of course its high sensitivity for Ca21 and the problem of confounding changes in [Ca21]i. Furthermore, its affinity for Zn21 (Kd for Zn21 about 2 nM) (Grynkiewicz et al., 1985) is too high for assessment of D[Zn21]i in neurons challenged with neurotoxic concentrations of extracellular Zn21 (see below).
LOW-AFFINITY Ca21 INDICATORS AS [Zn21]i INDICATORS Simons (1993) reported that a low-affinity Ca21 and Mg21 indicator, mag-fura-2, could be used to measure Zn21 in solution over the 1–100 nM range, even in the presence of moderate Ca21, reflecting the dye’s higher affinity for Zn21 (Kd 20 nM) over Ca21 or Mg21. We explored the use of another low-affinity Ca21 indicator, mag-fura-5 (Kd for Ca21 5 20 µM and for Mg21 5 2.6 mM) (Petrozzino et al., 1995; Del Bono and Stefani, 1993), for real-time measurements of D[Zn21]i in living cortical neurons (Canzoniero et al., 1997). We chose mag-fura-5 instead of mag-fura-2 because the former has a slightly further reduced affinity for Ca21 or Mg21 (Haugland, 1996). We estimated the Kd of mag-fura-5 for Zn21 to be ,27 nM from cuvette calibration experiments.
277
Neuronal Intracellular Free Zinc
Using mag-fura-5, we estimated that the D[Zn21]i in neuronal cell bodies induced by exposure to 100 µM glutamate plus 100 µM Zn21 in the absence of extracellular Ca21 or Mg21 was in the range of 40–50 nM (Fig. 1). Control experiments supported the conclusion that this mag-fura-5 ratio signal indeed reflected D[Zn21]i. The ratio signal was induced by application of a Zn21-selective ionophore, Na pyrithione, but only when extracellular Zn21 was present. Furthermore, this Na pyrithione/Zn21 signal was quenched rapidly upon subsequent addition of the specific cell-permeable Zn21 chelator (N,N,N8,N 8-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN). We considered it is unlikely that the signal was importantly confounded by detection of Ca21 or Mg21. In the absence of extracellular Ca21, prior studies have not suggested that intracellular Ca21 levels in neuronal soma would rise to the high micromolar levels needed to activate mag-fura-5 (estimated Kd for Ca21 approximately 20 µM, Petrozzino et al., 1995). Mg21 is a greater possibility, but arguing against the possibility of Ca21-triggered intracellular Mg21 release (Brocard et al., 1993), we saw similar changes in mag-fura-5 fluorescence in other experiments when neurons were exposed to Zn21 plus kainate instead of glutamate, and Brocard et al. (1993) did not observe any elevation of [Mg21]i after kainate stimulation.
The absence of extracellular Ca21 and Mg21 likely enhanced Zn21 influx over that which would occur under more physiological conditions, but in other experiments, we have found that including physiological levels of [Ca21]o and [Mg21]o typically reduces the increase in [Zn21]i by about half. Prior determinations of [Zn21]i in nonneuronal cells have generally reported a wide range of [Zn21]i. In erythrocytes, Simons (1991) using 65Zn21 flux estimated basal [Zn21]i to be from 1.5 to 32 pM, depending on the ionic composition of the extracellular solution; in human leukemic cells a basal [Zn21]i of approximately 1 nM was determined by 19F nuclear magnetic resonance (Adebodun and Post, 1995). In electrically stimulated chromaffin cells (Atar et al., 1995) [Zn21]i monitored by fura-2 increased from 0.4 to 2 nM. On the other side of the spectrum, in resting splenocytes and thymocytes Zinquin was utilized to arrive at an estimated [Zn21]i of approximately 20–50 µM (Zalewski et al., 1993) and [Zn21]i of 0.6–2.7 µM in hepatocytes removed from rats fed a high zinc diet (Brand and Kleineke, 1996). The comparatively modest increases in [Zn21]i seen in cortical neurons exposed to toxic concentrations of extracellular Zn21 may reflect the presence of powerful mechanisms for Zn21 homeostasis, i.e., buffering by metallothioneins (Maret, 1995; Ebadi et al., 1995; Masters et al., 1994) or extrusion via Zn21 transporters (Palmiter and Findley, 1995; Gunshin et al., 1997). Also, measurements of fluorescence shifts over neuronal cell bodies would not be expected to detect Zn21 entering in neurites, which might be substantial.
NOVEL Zn21-SENSITIVE FLUORESCENT DYES
FIG. 1. Glutamate-evoked increase in [Zn21]i. 100 µM glutamate was applied for 5 min together with 100 µM Zn21. TPEN (50 µM) was added at the indicated time. Each point is the mean 6 SEM of 87 neurons. The experiment was performed in a buffer lacking Ca21 and Mg21.
Other recently identified potential Zn21 indicators, Newport Green or APTRA-BTC (Haugland, 1996), have promising specificity but low-affinity (Kd for Zn21 approximately 1 and 1.4 µM, respectively). We tested Newport Green or APTRA-BTC. Both were successfully loaded into cortical neurons and exhibited strong responses after addition of Zn21 plus Na pyrithione. However, consistent with their low affinities for Zn21, few somatic changes were detectable after a depolarizing stimulus (500 µM kainate plus 300 µM Zn21 ). Perhaps these probes may be useful in evaluating changes of [Zn21]i in neuronal processes, if the changes there are larger than those in cell bodies. Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.
278
CONCLUSION In summary, mag-fura-5 appears to have utility for measuring [Zn21]i dynamically in living neurons. However, clearly such measurements will need to be interpreted cautiously and confirmed once truly Zn21selective ratioable dyes become available.
REFERENCES Adebodun, F., & Post, J. F. (1995) Role of intracellular free Ca(II) and Zn(II) in dexamethasone-induced apoptosis and dexamethasone resistance in human leukemic CEM cell lines. J. Cell. Physiol. 163, 80–86. Andrews, J. C., Nolan, J. P., Hammerstedt, R. H., & Bavister, B. D. (1995) Characterization of N-(6-methoxy-8 quinolyl)-p-toluenesulfonamide for the detection of zinc in living sperm cells. Cytometry 21, 153–159. Assaf, S. Y., & Chung, S. H. (1984) Release of endogenous Zn21 from brain tissue during activity. Nature 308, 734–736. Atar, D., Backx, P. H., Appel, M. M., Gao, W. D., & Marban, E. (1995) Excitation-transcription coupling mediated by zinc influx through voltage-dependent calcium channels. J. Biol. Chem. 270, 2473–2477. Berg, J. M., & Shi, Y. (1996) The galvanization of biology: A growing appreciation for the roles of zinc. Science 271, 1081–1085. Brand, I. A., & Kleineke, J. (1996) Intracellular zinc movement and its effect on the carbohydrate metabolism of isolated rat hepatocytes. J. Biol. Chem. 271, 1941–1949. Brocard, J. B., Rajdev, S., & Reynolds, I. J. (1993) Glutamate-induced increases in intracellular free Mg21 in cultured cortical neurons. Neuron 11, 751–757. Budde, T., Minta, A., White, J. A., & Kay, A. R. (1997) Imaging free zinc in synaptic terminals in live hippocampal slices. Neuroscience 79, 347–358. Canzoniero, L. M. T., Sensi, S. L., Yu, S. P., Ying, H. S., Koh, J. Y., Choi, D. W. (1997) Measurement of intracellular free zinc in cultured cortical neurons: inferred entry through calcium channels. 23, 473–511. Christine, C. W., & Choi, D. W. (1990) Effect on zinc on NMDA receptor-mediated channel currents in cortical neurons. J. Neurosci. 10, 108–116. Danscher, G. (1984) Do the Timm sulphide silver method and the selenium demonstrate zinc in the brain? In: The Neurobiology of Zinc (C. J. Frederickson, G. A. Howell, & E. J. Kasarkis, Eds.), pp 273–287. A. R. Liss, New York. Delbono, O., & Stefani, E. (1993) Calcium transients in single mammalian skeletal muscle fibres. J. Physiol. 463: 689–707. Denny, M. F., & Atchison, W. D. (1994) Methylmercury-induced elevations in intrasynaptosomal zinc concentrations: an 19F-NMR study. J. Neurochem. 63, 383–386. Draguhn, A., Verdorn, T. A., Ewert, M., Seeburg, P. H., & Sakmann, B. (1990) Functional and molecular distinction between recombinant rat GABAA receptor subtypes by Zn21. Neuron 5, 781–788. Ebadi, M., Iversen, P. L., Hao, R., Cerutis, D. R., Rojas, P., Happe, H. K., Murrin, L. C., & Pfeiffer, R. F. (1995) Expression and regulation of brain metallothionen. Neurochem. Int. 1, 1–22. Frederickson, C. J. (1989) Neurobiology of zinc and zinc-containing neurons. Int. Rev. Neurobiol. 31, 145–238. Frederickson, C. J., Kasarskis, E. J., Ringo, D., & Frederickson, R. E.
Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.
Canzoniero, Sensi, and Choi (1987) A quinoline fluorescence method for visualizing and assaying the histochemically reactive zinc (bouton zinc) in the brain. J. Neurosci. Methods 20, 91–103. Frederickson, C. J., & Moncrieff, D. W. (1994) Zinc-containing neurons. Biol. Signals 3, 127–139. Grynkiewicz, G., Poenie, M., & Tsien, R. Y. (1985) A new generation of Ca21 indicators with greatly improved fluorescence properties. J. Biol. Chem. 260, 3440–3350. Gunshin, H., Mackenzie, B., Berger, U. V., Gunshin, Y., Romero, M. F., Boron, W. F., Nussberger, S., Gollan, J. L., & Hediger, M. A. (1997) Cloning and characterization of a mammalian proton-coupled metal-ion transporter. Nature 388 (6641), 482–488. Harrison, N. L., Radke, H. K., Talukder, G., & Ffrench-Mullen, J. M. (1993) Zinc modulates transient outward current gating in hippocampal neurons. Receptor Channels 1, 153–163. Harrison, N. L., & Gibbons, S. J. (1994) Zn21: An endogenous modulator of ligand- and voltage-gated ion channels. Neuropharmacology 33, 935–952. Haugland, R. P. (1996) Handbook of Fluorescent Probes and Research Chemicals, (M. T. Z. Spence, Ed.), 6th ed., pp. 530–540. Molecular Probes, Eugene, Oregon. Hechtenberg, S., & Beyersmann, D. (1993) Differential control of free calcium and free zinc levels in isolated bovine liver nuclei. Biochem. J. 289, 757–760. Howell, G. A., Welch, M. G., & Frederickson, C. J. (1984) Stimulationinduced uptake and release of zinc in hippocampal slices. Nature 308(5961), 736–738. Koh, J. Y., & Choi, D. W. (1994) Zinc toxicity on cultured cortical neurons involvement of N-methyl-D-aspartate receptors. Neuroscience 60, 1049–1057. Koh, J. Y., Suh, S. W., Gwag, B. J., He, Y. Y., Hsu, C. Y., & Choi, D. W. (1996) The role of zinc in selective neuronal death after transient global cerebral ischemia. Science 272, 1013–1016. Kwok, W. M., & Kass, R. S. (1993) Block of cardiac ATP-sensitive K1 channels by external divalent cations is modulated by intracellular ATP. Evidence for allosteric regulation of the channel protein. J. Gen. Physiol. 102, 693–712. Maret, W. (1995) Metallothionein/disulfide interactions, oxidative stress, and the mobilization of cellular zinc. Neurochem. Int. 27, 111–117. Masters, B. A., Quaife, C. J., Erickson, J. C., Kelly, E. J., Froelick, G. J., Zambrowicz, B. P., Brinster, R. L., & Palmiter, R. D. (1994) Metallothionein III is expressed in neurons that sequester zinc in synaptic vesicles. J. Neurosci. 14, 5844–5857. Palmiter, R. D., & Findley, S. D. (1995) Cloning and functional characterization of a mammalian zinc transporter that confers resistance to zinc. EMBO J. 14, 639–649. Perez-Clausell, J., & Danscher, G. (1985) Intravesicular localization of zinc in rat telencephalic boutons: A histochemical study. Brain Res. 337, 91–98. Peters, S., Koh, J., & Choi, D. W. (1987) Zinc selectively blocks the action of NMDA on cortical neurons. Science 236, 589–593. Petrozzino, J. J., Pozzo Miller, L. D., & Connor, J. A. (1995) Micromolar Ca21 transients in dendritic spines of hippocampal pyramidal neurons in brain slice. Neuron 14, 1223–1231. Simons, T. J. (1991) Intracellular free zinc and zinc buffering in human red blood cells. J. Membr. Biol. 123, 63–71. Simons, T. J. (1993) Measurement of free Zn21 ion concentration with the fluorescent probe mag-fura-1 (furaptra). J. Biochem. Biophys. Methods 27, 25–37. Sloviter, R. S. (1985) A selective loss of hippocampal mossy fiber
Neuronal Intracellular Free Zinc Timm stain accompanies granule cell seizure activity induced by perforant path stimulation. Brain Res. 330, 150–153. Smart, T. G., Moss, S. J., Xie, X., & Huganir, R. L. (1991) GABAA receptors are differentially sensitive to zinc: dependence on subunit composition. Br. J. Pharmacol. 103, 1837–1839. Smart, T. G., Xie, X., & Krishek, B. J. (1994) Modulation of inhibitory and excitatory amino acid receptor ion channels by zinc. Progr. Neurobiol. 42, 393–441. Suh, S. W., Koh, J. H., & Choi, D. W. (1996) Extracellular zinc mediates selective neuronal death in hippocampus and amygdala following kainate-induced seizure. Soc. Neurosci. Abstr. 22, 823–826. Takahashi, K., & Akaike, N. (1991) Calcium antagonist effects on low-threshold (T-type) calcium current in rat isolated hippocampal CA1 pyramidal neurons. J. Pharmacol. Exp. Ther. 256, 169–175. Tonder, N., Johansen, F. F., Frederickson, C. J., Zimmer, J., & Diemer, N. H. (1990) Possible role of zinc in the selective degeneration of dentate hilar neurons after cerebral ischemia in the adult rat. Neurosci. Lett. 109, 247–252. Vallee, B. L., & Falchuk, K. H. (1993) The chemical basis of zinc physiology. Physiol. Rev. 73, 79–118. Vega, M. T., Villalobos, C., Garrido, B., Gandia, L., Bulbena, O., Garcia-Sancho, J., Garcia, A. G., Artalejo, A. R. (1994) Permeation
279 by zinc of bovine chromaffin cell calcium channels: Relevance to secretion. Pflugers Arch.- Eur. J. Physiol. 429, 231–239. Weiss, J. H., Hartley, D. M., Koh, J. Y., & Choi, D. W. (1993) AMPA receptor activation potentiates zinc neurotoxicity. Neuron 10, 43–49. Westbrook, G. L., & Mayer, M. L. (1987) Micromolar concentrations of Zn21 antagonize NMDA and GABA responses of hippocampal neurons. Nature 328, 640–643. White, J. A., Alonso, A., & Kay, A. R. (1993) A heart-like Na1 current in the medial entorhinal cortex. Neuron 11, 1037–1047. Yin, H. Z., & Weiss, J. H. (1995) Zn21 permeates Ca21 permeable AMPA/kainate channels and triggers selective neural injury. Neuroreport 6, 2553–2556. Yokoyama, M., Koh, J., & Choi, D. W. (1986) Brief exposure to zinc is toxic to cortical neurons. Neurosci. Lett. 71, 351–355. Zalewski, P. D., Forbes, I. J., & Betts, W. H. (1993) Correlation of apoptosis with change in intracellular labile Zn (II) using Zinquin [(2-methyl-8-p-toluesulphonamido-6-qinolyloxy)acetic acid]8 a new specific fluorescent probe for Zn(II). Biochem. J. 296, 403–408. Zalewski, P. D., Millard, S. H., Forbes, I. J., Kapaniris, O., Slavotinek, A., Betts, W. H., Ward, A. D., Lincoln, S. F., & Mahadevan, I. (1994) Video image analysis of labile zinc in viabile pancreatic islet cells using a specific fluorescent probe for zinc. J. Histochem. Cytochem. 42, 877–884.
Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.
MINIREVIEW Measurement of Intracellular Free Zinc in Living Neurons L. M. T. Canzoniero, S. L. Sensi, and D. W. Choi1 Center for the Study of Nervous System Injury and Department of Neurology, Washington University School of Medicine, St. Louis, Missouri 63110 Received August 21, 1997, accepted for publication August 21, 1997
Excessive Zn21 influx has been implicated in the pathogenesis of neuronal death after global ischemia or prolonged seizures, but little is presently known about cellular regulation of intracellular free Zn21 ([Zn21]i). In large part, this is because the tools currently available for measuring [Zn21]i are limited in comparison to those available for measuring [Ca21]i or other ions. We outline here approaches to this task that have been taken in the past, and summarize our recent experience using mag-fura-5 to measure [Zn21]i in living cortical neurons exposed to toxic levels of extracellular Zn21. r 1997 Academic Press
Key Words: fura; magfura; fluorescence; calcium channel; glutamate; neurotoxicity; ischemia.
Zn21 AS AN IMPORTANT REGULATOR OF BIOLOGICAL FUNCTIONS Zn21 is an essential component of many metalloproteins, including metalloenzymes and gene transcription factors (Vallee and Falchuk, 1993; Berg and Shi, 1996). Zn21 is also likely a mediator of central neural signaling at excitatory synapses (Frederickson, 1989; Frederickson and Moncrieff, 1994; Smart et al., 1994). Large amounts of chelatable Zn21 are present throughout the mammalian brain, predominantly stored in vesicles within excitatory nerve terminals (Danscher, 1984; Perez-Clausell and Danscher, 1985). This synaptic Zn21 can be released in a Ca21-dependent fashion with neuronal activity (Howell et al., 1984) and may achieve hundred micromolar concentrations in the extracellular space (Assaf and Chung, 1984). Once released, Zn21 may have functionally important effects on the behavior of multiple voltage- or ligand-gated channels (Smart et al., 1994; Harrison and Gibbons, 1994). Given the systematic colocalization of 1
To whom correspondence should be addressed at Department of Neurology, Box 8111, 660 S. Euclid, St. Louis, MO 63110. Fax: 314-362-9462. E-mail: [email protected]. 0969-9961/97 $25.00 Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.
Zn21 to glutamatergic terminals, its powerful ability to downmodulate N-methyl-D-aspartate (NMDA) receptors may be of particular significance (Peters et al., 1987; Westbrook and Mayer, 1987; Christine and Choi, 1990). Furthermore, Zn21 is also a potent blocker of certain forms of GABAA receptors lacking a g subunit (Westbrook and Mayer, 1987; Smart et al., 1991; Draguhn et al., 1990), TTX-insensitive voltage-gated Na1 channels (White et al., 1993), and T type Ca21 channels (Takahashi and Akaike, 1991) and can modulate the gating of certain K1 channels, especially hippocampal A currents (Harrison et al., 1993) and ATP-regulated K1 channels (Kwok and Kass, 1993). Excessive exposure to high concentration of Zn21 can kill cultured cortical neurons (Yokoyama et al., 1986). Previous studies suggest that this neurotoxicity is mediated by Zn21 influx, in large part through voltage-gated Ca21 channels (Weiss et al., 1993), as well as through NMDA receptor-mediated channels (Koh and Choi, 1994) and Ca21-permeable AMPA/kainate receptor-gated channels (Yin and Weiss, 1995). In vivo experiments have suggested that Zn21 can translocate from presynaptic terminals into the postsynaptic cell bodies of neurons injured after prolonged seizures (Sloviter, 1985; Friedrickson, 1989; Suh et al., 1996)
275
276 or transient global ischemia (Tonder et al., 1990; Koh et al., 1996). Blockade of ischemia-induced (Koh et al., 1996) or seizure-induced (Suh et al., 1996) Zn21 translocation by administration of the membrane-impermeable chelator CaEDTA markedly reduces resultant neuronal death.
Canzoniero, Sensi, and Choi
Zinquin fluorescence, even when the neurons were challenged with high concentrations of extracellular Zn21 in the presence of the Zn21-specific ionophore Na1 pyrithione. In contrast, we found Zinquin to work as an indicator of [Zn21]i in HEK-293 cells. Possibly, the failure of Zinquin to work as an indicator within cortical neurons reflects a failure of ethyl ester cleavage and subsequent dye trapping.
MEASUREMENT OF [Zn21]i It would be of interest to know what levels of intracellular cytosolic free Zn21 concentration ([Zn21]i ) are associated with toxic Zn21 influx. Unfortunately, the tools presently available for measuring dynamic changes in [Zn21]i are limited in comparison to those available for measuring intracellular free Ca21 or other ions. Most measurements of brain Zn21 to date have employed techniques requiring tissue fixation, such as Timm’s sulfide staining (Sloviter, 1985) or selenium (Danscher, 1984). In several studies a fluorescentspecific Zn21 dye (TS-Q), 6-methoxy-8-p-toluene sulfonamide quinoline, has been used to visualize Zn21 accumulation in cortical neurons and brain slices. While this Zn21-selective nonratioable fluorescent dye has provided valuable qualitative information about the location of chelatable Zn21 in cells and tissues (Frederickson et al., 1987; Weiss et al., 1993; Yin and Weiss, 1995; Koh et al., 1996), limitations of solubility (requiring very alkaline loading buffers) as well as possible complex interactions with Zn21 bound to membranes have precluded quantitative detection of changes of [Zn21]i in individual living cells (Andrews et al., 1995). Recently, other quinoline derivatives have been developed such as: (1) N-(6-methoxy-8-quinoylyl-carboxybenzoylsulfonamide) (TFLZn) and (2) D-methyl8-p-toluenesulphonamido-6-quinolyloxyacetic acid (Zinquin). Budde et al. (1997) used the water-soluble dye TFLZn to measure changes in [Zn21]i in mossy fiber boutons following electric stimulation in slices. However, in cultured neurons, TFLZn’s low affinity for Zn21 (Kd , 20 µM ) and poorly retention within cells represent major limitations. Zinquin has been successfully employed to examine [Zn21]i in thymocytes, human CLL cells, pancreatic islet cells, and hepatocytes (Zalewski et al., 1993, 1994; Brand and Kleineke, 1996). This new quinoline derivative is available as an ethyl ester and it is highly specific for Zn21. However, its fluorescence is not evenly distributed within cells and its loading capacity is poor. Working with cultured cortical neurons, we were unable to detect changes in Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.
FURA-2 AS Zn21 INDICATOR? Another approach to detecting [Zn21]i is to utilize fura-2-based Ca21-sensitive fluorescent dyes. Fura-2 binds transition metals such as Zn21 with much higher affinity than Ca21, producing a similar (although not identical) shift in emitted fluorescence (Grynkiewicz et al., 1985). Fura-2 as Zn21 indicator has been employed to study D[Zn21]i in response to electrical stimulation in cardiac myocytes (Atar et al., 1995), to characterize depolarization-induced Zn21 entry in chromaffin cells (Vega et al., 1994), to monitor nuclear and cytosolic [Zn21]i changes in bovine liver cells (Hechtenberg and Beyersmann, 1993), and to study the effect of methyl mercury on [Zn21]i in synaptosomal preparation (Denny and Atchison, 1994). However, the main drawback of fura-2 as an indicator of [Zn21]i is of course its high sensitivity for Ca21 and the problem of confounding changes in [Ca21]i. Furthermore, its affinity for Zn21 (Kd for Zn21 about 2 nM) (Grynkiewicz et al., 1985) is too high for assessment of D[Zn21]i in neurons challenged with neurotoxic concentrations of extracellular Zn21 (see below).
LOW-AFFINITY Ca21 INDICATORS AS [Zn21]i INDICATORS Simons (1993) reported that a low-affinity Ca21 and Mg21 indicator, mag-fura-2, could be used to measure Zn21 in solution over the 1–100 nM range, even in the presence of moderate Ca21, reflecting the dye’s higher affinity for Zn21 (Kd 20 nM) over Ca21 or Mg21. We explored the use of another low-affinity Ca21 indicator, mag-fura-5 (Kd for Ca21 5 20 µM and for Mg21 5 2.6 mM) (Petrozzino et al., 1995; Del Bono and Stefani, 1993), for real-time measurements of D[Zn21]i in living cortical neurons (Canzoniero et al., 1997). We chose mag-fura-5 instead of mag-fura-2 because the former has a slightly further reduced affinity for Ca21 or Mg21 (Haugland, 1996). We estimated the Kd of mag-fura-5 for Zn21 to be ,27 nM from cuvette calibration experiments.
277
Neuronal Intracellular Free Zinc
Using mag-fura-5, we estimated that the D[Zn21]i in neuronal cell bodies induced by exposure to 100 µM glutamate plus 100 µM Zn21 in the absence of extracellular Ca21 or Mg21 was in the range of 40–50 nM (Fig. 1). Control experiments supported the conclusion that this mag-fura-5 ratio signal indeed reflected D[Zn21]i. The ratio signal was induced by application of a Zn21-selective ionophore, Na pyrithione, but only when extracellular Zn21 was present. Furthermore, this Na pyrithione/Zn21 signal was quenched rapidly upon subsequent addition of the specific cell-permeable Zn21 chelator (N,N,N8,N 8-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN). We considered it is unlikely that the signal was importantly confounded by detection of Ca21 or Mg21. In the absence of extracellular Ca21, prior studies have not suggested that intracellular Ca21 levels in neuronal soma would rise to the high micromolar levels needed to activate mag-fura-5 (estimated Kd for Ca21 approximately 20 µM, Petrozzino et al., 1995). Mg21 is a greater possibility, but arguing against the possibility of Ca21-triggered intracellular Mg21 release (Brocard et al., 1993), we saw similar changes in mag-fura-5 fluorescence in other experiments when neurons were exposed to Zn21 plus kainate instead of glutamate, and Brocard et al. (1993) did not observe any elevation of [Mg21]i after kainate stimulation.
The absence of extracellular Ca21 and Mg21 likely enhanced Zn21 influx over that which would occur under more physiological conditions, but in other experiments, we have found that including physiological levels of [Ca21]o and [Mg21]o typically reduces the increase in [Zn21]i by about half. Prior determinations of [Zn21]i in nonneuronal cells have generally reported a wide range of [Zn21]i. In erythrocytes, Simons (1991) using 65Zn21 flux estimated basal [Zn21]i to be from 1.5 to 32 pM, depending on the ionic composition of the extracellular solution; in human leukemic cells a basal [Zn21]i of approximately 1 nM was determined by 19F nuclear magnetic resonance (Adebodun and Post, 1995). In electrically stimulated chromaffin cells (Atar et al., 1995) [Zn21]i monitored by fura-2 increased from 0.4 to 2 nM. On the other side of the spectrum, in resting splenocytes and thymocytes Zinquin was utilized to arrive at an estimated [Zn21]i of approximately 20–50 µM (Zalewski et al., 1993) and [Zn21]i of 0.6–2.7 µM in hepatocytes removed from rats fed a high zinc diet (Brand and Kleineke, 1996). The comparatively modest increases in [Zn21]i seen in cortical neurons exposed to toxic concentrations of extracellular Zn21 may reflect the presence of powerful mechanisms for Zn21 homeostasis, i.e., buffering by metallothioneins (Maret, 1995; Ebadi et al., 1995; Masters et al., 1994) or extrusion via Zn21 transporters (Palmiter and Findley, 1995; Gunshin et al., 1997). Also, measurements of fluorescence shifts over neuronal cell bodies would not be expected to detect Zn21 entering in neurites, which might be substantial.
NOVEL Zn21-SENSITIVE FLUORESCENT DYES
FIG. 1. Glutamate-evoked increase in [Zn21]i. 100 µM glutamate was applied for 5 min together with 100 µM Zn21. TPEN (50 µM) was added at the indicated time. Each point is the mean 6 SEM of 87 neurons. The experiment was performed in a buffer lacking Ca21 and Mg21.
Other recently identified potential Zn21 indicators, Newport Green or APTRA-BTC (Haugland, 1996), have promising specificity but low-affinity (Kd for Zn21 approximately 1 and 1.4 µM, respectively). We tested Newport Green or APTRA-BTC. Both were successfully loaded into cortical neurons and exhibited strong responses after addition of Zn21 plus Na pyrithione. However, consistent with their low affinities for Zn21, few somatic changes were detectable after a depolarizing stimulus (500 µM kainate plus 300 µM Zn21 ). Perhaps these probes may be useful in evaluating changes of [Zn21]i in neuronal processes, if the changes there are larger than those in cell bodies. Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.
278
CONCLUSION In summary, mag-fura-5 appears to have utility for measuring [Zn21]i dynamically in living neurons. However, clearly such measurements will need to be interpreted cautiously and confirmed once truly Zn21selective ratioable dyes become available.
REFERENCES Adebodun, F., & Post, J. F. (1995) Role of intracellular free Ca(II) and Zn(II) in dexamethasone-induced apoptosis and dexamethasone resistance in human leukemic CEM cell lines. J. Cell. Physiol. 163, 80–86. Andrews, J. C., Nolan, J. P., Hammerstedt, R. H., & Bavister, B. D. (1995) Characterization of N-(6-methoxy-8 quinolyl)-p-toluenesulfonamide for the detection of zinc in living sperm cells. Cytometry 21, 153–159. Assaf, S. Y., & Chung, S. H. (1984) Release of endogenous Zn21 from brain tissue during activity. Nature 308, 734–736. Atar, D., Backx, P. H., Appel, M. M., Gao, W. D., & Marban, E. (1995) Excitation-transcription coupling mediated by zinc influx through voltage-dependent calcium channels. J. Biol. Chem. 270, 2473–2477. Berg, J. M., & Shi, Y. (1996) The galvanization of biology: A growing appreciation for the roles of zinc. Science 271, 1081–1085. Brand, I. A., & Kleineke, J. (1996) Intracellular zinc movement and its effect on the carbohydrate metabolism of isolated rat hepatocytes. J. Biol. Chem. 271, 1941–1949. Brocard, J. B., Rajdev, S., & Reynolds, I. J. (1993) Glutamate-induced increases in intracellular free Mg21 in cultured cortical neurons. Neuron 11, 751–757. Budde, T., Minta, A., White, J. A., & Kay, A. R. (1997) Imaging free zinc in synaptic terminals in live hippocampal slices. Neuroscience 79, 347–358. Canzoniero, L. M. T., Sensi, S. L., Yu, S. P., Ying, H. S., Koh, J. Y., Choi, D. W. (1997) Measurement of intracellular free zinc in cultured cortical neurons: inferred entry through calcium channels. 23, 473–511. Christine, C. W., & Choi, D. W. (1990) Effect on zinc on NMDA receptor-mediated channel currents in cortical neurons. J. Neurosci. 10, 108–116. Danscher, G. (1984) Do the Timm sulphide silver method and the selenium demonstrate zinc in the brain? In: The Neurobiology of Zinc (C. J. Frederickson, G. A. Howell, & E. J. Kasarkis, Eds.), pp 273–287. A. R. Liss, New York. Delbono, O., & Stefani, E. (1993) Calcium transients in single mammalian skeletal muscle fibres. J. Physiol. 463: 689–707. Denny, M. F., & Atchison, W. D. (1994) Methylmercury-induced elevations in intrasynaptosomal zinc concentrations: an 19F-NMR study. J. Neurochem. 63, 383–386. Draguhn, A., Verdorn, T. A., Ewert, M., Seeburg, P. H., & Sakmann, B. (1990) Functional and molecular distinction between recombinant rat GABAA receptor subtypes by Zn21. Neuron 5, 781–788. Ebadi, M., Iversen, P. L., Hao, R., Cerutis, D. R., Rojas, P., Happe, H. K., Murrin, L. C., & Pfeiffer, R. F. (1995) Expression and regulation of brain metallothionen. Neurochem. Int. 1, 1–22. Frederickson, C. J. (1989) Neurobiology of zinc and zinc-containing neurons. Int. Rev. Neurobiol. 31, 145–238. Frederickson, C. J., Kasarskis, E. J., Ringo, D., & Frederickson, R. E.
Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.
Canzoniero, Sensi, and Choi (1987) A quinoline fluorescence method for visualizing and assaying the histochemically reactive zinc (bouton zinc) in the brain. J. Neurosci. Methods 20, 91–103. Frederickson, C. J., & Moncrieff, D. W. (1994) Zinc-containing neurons. Biol. Signals 3, 127–139. Grynkiewicz, G., Poenie, M., & Tsien, R. Y. (1985) A new generation of Ca21 indicators with greatly improved fluorescence properties. J. Biol. Chem. 260, 3440–3350. Gunshin, H., Mackenzie, B., Berger, U. V., Gunshin, Y., Romero, M. F., Boron, W. F., Nussberger, S., Gollan, J. L., & Hediger, M. A. (1997) Cloning and characterization of a mammalian proton-coupled metal-ion transporter. Nature 388 (6641), 482–488. Harrison, N. L., Radke, H. K., Talukder, G., & Ffrench-Mullen, J. M. (1993) Zinc modulates transient outward current gating in hippocampal neurons. Receptor Channels 1, 153–163. Harrison, N. L., & Gibbons, S. J. (1994) Zn21: An endogenous modulator of ligand- and voltage-gated ion channels. Neuropharmacology 33, 935–952. Haugland, R. P. (1996) Handbook of Fluorescent Probes and Research Chemicals, (M. T. Z. Spence, Ed.), 6th ed., pp. 530–540. Molecular Probes, Eugene, Oregon. Hechtenberg, S., & Beyersmann, D. (1993) Differential control of free calcium and free zinc levels in isolated bovine liver nuclei. Biochem. J. 289, 757–760. Howell, G. A., Welch, M. G., & Frederickson, C. J. (1984) Stimulationinduced uptake and release of zinc in hippocampal slices. Nature 308(5961), 736–738. Koh, J. Y., & Choi, D. W. (1994) Zinc toxicity on cultured cortical neurons involvement of N-methyl-D-aspartate receptors. Neuroscience 60, 1049–1057. Koh, J. Y., Suh, S. W., Gwag, B. J., He, Y. Y., Hsu, C. Y., & Choi, D. W. (1996) The role of zinc in selective neuronal death after transient global cerebral ischemia. Science 272, 1013–1016. Kwok, W. M., & Kass, R. S. (1993) Block of cardiac ATP-sensitive K1 channels by external divalent cations is modulated by intracellular ATP. Evidence for allosteric regulation of the channel protein. J. Gen. Physiol. 102, 693–712. Maret, W. (1995) Metallothionein/disulfide interactions, oxidative stress, and the mobilization of cellular zinc. Neurochem. Int. 27, 111–117. Masters, B. A., Quaife, C. J., Erickson, J. C., Kelly, E. J., Froelick, G. J., Zambrowicz, B. P., Brinster, R. L., & Palmiter, R. D. (1994) Metallothionein III is expressed in neurons that sequester zinc in synaptic vesicles. J. Neurosci. 14, 5844–5857. Palmiter, R. D., & Findley, S. D. (1995) Cloning and functional characterization of a mammalian zinc transporter that confers resistance to zinc. EMBO J. 14, 639–649. Perez-Clausell, J., & Danscher, G. (1985) Intravesicular localization of zinc in rat telencephalic boutons: A histochemical study. Brain Res. 337, 91–98. Peters, S., Koh, J., & Choi, D. W. (1987) Zinc selectively blocks the action of NMDA on cortical neurons. Science 236, 589–593. Petrozzino, J. J., Pozzo Miller, L. D., & Connor, J. A. (1995) Micromolar Ca21 transients in dendritic spines of hippocampal pyramidal neurons in brain slice. Neuron 14, 1223–1231. Simons, T. J. (1991) Intracellular free zinc and zinc buffering in human red blood cells. J. Membr. Biol. 123, 63–71. Simons, T. J. (1993) Measurement of free Zn21 ion concentration with the fluorescent probe mag-fura-1 (furaptra). J. Biochem. Biophys. Methods 27, 25–37. Sloviter, R. S. (1985) A selective loss of hippocampal mossy fiber
Neuronal Intracellular Free Zinc Timm stain accompanies granule cell seizure activity induced by perforant path stimulation. Brain Res. 330, 150–153. Smart, T. G., Moss, S. J., Xie, X., & Huganir, R. L. (1991) GABAA receptors are differentially sensitive to zinc: dependence on subunit composition. Br. J. Pharmacol. 103, 1837–1839. Smart, T. G., Xie, X., & Krishek, B. J. (1994) Modulation of inhibitory and excitatory amino acid receptor ion channels by zinc. Progr. Neurobiol. 42, 393–441. Suh, S. W., Koh, J. H., & Choi, D. W. (1996) Extracellular zinc mediates selective neuronal death in hippocampus and amygdala following kainate-induced seizure. Soc. Neurosci. Abstr. 22, 823–826. Takahashi, K., & Akaike, N. (1991) Calcium antagonist effects on low-threshold (T-type) calcium current in rat isolated hippocampal CA1 pyramidal neurons. J. Pharmacol. Exp. Ther. 256, 169–175. Tonder, N., Johansen, F. F., Frederickson, C. J., Zimmer, J., & Diemer, N. H. (1990) Possible role of zinc in the selective degeneration of dentate hilar neurons after cerebral ischemia in the adult rat. Neurosci. Lett. 109, 247–252. Vallee, B. L., & Falchuk, K. H. (1993) The chemical basis of zinc physiology. Physiol. Rev. 73, 79–118. Vega, M. T., Villalobos, C., Garrido, B., Gandia, L., Bulbena, O., Garcia-Sancho, J., Garcia, A. G., Artalejo, A. R. (1994) Permeation
279 by zinc of bovine chromaffin cell calcium channels: Relevance to secretion. Pflugers Arch.- Eur. J. Physiol. 429, 231–239. Weiss, J. H., Hartley, D. M., Koh, J. Y., & Choi, D. W. (1993) AMPA receptor activation potentiates zinc neurotoxicity. Neuron 10, 43–49. Westbrook, G. L., & Mayer, M. L. (1987) Micromolar concentrations of Zn21 antagonize NMDA and GABA responses of hippocampal neurons. Nature 328, 640–643. White, J. A., Alonso, A., & Kay, A. R. (1993) A heart-like Na1 current in the medial entorhinal cortex. Neuron 11, 1037–1047. Yin, H. Z., & Weiss, J. H. (1995) Zn21 permeates Ca21 permeable AMPA/kainate channels and triggers selective neural injury. Neuroreport 6, 2553–2556. Yokoyama, M., Koh, J., & Choi, D. W. (1986) Brief exposure to zinc is toxic to cortical neurons. Neurosci. Lett. 71, 351–355. Zalewski, P. D., Forbes, I. J., & Betts, W. H. (1993) Correlation of apoptosis with change in intracellular labile Zn (II) using Zinquin [(2-methyl-8-p-toluesulphonamido-6-qinolyloxy)acetic acid]8 a new specific fluorescent probe for Zn(II). Biochem. J. 296, 403–408. Zalewski, P. D., Millard, S. H., Forbes, I. J., Kapaniris, O., Slavotinek, A., Betts, W. H., Ward, A. D., Lincoln, S. F., & Mahadevan, I. (1994) Video image analysis of labile zinc in viabile pancreatic islet cells using a specific fluorescent probe for zinc. J. Histochem. Cytochem. 42, 877–884.
Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.