Protective Action of Zinc Against Glutamate Neurotoxicity in Cultured Retinal Neurons

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1 Protective Action of Zinc Against Glutamate Neurotoxicity in Cultured Retinal Neurons Masashi Kikuchi,* Satoshi Kashii* Yoshihito Honda* Hisamitsu Ujihara,\ Masashi Sasa,X Yutaka Tamura,% and Akinori Akaike\\ Purpose. To examine the effects of Zn a+ on glutamate-induced neurotoxicity in cultured retinal neurons. Methods. Primary cultures obtained from fetal rat retinas (16 to 19 days gestation) were used. The neurotoxic effects of excitatory amino acids were quantitatively assessed using the trypan blue exclusion method. Results. A brief exposure of retinal cultures to glutamate or N-methyl-D-aspartate (NMDA) induced delayed cell death. Zn 2+ at concentrations of 3 to 30 /xm ameliorated glutamate- and NMDA-induced neurotoxicity in a dose-dependent manner. By contrast, neurotoxicity induced by a 1-hour exposure to kainate was not affected by Zn 2+. Conclusions. These findings demonstrate that Zn 2+ protects retinal neurons from NMDA receptor-mediated glutamate neurotoxicity. Invest Ophthalmol Vis Sci. 1995;36: JL he concentration of Zn 2+ in ocular tissue is extremely high in comparison with other soft body tissue. Furthermore, Zn 2+ is the second most abundant trace element in the eye. 1 Among all tissues, the retina and choroid contain the highest Zn 2+ concentrations in ocular tissues in humans and in experimental animals. The mean concentrations of Zn 2+ in human retina and choroid have been calculated to be 464 fig/ g and 472 Mg/g dry weight, respectively, whereas those in most human organs were between 20 Mg/g an d 30 Mg/g dry weight. 2 In spite of the high concentration of Zn 2+ in the chorioretinal complex, its physiological role has not yet been clarified. Zn 2+ is a structural component of more than 50 different enzymes, including carbonic anhydrase, alkaline phosphatase, superoxide dismutase, catalase, and retinol dehydrogenase. It is not surprising to see the From the Defmrtments of * Ophthalmology, Faculty of Medicine, and \\Pharmacology, Faculty of Pharmaceutical Sciences, Kyoto University; f Pharmacology, School of Medicine, Yamaguchi University, Ube; Neurof)harmacology, Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University; and X Pharmacology, School of Medicine, Hiroshima University, Japan. Supported in part by a Grant-in-Aid for Scientific Research (A) from the Ministry of Education, Science and Culture, the Japanese Government. Submitted for publication August 23, 1994; revised Februaiy 7, 1995; accefded May 9, Proprietary interest category: N. Reprint requests: Satoshi Kashii, Department of Ophthalmology, Faculty of Medicine, Kyoto University, Kyoto 606, japan. multiplicity of the involvement of Zn 2+ in numerous biochemical processes through the proteins of which it is a part. Previous studies 3 of Zn 2+ in the eye have focused primarily on this enzymatic aspect. Recently, compelling evidence indicated that Zn 2+ is a signaling substance at excitatory synapses in the central nervous system, modulating glutamatergic neurotransmission. Friedrickson 4 has advocated the necessity of distinguishing between enzymatic Zn 2+ and neurosecretory Zn 2+. In the central nervous system, Zn 2+ has been reported' to play the role of modulator of ligand- and voltage-gated ion channels, including NMDA receptors. Our previous electrophysiological study on cultured retinal neurons demonstrated that depolarizing currents induced by N-methyl-D-aspartate (NMDA) were blocked by the application of Zn 2+. b In the cortical neurons, the protective action of Zn 2+ has been reported. 78 Therefore, we performed the current study to elucidate whether exogenously applied Zn 2+ protects cultured retinal neurons from neurotoxicity induced by glutamate. MATERIALS AND METHODS Cell Culture Primary cultures obtained from the retinas of fetal rats (16 to 19 days gestation) were used. Retinal tissue was 2048 Investigative Ophthalmology & Visual Science, September 1995, Vol. 36, No. 10 Copyright Association for Research in Vision and Ophthalmology

2 Zinc on Retinal Glutamate Receptor 2049 minced, mechanically dissociated, filtered through a stainless steel mesh, and plated as single-cell suspensions on plastic or glass coverslips placed in 60-mm dishes (4.5 to 6.0 X 10 cells/dish). Cultures were incubated in Eagle's minimal essential medium (Eagle's salts; Nissui, Tokyo, Japan) supplemented with 10% heat-inactivated fetal calf serum (1 to 9 days after plating), or 10% heat-inactivated horse serum (10 to 14 days after plating) containing 2 mm glutamine, 11 ram glucose, 24 mm sodium bicarbonate, and 10 mm 4-(2-hydroxy-ethyl)-l-piperazine-ethane-sulphonic acid (HEPES). Retinal cultures were maintained under conditions similar to those previously described. 9 After a 7- or 8-day culture, growth of the nonneuronal cells was terminated by the addition of 10" 5 M cytosine arabinoside. We used only those cultures maintained for 10 to 12 days in vitro and only isolated cells in this study. Clusters of cells were excluded from the results because cells located in the clusters could not be used for histologic and electrophysiological experiments. Most of the isolated cells were shaped like neurons. Our previous immunocytochemical study revealed that the isolated cells of the retinal cultures maintained in this study consisted mainly of amacrine cells. 9 In addition, our previous electrophysiological studies using a patch clamp technique demonstrated that all the retinal neurons tested responded to both NMDA and non-nmda receptor agonists. 6 Thus, according to Dixon and Copenhagen, 10 such neurons correspond to transient amacrine cells in the tiger salamander retina. All animals were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Measurement of Neurotoxicity The neurotoxic effects of excitatory amino acids (EAA) were quantitatively assessed by the trypan blue exclusion method as described."" 13 Experiments were performed in Eagle's minimal essential medium at 37 C. After the completion of drug treatment, cell cultures were stained with 1.5% trypan blue solution at room temperature for 10 minutes and were then fixed with isotonic formalin (ph 7.0, 2 C to 4 C). The fixed cultures were rinsed with physiological saline and examined under Hoffman modulation microscopy at X400. More than 200 cells were randomly counted to determine the viability of the cell culture. The viability of the cultures was calculated as the percentage of the ratio of the number of unstained cells (viable cells) against the total number of cells counted (viable cells plus nonviable cells). In each experiment, five coverglasses or coverslips were used to obtain mean values ± SEM of the cell viability. Excitatory amino acids and drugs were applied to the culture according to the method previously described by us. 12 ' 14 In brief, as for time dependence of the protective effects of Zn 2+, we have previously established a standard procedure for evaluation of the protective action of NMDA receptor antagonists in cultured cortical neurons. 13 In that study, the antagonists were added for 10 minutes before, during, or after glutamate exposure. Drugs induced maximum protection when they were added during glutamate exposure. Thus, we have used the same protocol to examine the effects of Zn 2+ and NMDA receptor antagonists in the current study. Zinc chloride was obtained from Nacarai Tesque (Kyoto, Japan). In our previous studies," 112 " 14 the neurotoxic effects of glutamate on cultured rat cortical and retinal neurons were studied in detail. A 10-minute exposure to 0.5 to 1 mm glutamate, followed by a 1-hour incubation with glutamate-free medium, was established as the appropriate conditions under which to examine glutamate neurotoxicity and drug-induced protection. In contrast to glutamate and NMDA, neurotoxicity induced by kainate was observed only after a 1-hour incubation at its concentration of 1 mm in cultured cortical neurons. Thus, we have used kainate, NMDA, and glutamate at concentrations of 1 mm. RESULTS Zn 2+ -Induced Protection From NMDA-induced Neurotoxicity Cultures were exposed to a solution containing either glutamate or NMDA for 10 minutes and were then incubated in EAA-free solution. Cell death does not occur immediately after exposure to EAA. Incubation of the cultures for more than 1 hour was required to observe marked reduction of the. Our earlier study 9 demonstrated that there was no significant difference between the values of reduction in cell viability for 1- hour incubation and 24-hour incubation. Therefore, drug-induced protection against the neurotoxicity induced by EAAs was determined after 10-minute exposure to EAA (1 mm) followed by 1-hour incubation with an EAA-free solution. Based on the previous study 9 on the ionic dependency of glutamate- and NMDA-induced neurotoxicity in cultured retinal neurons, glutamate, which acts on both NMDA and non-nmda receptors, was added to the standard medium containing Mg 2+, whereas special care was taken to use Mg 2+ -free medium for making the NMDA solution. Figure 1 shows an example of the effect of Zn 2+ on NMDA-induced neurotoxicity. The cells were exposed briefly (10 minutes) to NMDA (1 mm) and were incubated for 1 hour in an NMDA-free medium. NMDA treatment markedly reduced cell viability (Fig. IB).

3 2050 Investigative Ophthalmology & Visual Science, September 1995, Vol. 36, No. 10 Con1. Cont. Glutamale 10 MK-801 (MM) GlutamatB (ImM) CPP n2+ FIGURE 2. The protective effects of Zn2+ against glutamateinduced neurotoxicity. MK-801, a selective NMDA channel blocker, and CPP, a competitive antagonist for the NMDA receptor, inhibited glutamate-induced cell death. Zn2+ at a concentration lower than 1 /im was not effective for cell death induced by glutamate. At a concentration between 3 fim and 30 //M, Zn2+ demonstrated the protective effects in a concentration-dependent manner. ZnJ+ (300 //M) alone had no effect on cell viability. Error bars in this and the subsequent represent die SEM (N = 5). 50 um FIGURE i. Photomicrographs showing the effect of Zn2+ on NMDA-induced neurotoxicity. All cultures were photographed after trypan blue staining followed by formalin fixation using Hoffman modulation microscopy. Cells stained with trypan blue dye were regarded as nonviable. (A) Control (non treated) cells. Cells were stained without application of NMDA (B) Cells treated with NMDA (1 mm), followed by a 1-hour incubation with NMDA-free medium. Marked cell death occurred. (C) Cells treated with Zni+ (30 fm) plus NMDA (1 mm), followed by a 1-hour incubation with NMDA-free medium. Cell death was markedly reduced. Calibration bar = 50 fim. The addition of Zn y+ (30 fm) to the NMDA-containing medium and the NMDA-free incubation medium markedly reduced the cell death induced by NMDA (Fig. 1C). Figure 2 summarizes the protective effects of Zn i+ from glutamate-induced neurotoxicity. Marked reduction in cell viability was observed after a 10-minute exposure to glutamate, followed by a 1hour incubation in a glutamate-free medium. A noncompetitive antagonist, MK-801, and a competitive antagonist, CPP (3-[±]-2-carboxy-piperazin-4-yl)propyl-lphosphoric acid),15 for the NMDA receptor inhibited cell death. Zn a+ at concentrations lower than 1 JJM did not affect cell death induced by glutamate. However, in the media containing Zn 2+ at concentrations of more than 3 f/m, cell viability after exposure to glutamate was gradually restored to the level of nontreated cells with increasing concentrations of Zn a+. The protective effects of Zn 2+ against the glutamateinduced neurotoxicity was observed in a concentration-dependent manner at the starting concentration of 3 fim and reached its maximum at a concentration of 30 (JM. The Zn a+ concentration of 300 jim alone had no effect on cell viability, indicating that the Zn 2+ concentration of 300 fjm or less did not exhibit intrinsic neurotoxicity. Zn y+ also showed protective effects against the NMDA-induced neurotoxicity in a manner similar to those of glutamate (Fig. 3). Zn 2+ (30 to 300 ^M) almost completely blocked NMDA-induced neurotoxicity.

4 Zinc on Retinal Glutamate Receptor TABLE i. Effect of CNQX on Kainate Neurotoxicity Treatment Viability (%) % of Control.o CD Control (nontreated) 94.9 ± Kainate (1 mm)* 33.7 ± ± 5.1 Kainate (1 mm)* + CNQX (30//M)* 78.8 ± ± 1.2 * Kainate and CNQX were applied for 60 minutes. CNQX = 6-cyano-7-nitroquinoxaline-2,3-dione. Cont. NMDA(1mM) Zn 2+ (nm) FIGURE 3. The protective effects of Zn 2+ against NMDA-induced neurotoxicity. Zn 2+ showed protective effects against NMDA-induced neurotoxicity in a manner similar to those of glutamate (Fig. 2). Zn 2+ ( /zm) almost completely blocked NMDA-induced neurotoxicity. Effects of Zn 2+ on Kainate-Induced Neurotoxicity When kainate (1 mm) was applied for 10 minutes followed by a 1-hour incubation with kainate-free medium, cell viability was markedly decreased. However, this neurotoxicity induced by a brief exposure (10 minutes) to kainate was antagonized by MK-801, a selective NMDA blocker (Fig. 4). By contrast, the neurotoxicity induced by a 1-hour exposure to kainate was not affected by MK- 801, suggesting the involvement of NMDA receptors in the neurotoxicity induced by a brief exposure to kainate. A non-nmda receptorantagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), reduced kainate-induced neurotoxicity in this condition (Table 1). Thus, to examine kainate receptor-specific action, the effect of Zn 2+ was examined in the presence of 10 fjm MK-801 after a 1-hour exposure to kainate. Under this condition, Zn 2+ did not affect cell death induced by kainate (Fig. 4). DISCUSSION The results of the current study demonstrate that Zn 2+ protects cultured retinal neurons from NMDA receptor-mediated glutamate neurotoxicity. The competitive NMDA blocker, CPP, and the NMDA channel blocker, MK-801, inhibited glutamate neurotoxicity (Fig. 2). This result is consistent with our previous study showing that MK-801 inhibited both glutamateand NMDA-induced neurotoxicity. 9 These results indicate that NMDA receptor is a major cause of the delayed death induced by brief exposure to glutamate. Neurotoxicity induced by a brief exposure to glutamate was also markedly reduced by Zn 2+ at concentrations of more than 10 //M. Complete inhibition of glutamate neurotoxicity was induced by Zn 2+ at concentrations of more than 30 fjm. These Zn 2+ -induced effects were observed in a concentration-dependent fashion. By contrast, Zn 2+ did not affect cell death induced by a 1-hour exposure to kainate in the presence of MK-801. Cell death induced by a 10-minute exposure to kainate was antagonized with simultaneous application of MK-801, whereas neurotoxicity after a 1-hour exposure to kainate was not affected by MK However, neurotoxicity induced by kainate was inhibited by CNQX, a non-nmda receptor antagonist. This indicated that the cytotoxic effects of a 1- Cont. MK-801 MK-801 MK-801 Zn 2 + KA(10min) KA (60min) FIGURE 4. The effects of Zn 2+ on kainate-induced neurotoxicity. When kainate (1 mm) was applied for 10 minutes and was followed by a 1-hour incubation with kainate-free medium, cell viability was markedly decreased. However, this neurotoxicity induced by a brief exposure (10 minutes) to kainate was antagonized by MK-801. By contrast, neurotoxicity induced by a 1-hour exposure to kainate was not affected by MK-801. To examine the kainate receptor-specific action, the effects of Zn 2+ were examined in the presence of MK- 801 (10 fjm) after a 1-hour exposure to kainate. Under this condition, Zn 2+ did not affect cell death induced by kainate.

5 2052 Investigative Ophthalmology 8c Visual Science, September 1995, Vol. 36, No. 10 hour exposure to kainate was mediated by non-nmda receptors, though further study is necessary to ascertain the mechanisms of cytotoxic effects induced by a brief exposure to kainate. Considering that Zn 2+ failed to counteract the cell death induced by long-term exposure to kainate in the presence of MK-801, the protective action of Zn 2+ on the glutamate-induced neurotoxicity is selectively exerted by its action on the NMDA receptors. Our previous electrophysiological study using patch-clamp techniques demonstrated that 30 //M Zn 2+ suppressed 50% of the whole cell currents induced by NMDA (50 //M) at potentials more positive than 80 mv. h Starting at the same concentration of Zn 2+, neurotoxic effects of glutamate or NMDA were completely blocked, and cell viabilities were restored in the current study. The effect of Zn 2+ is thus not limited to glutamate-induced neuroexcitation but extends to neurotoxicity. Recent studies" 3 demonstrated that Zn 2+ is released with glutamate in the retina as well as in the brain. The estimated concentration of Zn 2+ in the synaptic clefts of the central nervous system during the intense presynaptic neuronal firing is at least 200 to 300 /AM, 4>17 " 19 which is sufficient to protect the deleterious effects of glutamate as demonstrated by the current study. The retina is rich in glutamatergic neurons. Therefore, an unusually high concentration of Zn 2+ distributed in the retina may contribute to the stabilization of postsynaptic neurons with glutamate-receptors. Zn 2+ selectively blocks the excitation mediated by the NMDA subtype of central glutamate receptors. 20 According to our previous patch-clamp study on cultured retinal neurons, 1 ' the inhibitory effect of Zn 2+ appeared not to depend on the membrane potential in comparison with that of Mg 2+. The inhibitory effect of Mg 2+ markedly decreased as the cell depolarized, indicating the relief of the Mg 2+ block of the NMDA channel by membrane depolarization. However, in the medium containing Zn 2+, there was a slight tendency for the actual currents to be depressed more than the predicted linear current-voltage relationship at potentials more positive than 80 mv, suggesting the presence of some voltage dependency of inhibition by Zn 2+ (30 //M). In cortical neurons, two major sites of action for Zn 2+ on the NMDA receptorchannel complex are suggested. One major site of action is a high-affinity, voltage-independent binding site near the external membrane surface, responsible for the reducing channel-opening frequency. The second site is a lower-affinity, voltage-dependent site within the NMDA channel itself responsible for a rapid flicker block of the open channel, distinct from the voltage-dependent Mg 2+ site. 20 ' 21 The retinal NMDA receptors also are assumed to possess the highaffinity and low affinity sites for Zn 2+. Thus, 30 fim Zn 2+ may act on both the voltage-independent and voltage-dependent NMDA receptor sites. If so, blockade of both sites of the NMDA receptor is necessary to induce the complete inhibition of NMDA receptormediated glutamate cytotoxicity by Zn 2+. Together with the finding that the protective effect of Zn 2+ was as potent as that of an NMDA receptor antagonist such as CPP, the protective action of Zn 2+ on glutamateinduced neurotoxicity is concluded to be produced by its selective blockade of the NMDA subtype of glutamate receptors. Key Wards excitatory amino acid, glutamate, N-methyl-i>aspartate, retinal cell culture, zinc References 1. Eckhert CD. 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