Protection of methylmercury effects on the in vivo dopamine release by NMDA receptor antagonists and nitric oxide synthase inhibitors

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1 Neuropharmacology 42 (2002) Protection of methylmercury effects on the in vivo dopamine release by NMDA receptor antagonists and nitric oxide synthase inhibitors L.R.F. Faro a, J.L.M. do Nascimento a, M. Alfonso b, R. Durán b, a Depto de Fisiologia, Centro de Ciências Biológicas, UFPA, Belém, PA, Brazil b Departamento de Biología Funcional y Ciencias de la Salud, Facultad de Ciencias, Universidad de Vigo, Vigo, Spain Received 11 April 2001; received in revised form 27 November 2001; accepted 10 January 2002 Abstract The possible protective effects of NMDA receptor antagonists dizocilpine (MK-801) and d( )-2-amino-5-phosphonopentanoic acid (AP5), and nitric oxide synthase (NOS) inhibitors l-nitro-arginine methyl ester (l-name) and 7-nitro-indazol (7-NI) on the methylmercury (MeHg)-induced dopamine (DA) release from rat striatum were investigated using in vivo microdialysis. Intrastriatal infusion of 400 µm or 4 mm MeHg increased the extracellular DA levels to 1941±199 and 7971±534% with respect to basal levels. Infusion of 400 µm or 4 mm MeHg in 400 µm MK-801 pretreated animals, increased striatal DA levels to 677±126 and 2926±254%, with respect to basal levels, these increases being 65 and 63% smaller than those induced by MeHg in non-pretreated animals. Infusion of 400 µm or 4 mm MeHg in 400 µm AP5 pretreated animals, increased striatal DA levels to 950±234 and 2251±254% with respect to basal levels, these increases being 51 and 72% smaller than those induced by MeHg in non-pretreated animals. Infusion of 400 µm MeHg in 100 µm l-name or 7-NI pretreated animals, increased the extracellular DA levels to 1159±90 and 981±292%, with respect to basal levels, these increases being 40 and 50% smaller than those induced by MeHg in non-pretreated animals. In summary, MeHg acts, at last in part, through an overstimulation of NMDA receptors with possible NO production to induce DA release, and administration of NMDA receptor antagonists and NOS inhibitors protects against MeHginduced DA release from rat striatum Elsevier Science Ltd. All rights reserved. Keywords: Methylmercury; Dopamine; NMDA antagonists; Nitric Oxide Synthase inhibitors; Microdialysis 1. Introduction The overall global increase of mercury, in various forms, in the environment due to its excessive use in industrial, agricultural, and mining practices has generated a serious toxicological problem and for this reason the screening of detoxicant agent is becoming extremely necessary (Week and Leicester, 1997). Methylmercury (MeHg) is a very dangerous toxicant which affects mainly the nervous system, creating rapid changes and disturbing both the structural and biochemical machinery in the cell (Chang, 1977). However, despite the broad reactivity of MeHg, selective impair- Corresponding author. Tel.: ; fax: address: rduran@uvigo.es (R. Durán). ment of cell functions is possible at low levels of exposure (Brookes, 1992). One of the biochemical effects of MeHg is an increase in neurotransmitter release. For example, it increases the in vivo dopamine (DA) release from rat striatum (Faro et al., 1997, 1998, 2000). In previous studies, we have observed that MeHg increased striatal DA levels, possibly through the membrane DA transporter (Faro et al., 2002). MeHg also increases glutamate release and inhibits its transport into cultured mouse spinal cord and rat cerebral cortical astrocyes thus increasing the extracellular concentration which leads to cell damage (Albrecht et al., 1993; Aschner et al., 1993, 2000; Brookes and Kristt, 1989). It has been reported that certain substances such as vitamins C and E and the monothiol glutathione prevent the effects of MeHg through its elimination from the /02/$ - see front matter 2002 Elsevier Science Ltd. All rights reserved. PII: S (02)

2 L.R.F. Faro et al. / Neuropharmacology 42 (2002) brain (Sood et al., 1993; Usuki et al., 2001). It has also been shown that these substances promote repair of the various components and enzymes in cells affected by MeHg (Vijayalakshmi et al., 1992). Recently, it has been found that the ionotropic glutamate receptor N-methyl-d-aspartate (NMDA) antagonist dizocilpine (MK-801) protects against cortical neuronal damage induced by MeHg (Miyamoto et al., 1999). These data suggest that glutamate receptors could be also involved in the effects produced by MeHg. The striatum is a brain area highly enriched in NMDA receptors (Greenamyre et al., 1995) and has been shown to have high nitric oxide synthase (NOS) activity (Bredt et al., 1991). The activation of NMDA receptors by endogenous glutamate increases Ca ++ conductance leading to a rise in the intracellular Ca ++ concentration. This Ca ++ binds to calmodulin and the Ca ++ /calmodulin complex activates NOS (Garthwaite et al., 1988). Stimulation of NMDA receptors by MeHg has been proposed as one possible neurotoxic mechanism underlying the effects of MeHg (Yamashita et al., 1997). Moreover, an overstimulation of NMDA receptors induced by MeHg could produce an activation of NOS and excessive nitric oxide (NO) production which could lead to neuronal death (Himi et al., 1996; Snyder and Bredt, 1992). Therefore, it was considered of interest to investigate the effects of the NMDA antagonists MK-801 (non-competitive antagonist) and d( )-2-amino-5-phosphonopentanoic acid (AP5) (competitive antagonist) on the MeHginduced in vivo DA release from rat striatum. In addition, to provide evidence for the proposed mechanism of the protective action of NMDA antagonists, the inhibitors of NOS, l-nitro-arginine methyl ester (l- NAME) and 7-nitro-indazol (7-NI) were also examined. 2. Methods 2.1. Animals, drug treatments and experimental groups Female adult Sprague Dawley rats (weighing between 240 and 260 g) were used in all the experiments. Animals were housed under controlled conditions of temperature (22±2 C) and light (light:dark 14:10 h), with free access to food and water. The experiments were performed according with the Guidelines of the European Union Council (86/609/EU) for the use of laboratory animals. The drugs were dissolved in the perfusion fluid and applied locally in the striatum through the dialysis probe. MeHg, l-name, and 7-NI were purchased from Sigma, St Louis (USA), MK-801 and d-ap5 were purchased from Tocris (USA). All other chemicals and reagents were of analytical grade. The experimental groups were as follows: (1) 400 µm MeHg; (2) 4 mm MeHg; (3) 400 µm MK-801; (4) 400 µm MeHg in 400 µm MK-801 pretreated animals; (5) 4 mm MeHg in 400 µm MK-801 pretreated animals; (6) 400 µm AP5; (7) 400 µm MeHg in 400 µm AP5 pretreated animals; (8) 4 mm MeHg in 400 µm AP5 pretreated animals; (9) 100 µm l-name; (10) 400 µm MeHg in 100 µm l-name pretreated animals; (11) 100 µm 7-NI; and (12) 400 µm MeHg in 100 µm 7-NI pretreated animals Microdialysis procedure For microdialysis sampling, animals were anesthetized (i.p.) with chloral hydrate (400 mg/kg) and placed in a stereotaxic apparatus (Narishige SR-6) for the implantation of a guide-cannula. A microdialysis probe (CMA/12, 3 mm membrane length, CMA/Microdialysis, Sweden) was implanted through the guide-cannula into the left striatum at the following coordinates from Bregma: A/P +2.0 mm; L +3.0 mm; V +6.0 mm. After experiments, rats were given an overdose of chloral hydrate, and the brains were fixed with 10% formalin via intracardiac perfusion. Coronal sections (30 µm) were made, stained with cresyl violet, and examined to determine the precise location of the dialysis probe. The experiments were carried out 24 h after implantation of the guide cannula. Continuous perfusion was performed with a Ringer s solution (147 mm NaCl, 4 mm KCl, 3.4 mm CaCl 2 ; ph 7.4) using a CMA/102 infusion pump (CMA/Microdialysis, Sweden) at a flow rate of 2 µl/min. At the beginning of our experiments with microdialysis, we have made controls with different compositions of Ringer medium in order to select the one most appropriate for our conditions. We also peformed periodical control experiments to confirm the basal values and that our conditions of microdialysis were correct. All experiments were made with awake, conscious, and freely-moving animals. The experiments were carried out over 4 h periods, sampling striatal dialysates every 15 min (30 µl). After collection of four basal samples (60 min), MeHg was infused during 60 min; after this, the medium was then switched back to the unmodified Ringer s solution and sampling was continued for an additional period of 120 min. In groups pretreated with antagonists or inhibitors, the drugs were infused from the beginning of experiment and then together with MeHg HPLC EC analysis The samples obtained from the microdialysis procedure (30 µl) were collected by means of a CMA/142 microsampler (CMA/Microdialysis, Sweden) and DA levels were quantified by High-Performance Liquid

3 614 L.R.F. Faro et al. / Neuropharmacology 42 (2002) Chromatography (HPLC) with electrochemical detection. The dialysates were injected (20 µl) into a Hewlett- Packard Series 1050 Liquid Chromatograph, using a Rheodyne 7125 injection valve. The isocratic separation of DA was achieved using Spherisorb ODS-1 reversedphase columns (10 µm particle size) according to Durán et al. (1998). The eluent (ph 4.0) was prepared as follows: 70 mm KH 2 PO 4, 1 mm octanesulfonic acid, 1 mm EDTA, and 7% methanol. Elution was carried out at a flow rate of 2 ml/min. The DA detection was achieved using an ESA Coulochem 5100A electrochemical detector (USA) at a potential of +400 mv Expression of results and statistics Values of extracellular DA were corrected using the percentage of relative recovery (the ratio between the concentration of a particular susbstance in the perfusate compared to its concentration in the medium outside the microdalysis probe) estimated by an in vitro method (Khan and Shuaib, 2001). The DA recovery was similar for the different probes used (approximately 15%). The averages of concentrations of DA in the three samples before drug administration were considered as basal levels. These basal levels were taken as 100% in order to compare the changes in DA release following drug administration. The results are shown as mean±s.e.m. of five six experiments, expressed as a percentage respect to basal levels. The rate of diffusion of MeHg through the microdialysis probe was also estimated in vitro. MeHg was dissolved in Ringer solution and pumped through a 1-ml glass syringe connected to the inlet of the microdialysis probe placed in a conical tube containing 1 ml of Ringer solution. Probes were perfused at a flow rate of 2 µl/min, as used during the experiments with freely moving rats. Under these conditions approximately 17% of MeHg diffused out the microdialysis probe in 1 h. Sample mercury levels were measured by Cold Vapor Atomic Absortion (USA E.P.A., 1979). Thus, with 400 µm MeHg in the perfusate, only nmoles/min of MeHg are available for diffusion through the dialysis membrane. Statistical evaluation of the results was performed by means of ANOVA and Student Newman Keuls multiple range test, considering the following significant differences: P 0.05, P 0.01, and P 0.001, with respect to basal. 3. Results 3.1. Effect of 400 mm and 4 mm MeHg on the basal DA release Intrastriatal infusion of 400 µm and 4 mm MeHg increased the extracellular DA levels to 1941±199 and 7971±534%, respectively, with respect to basal values (0.22±0.04 ng/15 min, n=15). The data for the effects of MeHg on striatal dopamine levels are plotted in Figs. 1 4 in order to compare with the data obtained under other experimental conditions (treatments with antagonists or inhibitors together with MeHg) Effect of NMDA receptor antagonists on MeHginduced DA release To investigate the possible protective action of NMDA antagonists, the effect of MeHg on DA release was studied in the presence of MK-801 or AP5. One hour infusion of 400 µm MK-801 had no significant effects on striatal DA levels (Fig. 1). Under 400 µm MK-801 pretreatment, infusion of 400 µm MeHg Fig. 1. Protective effects of 400 µm MK-801 pretreatment on (A) 400 µm and (B) 4 mm MeHg-induced DA release. MeHg infusion started at the time indicated by the arrows. In the pretreated group, MK-801 infusion started at the beginning of the experiment. The results are shown as mean±s.e.m. of five six experiments, expressed as a percentage of basal levels (100%). Basal levels were considered as the mean of substance concentrations in the three samples collected before drug administration. Significant differences: P 0.05, P 0.01 and P with respect to basal levels and a P 0.05, with respect to MeHg administration.

4 L.R.F. Faro et al. / Neuropharmacology 42 (2002) Fig. 3. Protective effects of 100 µm l-name pretreatment on 400 µm MeHg-induced DA release. MeHg infusion started at the time indicated by the arrows. In the pretreated group, l-name infusion started at the beginning of the experiment The results are shown as mean±s.e.m. of five six experiments, expressed as a percentage of basal levels (100%). Basal levels were considered as the mean of substance concentrations in the three samples collected before drug administration. Significant differences: P 0.05, P 0.01 and P 0.001, with respect to basal levels and a P 0.05, with respect to 400 µm MeHg. Fig. 2. Protective effects of 400 µm AP5 pretreatment on (A) 400 µm and (B) 4 mm MeHg-induced DA release. MeHg infusion started at the time indicated by the arrows. In the pretreated group, AP5 infusion started at the beginning of the experiment The results are shown as mean±s.e.m. of five six experiments, expressed as a percentage of basal levels (100%). Basal levels were considered as the mean of substance concentrations in the three samples collected before drug administration. Significant differences: P 0.05, P 0.01 and P with respect to basal levels and a P 0.05, with respect to MeHg administration. increased striatal DA levels to 677±126%, with respect to basal (Fig. 1A). The increase in extracellular DA levels induced by MeHg in MK-801 pretreated animals was 65% smaller than the increase produced in animals not pretreated with MK-801. In the same way, infusion of 4 mm MeHg in 400 µm MK-801 pretreated animals increased striatal DA levels to 2926±254% of basal (Fig. 1B), being this increase 63% smaller than that observed with 4 mm MeHg in non-pretreated animals. Intrastriatal infusion of 400 µm AP5 had no significant effects on striatal DA levels (Fig. 2). Infusion of 400 µm MeHg in 400 µm AP5 pretreated animals increased striatal DA levels to 950±234% with respect to basal (Fig. 2A), while infusion of 4 mm MeHg under 400 µm AP5 pretreatment increased the extracellular DA levels to 2251±254% with respect to basal (Fig. 2B). These increases produced by MeHg following AP5 pretreatment were 51 and 72% smaller than those produced by 400 µm and 4 mm MeHg, respectively, in animals not pretreated with AP5. Fig. 4. Protective effects of 100 µm 7-NI pretreatment on 400 µm MeHg-induced DA release. MeHg infusion started at the time indicated by the arrows. In the pretreated group, 7-NI infusion started at the beginning of the experiment The results are shown as mean±s.e.m. of five six experiments, expressed as a percentage of basal levels (100%). Basal levels were considered as the mean of substance concentrations in the three samples collected before drug administration. Significant differences: P 0.05, P 0.01 and P 0.001, with respect to basal levels and a P 0.05, with respect to 400 µm MeHg Effect of NOS inhibitors on MeHg-induced DA release To investigate if NO production could be implicated in the effects produced by MeHg on striatal DA levels, as well as the possible protective action of NOS inhibi-

5 616 L.R.F. Faro et al. / Neuropharmacology 42 (2002) tors on MeHg-induced DA release, we infused l-name or 7-NI through the microdialysis probe. Intrastriatal infusion of 100 µm l-name for 1 h decreased the extracellular DA levels 10±1% with respect to basal (Fig. 3). This value was considered as basal for the measurement of MeHg effects on DA release in l- NAME pretreated rats. Thus, with 100 µm l-name pretreatment, infusion of 400 µm MeHg increased striatal DA levels to 1159±90%, with respect to basal (Fig. 3), this increase being 40% smaller than that produced by 400 µm MeHg in animals not pretreated with l- NAME. Infusion of 100 µm 7-NI decreased the extracellular DA levels 21±1% with respect to basal levels (Fig. 4) and, in the same way, this value was considered as basal for the measurement of MeHg effects on DA release in 7-NI pretreated rats. Infusion of 400 µm MeHg in 100 µm 7-NI pretreated animals increased the extracellular levels to 981±292% with respect to basal (Fig. 4). This increase was 50% smaller than that produced by 400 µm MeHg in non-pretreated animals. Fig. 5 shows the comparative effects of the different treatments on MeHg-induced DA release. AP-5 and MK- 801 decreased the effect of 400 µm MeHg on striatal dopamine levels by 51 and 65% respectively. l-name and 7-NI decreased the effect of 400 µm MeHg on striatal dopamine levels by 40 and 50%, respectively. Moreover, the competitive and non-competitive glutamate receptor antagonists also decreased the effect of 4 mm MeHg on striatal dopamine levels by 72 and 63%, respectively. 4. Discussion The 1 h administration of MeHg increased striatal dopamine levels. Under our experimental conditions, the dopamine levels first increased and then recovered to the initial levels. This dopamine behaviour is consistent with the majority of microdialysis experiments, in which drugs are administered though a microdialysis probe. The purpose of the present study was to investigate the ability of certain substances to impair the MeHginduced DA release from rat striatum and their possible mechanisms of action. It has previously been shown that MK-801 has a protective effect in MeHg-induced neuronal injury in cerebral cortex in vitro and in systemic administration in vivo (Miyamoto et al., 1999). Recently, Miyamoto et al., (2001) reported that enhanced sensitivity of NMDA receptors is involved in the vulnerability of developing cortical neurons to MeHg. The authors concluded that the excytotoxic effects of MeHg in the cerebral cortex could be glutamate receptor-mediated. The effect of MeHg on DA release in the presence of the NMDA receptor antagonists MK-801 and AP5, was studied. Our findings demonstrate that pretreatment with 400 µm MK-801 decreases the DA release induced by 4 mm and 400 µm doses of MeHg (63 and 65%, respectively). These results indicate that DA release induced by MeHg could be partially mediated by the activation of NMDA glutamate receptors. The results obtained with AP5, a competitive NMDA receptor antagonist, confirm the data observed with MK- Fig. 5. Comparative effects of MK-801, AP5, l-name, and 7-NI on 400 µm and 4 mm MeHg-induced DA release. The protective effects obtained with the different drugs on MeHg-induced DA release were between 40 72%. P 0.05, comparing the protective effects of l-name and 7-NI, respectively, with the protective effect of MK-801 on 400 µm MeHg-induced DA release.

6 L.R.F. Faro et al. / Neuropharmacology 42 (2002) The increase in extracellular DA levels induced by MeHg was significantly smaller in AP5 pretreated rats than in non-pretreated animals. The results obtained show that pretreatment with high concentrations of NMDA antagonists protects against the increase in extracellular DA levels induced by MeHg in rat striatum. In our experimental conditions, there were no significant differences between the protective effects of competitive and non-competitive glutamate receptor antagonists. However, demonstrating the effects of blockade of NMDA receptors was not the aim of this work, but to prevent the MeHg-induced DA release. Glutamate and DA are closely related neurotransmitters in the CNS. It has been demostrated that glutamate agonists increase DA levels in the striatum (Jedema and Moghaddam, 1996; Arias et al., 2000). MeHg increases the extracellular concentration of glutamate, by stimulating its release and/or by inhibiting its uptake (Albrecht et al., 1993; Aschner et al., 1993, 2000). This increase in extracellular glutamate could overstimulate the NMDA receptors leading to excytotoxic damage (Shuman and Madison, 1994). The protective effects observed with MK-801 and AP5 pretreatment support the hypothesis that NMDA receptor activation by MeHg induces DA release. Since the activation of NMDA receptors is also bound to the production of NO (Shuman and Madison, 1994), the NOS inhibitors could produce similar protective effects to the NMDA receptor antagonists. Therefore, in order to study the protective role of NMDA receptors antagonists on MeHg-induced DA release and its possible mechanism of action, the NOS inhibitors l-name and 7-NI were used. Pretreatment with l-name reduced MeHg-induced DA release by 40%, while the specific neuronal NOS inhibits 7-NI reduced release by 50%. These data indicate that inhibition of NO production may protect against the MeHg-induced DA release. Moreover, these results support the hypothesis that MeHg could act through an overstimulation of NMDA receptors with consequent NOS activation and NO production to affect dopaminergic neurotransmission. The fact that we only observed a partial ( 40 70%) reduction in MeHg-induced DA release suggests that mechanisms other than NMDA or NOS activation may also be involved. MeHg is a highly reactive neurotoxicant and the greater part of its effects result from interaction with sulfhydril groups (-SH) on the surface of cell membrane, interfering with the membrane transport and metabolism (Mulaney et al., 1994). Thus, MeHg could interact with sulfhydril groups present in the protein structure of NMDA receptors thus modifying activity (Mulaney et al., 1994). Recently we have observed that MeHg increased the striatal DA release by a non-exocytotic process (Ca ++ -independent and unaffected by TTX) possibly mediated by the DA transporter (Faro et al., 2002). MeHg could interact directly with the sulfhydryl groups of the DA transporter altering its activity, but another possibility is that NO released by NMDA activation inhibits the DA transporter, thereby increasing the extracellular level of DA (Kiss and Vizi, 2001). Another possible mechanism by which MeHg could induce neurotransmitter release is through an increase in intracellular Ca ++ (Atchison, 1986). The possible activation of NMDA receptors by MeHg could lead to an elevation in intracellular Ca ++ through these receptors. An increase in intracellular Ca ++ could induce Ca ++ /calmodulim complex formation, which activates NOS and NO production (Garthwaite et al., 1988). The NO thus released could be toxic to the surrounding cells (Snyder and Bredt, 1992). Therefore, NMDA antagonism or NOS inhibition would decrease the DA release in accordance with our results. Another possible explanation could implicate the interaction of cortical glutamatergic and ascending dopaminergic pathways in the striatum. This glutamate dopamine interaction possibly occurs through non-synaptic actions (Kiss et al., 1999), and plays a very important role in the communication between glutamatergic and dopaminergic neurons (Vizi and Labos, 1984). Thus, glutamate has been implicated as a local regulator of DA release (Glowinski et al., 1988). MeHg increases the spontaneous release of DA and other transmitters, including glutamate (Albrecht et al., 1993; Aschner et al., 1993; Mulaney et al., 1994). If DA release in rat striatum is under the regulation of excitatory amino acids, and activation of NMDA receptors by endogenous glutamate can induce DA release (Zigmond et al., 1998), then the inhibition of NMDA receptors by MK-801 or AP5 would block the DA release induced by glutamate, decreasing the effects of MeHg on DA release. In conclusion, MeHg appears to act, at least in part, through an overstimulation of NMDA receptors, possibly leading to an increase in intracellular Ca ++ and NO production to induce DA release. The administration of NMDA antagonists and NOS inhibitors protects against MeHg-induced DA release from rat striatum. Acknowledgements This research was supported by grants from Xunta de Galicia (Spain). Lilian Faro acknowledges CNPq (Brazil) for a research grant. Authors wish to thank Dr J. L. Soengas for his help in the preparation of manuscript. 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