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Amantadine increases the extracellular dopamine levels in the striatum by re-uptake inhibition and by N-methyl-d-aspartate antagonism
BRAIN RESEARCH ELSEVIER
Brain Research 662 (1994) 255-258
Short communication
Amantadine increases the extracellular dopamine levels in the striatum by re-uptake inhibition and by N-methyl-D-aspartate antagonism Katsuhiro Mizoguchi *, Hideyasu Yokoo, Masami Yoshida, Takahiko Tanaka, Masatoshi Tanaka Department of Pharmacology, Kurume Unirersity School of Medicine, Kurume, 830, Japan Accepted 2 August 1994
Abstract
This study was performed to investigate the mechanism how amantadine increases the extracellular dopamine (DA) levels in the striatum in vivo. Local application of amantadine (1 mM, 40 min) to the striatum through the dialysis membrane significantly increased the extracellular DA levels. Coadministration of nomifensine (10 mM, 120 min), an inhibitor of neuronal DA uptake, into the perfusion fluid attenuated the amantadine-induced increase in DA outflow. The amantadine-induced increases in the extraceilular DA levels were also inhibited by co-perfusion with Ringer containing high Mg z+ (15 mM, 120 min) or with MK-801 (1 /zM, 80 min). These findings suggest that amantadine increases the extracellular DA levels in the striatum by inhibiting the re-uptake of DA and/or by blocking the channel in the N-methyl-D-aspartate (NMDA) receptor, which results in antagonism of NMDA receptor function.
Keywords: In vivo microdialysis; Striatum; Amantadine; Dopaminergic neuron; Re-uptake inhibition; N-Methyl-D-aspartate receptor
Amantadine has been used clinically for the treatment of Parkinson's disease. The clinical effectiveness of amantadine is generally attributed to its action on nigrostriatal dopaminergic neurons. It is generally accepted that amantadine acts presynaptically to enhance D A release [17] or inhibit DA uptake [3]. However, there have been little in vivo investigations concerning the action of amantadine on dopaminergic neurons in the striatum. It has been reported recently that memantine, an analogue of amantadine, bound to the MK-801 site in the N-methyl-D-aspartate (NMDA) receptor channel [8,9] and electrophysiologically blocked the N M D A receptor function [1]. In the present study, we examined the effect of amantadine on the extracellular D A levels in the striatum using in vivo microdialysis under conditions of inhibition of D A re-uptake or modification of N M D A receptor function with either MK-801 or high concentrations of magnesium. Male Wistar rats, weighing 250-300 g, were used. Animals were maintained on a 12 h light/dark cycle
* Corresponding author. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 4 ) 0 0 9 3 0 - 9
(lights on at 07.00 h) with free access to food and water. In accordance with the method as described previously [11,19], rats were anesthetized with pentobarbital sodium (45 m g / k g i.p.) and the dialysis probe, the tip of which consisted of a U-shaped dialysis membrane formed cellulose hollow fiber tubing (0.25 mm diameter; molecular weight cut off, 50,000), was implanted into the striatum (coordinates were A: 0.5 ram, L: 3.0 ram, V: 6.0 mm, from the bregma and the dura surface, according to the atlas of Paxinos and Watson [13]). The active surface of the dialysis probe was 6 mm. After the experiment, we determined the position of the dialysis probe histologically in Toluidine blue (0.5%)-stained frozen coronal sections [19]. Dialysis experiments were carried out 2 days after the operation. The dialysis probe was continuously perfused at a constant flow rate of 2 . 5 / x l / m i n with an artificial CSF (composition: NaC1 140 raM, KC1 3.35 mM, MgCI 2 1.15 mM, CaC12 1.26 mM, Na2HPO4 1.20 mM, N a H 2 P O 4 0.30 mM and p H 7.4). Experiments were started between 08.30 and 09.30 h. About 3 h after the beginning of the perfusion, stable basal levels of DA in the dialysates were ob-
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K. Mizoflucht et al. /'Hrain Research c,62 t1904~ 25~ 2~,';
tained and amantadine (1 mM) was infused into the striatum through the dialysis m e m b r a n e for 40 rain. After recovery of D A levels to the approximate basaline level, nomifensine (10/~M) was added to the perfusion fluid and maintained until the end of the experiment. Two h after the start of nomifensine infusion, amantadine (1 raM) was coadministered for 40 min and the effect of amantadine on D A release was reexamined in the presence of nomifensine. Similarly, after a stable D A peak was obtained, MK-801 (1 /~M) or high Mg2+(15 mM) were added to the Ringer until the end of the experiment, respectively. Amantadine (1 raM) was similarly coperfused for 40 rain at the time points of 80 min or 2 h after the start of the administration of MK-801 or high Mg 2+, respectively. The dialysates (50 /zl) were directly injected every 20 min using an automatized injector for analysis of D A into the high performance liquid chromatography (HPLC) coupled to an electrochemical detection system (EICOM, Kyoto, Japan), as described previously [11,19]. The mobile phase in the H P L C was 0.1 M sodium acetate buffer (pH 4.0) containing 0.6 mM octanesulfonic acid, 0.02 mM E D T A and 10% ( v / v ) methanol. D A was separated on an Eicompack M A - O D S column (4.6 mm O.D., 3.6 mm I . D . x 150 mm) at 25°C. The graphite working electrode was set at + 550 m V vs. an A g / A g C 1 reference electrode ( E I C O M ECD-100 electrochemical detector) and the flow rate ( E I C O M EP-10 pump) was 0.9 m l / m i n . Statistical analysis of the data was carried out using the one-way analysis of variance ( A N O V A ) for the effect of amantadine followed by S t u d e n t - N e w m a n Keuls test. The effect of p r e t r e a t m e n t was analysed by two way A N O V A followed by S t u d e n t - N e w m a n - K e uls test. The basal levels of D A in the striatum were determined from the mean of three samples obtained prior to administration of amantadine. In the pretreatment groups, the averaged basal levels of D A were determined from the mean of three samples prior to coperfusion of amantadine. Values are expressed as the mean + S.E.M. The basal level of D A in the striatum was 32.8 + 2.5 p g / s a m p l e (n = 15). Administration of amantadine (1 mM) to the perfusate significantly increased the extracellular D A levels in the striatum (F10,33 = 12.484, P < 0.001, Fig. 1). Addition of nomifensine (10/~M) to the perfusate increased extracellular D A levels 5 times higher than the basal levels and reached a plateau after 80-100 min (Data not shown). The amantadineinduced increase in extracelluar D A levels in the striaturn was significantly inhibited by p r e t r e a t m e n t with nomifensine ( F l o , 6 6 = 5.761, P < 0.001, Fig. 2). Administration of MK-801 (1 /~M) had no effect on the extracellular D A levels in the striatum (Data not shown). Addition of high Mg 2+ (15 raM) to the perfusate reduced the extracellular D A level to half of the
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Fig. 1. Effect of amantadine on the extracellular dopamine levels in the striatum. Each sample represents a 20-min collection period. The value indicates the mean+S.E.M, of four rats. P < 0:001 Vs. basal levels. basal levels and reached a plateau after 60-80 min (Data not shown). The amantadine-induced increases in extraceilular DA levels in the striatum were significantly attenuated in the presence of MK-80] or high Mg 2+ in the perfusion Ringer (F9.60 = 6.636. P < 0.001. Fig. 3, F10,6 6 = 5.761. P < 0.001. Fig. 4. respectively). The present study using in vivo microdialysis clearly demonstrates that the intrastriatal administration of amantadine increases the extracellular D A level in the striatum (Fig. 1). but had little effect on extracellular D O P A C levels (F~0.33 = 0.594. P = 0.8071. Data not shown). It has been known that both amphetamine and nomifensine [2,5] increase extracellular DA levels, but amphetamine decreases extraceltular D O P A C levels. while nomifensine has little effect on extracellular D O P A C levels [20]. Therefore, inhibition of D A re-uptake similar to that of nomifensine-like mechanism in the striatum is considered to be one of the mechanisms for the elevation of the extracellular D A level induced by amantadine because amantadine-induced increases in D A levels in the striatum were attenuated by pretreatment with nomifensine (Fig. 2) and amantadine itself had no effect on the extracellular striatal D O P A C levels. This result supports the previous findings that the tissue uptake of [SH]dopamine decreased following the administration of amantadine [3]. On the other hand, amantadine has been reported to increase the extracellular D A level by enhancing the release of D A in response to electrical stimulation of dopaminergic nerve terminals [17], but without identifying how amantadine enhances the DA release from nigrostriatal dopaminergic nerve terminals. In the present study, amantadine-induced increase in the extracellular D A levels in the striatum was inhibited by pretreatment with MK-80I (1 /.~M), which by itself had no effect on the extracellular baseline D A levels (Fig. 3). Pretreatment with Ringer containing high Mg 2+ (15 mM) also attenuated the facilitatory effect of amanta-
K. Mizoguchi et al. / Brain Research 662 (1994) 255-258
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dine on the extracellular DA levels in the striatum (Fig. 4). Electrophysiological and radioligand binding studies have previously shown that MK-801 antagonizes NMDA responses by binding inside the open channel in the NMDA receptor, thereby blocking transmembrane ion influxes [18]. Phencyclidine (PCP) also blocks the ion channel in a similar manner by binding to the same site as does MK-801 [4]. The ion channel of the NMDA receptor is also blocked by magnesium in a voltage-dependent fashion [12]. In addition, it has been shown that direct infusion of MK-801 increased the extracellular DA levels in the striatum in vivo [6]. Taken together, it mat be possible that amantadine increases the extracellular DA level by binding to the MK-801 site in the NMDA receptor and subsequently blocking the channel site, which resuits in enhancing DA release. This idea is supported by the findings that amantadine and memantine, an analogue of amantadine, displaced [3H]MK-801 in the
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postmortem human frontal cortex membranes [8,9] and that memantine blocked NMDA receptor channels in a manner similar to that of MK-801 as determined by electrophysical study [1]. It has also been demonstrated that amantadine bound to [3H]PCP site in a dose dependent manner [14]. This finding also supports the above hypothesis. Neuroprotective actions against NMDA-induced neurotoxicity via NMDA antagonistic actions by amantadine and memantine have been reported in previous studies [10,15,16]. Moreover, it has previously been shown that relatively high concentrations (raM range) as well as lower concentrations (0.1 mM) of glutamate receptor antagonists are potent to block excitatory amino acid (EAA) agonist-induced striatal DA release, but only higher concentrations of the antagonists increased basal striatal DA levels, but lower concentrations did not [7]. These findings further indicate the possibility that amantadine is a NMDA antagonist if amantadine was able to block NMDA-induced striatal DA release and only high concentrations (1 mM) and not lower concentrations of amantadine had an effect on basal striatal DA levels, We confirmed that high concentrations (1 raM) of amantadine significantly induced an increase in striatal DA levels (Fig. 1), but lower concentrations (0.1 raM) did not (data not shown). In conclusion, the clinical use of amantadine is a valuable supplement in the treatment of Parkinson's disease, with the effect of reducing the parkinsonian symptoms by increasing the extracellular DA levels via re-uptake inhibition and NMDA antagonistic action, which results in activating dopaminergic transmission. We wish to thank Prof. G.B. Glavin, Department of Pharmacology and Therapeutics, Faculty of Medicine,
258
K_ Mizoguchi et al. / Brain Research 062 (1994) 255 25¢~¢
University of Manitoba, Canada, for reviewing an earlier version of this manuscript. [1] Bormann, J., Memantine is a potent blocker of N-methyl-Daspartate (NMDA) receptor channels, Eur. J. Pharmacol.. 166 (1989) 591-592. [2] Braestrup, C., Biochemical differentiation of amphetamine vs methylphenidate and nomifensine in rats, J. Pharm. Pharmac., 29 (1977) 463-470. [3] Heikkila, R.E. and Cohen, G., Evaluation of amantadine as a releasing agent or uptake blocker for [3H]dopamine in rat brain slices, Ear. J. Pharmacol., 20 (1972) 156-160. [4] Honey, C.R., Milikovic, Z. and Macdonald, J.F., Ketamine and phencyclidine cause a voltage-dependent block of responses to L-aspartic acid, Neurosci. Lett., 61 (1985) 135-139. [5] Hyttel, J., Inhibition of [3H]dopamine accumulation in rat striatal synaptosomes by psychotropic drugs, Biochem. Pharmacol., 27 (1978) 1063-1068. [6] Imperato, A., Scrocco, M.G., Bacchi, S. and Angelucci, L., NMDA receptors and in vivo dopamine release in the nucleus accumbens and caudatus, Eur. J. Pharmacol., 187 (1990) 555556. [7] Keefe, K.A., Zigmond, M.J. and Abercrombie, E.D., Extracellular dopamine in striatum: influence of nerve impulse activity in medial forebrain bundle and local glutamatergic input, Neuroscience, 47 (1992) 325-332. [8] Kornbuber, J., Bormann, J., Retz, W., Hubers, M. and Riederer, P., Memantine displaces [3H]MK-801 at therapeutic concentrations in postmortem human frontal cortex, Eur. J. PharmacoL, 166 (1989) 589-590. [9] Kornhuber, J., Bormann, J., Hubers, M., Rusche, K. and Riederer, P., Effects of the 1-amino-adamantanes at the MK801-binding site of the NMDA-receptor-gated ion channel: a human postmortem brain study, Eur. J. Pharmacol., 206 (1991) 297-300. [10] Lustig, H.S., Ahern, K.von B. and Greenberg, D.A., Antiparkinsonian drugs and in vitro excitotoxicity, Brain Res., 597 (1992) 148-150.
[t 1] Mizoguchi, K., Yokoo, ti., Yoshida, M., Ianaka~ i . ;rod l;maka, M., Dopamine formation from ~-DOPA administered exogenously is independent of dopaminergic ncuromd actixity: studies with invivomicrodiatysis, Brain Res.,611(19¢}3)i52 154. [12] Nowak, L., Bregestovski, P. and Ashcr. P. Magnesium gales glutamate-activated channels in the mouse, ccnlI;d neurones. Nature, 3(17 (1984) 462-465. [13] Paxinos, G. and Watson, (_., The Rat B m m m St~'re{~tax~¢ ((~or(hnates, Academic Press, Sydney, 1982. [14] Quirion, R. and Pert, C.B., Amantadine modulates phencyclidine binding site sensitivity in rat brain. Erperientia, 38 (1982) 955-956. [15] Self el Nasr, M., Peruche, B., Rosberg, C., Mennel, tI.-D, and Krieglstein, J., Neuroprotective effect of mementine demonslrated in vivo and in vitro, Eur..I. Pharmacol. 185 (1990) 19-24.
[16] Vincent Chen, H-S., Pellegrini, J.W., Aggarwal, S.K., Lei, S.Z., Warach, S., Jensen, F.E. and Lipton, S.A., Open-channel block of N-methyl-D-aspartate (NMDA) responses by memantine: therapeutic advantage against NMDA receplor-mediated neurotoxicity, J. Neurosci.. 12 (1992) 4427-4436. [17] Von Voigtlander, P.F. and Moore, K.E., Dopamine: release from the brain in vivo by amantadine, Seienee, 174 (1971) 408-410. [18] Wong, E.H.F., Kemp, J.A., Priestly, T., Knight, A.R., Woodruff, G.N. and Iversen, L.L., The anticonvulsant MK-80I is a potent N-methyl-D-aspartate antagonist, Proc. Natl. Acad. Sci. USA, 83 (1986) 7104-7108. [19] Yoshida, M., Yokoo, H., Mizoguchi, K., Kawahara, H., Tsuda, A., Nishikawa, T. and Tanaka, M., Eating and drinking cause increased dopamine release in the nucleus accumbens and ventral tegmental area in the rat: measurement by in vivo microdialysis, Neurosci. Lett., 139 (1992) 73-76. [20] Zetterstrom, T., Sharp, T., Collin, A.K. and Ungerstedt, U., tn vivo measurement of extracellular dopamine and DOPAC in rat striatum after various dopamine-releasing drugs; implications for the origin of extracellular DOPAC, Eur. J. PharmacoL, 148 (1988) 327-334.
Brain Research 662 (1994) 255-258
Short communication
Amantadine increases the extracellular dopamine levels in the striatum by re-uptake inhibition and by N-methyl-D-aspartate antagonism Katsuhiro Mizoguchi *, Hideyasu Yokoo, Masami Yoshida, Takahiko Tanaka, Masatoshi Tanaka Department of Pharmacology, Kurume Unirersity School of Medicine, Kurume, 830, Japan Accepted 2 August 1994
Abstract
This study was performed to investigate the mechanism how amantadine increases the extracellular dopamine (DA) levels in the striatum in vivo. Local application of amantadine (1 mM, 40 min) to the striatum through the dialysis membrane significantly increased the extracellular DA levels. Coadministration of nomifensine (10 mM, 120 min), an inhibitor of neuronal DA uptake, into the perfusion fluid attenuated the amantadine-induced increase in DA outflow. The amantadine-induced increases in the extraceilular DA levels were also inhibited by co-perfusion with Ringer containing high Mg z+ (15 mM, 120 min) or with MK-801 (1 /zM, 80 min). These findings suggest that amantadine increases the extracellular DA levels in the striatum by inhibiting the re-uptake of DA and/or by blocking the channel in the N-methyl-D-aspartate (NMDA) receptor, which results in antagonism of NMDA receptor function.
Keywords: In vivo microdialysis; Striatum; Amantadine; Dopaminergic neuron; Re-uptake inhibition; N-Methyl-D-aspartate receptor
Amantadine has been used clinically for the treatment of Parkinson's disease. The clinical effectiveness of amantadine is generally attributed to its action on nigrostriatal dopaminergic neurons. It is generally accepted that amantadine acts presynaptically to enhance D A release [17] or inhibit DA uptake [3]. However, there have been little in vivo investigations concerning the action of amantadine on dopaminergic neurons in the striatum. It has been reported recently that memantine, an analogue of amantadine, bound to the MK-801 site in the N-methyl-D-aspartate (NMDA) receptor channel [8,9] and electrophysiologically blocked the N M D A receptor function [1]. In the present study, we examined the effect of amantadine on the extracellular D A levels in the striatum using in vivo microdialysis under conditions of inhibition of D A re-uptake or modification of N M D A receptor function with either MK-801 or high concentrations of magnesium. Male Wistar rats, weighing 250-300 g, were used. Animals were maintained on a 12 h light/dark cycle
* Corresponding author. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 4 ) 0 0 9 3 0 - 9
(lights on at 07.00 h) with free access to food and water. In accordance with the method as described previously [11,19], rats were anesthetized with pentobarbital sodium (45 m g / k g i.p.) and the dialysis probe, the tip of which consisted of a U-shaped dialysis membrane formed cellulose hollow fiber tubing (0.25 mm diameter; molecular weight cut off, 50,000), was implanted into the striatum (coordinates were A: 0.5 ram, L: 3.0 ram, V: 6.0 mm, from the bregma and the dura surface, according to the atlas of Paxinos and Watson [13]). The active surface of the dialysis probe was 6 mm. After the experiment, we determined the position of the dialysis probe histologically in Toluidine blue (0.5%)-stained frozen coronal sections [19]. Dialysis experiments were carried out 2 days after the operation. The dialysis probe was continuously perfused at a constant flow rate of 2 . 5 / x l / m i n with an artificial CSF (composition: NaC1 140 raM, KC1 3.35 mM, MgCI 2 1.15 mM, CaC12 1.26 mM, Na2HPO4 1.20 mM, N a H 2 P O 4 0.30 mM and p H 7.4). Experiments were started between 08.30 and 09.30 h. About 3 h after the beginning of the perfusion, stable basal levels of DA in the dialysates were ob-
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K. Mizoflucht et al. /'Hrain Research c,62 t1904~ 25~ 2~,';
tained and amantadine (1 mM) was infused into the striatum through the dialysis m e m b r a n e for 40 rain. After recovery of D A levels to the approximate basaline level, nomifensine (10/~M) was added to the perfusion fluid and maintained until the end of the experiment. Two h after the start of nomifensine infusion, amantadine (1 raM) was coadministered for 40 min and the effect of amantadine on D A release was reexamined in the presence of nomifensine. Similarly, after a stable D A peak was obtained, MK-801 (1 /~M) or high Mg2+(15 mM) were added to the Ringer until the end of the experiment, respectively. Amantadine (1 raM) was similarly coperfused for 40 rain at the time points of 80 min or 2 h after the start of the administration of MK-801 or high Mg 2+, respectively. The dialysates (50 /zl) were directly injected every 20 min using an automatized injector for analysis of D A into the high performance liquid chromatography (HPLC) coupled to an electrochemical detection system (EICOM, Kyoto, Japan), as described previously [11,19]. The mobile phase in the H P L C was 0.1 M sodium acetate buffer (pH 4.0) containing 0.6 mM octanesulfonic acid, 0.02 mM E D T A and 10% ( v / v ) methanol. D A was separated on an Eicompack M A - O D S column (4.6 mm O.D., 3.6 mm I . D . x 150 mm) at 25°C. The graphite working electrode was set at + 550 m V vs. an A g / A g C 1 reference electrode ( E I C O M ECD-100 electrochemical detector) and the flow rate ( E I C O M EP-10 pump) was 0.9 m l / m i n . Statistical analysis of the data was carried out using the one-way analysis of variance ( A N O V A ) for the effect of amantadine followed by S t u d e n t - N e w m a n Keuls test. The effect of p r e t r e a t m e n t was analysed by two way A N O V A followed by S t u d e n t - N e w m a n - K e uls test. The basal levels of D A in the striatum were determined from the mean of three samples obtained prior to administration of amantadine. In the pretreatment groups, the averaged basal levels of D A were determined from the mean of three samples prior to coperfusion of amantadine. Values are expressed as the mean + S.E.M. The basal level of D A in the striatum was 32.8 + 2.5 p g / s a m p l e (n = 15). Administration of amantadine (1 mM) to the perfusate significantly increased the extracellular D A levels in the striatum (F10,33 = 12.484, P < 0.001, Fig. 1). Addition of nomifensine (10/~M) to the perfusate increased extracellular D A levels 5 times higher than the basal levels and reached a plateau after 80-100 min (Data not shown). The amantadineinduced increase in extracelluar D A levels in the striaturn was significantly inhibited by p r e t r e a t m e n t with nomifensine ( F l o , 6 6 = 5.761, P < 0.001, Fig. 2). Administration of MK-801 (1 /~M) had no effect on the extracellular D A levels in the striatum (Data not shown). Addition of high Mg 2+ (15 raM) to the perfusate reduced the extracellular D A level to half of the
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Fig. 1. Effect of amantadine on the extracellular dopamine levels in the striatum. Each sample represents a 20-min collection period. The value indicates the mean+S.E.M, of four rats. P < 0:001 Vs. basal levels. basal levels and reached a plateau after 60-80 min (Data not shown). The amantadine-induced increases in extraceilular DA levels in the striatum were significantly attenuated in the presence of MK-80] or high Mg 2+ in the perfusion Ringer (F9.60 = 6.636. P < 0.001. Fig. 3, F10,6 6 = 5.761. P < 0.001. Fig. 4. respectively). The present study using in vivo microdialysis clearly demonstrates that the intrastriatal administration of amantadine increases the extracellular D A level in the striatum (Fig. 1). but had little effect on extracellular D O P A C levels (F~0.33 = 0.594. P = 0.8071. Data not shown). It has been known that both amphetamine and nomifensine [2,5] increase extracellular DA levels, but amphetamine decreases extraceltular D O P A C levels. while nomifensine has little effect on extracellular D O P A C levels [20]. Therefore, inhibition of D A re-uptake similar to that of nomifensine-like mechanism in the striatum is considered to be one of the mechanisms for the elevation of the extracellular D A level induced by amantadine because amantadine-induced increases in D A levels in the striatum were attenuated by pretreatment with nomifensine (Fig. 2) and amantadine itself had no effect on the extracellular striatal D O P A C levels. This result supports the previous findings that the tissue uptake of [SH]dopamine decreased following the administration of amantadine [3]. On the other hand, amantadine has been reported to increase the extracellular D A level by enhancing the release of D A in response to electrical stimulation of dopaminergic nerve terminals [17], but without identifying how amantadine enhances the DA release from nigrostriatal dopaminergic nerve terminals. In the present study, amantadine-induced increase in the extracellular D A levels in the striatum was inhibited by pretreatment with MK-80I (1 /.~M), which by itself had no effect on the extracellular baseline D A levels (Fig. 3). Pretreatment with Ringer containing high Mg 2+ (15 mM) also attenuated the facilitatory effect of amanta-
K. Mizoguchi et al. / Brain Research 662 (1994) 255-258
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dine on the extracellular DA levels in the striatum (Fig. 4). Electrophysiological and radioligand binding studies have previously shown that MK-801 antagonizes NMDA responses by binding inside the open channel in the NMDA receptor, thereby blocking transmembrane ion influxes [18]. Phencyclidine (PCP) also blocks the ion channel in a similar manner by binding to the same site as does MK-801 [4]. The ion channel of the NMDA receptor is also blocked by magnesium in a voltage-dependent fashion [12]. In addition, it has been shown that direct infusion of MK-801 increased the extracellular DA levels in the striatum in vivo [6]. Taken together, it mat be possible that amantadine increases the extracellular DA level by binding to the MK-801 site in the NMDA receptor and subsequently blocking the channel site, which resuits in enhancing DA release. This idea is supported by the findings that amantadine and memantine, an analogue of amantadine, displaced [3H]MK-801 in the
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Fig. 3. Effect of MK-801 on amantadine-induced increases in the extracellular dopamine levels in the striatum. Each sample represents a 20-rain collection period. The value indicates the mean+_ S.E.M. of four rats, P < 0,001 between absence (e) and presence (©) of MK-801.
so
;
~; 60 80 ,;0 ,;0 ,~o ,;o ,'80 2~o 2;0 210 m~.
Fig. 4. Effect of high Mg 2+ on amantadine-induced increases in the extracellular dopamine levels in the striatum. Each sample represents a 20-min collection period. The value indicates the mean+_ S.E.M. of four rats. P < 0.001 between absence (e) and presence (©) of high Mg 2+.
postmortem human frontal cortex membranes [8,9] and that memantine blocked NMDA receptor channels in a manner similar to that of MK-801 as determined by electrophysical study [1]. It has also been demonstrated that amantadine bound to [3H]PCP site in a dose dependent manner [14]. This finding also supports the above hypothesis. Neuroprotective actions against NMDA-induced neurotoxicity via NMDA antagonistic actions by amantadine and memantine have been reported in previous studies [10,15,16]. Moreover, it has previously been shown that relatively high concentrations (raM range) as well as lower concentrations (0.1 mM) of glutamate receptor antagonists are potent to block excitatory amino acid (EAA) agonist-induced striatal DA release, but only higher concentrations of the antagonists increased basal striatal DA levels, but lower concentrations did not [7]. These findings further indicate the possibility that amantadine is a NMDA antagonist if amantadine was able to block NMDA-induced striatal DA release and only high concentrations (1 mM) and not lower concentrations of amantadine had an effect on basal striatal DA levels, We confirmed that high concentrations (1 raM) of amantadine significantly induced an increase in striatal DA levels (Fig. 1), but lower concentrations (0.1 raM) did not (data not shown). In conclusion, the clinical use of amantadine is a valuable supplement in the treatment of Parkinson's disease, with the effect of reducing the parkinsonian symptoms by increasing the extracellular DA levels via re-uptake inhibition and NMDA antagonistic action, which results in activating dopaminergic transmission. We wish to thank Prof. G.B. Glavin, Department of Pharmacology and Therapeutics, Faculty of Medicine,
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