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l -DOPA induces Ca2+-dependent and tetrodotoxin-sensitive release of endogenous glutamate from rat striatal slices
167
Brain Research, 617 (1993) 167-170 © 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00
BRES 25732
L-DOPA induces Ca2+-dependent and tetrodotoxin-sensitive release of endogenous glutamate from rat striatal slices Y o s h i o G o s h i m a a K a o r u O h n o a, S h i n i c h i N a k a m u r a a, T a k e a k i M i y a m a e a, Y o s h i m i M i s u a and Akinori Akaike b a Department of Pharmacology, Yokohama City University School of Medicine, Yokohama (Japan) and 6 Department of Neuropharmacology, Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University, Fukuyama (Japan) (Accepted 6 April 1993)
Key words: L-DOPA; Striatum; Glutamate; Release; Parkinson's disease; Transmitter; Rat
L-DOPA (10-1000 /xM) concentration-dependently released glutamate (Glu) from superfused rat striatal slices. D-DOPA and dopamine (300 /zM) produced no effects. The L-DOPA-induced release of Glu was not affected by 3-hydroxybenzylhydrazine (20 ~zM), an L-aromatic amino acid decarboxylase inhibitor. L-DOPA methyl ester (200 #M), a selective L-DOPA antagonist, antagonized the effect of L-DOPA in a competitive manner. Ca 2+ deprivation and tetrodotoxin decreased L-DOPA (300 p.M)-induced release of Glu. These findings indicate that L-DOPA induces a transmitter-like release of Glu via activation of a recognition site for itself.
L-3,4-Dihydroxyphenylalanine (L-DOPA) is the most effective therapeutic agent for Parkinson's disease and believed to be an inert amino acid which exerts the actions entirely through its conversion to dopamine by L-aromatic amino acid decarboxylase (AADC). Contrary to this generally accepted idea, we have proposed that L-DOPA is a neurotransmitter or neuromodulator. L-DOPA is released in a transmitter-like manner in in vitro and in vivo rat striatum 4'13-a5. Exogenously applied L-DOPA at nano- or picomolar concentrations stereoselectively potentiates activities of presynaptic /3-adrenoceptors to facilitate the impulse-evoked release of noradrenaline and dopamine in the absence and even in the presence of an AADC inhibitor in rat brain slices 3'6'8'12. L-DOPA also facilitates the release of dopamine from the striatal slices of 1-methyl-4phenyl-l,2,3,6-tetrahydropyridine-treated C57 black mouse model for parkinsonism x'5. However, the propranolol-induced blockade of the facilitatory action of L-DOPA was noncompetitive. Moreover, L-DOPA did not displace specific binding of a /3-adrenoceptor ligand in rat membrane preparations. These findings sug-
gest that there exists a recognition site for L-DOPA itself which differs from presynaptic /3-adrenoceptors. This idea is also supported by the finding that the agonistic activities of L-DOPA are closely related to its catechol moiety, amino and carboxyl groups, and the action was competitively antagonized by several LDOPA analogues. Among these compounds tested, L-DOPA methyl ester is a potent L-DOPA antagonist at least in in vitro slice preparations 7. Furthermore, we found that unilateral microinjections of L-DOPA, but not D-DOPA, into the nucleus tractus solitarii (NTS) produced dose-dependent postsynaptic depressor and bradycardic responses in in vivo rats 1°. The important finding is that the actions of L-DOPA were completely antagonized by L-DOPA methyl ester while the actions of Glu were not affected by the same dose of this L-DOPA analogue ~°. These studies suggest that L-DOPA acts as a neurotransmitter or modulator in certain brain regions including the striatum. Since our previous study has demonstrated that L-DOPA facilitates the release of dopamine and noradrenaline, L-DOPA may affect the
Correspondence: Y. Misu, Department of Pharmacology, Yokohama City University School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama 236, Japan. Fax: (81)(045)785-3645.
168 release of Glu in the striatum. We, therefore, investigated whether or not L-DOPA itself induces release of endogenous Glu from rat striatal slices. Male Wistar rats (200-250 g) were decapitated and the brains were rapidly taken out into ice-cold Krebs medium with the following composition (mM): NaC1, 118.4; KCI, 4.74; CaCl2, 2.52; MgSO4, 1.18; KH2PO4, 1.19; NaHCO 3, 25; and glucose, 11.1. The striata were dissected out and 0.3-mm-thick slices were cut using a McIlwain tissue chopper. Slices (20 mg wet wt) were transferred to a chamber (0.45 ml) and superfused with Krebs medium, kept at 32.5°C and gassed with 95% 0 2 and 5% CO 2, at a flow rate of 0.45 ml/min. After a 64-rain equilibration period, superfusates were collected every 4 rain. L-DOPA was applied for 8 min, 72 min after the start of superfusion. The amount of L-DOPA-induced release of Glu was calculated as the total release minus the basal release in three successive samples for 12 rain and expressed as a percentage of the absolute value of the spontaneous release in the sample immediately before the L-DOPA application. Ca 2+ deprivation and pretreatment with L-DOPA methyl ester, 3-hydoxybenzylhydrazine (NSD-1015) or tetrodotoxin (TTX) were done 20 min before the LD O P A application and continued throughout the experiments. At the end of each experiment, slices were homogenized by sonication in 2 ml of ice-cold methanol. After centrifugation (9000 × g , 15 rain), the supernatant was diluted by two-fold and stored on ice until quantification of the amino acids. Endogenous amino acids were quantified by an HPLC-ECD-precolumn depricatization method as described previously 9. In brief, samples were mixed with 25 ~1 of o-phthalaldehyde solution. After 2 min, 50 tzl was injected into the HPLC apparatus. The derivatized amino acids were separated on a Wako-Pak 5C18 reverse-phase column (150 × 4.6 ram) at 40°C using a gradient of solvent A [tetrahydrofuran/methanol/0.1 M sodium acetate (pH 7.2), 3:10:90] and solvent B [tetrahydrofuran/methanol, 3:100 (vol./vol.)] at a flow rate of 1.2 ml/min. Eluted amino acids were quantified by using a spectrofluorometer (Hitachi 650-10LC). The detection limit for Glu at a signal:noise ratio of 2 was in the range of 0.05-0.1 pmol. All the test drugs used did not interfere with the peak of Glu. Results were expressed as mean _+ S.E.M. The statistical significance was determined by unpaired Student's t-test. The spontaneous release of Glu became stable 60 min after the start of superfusion. The amount of spontaneous release of Glu immediately before the application of L-DOPA was 0.69 _+ 0.03 p m o l / m i n / m g wet wt (n = 25). A 8-min application of L-DOPA (301000 p,M) released Glu in a concentration-dependent
4oi Y o
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Concentration (uM) Fig. 1. L-DOPA-induced release of endogenous Glu from rat superfused striatal slices in absence (©) and presence (e) of L-DOPA methyl ester (200 p,M). I.-DOPA, D-DOPA ([]) and dopamine (,x) were applied for 8 rain, 72 min after start of superfusion. Ordinate shows amount of e-DOPA-induced release of Glu. Each value represents percentage of absolute value of spontaneous release estimated in sample immediately before L-DOPA application. Values are mean _+S.E.M. of at least five determinations. * P < 0.05, ** P < 0.01, compared with L-DOPA 300/zM alone.
manner with an EDso value of 140/xM (Fig. 1). At the end of superfusion, the tissue content of Glu was 4.27_+0.17 n m o l / m g wet wt (n =5). The absolute amount of the evoked release of Glu induced by 300 /xM L-DOPA was 0.792 +_ 0.084 p m o l / m g wet wt (n = 15) and the fractional release of Glu expressed as a function of the tissue content was 0.018%. L-DOPA methyl ester (200 /~M) shifted the concentration-release curve for L-DOPA to the right without modifying the maximum effect (Fig. 1). o - D O P A (300 /xM) and dopamine (300 lxM) did not induce significant release of Glu. NSD-1015 (20 txM), an AADC inhibitor 6, did not affect the action of L-DOPA (Fig. 2). Deprivation of Ca 2+ and addition of q ~ X (0.3 izM) reduced the L-DOPA (300 /xM)-induced release of Glu by 70 and 43%, respectively. The spontaneous release and the tissue content of Glu at the end of experiments were not modified by these treatments. We demonstrated that micromolar concentrations of L-DOPA released endogenous Glu from superfused rat striatal slices. The e-DOPA-induced Glu release was transmitter-like because this was Ca2+-dependent and TTX-sensitive. Since the effect was neither mimicked by dopamine nor affected by AADC inhibition (99.6%) 6, the action of t,-DOPA is probably due to L-DOPA itself. Furthermore, the effect of L-DOPA was antagonized in a competitive fashion by L-DOPA methyl ester, the compound that we have evaluated as a potent competitive antagonist for the facilitatory effect of I,-DOPA on the impulse-evoked release of
169
,~3C o=
i! lC
C
NSD 20uM
Ca2" TTX free 30OHM
Fig. 2. Effects of NSD-1015, TTX and Ca 2+ deprivation on L-DOPA (300/zM)-induced Glu release. Treatment with NSD-1015 (20/~M) TTX (300 nM) or Ca 2÷ deprivation was performed 20 min before L-DOPA application. Data shown are mean+S.E.M, from at least five estimations. * P < 0.05, ** P < 0.01, compared with L-DOPA alone (C).
noradrenaline from superfused rat hypothalamic slices 7. The site of action of L-DOPA methyl ester is different from the carrier proteins for its transport system and from adrenergic and dopaminergic receptors 7. The specificity of L-DOPA methyl ester was also confirmed on a cardiovascular response in in vivo anesthetized rat l°. L-DOPA methyl ester showed no antagonism against the cardiovascular response induced by Glu microinjected into the NTS while the same dose of this ester compound completely antagonized that by LD O P A 1°. It is, therefore, likely that L-DOPA releases endogenous Glu from in vitro striatal slices via a recognition site for L-DOPA itself. This site was stereoselective in nature in common with many receptors since D-DOPA produced no effect. Ca 2+ deprivation from the medium decreased the L-DOPA-induced release by 70% which is analogous to kainate-induced release of Glu from rat striatal slices 2. This suggests that Glu is released by L-DOPA by a Ca2+-dependent excitation-secretion coupling process similar to that involved in transmitter release. LDOPA-induced Glu release showed a T T X sensitivity. This indicates that L-DOPA in part depolarizes neuronal cell soma a n d / o r dendrites which in turn leads to conduction of nerve impulses, terminal depolarization and release of Glu. However, q-TX at the concentration which almost completely blocks the impulseevoked release of Glu from slices of rat brain stem 9, decreased the release by only ~ 40%. This suggests that L-DOPA could also induce the release via a TTXinsensitive process: this release might be caused by
direct depolarizing action of L-DOPA on glutamatergic neuron terminals. The effective concentration range of L-DOPA to evoke the release of Glu seen in this study was comparable to that of kainate to evoke the release of Glu from rat striatal slices 2. The absolute amount of Glu released by L-DOPA (0.3 mM) in terms of percent increase over the spontaneous release was ~ 30% which was almost comparable to that obtained with kainate (0.5 mM) in rat striatal slices 2. It is an important issue to be solved whether or not Glu release is physiologically regulated by endogenous L-DOPA because L-DOPA is tonically released in a Tl'X-sensitive and Ca2+dependent manner in the striatum of freely moving rats 14. It could be possible that the release of Glu caused by exogenously applied L-DOPA affects the progress of Parkinson's disease during the chronic therapy because endogenous Glu has been implicated in the pathogenesis of neuronal cell death t6. The therapeutic plasma concentrations of L-DOPA are estimated to be 1-10 /~M in parkinsonian patients ~7. This concentration is ~ 1 / 1 0 of the EDso value of L-DOPA obtained in this study. It is, therefore, unlikely that L-DOPA at the therapeutic doses could produce neuroexcitatory a n d / o r neurotoxic actions on the striatal neurons. However, this might occur, if the tissue levels of AADC activity are extremely low. In fact, the mean activities of A A D C in the caudate putamen of patients with Parkinson's disease are only 5 - 1 5 % of those of control patients 11. This warrants further study to investigate whether or not the neuroexcitatory action of L-DOPA could have some adverse influence on the neurodegenerative process in Parkinson's disease on the long-term therapy. Our data represent the first evidence that L-DOPA depolarizes striatal neurons to induce release of excitatory amino acid Glu and further support a role of L-DOPA as an endogenous neuroactive substance in the central nervous system.
1 Arai, N., Misugi, K., Goshima Y. and Misu Y., Evaluation of a 1-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP)-treated C57 black mouse model for parkinsonism, Brain Res., 515 (1990) 57-63. 2 Ferkany, J.W. and Coyle, J.T., Kainic acid selectively stimulates the release of endogenous excitatory acidic amino acids, J. PharmacoL Exp. Ther., 225 (1983) 399-406. 3 Goshima, Y., Kubo, T. and Misu, Y., Biphasic actions of L-DOPA on the release of endogenous noradrenaline and dopamine from rat hypothalamic slices, Br. J. PharmacoL, 89 (1986) 229-234. 4 Goshima, Y., Kubo, T. and Misu, Y., Transmitter-like release of endogenous 3,4-dihydroxyphenylalanine from rat striatal slices, J. Neurochem., 50 (1988) 1725-1730. 5 Goshima, Y., Misu, Y., Arai, N. and Misugi, K., Nanomolar L-DOPA facilitates release of dopamine via presynaptic fl-adren-
170
6
7
8
9
10
oceptors - Comparative studies on the actions in striatal slices from control and l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP)-treated C57 black mice, an animal model for Parkinson's disease, Jpn. Z PharmacoL, 55 (1991) 93-100. Goshima, Y., Nakamura, S. and Misu, Y., L-DOPA facilitates the release of endogenous norepinephrine and dopamine via presynaptic/31- and /32-adrenoceptors under essentially complete inhibition of c-aromatic amino acid decarboxylase in rat hypothalamic slices, Jpn. J. Pharmacol., 53 (1990) 47-56. Goshima, Y., Nakamura, S. and Misu, Y., L-Dihydroxyphenylalanine methyl ester is a potent competitive antagonist of the c-dihydroxyphenylalanine-induced facilitation of the evoked release of endogenous norepinephrine from rat hypothalamic slices, J. Pharmacol. Exp. Ther., 258 (1991)466-471. Goshima, Y., Nakamura, S., Ohno, K. and Misu, Y., Picomolar concentrations of L-DOPA stereoselectively potentiate activities of presynaptic /3-adrenoceptors to facilitate the release of endogenous noradrenaline from rat hypothalamic slices, Neurosci. Lett., 129 (1991) 214-216. Kihara, M., Misu, Y. and Kubo, T., Release by electrical stimulation of endogenous glutamate, y-aminobutyric acid, and other amino acids from slices of the rat medulla oblongata, J. Neurochem., 52 (1989) 261-267. Kubo, T., Yue, J.-L., Goshima, Y., Nakamura, S. and Misu, Y., Evidence for c-DOPA systems responsible for cardiovascular
11
12
13
14
15
16 17
control in the nucleus tractus solitarii of the rat, Neurosci. Lett., 140 (1992) 153-156. Lloyd, K.G., Davidson, L. and Hornykiewicz, O., The neurochemistry of Parkinson's disease: Effect of L-DOPA therapy, J. Pharmacol. Exp. Ther., 195 (1975) 453-464. Misu, Y., Goshima, Y. and Kubo, T., Biphasic actions of c-DOPA on the release of endogenous dopamine via presynaptic receptors in rat striatal slices, Neurosci. Lett., 72 (1986) 194-198. Misu, Y.. Goshima, Y., Nakamura, S. and Kubo, T., Nicotine releases stereoselectively and CaZ+-dependently endogenous 3,4dihydroxyphenylalanine from rat striatal slices, Brain Res., 520 (1990) 334-337. Nakamura, S., Goshima, Y., Yue, J.-L., Miyamae, T. and Misu, Y., Transmitter-like basal and K+-evoked release of 3,4-dihydroxyphenylalanine from the striatum in conscious rats studied by microdialysis, J. Neurochem., 58 (1992) 270-275. Nakamura, S., Goshima, Y., Yue, J.-L., Miyamae, T. and Misu, Y., Transmitter-like DOPA is tonically released by nicotine in striata of conscious rats, Eur. J. PharmacoL, 222 (1992) 75-80. Olney, J.W., Excitotoxic amino acids and neuropsychiatric disorders, Annu. Ret,. Pharmacol. Toxicol., 30 (1990)47-71. Rossor, M.N., Watkins, J., Brown, M.J., Reid, J.L. and Dollery, C.T., Plasma levodopa, dopamine and therapeutic response following levodopa therapy of parkinsonian patients, Brain Res., 46 (1980) 385-392.
Brain Research, 617 (1993) 167-170 © 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00
BRES 25732
L-DOPA induces Ca2+-dependent and tetrodotoxin-sensitive release of endogenous glutamate from rat striatal slices Y o s h i o G o s h i m a a K a o r u O h n o a, S h i n i c h i N a k a m u r a a, T a k e a k i M i y a m a e a, Y o s h i m i M i s u a and Akinori Akaike b a Department of Pharmacology, Yokohama City University School of Medicine, Yokohama (Japan) and 6 Department of Neuropharmacology, Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University, Fukuyama (Japan) (Accepted 6 April 1993)
Key words: L-DOPA; Striatum; Glutamate; Release; Parkinson's disease; Transmitter; Rat
L-DOPA (10-1000 /xM) concentration-dependently released glutamate (Glu) from superfused rat striatal slices. D-DOPA and dopamine (300 /zM) produced no effects. The L-DOPA-induced release of Glu was not affected by 3-hydroxybenzylhydrazine (20 ~zM), an L-aromatic amino acid decarboxylase inhibitor. L-DOPA methyl ester (200 #M), a selective L-DOPA antagonist, antagonized the effect of L-DOPA in a competitive manner. Ca 2+ deprivation and tetrodotoxin decreased L-DOPA (300 p.M)-induced release of Glu. These findings indicate that L-DOPA induces a transmitter-like release of Glu via activation of a recognition site for itself.
L-3,4-Dihydroxyphenylalanine (L-DOPA) is the most effective therapeutic agent for Parkinson's disease and believed to be an inert amino acid which exerts the actions entirely through its conversion to dopamine by L-aromatic amino acid decarboxylase (AADC). Contrary to this generally accepted idea, we have proposed that L-DOPA is a neurotransmitter or neuromodulator. L-DOPA is released in a transmitter-like manner in in vitro and in vivo rat striatum 4'13-a5. Exogenously applied L-DOPA at nano- or picomolar concentrations stereoselectively potentiates activities of presynaptic /3-adrenoceptors to facilitate the impulse-evoked release of noradrenaline and dopamine in the absence and even in the presence of an AADC inhibitor in rat brain slices 3'6'8'12. L-DOPA also facilitates the release of dopamine from the striatal slices of 1-methyl-4phenyl-l,2,3,6-tetrahydropyridine-treated C57 black mouse model for parkinsonism x'5. However, the propranolol-induced blockade of the facilitatory action of L-DOPA was noncompetitive. Moreover, L-DOPA did not displace specific binding of a /3-adrenoceptor ligand in rat membrane preparations. These findings sug-
gest that there exists a recognition site for L-DOPA itself which differs from presynaptic /3-adrenoceptors. This idea is also supported by the finding that the agonistic activities of L-DOPA are closely related to its catechol moiety, amino and carboxyl groups, and the action was competitively antagonized by several LDOPA analogues. Among these compounds tested, L-DOPA methyl ester is a potent L-DOPA antagonist at least in in vitro slice preparations 7. Furthermore, we found that unilateral microinjections of L-DOPA, but not D-DOPA, into the nucleus tractus solitarii (NTS) produced dose-dependent postsynaptic depressor and bradycardic responses in in vivo rats 1°. The important finding is that the actions of L-DOPA were completely antagonized by L-DOPA methyl ester while the actions of Glu were not affected by the same dose of this L-DOPA analogue ~°. These studies suggest that L-DOPA acts as a neurotransmitter or modulator in certain brain regions including the striatum. Since our previous study has demonstrated that L-DOPA facilitates the release of dopamine and noradrenaline, L-DOPA may affect the
Correspondence: Y. Misu, Department of Pharmacology, Yokohama City University School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama 236, Japan. Fax: (81)(045)785-3645.
168 release of Glu in the striatum. We, therefore, investigated whether or not L-DOPA itself induces release of endogenous Glu from rat striatal slices. Male Wistar rats (200-250 g) were decapitated and the brains were rapidly taken out into ice-cold Krebs medium with the following composition (mM): NaC1, 118.4; KCI, 4.74; CaCl2, 2.52; MgSO4, 1.18; KH2PO4, 1.19; NaHCO 3, 25; and glucose, 11.1. The striata were dissected out and 0.3-mm-thick slices were cut using a McIlwain tissue chopper. Slices (20 mg wet wt) were transferred to a chamber (0.45 ml) and superfused with Krebs medium, kept at 32.5°C and gassed with 95% 0 2 and 5% CO 2, at a flow rate of 0.45 ml/min. After a 64-rain equilibration period, superfusates were collected every 4 rain. L-DOPA was applied for 8 min, 72 min after the start of superfusion. The amount of L-DOPA-induced release of Glu was calculated as the total release minus the basal release in three successive samples for 12 rain and expressed as a percentage of the absolute value of the spontaneous release in the sample immediately before the L-DOPA application. Ca 2+ deprivation and pretreatment with L-DOPA methyl ester, 3-hydoxybenzylhydrazine (NSD-1015) or tetrodotoxin (TTX) were done 20 min before the LD O P A application and continued throughout the experiments. At the end of each experiment, slices were homogenized by sonication in 2 ml of ice-cold methanol. After centrifugation (9000 × g , 15 rain), the supernatant was diluted by two-fold and stored on ice until quantification of the amino acids. Endogenous amino acids were quantified by an HPLC-ECD-precolumn depricatization method as described previously 9. In brief, samples were mixed with 25 ~1 of o-phthalaldehyde solution. After 2 min, 50 tzl was injected into the HPLC apparatus. The derivatized amino acids were separated on a Wako-Pak 5C18 reverse-phase column (150 × 4.6 ram) at 40°C using a gradient of solvent A [tetrahydrofuran/methanol/0.1 M sodium acetate (pH 7.2), 3:10:90] and solvent B [tetrahydrofuran/methanol, 3:100 (vol./vol.)] at a flow rate of 1.2 ml/min. Eluted amino acids were quantified by using a spectrofluorometer (Hitachi 650-10LC). The detection limit for Glu at a signal:noise ratio of 2 was in the range of 0.05-0.1 pmol. All the test drugs used did not interfere with the peak of Glu. Results were expressed as mean _+ S.E.M. The statistical significance was determined by unpaired Student's t-test. The spontaneous release of Glu became stable 60 min after the start of superfusion. The amount of spontaneous release of Glu immediately before the application of L-DOPA was 0.69 _+ 0.03 p m o l / m i n / m g wet wt (n = 25). A 8-min application of L-DOPA (301000 p,M) released Glu in a concentration-dependent
4oi Y o
T / I///
~20~ -~ 0 '-~
I
i
f
i lO
/ //
,V
S'
~J
30
100
300
1000
Concentration (uM) Fig. 1. L-DOPA-induced release of endogenous Glu from rat superfused striatal slices in absence (©) and presence (e) of L-DOPA methyl ester (200 p,M). I.-DOPA, D-DOPA ([]) and dopamine (,x) were applied for 8 rain, 72 min after start of superfusion. Ordinate shows amount of e-DOPA-induced release of Glu. Each value represents percentage of absolute value of spontaneous release estimated in sample immediately before L-DOPA application. Values are mean _+S.E.M. of at least five determinations. * P < 0.05, ** P < 0.01, compared with L-DOPA 300/zM alone.
manner with an EDso value of 140/xM (Fig. 1). At the end of superfusion, the tissue content of Glu was 4.27_+0.17 n m o l / m g wet wt (n =5). The absolute amount of the evoked release of Glu induced by 300 /xM L-DOPA was 0.792 +_ 0.084 p m o l / m g wet wt (n = 15) and the fractional release of Glu expressed as a function of the tissue content was 0.018%. L-DOPA methyl ester (200 /~M) shifted the concentration-release curve for L-DOPA to the right without modifying the maximum effect (Fig. 1). o - D O P A (300 /xM) and dopamine (300 lxM) did not induce significant release of Glu. NSD-1015 (20 txM), an AADC inhibitor 6, did not affect the action of L-DOPA (Fig. 2). Deprivation of Ca 2+ and addition of q ~ X (0.3 izM) reduced the L-DOPA (300 /xM)-induced release of Glu by 70 and 43%, respectively. The spontaneous release and the tissue content of Glu at the end of experiments were not modified by these treatments. We demonstrated that micromolar concentrations of L-DOPA released endogenous Glu from superfused rat striatal slices. The e-DOPA-induced Glu release was transmitter-like because this was Ca2+-dependent and TTX-sensitive. Since the effect was neither mimicked by dopamine nor affected by AADC inhibition (99.6%) 6, the action of t,-DOPA is probably due to L-DOPA itself. Furthermore, the effect of L-DOPA was antagonized in a competitive fashion by L-DOPA methyl ester, the compound that we have evaluated as a potent competitive antagonist for the facilitatory effect of I,-DOPA on the impulse-evoked release of
169
,~3C o=
i! lC
C
NSD 20uM
Ca2" TTX free 30OHM
Fig. 2. Effects of NSD-1015, TTX and Ca 2+ deprivation on L-DOPA (300/zM)-induced Glu release. Treatment with NSD-1015 (20/~M) TTX (300 nM) or Ca 2÷ deprivation was performed 20 min before L-DOPA application. Data shown are mean+S.E.M, from at least five estimations. * P < 0.05, ** P < 0.01, compared with L-DOPA alone (C).
noradrenaline from superfused rat hypothalamic slices 7. The site of action of L-DOPA methyl ester is different from the carrier proteins for its transport system and from adrenergic and dopaminergic receptors 7. The specificity of L-DOPA methyl ester was also confirmed on a cardiovascular response in in vivo anesthetized rat l°. L-DOPA methyl ester showed no antagonism against the cardiovascular response induced by Glu microinjected into the NTS while the same dose of this ester compound completely antagonized that by LD O P A 1°. It is, therefore, likely that L-DOPA releases endogenous Glu from in vitro striatal slices via a recognition site for L-DOPA itself. This site was stereoselective in nature in common with many receptors since D-DOPA produced no effect. Ca 2+ deprivation from the medium decreased the L-DOPA-induced release by 70% which is analogous to kainate-induced release of Glu from rat striatal slices 2. This suggests that Glu is released by L-DOPA by a Ca2+-dependent excitation-secretion coupling process similar to that involved in transmitter release. LDOPA-induced Glu release showed a T T X sensitivity. This indicates that L-DOPA in part depolarizes neuronal cell soma a n d / o r dendrites which in turn leads to conduction of nerve impulses, terminal depolarization and release of Glu. However, q-TX at the concentration which almost completely blocks the impulseevoked release of Glu from slices of rat brain stem 9, decreased the release by only ~ 40%. This suggests that L-DOPA could also induce the release via a TTXinsensitive process: this release might be caused by
direct depolarizing action of L-DOPA on glutamatergic neuron terminals. The effective concentration range of L-DOPA to evoke the release of Glu seen in this study was comparable to that of kainate to evoke the release of Glu from rat striatal slices 2. The absolute amount of Glu released by L-DOPA (0.3 mM) in terms of percent increase over the spontaneous release was ~ 30% which was almost comparable to that obtained with kainate (0.5 mM) in rat striatal slices 2. It is an important issue to be solved whether or not Glu release is physiologically regulated by endogenous L-DOPA because L-DOPA is tonically released in a Tl'X-sensitive and Ca2+dependent manner in the striatum of freely moving rats 14. It could be possible that the release of Glu caused by exogenously applied L-DOPA affects the progress of Parkinson's disease during the chronic therapy because endogenous Glu has been implicated in the pathogenesis of neuronal cell death t6. The therapeutic plasma concentrations of L-DOPA are estimated to be 1-10 /~M in parkinsonian patients ~7. This concentration is ~ 1 / 1 0 of the EDso value of L-DOPA obtained in this study. It is, therefore, unlikely that L-DOPA at the therapeutic doses could produce neuroexcitatory a n d / o r neurotoxic actions on the striatal neurons. However, this might occur, if the tissue levels of AADC activity are extremely low. In fact, the mean activities of A A D C in the caudate putamen of patients with Parkinson's disease are only 5 - 1 5 % of those of control patients 11. This warrants further study to investigate whether or not the neuroexcitatory action of L-DOPA could have some adverse influence on the neurodegenerative process in Parkinson's disease on the long-term therapy. Our data represent the first evidence that L-DOPA depolarizes striatal neurons to induce release of excitatory amino acid Glu and further support a role of L-DOPA as an endogenous neuroactive substance in the central nervous system.
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