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Keywords:

  • adenosine A2A receptor;
  • 6-hydroxydopamine (6-OHDA);
  • KW-6002;
  • MPTP;
  • neuroprotection;
  • Parkinson's disease.

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Note added during review
  7. Acknowledgements
  8. References

Adenosine A2A receptors are abundant in the caudate-putamen and involved in the motor control in several species. In MPTP-treated monkeys, A2A receptor-blockade with an antagonist alleviates parkinsonian symptoms without provoking dyskinesia, suggesting this receptor may offer a new target for the antisymptomatic therapy of Parkinson's disease. In the present study, a significant neuroprotective effect of A2A receptor antagonists is shown in experimental models of Parkinson's disease. Oral administration of A2A receptor antagonists protected against the loss of nigral dopaminergic neuronal cells induced by 6-hydroxydopamine in rats. A2A antagonists also prevented the functional loss of dopaminergic nerve terminals in the striatum and the ensuing gliosis caused by MPTP in mice. The neuroprotective property of A2A receptor antagonists may be exerted by altering the packaging of these neurotoxins into vesicles, thus reducing their effective intracellular concentration. We therefore conclude that the adenosine A2A receptor may provide a novel target for the long-term medication of Parkinson's disease, because blockade of this receptor exerts both acutely antisymptomatic and chronically neuroprotective activities.

Abbreviations used:
GFAP

glial fibrillary acidic protein

6-OHDA

6-hydroxydopamine

PBS

phosphate-buffered saline

PC-12

pheochromocytoma

TH

tyrosine hydroxylase

VMAT

vesicular monoamine transporter.

Most early Parkinson's disease patients respond well to current symptomatic treatment with dopamine replacement therapy, but disability increases with progression of the disease, when severe adverse reactions to the drugs emerge, including dyskinesia (Marsden et al. 1982). In addition, no treatment to date has succeeded in interfering with the basic pathogenic mechanism, which results in the death of the dopaminergic neurons of the substantia nigra (Dunnett and Björklund 1999; Mizuno et al. 1999). This death of nigral neurons may be due to the action of dopamine itself or an unidentified neurotoxin possibly related to compounds such as MPTP and 6-OHDA which cause selective destruction of these nigral neurons (Przedborski et al. 1995; Lotharius et al. 1999; Cochiolo et al. 2000). Therefore alternative approaches are needed for effective therapy of this disabling disease, perhaps involving novel drug targets.

Adenosine is known to act via four major receptor subtypes, A1, A2A, A2B and A3, which have been characterized according to their primary sequences (Fredholm et al. 1994). Adenosine A2A receptors are abundant in the caudate-putamen, nucleus accumbens, and olfactory tubercle in several species (Schiffmann et al. 1990; Svenningsson et al. 1999). In the caudate-putamen, adenosine A2A receptors are localized on several neurons and have been shown to modulate the neurotransmission of γ-aminobutyric acid (GABA), acetylcholine and glutamate (Kurokawa et al. 1996a; Mori et al. 1996; Richardson et al. 1997; Ochi et al. 2000). These actions of the A2A receptor contribute to the control of motor behavior (Kuwana et al. 1999), since A2A agonists inhibit locomotor activity and induce catalepsy in rodents (Shiozaki et al. 1999a,b). In contrast, adenosine A2A antagonists prevent the motor disturbances of dopamine D2 receptor null mice (Aoyama et al. 2000). Recently, the effects of A2A antagonists have been evaluated in parkinsonian monkeys (Kanda et al. 1998a,b; Kanda et al. 2000). It␣was demonstrated that an adenosine A2A receptor␣antagonist KW-6002 exhibits antiparkinsonian activity without producing hyperactivity and provoking dyskinesia. These results suggest that A2A antagonists have the potential to be a new class of antisymptomatic drugs for Parkinson's disease.

However, the involvement of adenosine A2A receptors in the dopaminergic neurodegeneration process in Parkinson's disease remains unknown. To address this question, we investigated whether adenosine A2A receptor blockade exerts a neuroprotective action in experimental models of Parkinson's disease. We used orally active adenosine A2A antagonists which we have recently developed (Nonaka et al. 1994a; Shimada et al. 1997; Ishiwata et al. 2000). In this report, these adenosine A2A receptor antagonists are shown to protect the nigral dopaminergic neurons from the degeneration induced by both MPTP and 6-OHDA.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Note added during review
  7. Acknowledgements
  8. References

Drugs

(E)-1,3-Diethyl-8-(3,4-dimethoxystyryl)-7-methyl-3,7-dihydro-1H-purine-2,6-dione (KW-6002, Kyowa Hakko Kogyo, Tokyo, Japan) (Shimada et al. 1997) (E)-1,3-dimethyl-8-(3,4,5-trimethoxystyryl)-7-methyl-3,7-dihydro-1H-purine-2,6-dione (KF18446, Kyowa Hakko Kogyo) (Ishiwata et al. 2000), 8-dicyclopropylmethyl-1,3-dipropylxanthine (KF15372, Kyowa Hakko Kogyo) (Kurokawa et al. 1996b) and 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, Kyowa Hakko Kogyo) (Kurokawa et al. 1996b) were suspended in 0.3% Tween 80 for oral administration at 10 mL/kg of body weight for mice and 5 mL/kg for rats, respectively. 2-[p-(− 2-carboxyethyl)-phenylethylamino]-5′-N-ethylcarboxamidoadenosine hydrochloride (CGS21680, Kyowa Hakko Kogyo) (Nonaka et al. 1994b) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride␣(MPTP, Research Biochemicals Inc.) were dissolved in saline. 6-hydroxydopamine hydrochloride (6-OHDA, Sigma Chemicals Co., St Louis, MO, USA) was dissolved in 0.05 (%w/v) ascorbic acid/saline. For in vitro experiments, KW-6002 was dissolved in 0.01% dimethylsulfoxide as shown previously (Shimada et al. 1997).

Experimental models for in vivo studies

All procedures used in this study were approved by our institutional Animal Research Committee and were conducted in accordance with the Guiding Principles for the Care and Use of Laboratory Animals endorsed by the Japanese Pharmacological Society. Male Sprague-Dawley rats (200–250 g, Charles River Japan Inc.) were anesthetized with sodium pentobarbital (40 mg/kg i.p.) and injected 6-OHDA (7 µg/3.5 µL/3.5 min, Sigma Chemical Co., St Louis, MO, USA) into left striatum (A + 0.2; L 3.0; V−5.0) (Paxinos and Watson 1986). Male C57BL/6NCrj mice (9–11 weeks old, Charles River Japan Inc.) were treated with MPTP (40 mg/kg, i.p) (Research biochemicals Inc.). Two or seven days after neurotoxins treatment, rats and mice were killed by decapitation and brains were stored at − 80°C until assay.

[3H]Mazindol binding

Binding of [3H]mazindol was measured according to the method described previously (Sundström et al. 1988) with some modifications. Briefly, each striatum was homogenized in microcentrifuge tube containing 300 µL of chilled buffer (mmol/L: NaCl 120, KCl 5, Tris 50; pH: 7.9) and centrifuged at 18 000 g for 5 min. The pellet was suspended in 300 µL of buffer and centrifuged again. Then the pellet was resuspended in 500 µL of buffer. Assay mixture (200 µL) was consisted of 100 µL of tissue suspension, [3H]mazindol (10 nmol/L, NENTM Life Science Products, Inc. Boston, MA, USA) and incubated at 0°C for 1 h in the presence (non-specific binding) or absence (total binding) of nomifensine (10 µmol/L). Then the mixture was immediately filtrated through GF-B glass-fiber filters (Whatman International Ltd, Maidstone, UK) and washed three times in 5 mL of buffer. The radioactivity was measured by liquid scintillation spectrometry. Protein concentration of each sample was assayed using DC protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA) with bovine serum albumin (Sigma) as a standard. Autoradiography for dopamine uptake sites was performed by incubating slide-mounted sections (10 µm) with [3H]mazindol (10 nmol/L) and desipramine (300 nmol/L) containing buffer at 0°C for 1 h. After washed in the buffer three times (10 s, 3 min, 3 min), the slides were immediately dried under cold dry air. Autoradiograms were generated by the apposition of the slides to imaging plates (BAS TR 2040, Fujifilm, Tokyo, Japan) for 48 h and analyzed by BAS-2000 (Fujifilm).

Assay of striatal dopamine content and tyrosine hydroxylase (TH) activity

Dopamine content was analyzed according to the previously described method (Warnhoff 1984; Kawasaki et al. 2000) with some modifications. Striatal tissues were sonicated in 1 mL of perchloroacid (0.2 mol/L) containing isoproterenol (500 nmol/L) as the internal standard. After removing 300 µL of suspension for protein assay, the rest was centrifuged at 18 000 g for 15 min 100 µL of supernatant was assayed by reverse-phase HPLC with electrochemical detection. The striatal TH activity was measured according to the previous report with some modifications (Mayer et al. 1986). Dissected striatum was rapidly homogenized in cold homogenization buffer [(mmol/L); sucrose 320, (EDTA)2Na 1, Tris-HCl (pH 8.5) 20, DTT 1]. Striatal homogenate (50 µL) was added into the 150 µL of incubation buffer [(mmol/L); [U-14C]tyrosine 0.0034, catalase solution (458 units), mercaptoethanol 100, 6-methylpterin 1, sodium acetate buffer (pH 6.1) 200] and incubated at 37°C for 5 min The reaction was terminated by the addition of 200 µL of perchloroacid (1 mol/L) and 50 µL of EDTA 2Na (1 mol/L) and chilled for 10 min Then, 1 mL of Tris-HCl (pH 8.3) (0.2 mol/L) and 150 µL of K2CO3 (1 mol/L) were added on and the samples were centrifuged at 1500 g for 10 min. Supernatant was␣then swirled with 250 mg of acid alumina for sticking [U-14C]DOPA. The alumina was washed twice by distilled water to remove residual [U-14C]-tyrosine. [U-14C]DOPA was eluted from alumina with 400 µL of HCl (0.5 mol/L). The radioactivity of elution was measured by liquid scintillation spectrometry. Protein concentration of each sample was assayed using DC protein assay kit and used for correction of the dopamine content and the specific activity of TH.

Immunohistochemistry

All animals were anesthetized with diethyl ether and intracardially perfused with 20 mL of saline and 40 mL of 4% paraformaldehyde in phosphate-buffered saline (PBS), pH 7.4. The brains were fixed again in 4% paraformaldehyde. Coronal sections (10 µm) were cut and blocked by 5% normal goat serum. Sections were incubated for 2 h with primary antiserum to TH (rabbit anti-TH antibody, 1 : 200; Pel-Freez, Rogers, AR, USA) and to glial fibrially acidic protein (GFAP) (rabbit anti-GFAP antiserum, 1: 200, Sigma). Then the sections were incubated for 1 h with secondary antibody conjugated biotin (Vectastain ABC-PO kit, Vector Laboratory, Burning, CA, USA). The immunostained sections were visualized using microscopy (ECLIPSE TE-300, Nikon, Tokyo, Japan). Observation for TH-positive cells by blinded investigators was made on three to five sections in each of six to eight of different brains. Every selected section showed almost similar shape of the both sides of hippocampus. TH-positive cells were counted only when their nuclei were optimally visualized. The number of TH-positive cells in lesioned side was showed as a percentage of the number of intact side in each section. It was averaged in each rat and then averaged in each treatment group.

Western blotting

Western blot analysis of GFAP content was performed using striatal homogenate. Samples containing 20 µg protein were loaded, separated on a 10% of polyacrylamide gel and transferred to a nitrocellulose membrane. The membrane was blocked in 10% of non-fat dry milk in PBS for 30 min and incubated with primary antiserum to GFAP overnight at 4°C. Then the membrane was rinsed, incubated with secondary antibody (anti-rabbit IgG, Sigma Chemicals, Co.) and blots were detected using enhanced chemiluminescence (ECF, Amersham Int. plc). The blots were quantified using FluorImager SI (Molecular Dynamics, Sunnyvale, CA, USA) and Fragment NT analysis (Molecular Dynamics).

Assay of striatal MPP+ levels

Ten animals per group were killed by decapitation 1, 2, 4 and 6 h after MPTP treatment. Their striatums were dissected and frozen until assay. The frozen striatums were homogenized in 400 µL of ice cold 0.1 mol/L PCA. The aliquot of homogenate was assayed for protein concentration. The homogenates were centrifuged and the level of MPP+ in supernatant was measured by HPLC coupled with a spectrophotometric detector. The mobile phase, composed of Na␣acetate 0.1 mol/acetonitrile(70/30) and triethylamine 0.1% was delivered at a flow rate of 1 mL/min. The column was a Bondasphere (3.9f × 150 mm, Waters Corporation, Massachusetts, USA). The wave length was set to 290 nm (L4000, HITACHI, Tokyo). A standard curve was prepared using known amounts of MPP+ dissolved in 0.1 mol/L perchloroacid. Values are expressed as ng/mg protein.

[3H]MPP+ and [3H]dopamine uptake

The ventral mesencephalon was removed from embryonic day 13 or 14 of Sprague–Dawley rats (Charles River Japan Inc., Yokohama, Japan) and cultured with Neurobasal medium (Life Technologies, Inc., Rockville, MD, USA) supplemented with 1 mmol/L l-glutamine, 100 µg/mL streptomycin, 100 U/mL penicillin, and B27 supplement (Life Technologies, Inc.). Pheochromo-cytoma (PC-12) cells were cultured as described previously (Nonaka et al. 1994a) with some modifications. These cells were seeded on 24-well plates and incubated for 30 min in a CO2 incubator with culture medium containing testing drugs and [3H]MPP+ (1 µCi/mL, 1 µm, NENTM. Life Science Products, Inc.) or [7,8-3H]dopamine (22 nmol/L, 1 µCi/well, Amersham Int. plc, Buckinghamshire, UK). To␣distinguish cytosolic and packaged level of [3H]MPP+, digitonin was used for permeabilization of plasma membrane, which allows free movement of small molecules through plasma membrane. In fact, after the treatment with digitonin (100 µmol/L, 2 min), PC-12 cells developed ATP dependent uptake of [3H]MPP+ into vesicles, which indicates that the digitonin-permeabilized cells had healthy vesicular uptake system and ATP could move across the plasma membrane (data not shown). It was also confirmed that this ATP dependent uptake of [3H]MPP+ was disappeared by the treatment with a inhibitor of vesicular monoamine transporter, reserpine (data not shown). For the detection of packaged level into vesicles, cells were incubated with culture medium containing testing drugs and [3H]MPP+ or [7,8-3H]dopamine. Then they were treated with digitonin (100 µmol/L) for 2 min and washed twice with chilled HEPES-Tris buffer (50 mmol/L HEPES-Tris, 6 mmol/L magnesium chloride, 0.32 mol/L sucrose (pH 8.4)). The resting radioactivity was measured after dissolving cells by 1% of sodium dodecyl sulfate.

Statistical analysis

Data were expressed as mean ± SEM values. Statistical significance (p < 0.05) for two groups was determined by Wilcoxon rank sum test. Statistical significance (p < 0.05) for multiple groups was determined by Kruskal–Wallis test, followed by Steel test.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Note added during review
  7. Acknowledgements
  8. References

Effects of KW-6002 on 6-OHDA-induced dopaminergic neurodegeneration in rats

We initially assessed the effect of A2A receptor blockade (using the selective antagonist KW-6002) on the 6-OHDA-induced dopaminergic neuronal degeneration in rats. A␣former study shows that KW-6002 enhances dopaminergic drug-induced rotation and the maximum effect is obtained at a dose of 3 or 10 mg, p.o. (Koga et al. 2000). In this experiment, KW-6002 (10 mg/kg, p.o.) was repeatedly administered once per day for 7 days, the first dose being 50 min before administration of the neurotoxin 6-OHDA into the striatum. One day after the final administration of KW-6002, the striatum was dissected and the number of dopamine uptake sites was investigated using [3H]mazindol binding, which served as an index of functional dopaminergic nerve terminals in the striatum. Intrastriatal injection of 6-OHDA reduced dopamine uptake sites to 51% of control; the effect was significantly inhibited by KW-6002 (Fig. 1a). On the other hand, administration of KW-6002 had no effect on [3H]mazindol binding in saline-injected rats (data not shown). As previously described (Przedborski et al. 1995), intrastriatal injection of 6-OHDA induced a reduction in the number of TH-positive dopaminergic cells in the substantia nigra (Fig. 1b). This retrograde destruction was also prevented by KW-6002 administration (Fig. 1b).

image

Figure 1. Induction of dopaminergic cell death in rats by unilateral intrastriatal injection of 6-OHDA and its inhibition by KW-6002. (a) Significant reduction of [3H]mazindol binding at 7 days after injection of 6-OHDA (7 µg/3.5 µL) is prevented by chronic administration of KW-6002 (10 mg/kg, p.o.) for 7 days (n = 8–10). (b) Significant loss of the number of TH-positive dopaminergic neurons in substantia nigra induced by 6-OHDA is prevented by KW-6002. Data represent mean ± SEM of the number of TH-positive cells in lesioned side as a percentage of intact side (n = 6–8). *p < 0.05, **p < 0.01, Wilcoxon rank sum test compared with 6-OHDA-treated group.

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Effects of KW-6002 on MPTP-induced dopaminergic neurodegeneration in mice

In line with previous reports (Cochiolo et al. 2000), MPTP (40 mg/kg, i.p) significantly decreased [3H]mazindol binding in striatum to approximately 50% of control (Fig. 2a). In this model, KW-6002 (10 mg/kg, p.o.) prevented the MPTP-induced reduction of [3H]mazindol binding (Figs 2b–d). As shown in Fig. 2(a), KW-6002 produced this neuroprotective effect if it was administered less than 1 h after the treatment with MPTP, although no significant effect was observed if KW-6002 was administered more than 5 h after MPTP treatment (Fig. 2a). Similar to [3H]mazindol binding, the MPTP-induced reduction in dopamine content and TH activity was prevented by acute administration of KW-6002 (Figs 2e and f).

image

Figure 2. Induction of dopaminergic dysfunction in mice by MPTP and its inhibition by KW-6002. (a) Time window of neuroprotective effects of KW-6002 (10 mg/kg, p.o.) on the reduction of [3H]mazindol binding by MPTP (n = 10). (b–d) Autoradiography of dopamine uptake sites of␣representative coronal sections including striatum determined by [3H]mazindol binding (b), control (c), MPTP (d) MPTP + KW-6002 (10 mg/kg, p.o.), and (e) and (f) KW-6002 10 mg/kg, p.o.; 1 h after MPTP treatment prevents reduction of dopamine content (e) and TH activity (f). Data represent mean ± SEM in each group. *p < 0.05, **p␣< 0.01, Steel test and ***p < 0.001, Wilcoxon rank sum test compared with MPTP-treated group.

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Role of adenosine receptors on MPTP-induced toxicity in mice

The direct involvement of the adenosine A2A receptor in MPTP-induced dopaminergic neurodegeneration was further confirmed using an adenosine A2A receptor antagonist KF18446 (10 mg/kg, p.o.) which was also neuroprotective as measured by maintenance of [3H]mazindol binding. On the other hand, adenosine A1 antagonists KF15372 and DPCPX had no such effect, although effective doses of them (Kurokawa et al. 1996b; Shiozaki et al. 1999a) were administered (Fig. 3a). An adenosine A2A receptor agonist CGS 21680 (10 µg/20 µL, i.c.v.) alone did not affect the MPTP-induced reduction of [3H]mazindol binding (Fig. 3b), although it did inhibit the protective effects of KW-6002 (1–10 mg/kg, p.o.) on MPTP-induced reduction of [3H]mazindol binding (Fig. 3c).

image

Figure 3. Effects of adenosine receptor agonist and antagonists on MPTP-induced reduction of [3H]mazindol binding in mice. (a) Effects on [3H]mazindol binding, of adenosine A2A antagonist, KF18446 (10 mg/kg, p.o.) and A1 antagonists, DPCPX and KF15372 (10 mg/kg, p.o.), which were administered at 1 h after MPTP treatment. (b) CGS 21680 (10 µg/20 µL, i.c.v.) alone does not affect MPTP toxicity. (c)␣Administration of KW-6002 at 1 h after MPTP treatment results in␣dose-dependent inhibition in MPTP toxicity. Pretreatment with CGS␣21680 (5 min before KW-6002 administration) reduces the neuroprotective effect of KW-6002. Data represent mean ± SEM in␣each group. *p < 0.05, **p < 0.01, Steel test and ***p < 0.001, Wilcoxon rank sum test compared with MPTP-treated group, †p < 0.05, Wilcoxon rank sum test compared with CGS 21680-untreated group.

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KW-6002 prevented gliosis in mice

In control sections, few GFAP-positive cells could be identified (Fig. 4a). MPTP treatment caused significant increase of GFAP positive cells in striatum. These cells showed hypertrophy with astrocytic processes, which is the typical characterization of activated astrocyte (Fig. 4b) (Hirsch et al. 1999). KW-6002 prevented the increase in the number of the GFAP-positive cells (Fig. 4c). Quantification of GFAP protein levels by image analysis of Western blots suggested that treatment of MPTP resulted in an increase of striatal GFAP protein levels to 176% of the␣control␣group (Figs 4d and e). Acute administration of KW-6002 1 h after MPTP treatment reduced the GFAP protein level to 131% of the control group (Figs 4d and e).

image

Figure 4. Induction of gliosis in striatum by MPTP treatment in mice and its inhibition by KW-6002. (a–c) The brains were taken up 48 h after MPTP treatment and the sections were immunostained with anti GFAP antibody. (a) Control, (b) MPTP, (c) MPTP + KW-6002. (d) and (e) Western blot analysis of GFAP expression in the striatum 48 h after MPTP treatment. (d) Typical GFAP bands are shown. (e)␣Quantification of GFAP expression level in striatum. Each column represents the mean ± SEM percentage of the GFAP intensity compared with that in control band. **p < 0.01, Wilcoxon rank sum test compared with control. †p < 0.05, Wilcoxon rank sum test compared with MPTP(+ vehicle)-treated group. Data represent mean ± SEM from 15 or 16 rats in each group.

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Effect of KW-6002 on MPP+ levels in MPTP-treated mice

We investigated concentration of MPP+ in striatum from 1 to 6 h after MPTP (40 mg/kg, i.p) treatment. MPTP treatment resulted in rapid increase of MPP+ content in the striatum, which might reach peak in less than 1 h and then decreased progressively from 1 to 6 h after MPTP treatment. Changes of MPP+ content values in the striatum over time after MPTP␣treatment were not altered prominently by KW-6002 (10 mg/kg, p.o.) administration (Fig. 5).

image

Figure 5. Content of MPP+ after MPTP treatment in striatum. Changes of MPP+ values (ng/mg protein) over time in the striatum of mice. Each time represents time (hours) after MPTP treatment. Data represent mean ± SEM from 10 mice in each group.

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Effect of adenosine A2A antagonist/agonist on [3H]MPP+ and [3H]dopamine uptake in PC-12 cells and primary cultural neurons

To investigate the mechanism of this neuroprotective effect, we examined the ability of the adenosine A2A receptor to modulate the packaging of dopamine and the toxin 1-methyl-4-phenylpyridinium (MPP+) into synaptic vesicles. The PC-12 cells express both the adenosine A2A receptor (Florio et al. 1999) and vesicular monoamine transporters (VMAT1) (Arvidsson et al. 1997), and have been frequently used as a model for catecholaminergic neurons.

Reserpine, a VMAT inhibitor decreased packaged levels of [3H]MPP+, indicating that most of packaged [3H]MPP+ is taken up into vesicles with VMAT (Fig. 6a). Similar with reserpine, both the adenosine A2A agonist CGS 21680 and 8-bromo cAMP significantly and concentration dependently reduced the packaged level of [3H]MPP+ (Figs 6b and c). This effect of CGS 21680 was diminished by the adenosine A2A antagonist KW-6002 (Fig. 6b).

image

Figure 6. Adenosine A2A receptor regulates [3H]MPP+ and [3H]dopamine packaging. (a) Reserpine, a vesicular monoamine transport inhibitor, reduces packaged [3H]MPP+ in PC-12 cells. (b) CGS 21680 reduces packaged [3H]MPP+ in PC-12 cells. The effect of CGS 21680 is inhibited by KW-6002. (c) 8-bromo-cAMP reduces packaged [3H]MPP+. (d) Reserpine reduces packaged [3H]dopamine in PC-12 cells. (e) CGS 21680 reduces packaged [3H]dopamine in PC-12 cells. The effect of CGS 21680 is inhibited by KW-6002. (f) CGS 21680 reduces [3H]dopamine packaged into the vesicles of primary cultured cells of rat ventral mesencephalon. Each column and bar represents the mean ± SEM percentage of the [3H]MPP+ and [3H]dopamine uptake compared with those of control (vehicle-treated wells). Data were determined from eight to nine separate wells in three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, Steel test compared with vehicle, †p < 0.05, ††p < 0.01, †††p < 0.001, Wilcoxon rank sum test compared with CGS 21680 alone group.

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Next we investigated the effects of the A2A agonist CGS 21680 on packaging of [3H]dopamine into PC-12 cells because dopamine is a candidate of intrinsic sources of oxygen radicals. In PC-12 cells, reserpine and CGS 21680 significantly reduced [3H]dopamine packaging into vesicles (Figs 6d and e). This effect of CGS 21680 was also diminished by coadministration of the adenosine A2A antagonist KW-6002 (Fig. 6e). The effects of CGS 21680 on [3H]MPP+ and [3H]dopamine uptake were almost similar (56.0 ± 3.7% and 54.2 ± 1.7% of control at a concentration of 10−6 mol/L CGS 21680).

In line with PC-12 cells, CGS 21680 significantly reduced [3H]dopamine packaging into vesicles of primary cultural neurons of rat mesencephalon (Fig. 6f).

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Note added during review
  7. Acknowledgements
  8. References

This study shows that adenosine A2A receptor antagonists can prevent dopaminergic neurodegeneration of experimental models of Parkinson's disease. We used both 6-OHDA and MPTP as dopaminergic neurotoxins, which act via distinct cell death pathways (Lotharius et al. 1999). Significant protection against the neurotoxins-induced dopaminergic neurodegeneration strongly supports a concept that adenosine A2A antagonists may be an useful drug for long-term medication of Parkinson's disease patients, because they have both antisymptomatic and neuroprotective properties in the experimental models.

The protective effect of the A2A antagonist, KW-6002 on MPTP-toxicity was observed when it was administered 1 h after MPTP-treatment (Fig. 2). Magnitude of this protective effect was similar to that being produced when KW-6002 was administered before MPTP treatment. In contrast, KW-6002 could not protect dopaminergic neurons when KW-6002 was administered 5 h after MPTP-treatment, even if it was followed by chronic treatments of KW-6002 for 7 days. In addition, we observed similar potencies in neuroprotective effect when KW-6002 was treated acutely or consecutively for 3 or 7 days (data not shown). These data suggest that KW-6002 produces the neuroprotective effect by inhibiting the early phase of the degeneration cascades induced by MPTP. Intrastriatal injection of MPP+ has been reported to cause transient increase of extracellular concentrations of striatal adenosine approximately for 3 h (Ballarin et al. 1989). Thus, time window of neuroprotective effect by KW-6002 seems to correspond with the period during which extracellular concentrations of striatal adenosine are maintained to increase after MPTP treatment.

The direct involvement of adenosine A2A receptor in MPTP-induced dopaminergic neurodegeneration was further confirmed using the adenosine A2A receptor antagonist KF18446 which was also neuroprotective as measured by maintenance of [3H]mazindol binding, although the adenosine A1 antagonists KF15372 and DPCPX were not (Fig. 3a). The neuroprotective effect of KW-6002 was diminished by the pretreatment with CGS 21680, a selective A2A agonist, although CGS21680 per se did not affect MPTP toxicity (Figs 3b and c). Taken together, these results confirm the involvement of adenosine A2A receptors in neuroprotective properties of KW-6002.

MPTP produces the toxicity after oxidized (by monoamine oxidase) to the highly toxic metabolite, MPP+ (Mizuno et al. 1999), and then transferred to the intracellular cytosols in dopaminergic neuronal cells through the dopamine transporter. It is unlikely that KW-6002 may act via inhibition of the monoamine oxidase or the dopamine transporter, because KW-6002 does not inhibit monoamine oxidase nor dopamine transporter in vitro (data not shown). It was also confirmed that KW-6002 does not affect the change of MPP+ content in the striatum over time after MPTP treatment (Fig. 5).

The means by which 6-OHDA and MPTP provoke damage to dopaminergic neurons may involve a complex interplay between dopaminergic neurons and environmental factors. Some previous reports have suggested that adenosine A2A receptors contribute to cerebral inflammation and excitotoxicity (Popoli et al. 1995; Okusa et al. 1999; Sullivan et al. 1999), and therefore adenosine may contribute to the pathological changes of Parkinson's disease by triggering the activation of surrounding glial cells, which are known to appear around degenerating dopaminergic neurons in Parkinson's disease (Hirsch et al. 1999). For instance, an adenosine A2A agonist enhances nitric oxide and cyclooxygenase production in vitro and intrastriatal administration of the A2A agonist induces gliosis in vivo (Fiebich et al. 1996). On the contrary, there is another recent report suggesting that adenosine may inhibit astroglial activation (Michael et al. 1999). We therefore investigated whether KW-6002 inhibits or aggravates MPTP-induced gliosis (Fig. 4). In our experimental condition, marked increase of GFAP-positive cells was observed in the striatum of MPTP-treated mice. These GFAP-positive cells showed characteristic features of activated astrocytes with astrocytic processes and hypertrophy (Hirsch et al. 1999). KW-6002 obviously decreased the number of GFAP-positive cells and MPTP-induced morphological changes. Therefore, adenosine A2A antagonists may inhibit the effect of dopaminergic neurotoxins via a direct action on glial cells in vivo. However we can not rule out the idea that the reduced gliosis may be a consequence of reduction in dopaminergic cell death.

There is another possibility that primal site of the neuroprotective action by adenosine A2A antagonists may be the nigral dopaminergic neurons, because recent literatures show the existence of functional A2A receptor in these cells (Okada et al. 1996; Chen et al. 2000). In dopaminergic neuronal cells, the adenosine receptor blockade may promote excretion of neurotoxin from the cytosol, perhaps via the VMAT. To examine this possibility, we tested the function of adenosine A2A receptors in the packaging of the neurotoxins into vesicles of rat PC-12 cells. A VMAT inhibitor, reserpine potentiates MPP+ and MPTP toxicity in vitro and in vivo (Liu et al. 1992; Varoqui and Erickson 1997). As with reserpine, both CGS 21680 and 8-bromo-cAMP decreased the packaged level of [3H]MPP+ into the vesicles and KW-6002 prevented this CGS 21680-induced reduction of [3H]MPP+ packaging (Fig. 6b). CGS 21680 caused similar reduction of [3H]dopamine packaging in PC-12 cells and this reduction was also inhibited by KW-6002 (Fig. 6e). In addition, in primary cultured neurons of rat ventral mesencephalon, CGS 21680 reduced [3H]dopamine packaged into synaptic vesicles (Fig. 6f). Since adenosine A2A receptors stimulate cAMP production, and VMAT activity is modulated (decreased) by a cAMP activated kinase (Nakanishi et al. 1995; Liu et al. 1999), blockade of adenosine A2A receptors may cause the enhancement of cytosolic MPP+ clearance via the release of inhibition of the VMAT function by A2A receptor stimulation and thus protect the neurons. Indeed in our experiment, addition of 8-bromo cAMP to mimic intercellular cAMP production dose dependently inhibits the packaging of MPP+ into synaptic vesicles (Fig. 6c).

Finally, our study does not rule out other possible mechanisms. It should be borne in mind that the vast majority of the A2A receptors in the striatum are on neurons targeted by the dopaminergic cells (Schiffmann et al. 1990). It is therefore possible that some indirect actions from these neurons might participate in the neuroprotective effect, including the inhibition of glutamatergic input from subthalamic nucleus to substantia nigra, which is overactivated by dopaminergic deficiency (Rodriguez et al. 1998).

In conclusion, we show that A2A receptor antagonists protect against dopaminergic neurodegeneration caused by MPTP and 6-OHDA. From these results, we think that adenosine A2A receptor may provide an ideal treatment target for the long-term medication of Parkinson's disease, because the blockade of it produces both antisymptomatic and neuroprotective effects.

Note added during review

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Note added during review
  7. Acknowledgements
  8. References

During the preparation of this paper, Chen et al. reported a similar neuroprotective effect of adenosine A2A receptor antagonists (Chen et al. 2001).

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Note added during review
  7. Acknowledgements
  8. References

We are grateful to Drs F. Suzuki and J. Shimada for providing adenosine antagonists. The authors also appreciate Dr P. J. Richardson for critical reading of this manuscript.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Note added during review
  7. Acknowledgements
  8. References
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