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Research report

The dopamine agonist pramipexole scavenges hydroxyl free radicals

induced by striatal application of 6-hydroxydopamine in rats: an in

vivo microdialysis study

a,b ,

_*

b c

Boris Ferger

, Peter Teismann , Joachim Mierau

Behavioural Neurobiology Laboratory, Swiss Federal Institute of Technology Zurich, Schorenstrasse 16, CH-8603 Schwerzenbach, Switzerland b

Institute of Pharmacology and Toxicology, Faculty of Pharmacy, Philipps-University of Marburg, Ketzerbach 63, 35032 Marburg, Germany c

Department of CNS Research, Boehringer Ingelheim Pharma KG, Binger Straße 173, 55216 Ingelheim, Germany Accepted 29 August 2000

Abstract

Hydroxyl free radical production seems to play an important role in the pathogenesis of Parkinson’s disease. In the present study, we investigated the dopamine agonists pramipexole and pergolide as well as the nitrone compound S-PBN (N-tert-butyl-a -(2-sulfophenyl)nit-rone) to reduce hydroxyl radical formation. Microdialysis experiments were carried out in non-anaesthetized Wistar rats. Salicylate was incorporated into the perfusion fluid to measure indirectly hydroxyl radicals indicated by 2,3-dihydroxybenzoic acid (2,3-DHBA). Local perfusion with 0.2 or 2 nmol / 2 ml / min 6-hydroxydopamine (6-OHDA) via the microdialysis probe significantly increased 2,3-DHBA levels 14-fold and 47-fold, respectively. Systemic application of either pergolide (0.05 mg / kg) or pramipexole (1 mg / kg) failed to significantly reduce 6-OHDA-induced hydroxyl radical production. In contrast, a 40 min pretreatment with pramipexole (2 and 10 nmol / 2

ml / min via the probe) before onset of 6-OHDA perfusion, significantly attenuated 2,3-DHBA levels compared with vehicle controls. S-PBN pretreatment (2 nmol / 2ml / min) was not effective to reduce 2,3-DHBA levels. In conclusion, pramipexole was able to reduce hydroxyl radical levels induced by 6-OHDA in vivo after local application. This property of pramipexole may be beneficial under conditions of enhanced hydroxyl radical formation in parkinsonian brains and may add to its well known dopamine D -like receptor2 agonistic effects.  2000 Elsevier Science B.V. All rights reserved.

Theme: Disorders of the nervous system Topic: Neurotoxicity

Keywords: Pramipexole; 6-Hydroxydopamine; Parkinson’s disease; Salicylate assay; Radical

1. Introduction factors may contribute to the progression of PD such as

oxidative stress, excitotoxicity, mitochondrial DNA dam-Parkinson’s disease (PD) is characterized by the pro- age, glial and inflammatory processes and apoptosis of gressive loss of dopaminergic neurons originating in the nigral neurons [24]. Potential protective strategies aim to substantia nigra pars compacta and the sustained decrease develop drugs which reduce the influence of these patho-in striatal dopampatho-ine content [3,10,32]. The malfunction of genic factors and most of them are interrelated.

the basal ganglia circuits is responsible for the cardinal We focused our interest on the investigation of oxidative motor symptoms of PD such as tremor at rest, muscular stress in PD. There are many lines of evidence that rigidity, bradykinesia / akinesia as well as stooped posture oxidative stress takes place in PD. The dopamine loss leads

and instability [36]. to a compensatory increase in dopamine turnover including

Although the cause of PD is still unknown [18], many enhanced dopamine metabolism and radical formation, because the enzymatic dopamine metabolism via mono-amine oxidase B (MAO-B) not only produces 3,4-dihydroxyphenylacetic acid (DOPAC) but also hydrogen

*Corresponding author. Tel.:141-1-655-7371; fax:141-1-655-7203.

E-mail address: [email protected] (B. Ferger). peroxide. Hydrogen peroxide itself is not a radical but

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reacts with iron ions to form hydroxyl radicals the most tracellular dopamine levels were measured in the third

reactive oxygen species. experiment.

The ‘gold standard’ for treatment of Parkinson’s disease today is levodopa, the precursor of dopamine, in

combina-tion with a peripheral decarboxylase inhibitor. For many 2. Material and methods years the safety of levodopa and its long-term benefit have

been topics of discussion. Levodopa significantly increased 2.1. In vitro Fenton system hydroxyl radical formation after systemic injection in

combination with the peripheral decarboxylase inhibitor Hydroxyl radicals were generated according to a previ-[37] and many in vitro studies reported toxic effects of ously published method [12,39]. In brief, a mixture of 0.3 levodopa (for review see [19]) and more recently the mM FeCl3,0.3 mM Na EDTA and 3 mM H O in 5 ml of2 2 2

induction of apoptosis [41]. Furthermore, long-term Tris-buffer adjusted to pH 7.4 in the presence of 0.5 mM levodopa therapy is accompanied with psychiatric and salicylic acid was incubated for 15 min at 378C. S-PBN, motor side effects (dyskinesias) and the efficacy of pramipexole or vehicle were co-incubated in this Fenton levodopa medication decreases after some years of treat- system in order to assess their possible radical scavenging ment. Therefore, delaying the onset of levodopa therapy or effects using the salicylate hydroxylation assay.

adjunctive medication with dopamine agonists or MAO

inhibitors may be beneficial in the early phase of PD. 2.2. Animals Dopamine agonists act on dopamine receptors to mimic

the effects of dopamine. In contrast to levodopa they do Adult male albino Wistar rats (Hsd / Cpb:WU, Fa. Har-not need surviving presynaptic dopaminergic neurons for lan-Winkelmann GmbH, Borchen, Germany) weighing uptake and metabolism. Pramipexole is a nonergot dopa- about 300 g at the time of microdialysis probe implanta-mine agonist with an azepine structure and exerts full tion, were used in this study. An ambient room tempera-intrinsic activity on D subfamily receptors, especially the2 ture was maintained at 23628C, the relative humidity at D3 receptor, with little interaction to adrenergic and 5565%. The animals were kept in a 12 h light–dark cycle serotoninergic receptors [26,30,31]. The mechanism of D2 (lights on at 07.00 a.m.–07.00 p.m.) and housed indi-autoreceptor mediated reduction of extracellular dopamine vidually in Plexiglas cages (40320324 cm) with light

 levels was postulated to protect nigral neurons against metal covering and fed with standard food (Altromin , Fa.

associated oxidative stress [23]. Altromin, Lage, Germany) and tap water. All efforts were

Indeed, pramipexole reduced extracellular dopamine made to minimize animal suffering, to reduce the number levels [4] and more recently neuroprotective effects of of animals used, and to utilize alternatives to in vivo pramipexole by additional mechanisms such as antioxidant techniques. The experimental protocols were approved by effects and induction of a trophic factor were claimed [5]. the appropriate institutional governmental agency

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To study the effects of pramipexole on hydroxyl free gierungsprasident Gießen, Germany). radicals in vitro, we used a cell-free Fenton system. Under

these conditions the potential antioxidant effects are direct- 2.3. In vivo microdialysis experiments ly related to the chemical structure of the compound and

not to indirect effects such as interaction with biological The microdialysis experiments were carried out as antioxidant mechanisms such as induction of antioxidant described earlier [13,38]. A guide cannula was implanted enzymes or reduction of the radical generating dopamine under chloral hydrate anaesthesia (400 mg / kg i.p.) and

metabolism. aimed at the head of the caudate nucleus (coordinates from

In vivo, two routes of administration (systemic and bregma: AP:11.25, ML:12.6, DV:22.5 according to the local) of pramipexole were applied to investigate the atlas of Paxinos and Watson [29]. On the following day a potential effect on hydroxyl radical levels. In the first microdialysis probe (CMA 12, membrane diameter 0.5 experiment, systemic application of pramipexole was mm, membrane length 4 mm, Carnegie Medicin, Stock-compared with pergolide on the reduction of basal hy- holm, Sweden) was introduced and the rats were placed in droxyl radical levels without any exogenous stimulation of a microdialysis system with balance arm for freely moving hydroxyl radical formation. In the second experiment, animals. The perfusion fluid contained 5 mM salicylic acid

1 1

systemic application of pramipexole was compared with dissolved in a modified Ringer solution (Na 147 mM, K

21 21 2

pergolide on the reduction of hydroxyl radical levels 4 mM, Ca 1.3 mM, Mg 1 mM, Cl 155.6 mM, flow

induced by striatal reverse dialysis with 6-OHDA. In the rate: 2 ml / min). After an equilibration period of approxi-third experiment, local application of pramipexole was mately 180 min a stable baseline was obtained and the compared with S-PBN (instead of pergolide, because of intraperitoneal injection of pramipexole (0.05, 0.2, and 1 the poor solubility of pergolide in the perfusion fluid) on mg / kg), pergolide (0.05 mg / kg) or saline was carried out. the reduction of hydroxyl radical levels again induced by The mean of three 20 min basal dialysate fractions were striatal reverse dialysis with 6-OHDA. Furthermore, ex- set as 100%.

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In experiments with exogenous hydroxyl radical stimu- column (12533 mm, pre-column 533 mm filled with lation the perfusion fluid was changed for 60 min to a fluid Nucleosil 120-3 C18 (Knauer, Berlin, Germany). Data additionally containing 0.2 or 2 nmol / 2 ml / min of 6- were calculated by an external standard calibration. hydroxydopamine for reverse dialysis to deliver 6-OHDA The detection limit for 2,3-DHBA was approximately into the surrounding tissue of the microdialysis probe. 0.1 nmol / l and its in vitro recovery (microdialysis probes Then, the perfusion fluid was changed back again to the CMA 12) was 15–20%. The values of 2,3-DHBA and perfusion fluid of the baseline recording, which was dopamine are expressed as percentages of three predrug without 6-OHDA. In control experiments modified Ringer dialysates and were not corrected for the recovery of the

solution was used instead of 6-OHDA. probes.

Samples were collected every 20 min in vials containing 10ml 0.4 M perchloric acid and were directly analyzed by

HPLC or stored at2708C until analysis. 3. Results

2.4. HPLC-analysis 3.1. In vitro studies

The HPLC system for determination of 2,3- and 2,5- Incubation of salicylic acid alone in the radical generat-DHBA and dopamine consisted of a solvent delivery ing system resulted in a similar increase in 2,3-DHBA and system (Waters model 600S in combination with Waters 2,5-DHBA levels and to a lesser extent in an increase in model 616, Millipore, Milford, USA), an autoinjector with catechol (data not shown), reflecting an enhanced hydroxyl cooling module set at 48C (Waters model 717 plus, radical formation. In control experiments without drug Millipore, Milford, USA), a column thermostat set at 228C incubation 2,3-DHBA levels (mean6S.E.M.) of 4.460.12

(Gynkotek model STH 585, Germering, Germany), an mM were obtained and set as 100%. Increasing

con-online degasser (Knauer model A1050, Berlin, Germany), centrations of pramipexole (0.5–5 mM) and S-PBN (0.5–5 a two channel electrochemical detector (BAS LC 4C, mM) led to a significant decrease in 2,3-DHBA levels Bioanalytical Systems, West Lafayette, USA) and a data (Fig. 1). In each concentration tested, pramipexole was collection and calculation unit controlled by Gynkosoft more effective than S-PBN (P,0.001). The highest con-software (Gynkotek, Germering, Germany). The detector centration of pramipexole (5 mM) reduced the increase of potentials were set at 1750 mV using a glassy carbon 2,3-DHBA to about 3.5% of the control values, whereas electrode and an Ag /AgCl reference electrode with a range S-PBN (5 mM) reduced the increase of 2,3-DHBA to of 1–10 nA / V. The mobile phase contained 0.14 g octane about 35% of the control values.

sulfonic acid sodium salt as an ion-pair reagent, 0.1 g

disodium EDTA, 6 ml triethylamine and 35 ml acetonitrile 3.2. Effects of saline, pramipexole and pergolide on 

in 1 l of millipore Q water adjusted to pH 2.95 with basal2,3-DHBA levels concentrated phosphoric acid. The eluent was delivered

with a flow rate of 0.5 ml / min onto a reversed-phase The perfusion with 5 mM salicylic acid produced stable

Fig. 1. In vitro effects of S-PBN and pramipexole on hydroxyl free radical levels in a Fenton system. 2,3-DHBA values indicate the alterations of hydroxyl free radical levels. Control values were obtained by incubating saline instead of pramipexole and were regarded as 100%. Data are given as mean6S.E.M. of n56 experiments. Statistical analysis was performed using Kruskal–Wallis H-test with subsequent Mann–Whitney U-test. * P,0.05, ** P,0.01, *** P,0.001 was considered significant by comparing drug values with controls.

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basal 2,3-DHBA levels, which were not significantly OHDA (0.2 nmol / 2ml / min) via the probe led to an about different between the experimental groups. After injection 14-fold increase in 2,3-DHBA levels in the saline treated of saline, pramipexole (1 mg / kg) or pergolide (0.05 mg / group and to an about 12-fold increase in the S-PBN kg) no significant changes in 2,3-DHBA levels were group. This reduction was not statistically significant. In observed compared to the corresponding pre-injection contrast, in the pramipexole (2 and 10 nmol / 2 ml / min) basal values (mean6S.E.M.) 53.4463.5, 55.6463.9 and treated groups the 6-OHDA-induced elevation in

2,3-57.9262.2 nmol / l, respectively (Fig. 2). DHBA levels was significantly attenuated to a 7.8- and

8.6-fold increase, respectively (Fig. 4A). 3.3. Effects of saline, pramipexole and pergolide on

6-OHDA-induced increase in 2,3-DHBA levels 3.5. Effects of local application of saline, S-PBN and pramipexole on6-OHDA-induced increase in dopamine Before onset of hydroxyl free radical stimulation with levels

6-OHDA 2,3-DHBA mean basal levels6S.E.M. in the

saline, pramipexole (0.05, 0.2 and 1 mg / kg i.p.) and The following dopamine mean basal levels6S.E.M. pergolide (0.05 mg / kg i.p.) treated groups of 53.4266.3, were obtained in the saline, S-PBN (2 nmol / 2ml / min) and 50.5663.0, 52.4965.8, 54.7865.3 nmol / l and 50.4466.0 pramipexole (2 and 10 nmol / 2 ml / min) treated groups of nmol / l, respectively were obtained. Perfusion with 6- 2.3160.3, 2.4360.2, 2.3460.5 and 2.2160.4 nmol / l, OHDA (0.2 nmol / 2ml / min) via the probe led to an about respectively. Perfusion with 6-OHDA (0.2 nmol / 2 ml / 14-fold increase in 2,3-DHBA levels in the saline treated min) led to an about 25.5-fold increase in dopamine levels group, to an about 12–13-fold increase in the pramipexole in the saline treated group compared to mean basal levels. treated groups and to an about 17-fold increase in the In the S-PBN treated group during 6-OHDA perfusion an pergolide treated group. There were no significant differ- attenuation to an 11-fold increase was observed. In con-ences between any of the experimental groups (Fig. 3). trast, pramipexole (2 and 10 nmol / 2ml / min) pretreatment significantly attenuated the 6-OHDA-induced increase in 3.4. Effects of local application of saline, S-PBN and dopamine levels to a 6.9- and 4.6-fold elevation, respec-pramipexole on6-OHDA-induced increase of 2,3-DHBA tively (Fig. 4B).

levels

Before onset of hydroxyl free radical stimulation with 4. Discussion

6-OHDA the following 2,3-DHBA mean basal

levels6S.E.M. were obtained in the saline, S-PBN (2 In the present study, pramipexole showed a pronounced nmol / 2 ml / min) and pramipexole (2 and 10 nmol / 2 ml / effect in reducing hydroxyl radical levels which were min) treated groups of 54.4265.1, 56.9162.9, 55.3462.8 generated in a cell-free in vitro Fenton system. In vivo, and 55.9664.9 nmol / l, respectively. Perfusion with 6- local application of pramipexole decreased the

6-OHDA-Fig. 2. In vivo effects of saline, pramipexole 1 mg / kg and pergolide 0.05 mg / kg on the extracellular concentration of 2,3-DHBA in striatal microdialysates. The heights of the columns represent the percentages of pre-drug levels of 2,3-DHBA; each column demonstrates a fraction of 20 min. The dialysate fractions 1–4 represent the basal concentration and were regarded as 100%. The following fractions (5–10) show the values after injection of saline, pramipexole or pergolide. Mean values6S.E.M. for rats (n56) are given. Statistical analysis was performed using ANOVA with Bonferroni’s-test for multiple comparisons between the groups. No significant differences were obtained.

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Fig. 3. In vivo effects of saline, pramipexole (0.05 mg / kg), pramipexole (0.2 mg / kg), pramipexole (1 mg / kg) and pergolide (0.05 mg / kg) on the extracellular concentration of 2,3-DHBA in striatal microdialysates. The dialysate fractions 1–4 represent the basal concentration and were regarded as 100%. In dialysate fractions 5 and 6, 6-hydroxydopamine (6-OHDA) (0.2 nmol / 2ml / min) was administered via the probe to stimulate hydroxyl radical formation. Mean values6S.E.M. for rats (n511–13) are given. ANOVA with Bonferroni’s-test for multiple comparisons showed no significant differences between the experimental groups.

induced hydroxyl radical formation which was paralleled malonate a massive overflow of dopamine was paralleled by an attenuation of the 6-OHDA-induced increase in by an increase in hydroxyl radical formation. Furthermore,

extracellular dopamine concentration. the lesion of the substantia nigra with 6-OHDA, which

Pramipexole was designed as a novel D2 receptor almost completely depleted striatal dopamine, prevented agonist [25]. Binding studies revealed a 5- to 10-fold the malonate-induced hydroxyl radical formation [11]. higher selectivity for human D receptors compared with3 Moreover, Lancelot et al. [22], reported that dopamine D2 like subtypes (D , D , D ) [26,31]. The dopamine2s 2l 4 release contributes to an enhanced hydroxyl radical forma-receptor agonistic properties of pramipexole may account tion after local glutamate application.

for the antagonism of parkinsonian symptoms in MPTP The antioxidant activity of many dopamine agonists pretreated monkeys [25], the improvement of parkinsonian seems to contribute to their protective effects as demon-symptoms in MPTP-induced hemiparkinsonian macaques strated for piribedil [7], bromocriptine [27,40], pergolide monkeys [9] and the symptomatic benefit in parkinsonian [15,28], apomorphine [14,33], ropinirole [17] and patients. Besides its postsynaptic effects, pramipexole pramipexole [6,16,42]. However, direct radical scavenging activates presynaptic dopamine autoreceptors and at- properties are difficult to observe under in vivo conditions. tenuates the extracellular dopamine concentration [4]. We demonstrated that pramipexole was only effective to However, the basal hydroxyl radical levels were not reduce hydroxyl radical levels when a local administration reduced after systemic administration in the present study. via the microdialysis probe was performed. The applied We think that the autoreceptor regulated dopamine release concentrations seem appropriate because the diffusion-seems not to play an important role to reduce hydroxyl controlled pretreatment of pramipexole via the probe was radicals under basal conditions where only low nanomolar carried out before the onset of 6-OHDA perfusion and concentrations of dopamine were measured in striatal pramipexole was not present in the perfusion fluid during

dialysates. Conversely, 6-OHDA produced a massive 6-OHDA delivery. This was done to avoid a direct

increase in the extracellular dopamine concentration (25.5- interaction of 6-OHDA with pramipexole before brain fold), which was significantly reduced by pretreatment entry.

with pramipexole. The role of excessive dopamine con- The concept of a radical scavenger is to protect bio-centrations seems to be critical for hydroxyl radical molecules such as lipids, proteins and nucleic acids from formation. In a previous study with local application of radical attacks. Indeed, in PD oxidative stress indicated by

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Fig. 4. In vivo effects of saline, S-PBN (2 nmol / 2ml / min), pramipexole (2 nmol / 2ml / min) and pramipexole (10 nmol / 2ml / min) on the extracellular concentration of 2,3-DHBA (A) and dopamine (B) in striatal microdialysates. The dialysate fractions 1–2 represent the basal concentration and were regarded as 100%. During fractions 3–4 S-PBN or pramipexole was perfused through the probe as pretreatment. In dialysate fractions 5 and 6, 6-hydroxydopamine (6-OHDA) (0.2 nmol / 2ml / min) was administered via the probe to stimulate hydroxyl radical formation. Mean values6S.E.M. for rats (n511–13) are given. Statistical analysis was performed using ANOVA with Bonferroni’s-test for multiple comparisons. * P,0.05, ** P,0.01, *** P,0.001 was considered significant by comparing drug pretreated animals with controls.

enhanced lipid peroxidation [8], oxidative protein altera- radical levels in vivo. In contrast, S-PBN was not able to tions [1] and oxidative DNA alterations [2,34] is evident. reduce 2,3-DHBA levels which is in line with the results of In respect of their radical scavenging activity, pramipexole the in vitro system in which pramipexole was more and S-PBN have to compete with biomolecules in the effective to decrease hydroxyl radical levels than S-PBN. reaction with hydroxyl radicals in order to reduce oxidative Although our study was not designed to measure stress and protect against cell damage. Therefore, only the neuroprotection by itself, our findings provide further higher brain concentrations which were achieved by local evidence that pramipexole exerts neuroprotective effects application of pramipexole were able to reduce hydroxyl which were reported by Hall et al. [16] in mice against

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High affinity binding for pramipexole, a dopamine D3 receptor [38] P. Teismann, B. Ferger, Comparison of the novel drug ensaculin ligand, in rat striatum, Neurosci. Lett. 219 (1996) 138–140. with MK-801 on the reduction of hydroxyl radical production in rat [32] P. Riederer, S. Wuketich, Time course of nigrostriatal degeneration striatum after local application of glutamate, Brain Res. 857 (2000)

in Parkinson’s disease. A detailed study of influential factors in 165–171.

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(1)

In experiments with exogenous hydroxyl radical stimu-

column (12533 mm, pre-column 533 mm filled with

lation the perfusion fluid was changed for 60 min to a fluid

Nucleosil 120-3 C18 (Knauer, Berlin, Germany). Data

additionally containing 0.2 or 2 nmol / 2

ml / min of 6-

were calculated by an external standard calibration.

hydroxydopamine for reverse dialysis to deliver 6-OHDA

The detection limit for 2,3-DHBA was approximately

into the surrounding tissue of the microdialysis probe.

0.1 nmol / l and its in vitro recovery (microdialysis probes

Then, the perfusion fluid was changed back again to the

CMA 12) was 15–20%. The values of 2,3-DHBA and

perfusion fluid of the baseline recording, which was

dopamine are expressed as percentages of three predrug

without 6-OHDA. In control experiments modified Ringer

dialysates and were not corrected for the recovery of the

solution was used instead of 6-OHDA.

probes.

Samples were collected every 20 min in vials containing

10 ml 0.4 M perchloric acid and were directly analyzed by

HPLC or stored at

2708C until analysis.

3. Results

2.4. HPLC-analysis

3.1. In vitro studies

The HPLC system for determination of 2,3- and 2,5-

Incubation of salicylic acid alone in the radical

generat-DHBA and dopamine consisted of a solvent delivery

ing system resulted in a similar increase in 2,3-DHBA and

system (Waters model 600S in combination with Waters

2,5-DHBA levels and to a lesser extent in an increase in

model 616, Millipore, Milford, USA), an autoinjector with

catechol (data not shown), reflecting an enhanced hydroxyl

cooling module set at 48C (Waters model 717 plus,

radical formation. In control experiments without drug

Millipore, Milford, USA), a column thermostat set at 228C

incubation 2,3-DHBA levels (mean6S.E.M.) of 4.460.12

(Gynkotek model STH 585, Germering, Germany), an

mM were obtained and set as 100%. Increasing

con-online degasser (Knauer model A1050, Berlin, Germany),

centrations of pramipexole (0.5–5 mM) and S-PBN (0.5–5

a two channel electrochemical detector (BAS LC 4C,

mM) led to a significant decrease in 2,3-DHBA levels

Bioanalytical Systems, West Lafayette, USA) and a data

(Fig. 1). In each concentration tested, pramipexole was

collection and calculation unit controlled by Gynkosoft

more effective than S-PBN (P,0.001). The highest

con-software (Gynkotek, Germering, Germany). The detector

centration of pramipexole (5 mM) reduced the increase of

potentials were set at

1750 mV using a glassy carbon

2,3-DHBA to about 3.5% of the control values, whereas

electrode and an Ag /AgCl reference electrode with a range

S-PBN (5 mM) reduced the increase of 2,3-DHBA to

of 1–10 nA / V. The mobile phase contained 0.14 g octane

about 35% of the control values.

sulfonic acid sodium salt as an ion-pair reagent, 0.1 g

disodium EDTA, 6 ml triethylamine and 35 ml acetonitrile

3.2. Effects of saline, pramipexole and pergolide on



in 1 l of millipore Q

water adjusted to pH 2.95 with

basal

2,3-DHBA levels

concentrated phosphoric acid. The eluent was delivered

with a flow rate of 0.5 ml / min onto a reversed-phase

The perfusion with 5 mM salicylic acid produced stable

(2)

basal 2,3-DHBA levels, which were not significantly

OHDA (0.2 nmol / 2

ml / min) via the probe led to an about

different between the experimental groups. After injection

14-fold increase in 2,3-DHBA levels in the saline treated

of saline, pramipexole (1 mg / kg) or pergolide (0.05 mg /

group and to an about 12-fold increase in the S-PBN

kg) no significant changes in 2,3-DHBA levels were

group. This reduction was not statistically significant. In

observed compared to the corresponding pre-injection

contrast, in the pramipexole (2 and 10 nmol / 2

ml / min)

basal values (mean6S.E.M.) 53.4463.5, 55.6463.9 and

treated groups the 6-OHDA-induced elevation in

2,3-57.9262.2 nmol / l, respectively (Fig. 2).

DHBA levels was significantly attenuated to a 7.8- and

8.6-fold increase, respectively (Fig. 4A).

3.3. Effects of saline, pramipexole and pergolide on

6-OHDA-induced increase in

2,3-DHBA levels

3.5. Effects of local application of saline, S-PBN and

pramipexole on

6-OHDA-induced increase in dopamine

Before onset of hydroxyl free radical stimulation with

levels

6-OHDA 2,3-DHBA mean basal levels6S.E.M. in the

saline, pramipexole (0.05, 0.2 and 1 mg / kg i.p.) and

The following dopamine mean basal levels6S.E.M.

pergolide (0.05 mg / kg i.p.) treated groups of 53.4266.3,

were obtained in the saline, S-PBN (2 nmol / 2

ml / min) and

50.5663.0, 52.4965.8, 54.7865.3 nmol / l and 50.4466.0

pramipexole (2 and 10 nmol / 2

ml / min) treated groups of

nmol / l, respectively were obtained. Perfusion with 6-

2.3160.3, 2.4360.2, 2.3460.5 and 2.2160.4 nmol / l,

OHDA (0.2 nmol / 2

ml / min) via the probe led to an about

respectively. Perfusion with 6-OHDA (0.2 nmol / 2

ml /

14-fold increase in 2,3-DHBA levels in the saline treated

min) led to an about 25.5-fold increase in dopamine levels

group, to an about 12–13-fold increase in the pramipexole

in the saline treated group compared to mean basal levels.

treated groups and to an about 17-fold increase in the

In the S-PBN treated group during 6-OHDA perfusion an

pergolide treated group. There were no significant differ-

attenuation to an 11-fold increase was observed. In

con-ences between any of the experimental groups (Fig. 3).

trast, pramipexole (2 and 10 nmol / 2

ml / min) pretreatment

significantly attenuated the 6-OHDA-induced increase in

3.4. Effects of local application of saline, S-PBN and

dopamine levels to a 6.9- and 4.6-fold elevation,

respec-pramipexole on

6-OHDA-induced increase of 2,3-DHBA

tively (Fig. 4B).

levels

Before onset of hydroxyl free radical stimulation with

4. Discussion

6-OHDA

the

following

2,3-DHBA

mean

basal

levels6S.E.M. were obtained in the saline, S-PBN (2

In the present study, pramipexole showed a pronounced

nmol / 2

ml / min) and pramipexole (2 and 10 nmol / 2

ml /

effect in reducing hydroxyl radical levels which were

min) treated groups of 54.4265.1, 56.9162.9, 55.3462.8

generated in a cell-free in vitro Fenton system. In vivo,

and 55.9664.9 nmol / l, respectively. Perfusion with 6-

local application of pramipexole decreased the

(3)

induced hydroxyl radical formation which was paralleled

malonate a massive overflow of dopamine was paralleled

by an attenuation of the 6-OHDA-induced increase in

by an increase in hydroxyl radical formation. Furthermore,

extracellular dopamine concentration.

the lesion of the substantia nigra with 6-OHDA, which

Pramipexole was designed as a novel D

receptor

almost completely depleted striatal dopamine, prevented

agonist [25]. Binding studies revealed a 5- to 10-fold

the malonate-induced hydroxyl radical formation [11].

higher selectivity for human D receptors compared with

Moreover, Lancelot et al. [22], reported that dopamine

D

like subtypes (D , D , D ) [26,31]. The dopamine

2s 2l 4

release contributes to an enhanced hydroxyl radical

forma-receptor agonistic properties of pramipexole may account

tion after local glutamate application.

for the antagonism of parkinsonian symptoms in MPTP

The antioxidant activity of many dopamine agonists

pretreated monkeys [25], the improvement of parkinsonian

seems to contribute to their protective effects as

demon-symptoms in MPTP-induced hemiparkinsonian macaques

strated for piribedil [7], bromocriptine [27,40], pergolide

monkeys [9] and the symptomatic benefit in parkinsonian

[15,28],

apomorphine

[14,33],

ropinirole

[17]

and

patients. Besides its postsynaptic effects, pramipexole

pramipexole [6,16,42]. However, direct radical scavenging

activates presynaptic dopamine autoreceptors and at-

properties are difficult to observe under in vivo conditions.

tenuates the extracellular dopamine concentration [4].

We demonstrated that pramipexole was only effective to

However, the basal hydroxyl radical levels were not

reduce hydroxyl radical levels when a local administration

reduced after systemic administration in the present study.

via the microdialysis probe was performed. The applied

We think that the autoreceptor regulated dopamine release

concentrations seem appropriate because the

diffusion-seems not to play an important role to reduce hydroxyl

controlled pretreatment of pramipexole via the probe was

radicals under basal conditions where only low nanomolar

carried out before the onset of 6-OHDA perfusion and

concentrations of dopamine were measured in striatal

pramipexole was not present in the perfusion fluid during

dialysates. Conversely, 6-OHDA produced a massive

6-OHDA delivery. This was done to avoid a direct

increase in the extracellular dopamine concentration (25.5-

interaction of 6-OHDA with pramipexole before brain

fold), which was significantly reduced by pretreatment

entry.

with pramipexole. The role of excessive dopamine con-

The concept of a radical scavenger is to protect

bio-centrations seems to be critical for hydroxyl radical

molecules such as lipids, proteins and nucleic acids from

formation. In a previous study with local application of

radical attacks. Indeed, in PD oxidative stress indicated by

(4)

enhanced lipid peroxidation [8], oxidative protein altera-

radical levels in vivo. In contrast, S-PBN was not able to

tions [1] and oxidative DNA alterations [2,34] is evident.

reduce 2,3-DHBA levels which is in line with the results of

In respect of their radical scavenging activity, pramipexole

the in vitro system in which pramipexole was more

and S-PBN have to compete with biomolecules in the

effective to decrease hydroxyl radical levels than S-PBN.

reaction with hydroxyl radicals in order to reduce oxidative

Although our study was not designed to measure

stress and protect against cell damage. Therefore, only the

neuroprotection by itself, our findings provide further

higher brain concentrations which were achieved by local

evidence that pramipexole exerts neuroprotective effects

application of pramipexole were able to reduce hydroxyl

which were reported by Hall et al. [16] in mice against

(5)

Verhalten bei Erkrankungen des extrapyramidalen Systems, Klin.

methamphetamine toxicity and in gerbils in the model of

Wochenschr. 38 (1960) 1236–1239.

forebrain ischemia with postischemic nigral degeneration.

[11] B. Ferger, O. Eberhardt, P. Teismann, C. de Groote, J.B. Schulz,

They postulated direct radical scavenging properties for

_{Malonate-induced generation of reactive oxygen species in rat}

pramipexole and an antioxidant capability indicated by a

striatum depends on dopamine release but not on NMDA-receptor

low oxidation potential. Inhibitory effects on MPTP-in-

activation, J. Neurochem. 73 (1999) 1329–1332.

[12] B. Ferger, C. Spratt, C.D. Earl, P. Teismann, W.H. Oertel, K.

duced dopamine reduction in mice [20], methamphetamine

Kuschinsky, Effects of nicotine on hydroxyl free radical formation

toxicity in mice [21], protection against 3-acetyl-pyridine

in vitro and on MPTP-induced neurotoxicity in vivo, Naunyn

[35] and against local application of MPP

[6] are further

_{Schmiedebergs Arch. Pharmacol. 358 (1998) 351–359.}

hints for additional effects of pramipexole not related to its

_{[13] B. Ferger, C. van Amsterdam, C. Seyfried, K. Kuschinsky, Effects}

D -like agonistic effects. The protection of cultured

2 of a-phenyl-tert-butylnitrone and selegiline on hydroxyl free radi-cals in rat striatum produced by local application of glutamate, J.

mesencephalic dopaminergic neurons against levodopa

Neurochem. 70 (1998) 276–280.

neurotoxicity may be mediated by pramipexole-mediated

[14] M. Gassen, Y. Glinka, B. Pinchasi, M.B. Youdim, Apomorphine is a

induction of trophic activity of pramipexole [5].

_{highly potent free radical scavenger in rat brain mitochondrial}

Taken together, pramipexole was able to reduce hy-

_{fraction, Eur. J. Pharmacol. 308 (1996) 219–225.}

droxyl radical levels induced by the Fenton reaction in

[15] M. Gomez-Vargas, S. Nishibayashi-Asanuma, M. Asanuma, Y.´ Kondo, E. Iwata, N. Ogawa, Pergolide scavenges both hydroxyl and

vitro and by 6-OHDA after local application in vivo. This

nitric oxide free radicals in vitro and inhibits lipid peroxidation in

property of pramipexole may be beneficial under

con-different regions of the rat brain, Brain Res. 790 (1998) 202–208.

ditions of enhanced hydroxyl radical formation in

par-[16] E.D. Hall, P.K. Andrus, J.A. Oostveen, J.S. Althaus, P.F. von

kinsonian brains and may add to its well known dopamine

_{Voigtlander, Neuroprotective effects of the dopamine D / D agonist}

2 3

D -like receptor agonistic effects.

2 pramipexole against postischemic or methamphetamine-induced degeneration of nigrostriatal neurons, Brain Res. 742 (1996) 80–88. [17] M. Iida, I. Miyazaki, K. Tanka, H. Kabuto, E. Iwata-Ichikawa, N. Ogawa, Dopamine D2 receptor-mediated antioxidant and neuro-protective effects of ropinirole, a dopamine agonist, Brain Res. 838

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