Research reportThe dopamine agonist pramipexole scavenges hydroxyl free radicals induced by striatal application of 6-hydroxydopamine in rats: an in vivo microdialysis study
Introduction
Parkinson’s disease (PD) is characterized by the progressive loss of dopaminergic neurons originating in the substantia nigra pars compacta and the sustained decrease in striatal dopamine content [3], [10], [32]. The malfunction of the basal ganglia circuits is responsible for the cardinal motor symptoms of PD such as tremor at rest, muscular rigidity, bradykinesia/akinesia as well as stooped posture and instability [36].
Although the cause of PD is still unknown [18], many factors may contribute to the progression of PD such as oxidative stress, excitotoxicity, mitochondrial DNA damage, glial and inflammatory processes and apoptosis of nigral neurons [24]. Potential protective strategies aim to develop drugs which reduce the influence of these pathogenic factors and most of them are interrelated.
We focused our interest on the investigation of oxidative stress in PD. There are many lines of evidence that oxidative stress takes place in PD. The dopamine loss leads to a compensatory increase in dopamine turnover including enhanced dopamine metabolism and radical formation, because the enzymatic dopamine metabolism via monoamine oxidase B (MAO-B) not only produces 3,4-dihydroxyphenylacetic acid (DOPAC) but also hydrogen peroxide. Hydrogen peroxide itself is not a radical but reacts with iron ions to form hydroxyl radicals the most reactive oxygen species.
The ‘gold standard’ for treatment of Parkinson’s disease today is levodopa, the precursor of dopamine, in combination with a peripheral decarboxylase inhibitor. For many years the safety of levodopa and its long-term benefit have been topics of discussion. Levodopa significantly increased hydroxyl radical formation after systemic injection in combination with the peripheral decarboxylase inhibitor [37] and many in vitro studies reported toxic effects of levodopa (for review see [19]) and more recently the induction of apoptosis [41]. Furthermore, long-term levodopa therapy is accompanied with psychiatric and motor side effects (dyskinesias) and the efficacy of levodopa medication decreases after some years of treatment. Therefore, delaying the onset of levodopa therapy or adjunctive medication with dopamine agonists or MAO inhibitors may be beneficial in the early phase of PD.
Dopamine agonists act on dopamine receptors to mimic the effects of dopamine. In contrast to levodopa they do not need surviving presynaptic dopaminergic neurons for uptake and metabolism. Pramipexole is a nonergot dopamine agonist with an azepine structure and exerts full intrinsic activity on D2 subfamily receptors, especially the D3 receptor, with little interaction to adrenergic and serotoninergic receptors [26], [30], [31]. The mechanism of D2 autoreceptor mediated reduction of extracellular dopamine levels was postulated to protect nigral neurons against associated oxidative stress [23].
Indeed, pramipexole reduced extracellular dopamine levels [4] and more recently neuroprotective effects of pramipexole by additional mechanisms such as antioxidant effects and induction of a trophic factor were claimed [5].
To study the effects of pramipexole on hydroxyl free radicals in vitro, we used a cell-free Fenton system. Under these conditions the potential antioxidant effects are directly related to the chemical structure of the compound and not to indirect effects such as interaction with biological antioxidant mechanisms such as induction of antioxidant enzymes or reduction of the radical generating dopamine metabolism.
In vivo, two routes of administration (systemic and local) of pramipexole were applied to investigate the potential effect on hydroxyl radical levels. In the first experiment, systemic application of pramipexole was compared with pergolide on the reduction of basal hydroxyl radical levels without any exogenous stimulation of hydroxyl radical formation. In the second experiment, systemic application of pramipexole was compared with pergolide on the reduction of hydroxyl radical levels induced by striatal reverse dialysis with 6-OHDA. In the third experiment, local application of pramipexole was compared with S-PBN (instead of pergolide, because of the poor solubility of pergolide in the perfusion fluid) on the reduction of hydroxyl radical levels again induced by striatal reverse dialysis with 6-OHDA. Furthermore, extracellular dopamine levels were measured in the third experiment.
Section snippets
In vitro Fenton system
Hydroxyl radicals were generated according to a previously published method [12], [39]. In brief, a mixture of 0.3 mM FeCl3, 0.3 mM Na2EDTA and 3 mM H2O2 in 5 ml of Tris-buffer adjusted to pH 7.4 in the presence of 0.5 mM salicylic acid was incubated for 15 min at 37°C. S-PBN, pramipexole or vehicle were co-incubated in this Fenton system in order to assess their possible radical scavenging effects using the salicylate hydroxylation assay.
Animals
Adult male albino Wistar rats (Hsd/Cpb:WU, Fa.
In vitro studies
Incubation of salicylic acid alone in the radical generating system resulted in a similar increase in 2,3-DHBA and 2,5-DHBA levels and to a lesser extent in an increase in catechol (data not shown), reflecting an enhanced hydroxyl radical formation. In control experiments without drug incubation 2,3-DHBA levels (mean±S.E.M.) of 4.4±0.12 μM were obtained and set as 100%. Increasing concentrations of pramipexole (0.5–5 mM) and S-PBN (0.5–5 mM) led to a significant decrease in 2,3-DHBA levels (
Discussion
In the present study, pramipexole showed a pronounced effect in reducing hydroxyl radical levels which were generated in a cell-free in vitro Fenton system. In vivo, local application of pramipexole decreased the 6-OHDA-induced hydroxyl radical formation which was paralleled by an attenuation of the 6-OHDA-induced increase in extracellular dopamine concentration.
Pramipexole was designed as a novel D2 receptor agonist [25]. Binding studies revealed a 5- to 10-fold higher selectivity for human D3
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