The dopamine agonist pramipexole scavenges hydroxyl free radicals induced by striatal application of 6-hydroxydopamine in rats: an in vivo microdialysis study

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-α-(2-sulfophenyl)nitrone) 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 μl/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 μl/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/2 μl/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 D₂-like receptor agonistic effects.

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.