|Mitochondria-targeted antioxidant effects of S(-) and R(+) pramipexole.|
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|PMID: 20137065 Owner: NLM Status: MEDLINE|
|BACKGROUND: Pramipexole exists as two isomers. The S(-) enantiomer is a potent D3/D2 receptor agonist and is extensively used in the management of PD. In contrast, the R(+) enantiomer is virtually devoid of any of the DA agonist effects. Very limited studies are available to characterize the pharmacological spectrum of the R(+) enantiomer of pramipexole.
RESULTS: Using differentiated SH-SY5Y neuroblastoma cells as an experimental model, here we show that S(-) and R(+) pramipexole are endowed with equipotent efficacy in preventing cell death induced by H2O2 and inhibiting mitochondrial reactive oxygen species generation. Both pramipexole enantiomers prevented mitochondrial ROS generation with a potency about ten times higher then that elicited for neuroprotection.
CONCLUSIONS: These results support the concept of both S(-) and R(+) pramipexole enantiomers as mitochondria-targeted antioxidants and suggest that the antioxidant, neuroprotective activity of these drugs is independent of both the chiral 6-propylamino group in the pramipexole molecule and the DA receptor stimulation.
|Giulia Ferrari-Toninelli; Giuseppina Maccarinelli; Daniela Uberti; Erich Buerger; Maurizio Memo|
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|Type: Journal Article; Research Support, Non-U.S. Gov't Date: 2010-02-05|
|Title: BMC pharmacology Volume: 10 ISSN: 1471-2210 ISO Abbreviation: BMC Pharmacol. Publication Date: 2010|
|Created Date: 2010-03-01 Completed Date: 2010-05-03 Revised Date: 2013-05-31|
Medline Journal Info:
|Nlm Unique ID: 100967806 Medline TA: BMC Pharmacol Country: England|
|Languages: eng Pagination: 2 Citation Subset: IM|
|Department of Biomedical Sciences and Biotechnologies and National Institute of Neuroscience, University of Brescia, Brescia, Italy.|
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administration & dosage*
Apoptosis / drug effects
Benzothiazoles / administration & dosage*
Cell Culture Techniques
Cell Survival / drug effects
Dopamine Antagonists / administration & dosage
Drug Delivery Systems
Hydrogen Peroxide / toxicity
Mitochondria / drug effects*, pathology
Neurons / drug effects*, pathology
Neuroprotective Agents / pharmacology
Oxidative Stress / drug effects
Reactive Oxygen Species / metabolism*
Thiazolidinediones / pharmacology
Tumor Cells, Cultured
|0/Antioxidants; 0/Benzothiazoles; 0/Dopamine Antagonists; 0/Neuroprotective Agents; 0/Reactive Oxygen Species; 0/Thiazolidinediones; 122320-73-4/rosiglitazone; 7722-84-1/Hydrogen Peroxide; 83619PEU5T/pramipexole|
Journal ID (nlm-ta): BMC Pharmacol
Publisher: BioMed Central
Copyright ?2010 Ferrari-Toninelli et al; licensee BioMed Central Ltd.
Received Day: 16 Month: 4 Year: 2009
Accepted Day: 5 Month: 2 Year: 2010
collection publication date: Year: 2010
Electronic publication date: Day: 5 Month: 2 Year: 2010
Volume: 10First Page: 2 Last Page: 2
Publisher Id: 1471-2210-10-2
PubMed Id: 20137065
|Mitochondria-targeted antioxidant effects of S(-) and R(+) pramipexole|
|Giulia Ferrari-Toninelli1||Email: email@example.com|
|Giuseppina Maccarinelli1||Email: firstname.lastname@example.org|
|Daniela Uberti1||Email: email@example.com|
|Erich Buerger2||Email: Erich.Buerger@bc.boehringer-ingelheim.com|
|Maurizio Memo1||Email: firstname.lastname@example.org|
1Department of Biomedical Sciences and Biotechnologies and National Institute of Neuroscience, University of Brescia, Brescia, Italy
2Department of Central Nervous System Research, Boehringer-Ingelheim Pharma, Biberach an der Riss, Germany
Parkinson's disease (PD) is the most common neurodegenerative movement disorder. The primary cause of the disorder is the progressive loss of the pigmented dopaminergic neurons in the substantia nigra pars compacta (SNpc) accompanied by the appearance of intracytoplasmic inclusions known as Lewy bodies [1-3].
To date, the etiopathogenesis of nigral dopaminergic neuron loss in PD is unknown. However, the presence of ongoing oxidative stress as the result of compromised antioxidant defence mechanisms and generation of radical oxygen species (ROS) in the SNpc of the parkinsonian brain are considered to be important Pathogenic mechanisms [3,4]. ROS can react with cellular macromolecules through oxidation and cause the cells to undergo dysfunction and eventually lead to necrosis or apoptosis. The control of the redox environment of the cell provides an additional regulation in the signal transduction pathways which are redox sensitive. Therefore, an effective anti-parkinsonian therapy should not only alleviate the disease associated symptoms, but should also stop the progressive dopaminergic cell death in the SN.
Modification of the rate of PD progression is currently a highly debated topic. Increased oxidative stress is indeed thought to be involved in the nigral cell death which is a well established peculiar neuropathological feature of PD. These mechanisms have been proven in vitro and in animal models, but their relevance in humans remains speculative [5,6]. However, several dopamine (DA) agonists of the DA D2-receptor family (including D2 and D3 subtypes) have recently been shown to possess neuroprotective properties in different in vitro and in vivo experimental PD models [7,8]. At cellular level, independent groups have demonstrated decreased free radical production and an amelioration of DA neuronal loss following DA agonist treatment [9-17]. Interestingly, not all the neuroprotective effects were clearly mediated by DA-receptor stimulation.
Pramipexole (2-amino-4,5,6,7-tetrahydro-6-propylaminobenzathiazole) is a non-ergot DA agonist that has been successfully applied to the treatment of Parkinson's disease. Pramipexole exhibits a high affinity for the D2 and D3 DA receptor subtypes but little or no affinity for the D1 receptor family. The neuroprotective effects elicited by this drug have directly and/or indirectly been associated with antioxidant effects, mitochondrial stabilization or induction of the antiapoptotic Bcl-2 family [18-21]. In particular, Le et al.,  reported that pramipexole protected DAergic MES 23.5 cell line against DA, 6-OH-DA and hydrogen peroxide (H2O2)-induced cytotoxicity possibly through antioxidant effects, and such neuroprotection was independent from DA receptor stimulation not being prevented by selective D2 or D3 antagonists. Similar results were obtained by Fujta et al.,  and Uberti et al , who demonstrated that pramipexole inhibited generation of H2O2-induced reactive oxygen species in PC12 cells and SH-SY5Y neuroblastoma cells, respectively, in a DA receptor independent way. Recent data also demonstrated neuroprotection by pramipexole against ?-amyloid ROS generation and toxicity [19,22].
Pramipexole exists as two isomers. The S(-) enantiomer is a potent D2/D3receptor agonist and is extensively used in the management of PD. In contrast, the R(+) enantiomer is virtually devoid of any of the DA agonist effects. Very limited studies are available to characterize the pharmacological spectrum of the R(+) enantiomer of pramipexole [19,22-27].
Here we show that S(-) and R(+) pramipexole are endowed with equipotent efficacy in preventing cell death induced by H2O2 and act as mitochondria-targeted antioxidants.
SH-SY5Y neuroblastoma cell lines were differentiated with 10 ?M all-trans retinoic acid for 1 week to acquire a neuronal phenotype. Cells were then challenged with 1 mM H2O2 for 5 min then cells returned to fresh medium for additional 24 h. H2O2 caused a reduction in cell viability of about 70% in comparison with untreated control cells (figure 1). As shown in figure 1A, treatment of the cells with increasing concentrations of S(-) or R(+) pramipexole dose-dependently prevented the viability impairment induced by H2O2. The tested drugs showed equipotent efficacy with calculated IC50 values of 8.8 ? 0.9 ?M and 9.2 ? 0.6 ?M for S(-) and R(+) pramipexole enantiomer, respectively. The neuroprotective effects of both pramipexole enantiomers were not prevented by preincubation of the cells with 10 ?M phenoxybenzamine (data not shown), 10 ?M haloperidol or 10 ?M (-) sulpiride (Figure 1B). Haloperidol and sulpiride treatments did not induce cell viability modifications (data not shown).
The effects of pramipexole and its R(+) enantiomer have been studied in an experimental model of mitochondrial ROS generation by video-rate confocal microscopy in living neuronal cells. This model allows detection of ROS levels specifically generated in mitochondria and is based on the CM-DCF formation after laser light stimulation [28-30]. Figure 2A, upper panel, shows the results from a representative experiment. Mitochondrial ROS generation was evaluated in cells after increasing exposure to laser at different intensity. In a parallel experiment (lower panels), cells were preincubated with Vitamin E (2 ?l/100 ml) for 30 min before the laser excitation. Fluorescence emission intensity was calculated as average grey level value per pixel and corrected for background. Data are reported in the graph reported in Figure 2B. The results clearly show that Vitamin E prevented laser-induced ROS generation in mitochondria of differentiated SH-SY5Y neuronal cells.
Using the same experimental paradigm, we tested the effects of different concentrations of S(-) and R(+) pramipexole in laser-induced mitochondria ROS generation. As shown in figure 3, both S(-) and R(+) pramipexole dose-dependently prevented laser-induced ROS generation in mitochondria of differentiated SH-SY5Y neuronal cells. When calculated as inhibition of ROS generation after 9% laser intensity, both drugs showed similar potency with IC50 values of 0.91 ? 0.14 ?M and 0.85 ? 0.21 ?M for S(-) and R(+) pramipexole enantiomer, respectively. The prevention of mitochondrial ROS generation induced by both pramipexole enantiomers was not affected by preincubation of the cells with 10 ?M phenoxybenzamine (data not shown), 10 ?M haloperidol or 10 ?M (-) sulpiride (Figure 4). Haloperidol and sulpiride treatments did not modify laser-induced increase in ROS production (data not shown).
Pramipexole exists as two stereoisomers. The S(-) enantiomer is a potent D2/D3 receptor agonist and is extensively used in the management of PD. In contrast, the R(+) enantiomer is virtually devoid of any of the DA agonist effects. A growing number of experimental data indicate an antioxidant property of pramipexole enantiomers, evidenced by equal antioxidant efficacy toward H2O2 and nitric oxide  and equipotent efficacy in preventing viability impairment induced by H2O2 and mithocondrial ROS generation (present results). We found S(-) and R(+) pramipexole enantiomers relatively weak H2O2 scavengers, with apparent IC50 values in the low micromolar range. Our data are consistent with previous data showing neuroprotection elicited by the S(-) and R(+) enantiomers against different neurotoxic agents [9,22-27]. In our study, S(-) and R(+) pramipexole enantiomer showed equipotent efficacy suggesting that the neuroprotective effects against H2O2 in differentiated SH-SY5Y neuroblastoma cells were DA receptor independent.
Although this study did not examine the precise site of action of pramipexole, several finding implicate the permeability transition pore (PTP) as a possible target of this drug [19,24,25]. Apart from binding to DA receptors, pramipexole has in fact been shown to enter and accumulate in mitochondria driven by the mitochondrial membrane potential . Targeting to mitochondria has also been recently demonstrated by patch clamp studies showing inhibition of PTP by pramipexole . PTP inhibition by pramipexole was further supported by experimental data obtained in functional intact mitochondria showing that this drug abolished Ca++-triggered swelling . By video-rate confocal microscopy in living neuronal cells, we found that both S(-) and R(+) pramipexole enantiomers prevented laser-induced ROS generation in mitochondria of differentiated SH-SY5Y neuronal cells. Interestingly, both pramipexole enantiomers prevented mitochondrial ROS generation with a potency about ten times higher then that elicited for neuroprotection.
The apparent discrepancy between the different potencies of pramipexole enantiomers in preventing mitochondrial ROS generation (about 0.9 ?M) and inhibiting H2O2-triggered viability impairment (about 8 ?M) may be related to the different experimental models. In fact, H2O2 itself is not a radical but reacts with iron to form hydroxyl radicals, the most reactive oxygen species. Thus, in our experimental paradigm, ROS are generated both intra- and extracellularly and causes apoptosis by the induction of several intracellular converging pathways involving lipid peroxidation, protein oxidation and DNA damage. We hypothesize that accumulation of promipexole enantiomers in the mitochondria [23,24] may limit their scavenger properties to specific subcellular compartments. Although accumulation of pramipexole into mitochondria has not been definitely established, the high potency of these drugs in preventing mitochondrial ROS generation strongly suggest the mitochondria as their primary site of action.
These results support the concept of both S(-) and R(+) pramipexole enantiomers as mitochondria-targeted antioxidants and suggest that the antioxidant, neuroprotective activity of these drugs is independent of both the chiral 6-propylamino group in the pramipexole molecule and the DA receptor stimulation.
The human neuroblastoma cell line SH-SY5Y was routinely cultured in Ham's F12 and Dulbecco modified Eagle's medium (DMEM) in a ratio of 1:1, supplemented with 10% (v/v) foetal calf serum, 2 mM glutamine, 50 ?g/ml penicillin, and 100 ?g/ml streptomycin and kept at 37?C in humidified 5% CO2/95% atmosphere. For differentiation, cultured cells were treated for one week with 10 ?M all trans retinoic acid. To obtain reproducible results, SH-SY5Y cells ranging from passage 18 to passage 25 were used for all the experiments.
S(-) and R(+) pramipexole were dissolved in water and added to the culture media 1 h before H2O2 pulse or laser light stimulation. IC50 values for S(-) and R(+) pramipexole enantiomer were calculated using at least 5 data points. Haloperidol and sulpiride were added to the culture media 1 h before pramipexole. Vitamin E (2 ng/100 ?l) was added to the culture media 30 min before the laser excitation. All drugs were from Sigma. S(-) and R(+) pramipexole were kindly supplied by Boehringer Ingelheim GmbH, Germany.
Cell viability was measured by quantitative colorimetric assay with MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), showing the mitochondrial activity of living cells. Differentiated SH-SY5Y neuronal cells in 96-well plates were challenged with H2O2 for 5 min, then 500 ?g/ml MTT was added in each well and cells were incubated at 37?C for additional 3 h. MTT was removed, and cells were lysed with dimethyl sulfoxide. The absorbance at 595 nm was measured using a Bio-Rad 3350 microplate reader. Control cells were treated in the same way without H2O2, and the values of different absorbances were expressed as a percentage of control. All the drugs and reagents concentrations are to be considered as final concentrations.
SH-SY5Y neuroblastoma cells were first differentiated in neuronal-like phenotype by treatment with retinoic acid for 7 days; then, according to Koopman et al. , cells were loaded with 5-choloromethyl-2',7'-dichlorodihydrofluorescein (CM-H2DCF) and its oxidative conversion into CM-DCF was monitored by video-rate confocal microscopy and real-time image averaging after increasing laser intensities. For mitochondria localization, cells were also loaded with Mitotracker Deep Red. Cells were then excited at increasing laser intensity and CM-DCF fluorescence intensity was selectively recorded in mitochondria. Fluorescence emission intensity was calculated as average grey level value per pixel and corrected for background.
Results were given as mean ? standard error mean values. Statistical significance of differences was determined by one way ANOVA, followed by Bonferroni's multiple comparison test as post-hoc analysis. A probability of less than 0.05 was considered as a significant difference.
GFT, GM and DU conducted and managed the study and analyzed the imaging data, MM drafted the manuscript. EB and MM participated in the design and coordination of the project. All authors cooperatively designed the project and discussed data interpretation. All authors participated in critical editing of the manuscript.
This research was supported in part by a grant from Boehringer Igelheim.
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