BACKGROUND
parkinson's disease  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  accompanied by the appearance of intracytoplasmic inclusions known as lewy bodies  <cit> .
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  in the snpc of the parkinsonian brain are considered to be important pathogenic mechanisms  <cit> .
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  <cit> .
however, several dopamine  agonists of the da d2-receptor family  have recently been shown to possess neuroprotective properties in different in vitro and in vivo experimental pd models  <cit> .
at cellular level, independent groups have demonstrated decreased free radical production and an amelioration of da neuronal loss following da agonist treatment  <cit> .
interestingly, not all the neuroprotective effects were clearly mediated by da-receptor stimulation.
pramipexole  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 d <dig> and d <dig> da receptor subtypes but little or no affinity for the d <dig> 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- <dig> family  <cit> .
in particular, le et al.,  <cit>  reported that pramipexole protected daergic mes  <dig>  cell line against da, 6-oh-da and hydrogen peroxide -induced cytotoxicity possibly through antioxidant effects, and such neuroprotection was independent from da receptor stimulation not being prevented by selective d <dig> or d <dig> antagonists.
similar results were obtained by fujta et al.,  <cit>  and uberti et al  <cit> , who demonstrated that pramipexole inhibited generation of h2o2-induced reactive oxygen species in pc <dig> cells and sh-sy5y neuroblastoma cells, respectively, in a da receptor independent way.
recent data also demonstrated neuroprotection by pramipexole against β-amyloid ros generation and toxicity  <cit> .
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  <cit> .
here we show that s and r pramipexole are endowed with equipotent efficacy in preventing cell death induced by h2o <dig> and act as mitochondria-targeted antioxidants.
RESULTS
neuroprotection against h2o2
sh-sy5y neuroblastoma cell lines were differentiated with  <dig> μm all-trans retinoic acid for  <dig> week to acquire a neuronal phenotype.
cells were then challenged with  <dig> mm h2o <dig> for  <dig> min then cells returned to fresh medium for additional  <dig> h. h2o <dig> caused a reduction in cell viability of about 70% in comparison with untreated control cells .
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 h2o <dig>  the tested drugs showed equipotent efficacy with calculated ic <dig> values of  <dig>  ±  <dig>  μm and  <dig>  ±  <dig>  μm for s and r pramipexole enantiomer, respectively.
the neuroprotective effects of both pramipexole enantiomers were not prevented by preincubation of the cells with  <dig> μm phenoxybenzamine ,  <dig> μm haloperidol or  <dig> μm  sulpiride .
haloperidol and sulpiride treatments did not induce cell viability modifications .
inhibition of mitochondrial ros generation
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  <cit> .
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 , cells were preincubated with vitamin e  for  <dig> 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  <dig>  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 ic <dig> values of  <dig>  ±  <dig>  μm and  <dig>  ±  <dig>  μm 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  <dig> μm phenoxybenzamine ,  <dig> μm haloperidol or  <dig> μm  sulpiride .
haloperidol and sulpiride treatments did not modify laser-induced increase in ros production .
discussion
pramipexole exists as two stereoisomers.
the s enantiomer is a potent d2/d <dig> 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 h2o <dig> and nitric oxide  <cit>  and equipotent efficacy in preventing viability impairment induced by h2o <dig> and mithocondrial ros generation .
we found s and r pramipexole enantiomers relatively weak h2o <dig> scavengers, with apparent ic <dig> 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  <cit> .
in our study, s and r pramipexole enantiomer showed equipotent efficacy suggesting that the neuroprotective effects against h2o <dig> 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  as a possible target of this drug  <cit> .
apart from binding to da receptors, pramipexole has in fact been shown to enter and accumulate in mitochondria driven by the mitochondrial membrane potential  <cit> .
targeting to mitochondria has also been recently demonstrated by patch clamp studies showing inhibition of ptp by pramipexole  <cit> .
ptp inhibition by pramipexole was further supported by experimental data obtained in functional intact mitochondria showing that this drug abolished ca++-triggered swelling  <cit> .
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  and inhibiting h2o2-triggered viability impairment  may be related to the different experimental models.
in fact, h2o <dig> 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  <cit>  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.
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.

 parkinson's disease 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 accompanied by the appearance of intracytoplasmic inclusions known as lewy bodies. 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 in the snpc of the parkinsonian brain are considered to be important pathogenic mechanisms. 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. however, several dopamine agonists of the da d2-receptor family have recently been shown to possess neuroprotective properties in different in vitro and in vivo experimental pd models. at cellular level, independent groups have demonstrated decreased free radical production and an amelioration of da neuronal loss following da agonist treatment. interestingly, not all the neuroprotective effects were clearly mediated by da-receptor stimulation. pramipexole 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 d and d da receptor subtypes but little or no affinity for the d 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- family. in particular, le et al., reported that pramipexole protected daergic mes cell line against da, 6-oh-da and hydrogen peroxide -induced cytotoxicity possibly through antioxidant effects, and such neuroprotection was independent from da receptor stimulation not being prevented by selective d or d 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 pc cells and sh-sy5y neuroblastoma cells, respectively, in a da receptor independent way. recent data also demonstrated neuroprotection by pramipexole against β-amyloid ros generation and toxicity. 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. here we show that s and r pramipexole are endowed with equipotent efficacy in preventing cell death induced by h2o and act as mitochondria-targeted antioxidants.  neuroprotection against h2o2 sh-sy5y neuroblastoma cell lines were differentiated with μm all-trans retinoic acid for week to acquire a neuronal phenotype. cells were then challenged with mm h2o for min then cells returned to fresh medium for additional h. h2o caused a reduction in cell viability of about 70% in comparison with untreated control cells. 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 h2o the tested drugs showed equipotent efficacy with calculated ic values of ± μm and ± μm for s and r pramipexole enantiomer, respectively. the neuroprotective effects of both pramipexole enantiomers were not prevented by preincubation of the cells with μm phenoxybenzamine, μm haloperidol or μm sulpiride. haloperidol and sulpiride treatments did not induce cell viability modifications. inhibition of mitochondrial ros generation 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. 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, cells were preincubated with vitamin e for 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 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 ic values of ± μm and ± μm 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 μm phenoxybenzamine, μm haloperidol or μm sulpiride. haloperidol and sulpiride treatments did not modify laser-induced increase in ros production. discussion pramipexole exists as two stereoisomers. the s enantiomer is a potent d2/d 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 h2o and nitric oxide and equipotent efficacy in preventing viability impairment induced by h2o and mithocondrial ros generation. we found s and r pramipexole enantiomers relatively weak h2o scavengers, with apparent ic 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. in our study, s and r pramipexole enantiomer showed equipotent efficacy suggesting that the neuroprotective effects against h2o 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 as a possible target of this drug. 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 and inhibiting h2o2-triggered viability impairment may be related to the different experimental models. in fact, h2o 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 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. 
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. 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 h2o and inhibiting mitochondrial reactive oxygen species generation. 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. the s enantiomer is a potent d3/d receptor agonist and is extensively used in the management of pd. both pramipexole enantiomers prevented mitochondrial ros generation with a potency about ten times higher then that elicited for neuroprotection.
 pramipexole exists as two isomers. the s enantiomer is a potent d3/d 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.  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 h2o 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.  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. 