Document Detail

Mitochondria-targeted antioxidant effects of S(-) and R(+) pramipexole.
Jump to Full Text
MedLine Citation:
PMID:  20137065     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
Giulia Ferrari-Toninelli; Giuseppina Maccarinelli; Daniela Uberti; Erich Buerger; Maurizio Memo
Related Documents :
2564615 - Behavioral recovery after irreversible inactivation of d-1 and d-2 dopamine receptors.
20837135 - Cytoplasmic tail of d1 dopaminergic receptor differentially regulates desensitization a...
15912275 - Imaging the dopamine system with in vivo [11c]raclopride displacement studies: understa...
23200895 - Pdgf α receptor is a mediator for cisplatin-induced met expression.
8940365 - Expression of gamma-aminobutyric acid and gonadotropin-releasing hormone during neurona...
7790855 - Differential regulation of d1 dopamine receptor- and of a2a adenosine receptor-stimulat...
Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2010-02-05
Journal Detail:
Title:  BMC pharmacology     Volume:  10     ISSN:  1471-2210     ISO Abbreviation:  BMC Pharmacol.     Publication Date:  2010  
Date Detail:
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    
Other Details:
Languages:  eng     Pagination:  2     Citation Subset:  IM    
Affiliation:
Department of Biomedical Sciences and Biotechnologies and National Institute of Neuroscience, University of Brescia, Brescia, Italy.
Export Citation:
APA/MLA Format     Download EndNote     Download BibTex
MeSH Terms
Descriptor/Qualifier:
Antioxidants / administration & dosage*
Apoptosis / drug effects
Benzothiazoles / administration & dosage*
Cell Culture Techniques
Cell Survival / drug effects
Dopamine Antagonists / administration & dosage
Drug Delivery Systems
Humans
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
Chemical
Reg. No./Substance:
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
Comments/Corrections

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine

Full Text
Journal Information
Journal ID (nlm-ta): BMC Pharmacol
ISSN: 1471-2210
Publisher: BioMed Central
Article Information
Download PDF
Copyright ?2010 Ferrari-Toninelli et al; licensee BioMed Central Ltd.
open-access:
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
ID: 2829550
Publisher Id: 1471-2210-10-2
PubMed Id: 20137065
DOI: 10.1186/1471-2210-10-2

Mitochondria-targeted antioxidant effects of S(-) and R(+) pramipexole
Giulia Ferrari-Toninelli1 Email: giuliaferraritoninelli@yahoo.it
Giuseppina Maccarinelli1 Email: maccarin@med.unibs.it
Daniela Uberti1 Email: uberti@med.unibs.it
Erich Buerger2 Email: Erich.Buerger@bc.boehringer-ingelheim.com
Maurizio Memo1 Email: memo@med.unibs.it
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

Background

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., [18] 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., [20] and Uberti et al [22], 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.


Results
Neuroprotection against H2O2

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).

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 [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).


Discussion

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 [24] 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 [24]. Targeting to mitochondria has also been recently demonstrated by patch clamp studies showing inhibition of PTP by pramipexole [25]. PTP inhibition by pramipexole was further supported by experimental data obtained in functional intact mitochondria showing that this drug abolished Ca++-triggered swelling [25]. 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.


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.


Methods
Cell culture

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.

Drug treatment

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.

Evaluation of cell viability

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.

Reactive oxygen species detection

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. [28], 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.

Statistical evaluation

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.


Authors' contributions

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.


Acknowledgements

This research was supported in part by a grant from Boehringer Igelheim.


References
Davie CA,A review of Parkinson's diseaseBr Med BullYear: 20088610912710.1093/bmb/ldn01318398010
McKeith IG,Mosimann UP,Dementia with Lewy bodies and Parkinson's diseaseParkinsonism and Related DisordersYear: 200410S15S1810.1016/j.parkreldis.2003.12.00515109582
Adams JD Jr,Odunze IN,Oxygen free radicals and Parkinson's diseaseFree Radic Biol MedYear: 19911016116910.1016/0891-5849(91)90009-R2016074
Olanow CW,A radical hypothesis for neurodegenerationTrends NeurosciYear: 19931643944410.1016/0166-2236(93)90070-37507613
Schapira AH,Neuroprotection in PD - a role for dopamine agonists?NeurologyYear: 200361S34S4214504378
Jenner P,Dopamine agonists, receptor selectivity and dyskinesia induction in Parkinson's diseaseCurr Opin NeurolYear: 200316S3S710.1097/00019052-200312001-0000215180131
Ferrari-Toninelli G,Bonini SA,Cenini G,Maccarinelli G,Grilli M,Uberti D,Memo M,Dopamine receptor agonists for protection and repair in Parkinson's diseaseCurr Top Med ChemYear: 200881089109910.2174/15680260878516140218691134
Radad K,Gille G,Rausch WD,Short review on dopamine agonists: insight into clinical and research studies relevant to Parkinson's diseasePharmacol RepYear: 200557670171216382188
Gassen M,Glinka Y,Pinchasi B,Youdim MB,Apomorphine is a highly potent free radical scavenger in rat brain mitochondrial fractionEur J PharmacolYear: 1996308221922510.1016/0014-2999(96)00291-98840135
Kondo T,Ito T,Sugita Y,Bromocriptine scavenges methamphetamine-induced hydroxyl radicals and attenuates dopamine depletion in mouse striatumAnn NY Acad SciYear: 19947382222297832431
Ogawa N,Tanaka K,Asanuma M,Kawai M,Masumizu T,Kohno M,Mori A,Bromocriptine protects mice against 6-hydroxydopamine and scavenges hydroxyl free radicals in vitroBrain ResYear: 199465720721310.1016/0006-8993(94)90969-57820619
Takashima H,Tsujihata M,Kishikawa M,Freed WJ,Bromocriptine protects dopaminergic neurons from levodopa-induced toxicity by stimulating D(2)receptorsExp NeurolYear: 19991599810410.1006/exnr.1999.712210486178
Ferger B,Teismann P,Mierau J,The dopamine agonist pramipexole scavenges hydroxyl free radicals induced by striatal application of 6-hydroxydopamine in rats: an in vivo microdialysis studyBrain ResYear: 200088321622310.1016/S0006-8993(00)02929-211074050
Uberti D,Carsana T,Francisconi S,Ferrari-Toninelli G,Canonico PL,Memo M,A novel mechanism for pergolide-induced neuroprotection: inhibition of NF-kappaB nuclear translocationBiochem PharmacolYear: 2004671743175010.1016/j.bcp.2004.01.01215081873
Iida M,Miyazaki I,Tanaka K,Kabuto H,Iwata-Ichikawa E,Ogawa N,Dopamine D2 receptor-mediated antioxidant and neuroprotective effects of ropinirole, a dopamine agonistBrain ResYear: 1999838515910.1016/S0006-8993(99)01688-110446316
Tanaka K,Miyazaki I,Fujita N,Haque ME,Asanuma M,Ogawa N,Molecular mechanism in activation of glutathione system by ropinirole, a selective dopamine D2 agonistNeurochem ResYear: 200126313610.1023/A:100767241423911358279
Uberti D,Bianchi I,Olivari L,Ferrari-Toninelli G,Bonini SA,Memo M,Dopaminergic agonists: possibile neurorescue drugs endowed with independent and synergistic multisites of actionNeurochem ResYear: 2007321726172910.1007/s11064-007-9350-917486445
Le WD,Jankovic J,Xie W,Appel SH,Antioxidant property of pramipexole independent of dopamine receptor activation in neuroprotectionJ Neural TransmYear: 200010711657310.1007/s00702007003011129106
Abramova NA,Cassarino DS,Khan SM,Painter TW,Bennett JP Jr,Inhibition by R(+) or S(-) pramipexole of caspase activation and cell death induced by methylpyridinium ion or beta amyloid peptide in SH-SY5Y neuroblastomaJ Neurosci ResYear: 20026749450010.1002/jnr.1012711835316
Fujita Y,Izawa Y,Ali N,Kanematsu Y,Tsuchiya K,Hamano S,Tamaki T,Yoshizumi M,Pramipexole protects against H2O2-induced PC12 cell deathNaunyn Schmiedebergs Arch PharmacolYear: 20063722576610.1007/s00210-005-0025-216362428
Iravani MM,Sadeghian M,Leung CC,Tel BC,Rose S,Shapira AH,Jenner P,Continuous subcutaneous infusion of pramipexole protects against lipopolysaccharide-induced dopaminergic cell death without affecting the inflammatory responseExp NeurolYear: 200821252253110.1016/j.expneurol.2008.04.03718571649
Uberti D,Bianchi I,Olivari L,Ferrari-Toninelli G,Canonico P,Memo M,Pramipexole prevents neurotoxicity induced by oligomers of beta-amyloidEur J PharmacolYear: 2007569319419610.1016/j.ejphar.2007.05.00917572405
Cassarino DS,Fall CP,Smith TS,Bennett JP Jr,Pramipexole reduces reactive oxygen species production in vivo and in vitro and inhibits the mitochondrial permeability transition produced by the parkinsonian neurotoxin methylpyridinium ionJ NeurochemYear: 1998712953019648878
Danzeisen R,Schwalenstoecker B,Gillardon F,Buerger E,Krzykalla V,Klinder K,Schild L,Hengerer B,Ludolph AC,Dorner-Ciossek C,Kussmaul L,Targeted antioxidant and neuroprotective properties of the dopamine agonist pramipexole and ist nondopaminergic enantiomer SND919CL2x [(+)2-amino-4,5,6,7-tetrahydro-6-L-propylamino-benzathiazole Dihydrochloride]J Pharmacol Exp TherYear: 200631618919910.1124/jpet.105.09231216188953
Sayeed I,Parvez S,Winkler-Stuck K,Seitz G,Trieu I,Wallesch CW,Sch?nfeld P,Siemen D,Patch clamp reveals powerful blockade of the mitochondrial permeability transition pore by the D2-receptor agonist pramipexoleFASEB JYear: 2006203556816407457
Gribkoff VK,Bozik ME,KNS-760704 [(6R)-4,5,6,7-tetrahydro-N6-propyl-2, 6-benzohiazole-diamine dihydrochloride monohydrate] for the treatment of Amyotrphic Lateral SclerosisCNS Neuroscience & TherYear: 20081421522610.1111/j.1755-5949.2008.00048.x
Gu M,Iravani M,Cooper JM,Jenner P,Shapira AHV,Pramipexole protects against apoptotic cell death by non-dopaminergic mechanismsJ NeurochemYear: 2004911075108110.1111/j.1471-4159.2004.02804.x15569251
Koopman WJH,Verkaart S,van Emst-de Vries SE,Grefte S,Smeitink JAM,Willems PHGM,Simultaneous quantification of oxidative stress and cell spreading using 5-(and 6-)-chloromethyl-2'-7'-dichlorofluoresceinCytometry Part AYear: 200669A1184119210.1002/cyto.a.20348
Gomes A,Fernandes E,Lima JLFC,Fluorescence probes used for detection of reactive oxygen speciesJ Biochem Biophys MethodsYear: 200565458010.1016/j.jbbm.2005.10.00316297980
Swift LM,Sarvazyan N,Localization of dichlorofluorescein in cardiac myocytes: Implications for assessment of oxidative stressAm J Physiol Heart Circ PhysiolYear: 2000278H982H99010710368

Figures

[Figure ID: F1]
Figure 1 

Neuroprotective effects of pramipexole (PPX) enatiomers against H2O2-induced cell death. A) Differentiated SH-SY5Y cells were exposed to different concentrations of S(-) PPX (gray bars) and R(+) PPX (black bars) for 1 h before being exposed to 1 mM H2O2 for 10 min. Cell viability was evaluated 24 h after by MTT assay B) Cells were exposed to 50 ?M ?M S(-) PPX or R(+) PPX in the presence or absence of 10 ?M haloperidol (H) or 10 ?M (-) sulpiride (S). Data represent means ? SEM of at least three different experiments and are from three separate cell preparations. *, p < 0.01 vs H2O2 alone values.



[Figure ID: F2]
Figure 2 

Detection of mitochondrial ROS generation. A) Upper panel. Representative pictures from cells exposed to increasing laser intensity, as indicated. CM-DCF fluorescence intensity (green) was selectively recorded in mitochondria (red). Lower panel. Cells were preincubated with Vitamin E (2 ng/100 ?l) for 30 min before the laser excitation (white bars in panel B). Fluorescence emission intensity was calculated as average grey level value per pixel and corrected for background. Bars in panel B represent the means ? SEM of at least three different experiments and are from three separate cell preparations. *, p < 0.01 vs the corresponding control values.



[Figure ID: F3]
Figure 3 

Inhibition of mitochondrial ROS generation by pramipexole (PPX) enantiomers. Cells were exposed to different concentrations, as indicated in the bottom, of S(-) PPX (upper panel) and R(+) PPX (lower panel) for 1 h before being exposed to laser. Mitochondrial ROS generation was evaluated as in figure 2. Data represent means ? SEM of at least three different experiments and are from three separate cell preparations. *, p < 0.05 and **, p < 0.001 vs the corresponding control values (black bars).



[Figure ID: F4]
Figure 4 

Lack of effect of DA receptor antagonists on the inhibition of mitochondrial ROS generation induced by pramipexole (PPX) enantiomers. Cells were exposed to 10 ?M S(-) PPX (gray bars) and R(+) PPX (white bars) for 1 h in the absence or presence of 10 ?M haloperidol (H) or 10 ?M (-) sulpiride (S) before being exposed to laser. Mitochondrial ROS generation was evaluated as in figure 2. Data represent means ? SEM of at least three different experiments and are from three separate cell preparations. *, p < 0.01 vs the corresponding control values (black bars).



Article Categories:
  • Research article


Previous Document:  Microglial responses around intrinsic CNS neurons are correlated with axonal regeneration.
Next Document:  Transcriptional response of Burkholderia cenocepacia J2315 sessile cells to treatments with high dos...