Activation of Pro-Apoptotic Cascade by Dopamine in Renal Epithelial Cells is Fully Dependent on Hydrogen Peroxide Generation by Monoamine Oxidases
Pascale Bianchi,
Marie-Hélène Séguélas,
Angelo Parini and
Claudie Cambon
INSERM U388, Toulouse, France.
Correspondence to Dr. Angelo Parini, INSERM U388, Bât L3, CHU Rangueil, 1 Av. J. Poulhès, 31403 Toulouse Cedex 4, France. Phone: 33-5-61-32-26-22; Fax: 33-5-62-17-25-54;
ABSTRACT. Dopamine plays a critical role in regulation of differentrenal functions, including glomerular filtration, renin secretion,and sodium excretion. Recent studies have shown that some ofthe dopamine effects in the proximal tubule may involve hydrogenperoxide (H2O2) generation by the catecholamine-degrading enzymemonoamine oxidases (MAO). The present study is an investigationof the potential role of H2O2 generated by MAO during dopaminedegradation in apoptosis of proximal tubule cells. Dopamineconcentrations between 50 and 200 µM induced apoptosisof rat proximal tubule and monoamine oxidase B-transfected HEK293 cells (+73% compared with untreated cells) but not in wild-typeHEK 293 cell lacking monoamine oxidases. Apoptosis of proximaltubule cells was preceded by an increase in the ratio of Bax/Bcl2proteins, the release of mitochondrial cytochrome c, caspase-3activation, and DNA fragmentation. All these events requireddopamine internalization into the cells, its metabolism by MAO,and H2O2 production, as they were prevented by the dopamineuptake inhibitor GBR-12909, the irreversible MAO inhibitor pargyline,or the antioxidant N-acetylcysteine. These results show that,in renal proximal tubule cells, dopamine induces oxidative stress,activation of pro-apoptotic cascade, and cell apoptosis exclusivelyby mechanisms involving H2O2 production by monoamine oxidases.E-mail: parini@toulouse.inserm.fr
Dopamine regulates a variety of functions in the central nervoussystem and in the peripheral organs. It is commonly acceptedthat the effects of dopamine require stimulation of specificG-coupled membrane receptors and the subsequent activation orinhibition of intracellular signaling cascades (1–3).In kidney, dopamine plays a critical role in the regulationof different renal functions, including glomerular filtration,renin production, and sodium excretion (4). Most of the renaldopamine is produced by proximal tubule cells (5). As shownfor dopaminergic presynaptic endings, proximal tubule cellscontain all the enzyme machinery necessary for dopamine synthesis(6,7). On the other hand, these cells are also one of the majortargets of dopamine for regulation of tubule sodium reabsorptionby a mechanism involving D1 and D2-like receptor stimulation(8). The availability of renal dopamine depends in part on theactivity of the mitochondrial enzyme monoamine oxidases (MAO:EC 1.4.3.4.) (5). These enzymes, which are highly expressedin renal proximal tubule, catalyze the oxidative deaminationof neurotransmitters (i.e., noradrenaline, dopamine, and serotonin)and different exogenous and endogenous amines. The two isoenzymesMAO-A and -B are irreversibly and specifically inhibited bypropargylamine derivatives such as pargyline (9).
Hydrogen peroxide (H2O2) is one of the reaction products generatedby MAO during substrate degradation. Except for the potentialcytotoxic effect of H2O2 in nigral cell degeneration in Parkinsondisease (10), the cell events following MAO-dependent H2O2 productionin physiologic and pathologic conditions are still unclear.We recently demonstrated that H2O2 generated by MAO may mediatesome of the dopamine effects in the renal proximal tubule (11).Indeed, we showed that, independently of receptor stimulation,dopamine induces proliferation of proximal tubule cells by amechanism requiring dopamine degradation by monoamine oxidases,hydrogen peroxide generation, and ERK activation (11).
The fact that previous studies showed that H2O2 behaves as aproliferative or apoptotic factor at low or high concentrations,respectively (19), suggests that, at concentrations higher thanthose inducing proliferation of proximal tubule cells, H2O2produced by MAO may have apoptotic properties. This possibilityis supported by studies showing that, in neuronal cells, dopaminemay behave as a pro-apoptotic factor. Converging evidence indicatedthat the pro-apoptotic activity of dopamine was mediated byreactive oxygen species (ROS) (12). However, the mechanismsof ROS generation by dopamine, the intracellular events leadingto cell apoptosis, and the physiopathologic relevance remainunclear.
To date, the potential pro-apoptotic activity of dopamine inkidney and the cell mechanisms involved have not been described.In the present study, we investigated the effect of dopaminein apoptosis of renal proximal tubule cells and the involvementof H2O2 generated by MAO. To define the specific role of MAOin the dopamine effects, experiments were performed in rat proximaltubule cells and in MAO-deficient (wild-type) and MAO-B–transfectedHEK 293 cells. Our results show for the first time that dopamineinduces apoptosis of rat renal proximal tubule and MAO-B–transfectedcells. This effect is fully dependent on H2O2 generation byMAO and involves a sequential decrease of the Bcl-2/Bax ratio,cytochrome c release from mitochondria, and caspase-3 activation.
Materials
HBSS medium, DMEM/Ham’s F-12 medium, nonessential aminoacid mixture, penicillin, streptomycin, EGF, and FCS were fromLife Technologies-BRL (Eragny, France); Percoll was from Pharmacia(Uppsala, Sweden); polyvinylidene difluoride (PVDF), membranewere from NEN Life Science Products (Boston, MA); goat polyclonalanti-cytochrome c, anti-Bcl-2, anti-actin antibody, rabbit polyclonalanti-procaspase-3 antibody, mouse monoclonal anti-Bax antibody,anti-rabbit, anti-mouse, and anti-goat IgG-HRP conjugated werefrom Santa Cruz Biotechnology (Santa Cruz, CA); Kit ECL detectorreagents were from Amersham (Buckinghamshire, UK); GBR-12909dihydrochloride was from TOCRIS (Ballwin, MO); Bio-Rad DC proteinassay reagents were from Bio-Rad Laboratories (Ivry-sur-Seine,France); SYTO-13 and propidium iodide were from Molecular Probes(Oregon, WA); Wizard genomic DNA purification kit and apoptosisdetection system fluorescein kit were from Promega (Madison,WI); and Ac-DEVD-AMC was from Bachem (Switzerland). Other chemicalswere purchased from Sigma Chemical Co. (St Louis, MO).
Primary Culture of Rat Proximal Tubule Cells
The cells were isolated from Sprague-Dawley rats (40 g), asdescribed by Vinay et al. (13). Briefly, the kidneys were removedaseptically, decapsulated, and minced coarsely in HBSS supplementedwith 10 mM Hepes and 5 mM D-glucose, pH 7.4. Cortex was separatedfrom the medulla and incubated in HBSS supplemented with 0.48U/ml collagenase and 0.1% BSA in a flask under gentle stirringfor 40 min at 37°C under a 5% CO2 atmosphere. To separatehomogeneous populations of nephron segments, the mixture oftubules was suspended in 42% Percoll made isotonic with 10xconcentrated KHB (Krebs Henseleit Buffer: 1.18 M NaCl, 47 mMKCl, 100 mM Hepes, 200 mM cyclamic acid, 1.26 mM MgSO4, 11.4mM KH2PO4, 50 mM glucose) and was centrifuged (17,000 rpm, 30min, 4°C). The F4 layer, composed by proximal tubules, wassuspended in culture medium (DMEM/Ham’s F-12 medium supplementedwith 25 mM Hepes, 25 mM NaHCO3, 4 mM glutamine, 20 nM sodiumselenite, 10 ml/L of a 100x nonessential amino acid mixture,50 U/ml penicillin, 50 µg/ml, streptomycin, 10 µg/mlinsulin, 5 nM transferrin, 0.1 nM dexamethasone, 10 ng/ml EGF,5 µg/ml triodothyronine) and plated at 6 mg, 1 mg, and0.6 mg of protein in 150-mm or 60-mm Petri dishes or six-wellplates, respectively, that had been coated with collagen typeI from calf skin. FCS 5% was added to the culture medium untilthe first change of medium (2 d after seeding). The experimentswere carried out at day 5.
Transfection and Culture of HEK 293 Cells
HEK cells were obtained from the American Type Tissue Cultureand grown in DMEM (Dulbecco modified Eagle medium) at 37°C(5% CO2) supplemented with 10% fetal calf serum, penicillin(100 units/ml), streptomycin (100 µg/ml), and fungizone(0.25 µg/ml). Human MAO-B cDNA was provided by Dr. JeanShih in the pECE vector. pECE MAO-B was restricted with KpnIand XbaI (sites in the pECE polylinker), and the insert waspurified and ligated into the KpnI, XbaI sites of pCDNAIII.Cells were stably transfected with DNA by calcium phosphatecoprecipitation as described previously (14).
Evaluation of Apoptosis and Necrosis
Morphologic changes of the nucleus were detected as describedby Meilhac et al. (15). Necrosis and apoptosis were evaluatedconcomitantly on intact cultured cell (grown in six-well plates)after fluorescence straining using vital fluorescence dyes,0.6 µM SYTO-13 (a permeant DNA intercalating green-coloredprobe) and 15 µM propidium iodide (a nonpermeant intercalatingorange probe), and counted using an inverted fluorescence microscope(Fluovert FU, Leitz). Normal nuclei exhibited loose chromatincolored green by SYTO-13; apoptotic nuclei exhibited condensedgreen-colored chromatin (postapoptotic necrosis being characterizedby nuclei exhibiting the same apoptotic morphologic features,but with orange colored) chromatin; necrotic cells exhibitedorange-colored nuclei with loose chromatin.
DNA fragmentation was visualized in situ by TUNEL (teminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling) using the ApoptosisDetection System-Fluorescein kit (Promega, Madison, WI). Briefly,after growing on glass slides (Superfrost plus), cells werefixed in 4% buffered paraformaldehyde. After permeabilizingthe cells with Triton X-100, the slides were covered by 45 µlof Equilibration buffer, 5 µl of Nucleotide Mix, and 1µl of TdT enzyme and incubated at 37°C for 1 h, washedfour times, and all cells were strained with propidium iodidefor 15 min. Green fluorescence of apoptotic cells (fluorescein-12-dUTP)was counted on a red background (propidium iodide) under fluorescencemicroscopy. The positive control was treated by DNAse I (1 unit/ml)before being processed through the TUNEL procedure.
Visualization of DNA Ladder
To visualize DNA laddering, DNA was extracted using the Wizardgenomic DNA purification kit of Promega according to the manufacturer’sinstructions. Briefly, cells were harvested and centrifugedat 14,000 x g for 10 s at room temperature. Cell pellets weresuspended in 600 µl of nucleus lysis solution and incubatedfor 15 min at 37°C with 3 µl of RNase solution. DNAwas then extracted with protein precipitation solution, precipitatedin isopropanol, washed with 70% ethanol, centrifuged at 14,000x g for 1 min, and resuspended in DNA rehydratation solution.After quantification of DNA, 5 µg of DNA per lane waselectrophoresed in a 1.2% agarose gel for 1 h at 50 V and thegel was visualized with ethidium bromide.
Analysis of Caspase-3 Activity
Cells (grown on 150-mm dishes) were washed and collected bycentrifugation at 700 x g for 5 min at 4°C. Cell pelletswere resuspended in lysis buffer (100 mM Hepes-KOH, pH 7.4,42 mM KCl, 5 mM MgCl2, 1 mM DTT, 0.5% CHAPS, 1 mM phenylmethylsulfonylfluoride, and 2 µg/ml leupeptin) and left on ice for 30min. After sonication, lysates were centrifuged at 10,000 xg for 10 min at 4°C. Supernatants (100 µg) were usedfor caspase-3 assay in lysis buffer containing the specificsubstrate Ac-DEVD-AMC (20 µM) at 30°C for 5 h. Substratecleavage was monitored fluometrically using a Jobin-Yvon spectrophotometerwith excitation and emission wavelengths of 351 and 430 nm.
Western Blot Analysis
Confluent rat proximal tubule plates (60 mm) were appropriatelyconditioned and pretreated with or without various inhibitors.At specified times after DA treatment, cells were lysed in buffercontaining 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1mM EGTA, 1% Triton X100, 2 mM Na3VO4, 10 µg/ml PMSF, 3µg/ml aprotinin, and 3 µg/ml leupeptin. After sonication,30 µg of proteins were loaded on a 10% (for pro-caspase-3)or on a 12% (for Bax and Bcl-2) polyacrylamide gel and transferredto PVDF membrane. The membrane was blocked with 1% BSA in TBS-Tween20 (0.1%) (TBST) overnight at 4°C. Polyclonal anti-pro-caspase-3(1:500), monoclonal anti-Bax (1:100), or polyclonal anti-Bcl-2(1:200) was used as primary antibody. After incubation withappropriate HRP-linked secondary antibody (1:10,000, 30 min,room temperature), proteins were detected using the ECL reaction.The blot was stripped completely of antibodies before reprobingwith a polyclonal anti-actin (1:1000) antibody used as a standard.
Localization of Cytochrome c
Cells (detached and attached) were washed with PBS and collectedby centrifugation at 700 x g for 5 min at 4°C. The cellpellet was resuspended in extraction buffer containing 220 mMmannitol, 68 mM sucrose, 50 mM PIPES-KOH, pH 7.4, 50 mM KCl,5 mM EGTA, 2 mM MgCl2, 1 mM dithiothreitol (DTT), and proteaseinhibitors. After 30 min incubation on ice, cells were homogenizedwith a glass Dounce. Cell homogenates were spun at 14,000 xg for 15 min at 4°C, and supernatants were removed and storedat -80°C until analysis by gel electrophoresis. Fifty microgramsof cytosolic protein extracts were loaded onto each lane of12% SDS-polyacrylamide gel, separated, and then blotted ontoPVDF membrane. Purified horse heart cytochrome c was used asstandard. Goat anti-cytochrome c monoclonal antibody was usedas primary antibody (1:500). The specific protein complex formedon appropriate secondary antibody treatment (1:10,000) was identifiedusing the ECL reaction.
Statistical Analyses
All values are presented as mean ± SEM. Student’sunpaired t test (Prism GraphPad, San Diego, CA) was used forstatistical analyses, and differences were considered significantwhen P < 0.05.
Dopamine Induces Renal Epithelial Cell Apoptosis through MAO-Dependent H2O2 Production
In a first series of experiments, we investigated the abilityof dopamine to induce apoptosis of renal epithelial cells. Cellapoptosis was evaluated by analysis of DNA fragmentation andthe measure of cell incorporation of two fluorescence dyes:SYTO-13, a permeant DNA intercalating green-colored probe, andpropidium iodide, a nonpermeant intercalating orange probe.Apoptotic and necrotic cells were discriminated by green (SYTO-13)and orange (propidium iodide) staining, respectively. Cell treatmentwith dopamine concentrations (from 50 to 200 µM) for 24h induced a significant increase (+73%) in the number of SYTO-13–stainedcells as compared with untreated cells (Figure 1, A and B).In contrast, dopamine treatment did not increase cell stainingby propidium iodide, the marker of necrotic cells (Figure 1A).The increase in cell apoptotis by dopamine was also confirmedby TUNEL staining (Figure 2, A and B) and DNA laddering (Figure 2C).Cell treatment with dopamine increased the number of TUNEL-positivecells to an extent (+75%) similar to that defined by SYTO-13staining (Figure 2, A and B) and also induced DNA laddering(Figure 2C). As shown in Figures 1B, 2B, and 2C, SYTO-13 andTUNEL staining and DNA laddering were prevented by cell treatmentwith the dopamine uptake inhibitor GBR-12909, the irreversibleMAO inhibitor pargyline, or the antioxidant N-acetylcysteine(NAC) and pyruvate.
Figure 1. Morphologic analysis of dopamine-induced proximal tubule cell apoptosis. Cell nuclei were double-stained with SYTO-13 and propidium iodide, discriminating normal cells and cells undergoing apoptosis or necrosis by fluorescence microscopy. (A) Photomicrographs of proximal tubule cell primary cultures pretreated for 30 min with or without 1 µM pargyline (P), 1 mM N-acetylcysteine (NAC), or 3 µM GBR-12909 (GBR) before addition of 100 µM dopamine (DA) for 24 h. (B) Quantification of proximal tubule cell stained by SYTO-13. (Right panel) Quantification of apoptotic cells presented in A plus cells pretreated with 10 mM pyruvate. (Left panel) Percentage of apototic cells after incubation with increasing concentrations of dopamine. The data result from counting of three fields of approximately100 cells each per dish. Values are mean ± SEM of the number of morphologically apoptotic cells relative to the control from three separate experiments. *** P< 0.001.
Figure 2. DNA fragmentation of dopamine-treated proximal tubule cells. Proximal tubule cell primary cultures were pretreated with or without 1 µM pargyline (P), 1 mM N-acetylcysteine (NAC), or 3 µM GBR-12909 (GBR) for 30 min before addition of 100 µM dopamine (DA) for 24 h. DNA fragmentation was visualized in situ by the TUNEL procedure and by electrophoresis. (A) Fluorescence microscopy of TUNEL staining. (B) Quantification of TUNEL-positive cells. Values are mean ± SEM of TUNEL fluorescent cells relative to control from three separate experiments. The data result from counting three fields of approximately100 cells each per slide. (C) DNA laddering is visualized by electrophoresis of DNA extracted from cells incubated for 24 h with various concentrations of dopamine or in the presence of inhibitors prior to 100 µM dopamine addition (+) or not (-). The results are representative of three different experiments. M, 100 bp marker. *** P < 0.001.
To specify the role of MAO-dependent H2O2 production from dopaminein cell apoptosis, experiments were performed in wild-type HEK293 cells lacking MAO and in HEK 293 cells stably transfectedwith human MAO B cDNA (HEK-MAO-B) (11). In wild-type HEK 293cells, dopamine treatment did not modify the number of SYTO-13–positivecells (Figure 3). In contrast, incubation of HEK-MAO-B cellswith 100 µM dopamine for 24 h increased the number ofSYTO-13 dyed cells by 80% compared with untreated cells (Figure 3).As observed in proximal tubule cells, SYTO-13 staining wasabolished by cell incubation with pargyline, NAC, and GBR-12909(Figure 3). These results support the exclusive role of MAO-dependentH2O2 production on dopamine-induced renal epithelial cell apoptosis.
Figure 3. Effect of dopamine on HEK 293 cell apoptosis. HEK 293 wild-type (HEK-WT) and HEK 293 cells stably transfected with human MAO-B cDNA (HEK-MAO B) were pretreated with or without 1 µM pargyline (P), 1 mM N-acetylcysteine (NAC), or 3 µM GBR-12909 for 30 min before addition of 100 µM dopamine (DA) for 24 h. Cell nuclei were double-stained with SYTO-13 and propidium iodide. Values are mean ± SEM of the number of morphologically apoptotic cells relative to the control from three separate experiments. *** P < 0.001). Similar have been obtained using MAO-B transfected HEK 293 pools.
Effect of Dopamine on Bcl-2 and Bax Expression, Mitochondrial Cytochrome c Release, and Caspase-3 Activation in Proximal Tubule Cells
Members of the Bcl-2 protein family play a critical role intriggering mitochondrial dysfunction associated to apoptoticprocesses (16–19). We thus examined the effect of dopamineon two members of the Bcl-2 family, the anti-apoptotic proteinBcl-2 and the pro-apoptotic protein Bax. Western blot analysisshowed that incubation of proximal tubule cells with dopamineinduced a concomitant increase in Bax and decrease in Bcl-2proteins (Figure 4A). This effect started between 1 and 3 hafter cell incubation with dopamine and persisted for up to16 h incubation, the longest time tested. Regulation of bothBax and Bcl-2 proteins by dopamine was dose-dependent, peakingbetween 100 and 200 µM (Figure 4A).
Figure 4. Effect of dopamine on Bcl-2 and Bax expression in proximal tubule cells. (A) Cells were treated with dopamine (DA) for the indicated concentration and time. (B) After pretreatment with 1 µM pargyline, 1 mM N-acetylcysteine, or 3 µM GBR-12909, cells were treated with 100 µM dopamine (+) or not (-) for 16 h. The protein expression of Bax and Bcl-2 was determined by Western blot in cell lysates. Antibody against actin was used to confirm equal loading of the extracts. (C) Quantification of Bax (left panel) and Bcl-2 (right panel) protein expression in cells treated with 100 µM dopamine for 16 h in the presence of 1 µM pargyline, 1 mM N-acetylcysteine, or 3 µM GBR-12909. The histograms are representative of three different experiments. *** P < 0.001 compared with control.
As Bax and Bcl-2 are known to respectively induce and inhibitmitochondrial cytochrome c release (16–19), we nextinvestigated the effect of dopamine on the mitochondrial andcytosolic distribution of cytochrome c. Cell treatment withdopamine for 16 h led to a dose-dependent increase in cytosoliccytochrome c (Figure 5A). Moreover, dopamine treatment alsodecreased the amount of cytochrome c in the mitochondrial fraction(Figure 5B).
Figure 5. Effect of dopamine on cytochrome c release in proximal tubule cells. Cells were treated for 16 h with increasing dopamine (DA) concentrations (left panel) or in the presence (right panel) of 1 µM pargyline, 1 mM N-acetylcysteine, or 3 µM GBR-12909 µM before 100 µM dopamine (+) or not (-). After treatments, the cytosolic fraction was separated from the mitochondria fraction as described in Materials and Methods. The level of cytochrome c was determined by Western blot in cytosol (A) and mitochondria (B). Western blot for actin and HSP60 was carried out to confirm equal loading of the extracts. HSP 60, a mitochondrial protein, was used in the cytosolic fraction to check for non-contamination by mitochondria. The results are representative of three different experiments.
Release of mitochondrial cytochrome c is an upstream step ofprocaspase cleavage and generation of active caspases (20,21). Among the different caspases, caspase-3 represents the finaleffector leading to endonuclease activation and cell apoptosis(22). To determine the involvement of caspases in dopamine-dependentcell apoptosis, we studied the effect of dopamine in the activationof caspase-3. Caspase-3 activation was evaluated by Westernblot of procaspase-3 and the measure of caspase-3 activity.Treatment of proximal tubule cells with dopamine for 16 h induceda concentration-dependent decrease in procaspase-3 protein (Figure 6A)and an increase in caspase-3 activity (4.2-fold comparedwith untreated cells; Figure 6C).
Figure 6. Effect of dopamine on caspase-3 activation in proximal tubule cells. (A) Level of caspase-3 in proximal tubule cells treated with increasing dopamine concentrations for 16 h. (B) Cells were pretreated with 1 µM pargyline, 1 mM N-acetylcysteine, or 3 µM GBR-12909 before 100 µM dopamine (DA) (+) addition for 16 h or not (-). (A and B) Western blot analyses. The data are representative of three independent experiments. (C) Caspase-3 activity was measured fluorometrically with 20 µM Ac-DEVD-AMC as described in Materials and Methods. Proximal tubule cells were treated by DA for 16 h in the same conditions in A and C and in B and D. Data are means ± SEM from three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001.
As shown in Figures 4B, 5, 6B, and 6D, the effects of dopaminetreatment on Bax and Bcl-2 expression, mitochondrial cytochromec release, and activation of caspase-3 were prevented by pargyline,NAC, or GBR-12909, indicating that dopamine needs to be internalizedand metabolized by MAO to activate the pro-apoptotic cascade.
During the last years, most of the studies on the renal effectsof dopamine concerned the regulation of fluid and sodium homeostasis.Our results show for the first time that, in proximal tubulecells, dopamine may also have pro-apoptotic properties.
Using two models of cells expressing MAO, proximal tubule andHEK 293-MAO-B–transfected cells (HEK-MAO-B), we have beenable to show that apoptosis by dopamine is entirely dependenton H2O2 production by MAO. Indeed, cell apoptosis required sequentialdopamine internalization into the cells, degradation by MAO,and H2O2 production as shown by the prevention of apoptosisby the dopamine transporter inhibitor GBR-12909, the MAO inhibitorpargyline, and the antioxidant NAC. The use of wild-type andtransfected HEK cells clearly discriminated apoptotic activityinvolving MAO from that related to dopamine autoxidation (23).Indeed, dopamine concentrations inducing maximal cell apoptosisin proximal tubule and MAO-transfected HEK 293 cells (up to200 µM) were without significant effects in wild-typeHEK 293 cells lacking MAO. In contrast, at a dopamine concentrationof 500 µM, cell apoptosis was also observed in MAO-deficientcells (Bianchi et al., unpublished results). In addition, apoptosisof proximal tubules and MAO-transfected HEK cells induced by500 µM dopamine was only partially prevented by the MAOinhibitor pargyline (Bianchi et al., unpublished results). Theseresults indicate that, within the same cell type, the mechanismsof cell apoptosis by dopamine differ in a narrow dopamine concentrationrange. It is noteworthy that a dopamine concentration of 10µM, slightly lower than that triggering cell apoptosis(50 µM), induces proximal tubule and HEK-MAO-B cell proliferationthat is receptor-independent and requires H2O2 production byMAO (11,24). Overall, these results underline the complexityof dopamine effects and may represent a starting point for understandingof the receptor-independent effects of dopamine in vitro andin vivo not only in kidney but also in others cell types targetedby dopamine and expressing MAO.
In the concentration range in which cell apoptosis by dopamineis selectively dependent on MAO activity, cell death is precededby sequential activation of pro-apoptotic factors. Modificationof the expression of members of the Bcl2 protein family appearsto be the early event. Indeed, we showed that cell incubationwith dopamine for 3 h led to the increase in Bax/Bcl-2 ratio.This was related to both the rise in the amount of pro-apoptoticfactor Bax and the decrease in the expression of the anti-apoptoticprotein Bcl2. This phenomenon was followed by mitochondrialcytochrome c release and the subsequent activation of caspase-3.All these events were prevented by the MAO inhibitor pargylineand the antioxidant NAC, indicating the pivotal role of H2O2produced by MAO in triggering dopamine-dependent cell apoptosis.The mitochondrial toxicity of H2O2 produced by MAO may be particularlyintense in relation to the location of these enzymes in theouter mitochondrial membrane. The H2O2 produced by MAO may quicklydiffuse into the mitochondria, participate in the disruptionof the mitochondrial membrane, and/or behave as a positive feedbackon the respiratory chain enzymes to generate larger amountsof ROS. This possibility is also supported by previous studiesshowing that, in isolated mitochondria, metabolism of dopamineand other substrates by MAO suppresses mitochondrial electrontransport (25,26).
In neuronal pheochromocytoma cells, expression of the MAO genesincreased significantly during apoptosis, indicating that MAOmay be a target gene of pro-apoptotic signal transduction (27).On the basis of these results and the findings of the presentstudy, it is conceivable that MAO may be simultaneously a keyelement for the induction of apoptosis and the target of pro-apoptoticfactors, increasing its expression and, consequently, its apoptoticpotential.
The demonstration that dopamine induces oxidative stress andapoptosis in neuronal cells (2,28,29) suggested its involvementin neurodegenerative diseases (30,31). Our results, showingthat dopamine also induces apoptosis in renal epithelial cells,may have an impact on the understanding of the cell mechanismsleading to the development of renal disorders. Indeed, the apoptosisof proximal tubule cells is one of the major events precedingacute renal failure after ischemia-reperfusion and toxic kidneyinjury. We have recently shown that, in a rat model of unilateralrenal ischemia-reperfusion, H2O2 generated by MAO plays a majorrole in the oxidative stress, cell apoptosis, and necrosis occurringafter reperfusion (32). It is conceivable that dopamine andperhaps other MAO substrates, which increase in the early reperfusionperiod, contribute to proximal tubule cell damage and the developmentof renal failure.
In conclusion, our results stress the critical role of the MAO-dependentH2O2 production on cell apoptosis and open new avenues for theunderstanding of the mechanisms of action of dopamine in renalphysiologic and pathologic situations. It is conceivable thatMAO inhibitors may represent an efficient therapeutic approachto prevent cell apoptosis in renal chronic and acute diseases.
Acknowledgments
We thank Prof. T. Levade, his team, and Dr. I. Escargueil-Blancfor the apoptosis protocols used. This work was supported bythe Institut National de la Santé et de la RechercheMédicale.
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Received for publication August 9, 2002.
Accepted for publication December 27, 2003.
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Abstract
Introduction
B in rats. Mol Pharmacol 56: 254–264, 1999
