The Parkinsonism-inducing Drug 1-Methyl-4-phenylpyridinium Triggers Intracellular Dopamine Oxidation
2000; Elsevier BV; Volume: 275; Issue: 49 Linguagem: Inglês
10.1074/jbc.m005385200
ISSN1083-351X
AutoresJulie Lotharius, Karen L. O’Malley,
Tópico(s)Neurological disorders and treatments
ResumoUptake of the Parkinsonism-inducing toxin, 1-methyl-4-phenylpyridinium (MPP+), into dopaminergic terminals is thought to block Complex I activity leading to ATP loss and overproduction of reactive oxygen species (ROS). The present study indicates that MPP+-induced ROS formation is not mitochondrial in origin but results from intracellular dopamine (DA) oxidation. Although a mean lethal dose of MPP+ led to ROS production in identified dopaminergic neurons, toxic doses of the Complex I inhibitor rotenone did not. Concurrent with ROS formation, MPP+ redistributed vesicular DA to the cytoplasm prior to its extrusion from the cell by reverse transport via the DA transporter. MPP+-induced DA redistribution was also associated with cell death. Depleting cells of newly synthesized and/or stored DA significantly attenuated both superoxide production and cell death, whereas enhancing intracellular DA content exacerbated dopaminergic sensitivity to MPP+. Lastly, depleting cells of DA in the presence of succinate completely abolished MPP+-induced cell death. Thus, MPP+neurotoxicity is a multi-component process involving both mitochondrial dysfunction and ROS generated by vesicular DA displacement. These results suggest that in the presence of a Complex I defect, misregulation of DA storage could lead to the loss of nigrostriatal neurons in Parkinson's disease. Uptake of the Parkinsonism-inducing toxin, 1-methyl-4-phenylpyridinium (MPP+), into dopaminergic terminals is thought to block Complex I activity leading to ATP loss and overproduction of reactive oxygen species (ROS). The present study indicates that MPP+-induced ROS formation is not mitochondrial in origin but results from intracellular dopamine (DA) oxidation. Although a mean lethal dose of MPP+ led to ROS production in identified dopaminergic neurons, toxic doses of the Complex I inhibitor rotenone did not. Concurrent with ROS formation, MPP+ redistributed vesicular DA to the cytoplasm prior to its extrusion from the cell by reverse transport via the DA transporter. MPP+-induced DA redistribution was also associated with cell death. Depleting cells of newly synthesized and/or stored DA significantly attenuated both superoxide production and cell death, whereas enhancing intracellular DA content exacerbated dopaminergic sensitivity to MPP+. Lastly, depleting cells of DA in the presence of succinate completely abolished MPP+-induced cell death. Thus, MPP+neurotoxicity is a multi-component process involving both mitochondrial dysfunction and ROS generated by vesicular DA displacement. These results suggest that in the presence of a Complex I defect, misregulation of DA storage could lead to the loss of nigrostriatal neurons in Parkinson's disease. dopamine 1-methyl-4-phenyl-l,2,3,6-tetrahydropyridine l-methyl-4-phenylpyridinium DA transporter vesicular monoamine transporter dl-α-methyl-p-tyrosine dihydroethidium 5,7-dihydroxytryptamine dihydrorhodamine rhodamine 123 tyrosine hydroxylase high performance liquid chromatography analysis of variance reactive oxygen species The neuropathological hallmark of Parkinson's disease is an irreversible loss of dopamine (DA)1-containing neurons in the substantia nigra. Although the underlying cellular and molecular events in Parkinson's disease are still unknown, post-mortem studies suggest that both oxidative stress and impaired energy metabolism are involved (for review see Ref. 1Bowling A.C. Beal M.F. Life Sci. 1995; 56: 1151-1171Crossref PubMed Scopus (319) Google Scholar). Because the neurotoxin 1-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP) has been shown to produce Parkinsonian symptoms in primates and rodents (for review see Ref. 2Przedborski S. Jackson-Lewis V. Move. Disord. 1998; 13 (Suppl. 1): 35-38PubMed Google Scholar), it has been extensively used as an animal model of Parkinson's disease. Thus, elucidation of its mode of action has been of paramount importance in understanding and potentially treating this disorder. Toward this end, studies have indicated that l-methyl-4-phenylpyridinium (MPP+), the active metabolite of MPTP, can block electron transport by binding to the same site as the classic Complex I inhibitor, rotenone, leading to a loss of ATP production (3Nicklas W.J. Vyas I. Heikkila R.E. Life Sci. 1985; 36: 2503-2508Crossref PubMed Scopus (1096) Google Scholar, 4Ramsay R.R. Salach J.I. Singer T.P. Biochem. Biophys. Res. Commun. 1986; 134: 743-748Crossref PubMed Scopus (230) Google Scholar, 5Ramsay R.R. Krueger M.J. 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A. 1985; 82: 2173-2177Crossref PubMed Scopus (1154) Google Scholar), MPP+ also undergoes high affinity uptake by the vesicular monoamine transporter (VMAT2; Refs. 21Del Zompo M. Piccardi M.P. Ruiu S. Corsini G.U. Vaccari A. Eur. J. Pharmacol. 1991; 202: 293-294Crossref PubMed Scopus (12) Google Scholar, 22Del Zompo M. Piccardi M.P. Ruiu S. Corsini G.U. Vaccari A. Brain Res. 1992; 571: 354-357Crossref PubMed Scopus (18) Google Scholar, 23Peter D. Jimenez J. Liu Y. Kim J. Edwards R.H. J. Biol. Chem. 1994; 269: 7231-7237Abstract Full Text PDF PubMed Google Scholar). Indeed, fractionation studies in chromaffin cells have shown that MPP+ is predominantly localized within catecholaminergic vesicles with only negligible amounts found in mitochondria (24Reinhard Jr., J.F. Diliberto Jr., E.J. Viveros O.H. Daniels A.J. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 8160-8164Crossref PubMed Scopus (102) Google Scholar). Because VMAT2 overexpressing cell lines are resistant to MPP+ (25Liu Y. Peter D. Roghani A. Schuldiner S. Prive G.G. Eisenberg D. Brecha N. Edwards R.H. Cell. 1992; 70: 539-551Abstract Full Text PDF PubMed Scopus (525) Google Scholar, 26Liu Y. Roghani A. Edwards R.H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9074-9078Crossref PubMed Scopus (109) Google Scholar), sequestration within vesicles appears to play a protective role. Consistent with this hypothesis, heterozygous VMAT2 knock-out mice display increased susceptibility to MPTP (27Takahashi N. Miner L.L. Sora I. Ujike H. Revay R.S. Kostic V. Jackson-Lewis V. Przedborski S. Uhl G.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9938-9943Crossref PubMed Scopus (342) Google Scholar, 28Gainetdinov R.R. Fumagalli F. Wang Y.M. Jones S.R. Levey A.I. Miller G.W. Caron M.G. J. Neurochem. 1998; 70: 1973-1978Crossref PubMed Scopus (145) Google Scholar). A consequence of sequestration, however, might be the displacement of DA from vesicular stores. Once in the cytoplasm, DA would be readily autoxidized (for review see Ref. 29Fornstedt B. Acta Neurol. Scandinavica. Suppl. 1990; 129: 12-14PubMed Google Scholar) or deaminated (30Maker H.S. Weiss C. Silides D.J. Cohen G. J. Neurochem. 1981; 36: 589-593Crossref PubMed Scopus (208) Google Scholar) to produce hydrogen peroxide, superoxide, and reactive quinone species capable of covalently modifying cellular macromolecules (31Graham D.G. Mol. Pharmacol. 1978; 14: 633-643PubMed Google Scholar). Thus, MPP+-mediated displacement of DA from secretory vesicles could not only lead to cytoplasmic DA oxidation and generation of free radicals but also to extracellular DA release and oxidation, both of which could contribute to dopaminergic neuronal degeneration. Given the alternative and possibly overlapping hypotheses as to the source and sequelae of MPP+-induced free radicals, the present study used free radical sensitive fluorophores together with pharmacological and molecular techniques to determine the role of DA in MPP+-mediated toxicity in primary dopaminergic neurons. Cultures of murine (CF1; Charles River Laboratories) mesencephalic neurons were prepared as described previously (32Lotharius J. Dugan L.L. O'Malley K.L. J. Neurosci. 1999; 19: 1284-1293Crossref PubMed Google Scholar). For immunocytochemistry, cells were plated at a density of 100,000 cells/glass microwell plate (1.25 × 103 cells/mm2). For DA release/content assays, cells were plated at a density of 400,000 cells/16-mm well (2 × 103 cells/mm2). Experiments were conducted after 6 days in vitro unless otherwise specified. MPP+ iodide,dl-α-methyl-p-tyrosine (MPT), mazindol, nifedipine, and reserpine were purchased from Research Biochemicals International (Natick, MA). Dihydroethidium (DHE), rotenone, 3-hydroxytyramine (dopamine), 5,7-dihydroxytryptamine (DHT), and succinate were obtained from Sigma. Dihydrorhodamine (DHR) and rhodamine 123 (R123) were purchased from Molecular Probes (Eugene, OR). The C3 isomer of malonic C60 was a kind gift from Dr. L. Dugan (Department of Neurology, Washington University School of Medicine; Ref. 33Dugan L.L. Turetsky D.M. Du C. Lobner D. Wheeler M. Almli C.R. Shen C.K. Luh T.Y. Choi D.W. Lin T.S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9434-9439Crossref PubMed Scopus (709) Google Scholar). [7,8-3H]Dopamine was purchased from Amersham Pharmacia Biotech. C3 was dissolved in water, DHT was dissolved in 1% ascorbic acid, and mazindol was first dissolved in 50 mm HCl and then diluted in medium. Reserpine, rotenone, and the fluorophores were dissolved in Me2SO as stock solutions and then diluted in medium such that the final concentration of Me2SO was 515 nm). Cells were then fixed and stained for TH, and field relocation was used to confirm that DHT cells were indeed dopaminergic. To exclude the possibility that enhanced DHE fluorescence was an artifact of decreased mitochondrial membrane potential (ΔΨm; Ref. 35Budd S.L. Castilho R.F. Nicholls D.G. FEBS Lett. 1997; 415: 21-24Crossref PubMed Scopus (204) Google Scholar), we monitored changes in ΔΨm following both rotenone and MPP+treatment using the fluorescent, potentiometric dye R123. Cells were stained with DHT and 0.3 μm R123 and imaged exactly as described previously (32Lotharius J. Dugan L.L. O'Malley K.L. J. Neurosci. 1999; 19: 1284-1293Crossref PubMed Google Scholar). To measure extracellular and intracellular DA levels, all drug treatments were performed in 200 μl of Krebs-Ringer solution/0.01%l-cysteine. To measure extracellular DA, the culture medium was removed and filtered with a 0.1-μm nylon filter. For measurement of intracellular DA, 175 μl of 0.1 nperchloric acid/0.01% l-cysteine was added to each well after medium removal. Cells were freeze-thawed, and the perchloric acid lysates were filtered. Samples were stored on ice or frozen at −80 °C until use. A 100-μl aliquot from each sample was then analyzed by high performance liquid chromatography (HPLC) using a Coulochem electrochemical detector (ESA, Bedford, MA) and an ESA catecholamine HR-80 reverse phase column (37Owens G.C. Johnson R. Bunge R.P. O'Malley K.L. J. Neurochem. 1991; 56: 1030-1036Crossref PubMed Scopus (18) Google Scholar, 38Tang L. Todd R.D. O'Malley K.L. J. Pharmacol. Exp. Ther. 1994; 270: 475-479PubMed Google Scholar). The mobile phase consisted of 100 mmNaH2PO4·H20, 80 mg/liter EDTA, 250 mg/liter heptasulfonic acid, and 6% methanol titrated to pH 2.5. Peaks were identified by retention times set to known standards. To measure DA release, cells were loaded with 2.4 μCi/ml [3H]DA/Krebs-Ringer solution for 20 min at 37 °C and washed 3 times for 3 min. Radioactive counts from a wash sample were measured using a Beckman scintillation counter and used as a control for basal levels of [3H]DA release. Cells were then treated with 1 μm MPP+ for 10 min, and the amount of [3H]DA released during this time period was counted. Cultures were then washed extensively and maintained in [3H]DA-free Krebs-Ringer solution. Extracellular [3H]DA was measured after an additional 10, 30, and 50 min. The amount of spontaneous [3H]DA released at equivalent time periods in vehicle-treated wells was subtracted from the amount of [3H]DA released at each treatment time point. Following medium collection, cells were lysed in 0.1n perchloric acid by freeze-thawing, and residual, intracellular [3H]DA was measured. To determine dopaminergic cell viability following MPP+ treatment, mesencephalic cultures were processed for TH immunoreactivity and counted as described previously (32Lotharius J. Dugan L.L. O'Malley K.L. J. Neurosci. 1999; 19: 1284-1293Crossref PubMed Google Scholar). Briefly, six consecutive fields were assayed per dish leading to the quantification of 200–300 TH neurons/experiment. Experiments were repeated 3–5 times using cultures isolated from independent dissections. Descriptive statistics (means ± S.E.) of cell survival counts were calculated with the statistical package GraphPad Prism Software. Cell survival curves were generated using the means ± S.E. with data collected from at least three separate experiments. The significance of effects between control and drug conditions was determined by one- or two-way factor ANOVA as indicated and post-hoc Student's t tests (GraphPad Prism Software). Overproduction of ROS has been proposed to be an important mechanism underlying the cytotoxicity of MPP+ (32Lotharius J. Dugan L.L. O'Malley K.L. J. Neurosci. 1999; 19: 1284-1293Crossref PubMed Google Scholar, 39Przedborski S. Kostic V. Jackson-Lewis V. Naini A.B. Simonetti S. Fahn S. Carlson E. Epstein C.J. Cadet J.L. J. Neurosci. 1992; 12: 1658-1667Crossref PubMed Google Scholar, 40Przedborski S. Jackson-Lewis V. Yokoyama R. Shibata T. Dawson V.L. Dawson T.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4565-4571Crossref PubMed Scopus (592) Google Scholar, 41Akaneya Y. Takahashi M. Hatanaka H. Neurosci. Lett. 1995; 193: 53-56Crossref PubMed Scopus (52) Google Scholar). In support of this model, our previous studies have shown that doses of MPP+ capable of killing 70% of cultured dopaminergic neurons produced an early, 3-fold increase in superoxide radicals (32Lotharius J. Dugan L.L. O'Malley K.L. J. Neurosci. 1999; 19: 1284-1293Crossref PubMed Google Scholar). Given that mitochondria are a major cellular source of ROS (for review see Ref. 42Richter C. Gogvadze V. Laffranchi R. Schlapbach R. Schweizer M. Suter M. Walter P. Yaffee M. Biochim. Biophys. Acta. 1995; 1271: 67-74Crossref PubMed Scopus (494) Google Scholar) and that MPP+ is thought to block Complex I activity, it has been hypothesized that MPP+-induced ROS are mitochondrial in origin. Because MPP+ and rotenone are thought to bind to the same site on Complex I (5Ramsay R.R. Krueger M.J. Youngster S.K. Gluck M.R. Casida J.E. Singer T.P. J. Neurochem. 1991; 56: 1184-1190Crossref PubMed Scopus (190) Google Scholar, 7Krueger M.J. Singer T.P. Casida J.E. Ramsay R.R. Biochem. Biophys. Res. Commun. 1990; 169: 123-128Crossref PubMed Scopus (71) Google Scholar), rotenone should also induce superoxide production. Although high concentrations of rotenone have been shown to induce superoxide production in vitro and in vivo(8Takeshige K. Minakami S. Biochem. J. 1979; 180: 129-135Crossref PubMed Scopus (260) Google Scholar, 9Turrens J.F. Boveris A. Biochem. J. 1980; 191: 421-427Crossref PubMed Scopus (1367) Google Scholar, 10Hasegawa E. Takeshige K. Oishi T. Murai Y. Minakami S. Biochem. Biophys. Res. Commun. 1990; 170: 1049-1055Crossref PubMed Scopus (312) Google Scholar, 43Packer M.A. Miesel R. Murphy M.P. Biochem. Pharmacol. 1996; 51: 267-273Crossref PubMed Scopus (45) Google Scholar), other studies have suggested the converse (44Budd S.L. Nicholls D.G. J. Neurochem. 1996; 67: 2282-2291Crossref PubMed Scopus (396) Google Scholar, 45Rottenberg H. Wu S. Biochim. Biophys. Acta. 1998; 1404: 393-404Crossref PubMed Scopus (227) Google Scholar, 46Chinopoulos C. Tretter L. Adam-Vizi V. J. Neurochem. 1999; 73: 220-228Crossref PubMed Scopus (142) Google Scholar). To determine whether rotenone mimicked MPP+-induced changes in mitochondrial function, we monitored mitochondrial membrane potential (ΔΨm) and free radical production in response to this drug. Specific changes in dopaminergic neurons were determined by either preloading cells with the monoaminergic-specific, autofluorescent dye DHT as described previously (32Lotharius J. Dugan L.L. O'Malley K.L. J. Neurosci. 1999; 19: 1284-1293Crossref PubMed Google Scholar) or by post-hoc identifying cells using TH immunocytochemistry. For example, the ethidium cation released from DHE, a dye widely used to monitor superoxide production (34Bindokas V.P. Jordan J. Lee C.C. Miller R.J. J. Neurosci. 1996; 16: 1324-1336Crossref PubMed Google Scholar, 35Budd S.L. Castilho R.F. Nicholls D.G. FEBS Lett. 1997; 415: 21-24Crossref PubMed Scopus (204) Google Scholar, 36Benov L. Sztejnberg L. Fridovich I. Free Radic. Biol. Med. 1998; 25: 826-831Crossref PubMed Scopus (428) Google Scholar), can be stably intercalated into DNA. This property allowed DHE-treated cultures to be subsequently stained for TH. Fields containing TH positive cells were then imaged by confocal microscopy. Low doses (5–50 nm) of rotenone killed dopaminergic neurons (LD50 = 20 ± 5.2 nm) with a similar time course and morphology to MPP+ (Fig. 1 and Ref. 32Lotharius J. Dugan L.L. O'Malley K.L. J. Neurosci. 1999; 19: 1284-1293Crossref PubMed Google Scholar). Despite the loss of cell viability, rotenone-treated dopaminergic neurons did not exhibit an increase in intracellular superoxide levels at any time point studied (Fig. 2, A and C). Moreover, subtoxic doses of rotenone as well as doses capable of killing up to 75% of the dopaminergic population had no effect on superoxide generation (not shown). In contrast, dopaminergic neurons treated with a mean lethal dose of MPP+ exhibited a robust increase in superoxide that reached a plateau between 1 and 3 h (Fig. 2, A and B, and Ref. 32Lotharius J. Dugan L.L. O'Malley K.L. J. Neurosci. 1999; 19: 1284-1293Crossref PubMed Google Scholar). DHE fluorescence was punctate in appearance and localized to mitochondria.Figure 2MPP+ but not rotenone induces ROS although neither drug depolarizes ΔΨm. A, mesencephalic cultures were exposed to 1 μmMPP+ or 20 nm rotenone for 20 min, 1 h, 3 h, and 6 h and incubated with DHE as described under "Experimental Procedures." Cells were subsequently stained for TH, and fields containing dopaminergic neurons were imaged by confocal microscopy. Left panels, transmitted light images.Right panels, same fields showing DHE fluorescence at 3 h. B, mesencephalic cultures were treated with 1 μm MPP+ for 0.5, 1, 3, and 6 h and incubated with DHE or R123 as described under "Experimental Procedures." The fluorescence intensity of 30–60 TH immunoreactive neurons was averaged and standardized to the basal fluorescence from vehicle-treated TH-positive cells. C, same as B, but cells were treated with 20 nm rotenone. D, DHE results from B and C were compared.a, p < 0.01 compared with vehicle-treated cultures (one-way ANOVA with post-hoc Student's ttest). Error bars of less than 2% are buried within the symbols.View Large Image Figure ViewerDownload (PPT) To exclude the possibility that enhanced DHE fluorescence was an artifact of mitochondrial membrane depolarization and dequenching of the dye (35Budd S.L. Castilho R.F. Nicholls D.G. FEBS Lett. 1997; 415: 21-24Crossref PubMed Scopus (204) Google Scholar), we monitored changes in ΔΨm following both rotenone and MPP+ treatment using the fluorescent, potentiometric dye R123. No significant change in ΔΨmwas observed in cells treated with MPP+ (Fig. 2 B), whereas rotenone-treated cells displayed a small, significant increase in ΔΨm after 3 h of drug exposure (Fig. 2 C). The latter finding is similar to reports by others suggesting that low (nM) to moderate ( 16 h (not shown). Concomitantly, extracellular DA levels rose (Fig. 4 A). Brief (5 min) pretreatment with 10 μm reserpine, a VMAT2 inhibitor, almost completely blocked MPP+-induced DA release (Fig. 4 B), whereas lower doses of reserpine (10 and 100 nm) did not. In agreement with striatal slice studies (14Schmidt C.J. Matsuda L.A. Gibb J.W. Eur. J. Pharmacol. 1984; 103: 255-260Crossref PubMed Scopus (43) Google Scholar, 53Pileblad E. Carlsson A. Neuropharmacology. 1985; 24: 689-692Crossref PubMed Scopus (75) Google Scholar), pretreatment with the general calcium channel blocker, CdCl2 (100 μm), or the specific calcium channel blocker, nifedipine, did not affect MPP+-induced DA release excluding a Ca2+-dependent, exocytic process (Fig. 4 B). In contrast, 10 μmmazindol, a DAT inhibitor, significantly blocked toxin-mediated release suggesting that, upon vesicular displacement, DA is extruded from the cell by reverse transport via the plasma membrane DAT (Fig. 4 C). Predictably, DAT blockade was accompanied by an increase in intracellular DA content (Fig. 4 D). Thus, in this paradigm, MPP+ redistributes vesicular DA to the cytoplasm followed by its outward transport through the DAT. Because redistributed DA can be easily autoxidized to produce a variety of ROS, including superoxide anions, we hypothesized that MPP+-induced superoxide production arises from DA metabolism. To test this hypothesis, cells were depleted of both stored and newly synthesized DA using the VMAT2 blocker reserpine and the TH inhibitor MPT. Incubation of cells with either 100 μm MPT or 10 nm reserpine for 3 h led to 75–80% depletion of intracellular DA (DA content with MPT was 24.72% ± 5.91 of control, with reserpine 22.54% ± 2.02 of control). Because reserpine and MPT concentrations exceeding 100 nm and 1 mm, respectively, were toxic over a prolonged period of time, the minimum effective dose needed to deplete DA was used. MPP+ treatment resulted in a significant increase in superoxide that could be fully blocked by the general free radical scavenger, carboxyfullerene isomer C3 (Fig. 5, A and B, and Ref. 33Dugan L.L. Turetsky D.M. Du C. Lobner D. Wheeler M. Almli C.R. Shen C.K. Luh T.Y. Choi D.W. Lin T.S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9434-9439Crossref PubMed Scopus (709) Google Scholar). Interestingly, treatment with reserpine or MPT also blocked DHE fluorescence, suggesting that redistributed DA is the major contributor to MPP+-generated superoxide (Fig. 5,A and B). In this set of experiments, MPP+ led to a lower increase in superoxide formation than previously observed (Fig. 2 and Ref. 32Lotharius J. Dugan L.L. O'Malley K.L. J. Neurosci. 1999; 19: 1284-1293Crossref PubMed Google Scholar), which was correlated with decreased toxicity (<50%; not shown). Presumably this was due to lot-to-lot variation in the toxin because 1 μmMPP+ typically killed 50–70% of dopaminergic neurons. To further test the hypothesis that DA oxidati
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