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Dopamine but not 3,4-dihydroxy phenylacetic acid (DOPAC) inhibits brain respiratory chain activity by autoxidation and mitochondria catalyzed oxidation to quinone products: Implications in Parkinson's disease
BR A I N R ES E A RC H 1 1 3 9 ( 2 00 7 ) 1 9 5 –2 00
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Research Report
Dopamine but not 3,4-dihydroxy phenylacetic acid (DOPAC) inhibits brain respiratory chain activity by autoxidation and mitochondria catalyzed oxidation to quinone products: Implications in Parkinson's disease Sirsendu Jana a , Arpan Kumar Maiti a , Maria Bindu Bagh a , Kalpita Banerjee a , Amitabha Das a , Arun Roy b , Sasanka Chakrabarti a,⁎ a
Department of Biochemistry, Institute of Post Graduate Medical Education & Research (Dr. B.C. Roy Post Graduate Institute of Basic Medical Sciences) 244B, Acharya J.C. Bose Road, Kolkata-700020, India b Department of Microbiology, Vijaygarh Jyotish Ray College (Affiliated to the University of Calcutta) 8/2 Bejoygarh, Jadavpur, Kolkata-700032, India
A R T I C LE I N FO
AB S T R A C T
Article history:
This study reveals that, in contrast to dopamine (DA), 3,4 dihydroxyphenylacetic acid
Accepted 30 September 2006
(DOPAC) during in vitro incubation up to 2 h causes only marginal inhibition of rat brain
Available online 8 February 2007
mitochondrial respiratory chain activity, a minimal loss of protein free thiols and very little quinoprotein adduct formation. The damaging effects of DA on brain mitochondria are,
Keywords:
however, conspicuous and apparently mediated by quinone oxidation products generated by
Dopamine
autoxidation of DA as well as catalyzed by a mitochondrial activity, inhibitable by clorgyline
Quinone
(2.5–10 μM) and cyanide (1 mM).
Parkinson's disease
© 2006 Elsevier B.V. All rights reserved.
Mitochondria Clorgyline
1.
Introduction
Mitochondrial dysfunction is considered to be a crucial step in the network of cellular damage pathways that culminate in the death of nigral dopaminergic neurons in sporadic form of Parkinson's disease (PD), and in a large measure such mitochondrial dysfunctions are caused by the toxic actions of dopamine (DA) oxidation products within the neurons (Przedborski et al., 1995; Berman and Hastings, 1999; Greenamyre et al., 1999; Beal, 2003; Moore et al., 2005; Jenner and Olanow, 2006). Studies with in vitro systems and animal models have demonstrated the deleterious effects
of dopamine oxidation products on mitochondrial respiration, transmembrane potential and permeability transition pores (Berman and Hastings, 1999; Gluck et al., 2002; Khan et al., 2005). Autoxidation or monoamine oxidase (MAO) catalyzed oxidation of DA gives rise to H 2O 2 , toxic oxyradicals, an array of reactive quinone products and 3,4 dihydroxyphenyl acetic acid (DOPAC) which have all been implicated in causing mitochondrial injury (Berman and Hastings, 1999; Gluck et al., 2002; Gluck and Zeevalk, 2004; Khan et al., 2005). Thus, mitochondrial dysfunction and the degeneration of dopaminergic neurons in PD may be related to intrinsic mitochondrial damage by intraneuronal
⁎ Corresponding author. E-mail address: [email protected] (S. Chakrabarti). 0006-8993/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2006.09.100
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oxidative catabolism of DA (Cohen et al., 1997; Gluck et al., 2002). In substantia nigra of PD subjects, an elevated level of iron and a decrease in GSH and ferritin content have been reported which may further aggravate oxidative damage by DA (Riederer et al., 1989; Dexter et al., 1994; Li et al., 1998). In our earlier study, we have demonstrated that DA can inhibit rat brain mitochondrial electron transport chain (ETC) activity at several levels during extended incubation and the damage apparently is mediated by toxic quinones and not by H2O2 or reactive oxyradicals (Khan et al., 2005). Furthermore, clorgyline, an inhibitor of MAO-A, which can block the conversion of DA to DOPAC, provides partial protection against DA induced impairment of brain mitochondrial ETC activity under our conditions of incubation (Khan et al., 2005). While probing further the actions of DA and DOPAC on mitochondria and the cause of clorgyline protection on DA mediated impairment of mitochondrial respiratory functions in this study, we have noticed an interesting mitochondrial activity related to DA catabolism to quinone products. A body of experimental and postmortem evidence indicates that toxic quinones are involved in the pathogenesis of sporadic PD. The administration of neurotoxic doses of DA in striatum leads to increased formation of protein bound cysteinyl–DA adducts, and the postmortem samples of substantia nigra of PD patients contain an elevated levels of cysteinyl adducts of DA or DOPA formed by the reactions of quinone derivatives of DA or DOPA with cysteine or glutathione (Hastings et al., 1996; Spencer et al., 1998). Cysteinyl adducts of DA can undergo mitochondria catalyzed oxidation to produce several toxins of respiratory chain (Li and Dryhurst, 1997; Li et al., 1998). The present study on mitochondrial catabolism of DA and the effect of DA or its catabolites on electron transport chain (ETC) activity may, therefore, have potential implications in the context of the pathogenesis of PD.
2.
Results
The data presented in Fig. 1 show that, in the presence of 400 μM of DA, complex I and complex IV activities of rat brain mitochondria were decreased by nearly 32% and 57%, respectively, whereas DOPAC (400 μM) caused only a marginal, although statistically significant, inhibition in both the cases. When incubation of mitochondria was carried out in 25 mM HEPES buffer, pH 7.4, instead of 50 mM phosphate buffer, DA again produced very significant inhibition of complex I and complex IV activities while DOPAC failed to produce any effect on the latter (data not shown). When mitochondria were pre-incubated with DA (400 μM) or DOPAC (400 μM) for 2 h, subsequent reduction of MTT by mitochondria in the presence of succinate as the respiratory substrate was inhibited by more than 78% by DA and only 13% by DOPAC (Fig. 2). Reduced glutathione (5 mM) gave almost complete protection against DA induced inhibition of mitochondrial MTT reduction ability and complex I and complex IV activities (Figs. 1 and 2). In the present study, a high concentration of reduced glutathione (5 mM) was added to the incubation mixture to compensate for any loss
Fig. 1 – Effects of DA and DOPAC on rat brain mitochondrial complex I and complex IV activities. Rat brain mitochondria were incubated without or with DA (400 μM) or with DOPAC (400 μM) in the presence or absence of GSH (5 mM) for 2 h at 37 °C. Complex I and complex IV activities were measured in mitochondrial suspensions at the end of the incubation as described in the text. The values are means ± SEM of 12 observations for complex I (A) and 10 observations for complex IV (B). GSH alone did not have any effect on mitochondrial complex I and complex IV activities (data not shown). Statistical significance was calculated by Student's t test, paired. Ψp < 0.001 vs. control, ♦p < 0.01 vs. control, ♦♦p < 0.05 vs. control, *p < 0.001 vs. mitochondria + DA.
by oxidation during prolonged incubation period of 2 h. We verified that only 10% of the reduced glutathione was lost by oxidation during incubation for 2 h under our experimental conditions and furthermore the pH of the incubation mixture was not altered noticeably in any of the tubes at the end of the incubation. A significant formation of quinoprotein adducts (protein–cysteinyl catechols) in mitochondrial proteins was observed in the presence of DA but not DOPAC and such DA induced quinoprotein formation, in turn, was inhibited by GSH (Table 1). Results presented in Fig. 3 show that DA at a concentration of 400 μM caused a significant (∼ 32%) reduction in mitochondrial protein thiols during in vitro incubation, whereas DOPAC at a similar concentration caused only a marginal loss of protein thiols. During in vitro incubation, DA produced quinone products as a result of autoxidation but, in the presence of co-incubated mitochondria, quinone production was enhanced significantly in pro-
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Fig. 2 – Effects of DA and DOPAC on rat brain mitochondrial MTT reduction. Intact mitochondria from rat brain were incubated in isotonic buffer A either alone (control) or with DA (400 μM) or DOPAC (400 μM) in the presence or absence of GSH (5 mM) for 2 h at 37 °C followed by the measurement of mitochondrial MTT reduction in the presence of 10 mM succinate. GSH alone was without effect on mitochondrial MTT reduction (data not shown). Each value expressed as percentage of control represents mean ± SEM of 5 observations. Statistical significance was calculated by Student's t test, paired. Ψp < 0.001 vs. control, ♦p < 0.01 vs. control, *p < 0.001 vs. mitochondria + DA.
portion to increasing mitochondrial protein added (Table 2). Moreover, the mitochondria catalyzed quinone production from incubated DA was remarkably inhibited by clorgyline (2.5–10 μM) and NaCN (1 mM) present in the incubation medium (Table 3). Heat-inactivated mitochondria, on the other hand, failed to increase the rate of quinone production from incubated DA (Table 3). We further observed that the mitochondria catalyzed conversion of DA to quinone products in 25 mM HEPES buffer, pH 7.4, was somewhat less than that seen in 50 mM phosphate buffer (results not shown). However, a proportionate increase in quinone conversion with increasing amount of mitochondrial protein added as well as inhibition of mitochondrial activity by clorgyline was consistently observed in 25 mM HEPES buffer, pH 7.4, as
Table 1 – Production of quinoprotein adducts during incubation of rat brain mitochondria with DA or DOPAC Incubation mixture Mitochondria in buffer (control) Mitochondria + DA Mitochondria + DA + GSH Mitochondria + DOPAC
Fig. 3 – Loss of protein thiols during incubation of mitochondria with DA or DOPAC. Rat brain mitochondria were incubated without or with DA (400 μM) or DOPAC (400 μM) for 2 h at 37 °C followed by the measurement of protein thiols using Ellman's reagent as detailed in the text. Results are the means ± SEM of 6 observations. Statistical significance was calculated by Student's t test, paired. *p < 0.001 vs. control, **p < 0.01 vs. control.
was seen in the case of 50 mM phosphate buffer (data not produced).
3.
Discussion
Our results clearly indicate that in equimolar concentration DOPAC causes a much lesser degree of inhibition of mitochondrial ETC activity than DA as assessed by the measurement of complex I and complex IV activities and MTT reduction ability of rat brain mitochondria (Figs. 1 and 2). Such data, therefore, imply that under our conditions of incubation DOPAC derived from MAO catalyzed DA oxidation can contribute only marginally to the inhibition of mitochondrial respiratory functions when mitochondria are co-incubated with DA. The primary damaging species in this process are the quinone oxidation products of DA and not ROS as has been demonstrated in our earlier published study (Khan et al., 2005). Since the quinones presumably exert their damaging functions through the formation of covalent adducts with the protein nucleophiles (e.g. the free thiols), it is anticipated that the formation of quinoprotein adducts and the inactivation of
Absorbance at 520 nm 0.084 ± 0.003 0.364 ± 0.004* 0.096 ± 0.003▴ 0.101 ± 0.004
Rat brain mitochondria were incubated without or with DA (400 μM) in the presence or absence of GSH (5 mM) or DOPAC (400 μM) for 2 h at 37 °C followed by the measurement of quinoprotein adducts by NBT-glycinate assay as described in the text. Absorbance at 530 nm was taken as the measure of protein bound quinones. The values (normalized to identical mitochondrial protein per tube) are the means ± SEM of 4 observations. Statistical significance was calculated by Student's t test, paired. * p < 0.001 vs. control. ▴ p < 0.001 vs. mitochondria + DA.
Table 2 – Production of quinones from DA in the presence of brain mitochondria Incubation mixture DA in buffer alone DA + mitochondria (50 μg protein) DA + mitochondria (100 μg protein) DA + mitochondria (200 μg protein)
Absorbance at 303 nm 0.080 ± 0.006 0.157 ± 0.027 0.240 ± 0.050 0.359 ± 0.023
Dopamine (400 μM) was incubated alone or with rat brain mitochondria (50–200 μg protein) in 50 mM phosphate buffer, pH 7.4 for 2 h at 37 °C followed by the measurement of quinones at 303 nm. Turbidity due to mitochondria was corrected by using the appropriate control. Values are the means ± SEM of 5 observations.
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Table 3 – Effects of clorgyline and cyanide on quinone formation during incubation of rat brain mitochondria with DA Incubation mixture DA in buffer alone DA + mitochondria DA + mitochondria + clor (2.5 μM) DA + mitochondria + clor (5 μM) DA + mitochondria + clor (10 μM) DA + mitochondria + NaCN (1 mM) DA + mitochondria (heat treated)
Absorbance at 303 nm 0.052 ± 0.005 0.283 ± 0.023▴ 0.167 ± 0.028φ 0.132 ± 0.026* 0.091 ± 0.022* 0.059 ± 0.015* 0.058 ± 0.008*
Dopamine (400 μM) was incubated alone or with rat brain mitochondria (intact or heat treated) in the presence or absence of clorgyline (Clor) or NaCN in 50 mM phosphate buffer, pH 7.4 for 2 h at 37 °C followed by the measurement of quinones at 303 nm. Turbidity due to mitochondria was corrected by using the appropriate control. The values (normalized to identical mitochondrial protein per tube) are the means ± SEM of 6 observations. Statistical significance was calculated by Student's t test, paired. ▴ p < 0.001 vs. DA in buffer alone. * p < 0.001 vs. DA + mitochondria. φ p < 0.01 vs. DA + mitochondria.
protein thiol groups will also be much less if mitochondria are incubated with DOPAC instead of DA. Our data presented in Table 1 and Fig. 3 precisely confirm this. In agreement with our data, others have also reported very little effect of DOPAC on mitochondrial complex I activity (Przedborski et al., 1995). Our results, however, differ clearly from those reported from another major study in which DA (IC 50 7 mM) and DOPAC (IC50 7.4 mM) have been shown to be equipotent in inhibiting mitochondrial respiration during in vitro incubation (Gluck and Zeevalk, 2004). The discrepancy may have resulted from the fact that the former study (Gluck and Zeevalk, 2004) has used a brief incubation period of 15 min and a very high concentration of DA or DOPAC (5–20 mM) in contrast to our study where a low concentration of DA or DOPAC (400 μM) and an extended incubation of 2 h have been utilized. However, it will be pertinent here to discuss at some length the reasons for such conflicting results of these two studies. It has been suggested that DA, DOPAC, and some other catecholamines cause mitochondrial respiratory chain inhibition by several mechanisms (MAO-dependent as well as MAO-independent) which include toxic actions of ROS derived from MAO catalyzed oxidation of DA, oxidation of catechol ring to quinone derivatives and formation of DA–cysteinyl adducts or DOPAC–cysteinyl adducts with respiratory chain protein components and direct adverse effect of aromatic carboxylate ions on mitochondria (Gluck et al., 2002; Gluck and Zeevalk, 2004). It is presumable that each mechanism will contribute to a varying extent to the damage process based on the nature and the concentration of the particular catecholamine or its derivative added to the incubation system as well as on the duration of such incubation (Gluck et al., 2002; Gluck and Zeevalk, 2004; Khan et al., 2005). The duration of incubation may be of particular importance in determining which of the proposed damage mechanisms will be operative because of the obvious differences in the rates of MAO catalyzed DA oxidation, DA autoxidation, ROS-mediated reactions, quinone mediated reactions and aromatic carboxylate ion mediated actions. Thus, the effects of DA or DOPAC on brain mitochon-
drial respiratory chain function during in vitro incubation are likely to be both concentration dependent and incubation time dependent, and this probably accounts for the difference in the results between the present study and the earlier published report (Gluck and Zeevalk, 2004). However, we have thought that our incubation protocol with micromolar concentration of DA or DOPAC with extended time of incubation will serve as a better model to understand the deleterious action of the latter under in vivo conditions where mitochondria in nigral dopaminergic neurons are continuously exposed to a low concentration of DA and its catabolites. When the results of the present study are compared with those of our earlier published data on DA-mediated inhibition of mitochondrial respiratory chain activities, a somewhat surprising fact is noticeable concerning the protection provided by clorgyline against the latter (Khan et al., 2005). The protection caused by clorgyline, a specific inhibitor of MAO-A, indirectly implicates DA catabolites such as H2O2 and DOPAC in DA mediated brain mitochondrial ETC impairment. However, the involvement of H2O2, OH radicals and Fenton's chemistry has been excluded in the inactivation of mitochondrial ETC activity by DA in our previous study because of the failure of the radical scavengers, metal chelators and catalase to prevent DA mediated mitochondrial damage (Khan et al., 2005). On the other hand, the present study also rules out any significant involvement of DOPAC in ETC impairment when mitochondria are incubated with DA under our experimental conditions (Figs. 1 and 2). This has led us to believe that clorgyline may have other effects on DA catabolism by mitochondria which could account for its protective action on DA-mediated mitochondrial ETC inhibition. The data presented in Tables 2 and 3 imply that, apart from autoxidation, DA can be converted to quinones by some mitochondrial activity which can be inhibited by clorgyline and cyanide. Furthermore, heat inactivation leads to the loss of this apparent enzyme activity in mitochondria (Table 3). The enzyme tyrosinase can convert DA to quinone products and is expressed at very low levels in brain (Greggio et al., 2005). However, DA oxidation in vitro by exogenously added purified tyrosinase cannot be prevented by clorgyline (data not shown). It appears that the mitochondrial activity converting DA to its quinone products as reported by us is different from tyrosinase. Quinone products of DA oxidation comprise of several related compounds such as o-quinone, aminochrome, indolequinone, dihydroxyindole etc. which undergo further reaction to form neuromelanin, and from our limited data it is not possible to identify either the particular reaction catalyzed by this mitochondrial activity or the product formed in this reaction, and a highly specific inhibitor for this activity when available may help us to resolve some of these issues. In this context, it may be relevant to refer to earlier studies where some uncharacterized mitochondrial activity has been shown to convert DA derivative, such as 5-S-cysteinyl dopamine, to its o-quinone product which is further oxidized to 7-(2-aminoethyl)-3,4-dihydro-5-hydroxy-2H-1,4-benzothiazine-3-carboxylic acid (DHBT-1) that inhibits mitochondrial complex I activity irreversibly (Li and Dryhurst, 1997; Li et al., 1998). DHBT-1, in turn, can undergo further mitochondria catalyzed oxidation to an o-quinoneimine intermediate followed by intramolecular rearrangement and decarboxylation to pro-
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duce several mitochondrial toxins (Li and Dryhurst, 1997; Li et al., 1998). It is necessary to explore if the mitochondrial activity mentioned in our study is related to those involved in the metabolism of 5-S-cysteinyl dopamine and DHBT-1. It will be, furthermore, interesting to elucidate the relationship of this enzyme to macrophage migration inhibitory factor (MIF) which is highly expressed in brain and has multiple immune and catalytic functions including the ability to convert quinone oxidation products of DA to dihydroxy indole derivatives (Matsunaga et al., 1999). The importance of this proposed enzyme activity in mitochondria, however, is indicated from the fact that clorgyline which inhibits this enzyme provides partial protection against DA induced mitochondrial injury (Khan et al., 2005). It will be, therefore, interesting to purify and characterize this mitochondrial activity and to understand its role in the cytotoxic actions of DA relevant to the pathogenesis of sporadic Parkinson's disease.
4.
Experimental procedure
Albino rats of Charles-Foster strain maintained as per the guidelines of the Animal Ethical Committee of the Institute were used for this study. Mitochondria were isolated from rat brain by a method based on differential centrifugation and digitonin treatment as published earlier (Berman and Hastings, 1999; Khan et al., 2005). The final mitochondrial pellet was resuspended in isotonic buffer A (145 mM KCl, 50 mM sucrose, 5 mM NaCl, 1 mM EGTA, 1 mM magnesium chloride, 10 mM phosphate buffer, pH 7.4) or in 50 mM phosphate buffer, pH 7.4 depending on subsequent experimental procedures. Furthermore, isolated mitochondria were checked for membrane integrity by assaying citrate synthase activity in the presence of 0.1% Triton-X 100 (Clark et al., 1997). Frozen and thawed samples of mitochondria in 50 mM phosphate buffer, pH 7.4 were incubated for 2 h at 37 °C in the presence or absence of DA (400 μM) or DOPAC (400 μM) with or without GSH (5 mM) in a total volume of 400 μl. At the end of the incubation, the mitochondria were washed with an excess of ice-cold 50 mM phosphate, pH 7.4, collected by centrifugation at 4 °C and resuspended in an appropriate volume of the same buffer. An aliquot of the mitochondrial suspension (10–30 μg of protein) was utilized to measure complex I or complex IV activities. Complex I activity was assayed by using ferricyanide as the electron acceptor (Hatefi, 1978). The assay was carried out at 30 °C in a reaction system containing 0.17 mM NADH, 0.6 mM potassium ferricyanide, 0.1% (v/v) Triton-X 100 in 50 mM phosphate buffer, pH 7.4. The rate of oxidation of NADH was monitored by the decrease in absorbance at 340 nm after the addition of mitochondrial suspension to the sample cuvette (Clark et al., 1997; Khan et al., 2005). The activity of complex IV was assayed following the oxidation of reduced cytochrome c (ferrocytochrome c) at 550 nm in 10 mM phosphate buffer, pH 7.4 at room temperature (Wharton and Tzagoloff, 1967). In the blank cuvette, ferricyanide (1 mM) was added to oxidize ferrocytochrome c and the reaction initiated in the sample cuvette by the addition of mitochondrial suspension. The activity of the enzyme was calculated from the first order rate constant and the concentration of reduced cytochrome c in the sample cuvette as published earlier (Khan et al., 2005).
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For measurements of MTT reduction ability, freshly isolated mitochondria were suspended in isotonic buffer A with DA (400 μM) or DOPAC (400 μM) or other additions and identical incubation and washing protocols were followed as described above. An aliquot of mitochondrial suspension (200 μl) was added to 800 μl isotonic buffer A, pH 7.4 containing 10 mM succinate and MTT (0.5 mg/ml) and incubated at 37 °C for 15 min. The reaction was terminated with 500 μl of lysis buffer (45% dimethylformamide and 10% SDS, pH 4.7). Absorbance was taken after 5 min and the difference in absorbance values at 550 nm and 620 nm was noted (Cohen et al., 1997). For some other experiments, fresh mitochondria were incubated in 50 mM phosphate buffer at pH 7.4 in the presence of DA (400 μM) or DOPAC (400 μM) along with other additions as necessary for 2 h at 37 °C followed by an identical washing and centrifugation protocol described for earlier experiments. The mitochondrial pellet was resuspended in 50 mM phosphate buffer pH 7.4 and used for the measurement of quinoprotein adducts and protein thiols. Quinoprotein adduct formation was measured by spectrophotometric Nitro blue tetrazolium (NBT)-glycinate assay as published earlier (Khan et al., 2001). The protein thiol content was estimated by using Ellman's reagent (Habeeb, 1972). Quinone formation from DA was measured spectrophotometrically at 303 nm after incubating DA (400 μM) in 50 mM phosphate buffer, pH 7.4 at 37 °C with or without co-incubated mitochondria (intact or heat-inactivated) in the presence or absence of clorgyline (2.5–10 μM) or NaCN (1 mM). Appropriate blanks were kept, and turbidity due to mitochondria was suitably corrected for this assay. Quinones derived from DA oxidation constitute an array of compounds that undergo further oxidation and polymerization to produce black melanin pigments during the period of incubation. Under our assay condition, the pigments showed a general increase in absorbance compared to unoxidized DA during wavelength scanning in the region 280–560 nm with no clear absorption peak, and we chose to monitor the formation of quinone products formation by measuring the increase in absorbance at 303 nm where unoxidized DA had very little absorbance. Protein was estimated after solubilizing the membranes in 1% SDS by the method of Lowry et al. (1951). Some of the experiments were repeated with mitochondria incubated in 25 mM HEPES buffer, pH 7.4 instead of 50 mM phosphate buffer, pH 7.4 in order to verify if the buffer composition of the incubation mixture could modify the effects of DA or DOPAC on mitochondrial respiration.
Acknowledgments This work was supported by a research grant from Life Sciences Research Board, Defence Research and Development Organisation, Government of India (No. LSRB-93/EPB/2006). REFERENCES
Beal, M.F., 2003. Mitochondrial oxidative damage and inflammation in Parkinson's disease. Ann. N. Y. Acad. Sci. 991, 120–131. Berman, S.B., Hastings, T.G., 1999. Dopamine oxidation alters mitochondrial respiration and induces permeability transition
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in brain mitochondria: implications for Parkinson's disease. J. Neurochem. 73, 1127–1137. Clark, J.B., Bates, T.E., Boakye, P., Kuimov, A., Land, J.M., 1997. Investigation of mitochondrial defects in brain and skeletal muscle. In: Turner, A.J., Bachelard, H.S. (Eds.), Neurochemistry: A Practical Approach. Oxford Univ. Press Inc, New York, pp. 151–174. Cohen, G., Farooqui, R., Kesler, N., 1997. Parkinson disease: a new link between monoamine oxidase and mitochondrial electron flow. Proc. Natl. Acad. Sci. U. S. A. 94, 4890–4894. Dexter, D.T., Sian, J., Rose, S., Hindmarsh, J.G., Mann, V.M., Cooper, J.M., Wells, F.R., Daniel, S.E., Lees, A.J., Schapira, A.H.V., Jenner, P., Marsden, C.D., 1994. Indices of oxidative stress and mitochondrial function in individuals with incidental Lewy body disease. Ann. Neurol. 35, 38–44. Gluck, M., Ehrhart, J., Jayatilleke, E., Zeevalk, G.D., 2002. Inhibition of brain mitochondrial respiration by dopamine: involvement of H2O2 and hydroxyl radicals but not glutathione-protein-mixed disulfides. J. Neurochem. 82, 66–74. Gluck, M.R., Zeevalk, G.D., 2004. Inhibition of brain mitochondrial respiration by dopamine and its metabolites: implications for Parkinson's disease and catecholamine-associated diseases. J. Neurochem. 91, 788–795. Greenamyre, J.T., MacKenzie, G., Peng, T., Stephans, S.E., 1999. Mitochondrial dysfunction in Parkinson's disease. Biochem. Soc. Symp. 66, 85–97. Greggio, E., Bergantino, E., Carter, D., Ahmad, R., Costin, G.E., Hearing, V.J., Clarimon, J., Singleton, A., Erola, J., Hellstrom, O., Tienari, P.J., Miller, D.W., Beilina, A., Bubacco, L., Cookson, M.R., 2005. Tyrosinase exacerbates dopamine toxicity but is not genetically associated with Parkinson's disease. J. Neurochem. 93, 246–256. Habeeb, A.F.S.A., 1972. Reaction of protein sulfhydryl groups with Ellman's reagent. Methods Enzymol. 25, 457–464. Hastings, T.G., Lewis, D.A., Zigmond, M.J., 1996. Role of oxidation in the neurotoxic effects of intrastriatal dopamine injections. Proc. Natl. Acad. Sci. U. S. A. 93, 1956–1961. Hatefi, Y., 1978. Preparation and properties of NADH:ubiquinone oxidoreductase (complex I) E.C. 1,6,5,3. Methods Enzymol. 53, 11–15. Jenner, P., Olanow, C.W., 2006. The pathogenesis of cell death in Parkinson's disease. Neurology 66, S24–S36. Khan, F.H., Saha, M., Chakrabarti, S., 2001. Dopamine induced
protein damage in mitochondrial–synaptosomal fraction of rat brain. Brain Res. 895, 245–249. Khan, F.H., Sen, T., Maiti, A.K., Jana, S., Chatterjee, U., Chakrabarti, S., 2005. Inhibition of rat brain mitochondrial electron transport chain activity by dopamine oxidation products during extended in vitro incubation: implications for Parkinson's disease. Biochim. Biophys. Acta 1741, 65–74. Li, H., Dryhurst, G., 1997. Irreversible inhibition of mitochondrial complex I by 7-(2-aminoethyl)-3, 4-dihydro-5-hydroxy-2H-1,4-benzothiazine-3-carboxylic acid (DHBT-1): a putative nigral endotoxin of relevance to Parkinson's disease. J. Neurochem. 69, 1530–1541. Li, H., Shen, X.M., Dryhurst, G., 1998. Brain mitochondria catalyze the oxidation of 7-(2-aminoethyl)-3, 4-dihydro-5-hydroxy-2H-1,4-benzothiazine-3-carboxylic acid (DHBT-1) to intermediates that irreversibly inhibit complex I and scavenge glutathione: potential relevance to the pathogenesis of Parkinson's disease. J. Neurochem. 71, 2049–2062. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with Folin-Phenol reagent. J. Biol. Chem. 193, 265–275. Matsunaga, J., Sinha, D., Pannell, L., Santis, C., Solano, F., Wistow, G.J., Hearing, V.J., 1999. Enzyme activity of macrophage migration inhibitory factor toward oxidized catecholamines. J. Biol. Chem. 274, 3268–3271. Moore, D.J., West, A.B., Dawson, V.L., Dawson, T.M., 2005. Molecular pathophysiology of Parkinson's disease. Annu. Rev. Neurosci. 28, 57–87. Przedborski, S., Jacobson-Lewis, V., Fahn, S., 1995. Antiparkinsonian therapies and brain mitochondrial complex-I activity. Mov. Disord. 10, 312–317. Riederer, P., Sofie, E., Rausch, W.D., Schmidt, B., Reynolds, G.P., Jellinger, K., Youdim, M.B.H., 1989. Transition metals, ferritin, glutathione and ascorbic acid in parkinsonian brains. J. Neurochem. 52, 515–520. Spencer, J.P.E., Jenner, P., Daniel, S.E., Lees, A.J., Marsden, D.C., Halliwell, B., 1998. Conjugates of catecholamines with cysteine and GSH in Parkinson's disease: possible mechanism of formation involving reactive oxygen species. J. Neurochem. 71, 2112–2122. Wharton, D.C., Tzagoloff, A., 1967. Cytochrome oxidase from beef heart mitochondria. Methods Enzymol. 10, 245–250.
a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
Dopamine but not 3,4-dihydroxy phenylacetic acid (DOPAC) inhibits brain respiratory chain activity by autoxidation and mitochondria catalyzed oxidation to quinone products: Implications in Parkinson's disease Sirsendu Jana a , Arpan Kumar Maiti a , Maria Bindu Bagh a , Kalpita Banerjee a , Amitabha Das a , Arun Roy b , Sasanka Chakrabarti a,⁎ a
Department of Biochemistry, Institute of Post Graduate Medical Education & Research (Dr. B.C. Roy Post Graduate Institute of Basic Medical Sciences) 244B, Acharya J.C. Bose Road, Kolkata-700020, India b Department of Microbiology, Vijaygarh Jyotish Ray College (Affiliated to the University of Calcutta) 8/2 Bejoygarh, Jadavpur, Kolkata-700032, India
A R T I C LE I N FO
AB S T R A C T
Article history:
This study reveals that, in contrast to dopamine (DA), 3,4 dihydroxyphenylacetic acid
Accepted 30 September 2006
(DOPAC) during in vitro incubation up to 2 h causes only marginal inhibition of rat brain
Available online 8 February 2007
mitochondrial respiratory chain activity, a minimal loss of protein free thiols and very little quinoprotein adduct formation. The damaging effects of DA on brain mitochondria are,
Keywords:
however, conspicuous and apparently mediated by quinone oxidation products generated by
Dopamine
autoxidation of DA as well as catalyzed by a mitochondrial activity, inhibitable by clorgyline
Quinone
(2.5–10 μM) and cyanide (1 mM).
Parkinson's disease
© 2006 Elsevier B.V. All rights reserved.
Mitochondria Clorgyline
1.
Introduction
Mitochondrial dysfunction is considered to be a crucial step in the network of cellular damage pathways that culminate in the death of nigral dopaminergic neurons in sporadic form of Parkinson's disease (PD), and in a large measure such mitochondrial dysfunctions are caused by the toxic actions of dopamine (DA) oxidation products within the neurons (Przedborski et al., 1995; Berman and Hastings, 1999; Greenamyre et al., 1999; Beal, 2003; Moore et al., 2005; Jenner and Olanow, 2006). Studies with in vitro systems and animal models have demonstrated the deleterious effects
of dopamine oxidation products on mitochondrial respiration, transmembrane potential and permeability transition pores (Berman and Hastings, 1999; Gluck et al., 2002; Khan et al., 2005). Autoxidation or monoamine oxidase (MAO) catalyzed oxidation of DA gives rise to H 2O 2 , toxic oxyradicals, an array of reactive quinone products and 3,4 dihydroxyphenyl acetic acid (DOPAC) which have all been implicated in causing mitochondrial injury (Berman and Hastings, 1999; Gluck et al., 2002; Gluck and Zeevalk, 2004; Khan et al., 2005). Thus, mitochondrial dysfunction and the degeneration of dopaminergic neurons in PD may be related to intrinsic mitochondrial damage by intraneuronal
⁎ Corresponding author. E-mail address: [email protected] (S. Chakrabarti). 0006-8993/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2006.09.100
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oxidative catabolism of DA (Cohen et al., 1997; Gluck et al., 2002). In substantia nigra of PD subjects, an elevated level of iron and a decrease in GSH and ferritin content have been reported which may further aggravate oxidative damage by DA (Riederer et al., 1989; Dexter et al., 1994; Li et al., 1998). In our earlier study, we have demonstrated that DA can inhibit rat brain mitochondrial electron transport chain (ETC) activity at several levels during extended incubation and the damage apparently is mediated by toxic quinones and not by H2O2 or reactive oxyradicals (Khan et al., 2005). Furthermore, clorgyline, an inhibitor of MAO-A, which can block the conversion of DA to DOPAC, provides partial protection against DA induced impairment of brain mitochondrial ETC activity under our conditions of incubation (Khan et al., 2005). While probing further the actions of DA and DOPAC on mitochondria and the cause of clorgyline protection on DA mediated impairment of mitochondrial respiratory functions in this study, we have noticed an interesting mitochondrial activity related to DA catabolism to quinone products. A body of experimental and postmortem evidence indicates that toxic quinones are involved in the pathogenesis of sporadic PD. The administration of neurotoxic doses of DA in striatum leads to increased formation of protein bound cysteinyl–DA adducts, and the postmortem samples of substantia nigra of PD patients contain an elevated levels of cysteinyl adducts of DA or DOPA formed by the reactions of quinone derivatives of DA or DOPA with cysteine or glutathione (Hastings et al., 1996; Spencer et al., 1998). Cysteinyl adducts of DA can undergo mitochondria catalyzed oxidation to produce several toxins of respiratory chain (Li and Dryhurst, 1997; Li et al., 1998). The present study on mitochondrial catabolism of DA and the effect of DA or its catabolites on electron transport chain (ETC) activity may, therefore, have potential implications in the context of the pathogenesis of PD.
2.
Results
The data presented in Fig. 1 show that, in the presence of 400 μM of DA, complex I and complex IV activities of rat brain mitochondria were decreased by nearly 32% and 57%, respectively, whereas DOPAC (400 μM) caused only a marginal, although statistically significant, inhibition in both the cases. When incubation of mitochondria was carried out in 25 mM HEPES buffer, pH 7.4, instead of 50 mM phosphate buffer, DA again produced very significant inhibition of complex I and complex IV activities while DOPAC failed to produce any effect on the latter (data not shown). When mitochondria were pre-incubated with DA (400 μM) or DOPAC (400 μM) for 2 h, subsequent reduction of MTT by mitochondria in the presence of succinate as the respiratory substrate was inhibited by more than 78% by DA and only 13% by DOPAC (Fig. 2). Reduced glutathione (5 mM) gave almost complete protection against DA induced inhibition of mitochondrial MTT reduction ability and complex I and complex IV activities (Figs. 1 and 2). In the present study, a high concentration of reduced glutathione (5 mM) was added to the incubation mixture to compensate for any loss
Fig. 1 – Effects of DA and DOPAC on rat brain mitochondrial complex I and complex IV activities. Rat brain mitochondria were incubated without or with DA (400 μM) or with DOPAC (400 μM) in the presence or absence of GSH (5 mM) for 2 h at 37 °C. Complex I and complex IV activities were measured in mitochondrial suspensions at the end of the incubation as described in the text. The values are means ± SEM of 12 observations for complex I (A) and 10 observations for complex IV (B). GSH alone did not have any effect on mitochondrial complex I and complex IV activities (data not shown). Statistical significance was calculated by Student's t test, paired. Ψp < 0.001 vs. control, ♦p < 0.01 vs. control, ♦♦p < 0.05 vs. control, *p < 0.001 vs. mitochondria + DA.
by oxidation during prolonged incubation period of 2 h. We verified that only 10% of the reduced glutathione was lost by oxidation during incubation for 2 h under our experimental conditions and furthermore the pH of the incubation mixture was not altered noticeably in any of the tubes at the end of the incubation. A significant formation of quinoprotein adducts (protein–cysteinyl catechols) in mitochondrial proteins was observed in the presence of DA but not DOPAC and such DA induced quinoprotein formation, in turn, was inhibited by GSH (Table 1). Results presented in Fig. 3 show that DA at a concentration of 400 μM caused a significant (∼ 32%) reduction in mitochondrial protein thiols during in vitro incubation, whereas DOPAC at a similar concentration caused only a marginal loss of protein thiols. During in vitro incubation, DA produced quinone products as a result of autoxidation but, in the presence of co-incubated mitochondria, quinone production was enhanced significantly in pro-
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Fig. 2 – Effects of DA and DOPAC on rat brain mitochondrial MTT reduction. Intact mitochondria from rat brain were incubated in isotonic buffer A either alone (control) or with DA (400 μM) or DOPAC (400 μM) in the presence or absence of GSH (5 mM) for 2 h at 37 °C followed by the measurement of mitochondrial MTT reduction in the presence of 10 mM succinate. GSH alone was without effect on mitochondrial MTT reduction (data not shown). Each value expressed as percentage of control represents mean ± SEM of 5 observations. Statistical significance was calculated by Student's t test, paired. Ψp < 0.001 vs. control, ♦p < 0.01 vs. control, *p < 0.001 vs. mitochondria + DA.
portion to increasing mitochondrial protein added (Table 2). Moreover, the mitochondria catalyzed quinone production from incubated DA was remarkably inhibited by clorgyline (2.5–10 μM) and NaCN (1 mM) present in the incubation medium (Table 3). Heat-inactivated mitochondria, on the other hand, failed to increase the rate of quinone production from incubated DA (Table 3). We further observed that the mitochondria catalyzed conversion of DA to quinone products in 25 mM HEPES buffer, pH 7.4, was somewhat less than that seen in 50 mM phosphate buffer (results not shown). However, a proportionate increase in quinone conversion with increasing amount of mitochondrial protein added as well as inhibition of mitochondrial activity by clorgyline was consistently observed in 25 mM HEPES buffer, pH 7.4, as
Table 1 – Production of quinoprotein adducts during incubation of rat brain mitochondria with DA or DOPAC Incubation mixture Mitochondria in buffer (control) Mitochondria + DA Mitochondria + DA + GSH Mitochondria + DOPAC
Fig. 3 – Loss of protein thiols during incubation of mitochondria with DA or DOPAC. Rat brain mitochondria were incubated without or with DA (400 μM) or DOPAC (400 μM) for 2 h at 37 °C followed by the measurement of protein thiols using Ellman's reagent as detailed in the text. Results are the means ± SEM of 6 observations. Statistical significance was calculated by Student's t test, paired. *p < 0.001 vs. control, **p < 0.01 vs. control.
was seen in the case of 50 mM phosphate buffer (data not produced).
3.
Discussion
Our results clearly indicate that in equimolar concentration DOPAC causes a much lesser degree of inhibition of mitochondrial ETC activity than DA as assessed by the measurement of complex I and complex IV activities and MTT reduction ability of rat brain mitochondria (Figs. 1 and 2). Such data, therefore, imply that under our conditions of incubation DOPAC derived from MAO catalyzed DA oxidation can contribute only marginally to the inhibition of mitochondrial respiratory functions when mitochondria are co-incubated with DA. The primary damaging species in this process are the quinone oxidation products of DA and not ROS as has been demonstrated in our earlier published study (Khan et al., 2005). Since the quinones presumably exert their damaging functions through the formation of covalent adducts with the protein nucleophiles (e.g. the free thiols), it is anticipated that the formation of quinoprotein adducts and the inactivation of
Absorbance at 520 nm 0.084 ± 0.003 0.364 ± 0.004* 0.096 ± 0.003▴ 0.101 ± 0.004
Rat brain mitochondria were incubated without or with DA (400 μM) in the presence or absence of GSH (5 mM) or DOPAC (400 μM) for 2 h at 37 °C followed by the measurement of quinoprotein adducts by NBT-glycinate assay as described in the text. Absorbance at 530 nm was taken as the measure of protein bound quinones. The values (normalized to identical mitochondrial protein per tube) are the means ± SEM of 4 observations. Statistical significance was calculated by Student's t test, paired. * p < 0.001 vs. control. ▴ p < 0.001 vs. mitochondria + DA.
Table 2 – Production of quinones from DA in the presence of brain mitochondria Incubation mixture DA in buffer alone DA + mitochondria (50 μg protein) DA + mitochondria (100 μg protein) DA + mitochondria (200 μg protein)
Absorbance at 303 nm 0.080 ± 0.006 0.157 ± 0.027 0.240 ± 0.050 0.359 ± 0.023
Dopamine (400 μM) was incubated alone or with rat brain mitochondria (50–200 μg protein) in 50 mM phosphate buffer, pH 7.4 for 2 h at 37 °C followed by the measurement of quinones at 303 nm. Turbidity due to mitochondria was corrected by using the appropriate control. Values are the means ± SEM of 5 observations.
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Table 3 – Effects of clorgyline and cyanide on quinone formation during incubation of rat brain mitochondria with DA Incubation mixture DA in buffer alone DA + mitochondria DA + mitochondria + clor (2.5 μM) DA + mitochondria + clor (5 μM) DA + mitochondria + clor (10 μM) DA + mitochondria + NaCN (1 mM) DA + mitochondria (heat treated)
Absorbance at 303 nm 0.052 ± 0.005 0.283 ± 0.023▴ 0.167 ± 0.028φ 0.132 ± 0.026* 0.091 ± 0.022* 0.059 ± 0.015* 0.058 ± 0.008*
Dopamine (400 μM) was incubated alone or with rat brain mitochondria (intact or heat treated) in the presence or absence of clorgyline (Clor) or NaCN in 50 mM phosphate buffer, pH 7.4 for 2 h at 37 °C followed by the measurement of quinones at 303 nm. Turbidity due to mitochondria was corrected by using the appropriate control. The values (normalized to identical mitochondrial protein per tube) are the means ± SEM of 6 observations. Statistical significance was calculated by Student's t test, paired. ▴ p < 0.001 vs. DA in buffer alone. * p < 0.001 vs. DA + mitochondria. φ p < 0.01 vs. DA + mitochondria.
protein thiol groups will also be much less if mitochondria are incubated with DOPAC instead of DA. Our data presented in Table 1 and Fig. 3 precisely confirm this. In agreement with our data, others have also reported very little effect of DOPAC on mitochondrial complex I activity (Przedborski et al., 1995). Our results, however, differ clearly from those reported from another major study in which DA (IC 50 7 mM) and DOPAC (IC50 7.4 mM) have been shown to be equipotent in inhibiting mitochondrial respiration during in vitro incubation (Gluck and Zeevalk, 2004). The discrepancy may have resulted from the fact that the former study (Gluck and Zeevalk, 2004) has used a brief incubation period of 15 min and a very high concentration of DA or DOPAC (5–20 mM) in contrast to our study where a low concentration of DA or DOPAC (400 μM) and an extended incubation of 2 h have been utilized. However, it will be pertinent here to discuss at some length the reasons for such conflicting results of these two studies. It has been suggested that DA, DOPAC, and some other catecholamines cause mitochondrial respiratory chain inhibition by several mechanisms (MAO-dependent as well as MAO-independent) which include toxic actions of ROS derived from MAO catalyzed oxidation of DA, oxidation of catechol ring to quinone derivatives and formation of DA–cysteinyl adducts or DOPAC–cysteinyl adducts with respiratory chain protein components and direct adverse effect of aromatic carboxylate ions on mitochondria (Gluck et al., 2002; Gluck and Zeevalk, 2004). It is presumable that each mechanism will contribute to a varying extent to the damage process based on the nature and the concentration of the particular catecholamine or its derivative added to the incubation system as well as on the duration of such incubation (Gluck et al., 2002; Gluck and Zeevalk, 2004; Khan et al., 2005). The duration of incubation may be of particular importance in determining which of the proposed damage mechanisms will be operative because of the obvious differences in the rates of MAO catalyzed DA oxidation, DA autoxidation, ROS-mediated reactions, quinone mediated reactions and aromatic carboxylate ion mediated actions. Thus, the effects of DA or DOPAC on brain mitochon-
drial respiratory chain function during in vitro incubation are likely to be both concentration dependent and incubation time dependent, and this probably accounts for the difference in the results between the present study and the earlier published report (Gluck and Zeevalk, 2004). However, we have thought that our incubation protocol with micromolar concentration of DA or DOPAC with extended time of incubation will serve as a better model to understand the deleterious action of the latter under in vivo conditions where mitochondria in nigral dopaminergic neurons are continuously exposed to a low concentration of DA and its catabolites. When the results of the present study are compared with those of our earlier published data on DA-mediated inhibition of mitochondrial respiratory chain activities, a somewhat surprising fact is noticeable concerning the protection provided by clorgyline against the latter (Khan et al., 2005). The protection caused by clorgyline, a specific inhibitor of MAO-A, indirectly implicates DA catabolites such as H2O2 and DOPAC in DA mediated brain mitochondrial ETC impairment. However, the involvement of H2O2, OH radicals and Fenton's chemistry has been excluded in the inactivation of mitochondrial ETC activity by DA in our previous study because of the failure of the radical scavengers, metal chelators and catalase to prevent DA mediated mitochondrial damage (Khan et al., 2005). On the other hand, the present study also rules out any significant involvement of DOPAC in ETC impairment when mitochondria are incubated with DA under our experimental conditions (Figs. 1 and 2). This has led us to believe that clorgyline may have other effects on DA catabolism by mitochondria which could account for its protective action on DA-mediated mitochondrial ETC inhibition. The data presented in Tables 2 and 3 imply that, apart from autoxidation, DA can be converted to quinones by some mitochondrial activity which can be inhibited by clorgyline and cyanide. Furthermore, heat inactivation leads to the loss of this apparent enzyme activity in mitochondria (Table 3). The enzyme tyrosinase can convert DA to quinone products and is expressed at very low levels in brain (Greggio et al., 2005). However, DA oxidation in vitro by exogenously added purified tyrosinase cannot be prevented by clorgyline (data not shown). It appears that the mitochondrial activity converting DA to its quinone products as reported by us is different from tyrosinase. Quinone products of DA oxidation comprise of several related compounds such as o-quinone, aminochrome, indolequinone, dihydroxyindole etc. which undergo further reaction to form neuromelanin, and from our limited data it is not possible to identify either the particular reaction catalyzed by this mitochondrial activity or the product formed in this reaction, and a highly specific inhibitor for this activity when available may help us to resolve some of these issues. In this context, it may be relevant to refer to earlier studies where some uncharacterized mitochondrial activity has been shown to convert DA derivative, such as 5-S-cysteinyl dopamine, to its o-quinone product which is further oxidized to 7-(2-aminoethyl)-3,4-dihydro-5-hydroxy-2H-1,4-benzothiazine-3-carboxylic acid (DHBT-1) that inhibits mitochondrial complex I activity irreversibly (Li and Dryhurst, 1997; Li et al., 1998). DHBT-1, in turn, can undergo further mitochondria catalyzed oxidation to an o-quinoneimine intermediate followed by intramolecular rearrangement and decarboxylation to pro-
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duce several mitochondrial toxins (Li and Dryhurst, 1997; Li et al., 1998). It is necessary to explore if the mitochondrial activity mentioned in our study is related to those involved in the metabolism of 5-S-cysteinyl dopamine and DHBT-1. It will be, furthermore, interesting to elucidate the relationship of this enzyme to macrophage migration inhibitory factor (MIF) which is highly expressed in brain and has multiple immune and catalytic functions including the ability to convert quinone oxidation products of DA to dihydroxy indole derivatives (Matsunaga et al., 1999). The importance of this proposed enzyme activity in mitochondria, however, is indicated from the fact that clorgyline which inhibits this enzyme provides partial protection against DA induced mitochondrial injury (Khan et al., 2005). It will be, therefore, interesting to purify and characterize this mitochondrial activity and to understand its role in the cytotoxic actions of DA relevant to the pathogenesis of sporadic Parkinson's disease.
4.
Experimental procedure
Albino rats of Charles-Foster strain maintained as per the guidelines of the Animal Ethical Committee of the Institute were used for this study. Mitochondria were isolated from rat brain by a method based on differential centrifugation and digitonin treatment as published earlier (Berman and Hastings, 1999; Khan et al., 2005). The final mitochondrial pellet was resuspended in isotonic buffer A (145 mM KCl, 50 mM sucrose, 5 mM NaCl, 1 mM EGTA, 1 mM magnesium chloride, 10 mM phosphate buffer, pH 7.4) or in 50 mM phosphate buffer, pH 7.4 depending on subsequent experimental procedures. Furthermore, isolated mitochondria were checked for membrane integrity by assaying citrate synthase activity in the presence of 0.1% Triton-X 100 (Clark et al., 1997). Frozen and thawed samples of mitochondria in 50 mM phosphate buffer, pH 7.4 were incubated for 2 h at 37 °C in the presence or absence of DA (400 μM) or DOPAC (400 μM) with or without GSH (5 mM) in a total volume of 400 μl. At the end of the incubation, the mitochondria were washed with an excess of ice-cold 50 mM phosphate, pH 7.4, collected by centrifugation at 4 °C and resuspended in an appropriate volume of the same buffer. An aliquot of the mitochondrial suspension (10–30 μg of protein) was utilized to measure complex I or complex IV activities. Complex I activity was assayed by using ferricyanide as the electron acceptor (Hatefi, 1978). The assay was carried out at 30 °C in a reaction system containing 0.17 mM NADH, 0.6 mM potassium ferricyanide, 0.1% (v/v) Triton-X 100 in 50 mM phosphate buffer, pH 7.4. The rate of oxidation of NADH was monitored by the decrease in absorbance at 340 nm after the addition of mitochondrial suspension to the sample cuvette (Clark et al., 1997; Khan et al., 2005). The activity of complex IV was assayed following the oxidation of reduced cytochrome c (ferrocytochrome c) at 550 nm in 10 mM phosphate buffer, pH 7.4 at room temperature (Wharton and Tzagoloff, 1967). In the blank cuvette, ferricyanide (1 mM) was added to oxidize ferrocytochrome c and the reaction initiated in the sample cuvette by the addition of mitochondrial suspension. The activity of the enzyme was calculated from the first order rate constant and the concentration of reduced cytochrome c in the sample cuvette as published earlier (Khan et al., 2005).
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For measurements of MTT reduction ability, freshly isolated mitochondria were suspended in isotonic buffer A with DA (400 μM) or DOPAC (400 μM) or other additions and identical incubation and washing protocols were followed as described above. An aliquot of mitochondrial suspension (200 μl) was added to 800 μl isotonic buffer A, pH 7.4 containing 10 mM succinate and MTT (0.5 mg/ml) and incubated at 37 °C for 15 min. The reaction was terminated with 500 μl of lysis buffer (45% dimethylformamide and 10% SDS, pH 4.7). Absorbance was taken after 5 min and the difference in absorbance values at 550 nm and 620 nm was noted (Cohen et al., 1997). For some other experiments, fresh mitochondria were incubated in 50 mM phosphate buffer at pH 7.4 in the presence of DA (400 μM) or DOPAC (400 μM) along with other additions as necessary for 2 h at 37 °C followed by an identical washing and centrifugation protocol described for earlier experiments. The mitochondrial pellet was resuspended in 50 mM phosphate buffer pH 7.4 and used for the measurement of quinoprotein adducts and protein thiols. Quinoprotein adduct formation was measured by spectrophotometric Nitro blue tetrazolium (NBT)-glycinate assay as published earlier (Khan et al., 2001). The protein thiol content was estimated by using Ellman's reagent (Habeeb, 1972). Quinone formation from DA was measured spectrophotometrically at 303 nm after incubating DA (400 μM) in 50 mM phosphate buffer, pH 7.4 at 37 °C with or without co-incubated mitochondria (intact or heat-inactivated) in the presence or absence of clorgyline (2.5–10 μM) or NaCN (1 mM). Appropriate blanks were kept, and turbidity due to mitochondria was suitably corrected for this assay. Quinones derived from DA oxidation constitute an array of compounds that undergo further oxidation and polymerization to produce black melanin pigments during the period of incubation. Under our assay condition, the pigments showed a general increase in absorbance compared to unoxidized DA during wavelength scanning in the region 280–560 nm with no clear absorption peak, and we chose to monitor the formation of quinone products formation by measuring the increase in absorbance at 303 nm where unoxidized DA had very little absorbance. Protein was estimated after solubilizing the membranes in 1% SDS by the method of Lowry et al. (1951). Some of the experiments were repeated with mitochondria incubated in 25 mM HEPES buffer, pH 7.4 instead of 50 mM phosphate buffer, pH 7.4 in order to verify if the buffer composition of the incubation mixture could modify the effects of DA or DOPAC on mitochondrial respiration.
Acknowledgments This work was supported by a research grant from Life Sciences Research Board, Defence Research and Development Organisation, Government of India (No. LSRB-93/EPB/2006). REFERENCES
Beal, M.F., 2003. Mitochondrial oxidative damage and inflammation in Parkinson's disease. Ann. N. Y. Acad. Sci. 991, 120–131. Berman, S.B., Hastings, T.G., 1999. Dopamine oxidation alters mitochondrial respiration and induces permeability transition
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in brain mitochondria: implications for Parkinson's disease. J. Neurochem. 73, 1127–1137. Clark, J.B., Bates, T.E., Boakye, P., Kuimov, A., Land, J.M., 1997. Investigation of mitochondrial defects in brain and skeletal muscle. In: Turner, A.J., Bachelard, H.S. (Eds.), Neurochemistry: A Practical Approach. Oxford Univ. Press Inc, New York, pp. 151–174. Cohen, G., Farooqui, R., Kesler, N., 1997. Parkinson disease: a new link between monoamine oxidase and mitochondrial electron flow. Proc. Natl. Acad. Sci. U. S. A. 94, 4890–4894. Dexter, D.T., Sian, J., Rose, S., Hindmarsh, J.G., Mann, V.M., Cooper, J.M., Wells, F.R., Daniel, S.E., Lees, A.J., Schapira, A.H.V., Jenner, P., Marsden, C.D., 1994. Indices of oxidative stress and mitochondrial function in individuals with incidental Lewy body disease. Ann. Neurol. 35, 38–44. Gluck, M., Ehrhart, J., Jayatilleke, E., Zeevalk, G.D., 2002. Inhibition of brain mitochondrial respiration by dopamine: involvement of H2O2 and hydroxyl radicals but not glutathione-protein-mixed disulfides. J. Neurochem. 82, 66–74. Gluck, M.R., Zeevalk, G.D., 2004. Inhibition of brain mitochondrial respiration by dopamine and its metabolites: implications for Parkinson's disease and catecholamine-associated diseases. J. Neurochem. 91, 788–795. Greenamyre, J.T., MacKenzie, G., Peng, T., Stephans, S.E., 1999. Mitochondrial dysfunction in Parkinson's disease. Biochem. Soc. Symp. 66, 85–97. Greggio, E., Bergantino, E., Carter, D., Ahmad, R., Costin, G.E., Hearing, V.J., Clarimon, J., Singleton, A., Erola, J., Hellstrom, O., Tienari, P.J., Miller, D.W., Beilina, A., Bubacco, L., Cookson, M.R., 2005. Tyrosinase exacerbates dopamine toxicity but is not genetically associated with Parkinson's disease. J. Neurochem. 93, 246–256. Habeeb, A.F.S.A., 1972. Reaction of protein sulfhydryl groups with Ellman's reagent. Methods Enzymol. 25, 457–464. Hastings, T.G., Lewis, D.A., Zigmond, M.J., 1996. Role of oxidation in the neurotoxic effects of intrastriatal dopamine injections. Proc. Natl. Acad. Sci. U. S. A. 93, 1956–1961. Hatefi, Y., 1978. Preparation and properties of NADH:ubiquinone oxidoreductase (complex I) E.C. 1,6,5,3. Methods Enzymol. 53, 11–15. Jenner, P., Olanow, C.W., 2006. The pathogenesis of cell death in Parkinson's disease. Neurology 66, S24–S36. Khan, F.H., Saha, M., Chakrabarti, S., 2001. Dopamine induced
protein damage in mitochondrial–synaptosomal fraction of rat brain. Brain Res. 895, 245–249. Khan, F.H., Sen, T., Maiti, A.K., Jana, S., Chatterjee, U., Chakrabarti, S., 2005. Inhibition of rat brain mitochondrial electron transport chain activity by dopamine oxidation products during extended in vitro incubation: implications for Parkinson's disease. Biochim. Biophys. Acta 1741, 65–74. Li, H., Dryhurst, G., 1997. Irreversible inhibition of mitochondrial complex I by 7-(2-aminoethyl)-3, 4-dihydro-5-hydroxy-2H-1,4-benzothiazine-3-carboxylic acid (DHBT-1): a putative nigral endotoxin of relevance to Parkinson's disease. J. Neurochem. 69, 1530–1541. Li, H., Shen, X.M., Dryhurst, G., 1998. Brain mitochondria catalyze the oxidation of 7-(2-aminoethyl)-3, 4-dihydro-5-hydroxy-2H-1,4-benzothiazine-3-carboxylic acid (DHBT-1) to intermediates that irreversibly inhibit complex I and scavenge glutathione: potential relevance to the pathogenesis of Parkinson's disease. J. Neurochem. 71, 2049–2062. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with Folin-Phenol reagent. J. Biol. Chem. 193, 265–275. Matsunaga, J., Sinha, D., Pannell, L., Santis, C., Solano, F., Wistow, G.J., Hearing, V.J., 1999. Enzyme activity of macrophage migration inhibitory factor toward oxidized catecholamines. J. Biol. Chem. 274, 3268–3271. Moore, D.J., West, A.B., Dawson, V.L., Dawson, T.M., 2005. Molecular pathophysiology of Parkinson's disease. Annu. Rev. Neurosci. 28, 57–87. Przedborski, S., Jacobson-Lewis, V., Fahn, S., 1995. Antiparkinsonian therapies and brain mitochondrial complex-I activity. Mov. Disord. 10, 312–317. Riederer, P., Sofie, E., Rausch, W.D., Schmidt, B., Reynolds, G.P., Jellinger, K., Youdim, M.B.H., 1989. Transition metals, ferritin, glutathione and ascorbic acid in parkinsonian brains. J. Neurochem. 52, 515–520. Spencer, J.P.E., Jenner, P., Daniel, S.E., Lees, A.J., Marsden, D.C., Halliwell, B., 1998. Conjugates of catecholamines with cysteine and GSH in Parkinson's disease: possible mechanism of formation involving reactive oxygen species. J. Neurochem. 71, 2112–2122. Wharton, D.C., Tzagoloff, A., 1967. Cytochrome oxidase from beef heart mitochondria. Methods Enzymol. 10, 245–250.