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Chronic Inhibition of the High-Affinity Dopamine Uptake System Increases Oxidative Damage to Proteins in the Aged Rat Substantia Nigra
Free Radical Biology & Medicine, Vol. 23, No. 1, pp. 1–7, 1997 Copyright q 1997 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/97 $17.00 / .00
PII S0891-5849(96)00405-4
Original Contribution CHRONIC INHIBITION OF THE HIGH-AFFINITY DOPAMINE UPTAKE SYSTEM INCREASES OXIDATIVE DAMAGE TO PROTEINS IN THE AGED RAT SUBSTANTIA NIGRA
Marina Romero-Ramos, Jose A. Rodrı´guez-Go´mez, Jose L. Venero, Josefina Cano, and Alberto Machado Departamento de Bioquı´mica, Bromatologı´a y Toxicologı´a, Facultad de Farmacia, Universidad de Sevilla, Sevilla 41012, Spain (Received 22 April 1996; Revised 1 August 1996; Accepted 1 August 1996)
Abstract—The effect of chronic treatment of aged rats with nomifensine has been studied in the rat nigrostriatal dopaminergic system. The rat substantia nigra suffers an oxidative damage during aging that results in both an increase in carbonyl groups of its total proteins and the oxidative inactivation of tyrosine hydroxylase (TH) enzyme,1 which are partially reversed by chronic treatment with deprenyl. Different mechanisms may account for this effect, including inhibition of the high-affinity dopamine uptake system. We treated aged rats chronically with nomifensine for 2 months and found some significant effects. Nomifensine treatment significantly increased TH enzyme amount in substantia nigra (39.2%), which was accompanied by a significant increase in TH enzyme activity (47.8%). However, these effects were not observed in the terminal field (striatum). As a further step we quantified the oxidative level of proteins by measuring the number of carbonyl groups coupled either to total proteins or specifically to TH enzyme. The proteins of aged rat substantia nigra showed a significant increase of carbonyl groups following nomifensine treatment. The number of carbonyl groups coupled to nigral TH enzyme also increased in the nomifensinetreated animals. However, this increase was lower than that found in the total homogenate proteins. All these results show that the oxidative damage produced during aging in tyrosine hydroxylase enzyme and total proteins is not reduced by nomifensine treatment. On the contrary, the nomifensine treatment increased the oxidative damage to proteins. These results suggest the capability of deprenyl to induce TH enzyme could be due to inhibition of the high-affinity dopamine uptake system, but its ability to protect against oxidative damage is not produced by this mechanism. q 1997 Elsevier Science Inc. Keywords—Aging, Nomifensine, Substantia nigra, Oxidative stress, Tyrosine hydroxylase, Free radicals
ment of aged rats with deprenyl for 2 months produced a significant protection against the agedinduced oxidative damage in substantia nigra.1 Deprenyl has been reported as a safe therapeutic agent for a variety of neurodegenerative diseases and possibly for aging. It has several marked effects that facilitate the activity of dopaminergic neurons in the nigrostriatal system selectively.3 At the same time, multisite studies treating Parkinson’s patients with deprenyl have indicated its therapeutic benefits in producing a delay in the onset of disability4 and possibly increasing survival of treated patients.5 It slows down the age-related decline of performance in behavioral tests3 and extends6 life-span considerably.
INTRODUCTION
The rat substantia nigra suffers oxidative damage during aging that results in both an increase in carbonyl groups of its total proteins and the oxidative inactivation of tyrosine hydroxylase enzyme (TH).1 This is in accord with the production of oxidants generated during normal metabolism, which appear to play a significant role in the processes of aging.2 In addition, we have recently shown that chronic treatAddress correspondence to: Alberto Machado, Departamento de Bioquı´mica, Bromotologı´a y Toxicologı´a, Facultad de Farmacia, Universidad de Sevilla, c/ Prof. Garcı´a Gonza´lez s/n, Sevilla, 41012, Spain. 1
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Moreover, deprenyl treatment prevents age-related pigment changes in the substantia nigra.7 Many actions have been described for deprenyl that could be responsible for its therapeutic and/or protective effects against oxidative stress in substantia nigra above described. Deprenyl is a selective inhibitor of monoamine oxidase B (MAO B);8 however, it could also inhibit monoamine oxidase A (MAO A), depending on dose and route of administration.9,10 Deprenyl increases the enzymatic activities of both superoxide dismutase and catalase—enzymes involved in the scavenging of free radicals—in different brain regions including striatum and substantia nigra.11 Deprenyl also has an inhibitory effect upon the dopamine uptake system.3 In order to know which of these actions of deprenyl are involved in its protective actions against the oxidative damage produced by aging in substantia nigra, we have studied the effect of chronic treatment of aged rats with a specific dopamine carrier blocker, nomifensine, on the nigrostriatal dopaminergic system. For that, 22-month old rats were treated with nomifensine (2.5 mg free base kg01) for 2 months. Then, we studied levels of dopamine (DA) and its metabolites and the activity and content of TH, the step-limiting enzyme in the biosynthesis of dopamine and protein marker of dopaminergic neurons. The carbonyl derivatives resulting from oxidative damage to proteins12 were measured in homogenate proteins and TH. All these measurements were carried out in the substantia nigra and striatum of treated and untreated aged animals. Nomifensine seems not to have a protector capacity against the age-induced oxidative damage produced in the nigrostriatal dopaminergic system. Unexpectedly, levels of carbonyl groups of protein homogenates and TH enzyme increased significantly in nomifensine-treated animals. This nomifensine-induced effect on TH oxidation was accompanied by an increased TH enzyme expression in substantia nigra.
MATERIALS AND METHODS
Animals, treatment and dissection Twenty-two-month-old male Wistar rats were used in this study. They were divided into two groups: control animals (n Å 5) and experimental animals (n Å 5). Rats were given either IP injections of physiological saline solution or (0)nomifensine (2.5 mg free base kg01) three times a week for 2 months. The animals were kept under control environmental conditions. Food and tap water were allowed ad lib. The day following the last injection, animals were decapitated between 10:00 and 11:00 h, and the brains removed and placed on an ice-cold plate. The rats did not show brain
tumors because these pathological lesions can affect forebrain cell functions. There were no differences between saline- and nomifensine-treated rats in terms of body weight gain and behavior. The substantia nigra and striatum were dissected according to the atlas of Paxinos and Watson.13 After dissection, the two brain areas were immediately frozen in liquid N2 until assay. (0)Nomifensine was purchased from Research Biochemicals Inc., Natick, MA, and it was dissolved in 0.9% NaCl. Measurement of biogenic amines and their metabolites Analyses were performed by HPLC with electrochemical detection as described previously by Castan˜o et al.14 Samples were homogenized (5 w/v) in 20 mM Tris-HCl buffer by ultrasonic disintegration over ice using a Labsonic 1510. For this measurement an aliquot was further diluted 1:2 with 0.25 N perchloric acid. Samples were centrifuged at 12,000 1 g for 15 min at /47C and the supernatant was then filtered through a 0.2 mm filter and injected onto HPLC. Tyrosine hydroxylase activity assay TH activity was measured according to a modification of a previously published procedure.15 An aliquot coming from the Tris-HCl buffer homogenate was further diluted 1:60 with 30 mM Tris-acetic acid containing 0.1% Triton X-100 and incubated with 2.5 nmol of tyrosine-HCl (containing 0.4 mCi/nmol of L-[ring-3,503 H]tyrosine), 50 nmol of the cofactor 6(R)-L-erythro5,6,7,8-tetrahydrobiopterin, 5000 units of catalase and 5 nM DTT in 100 mM potassium phosphate, pH 6.0. The released [3H]OH was separated by an aqueous slurry of activated charcoal, and the radioactivity was determined by liquid scintillation counting. Quantification of tyrosine hydroxylase Another aliquot coming from the Tris-buffer homogenate was further diluted 1:2 with 80 mM TrisClH, 0.4 mM DTT, 16% sucrose, pH 7,4 plus protease inhibitors: leupeptin, 0.5 mg l01, pepstatin, 0.7 mg l01 and phenylmethyl-sulfonyl fluoride (PMSF), 40 mg l01. Analyses were performed by SDS-PAGE as describe Laemmli.16 After electrophoresis, the gels were Western blotted. Blots were incubated with anti-TH antibody (monoclonal antibody from Boehringer, Germany) followed by antimouse Ig-POD (Sheep antimouse Ig-G peroxidase conjugate from Boehringer, Germany). Specific immunolabeling was visualized using the horseradish peroxidase conjugate substrate kit
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Table 1. Effect of Nomifensine Treatment on the Concentration of Dopamine and Its Metabolites in Rat Striatum and Substantia Nigra Striatum Control DA DOPAC 3-MT HVA
6717.8 1982.4 276.1 559.9
{ { { {
706.5 220.7 42.3 121.9
Substantia Nigra Nomifensine
Control
Nomifensine
{ { { {
300.9 { 46.0 83.3 { 25.2 ND 70.7 { 8.1
494.1 { 137.1 91.3 { 24.0 ND 65.5 { 13.1
7821.7 1473.2 308.4 515.9
516.1 170.4* 46.7 92.3
Results are given in ng/g tissue as means { SD for n (number of animals assayed) Å 5. Statistical significance: Student’s t-test: *p õ .05 as compared with control group.
(Bio-Rad, California), and analyzed by densitometry using a laser computing densitometer (Molecular Dynamics, California). Determination of carbonyl groups of proteins in homogenates Carbonyl groups were determined by measurement of incorporation of tritium [3H] after reduction with sodium boro [3H] hydride as described by Lenz et al.17 Determination of tyrosine hydroxylase enzyme carbonyl groups For this purpose, the homogenates incubated with sodium boro [3H] hydride were subjected to SDSPAGE and Western blotted. Blots were incubated with anti-TH antibody followed by antimouse Ig-biotin (sheep antimouse IgG biotin conjugate from Boehringer, Germany). Blots were then incubated with streptavidin–biotinylated alkaline phosphatase complex (Bio-Rad, California) and visualized by BCIP/ NBT color development solution (Bio-Rad, California). The radioactivity incorporated into TH was determinated by cutting out the protein bands and counting the c.p.m. after the addition of 5 ml of Ready-protein liquid scintillation cocktail (Beckman Instruments, USA). Prior the cutting, band intensity was measured in the membrane by densitometric scanning and results were expressed by c.p.m. per optical density. Measurement of protein content The protein content of homogenates was determined using the procedure of Lowry et al.18 Statistical analyses A Student’s t-test was used. When the p value was õ .05 (*) or õ .01 (**), the difference was considered significant.
RESULTS
Levels of dopamine and their metabolites following chronic treatment with nomifensine In substantia nigra, the nomifensine-treated rats did not differ from control animals in terms of catecholamine levels. Thus, levels of dopamine (DA) and its metabolites remained unchanged following chronic treatment with nomifensine (Table 1). However, there was a significant decrease in the (3,4-dihydroxyphenylacetic acid (DOPAC)/DA ratio following nomifensine treatment (0.29 { 0.06 to 0.15 { 0.02 from untreated and treated rat respectively p õ .05) (Table 1). Striatum showed a rather different neurochemical profile following treatment with nomifensine. While DA levels remained to control levels, the levels of the main acid DA metabolite, the DOPAC, were significantly decreased (025.6% of controls) in the nomifensine-injected animals (Table 1). The striatal DOPAC/ DA ratio decreased statistically in treated animals (0.29 { 0.006 to 0.18 { 0.02 from untreated and treated rat respectively p õ .01). Effect of nomifensine treatment on TH enzyme quantity and activity As seen in Figs. 1 and 2, TH enzyme activity was significantly induced following chronic treatment with nomifensine in substantia nigra (/39.2% of controls) but not in striatum. This change in TH enzyme activity was accompanied by a significant increase in TH enzyme amount in substantia nigra (/47.8% control levels) without any change in striatum (Figs. 1 and 2). Effect of nomifensine treatment on carbonyl groups in homogenates and in TH enzyme in striatum and substantia nigra The carbonyl groups of proteins have been widely used as a measure of proteins modified by oxidation. Animals treated with nomifensine showed a significant increase in the number of carbonyl groups coupled to
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Fig. 1. Tyrosine hydroxylase enzyme activity and content in aged rat striatum and substantia nigra following chronic treatment with nomifensine. TH activity was measured in vitro and TH content was obtained by Western blotting, as described in Materials and Methods. Loading gel conditions were determinated in a previous analyses using increasing amount of protein. The optimum amount were 10 mg and 40 mg for striatum and substantia nigra respectively. All data are mean { SD for n (number of animals assayed) Å 5. Statistical significance: Student’s t-test * p õ .05; ** p õ .01 as compared with controls.
nigral protein homogenates (/83.3% of controls) (Table 2). Considering that nomifensine treatment strongly increased the number of carbonyl groups in nigral protein homogenates, we further wanted to know whether this effect included TH enzyme protein. As seen in Table 2, the number of carbonyl groups specifically coupled to TH enzyme was significantly increased in substantia nigra (/35.8% of controls) of nomifensinetreated rats. Contrary to that found in substantia nigra, chronic treatment with nomifensine was ineffective in altering the number of carbonyl groups coupled to striatal protein homogenates (Table 2).
DISCUSSION
The possibility that deprenyl protects the dopaminergic system against the age-induced increase in oxidized proteins by its inhibitory action on the DA-uptake system1 prompted us to study the effect of a specific antagonist of the high affinity DA transport system, nomifensine. Moreover, drugs blocking the catecholamine transport system have been extensively studied
and many of them are used as therapeutic agents.19 Therefore, the present study analyzed the modifications of DA metabolism produced by chronic treatment with nomifensine. In striatum, these included a significant decrease in DOPAC levels with no significant rise in dopamine content. Similar, but lower effects, were found in substantia nigra. The nomifensine treatment produced a nonsignificant increase in DA along with no change in DOPAC concentrations. However, the DOPAC/DA ratio was significantly decreased in both brain areas examined, thus indicating a decreased intraneuronal DA metabolism in the rat dopaminergic nigrostriatal system.20 Inhibition of the high-affinity DA transport system is supposed to increase extracellular DA levels, and, hence, the extraneuronal DA metabolism. However, while HVA levels remained to control levels, HVA/DA ratio decreased in nomifensine-treated rats. Taken together, our neurochemical analysis suggests a downregulation of the dopaminergic system following chronic inhibition of the high-affinity DA transport system, with decreases in DA turnover, both extraneuronally and intraneuronally as deduced from the DOPAC/DA and HVA/DA ratios. However, other
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Fig. 2. Western blot analysis of TH amount in substantia nigra. Nomifensine treatment-induced TH enzyme amount in substantia nigra (N1 and N2) as compared with control animals (C1 and C2).
possibilities should not be excluded, and thus, nomifensine treatment may induce downregulation of MAO enzyme. Under these conditions, higher DA levels in the synaptic cleft has necessarily not to be associated to higher HVA levels. If we compare the nomifensine effect to that produced by deprenyl, some differences are apparent. Deprenyl treatment1 produced more extensive changes than nomifensine treatment. Deprenyl produced a change in 3-methoxytyramine and HVA, levels probably due to its inhibitory action on MAO enzymes. Deprenyl also produced higher changes in DA and DOPAC concentrations although in the same direction as that produced by nomifensine. The higher effect of deprenyl must be produced by a combination of the effects of inhibition of both MAO enzymes and DA uptake system. The nomifensine treatment produced an increase in TH activity and amount in substantia nigra (39 and 47%, respectively), with no change in striatum. In addition, the number of carbonyl groups coupled to protein homogenates was increased in substantia nigra
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while striatum remained at control levels. This increase was also evident in the number of carbonyl groups specifically coupled to TH enzyme in substantia nigra, although this increase was lower than that found for the total homogenate proteins. These results contrast to those seen after chronic treatment with deprenyl, which protected the substantia nigra against the oxidative damage produced by aging. In contrast, and unexpectedly, chronic treatment of aged rats with nomifensine exacerbated the age-induced oxidative stress that normally occurs in substantia nigra. However, and similarly to deprenyl treatment, the nomifensine treatment induced TH enzyme and activity in substantia nigra. All these results suggest that the effect of nomifensine treatment should be produced by its specific inhibitory action on the high-affinity DA uptake system. Some of the factors that increase TH enzyme protein are those which increase DA release, e.g., reserpine,21 a sympatholytic drug that blocks vesicular storage of catecholamines and thereby leads to transmitter degradation and depletion and cocaine,22 which has been shown to inhibit DA, serotonin and noradrenaline uptake.23–25 The induction of TH enzyme in substantia nigra has also been found with deprenyl treatment;1 it could also be the result of the inhibitory action described for some metabolites of deprenyl (i.e., 1-methamphetamine and 1-amphetamine).3 However, the protective effect of deprenyl against the oxidative damage produced in substantia nigra by aging does not seem to be a result of its inhibitory effect upon the dopamine uptake system.3 It could be produced by its inhibitory action ascribed to MAO enzymes8,26 or/along with the induction of superoxide dismutase and catalase, enzymes involved in the scavenging of free radicals in the nigrostriatal system.11 The increase in oxidized proteins that occurs during aging could be produced either by greater damage induced by oxygen reactive species or alternatively by a decrease in the rates of proteolytic degradation of the oxidized proteins27 with the decrease in protein turnover. Either system could explain the accumulation of Table 2. Measurements of Carbonyl Groups in Aged Rats after Treatment with Nomifensine Striatum Homogenates
Substantia Nigra Homogenates
Control 144.99 { 26.04 138.77 { 36.12 Nomifensine 129.35 { 10.72 254.42 { 60.31*
TH Enzyme 5.07 { 0.27 6.89 { 0.14†
Results are given in cpm/mg protein for homogenates and cpm/ O.D for TH enzyme as means { SD for n (number of animals assayed) Å 5. Statistical significance: Student’s t-test: *p õ .05, †p õ .01 as compared with control group.
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inactivated enzymes in this process. We found a significant increase in the number of carbonyl groups coupled to nigral proteins. If we a consider a diminished DA turnover following chronic treatment with nomifensine as indicated by our neurochemical analysis, the higher oxidation rate of the general nigral protein homogenates cannot be associated to oxidation of DA by monoamine oxidase.28 DA deamination by monoamine oxidase produces hydrogen peroxide, a precursor of highly oxidizing tissue-damaging radicals (hydroxyl radicals).29,30 However, a higher degree of nigral protein oxidation following nomifensine treatment may also derive from DA auto-oxidation products, which are more likely to occur with a diminished DA turnover. It is generally assumed that a decrease in turnover rate produces a greater chemicals modifications. DA readily oxidizes at a physiological pH to form potentially toxic metabolites including hydroxyl radical, superoxide radical, hydrogen peroxide, and quinones.31–33 Strikingly, Hastings and Zigmond (1994)34 have demonstrated that incubation of [3H]DA with neostriatal slices results in the binding of tritium to acid-insoluble proteins.34 Acid hydrolysis of these proteins revealed the presence of cysteinyl-DA and cysteinyl-DOPAC.34 The authors conclude that under conditions in which the ratio of available DA to antioxidant capacity is sufficiently high, reactive metabolites formed from DA autooxidation that include free radicals could result in the binding of catechols to protein and also its oxidation. Aging is known to be associated to a poorer antioxidant capacity, which, together with the nomifensine-derived effects reported here, may trigger the subsequent formation of DA- and DOPAC-protein conjugates in substantia nigra. Particularly interesting is the specific partial protection given by nomifensine against the age-induced declines in TH enzyme activity in the nigrostriatal system in spite of showing higher number of carbonyl groups. Different mechanisms may account for this effect. First, our Western analysis strongly suggest chronic treatment with nomifensine induces TH enzyme expression. It is, therefore, plausible that the newly synthesized TH enzyme is not yet inactivated by oxidation. This would explain why TH enzyme activity is higher in the treated rats and why the number of drug-induced carbonyl groups is lower in TH enzyme than that found in the general protein homogenates. Alternatively, further protein modifications on TH enzyme following nomifensine treatment may not affect the active site of the protein, thus, not affecting the catalytic activity. Taken together, all these results show that long-lasting inhibition of the high affinity DA uptake system produces an increase in oxidative damage of substantia nigra. This is also in agreement with the possibility that
increased oxidative stress may play a role in the natural history of Parkinson’s disease, as has been suggested on many times, e.g., Spina and Cohen.28 Contrary to expected, chronic treatment of aged rats with nomifensine (2.5 mg kg01) for 2 months conferred a partial protection against the oxidative inactivation of TH enzyme in terms of enzymatic activity in this brain area. This partial protection was probably due to the induction of TH enzyme expression in the aged substantia nigra following the treatment with nomifensine. These results could explain the induction of TH enzyme described for deprenyl treatment but not its ability to protect against the oxidative damage to the total proteins in substantia nigra. In addition, all these results suggest that the possible use of specific DA uptake inhibitors in aged patients could increase the Parkinson incidence, assuming oxidative stress as a major cause in the ethiology of Parkinson disease.35 It remains to be established whether other biogenic amine uptake blockers also induce protein oxidation after long-lasting treatments. This is an important issue because serotonin and noradrenaline uptake blockers are widely used in the treatment of depressive disorders.36 Acknowledgements — This work was supported by grant FIS 96/ 1142 from Fondo de Investigacion Sanitaria.
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Original Contribution CHRONIC INHIBITION OF THE HIGH-AFFINITY DOPAMINE UPTAKE SYSTEM INCREASES OXIDATIVE DAMAGE TO PROTEINS IN THE AGED RAT SUBSTANTIA NIGRA
Marina Romero-Ramos, Jose A. Rodrı´guez-Go´mez, Jose L. Venero, Josefina Cano, and Alberto Machado Departamento de Bioquı´mica, Bromatologı´a y Toxicologı´a, Facultad de Farmacia, Universidad de Sevilla, Sevilla 41012, Spain (Received 22 April 1996; Revised 1 August 1996; Accepted 1 August 1996)
Abstract—The effect of chronic treatment of aged rats with nomifensine has been studied in the rat nigrostriatal dopaminergic system. The rat substantia nigra suffers an oxidative damage during aging that results in both an increase in carbonyl groups of its total proteins and the oxidative inactivation of tyrosine hydroxylase (TH) enzyme,1 which are partially reversed by chronic treatment with deprenyl. Different mechanisms may account for this effect, including inhibition of the high-affinity dopamine uptake system. We treated aged rats chronically with nomifensine for 2 months and found some significant effects. Nomifensine treatment significantly increased TH enzyme amount in substantia nigra (39.2%), which was accompanied by a significant increase in TH enzyme activity (47.8%). However, these effects were not observed in the terminal field (striatum). As a further step we quantified the oxidative level of proteins by measuring the number of carbonyl groups coupled either to total proteins or specifically to TH enzyme. The proteins of aged rat substantia nigra showed a significant increase of carbonyl groups following nomifensine treatment. The number of carbonyl groups coupled to nigral TH enzyme also increased in the nomifensinetreated animals. However, this increase was lower than that found in the total homogenate proteins. All these results show that the oxidative damage produced during aging in tyrosine hydroxylase enzyme and total proteins is not reduced by nomifensine treatment. On the contrary, the nomifensine treatment increased the oxidative damage to proteins. These results suggest the capability of deprenyl to induce TH enzyme could be due to inhibition of the high-affinity dopamine uptake system, but its ability to protect against oxidative damage is not produced by this mechanism. q 1997 Elsevier Science Inc. Keywords—Aging, Nomifensine, Substantia nigra, Oxidative stress, Tyrosine hydroxylase, Free radicals
ment of aged rats with deprenyl for 2 months produced a significant protection against the agedinduced oxidative damage in substantia nigra.1 Deprenyl has been reported as a safe therapeutic agent for a variety of neurodegenerative diseases and possibly for aging. It has several marked effects that facilitate the activity of dopaminergic neurons in the nigrostriatal system selectively.3 At the same time, multisite studies treating Parkinson’s patients with deprenyl have indicated its therapeutic benefits in producing a delay in the onset of disability4 and possibly increasing survival of treated patients.5 It slows down the age-related decline of performance in behavioral tests3 and extends6 life-span considerably.
INTRODUCTION
The rat substantia nigra suffers oxidative damage during aging that results in both an increase in carbonyl groups of its total proteins and the oxidative inactivation of tyrosine hydroxylase enzyme (TH).1 This is in accord with the production of oxidants generated during normal metabolism, which appear to play a significant role in the processes of aging.2 In addition, we have recently shown that chronic treatAddress correspondence to: Alberto Machado, Departamento de Bioquı´mica, Bromotologı´a y Toxicologı´a, Facultad de Farmacia, Universidad de Sevilla, c/ Prof. Garcı´a Gonza´lez s/n, Sevilla, 41012, Spain. 1
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Moreover, deprenyl treatment prevents age-related pigment changes in the substantia nigra.7 Many actions have been described for deprenyl that could be responsible for its therapeutic and/or protective effects against oxidative stress in substantia nigra above described. Deprenyl is a selective inhibitor of monoamine oxidase B (MAO B);8 however, it could also inhibit monoamine oxidase A (MAO A), depending on dose and route of administration.9,10 Deprenyl increases the enzymatic activities of both superoxide dismutase and catalase—enzymes involved in the scavenging of free radicals—in different brain regions including striatum and substantia nigra.11 Deprenyl also has an inhibitory effect upon the dopamine uptake system.3 In order to know which of these actions of deprenyl are involved in its protective actions against the oxidative damage produced by aging in substantia nigra, we have studied the effect of chronic treatment of aged rats with a specific dopamine carrier blocker, nomifensine, on the nigrostriatal dopaminergic system. For that, 22-month old rats were treated with nomifensine (2.5 mg free base kg01) for 2 months. Then, we studied levels of dopamine (DA) and its metabolites and the activity and content of TH, the step-limiting enzyme in the biosynthesis of dopamine and protein marker of dopaminergic neurons. The carbonyl derivatives resulting from oxidative damage to proteins12 were measured in homogenate proteins and TH. All these measurements were carried out in the substantia nigra and striatum of treated and untreated aged animals. Nomifensine seems not to have a protector capacity against the age-induced oxidative damage produced in the nigrostriatal dopaminergic system. Unexpectedly, levels of carbonyl groups of protein homogenates and TH enzyme increased significantly in nomifensine-treated animals. This nomifensine-induced effect on TH oxidation was accompanied by an increased TH enzyme expression in substantia nigra.
MATERIALS AND METHODS
Animals, treatment and dissection Twenty-two-month-old male Wistar rats were used in this study. They were divided into two groups: control animals (n Å 5) and experimental animals (n Å 5). Rats were given either IP injections of physiological saline solution or (0)nomifensine (2.5 mg free base kg01) three times a week for 2 months. The animals were kept under control environmental conditions. Food and tap water were allowed ad lib. The day following the last injection, animals were decapitated between 10:00 and 11:00 h, and the brains removed and placed on an ice-cold plate. The rats did not show brain
tumors because these pathological lesions can affect forebrain cell functions. There were no differences between saline- and nomifensine-treated rats in terms of body weight gain and behavior. The substantia nigra and striatum were dissected according to the atlas of Paxinos and Watson.13 After dissection, the two brain areas were immediately frozen in liquid N2 until assay. (0)Nomifensine was purchased from Research Biochemicals Inc., Natick, MA, and it was dissolved in 0.9% NaCl. Measurement of biogenic amines and their metabolites Analyses were performed by HPLC with electrochemical detection as described previously by Castan˜o et al.14 Samples were homogenized (5 w/v) in 20 mM Tris-HCl buffer by ultrasonic disintegration over ice using a Labsonic 1510. For this measurement an aliquot was further diluted 1:2 with 0.25 N perchloric acid. Samples were centrifuged at 12,000 1 g for 15 min at /47C and the supernatant was then filtered through a 0.2 mm filter and injected onto HPLC. Tyrosine hydroxylase activity assay TH activity was measured according to a modification of a previously published procedure.15 An aliquot coming from the Tris-HCl buffer homogenate was further diluted 1:60 with 30 mM Tris-acetic acid containing 0.1% Triton X-100 and incubated with 2.5 nmol of tyrosine-HCl (containing 0.4 mCi/nmol of L-[ring-3,503 H]tyrosine), 50 nmol of the cofactor 6(R)-L-erythro5,6,7,8-tetrahydrobiopterin, 5000 units of catalase and 5 nM DTT in 100 mM potassium phosphate, pH 6.0. The released [3H]OH was separated by an aqueous slurry of activated charcoal, and the radioactivity was determined by liquid scintillation counting. Quantification of tyrosine hydroxylase Another aliquot coming from the Tris-buffer homogenate was further diluted 1:2 with 80 mM TrisClH, 0.4 mM DTT, 16% sucrose, pH 7,4 plus protease inhibitors: leupeptin, 0.5 mg l01, pepstatin, 0.7 mg l01 and phenylmethyl-sulfonyl fluoride (PMSF), 40 mg l01. Analyses were performed by SDS-PAGE as describe Laemmli.16 After electrophoresis, the gels were Western blotted. Blots were incubated with anti-TH antibody (monoclonal antibody from Boehringer, Germany) followed by antimouse Ig-POD (Sheep antimouse Ig-G peroxidase conjugate from Boehringer, Germany). Specific immunolabeling was visualized using the horseradish peroxidase conjugate substrate kit
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Table 1. Effect of Nomifensine Treatment on the Concentration of Dopamine and Its Metabolites in Rat Striatum and Substantia Nigra Striatum Control DA DOPAC 3-MT HVA
6717.8 1982.4 276.1 559.9
{ { { {
706.5 220.7 42.3 121.9
Substantia Nigra Nomifensine
Control
Nomifensine
{ { { {
300.9 { 46.0 83.3 { 25.2 ND 70.7 { 8.1
494.1 { 137.1 91.3 { 24.0 ND 65.5 { 13.1
7821.7 1473.2 308.4 515.9
516.1 170.4* 46.7 92.3
Results are given in ng/g tissue as means { SD for n (number of animals assayed) Å 5. Statistical significance: Student’s t-test: *p õ .05 as compared with control group.
(Bio-Rad, California), and analyzed by densitometry using a laser computing densitometer (Molecular Dynamics, California). Determination of carbonyl groups of proteins in homogenates Carbonyl groups were determined by measurement of incorporation of tritium [3H] after reduction with sodium boro [3H] hydride as described by Lenz et al.17 Determination of tyrosine hydroxylase enzyme carbonyl groups For this purpose, the homogenates incubated with sodium boro [3H] hydride were subjected to SDSPAGE and Western blotted. Blots were incubated with anti-TH antibody followed by antimouse Ig-biotin (sheep antimouse IgG biotin conjugate from Boehringer, Germany). Blots were then incubated with streptavidin–biotinylated alkaline phosphatase complex (Bio-Rad, California) and visualized by BCIP/ NBT color development solution (Bio-Rad, California). The radioactivity incorporated into TH was determinated by cutting out the protein bands and counting the c.p.m. after the addition of 5 ml of Ready-protein liquid scintillation cocktail (Beckman Instruments, USA). Prior the cutting, band intensity was measured in the membrane by densitometric scanning and results were expressed by c.p.m. per optical density. Measurement of protein content The protein content of homogenates was determined using the procedure of Lowry et al.18 Statistical analyses A Student’s t-test was used. When the p value was õ .05 (*) or õ .01 (**), the difference was considered significant.
RESULTS
Levels of dopamine and their metabolites following chronic treatment with nomifensine In substantia nigra, the nomifensine-treated rats did not differ from control animals in terms of catecholamine levels. Thus, levels of dopamine (DA) and its metabolites remained unchanged following chronic treatment with nomifensine (Table 1). However, there was a significant decrease in the (3,4-dihydroxyphenylacetic acid (DOPAC)/DA ratio following nomifensine treatment (0.29 { 0.06 to 0.15 { 0.02 from untreated and treated rat respectively p õ .05) (Table 1). Striatum showed a rather different neurochemical profile following treatment with nomifensine. While DA levels remained to control levels, the levels of the main acid DA metabolite, the DOPAC, were significantly decreased (025.6% of controls) in the nomifensine-injected animals (Table 1). The striatal DOPAC/ DA ratio decreased statistically in treated animals (0.29 { 0.006 to 0.18 { 0.02 from untreated and treated rat respectively p õ .01). Effect of nomifensine treatment on TH enzyme quantity and activity As seen in Figs. 1 and 2, TH enzyme activity was significantly induced following chronic treatment with nomifensine in substantia nigra (/39.2% of controls) but not in striatum. This change in TH enzyme activity was accompanied by a significant increase in TH enzyme amount in substantia nigra (/47.8% control levels) without any change in striatum (Figs. 1 and 2). Effect of nomifensine treatment on carbonyl groups in homogenates and in TH enzyme in striatum and substantia nigra The carbonyl groups of proteins have been widely used as a measure of proteins modified by oxidation. Animals treated with nomifensine showed a significant increase in the number of carbonyl groups coupled to
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Fig. 1. Tyrosine hydroxylase enzyme activity and content in aged rat striatum and substantia nigra following chronic treatment with nomifensine. TH activity was measured in vitro and TH content was obtained by Western blotting, as described in Materials and Methods. Loading gel conditions were determinated in a previous analyses using increasing amount of protein. The optimum amount were 10 mg and 40 mg for striatum and substantia nigra respectively. All data are mean { SD for n (number of animals assayed) Å 5. Statistical significance: Student’s t-test * p õ .05; ** p õ .01 as compared with controls.
nigral protein homogenates (/83.3% of controls) (Table 2). Considering that nomifensine treatment strongly increased the number of carbonyl groups in nigral protein homogenates, we further wanted to know whether this effect included TH enzyme protein. As seen in Table 2, the number of carbonyl groups specifically coupled to TH enzyme was significantly increased in substantia nigra (/35.8% of controls) of nomifensinetreated rats. Contrary to that found in substantia nigra, chronic treatment with nomifensine was ineffective in altering the number of carbonyl groups coupled to striatal protein homogenates (Table 2).
DISCUSSION
The possibility that deprenyl protects the dopaminergic system against the age-induced increase in oxidized proteins by its inhibitory action on the DA-uptake system1 prompted us to study the effect of a specific antagonist of the high affinity DA transport system, nomifensine. Moreover, drugs blocking the catecholamine transport system have been extensively studied
and many of them are used as therapeutic agents.19 Therefore, the present study analyzed the modifications of DA metabolism produced by chronic treatment with nomifensine. In striatum, these included a significant decrease in DOPAC levels with no significant rise in dopamine content. Similar, but lower effects, were found in substantia nigra. The nomifensine treatment produced a nonsignificant increase in DA along with no change in DOPAC concentrations. However, the DOPAC/DA ratio was significantly decreased in both brain areas examined, thus indicating a decreased intraneuronal DA metabolism in the rat dopaminergic nigrostriatal system.20 Inhibition of the high-affinity DA transport system is supposed to increase extracellular DA levels, and, hence, the extraneuronal DA metabolism. However, while HVA levels remained to control levels, HVA/DA ratio decreased in nomifensine-treated rats. Taken together, our neurochemical analysis suggests a downregulation of the dopaminergic system following chronic inhibition of the high-affinity DA transport system, with decreases in DA turnover, both extraneuronally and intraneuronally as deduced from the DOPAC/DA and HVA/DA ratios. However, other
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Fig. 2. Western blot analysis of TH amount in substantia nigra. Nomifensine treatment-induced TH enzyme amount in substantia nigra (N1 and N2) as compared with control animals (C1 and C2).
possibilities should not be excluded, and thus, nomifensine treatment may induce downregulation of MAO enzyme. Under these conditions, higher DA levels in the synaptic cleft has necessarily not to be associated to higher HVA levels. If we compare the nomifensine effect to that produced by deprenyl, some differences are apparent. Deprenyl treatment1 produced more extensive changes than nomifensine treatment. Deprenyl produced a change in 3-methoxytyramine and HVA, levels probably due to its inhibitory action on MAO enzymes. Deprenyl also produced higher changes in DA and DOPAC concentrations although in the same direction as that produced by nomifensine. The higher effect of deprenyl must be produced by a combination of the effects of inhibition of both MAO enzymes and DA uptake system. The nomifensine treatment produced an increase in TH activity and amount in substantia nigra (39 and 47%, respectively), with no change in striatum. In addition, the number of carbonyl groups coupled to protein homogenates was increased in substantia nigra
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while striatum remained at control levels. This increase was also evident in the number of carbonyl groups specifically coupled to TH enzyme in substantia nigra, although this increase was lower than that found for the total homogenate proteins. These results contrast to those seen after chronic treatment with deprenyl, which protected the substantia nigra against the oxidative damage produced by aging. In contrast, and unexpectedly, chronic treatment of aged rats with nomifensine exacerbated the age-induced oxidative stress that normally occurs in substantia nigra. However, and similarly to deprenyl treatment, the nomifensine treatment induced TH enzyme and activity in substantia nigra. All these results suggest that the effect of nomifensine treatment should be produced by its specific inhibitory action on the high-affinity DA uptake system. Some of the factors that increase TH enzyme protein are those which increase DA release, e.g., reserpine,21 a sympatholytic drug that blocks vesicular storage of catecholamines and thereby leads to transmitter degradation and depletion and cocaine,22 which has been shown to inhibit DA, serotonin and noradrenaline uptake.23–25 The induction of TH enzyme in substantia nigra has also been found with deprenyl treatment;1 it could also be the result of the inhibitory action described for some metabolites of deprenyl (i.e., 1-methamphetamine and 1-amphetamine).3 However, the protective effect of deprenyl against the oxidative damage produced in substantia nigra by aging does not seem to be a result of its inhibitory effect upon the dopamine uptake system.3 It could be produced by its inhibitory action ascribed to MAO enzymes8,26 or/along with the induction of superoxide dismutase and catalase, enzymes involved in the scavenging of free radicals in the nigrostriatal system.11 The increase in oxidized proteins that occurs during aging could be produced either by greater damage induced by oxygen reactive species or alternatively by a decrease in the rates of proteolytic degradation of the oxidized proteins27 with the decrease in protein turnover. Either system could explain the accumulation of Table 2. Measurements of Carbonyl Groups in Aged Rats after Treatment with Nomifensine Striatum Homogenates
Substantia Nigra Homogenates
Control 144.99 { 26.04 138.77 { 36.12 Nomifensine 129.35 { 10.72 254.42 { 60.31*
TH Enzyme 5.07 { 0.27 6.89 { 0.14†
Results are given in cpm/mg protein for homogenates and cpm/ O.D for TH enzyme as means { SD for n (number of animals assayed) Å 5. Statistical significance: Student’s t-test: *p õ .05, †p õ .01 as compared with control group.
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inactivated enzymes in this process. We found a significant increase in the number of carbonyl groups coupled to nigral proteins. If we a consider a diminished DA turnover following chronic treatment with nomifensine as indicated by our neurochemical analysis, the higher oxidation rate of the general nigral protein homogenates cannot be associated to oxidation of DA by monoamine oxidase.28 DA deamination by monoamine oxidase produces hydrogen peroxide, a precursor of highly oxidizing tissue-damaging radicals (hydroxyl radicals).29,30 However, a higher degree of nigral protein oxidation following nomifensine treatment may also derive from DA auto-oxidation products, which are more likely to occur with a diminished DA turnover. It is generally assumed that a decrease in turnover rate produces a greater chemicals modifications. DA readily oxidizes at a physiological pH to form potentially toxic metabolites including hydroxyl radical, superoxide radical, hydrogen peroxide, and quinones.31–33 Strikingly, Hastings and Zigmond (1994)34 have demonstrated that incubation of [3H]DA with neostriatal slices results in the binding of tritium to acid-insoluble proteins.34 Acid hydrolysis of these proteins revealed the presence of cysteinyl-DA and cysteinyl-DOPAC.34 The authors conclude that under conditions in which the ratio of available DA to antioxidant capacity is sufficiently high, reactive metabolites formed from DA autooxidation that include free radicals could result in the binding of catechols to protein and also its oxidation. Aging is known to be associated to a poorer antioxidant capacity, which, together with the nomifensine-derived effects reported here, may trigger the subsequent formation of DA- and DOPAC-protein conjugates in substantia nigra. Particularly interesting is the specific partial protection given by nomifensine against the age-induced declines in TH enzyme activity in the nigrostriatal system in spite of showing higher number of carbonyl groups. Different mechanisms may account for this effect. First, our Western analysis strongly suggest chronic treatment with nomifensine induces TH enzyme expression. It is, therefore, plausible that the newly synthesized TH enzyme is not yet inactivated by oxidation. This would explain why TH enzyme activity is higher in the treated rats and why the number of drug-induced carbonyl groups is lower in TH enzyme than that found in the general protein homogenates. Alternatively, further protein modifications on TH enzyme following nomifensine treatment may not affect the active site of the protein, thus, not affecting the catalytic activity. Taken together, all these results show that long-lasting inhibition of the high affinity DA uptake system produces an increase in oxidative damage of substantia nigra. This is also in agreement with the possibility that
increased oxidative stress may play a role in the natural history of Parkinson’s disease, as has been suggested on many times, e.g., Spina and Cohen.28 Contrary to expected, chronic treatment of aged rats with nomifensine (2.5 mg kg01) for 2 months conferred a partial protection against the oxidative inactivation of TH enzyme in terms of enzymatic activity in this brain area. This partial protection was probably due to the induction of TH enzyme expression in the aged substantia nigra following the treatment with nomifensine. These results could explain the induction of TH enzyme described for deprenyl treatment but not its ability to protect against the oxidative damage to the total proteins in substantia nigra. In addition, all these results suggest that the possible use of specific DA uptake inhibitors in aged patients could increase the Parkinson incidence, assuming oxidative stress as a major cause in the ethiology of Parkinson disease.35 It remains to be established whether other biogenic amine uptake blockers also induce protein oxidation after long-lasting treatments. This is an important issue because serotonin and noradrenaline uptake blockers are widely used in the treatment of depressive disorders.36 Acknowledgements — This work was supported by grant FIS 96/ 1142 from Fondo de Investigacion Sanitaria.
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