Alpha-lipoic acid differently affects the reserpine-induced oxidative stress in the striatum and prefrontal cortex of rat brain

Neuroscience 146 (2007) 1758 –1771

ALPHA-LIPOIC ACID DIFFERENTLY AFFECTS THE RESERPINE-INDUCED OXIDATIVE STRESS IN THE STRIATUM AND PREFRONTAL CORTEX OF RAT BRAIN Key words: ␣-lipoic acid, glutathione, reserpine, S-nitrosothiols, Parkinson’s disease, nitric oxide.

A. BILSKA,a M. DUBIEL,a M. SOKOŁOWSKA-JEZ˙EWICZ,a E. LORENC-KOCIb* AND L. WŁODEKa a

The Chair of Medical Biochemistry, Collegium Medicum, Jagiellonian University, 7, Kopernika Street, PL-31-034 Kraków, Poland

A growing body of evidence from clinical and experimental studies clearly indicates, that oxidative stress may contribute importantly to the loss of nigrostriatal dopaminergic neurons in the course of Parkinson’s disease (PD) (Fahn and Cohen, 1992; Jenner, 2003). In the dopaminergic system, dopamine (DA) itself may be one of the key contributors to oxidative stress. The postmortem examination of parkinsonian brains revealed a several-fold increase in DA catabolism in the surviving dopaminergic neurons of the substantia nigra pars compacta (SNc) suggesting the involvement of DA in the pathological process (Hornykiewicz and Kish, 1987; Antkiewicz-Michaluk et al., 1999). Moreover, it has been demonstrated that DA and its precursor L-DOPA are toxic to neurons both in culture (Tanaka et al., 1991; Mena et al., 1992) and in vivo (Filloux and Townsend, 1993; Hastings et al., 1996). It is well known that the oxidative deamination of DA catalyzed by a mitochondrial enzyme monoamine oxidase (MAO) as well as its autoxidation is accompanied by formation of hydrogen peroxide, which may be converted to highly toxic hydroxyl radical by the iron-mediated Fenton reactions (Cohen, 1983; Graham, 1978; Dexter et al., 1989; Chiueh et al., 1992). Hydroxyl radicals are able to initiate lipid peroxidation, to oxidize amino acid side chains in proteins and to cause DNA strand breaks (Dexter et al., 1994; Alam et al., 1997a,b; Zhang et al., 1999). In addition to reactive oxygen species, also redox-labile DA-derived o-quinones, that are formed during DA oxidation, may lead to damage of the vital molecules (Fornstedt et al., 1986, 1989; Hastings et al., 1996; Dagnino-Subiabre et al., 2000). Reduced glutathione (GSH), that is the most abundant cellular nonprotein thiol in the mammalian brain plays a very important role in the protection of cells against deleterious effects of free radicals. GSH is considered to be the major regulator of the intracellular redox state acting either as an antioxidant by scavenging reactive oxygen species (Dringen 2000), or as a substrate in various enzymatic antioxidant defense mechanisms (Dringen et al., 2005; Hayes et al., 2005). Hydrogen peroxide generated in brain cells under physiological conditions is effectively neutralized by glutathione peroxidase (GPx) localized within cytosol and mitochondria, as well as by the peroxisomal enzyme catalase (Dringen et al., 2005). GPx detoxifies H2O2 with GSH serving as an electron donor in the reduction reaction, producing glutathione disulfide (GSSG) and

b

Department of Neuropsychopharmacology, Institute of Pharmacology, Polish Academy of Sciences, 12, Sme˛tna Street, PL-31-343 Kraków, Poland

Abstract—Antioxidative properties of ␣-lipoic acid (LA) are widely investigated in different in vivo and in vitro models. The aim of this study was to examine whether LA attenuates oxidative stress induced in rats by reserpine, a model substance frequently used to produce Parkinsonism in animals. Male Wistar rats were treated with reserpine (5 mg/kg) and LA (50 mg/kg) separately or in combination. The levels of reduced glutathione (GSH), glutathione disulfide (GSSG), nitric oxide (NO) and S-nitrosothiols as well as activities of glutathione peroxidase (GPx), glutathione-S-transferase (GST) and L-␥-glutamyl transpeptidase (␥-GT) were determined in the striatum and prefrontal cortex homogenates. In the striatum and prefrontal cortex a single dose of reserpine significantly enhanced levels of GSSG and NO but not that of S-nitrosothiols when compared with control. In the striatum, LA administered jointly with reserpine markedly increased the concentration of GSH and decreased GSSG level. In the prefrontal cortex, such treatment produced only an increasing tendency in GSH level but caused no changes in GSSG content. In both structures LA injected jointly with reserpine markedly decreased NO concentrations but did not cause significant changes in S-nitrosothiol levels when compared with control. Enzymatic activities of GPx and GST were intensified by LA in the striatum. In the prefrontal cortex, GPx activity was not altered, while that of GST was decreased. ␥-GT activity was attenuated by reserpine in the striatum while LA reversed this effect. Such changes were not observed in the prefrontal cortex. The mode of LA action in the striatum during the reserpine-evoked oxidative stress strongly suggests that this compound may be of therapeutic value in the treatment of Parkinson’s disease. © 2007 IBRO. Published by Elsevier Ltd. All rights reserved. *Corresponding author. Tel: ⫹48-12-6623272; fax: ⫹48-12-63745-00. E-mail address: [email protected] (E. Lorenc-Koci). Abbreviations: CDNB, 1-chloro-2,4-dinitrobenzene; DA, dopamine; DAN, 2,3-diaminonaphthalene; DHLA, dihydrolipoic acid; DTNB, 5,5=-dithio-bis2-nitrobenzoic acid; EDTA, ethylenediaminetetra-acetate; GPx, glutathione peroxidase; GR, glutathione disulfide reductase; GSH, reduced glutathione; GSNO, S-nitrosoglutathione; GSSG, oxidized glutathione, glutathione disulfide GST, glutathione-S-transferase; ␥-GT, L-␥-glutamyl transpeptidase; LA, ␣-lipoic acid; MAO, monoamine oxidase; NA, noradrenaline; NO, nitric oxide; NQO, NAD(P)H:quinone oxidoreductase; PD, Parkinson’s disease; PSSG, protein-glutathione mixed disulfide; SNc, substantia nigra pars compacta; tBOOH, t-butyl hydroperoxide; TCA, trichloroacetic acid; TH, tyrosine hydroxylase.

0306-4522/07$30.00⫹0.00 © 2007 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2007.04.002

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water as final products. GSH is regenerated from GSSG in the reaction catalyzed by glutathione disulfide reductase (GR) that requires NADPH. However, when NADPH is deficient or GR has insufficient activity an excess of GSSG can be transported from the cell into the extracellular space by means of specific transporters termed as multidrug resistance protein (MRP) (Hirrlinger et al., 2001; Minich et al., 2006). However, it leads to the loss of GSH from system. GSSG can also react with protein sulfhydryl groups forming protein-glutathione mixed disulfides (PSSGs) (Klatt and Lamas, 2000; Giustarini et al., 2004). This redox-sensitive posttranslational modification referred to as S-glutationylation may potentially serve to protect sulfhydryl groups in proteins from their irreversible oxidation. On the other hand, such modulation may render these proteins inactive and thus compromise their cellular function. Therefore, S-glutationylated proteins which are accumulated under oxidative/nitrosative stress conditions can be readily reduced to free ⫺SH groups by glutaredoxin (thioltransferase), the enzyme that requires optimal cellular GSH level for its efficient functioning (Daily et al., 2001; Kenchappa and Ravindranath, 2003). Apart from GP, GSH is also a substrate for glutathioneS-transferases (GSTs) and L-␥-glutamyl transpeptidase (␥GT). GSTs are enzymes that detoxify electophilic xenobiotics and inactivate endogenous substances, such as quinones, ␣,␤-unsaturated aldehydes and hydroperoxides formed as secondary metabolites during oxidative stress (Dagnino-Subiabre et al., 2000; Hayes et al., 2005). ␥-GT is a membrane-bound enzyme attached to the outer side of plasma membrane that is responsible for extracellular cleavage of ␥-glutamyl bound in GSH molecule (Dringen et al., 1997). The cysteinylglicyne (CysGly), the product of the ␥-GT-catalyzed reaction, is reused by astrocytes and neurons for GSH synthesis (Dringen et al., 2000). Activity of ␥-GT was markedly enhanced in the SN of parkinsonian patients (Sian et al., 1994). Moreover, GSH reacts with nitric oxide (NO) and forms S-nitrosothiols, mainly S-nitrosoglutathione (GSNO), that is a much more potent antioxidant than GSH itself. It has been recently demonstrated that GSNO protects nigral DA neurons against iron-induced oxidative stress (Chiueh and Rauhala, 1999). The abovementioned data distinctly show that GSH and enzymes involved in its metabolism as well as GSNO are of crucial importance for maintenance DA homeostasis. Therefore, it seems that raising cellular GSH content and modulation of activities of the GSH-related enzymes in brain dopaminergic structures may be beneficial in preventing the effects of the DA-mediated oxidative stress. However, manipulation with brain GSH levels using either precursors (Anderson and Meister, 1989) or prodrugs of GSH are faced with difficulties (Pileblad and Magnusson, 1992; Anderson, 1998). For this reason, attention has been focused on ␣-lipoic acid (LA), a dithiol compound naturally occurring in biological systems. LA has been known for a long time as an essential cofactor for mitochondrial bioenergetic enzymes but recent in vitro and in vivo studies suggest that this compound acts also as a potent antioxidant and detoxifying agent in heavy

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metals poisoning (Smith et al., 2004; Packer et al., 1997; Suh et al., 2004). In mammalian cells, exogenously supplied LA is rapidly reduced to dihydrolipoic acid (DHLA) by NADH-dependent mitochondrial dihydrolipoate dehydrogenase and by cytoplasmic NADPH-dependent reductases: GR and thioredoxin reductase (Biewenga et al., 1997; Bustamante et al., 1998). Both LA and DHLA can readily cross the blood– brain barrier (Handelman et al., 1994) and act as a redox couple that is characterized by a very low reduction potential (E0=⫽⫺0.32V). Due to these properties DHLA is capable of regenerating other low molecular weight antioxidants, such as GSH, coenzyme Q10 as well as vitamins A and C (Biewenga et al., 1997; Kozlov et al., 1999). Moreover, thiol groups of LA and DHLA make them capable of scavenging a variety of reactive oxygen (ROS) and nitrogen species with capacity higher or comparable to that of GSH (Dikalov et al., 1996; Smith et al., 2004). All the abovementioned properties makes LA a very promising drug for treatment of neurological diseases whose etiology is related to oxidative stress (Biewenga et al., 1997; Farr et al., 2003; Bharath et al., 2002; Panigrahi et al., 1996). The aim of the present study was to find out whether LA alleviates effects of DA-mediated oxidative stress induced in rats by reserpine (Spina and Cohen, 1988,1989), a model substance frequently used to produce Parkinsonism in animals (Colpaert, 1987; Fornstedt and Carlsson, 1989; Elverfors and Nissbrandt, 1991; Lorenc-Koci et al., 1995). Reserpine prevents the storage of DA in neuronal transmitter vesicles by interfering with the vesicular monoamine transporter (VMAT). As a result, the oxidative catabolism of cytosolic DA by MAO is accelerated, being followed by DA disappearance and simultaneous formation of acidic metabolites and a cellular oxidant hydrogen peroxide. This action mimics, to some extent, the increased turnover of DA in the surviving dopaminergic terminals in the course of PD. To assess the usefulness of LA in the protection of dopaminergic system against the oxidative stress, we investigated effects of this compound, administered alone and in combination with reserpine, on levels of total (GSH⫹GSSG), reduced (GSH) and oxidized (GSSG) glutathione, NO and S-nitrosothiols, mainly GSNO, as well as on activity of GSH-related enzymes such as GP, GST and ␥-GT in the striatum and prefrontal cortex. In addition, to provide an evidence supporting neuroprotective effect of LA toward tyrosine hydroxylase (TH), the initial and ratelimiting enzyme in the biosynthesis of DA, we determined levels of PSSGs in the striatal and prefrontal cortex homogenates incubated in vitro with reserpine, t-butyl hydroperoxide (tBOOH) and LA, separately or in combination. TH is an oxidatively labile enzyme whose level of activity is determined by the redox status of its cysteine sulfhydryl groups. Sulfhydryl oxidants such as peroxynitrite and catechol-quinones reduce TH activity to an extent that is proportional to cysteine modification (Kuhn et al., 1999a,b). Recently, it has been demonstrated that this enzyme is regulated by S-glutathionylation. When six of the seven cysteines in TH are S-glutathionylated, its activity is lowered by 70 – 80% (Borges et al., 2002). Taking into

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account the above-described regulation of TH activity and fact that reserpine increases GSSG contents in the examined structures, we used PSSG level as an indirect measure of TH activity. It seems that this set of experiments may provide a fresh insight into mechanism of LA action in the dopaminergic system.

Chemicals

and the mixture was centrifuged at 12,000⫻g for 10 min. The supernatant was neutralized by the addition of appropriate volume of 5 M NaOH. For determination of the total glutathione content, 10 ␮l of the neutralized TCA-supernatant was added to the reaction mixture containing 0.6 mM DTNB, 69 mM phosphate buffer (pH 7.5), 3.45 mM EDTA, GR (1.512 U) and 0.22 mM NADPH in a final volume of 1 ml. GSH content was determined from a change in an absorbance at 412 nm measured at 1 and 2 min after the addition of GR. Total GSH content was determined from a standard curve for the GSH and was expressed in nmol per mg of protein. Oxidized glutathione (GSSG) was measured according to the method of Griffith (1980). Briefly, GSH contained in 100 ␮l of the diluted TCA-supernatant (1:4) was derivatized with 2 ␮l of 2-vinylpyridine in the presence of 6 ␮l of triethanolamine at room temperature for 1 h, and the reaction was carried out as above. GSH content was calculated by subtracting GSSG level (1 mol GSSG⫽2 mol of GSH determined) from total GSH level.

L-␥-Glutamyl-p-nitroanilide

Determination of nitrites concentration

EXPERIMENTAL PROCEDURES All the experiments were carried out according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals (publication no. 85–23, revised 1985) and were approved by the internal Bioethics Commission. All efforts were made to minimize the number of animals used and their suffering.

was obtained from Boehringer GmbH (Mannheim, Germany), trichloroacetic acid (TCA), 5,5=-dithio-bis2-nitrobenzoic acid (DTNB), reduced GSH, NADPH, GR, 2-vinylpyridie, 2,3-diaminonaphthalene (DAN), GSNO, glycyl-glycine, N-1-naphthyl-ethylenodiamine, mercury chloride (II) and sulfanilamide were purchased from Sigma Chemical Company (Steinheim, Germany). All the other reagents were of analytical grade and were obtained from Polish Reagent Company (P.O.CH., Gliwice, Poland). The drug LA was a gift from Hexal A.G. (Holzkirchen, Germany). Reserpine from Polfa (Warszawa, Poland) was dissolved in a solution containing 0.25% citric acid, 2% benzyl alcohol and 10% Tween 80.

Animals and experimental procedures The experiments were performed on four groups of males Wistar rats weighing 260 –290 g which were injected intraperitoneally with LA (50 mg/kg) and reserpine (5 mg/kg) alone and in combination. LA was administered twice, 30 min before and after reserpine injection. Control groups instead of LA or reserpine received i.p. 0.9% solution of NaCl in the same time intervals. Rats were killed by decapitation 90 min after the second injection. Their brains were rapidly removed; the prefrontal cortex and the striatum were dissected on an ice-chilled plate. Then, the tissues were immediately frozen on dry ice and stored at ⫺80 °C until further processing.

Preparation of brain homogenates Homogenates of brain tissue assigned for determination of concentration of nitrite (as a measure of NO level), S-nitrosothiols and GSH, and enzymatic activity of ␥-GT, GPx and GST were prepared in the same way. To prevent undesirable changes in the examined brain tissue that could happen ex vivo during the postmortem period, all experimental procedures such as thawing, weighing, homogenization and centrifugation were carried out extremely fast at 4 °C. Tissue samples of the striatum and prefrontal cortex were homogenized in four volumes of 0.1 M phosphate buffer, pH 7.4 using a Polytron disintegrator. Crude tissue homogenates containing membranes, and all cellular components were kept on ice until further processing.

Glutathione determination Total glutathione content (GSH and GSSG) was measured using a method described by Tietze (1969). Reduction of DTNB by GSH resulted in formation of the chromophoric product 2-nitro-5-thiobenzoic acid (TNB), which was detected spectrophotometrically at 412 nm. To 19 volumes of crude homogenates, one volume of 50% TCA was added (its final concentration in a sample was 245 mM)

The level of nitrites (the final products of NO metabolism) was determined using Roche’s test “nitric oxide colorimetric assay” (cat. No. 1 756 281). The test is based on the following reaction: nitrites⫹sulfanilamide⫹N-(1-naphthyl)-ethylenediamine dihydrochloride and yields a reddish–violet diazo dye whose absorbance is measured in the visible range at 540 nm. To determine the nitrite level in the brain samples, 100 ␮l of an appropriate homogenate was added to 400 ␮l of redistilled water, and then the mixture was placed in a hot waterbath (100 °C) for 15 min to stop all enzymatic processes. After cooling, 30 ␮l of the reagents named Carrez I (0.36 M K4[Fe(CN)6]⫻3 H2O) and 30 ␮l of Carrez II (1 M ZnSO4⫻7 H2O) was added to each sample. Next, the samples were alkalized to pH 8 by adding 4 ␮l of 10 M NaOH and centrifuged at 10,000⫻g before further use. For nitrite determination, 75 ␮l of supernatant and 75 ␮l of redistilled water were placed in microplate wells. In blank samples, redistilled water was used instead of supernatant. The samples were incubated at room temperature for 30 min, and then the absorbance at 540 nm was measured. Finally, the colored reaction was developed in the examined samples by introducing to each well 50 ␮l of 1% solution of sulfanilamide in 2.5% H3PO4 and 50 ␮l of 0.1% solution of N-(1-naphthyl)-ethylenediamine dihydrochloride in 2.5% of H3PO4. After mixing, microplates were allowed to stand in the dark for 15 min and absorbance was again measured at 540 nm. The results were calculated according to standard curves obtained for solutions of sodium nitrite (6 – 600 ␮M), using the change in absorbance measured before and after incubation with sulfanilamide and N-(1-naphthyl)-ethylenediamine dihydrochloride. Concentrations of nitrites as a measure of NO in the examined tissue samples were calculated and expressed in nmol per mg of protein.

Determination of S-nitrosothiols The level of S-nitrosothiols (mainly GSNO) was determined using the improved method described by Marzinzig et al. (1997). This method is based on the homolytic degradation of S-nitrosothiols under influence of mercury ions (Hg⫹2). NO released during this reaction immediately reacts with DAN and forms fluorescent product 2,3-naphthotrizole (NAT). The fluorescence of samples containing DAN and mercury ions diminished by fluorescence of samples devoid of these ions allows for establishing the concentration of S-nitrosothiols. In order to determine the level of Snitrosothiols, 50 ␮l of the appropriate brain homogenate was added to 700 ␮l of redistilled water and 250 ␮l of 1.58 mM DAN in 0.62 M HCl. Samples containing 50 ␮l of 1.11 mM HgCl2 and 200 ␮l of 1.58 mM DAN in 0.62 M HCl were prepared in parallel. All the examined samples were incubated at room temperature in

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the dark for 10 min. The reaction was stopped by addition of 50 ␮l of 10 M NaOH. Fluorescence measurements were performed at excitation wavelength of 365 nm and emission wavelength of 410 nm. The S-nitrosothiol level was calculated according to standard curve obtained for 10 ␮l GSNO and was expressed in nmol per mg of protein.

Millimolar absorbance index of the conjugate is 9.6 mmol⫺1 · cm⫺1. The difference between an increase in absorbance for the test sample and control sample divided by molar absorbance index was a measure of enzymatic activity. Enzymatic activity was expressed in micromoles of 2,4-dinitrophenyl-S-GSH formed within 1 min per of protein.

Activity of ␥-GT

Determination of PSSGs

The activity of ␥-GT was estimated by the method of Orlowski and Meister (1966) in which the colorless substrate, L-␥-glutamyl-pnitroanilide, was enzymatically converted by ␥-GT to p-nitroaniline. The latter compound exhibits a maximum absorbance at 410 nm. Activity of ␥-GT was assayed by the incubation (5 min, 37 °C) of 50 ␮l of the appropriate homogenate together with (mM) L-␥-glutamyl-p-nitroanilide (5), MgCl2 (11), glycyl-glycine (22) and Tris–HCl buffer pH 9 (111) in a final volume of 1 ml. The reaction was stopped by the addition of 1 ml of 1.5 M acetic acid, and then the mixture was centrifuged at 12,000⫻g for 5 min. The absorbance of p-nitroaniline formed during 5 min of incubation was measured at 410 nm. The rate of generation of p-nitroaniline was found to be linear over that time. The activity of the enzyme was expressed as nmol of the product, formed during 1 min of incubation per mg of protein.

Activity of GPx The activity of GPx was estimated by the method of Flohe and Gunzler (1984). This method is based on GSH oxidation by hydrogen peroxide that is catalyzed by GPx. This reaction yields GSSG which is then reduced by GR to GSH at the expanse of NADPH oxidation. NADPH oxidation causes a decrease in absorbance at 340 nm, which can be measured spectrophotometrically. To a thermostated spectrophotometric cuvette kept at 37 °C, the following reagents were added: 500 ␮l of 0.1 M phosphate buffer pH 7.0 containing 0.1 mM EDTA, 100 ␮l of homogenate diluted 50-fold, 100 ␮l of GR solution of 2.4 U/ml final activity, 100 ␮l of 10 mM GSH solution and 100 ␮l of 1.5 mM NADPH solution in 0.1% NaHCO3 solution. The reaction was initiated by the addition of 100 ␮l of 1.5 mM H2O2 heated to 37 °C. Then a decline in absorbance was measured at 340 nm for 5 min. Control sample was prepared in the same way, but 100 ␮l of redistilled water was added instead of homogenate. The decline in absorbance was measured at 340 nm for 5 min. The difference between absorbance decrement (⌬A340/minute) in the homogenate-containing sample and control sample (without homogenate) was calculated. The difference between the absorbance change rates ␯ is a measure of GPx activity in the sample. A unit of the enzyme’s activity corresponds to such amount of the enzyme that oxidizes 1 ␮mol GSH (0.5 ␮mol NADPH) per minute. As millimolar absorbance index of NADPH at 340 nm is ␧⫽6.22 mmol⫺1 · 1 cm⫺1, enzyme’s activity (in units per ml)⫽2␯/ 6.22.

Activity of GST The activity of GST was estimated by the method of Habig et al. (1974). Glutathione transferase catalyzes reaction between GSH and 1-chloro-2,4-dinitrobenzene (CDNB), yielding colored conjugate (2,4-dinitrophenyl-S-glutathione). The rate of conjugate formation is measured colometrically at 340 nm. To a spectrophotometric cuvette thermostated at 30 °C, the following reagents were added: 850 ␮l of 0.1 M phosphate buffer pH 6.5 equilibrated at this temperature, 50 ␮l of 20 mM GSH solution and 50 ␮l of 20 mM CDNB solution. The reaction was initiated by addition of 50 ␮l of homogenate diluted 50-fold. Absorbance was measured at 340 nm and absorbance increase rate (absorbance increase per minute, ⌬A) was calculated. Absorbance of control sample in which homogenate was replaced by 50 ␮l of the buffer was measured analogously.

The level of PSSGs was measured according to the method described by Rossi et al. (1995). It is based on the use of protein sulfhydryl groups as an endogenous reductant and on the spectrophotometric determination of GSH. The procedure relies on the observation that acid-precipitated proteins rapidly release GSH from PSSGs when brought to neutral pH. To determine the PSSG level in brain samples, under in vitro conditions, first 125 ␮l of crude striatal and prefrontal cortex homogenates were incubated for 20 min with 50 ␮l of reserpine (5.8 mM final concentration) and LA (34.3 mM final concentration) alone or in combination. Control homogenates instead of reserpine were incubated for the same time with 50 ␮l of their solvents i.e. mixture of citric acid (0.05 mM final concentration) with benzyl alcohol (8% w/v final concentration). Additionally, 125 ␮l of striatal and prefrontal cortex homogenates were incubated with 50 ␮l of tBOOH (1 mM final concentration) alone or in combination with LA (34.3 mM final concentration). Next, protein in the incubated samples was precipitated with 6.75 ␮l of 50% TCA. Acidprecipitated proteins were thoroughly washed twice with 2.5% TCA and then suspended in 125 ␮l of 0.1 M phosphate buffer (pH⫽7.4). Neutral pH was reached by adding 5 ␮l of NaOH. Such samples were incubated for 1 h at room temperature. After that time they were again precipitated with 6.75 ␮l of 50% TCA and centrifuged. The amount of GSH released from denatured proteins was a measure of PSSG level. GSH determined in the supernatants using the method described by Tietze (1969) was expressed in nmol per mg of protein.

Statistics The results are presented as the mean⫾S.E.M. for each group. Statistically significant differences between groups were calculated using two-way ANOVA, followed (if significant) by an LSDtest.

RESULTS The effect of acute i.p. administration of LA and reserpine, separately and jointly, on the striatal levels of the total, reduced and oxidized glutathione The total glutathione (GSH⫹GSSG) levels in the striatum did not differ significantly between groups of rats treated with LA or reserpine alone. However, the combined administration of both these compounds caused a significant increase in its concentration when compared with the control (by 16.7%) and the reserpine-treated (by 17.1%) groups (Fig. 1A). When the GSH was analyzed, it was found that its level was significantly decreased in the reserpine-treated group (by 14.5% of LA-treated group), but it was markedly enhanced in that receiving LA jointly with reserpine (by 15.4% of control and by 29.3% of the reserpine-treated groups, respectively; Fig. 1B). The striatal concentration of the GSSG was not changed in the group treated with LA alone while in that administered reserpine it was considerably elevated (by 172% of control and 183% of LA-treated group). Pretreatment of rats receiving reser-

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Fig. 1. The effect of LA (50 mg/kg i.p.) and reserpine (5 mg/kg i.p.) administration, alone or in combination, on levels of total (A, D), reduced (B, E) and oxidized (C, F) glutathione in the striatum (A–C) and prefrontal cortex (D–F) of rat brain. The results are presented as the mean and S.E.M., n⫽8 –10 for each group of rats. (A) In the striatum a two-way ANOVA showed a lack of reserpine (R) effect [F(1,28)⫽1.708, NS], an overall treatment effect of LA (L) [F(1,28)⫽4.909, P⬍0.05] on total glutathione and no interaction between LA and reserpine (LR) [F(1,28)⫽1.927, NS]. (B) No effect of reserpine (R) [F(1,28)⫽0.0006, NS], a significant effect of LA (L) [F(1,28)⫽9.918, P⬍0.005] on reduced glutathione and interaction between LA and reserpine (LR) [F(1,28)⫽6.741, P⬍0.05]. (C) A significant general treatment effect of reserpine [F(1,28)⫽23.180, P⬍0.00005] and LA [F(1,28)⫽9.526, P⬍0.005] on oxidized glutathione, as well as interaction between these two compounds [F(1,28)⫽8.504, P⬍0.01]. (D, E) In prefrontal cortex a two-way ANOVA showed lack of reserpine [F(1,36)⫽0.322, NS], [F(1,36)⫽0.103, NS] and LA [F(1,36)⫽3.648, P⫽0.064], [F(1,36)⫽3.806, P⫽0.058] effects on total and reduced glutathione as well as no interaction between these two compounds [F(1,36)⫽3.585, P⫽0.066], [F(1,36)⫽3.657, P⫽0.064], respectively. (F) An overall treatment effect of reserpine [F(1,36)⫽12,801, P⬍0.001], no effect of lipoic acid [F(1,36)⫽0.152, NS] on oxidized glutathione and no interaction between these compounds [F(1,36)⫽0.460, NS]. Symbols indicate significance of differences in LSD test, * P⬍0.05, ** P⬍0.01, *** P⬍0.0001 vs. control; ⌬ P⬍0.05, ⌬⌬ P⬍0.01, ⌬⌬⌬ P⬍0.0001 vs. LA; and # P⬍0.05, ### P⬍0.001 vs. reserpine-treated groups.

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pine with LA prevented the increase in the GSSG content. Its level was markedly lower than in the reserpine-treated group and approximated that observed in the control and LA-treated groups (Fig. 1C). The effect of acute i.p. administration of LA and reserpine, alone and in combination, on the levels of the total, reduced and oxidized glutathione in the rat prefrontal cortex In the prefrontal cortex, concentrations of the total (GSH⫹GSSG) and reduced form of glutathione were significantly changed neither by LA nor by reserpine. However, when these compounds were administered jointly an increasing tendency in the levels of total and reduced glutathione was observed (by 34.8 and 36% of the reserpine-treated group; Fig. 1D, 1E). Administration of LA alone did not affect GSSG content in the prefrontal cortex. Reserpine, similarly like in the

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striatum, increased GSSG level markedly (by 62% of control and 73% of LA-treated groups; Fig. 1F). However, when LA and reserpine were administered jointly, GSSG level contrary to the striatum, was still significantly higher than in the control and LA-treated groups (by 85 and 97% respectively; Fig. 1F). The effect of acute i.p. administration of LA and reserpine, separately and jointly, on the striatal and prefrontal cortex levels of NO and S-nitrosothiols The levels of NO in the striatum and prefrontal cortex were not altered significantly by LA given alone whereas reserpine enhanced their contents markedly both when compared with the control and LA-treated groups (Fig. 2A, 2C). LA administered jointly with reserpine lowered NO levels almost to the control values in either structure (Fig. 2A, 2C).

Fig. 2. The influence of LA (50 mg/kg i.p.) and reserpine (5 mg/kg i.p.) administration, alone or in combination, on levels of NO (A, C) and S-nitrosothiols (B, D) in the rat striatum (A, B) and prefrontal cortex (C, D). The results are presented as the mean and S.E.M., n⫽8 –10 for each group of rats. (A) In the striatum a two-way ANOVA showed an overall treatment effect of reserpine (R) [F(1,32)⫽6.941, P⬍0.01], a lack of LA (L) effect [F(1,32)⫽2.926, P⫽0.097] on NO level and an interaction between LA and reserpine (LR) [F(1,32)⫽9.897, P⬍0.005]. (B) No effect of reserpine (R) [F(1,20)⫽1.199, NS], a significant effect of LA (L) [F(1,20)⫽6.356, P⬍0.02] on S-nitrosothiols and no interaction between these two compounds (LR) [F(1,20)⫽0.314, NS]. (C) In the prefrontal cortex a two-way ANOVA showed an overall treatment effects of reserpine (R) [F(1,20)⫽6.823, P⬍0.01] and LA (L) [F(1,32)⫽9.069, P⬍0.005] on NO level and no interaction between these two compounds (LR) [F(1,32)⫽0.540, NS]. (D) An overall treatment effect of reserpine (R) [F(1,20)⫽10.229, P⬍0.005], a lack of LA (L) effect [F(1,20)⫽2.902, NS] on S-nitrosothiols and no interaction between LA and reserpine (LR) [F(1,20)⫽1.573, NS]. Significance of differences in LSD test, * P⬍0.01, *** P⬍0.0005 vs. control; ⌬ P⬍0.05, ⌬⌬ P⬍0.01 vs. LA; and # P⬍0.05, ## P⬍0.005 vs. reserpine-treated groups.

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In the striatum, level of S-nitrosothiols was not affected by LA administered alone but in the prefrontal cortex such treatment produced a marked decrease in their content (by 40% of control; Fig. 2B, 2C). In both structures, reserpine administered alone did not evoke significant changes in the concentrations of S-nitrosothiols when compared with appropriate controls, but their levels were markedly higher than in the LA-treated groups. LA given jointly with reserpine did not produce significant changes in the contents of S-nitrosothiols in either structure when compared with appropriate controls. However, in the striatum S-nitrosothiol level was lower than in the reserpine-treated group while in the prefrontal cortex it was higher than in the LA-treated group (Fig. 2B, 2C). The enzymatic activities of GPx, GST and ␥-GT in the striatum after acute, i.p. administration of LA and reserpine, alone or in combination GPx activity in the striatum was slightly but significantly increased by reserpine while the combined administration of LA and reserpine evoked dramatic increase in the activity of this enzyme (by 185% of control, 155% of LA- and 112% of reserpine-treated groups; Fig. 3A). The striatal activity of GST was markedly enhanced by LA administered alone (by 88% of control) as well as by its combination with reserpine (by 59% of control and 73% of reserpine-treated group; Fig. 3B). Enzymatic activity of ␥-GT was not changed by treatment with LA whereas reserpine alone decreased it significantly in comparison to the control group (by 28.4%). Administration of LA before reserpine prevented the lowering in the ␥-GT enzymatic activity in the rat striatum (Fig. 3C). The enzymatic activities of GPx, GST and ␥-GT in the rat prefrontal cortex after acute, i.p. administration of LA and reserpine, alone and in combination Enzymatic activity of GPx in the prefrontal cortex was markedly decreased by reserpine but only when compared with the lipoate-treated group. Lipoate administered jointly with reserpine maintained activity of this enzyme at control level (Fig. 3D). GST activity was significantly lower in groups receiving reserpine alone and in combination with LA than in control. Also in rats treated only with LA, a downward tendency in GST activity was observed (Fig. 3E). Activity of ␥-GT was not altered in any of the examined groups (Fig. 3F). PSSG levels in the striatal and prefrontal cortex homogenates incubated in vitro with reserpine, tBOOH and LA, separately and jointly In order to optimize the analytical assay for in vitro formation of PSSGs in the striatal and prefrontal cortex homogenates incubated with reserpine, tBOOH and LA, separately or jointly, first different concentrations of the above compounds and different reaction time were studied. For the final experiment, the optimal conditions were selected.

Reserpine incubated for 20 min with the striatal homogenates evoked a significant increase in PSSG level (by 32.7% of control) while LA at the same conditions lowered it markedly (by 37.1% of control and 52.6% the reserpinetreated homogenates). However, the most pronounced decrease in PSSG level was observed when reserpine and LA were incubated jointly (by 87.5% and 73.6% of the reserpine- and LA-treated homogenates, respectively) (Fig. 4A). In contrast to striatal homogenates, incubation of the prefrontal cortex homogenates with reserpine and LA both separately and jointly did not cause significant changes in PSSG levels although some slight decreasing tendency in homogenates incubated with LA alone and in combination with reserpine was observed (Fig. 4C). In additional experiments, it was also found that that incubation of tBOOH with both striatal and prefrontal cortex homogenates produced significant increases in PSSG levels when compared with control while the combined administration of this compound with LA decreased it distinctly (by 33.6 and 43.4% of the tBOOHtreated striatal and prefrontal cortex homogenates, respectively) (Fig. 4B, 4C).

DISCUSSION The present study demonstrated for the first time that LA affected in a different manner the GSH defense system in the examined dopaminergic structures of rat brain under conditions of the reserpine-induced oxidative stress. The striatum and the prefrontal cortex are brain regions that differ markedly in respect of DA concentrations. The striatum densely innervated by dopaminergic projection arising from the SNc is the richest in DA content brain structure. Contrary to the striatum, the prefrontal cortex, that receives spare DA innervation from the ventral tegmental area (VTA), is a brain region characterized by the lowest level of DA, so it may be considered as a negative control. Reserpine is known to exert its monoamine-depleting action by blocking the ATP-dependent uptake mechanism of the storage organelles. Such reserpine activity results in the accelerated presynaptic turnover of the cytoplasmic DA that is associated with the increased production of hydrogen peroxide thereby causing distinct alternation in the redox status of dopaminergic nerve terminals, measured as concentrations of GSH and GSSG (Spina and Cohen, 1988, 1989). Administration of reserpine to rats increased dramatically levels of GSSG and significantly decreased its reduced form in the striatum. Similar effects, although much less pronounced were observed in the prefrontal cortex. Previously, a considerable rise in the striatal GSSG level was reported by Spina and Cohen (1989) in the reserpine-treated mice. Moreover, in that study it was demonstrated that inhibition of MAO type A by clorgyline, blocked the formation of GSSG confirming that its increase was related to the oxidative DA catabolism and was restricted mainly to the nigrostriatal, dopaminergic terminals. However, in contrast to our data, those authors did not find any changes in GSSG content in the mouse frontal cortex.

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Fig. 3. The effect of LA (50 mg/kg i.p.) and reserpine (5 mg/kg i.p.) administration, alone or in combination, on enzymatic activities of GPx (A, D), GST (B, E) and ␥-GT (C, F) in the striatum (A–C) and prefrontal cortex (D–F) of rat brain. The results are presented as the mean and S.E.M., n⫽8 –10 for each group of rats. (A) In the striatum a two-way ANOVA showed significant treatment effect of reserpine (R) [F(1,20)⫽86.886, P⬍0.0001], and LA (L) [F(1,20)⫽52.866, P⬍0.0001] on GPx activity as well as interaction between these two compounds (LR) [F(1,20)⫽38.535, P⬍0.0001]. (B) No effect of reserpine (R) [F(1,28)⫽0.753, NS], a significant effect of LA (L) [F(1,28)⫽13.605, P⬍0.001] on GST activity and no interaction between these two compounds (LR) [F(1,28)⫽0.628, NS]. (C) No effect of reserpine (R) [F(1,28)⫽1.899, NS] and LA (L) [F(1,28)⫽0.433, NS]on ␥-GT activity and a significant interaction between LA and reserpine (LR) [F(1,28)⫽6.153, P⬍0.05]. (D) In the prefrontal cortex a two-way ANOVA showed no effect of reserpine (R) [F(1,20)⫽1.462, NS], a significant effect of LA (L) [F(1,20)⫽5.082, P⬍0.05] on GPx activity and no interaction between these two compounds (LR) [F(1,20)⫽0.939, NS]. (E) An overall treatment effect of reserpine (R) [F(1,20)⫽7.428, P⬍0.01], a lack of LA (L) effect [F(1,20)⫽0.787, NS] on GST activity and no interaction between LA and reserpine (LR) [F(1,20)⫽2.175, NS]. Symbols indicate significance of differences in LSD test, * P⬍0.05, ** P⬍0.01, *** P⬍0.0001 vs. control; ⌬ P⬍0.05, ⌬⌬ P⬍0.01, ⌬⌬⌬ P⬍0.0001 vs. LA; and # P⬍0.05, ### P⬍0.001 vs. reserpine-treated groups.

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Fig. 4. Concentrations of PSSG in the striatal (A, B) and prefrontal cortex (C, D) homogenates incubated in vitro with reserpine (R), tBOOH (tB) and LA (L) alone or in combination. Data are presented as the mean and S.E.M, n⫽6 –10 per each group. (A) In the striatal homogenates, a two-way ANOVA showed a lack of reserpine effect [F(1,20)⫽)0.732, NS], an overall treatment effect of LA [F(1,20)⫽91.087, P⬍0.00001] on PSSG level and an interaction between reserpine and LA [F(1,20)⫽24.248, P⬍0.0001]. (B) An overall treatment effect of tBOOH [F(1,20)⫽318.317, P⬍0.0001] and LA [F(1,20)⫽61.092, P⬍0.0001] on PSSG level as well as interaction between these two compounds [F(1,20)⫽11.219, P⬍0.005] (C) In the prefrontal cortex homogenates, a two-way ANOVA showed a lack of both reserpine [F(1,31)⫽0.134, NS] and LA [F(1,31)⫽3.395, P⫽0.075] effects on PSSG as well as no interaction between reserpine and LA [F(1,31)⫽0.103, NS]. (D) No effect of tBOOH [F(1,25)⫽0.534, NS], a significant effect of LA [F(1,25)⫽84.363, P⬍0.00001] on PSSG and interaction between tBOOH and LA [F(1,25)⫽31.862, P⬍0.0001]. Symbols indicate significance of differences in LSD test, * P⬍0.01, ** P⬍0.001, *** P⬍0.0001 vs. control; ⌬⌬ P⬍0.005, ⌬⌬⌬ P⬍0.0001 vs. LA; ### P⬍0.0001 vs. reserpine- or tBOOH-treated groups.

Apart from the changes in DAergic system, reserpine evokes also depletion of noradrenaline (NA), so one can expect that an increase in GSSG level may also occur in terminals of noradrenergic projections innervating both examined brain regions. It is well known that NA concentration in the prefrontal cortex is much higher than in the striatum. Moreover, it has been evidenced that NA autoxidation to NA-orthoquinone and hydroquinone is a potent source of free radicals (Graham, 1978; Linderson et al., 1994). Hence, an enhancement of GSSG content in that structure, may be in part, a consequence of the latter reaction. On the other hand, it is worth underlining that even though NA concentration is higher in the prefrontal cortex, the total concentration of reactive oxygen species generated in that structure in the presence of reserpine, especially hydrogen peroxide, paralleled by GSSG level, was considerably lower than in the striatum.

LA given twice, before and after reserpine, decreased GSSG concentration in the rat striatum to almost control level while in the prefrontal cortex its content was not changed by this drug although an increase in GSH level was found in either structure. Restoring the striatal pool of GSH by LA can be partially explained by the difference in the redox potential between LA/DHLA (E0⫽⫺0.32V) and GSSG/GSH (E0⫽⫺0.24V) systems (Smith et al., 2004). However, a marked increase in GSH content induced by this drug cannot result exclusively from the enhanced GSSG reduction because, as our calculations showed, the striatal concentration of GSSG in the reserpine-treated group constituted 7.7% of total pool of glutathione while GSH increment caused by the combined treatment was much higher (15 and 29% of total glutathione levels for control and the reserpine-treated groups, respectively). An increase in GSH content that reached even 70% of control

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values was also found in some cell lines treated with LA (Busse et al., 1992). All these results suggest that LA not only restores GSH level but also stimulates its synthesis. In line with the latter view, it has been demonstrated that LA increases de novo synthesis of cellular GSH by improving cystine utilization (Han et al., 1997). As in our study the enhancement of GSH content was observed 2 h after the combined injection of LA and reserpine, it is reasonable to assume that the abovementioned mechanism may play a pivotal role in that process. However, it is difficult to explain why LA did not decrease the GSSG level enhanced by reserpine in the prefrontal cortex, although it caused some slight but nonsignificant increase in GSH content. It seems that structure-dependent concentration of catecholamines that is closely related to the level of oxidative stress evoked by reserpine may play a decisive role here. Our results, to some extent, support this view by showing that the coadministration of LA and reserpine differentially affects activity of GPx, an enzyme that together with catalase, provides the first line of defense against hydrogen peroxide. In the GPx-catalyzed reaction, GSH acts as a cofactor mediating hydrogen peroxide reduction with concomitant formation of GSSG. In the striatum, activity of this enzyme was significantly enhanced by reserpine alone and further intensified when this compound was administered jointly with LA. The LA-induced increase in GPx activity in that structure indicates that under conditions of oxidative stress this enzyme effectively scavenges an excess of hydrogen peroxide produced during DA catabolism protecting the nigrostriatal dopaminergic terminals against its toxicity. Moreover, since the GSSG level in the striatum was significantly decreased by LA upon the combined treatment, reaching a value approximating that found in control group, we assumed, although we did not measure it, that also activity of GR which in the presence of NADPH regenerates GSH from GSSG, was up-regulated in that structure. In line with the abovementioned supposition, an enhancement of GR activity in different brain regions in response to LA has been described in old rats (Arivazhagan et al., 2001, 2002). However, in the prefrontal cortex the basal GPx activity, which was markedly higher than in the striatum, was not altered by LA supplementation, neither when it was administered alone nor in combination with reserpine. Hence, we believed that physiological activity of GPx in that structure was sufficient to remove hydrogen peroxide generated during DA and NA metabolism that due to low content of these neurotransmitters was less extensive than in the striatum, even under conditions of oxidative stress. However, the increased concentration of GSSG in the prefrontal cortex let us suppose that also activity of GR was not affected by LA. What is more, it may signify that in contrast to GPx, the basal GR activity was too low to reduce the almost double amount of GSSG produced as a result of reserpine action. Besides the influence of LA on GPx activity, this compound also affects enzymes of phase II of detoxification, such as GST and a flavoenzyme NAD(P)H:quinone oxidoreductase (NQO), also referred to as DT-diaphorase

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(Flier et al., 2002). GSTs are a multigene family of isoenzymes that catalyze reactions of detoxification of electrophilic xenobiotics by their coupling with GSH yielding Sconjugates (Hayes et al., 2005). Also o-quinones that are formed during autoxidation of catecholamines are conjugated with GSH in the presence of class Mu GST, in particular by isoenzyme M2-2 (Baez et al., 1997; SeguraAguilar et al., 1997). This isoenzyme preventing redox cycling of catecholamine-derived o-quinones provides efficient protection against their deleterious activity (Dagnino-Subiabre et al., 2000). In addition to GST, also NQO via two-electron reduction of the redox-labile o-quinones is implicated in their detoxification (van Muiswinkel et al., 2000). Recently, it has been demonstrated that enzymatic activities of GST and NQO are induced by LA in cultured astroglial cells (Flier et al., 2002). Moreover, it has been found that the enhanced activities of both these enzymes contribute to antioxidant effects of estradiol in the rat striatum but not in the cortex (Stakhiv et al., 2006). Like in our study, LA administered both alone and jointly with reserpine, through increasing GST activity in the striatum may protect this structure against noxious effects of DAderived quinones. However, in the prefrontal cortex LA does not seem to fulfill this function since activity of this enzyme was decreased. ␥-GT, an enzyme responsible for the extracellular cleavage of ␥-glutamyl bound within the GSH molecule, whose activity is markedly enhanced in the SN of parkimsonian patients (Sian et al., 1994), has been also examined in our study. Reserpine administration lowered activity of this enzyme in the striatum while LA reversed this effect. Activity of ␥-GT in the prefrontal cortex was affected neither by reserpine nor by LA administered alone or in combination. All these results indicate that enzymes implicated in glutathione metabolism are regulated by LA in a different way in the striatum and prefrontal cortex under conditions of reserpine-induced oxidative stress. Nowadays, there is a great controversy regarding the possible contribution of NO to the neurodegeneration of DA neurons in PD. Several studies have suggested that NO is a toxic molecule mediating death of DA cells (Przedborski et al., 1996; LaVoie and Hastings, 1999) whereas other researchers believe that its antioxidant and antiapoptotic properties could mediate neuroprotection (Kagan et al., 2001; Sharpe et al., 2003; Rauhala et al., 2005). Namely, NO generated by NO synthase (NOS) or released from GSNO my up-regulate antioxidative thioredoxin system through a cGMP-dependent mechanism (Andoh et al., 2003; Arner and Holmgren, 2000). Moreover, NO as a nitrogen-centered radical can exert a direct antioxidant effect through its reaction with oxygen-centered radicals and iron, blocking iron-induced hydroxyl radical generation (Kanner et al., 1991) and lipid peroxidation (Rauhala et al., 1996). In addition, GSNO, that is thought to be an NO storage and transport form, protects brain cells against peroxynitrate-induced lipid peroxidation and ironinduced oxidative stress both in vitro and in vivo (Rauhala et al., 1998; Chiueh and Rauhala, 1999). In our study, the concentrations of NO in the striatum and prefrontal cortex

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increased in response to the reserpine-induced oxidative stress while LA decreased their contents in either structure to control levels. It should be added that LA is not a direct NO scavenger, but it can scavenge NO acting via DHLA (k⫽3.19 M⫺1 s⫺1) (Vriesman et al., 1997). Incidentally, it is worth mentioning that LA is reduced in the cells to DHLA by NADH-dependent enzymes such as dihydrolipoate dehydrogenase, GR and thioredoxin reductase (Biewenga et al., 1997; Bustamante et al., 1998). It seems that at least dihydrolipoate dehydrogenase contributes to LA effect upon NO concentrations in the preset study, however, it cannot be excluded that also two other enzymes may play some role here. In both examined structures, reserpine did not cause significant changes in S-nitrosothiol levels when compared with control, but their concentrations were markedly higher than in the LA-treated groups. LA given jointly with reserpine did not affect significantly S-nitrosothiol levels when compared with appropriate controls in either structure. However, in the striatum its concentration enhanced by reserpine was lowered by LA to control level while in the prefrontal cortex it was unchanged. It has been demonstrated that GPx can catalyze decomposition of S-nitrosothiols (Hou et al., 1996). Hence, it is likely that different activity of this enzyme found in the present study may have some implications for S-nitrosothiols levels in the examined brain structures. Interestingly, GPx activity corresponded well to S-nitrosothiol levels both in the striatum and prefrontal cortex of all examined groups. On the other hand, S-nitrosothiols can be formed under various conditions and in different reactions (Gow et al., 1997; Balazy et al., 1998; Van der Vliet et al., 1998), therefore, it is difficult unequivocally explain the difference in their concentrations between the striatum and prefrontal cortex. However, since in the prefrontal cortex LA did not evoke stimulation of enzymes of GSH defense system (GPx and GST) one can speculate that GSNO, that is the most abundant endogenous S-nitrosothiol and a very potent antioxidant, may contribute to neuroprotection in this structure against consequences of the reserpine-induced oxidative stress. TH is an enzyme that plays a crucial role in the DA homeostasis. As mentioned in the introduction, TH activity can be markedly reduced by S-glutathionylation (Borges et al., 2002). Hence, to show neuroprotective effect of LA on this enzyme, we determined PSSG level and recognized it as a marker of TH activity. At physiological conditions, protein sulfhydryl groups are typically maintained in the reduced state which means that functions of these proteins are preserved. In contrast, accumulation of PSSG at a variety of oxidative conditions denotes that some of these proteins lost their physiological functions (Giustarini et al., 2004). One of several mechanisms that have been proposed for PSSG formation is a thiol/disulfide exchange reaction. This mechanism is particularly interesting in relevance to our study because reserpine markedly enhances GSSG level which makes formation of PSSGs more likely. In fact reserpine incubated with the striatal homogenates produced a significant increase in PSSG content while LA reduced it drastically. These results

clearly demonstrate that LA restores protein functions inhibited earlier by S-glutathionylation. This effect refers also to TH that occurs at a high concentration in the striatal dopaminergic terminals. A very low level of PSSGs in the striatal homogenates after combined incubation with reserpine and LA suggests that most TH is in an active form. Besides TH, also activity of ␥-GT which was examined in our study can be regulated by S-glutathionylation (Dominici et al., 1999). It seems that our results confirm such regulation because activity of this enzyme was reduced by reserpine while LA restored its function to control level. In contrast to striatal homogenates, incubation of reserpine with prefrontal cortex homogenates did not evoke any increase in PSSG content although this drug caused some slight enhancement of GSSG level. We suppose that the level of oxidative stress produced in that structure by reserpine was too low to alter functions of proteins containing sulfhydryl groups. This assumption is in line with the lack of reserpine effect on ␥-GT activity in the prefrontal cortex. On the other hand, tBOOH, a widely used oxidant, induced significant increases in PSSG levels both in the striatal and prefrontal cortex homogenates while LA attenuated these effects in either homogenate. Glutaredoxin plays a very important role in maintenance of physiological functions of sulfhydryl proteins. This enzyme requires, however, an optimal concentration of GSH to be effective. Hence, the cooperation of glutaredoxin with LA that regenerates GSH from GSSG is of key significance for regeneration of PSSGs. It seems that such cooperation exists in the striatum in our study. It has been described that PSSG formation can be promoted by NO, through various mechanisms including the formation of GSNO and thiyl radical (Klatt and Lamas, 2000; Okamoto et al., 2001). Hence, decreasing of NO content by LA during reserpine-induced oxidative stress can be considered to be neuroprotective.

CONCLUSION In conclusion, our results confirm a beneficial effect of LA in alleviating results of oxidative stress associated with the enhanced DA catabolism in the rat striatum. Administration of this drug strengthens the GSH defense system of that structure by increasing both GSH level as well as activities of two important detoxifying enzymes, i.e. GPx and GST. LA protects also TH against loss of its physiological function. It seems that such a mode of action makes LA a useful drug for treatment of PD since oxidative stress plays a central role in the pathogenesis of this disease. To make a better use of unique properties of LA, recently a series of multifunctional co-drugs obtained by joining L-DOPA and DA with (R)-LA was synthesized to overcome the prooxidant effect associated with L-DOPA replacement therapy (Di Stefano et al., 2006). It seems that such a form of L-DOPA treatment will be more efficient and safer for PD patients. Acknowledgments—This work was supported by the Polish Ministry of Education and Sciences, grant no. 2PO5F 010 27.

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(Accepted 1 April 2007) (Available online 2 May 2007)