(l-DOPA) and Inhibition of l-DOPA Decarboxylase in the Rat Striatum

1372

Chem. Res. Toxicol. 2003, 16, 1372-1384

The Parkinsonian Neurotoxin 1-Methyl-4-Phenylpyridinium (MPP+) Mediates Release of L-3,4-Dihydroxyphenylalanine (L-DOPA) and Inhibition of L-DOPA Decarboxylase in the Rat Striatum: A Microdialysis Study Steven B. Foster, Monika Z. Wrona, Jilin Han, and Glenn Dryhurst* Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma 73019-0370 Received April 3, 2003

Reactive oxygen species (ROS) and reactive nitrogen species (RNS), particularly peroxynitrite, have been implicated as key participants in the dopaminergic neurotoxicity of 1-methyl-4phenylpyridinium (MPP+). However, on the basis of available information, it is not clear whether the MPP+-induced overproduction of ROS and RNS occurs in the intraneuronal and/ or extracellular compartment. Early steps in the neurotoxic mechanism evoked by MPP+ include a profound dopaminergic energy impairment, which mediates a massive release of dopamine (DA), glutathione (GSH), and cysteine (CySH). In the event that MPP+ mediates extracellular generation of ROS (such as superoxide and/or hydroxyl radicals) and/or peroxynitrite, released DA, GSH, and CySH should be oxidized forming thioethers of DA and disulfides. Using microdialysis experiments in which MPP+ was perfused into the striatum of awake rats, the present study was unable to detect the presence of such biomarkers of extracellular ROS and/ or RNS generation. However, MPP+ induced a transient, concentration-dependent rise of extracellular L-3,4-dihydroxyphenylalanine (L-DOPA), identified on the basis of dialysate analysis using several HPLC methods and its conversion to DA by purified L-DOPA decarboxylase (DDC). Methamphetamine (30 mg/kg, i.p.) similarly caused a significant but transient rise of L-DOPA in the rat striatum. Antioxidants such as salicylate and mannitol had no effect on the MPP+-mediated elevation of extracellular L-DOPA, suggesting that it is not formed by nonenzymatic hydroxylation of L-tyrosine by ROS or RNS. Rather, in vivo, but not in vitro, MPP+ caused rapid inhibition of DDC, which appears to result in intraneuronal accumulation and subsequent release of L-DOPA. Because L-DOPA can mediate L-glutamate release, as well as be an excitotoxin, the possibility is raised that L-DOPA may play a role in the dopaminergic neurotoxicity of MPP+.

Introduction MPTP1

has been used extensively to produce an experimental animal model of PD (1). Thus, MPTP mediates selective degeneration of nigrostriatal DA * To whom correspondence should be addressed. Tel: (405)325-4811. Fax: (405)325-6111. E-mail: [email protected]. 1 Abbreviations: aCSF, artificial cerebrospinal fluid; AMPA, R-amino3-hydroxy-5-methylisoxazole-4-propionic acid; CySH, L-cysteine; DA, dopamine; DAQ, dopamine-o-quinone; DAT, dopamine plasma membrane transporter; DDC, L-DOPA decarboxylase; 2,3-DHBA, 2,3dihydroxybenzoic acid; 2,5-di-S-CyS-DA, 2,5-di-S-cysteinyldopamine; 2,5-di-S-GS-DA, 2,5-di-S-glutathionyldopamine; 2,5-di-S-GS-DOPAC, 2,5-di-S-glutathionyl-3,4-dihydroxyphenylacetic acid; DOPAC, 3,4dihydroxyphenylacetic acid; DTT, dithiothreitol; FDNB, 1-fluoro-2,4dinitrobenzene; Glu, L-glutamate; GSH, glutathione; GSSCy, glutathione cysteine disulfide; GSSG, glutathione disulfide; 5-HIAA, 5-hydroxyindole-3-acetic acid; HO•, hydroxyl radical; HVA, homovanillic acid; 5-HT, 5-hydroxytryptamine (serotonin); iNOS, inducible nitric oxide synthase; L-DOPA, L-3,4-dihydroxyphenylalanine; MAO, monoamine oxidase; MeCN, acetonitrile; MeOH, methanol; MPP+, 1-methyl4-phenylpyridinium; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; 3-MT, 3-methoxytyramine; nNOS, neuronal nitric oxide synthase; •NO, nitric oxide; O2-•, superoxide anion radical; PD, Parkinson’s disease; 5-S-CyS-DA, 5-S-cysteinyldopamine; 5-S-GS-DA, 5-S-glutathionyldopamine; 5-S-GS-DOPAC, 5-S-glutathionyl-3,4-dihydroxyphenylacetic acid; SN, substantia nigra; SOS, sodium 1-octanesulfonic acid; TFA, trifluoroacetic acid; TH, tyrosine hydroxylase; 2,5,6-tris-S-GS-DA, 2,5,6-tris-S-glutathionyldopamine.

neurons (2) and, in humans, a clinical syndrome indistinguishable from PD (3). The first step in the mechanism underlying the dopaminergic neurotoxicity of MPTP is its oxidation by MAO-B in astrocytes to its active metabolite MPP+ (4). Following release from astrocytes, extracellular MPP+ is transported into dopaminergic neurons by the DAT (5). Then, in an energy-dependent process, MPP+ is concentrated into mitochondria (6) where it reversibly inhibits complex I respiration (7) and hence ATP synthesis (8). By blocking the mitochondrial electron transport chain (9) and other mechanisms (10), MPP+ stimulates increased intraneuronal production of superoxide (O2-•), a key participant in the neurotoxic mechanism (11). Part of this intraneuronal O2-• reacts with •NO forming peroxynitrite, which nitrates and inactivates TH (12-14) and damages DNA (15). However, nigrostriatal DA neurons do not express nNOS (16). Thus, the •NO required for intraneuronal peroxynitrite synthesis must be produced by other cells. In the striatum, MPP+ mediates •NO production by nNOS positive neurons that colocalize with somatostatin and neuropeptide Y (16, 17). In the SN, the source of •NO appears to be activated microglia, which express iNOS (18). Thus,

10.1021/tx030015l CCC: $25.00 © 2003 American Chemical Society Published on Web 10/02/2003

MPP+ Mediates Release of Striatal L-DOPA

MPTP/MPP+ must mediate significant trafficking of •NO from nNOS positive neurons in the striatum into dopaminergic terminals and from activated microglia into neurons in the SN where it reacts with O2-• forming intraneuronal peroxynitrite. In fact, MPTP/MPP+ induces a strong glial reaction in both the striatum and the SN (18), which could also result in their generation of O2-• (19). Thus, released •NO might react with O2-• in the extracellular compartment to form peroxynitrite, which could damage dopaminergic terminals and cell bodies. Microdialysis experiments reveal that perfusion of MPP+ into the rat striatum results in the nonenzymatic hydroxylation of coadministered salicylate to 2,3-DHBA by HO• (20) or, more probably, peroxynitrite (21, 22), which possesses HO•-like activity (23). However, it is not clear whether this HO•- and/or peroxynitrite-mediated salicylate hydroxylation occurs extracellularly or intraneuronally. In the event that MPP+ induces the generation of extracellular HO• or peroxynitrite, these oxidants would be expected to react with endogenous species to form characteristic metabolites. In view of the possibility that MPTP/MPP+ evokes nigrostriatal dopaminergic neurotoxicity by a mechanism similar to that underlying PD, such metabolites might be of interest, for example, as clinical biomarkers of early stage PD. Furthermore, such metabolites might play roles in the neurotoxic process. In this regard, it is known that peroxynitrite can nitrate L-tyrosine, present in the brain at low micromolar extracellular concentrations (24). Furthermore, peroxynitrite can hydroxylate L-tyrosine (25, 26) as can HO• (27). However, in vitro, nitration of L-tyrosine by peroxynitrite is completely blocked by equimolar or higher concentrations of DA, which is preferentially oxidized to DAQ (28). This is of interest because MPP+ evokes a very rapid and massive release of DA (29). Furthermore, when the oxidation of DA by peroxynitrite occurs in the presence of GSH, also released by MPP+ (30), DAQ is scavenged to form glutathionyl conjugates of DA (31, 32). Similarly, CySH, also released by MPP+ (30), reacts with DAQ forming cysteinyl conjugates of DA (33), which have neurotoxic properties (34). Incubation of DA with GSH or CySH and a HO•-generating system also leads to formation of DA thioethers (35). Thus, generation of extracellular HO• and/or peroxynitrite by MPP+ would be expected to result in formation of characteristic thioethers of DA together with disulfides formed by oxidation of GSH and CySH. When incubated for 48 h with cultures of rat mesencephalic neurons, quite low concentrations of MPP+ (510 µM) cause the selective destruction of dopaminergic neurons (36, 37). However, in microdialysis experiments, 1-10 mM concentrations of MPP+ are usually perfused into the awake rat striatum for 15-30 min in order to cause significant dopaminergic neurotoxicity in the vicinity of the probe (20-22, 38, 39). Thus, in this paper, the results of microdialysis experiments are presented indicating that perfusion of such neurotoxic concentrations of MPP+ into the striatum of awake rats does not lead to formation of metabolites expected as a result of the oxidation of released DA, GSH, and CySH by peroxynitrite or HO•. However, MPP+ induces a concentrationdependent rise of extracellular L-DOPA. This rise of extracellular L-DOPA does not appear to be caused by nonenzymatic hydroxylation of L-tyrosine by HO• or peroxynitrite but rather by inhibition of DDC.

Chem. Res. Toxicol., Vol. 16, No. 10, 2003 1373

Materials and Methods Animals. Adult male albino Sprague-Dawley rats (Harlan Sprague-Dawley, Madison, WI) weighing 330-350 g were used. During surgery, animals were maintained under ketamine anesthesia (85 mg/kg, i.p.). Animals were placed in a stereotaxic frame with the nose bar positioned 3.3 mm below the interaural line. A midsagital incision was made with a surgical scalpel, and the skull was exposed. Following bone trapenization, a CMA (Stockholm, Sweden) type 12 microdialysis probe guide and guide cannula were implanted into the right striatum (2.8 mm lateral and 0.5 mm anterior to bregma) with the tip positioned 3.4 mm below dura and secured to the skull with three screws and cranioplastic cement. In some experiments, probe guides/ guide cannulas were implanted into both the left and the right striatum using the coordinates described above. Animals were allowed 3 days to recover from surgery prior to microdialysis experiments. During this period, they were housed individually with free access to food and water with a 12 h light-dark cycle (lights on at 7:00 am). On the day of experiments using MPP+, the guide cannula was replaced by a microdialysis probe (CMA 12, 4 mm membrane) with the tip positioned 7.4 mm below dura. Throughout microdialysis experiments, rats were housed in a 40 cm diameter Plexiglas bowl seated on a BAS (Bioanalytical Systems, West Lafayette, IN) Raturn and hence were able to move freely and had free access to food and water. For experiments with methamphetamine (MA), guide cannulas were stereotaxically implanted into both the right and the left striata as described above. On the day of the experiment, the guide cannula in the right striatum was replaced by a CMA 12 (4 mm tip) probe with the tip positioned 7.4 mm below dura. The left guide cannula was replaced with a Teflon-coated copper/constantan temperature probe (Type T, Physitemp Instruments, Clifton, NJ) with the tip positioned 7.4 mm below dura. Brain temperature was monitored using a Physitemp Thermalert TH-8. All animal experiments were approved by the Institutional Animal Care and Use Committee at the University of Oklahoma. Microdialysis Procedure to Monitor Biogenic Amines and Metabolites. Implanted microdialysis probes were initially perfused with aCSF (147 mM NaCl, 2.7 mM KCl, 1.2 mM CaCl2, and 0.85 mM MgCl2 in deionized water) at a flow rate of 1.5 µL/min using a CMA 100 microinjection pump. Dialysate was collected in a CMA 170 refrigerated (4 °C) microfraction collector in which each vial, except where noted otherwise, contained a volume of 0.2 M HCl equivalent to one-fourth the volume of dialysate to be collected. Basal dialysate levels of DA, 5-HT, and their metabolites were determined in fractions collected at 15 min intervals (22.5 µL). After analysis of dialysates gave at least three consecutive constant basal levels of these neurochemicals (ca. 3 h), a BAS Uniswitch Syringe Selector switched the perfusion solution to aCSF containing MPP+ (0.7, 1.3, 2.5, or 10 mM) for 30 min after which the perfusate was switched back to aCSF alone. Upon initiation of MPP+ perfusion, dialysate samples were collected at 5 min intervals (7.5 µL) for 3 h and subsequently at 10 min intervals (15 µL) for at least another 3 h. In many studies, an identical microdialysis experiment was carried out on the same animal 24 h after the initial experiment. In some experiments, aCSF containing sodium salicylate or mannitol was perfused prior to, during, and after MPP+ perfusion. MA (30 mg free base/kg, i.p.) was administered when the concentration of DA, 5-HT, and their metabolites in dialysate samples had reached constant basal levels. After MA administration, two plastic bags containing crushed ice were placed in the bottom of the Raturn bowl when the brain temperature of rats reached 41.5 °C. This ice was removed when the brain temperature had declined to 40.0 °C. In vitro recoveries of microdialysis probes were measured before and after each experiment on both day 1 and day 2. Typical in vitro recoveries were 23% for DA and 28% for L-DOPA. When microdialysis experiments were completed,

1374 Chem. Res. Toxicol., Vol. 16, No. 10, 2003 correct placement of probes was verified as described by Santiago et al. (40). Microdialysis Procedure to Monitor Thiols and Disulfides. The microdialysis procedure described above was also employed in experiments designed to measure dialysate levels of thiols and disulfides except that the aCSF (without and with MPP+) was thoroughly deoxygenated by argon sparging (30 min) to minimize autoxidation of thiols. Vials in the microfraction collector contained 1.5 µL of a thoroughly deoxygenated solution of iodoacetic acid (100 mM) and m-cresol purple (0.2 mM) in 0.24 M NaHCO3/0.12 M NaOH buffer at pH 10. Under these conditions, iodoacetic acid rapidly scavenges free thiol groups, forming S-carboxymethyl derivatives, and prevents their autoxidation to disulfides (41). Vials were briefly sparged with argon and capped with a septum to minimize access of oxygen. A short length of peek tubing (0.12 mm i.d.) was used to deliver dialysate for 10 min (15 µL) through septa into each vial. After 30 min of reaction with iodoacetic acid, 1.5 µL of a 10% solution (v/v) of FDNB in ethanol was added to each vial in order to form N-dinitrophenyl derivatives (41). Vials were stored overnight at 4 °C prior to HPLC analysis. Drugs and Chemicals. DA, L-DOPA, 3-MT, 5-HT, DOPAC, HVA, 5-HIAA, DTT, 3,4-dihydroxybenzylamine, diethylamine, iodoacetic acid, m-cresol purple, FDNB, GSH, CySH, cystine, GSSG, GSSCy, GSH sulfonic acid, cysteine sulfinic acid, cysteic acid, aluminum oxide, mannitol, salicylic acid (sodium salt), SOS, ethylenediaminetetraacetic acid (EDTA, disodium salt), and 2,3-DHBA were purchased from Sigma (St. Louis, MO). MPP+ (iodide salt) and S-(+)-MA (hydrochloride salt) were obtained from Research Biochemicals International (RBI, Natick, MA). Glutathionyl and cysteinyl conjugates of DA and DOPAC were synthesized and purified as described elsewhere (32, 33). Purified DDC was a generous gift from Professor Carla Borri Voltattorni (University of Perugia, Italy). All other chemicals used were of the highest purity commercially available. HPLC with Electrochemical Detection (EC) Methods for Analysis of Dialysates for DA, 5-HT, and Metabolites. HPLC-EC method I was used to analyze dialysate fractions for DA, 5-HT, and their normal metabolites and to search for glutathionyl and cysteinyl conjugates of DA and DOPAC. This method employed a BAS 200B HPLC system equipped with a BAS Unijet reversed phase microbore column (ODS-18, 100 mm × 1 mm, 3 µm particle size), Unijet guard column (ODS-18, 10 mm × 1 mm, 3 µm particle size), and a glassy carbon detector electrode (3 mm diameter) set at +750 mV with respect to a Ag/AgCl reference electrode. Vials containing dialysate fractions were loaded into a CMA/200 refrigerated autosampler (4 °C) equipped with a CMA/240 online injector and a 5.2 µL sample loop. The mobile phase consisted of 890 mL of deionized water, 100 mL of MeOH, 10 mL of MeCN, 30 mL of concentrated ammonium hydroxide (NH4OH, ca. 14.8 M), and 31 mL of concentrated TFA. After filtration (0.22 µm type HA membrane filter, Millipore, Bedford, MA), the apparent pH was adjusted to 2.60 with additional TFA. The flow rate was 40 µL/min. Neurochemical concentrations (expressed as fmol/min) were determined based on calibration curves obtained with standard mixtures of known compounds. At the beginning of each day’s chromatographic analysis, standard mixtures (three different concentrations, three times each) were injected to check calibration curves. During the course of autoinjection of dialysate fractions, standard mixtures were injected as every fifth sample to monitor and correct for possible chromatographic drift and detector response deviations. Using this method, basal neurochemical levels were measured as follows (fmol/min ( SEM): DA (28 ( 20), L-DOPA (8 ( 6), DOPAC (3468 ( 1159), HVA (2211 ( 489), and 5-HIAA (1864 ( 270). Basal levels of 5-HT and 3-MT were at or below their detection limits, ca. 5 and 7 fmol/min, respectively. HPLC-EC method I was also employed to determine 2,3-DHBA (and 2,5-DHBA) in dialysates using the method described by Rose et al. (22).

Foster et al. HPLC-EC methods II-IV all employed a BAS PM-80 pump, an LC-4C amperometric detector with a glassy carbon detector electrode (3 mm diameter), and a DA-5 data acquisition system. HPLC-EC method II used a Rheodyne (Cotati, CA) 9125 injector with a 5.0 µL sample loop. The glassy carbon detector electrode was set at +750 mV vs Ag/AgCl reference electrode. A BAS Unijet microbore column (ODS-18, 100 mm × 1 mm, particle size 3 µm) was used. The mobile phase was prepared by adding 50 mL of MeOH, 20 mL of NH4OH, and 21 mL of TFA to 950 mL of deionized water. After filtration, the apparent pH was adjusted to 2.5 with additional TFA. The flow rate was 30 µL/ min. HPLC-EC method III used a Rheodyne 7125 injector and a 20 µL sample loop. The glassy carbon detector electrode was set at +850 mV vs Ag/AgCl. This system was equipped with a Phenomenex (Torrence, CA) IB-SIL reversed phase column (ODS-18-BD, 100 mm × 3.2 mm, 3 µm). The mobile phase was 5% (v/v) MeOH in deionized water adjusted to pH 2.8 with formic acid. The flow rate was 0.3 mL/min. HPLC-EC method IV used a Rheodyne 9125 injector and 5.0 µL sample loop. The glassy carbon detector electrode was set at +850 mV vs Ag/AgCl. This system was equipped with a BAS Unijet reversed phase microbore column (ODS-18, 100 mm × 1 mm, 3 µm) and Unijet guard column (ODS-18, 10 mm × 1 mm, 3 µm). The mobile phase was prepared by adding 0.85 mL of diethylamine, 59.2 mg of SOS, 187 mg of Na2EDTA dihydrate, and 21 g of citric acid monohydrate to 970 mL of deionized water. After filtration, the pH of this solution was adjusted to 2.40 with HClO4 and then 30 mL of MeCN was added. The flow rate was 72 µL/min. HPLC-EC method V used a Rheodyne 7125 injector with a 20.0 µL sample loop. The glassy carbon detector electrode was set at +850 mV vs Ag/AgCl. A Phenomenex IB-SIL column (ODS-18-BD, 100 mm × 3.2 mm, 3 µm) was used. The mobile phase was the same as that described for method IV except 15 mL of MeOH was added to 1 L of the mobile phase. The flow rate was 0.35 mL/min. For all chromatography, mobile phases were degassed by stirring under vacuum for approximately 30 min prior to use. HPLC-EC methods I-IV were used to initially identify L-DOPA in rat striatal dialysates on the basis of both chromatographic retention time (tR) and coelution with the authentic compound. Methods IV and V were also employed in assays for the activity of purified DDC and the activity of this enzyme in rat brain tissue samples. HPLC Method for Thiols and Disulfides. A modification of the HPLC methods of Reed et al. (41) and Wang and Cynader (42) was employed in which N-dinitrophenyl derivatives of S-carboxymethylated GSH and CySH and of GSSG, cystine, GSSCy, cysteine sulfinic acid, cysteic acid, and GSH sulfonic acid were detected as N-dinitrophenyl derivatives based on their UV absorbance at 365 nm. HPLC employed a BAS 200B instrument equipped with a Waters (Bedford, MA) YMC amine microbore column (150 mm × 1 mm, 120 Å, 3 µm particle size) and a BAS model UV-116A UV-visible detector set at 365 nm. Mobile phase A was 4:1 (v/v) ethanol in deionized water. Mobile phase B was prepared by mixing 272 g of sodium acetate trihydrate, 122 mL of deionized water, and 378 mL of glacial acetic acid. Then, 200 mL of this solution was added to 800 mL of mobile phase A to give mobile phase B. These mobile phase solutions were filtered and vacuum degassed prior to use. A binary gradient was used as follows: 0-10 min, 80% solvent A and 20% solvent B; 10-15 min linear gradient to 5% solvent A and 95% solvent B; 15-25 min, 5% solvent A and 95% solvent B. Between 25 and 30 min, a linear gradient returned the mobile phase to 80% solvent A and 20% solvent B, which was then maintained for another 20 min before injection of the next sample. The flow rate was 70 µL/min. Enzymatic Confirmation of L-DOPA in Dialysates. Dialysate fractions (7.5 µL) from rat brains collected during the period when maximal concentrations of putative L-DOPA were formed in response to perfusion of MPP+ were collected in vials

MPP+ Mediates Release of Striatal L-DOPA maintained at 4 °C, which contained 0.75 µL of 4.7 mM ascorbic acid. Dialysates from four rats were then pooled (170 µL), and 14.5 µL of 1 M Tris-HCl buffer (pH 7.3) was added to give a final pH of 7.2. One fraction (74 µL) was incubated with a solution containing purified DDC (6.0 µL of 50 mM potassium phosphate buffer, pH 6.8, containing 0.1 mM DTT and 3 µg of DDC protein) for 20 min at 20 °C. A second 74 µL fraction was incubated with a solution containing heat-denatured DDC (30 min at 100 °C) under otherwise identical conditions. Reactions were terminated after 20 min by addition of 8.0 µL of 9.1 M HClO4 solution. The resulting solutions were treated with 160 µL of 0.75 M Tris-HCl buffer (pH 8.8) containing 0.43 mM ascorbate, to give a final pH of 7.2, and then, 10.0 mg of alumina was added. Vials were then placed in a Vortex Test Tube Mixer (model K-500-4, Scientific Industries, Bohemia, NY) and shaken on high speed for 10 min and then centrifuged at 12 000g for 10 min, and the supernatants were discarded. The alumina was washed three times with 360 µL of deionized water using the same procedure. After the final wash, catechols adsorbed on the alumina were eluted by adding 30 µL of a solution containing 0.5 M acetic acid and 0.4 M HCl in deionized water and shaking for 10 min. After centrifugation (12 000g, 5 min), aliquots (5.0 µL) of the supernatants from experimentals (dialysate incubated with DDC) and controls (dialysate incubated with heat denatured DDC) were analyzed for concentrations of l-DOPA, DA, and DOPAC using HPLC method IV. On the basis of experiments in which rat brain dialysate samples were replaced by aCSF containing 0.43 mM ascorbate, 1.71 µM DA, 0.63 µM L-DOPA, and 0.63 µM DOPAC and carried through the entire procedure described above, the recoveries (n ) 16) of DA, L-DOPA, and DOPAC from alumina were 77 ( 6, 72 ( 5, and 63 ( 5%, respectively. These recoveries were not affected by inclusion of 10 mM MPP+ in the original aCSF solution. Assay for the Activity of Purified DDC. An in vitro procedure was employed to assay the activity of purified DDC and the effect of MPP+ on this activity. The assay solution contained l-DOPA (1.5 mM), DOPAC (1.0 µM, internal standard), and ascorbate (0.43 mM) dissolved in 917 µL of aCSF and 75.5 µL of 0.75 M Tris-HCl buffer (pH 7.8) to give a final pH of 7.2. Reactions were initiated by addition of 37.5 µg of DDC protein or heat-denatured (30 min, 100 °C) DDC protein in 7.5 µL of 50 mM potassium phosphate buffer (pH 6.8) containing 0.1 mM DTT. Aliquots (75 µL) were removed from the reaction mixture immediately after addition of DDC protein and then at 5 min intervals and added to 7.5 µL of ice-cold 9.1 M HClO4 solution to terminate the reaction. Vials containing these solutions were centrifuged (14 000g, 20 min, 4 °C), and aliquots (5 µL) of the supernatants were analyzed for concentrations of L-DOPA, DA, and DOPAC using HPLC method IV. The effect of MPP+ on the activity of DDC was studied by including 15 mM MPP+ in the initial assay solution. The activity of DDC was expressed as nmol DA formed/µg protein/min. Effect of MPP+ on Rat Striatal DDC Activity. To assess the effect of MPP+ perfusion on the activity of DDC in vivo, microdialysis probes were implanted in both the right and the left striata of rats. On the day of the experiment, aCSF was perfused (1.5 µL/min) into the left and right striatum for ca. 3 h until HPLC analysis of dialysates (collected at 15 min intervals) gave at least three consecutive constant basal levels of DA, 5-HT, and their metabolites. At this point, the right striatum was perfused for 30 min with aCSF containing 10 mM MPP+, and the left striatum was perfused with aCSF alone. The perfusate into the right striatum was then changed back to aCSF, which continued to be perfused into the left striatum. After 10 min, perfusions were terminated, microdialysis probes were removed, and rats were killed by decapitation. Brains were removed and placed in an ice-cold BAS coronal brain matrix apparatus, and a 2 mm coronal slice was made 0.75 mm posterior and 0.75 mm anterior to the two microdialysis probe tracks (0.5 mm diameter). The slice was then placed on an icecold glass plate, and a 2 mm × 4 mm area of striatal tissue

Chem. Res. Toxicol., Vol. 16, No. 10, 2003 1375 centered around each microdialysis probe hole was excised with a punch (constructed in this laboratory). The activity of striatal DDC was run under saturating conditions with respect to L-DOPA using a modification of the method described by Hadjiconstantinou et al. (43). Briefly, excised tissue was weighed (16 ( 0.6 mg, n ) 6) and homogenized in ice-cold 0.25 M sucrose (1.0 mg tissue/7.0 µL sucrose solution). A 10 µL sample of the homogenate was then diluted to 40 µL with ice-cold 0.25 M sucrose, and 10 µL of this solution was added to 200 µL of the assay mixture and incubated for 20 min at 37 °C. The assay mixture consisted of 50 mM sodium phosphate buffer (pH 7.2) containing 0.1 mM Na2EDTA, 0.17 mM ascorbate, 1.0 mM β-mercaptoethanol, 0.1 mM pargyline, 0.01 mM pyridoxal 5′-phosphate, and 0.2 mM L-DOPA. The reaction was terminated by adding 21 µL of ice-cold 9.1 M HClO4 solution containing 410 pmol of 3,4-dihydroxybenzylamine (internal standard; final concentration, 1.77 µM). The pH of this solution was adjusted to 8.0 by addition of 450 µL of 0.75 M Tris-HCl (pH 8.8) containing 0.43 mM ascorbate. Aluminum oxide (10.0 mg) was then added to the resulting solutions, contained in 1.5 mL polypropylene microcentrifuge tubes, to adsorb catechols. The reaction vials were placed in a Vortex Test Tube Mixer and shaken at the high speed setting for 10 min and then centrifuged for 3 min at 12 000g, and the supernatant was discarded. The alumina was washed three times with 300 µL of water using the same procedure. After the final wash, compounds adsorbed on the alumina were eluted by adding 30 µL of a solution containing 0.5 M acetic acid and 0.4 M HCl, shaking for 10 min, and centrifuging for 5 min at 12 000g. Aliquots (20 µL) of the supernatant were then analyzed for the concentration of DA using HPLC method V. The activity of DDC was expressed as nmol DA formed/mg total protein/20 min. The extraction recoveries for DA and 3,4-dihydroxybenzylamine from alumina were 75 ( 4 and 69 ( 5%, respectively. Protein was determined using the Coomassie Plus Protein Assay Reagent Kit (Pierce, Rockford, IL) using bovine serum albumin as the standard. Calculations and Statistics. Neurochemical concentrations measured in microdialysis experiments are expressed as efflux in fmol/min ( SEM based on at least three replicate experiments. The effects of MPP+ on neurochemical levels compared efflux data before, during, and after perfusion using one way ANOVA followed by Dunnett’s post hoc test. A p value < 0.05 was taken as significant.

Results MPP+

Effects of Perfusion on Dialysate Levels of DA, 5-HT, and Metabolites. HPLC-EC method I was used to analyze rat striatal dialysate samples for the effects of MPP+ perfusion on the concentrations of DA and its normal metabolites (DOPAC, HVA, and 3-MT), 5-HT, and 5-HIAA and to search for glutathionyl and cysteinyl conjugates of DA and DOPAC. A chromatogram of a standard mixture of DA, 5-HT, their normal metabolites together with 5-S-, 2,5-di-S-, and 2,5,6-tris-SGS-DA, 5-S- and 2,5-di-S-GS-DOPAC, and 5-S- and 2,5di-S-CyS-DA is shown in Figure 1A. A representative chromatogram of a rat striatal dialysate collected immediately prior to perfusion of MPP+ (Figure 1B) shows peaks corresponding to basal levels of DA, its metabolites, DOPAC and HVA, and 5-HIAA. Perfusion of MPP+ evoked a massive increase of dialysate levels of DA and other characteristic changes in the concentrations of its metabolites. However, MPP+ also evoked the appearance of a compound in dialysates having a peak chromatographic retention time of 3.66 min (Figure 1C), identical to that of 2,5,6-tris-S-GS-DA (Figure 1A). Several factors, however, tended to argue against this peak being that of 2,5,6-tris-S-GS-DA. For example, peaks corresponding to

1376 Chem. Res. Toxicol., Vol. 16, No. 10, 2003

Foster et al. Table 1. Chromatographic Retention Times for DA, 2,5,6-tris-S-GS-DA, L-DOPA, and the Compound Present in Rat Striatal Dialysates in Response to Perfusion of MPP+a retention time/minc HPLC-EC methodb

DA

2,5,6-tris-S-GS-DA

L-DOPA

dialysate compd

I II III IV

4.02 4.58 3.74 5.25

3.66 9.09 4.23 4.09

3.66 4.97 3.41 3.90

3.66 4.99 3.43 3.90

a Dialysate samples were collected between 15 and 20 min (5 min collection time, 7.5 µL) following 30 min of perfusion of 10 mM MPP+ dissolved in aCSF. b Experimental conditions for HPLC-EC methods I-IV are provided in the Materials and Methods. c Data are means of at least three replicate measurements.

Figure 1. HPLC-EC chromatograms (method I) of (A) a standard mixture of DA, 5-HT, their metabolites, and glutathionyl and cysteinyl conjugates of DA and DOPAC; (B) rat striatal dialysate collected immediately prior to perfusion of MPP+; and (C) rat striatal dialysate collected from 15 to 20 min after a 30 min perfusion of 2.5 mM MPP+ was terminated.

5-S- and 2,5-di-S-GS-DA, obligatory precursors of 2,5,6tris-S-GS-DA when DA is oxidized in the presence of GSH (32), were never observed in chromatograms of striatal dialysate samples collected during or after perfusions of MPP+ (0.7-10 mM). Similarly, peaks corresponding to cysteinyl conjugates of DA were never detected in dialysates. Such conjugates would be expected both as a consequence of the oxidation of released DA in the presence of CySH (33) and as a result of the degradation of extracellular glutathionyl conjugates of DA by the action of γ-glutamyl transpeptidase and dipeptidases (44). Subsequent chromatographic analysis of dialysates using HPLC-EC methods II-IV indicated that the unknown compound exhibited different retention times to 2,5,6-tris-S-GS-DA but the same retention times as L-DOPA (Table 1). Furthermore, the peak in dialysates comigrated with added L-DOPA using HPLC methods I-IV. In subsequent studies, dialysate containing putative L-DOPA was incubated with purified DDC at pH 6.8

Figure 2. HPLC-EC chromatograms of pooled dialysates from four rats collected for 26 min after a 30 min perfusion of 10 mM MPP+ was terminated. (A) One fraction of this dialysate was incubated for 20 min at pH 6.8 with heat-denatured DDC. Then, catechols were extracted by adsorption onto alumina. After the alumina was washed with water, catechols were acid eluted (0.5 M acetic acid plus 0.4 M HCl in water) and an aliquot (5.0 µL) of the eluent was analyzed by HPLC method IV. (B) A second identical fraction of the dialysate was carried through the same procedure as (A) except active purified DDC was employed.

for 20 min. The resultant solution was then treated with alumina to extract catechols. After the alumina was washed, catechols were desorbed with acid and the eluents were analyzed by HPLC. When carried through such a procedure but using heat-denatured DDC, such eluents exhibited chromatographic peaks for DA and DOPAC in addition to a peak at the same retention time as L-DOPA (Figure 2A). However, when the dialysate was incubated with active DDC, chromatograms showed that



Figure 3. Time-dependent effects of a 30 min perfusion of MPP+ (2.5 mM) into rat striatum on dialysate levels of DA, DOPAC, HVA, 3-MT, L-DOPA, 5-HT, and 5-HIAA on day 1 and an identical perfusion on day 2. Horizontal black bar shows the period during which MPP+ entered the brain. *p < 0.05; **p < 0.01 as compared to basal levels measured on day 1 or day 2. #p < 0.05 when basal dialysate concentration on day 2 was compared to day 1. Data are means ( SEM (n ) 4).

the L-DOPA peak was quantitatively transformed into DA (Figure 2B).

In subsequent microdialysis studies, various concentrations of MPP+ were perfused for 30 min into the


striata of awake rats on day 1 and dialysate levels of L-DOPA, DA, 5-HT, and their metabolites were measured using HPLC-EC method I. Twenty-four hours later (day 2), an identical microdialysis experiment was carried out on the same animal. Previous studies using a similar two day test/challenge paradigm (38) have shown that a significantly reduced efflux of DA in response to the MPP+ perfusion on day 2 as compared to that on day 1 provides a reliable indication that the day 1 perfusion caused neurotoxic damage to dopaminergic terminals (39). However, the present study differed in two ways from these previous studies. First, the same concentration of MPP+ was perfused on day 2 as on day 1. The rationale for this was to interrogate the same volume of striatal tissue on both day 2 and day 1. Second, dialysate samples were collected at much shorter time intervals (5 min) in order to obtain a more detailed temporal profile of the extracellular changes evoked during perfusions of MPP+ and after they were terminated. The time-dependent effects evoked by 30 min perfusions of MPP+ (2.5 mM) on day 1 into the rat striatum on dialysate neurochemical levels are presented in Figure 3. Thus, MPP+ evoked an almost instantaneous and massive efflux of DA that after attaining maximal values, declined back toward basal levels (Figure 3A). Perfusion of MPP+ also evoked an almost immediate decrease of dialysate concentrations of DOPAC and HVA (Figure 3B,C), an effect that persisted for many hours after the perfusion was discontinued, presumably the result of the inhibition of MAO-A and -B by MPP+ (45). Dialysate levels of 3-MT increased when MPP+ perfusions were initiated but then declined when the perfusions were discontinued (Figure 3D). However, both during and after MPP+ perfusion, dialysate concentrations of 3-MT remained significantly elevated above virtually undetectable basal levels indicating a shift of DA metabolism from the MAO-B pathway, inhibited by MPP+, to that catalyzed by catechol-O-methyltransferase. Perfusion of MPP+ on day 1 evoked a delayed increase of dialysate levels of L-DOPA (Figure 3E). As with DA, this rise of extracellular L-DOPA was only transient. Perfusion of MPP+ on day 1 also evoked an almost immediate but transient efflux of 5-HT (Figure 3F), although much smaller than that of DA (Figure 3A), and a profound but persistent decrease of dialysate levels of 5-HIAA (Figure 3G) presumably caused by inhibition of MAO. Perfusion of MPP+ (2.5 mM) on day 2 resulted in a greatly attenuated release of DA (Figure 3A), L-DOPA (Figure 3E), and 5-HT (Figure 3F) as compared to day 1. Prior to MPP+ perfusion on day 2, dialysate concentrations of DOPAC (Figure 3B), HVA (Figure 3C), and 5-HIAA (Figure 3G) were all appreciably lower than basal levels of these metabolites measured on day 1. These effects are consistent with neurotoxic damage to dopaminergic and serotonergic systems in the striatum by MPP+ (2.5 mM) perfusion on day 1 (39). Perfusion of increasing concentrations of MPP+ (0.710 mM) into the rat striatum on day 1 caused correspondingly increased maximal releases of DA, L-DOPA, and 5-HT (Figure 4). However, the larger the release of DA, L-DOPA, and 5-HT on day 1, the smaller their release caused by the day 2 perfusion of MPP+ (Figure 4). These observations indicate that the severity of dopaminergic and serotonergic damage increases with increasing concentration of MPP+ perfused on day 1.

Foster et al.

Figure 4. Maximum efflux of (A) DA, (B) L-DOPA, and (C) 5-HT evoked by 30 min perfusions of MPP+ (0-10 mM) into the awake rat striatum on day 1 and day 2. A significant difference between DA, L-DOPA, or 5-HT efflux vs basal efflux is indicated by *p < 0.05, **p < 0.01, and ***p < 0.001. A significant difference between DA, L-DOPA, or 5-HT efflux on day 1 or day 2 as compared to that evoked by the immediate lower concentration is indicated by xp < 0.05 and xxp < 0.01. A significant difference between the day 2 and the day 1 efflux is indicated by #p < 0.05, ##p < 0.01, and ###p < 0.001. Data are means ( SEM (n ) 4).



Table 2. Maximum Extracellular Neurochemical Changes Evoked by Perfusion of MPP+ (2.5 mM; 30 min) into the Awake Rat Striatum on Day 1 and on Day 2 after Perfusion of aCSF Alone on Day 1 max neurochemical concna (fmol/min) for

neurochemical L-DOPA

DA DOPAC 3-MT HVA 5-HT 5-HIAA

day 1 perfusionb of MPP+

day 2 perfusionb of MPP+ one day after aCSF perfusion

327 ( 111 6766 ( 2031 3713 ( 1478 600 ( 109 2092 ( 683 198 ( 69 1904 ( 255

341 ( 119 6886 ( 2043 3521 ( 1646 685 ( 115 2257 ( 715 179 ( 36 1842 ( 340

a Measured at time of maximum release. b Data are means ( SEM (n ) 4).

Using the preceding two day experimental paradigm, it was possible that physical damage caused by insertion of the microdialysis probe on day 1 might have affected the MPP+-mediated release of DA on day 2. This was investigated by perfusing aCSF alone on day 1 followed by MPP+ (2.5 mM; 30 min) on day 2 and comparing the DA release and other neurochemical changes on day 2 to those observed when MPP+ was perfused on day 1. The results (Table 2) indicated that the neurochemical effects evoked by MPP+ perfusion on day 2, aCSF alone having been perfused on day 1, were indistinguishable from those evoked by MPP+ perfusion on day 1. These results indicate that there was no significant dopaminergic or serotonergic terminal damage caused by probe insertion on day 1 that influences DA or 5-HT release by MPP+ on day 2. Effects of Antioxidants on MPP+-Induced Extracellular Chemistry and Neurotoxicity. Coperfusion of salicylate (2.5 mM) and MPP+ (10 mM) into the rat striatum resulted in the appearance of 2,3-DHBA in dialysates (Figure 5) together with 2,5-DHBA (data not shown) indicative of extracellular and/or intracellular HO• and/or peroxynitrite generation (22). However, the appearance of 2,3-DHBA in dialysates was delayed such that it was first detected when the perfusion of MPP+ was discontinued (Figure 5). Furthermore, 2,3-DHBA appeared in dialysates later than L-DOPA and as extracellular DA levels were decreasing (Figure 3A,E). Perfusion of MPP+ on day 1 evoked the same release of DA, L-DOPA, and 5-HT in the presence of salicylate as in its absence (Table 3). Salicylate also failed to provide any protection against the dopaminergic or serotonergic neurotoxicity of MPP+ based on the fact that the day 2 perfusion resulted in greatly attenuated release of DA, L-DOPA, and 5-HT as compared to day 1 (Table 3). A high concentration (100 mM) of mannitol, which is believed to act exclusively as an extracellular antioxidant (46), also failed to affect the release of DA, L-DOPA, and 5-HT in response to a 30 min perfusion of MPP+ on day 1 or attenuate the dopaminergic and serotonergic neurotoxicity, based on a greatly reduced release of DA and 5-HT on day 2 (Table 3). Effect of MPP+ Perfusion on Extracellular Disulfide Formation. In the event that MPP+ mediates the generation of extracellular HO• or peroxynitrite, then GSH and CySH, both released by MPP+ (30), should be at least partially oxidized to disulfides and possibly to sulfinic and sulfonic acids. This was investigated by

Figure 5. Time-dependent effects of a 30 min coperfusion of MPP+ (10 mM) and salicylate (2.5 mM) into the awake rat striatum on 2,3-DHBA levels in dialysates. Salicylate alone was also perfused for ca. 3 h prior to MPP+ and at all times after the perfusion of MPP+ was terminated. *p < 0.05 and **p < 0.01 as compared to basal efflux of 2,3-DHBA. Data are means ( SEM (n ) 4). Table 3. Effect of Salicylate and Mannitol on the Maximal Release of DA, L-DOPA, and 5-HT on Day 1 and Day 2 Evoked by Perfusion of 10 mM MPP+ for 30 min into the Striata of Awake Rats max release (fmol/min) ofb antioxidanta

(concn) day

0 salicylate (2.5 mM) mannitol (100 mM)

1 2 1 2 1 2

DA

L-DOPA

5-HT

8,596 ( 1268 606 ( 108 324 ( 110 0***c 6 ( 10** 43 ( 69** 8,273 ( 1,524 643 ( 111 316 ( 78 3 ( 5** 2 ( 3** 6 ( 6** 8,841 ( 892 723 ( 200 283 ( 53 0***c 5 ( 6* 7 ( 6**

a Salicylate or mannitol dissolved in aCSF at the indicated concentration was perfused for ca. 3 h before MPP+ perfusion, during, and for several hours after this perfusion. b Data are means ( SEM (n g 3) for release. c No release was observed with any animal; a significant difference between the day 1 and the day 2 release is indicated by *p < 0.05, **p < 0.01, and ***p < 0.001.

analyzing rat striatal dialysates, both during and after MPP+ perfusion, using an HPLC with UV detection method (41, 42). In this method, reduced GSH and CySH in dialysates were trapped by iodoacetic acid forming S-carboxymethyl derivatives. These derivatives together with GSSG, cystine, GSSCy, cysteine sulfinic acid, cysteic acid, GSH sulfonic acid, and other acidic amino acids such as Glu, L-aspartic acid (Asp), and γ-glutamyl dipeptides (e.g., γ-glutamylglutamine and γ-glutamylglycine) were then converted to N-dinitrophenyl derivatives, which were analyzed by HPLC with UV detection. The limit of detection for GSH, CySH, GSSG, and cystine was ca. 1 µM. Because extracellular concentrations of GSH and CySH in the rat striatum are approximately 2 µM (30, 47) and typical in vitro microdialysis probe recoveries for these compounds were ca. 20%, basal concentrations of these thiols and their disulfides in dialysates were below the detection limit of the HPLC-UV method. However,


perfusion of, for example, 2.5 mM MPP+ for 30 min into the rat striatum resulted in a delayed and massive rise of dialysate concentrations of GSH and CySH from undetectable basal levels to 7.2 ( 0.4 and 2.3 ( 0.6 µM, respectively. These results agree with those presented in an earlier paper (30). Similarly, MPP+ perfusion evoked a delayed and massive rise of dialysate concentrations of Glu and Asp in agreement with the paper of Carboni et al. (48). However, GSSG, cystine, GSSCy, and sulfinic and sulfonic acids of GSH and CySH were not detected in dialysate samples collected prior to, during MPP+ perfusions, or after perfusions were terminated. Effects of MPP+ on the Activity of DDC in Vitro and in Vivo. The activity of purified DDC measured at pH 7.2 in the presence of a saturating concentration of L-DOPA was 5.6 ( 0.4 nmol DA formed/µg protein/min (n ) 12). Heat-denatured DDC was unable to convert L-DOPA into DA. When 15 mM MPP+ was included with L-DOPA in the assay solution, the measured activity of purified DDC was 5.3 ( 0.6 nmol DA formed/µg protein/ min (n ) 12). These results indicate that in vitro, MPP+ has no effect on the activity of DDC. Nevertheless, the marked elevation of extracellular L-DOPA in the rat striatum evoked by MPP+ (Figure 3E), which appeared to be unrelated to the nonenzymatic hydroxylation of L-tyrosine by HO• or peroxynitrite (Table 3), suggested that in vivo DDC might be inhibited. This was explored by terminating microdialysis experiments 10 min after a 30 min perfusion of MPP+ into the right striatum of rats had been discontinued. This time corresponded to the point when extracellular concentrations of L-DOPA were highest (Figure 3E). Tissue in the vicinity of the microdialysis probe was then excised and, after homogenization, assayed for the activity of DDC. Control tissue was excised from the vicinity of an identical microdialysis probe, positioned in the left striatum, that was perfused with aCSF alone. The activity of the DDC in the striatal tissue perfused with MPP+ was 0.36 ( 0.04 nmol DA formed/mg total protein/20 min (n ) 3), which was significantly (p < 0.001) lower than that in control tissue, 0.86 ( 0.04 nmol DA formed/mg total protein/20 min. Effect of MA on Dialysate Levels of DA, Its Metabolites, and L-DOPA. The preceding results indicated that MPP+ mediates a significant rise of extracellular levels of L-DOPA in the rat striatum. In an effort to assess whether this effect is evoked by other dopaminergic neurotoxins, preliminary experiments were carried out with MA (49). The neurotoxic processes triggered by MA are virtually identical to those implicated with the dopaminergic neurotoxicity of MPP+. These include an initial rapid MA-induced neuronal energy impairment (50), which mediates a massive release of DA (51), and elevated production of O2-• (52), •NO, peroxynitrite (53), and/or HO• (54). However, unlike MPP+, the neurotoxicity of MA is highly dependent on the hyperthermia induced by this drug (55). A single 30 mg/kg i.p. dose of MA, previously shown to be neurotoxic (56, 57), induced significant hyperthermia (Figure 6A) and an almost immediate release of DA (Figure 6B). The maximum release of DA was approximately one-third that caused by perfusion of 0.7 mM MPP+ (Figure 4A). MA also induced a rise of L-DOPA (Figure 6C), the maximum release also being approximately one-third that caused by perfusion of 0.7 mM MPP+ (Figure 4B). Injection of MA induced a fall of

Foster et al.

Figure 6. Time-dependent effects of MA (30 mg/kg, i.p.) injection into the rat on (A) striatal temperature, (B) dialysate levels of DA, and (C) dialysate levels of L-DOPA. The arrow shows the time when MA was injected. A significant difference from basal temperature or neurochemical efflux is indicated by *p < 0.05 and **p < 0.001. Data are means ( SEM (n ) 6).

dialysate levels of DOPAC, an initial rise then timedependent decrease of 3-MT (data not shown) similar to those evoked by MPP+ perfusion. However, dialysate levels of HVA did not change significantly from basal levels following MA injection. HPLC-EC (method I) analysis of rat striatal dialysates after MA administra-


tion did not detect the presence of glutathionyl or cysteinyl conjugates of DA or DOPAC.

Discussion The initial steps in the neurotoxic mechanism evoked by MPP+ involve its rapid DAT-mediated uptake into dopaminergic neurons (5), concentration into mitochondria (6), and inhibition of complex I (7). The resultant neuronal energy impairment, depolarization (58), interference by MPP+ with the vesicular storage of DA (59), and reversal of the DAT (60) all contribute to the massive almost instantaneous release of DA (Figure 3A). However, the transport process that concentrates MPP+ into mitochondria is energy dependent (6). Thus, when dopaminergic mitochondria become severely deenergized, MPP+ is presumably released into the cytoplasm and then transported out of these neurons by the reversed DAT. MPP+ therefore induces a transient dopaminergic energy impairment (61). As the MPP+-induced energy impairment subsides, increasing ATP production would initiate repolarization of the neuronal membrane, which in turn would return the reversed DAT to its normal function with resultant reuptake of released DA (62). Such a sequence is consistent with the massive release of DA evoked by MPP+ perfusion followed by the fall of extracellular DA concentrations when the perfusion is discontinued (Figure 3A). That the latter effect is probably indicative of the DAT-mediated reuptake rather than metabolism of released DA is supported by the observation that as extracellular DA concentrations fall, so also do those of its metabolites DOPAC, HVA, and 3-MT (Figure 3). The present study indicates that perfusion of g0.7 mM MPP+ on day 1 produces an efflux of DA on day 2 that is inversely proportional to the concentration perfused on day 1. This observation indicates that perfusion of g0.7 mM MPP+ for 30 min on day 1 evokes neurotoxic damage to dopaminergic terminals as demonstrated by Santiago et al. (39). The decreased release of 5-HT in response to perfusion of g1.3 mM MPP+ on day 2 as compared to day 1 (Figure 4C) suggests that at higher concentrations MPP+ is also a serotonergic neurotoxin as proposed by others (63). HPLC analyses of striatal dialysate samples collected both during MPP+ perfusion and after its termination were unable to detect the presence of glutathionyl or cysteinyl conjugates of DA or disulfides such as GSSG and cystine. The presence of such compounds in dialysates would be expected if MPP+ mediates extracellular generation of HO• and/or peroxynitrite, which should oxidize released DA, GSH, and CySH. Furthermore, high concentrations of mannitol, which probably acts exclusively as an extracellular antioxidant (46), failed to attenuate either L-DOPA formation or the dopaminergic (and apparent serotonergic) neurotoxicity of MPP+ (Table 3). These observations suggest that MPP+ does not mediate significant fluxes of extracellular HO• or peroxynitrite. Nevertheless, MPP+ clearly induces the hydroxylation of coperfused salicylate to 2,3-DHBA (Figure 5) indicative of HO• (20) and/or peroxynitrite production (21, 22). Interestingly, 2,3-DHBA initially appears in dialysates after MPP+ perfusions are discontinued (Figure 5) at a time when extracellular DA has declined significantly from its peak concentration (Figure 3A). These observations argue against the idea that autoxi-


dation of released DA produces O2-•, H2O2, and hence HO•, which hydroxylates extracellular salicylate. In view of the appearance of 2,3-DHBA in rat striatal dialysates only after MPP+ perfusions are terminated, it is interesting that the massive rise of extracellular GSH is similarly delayed (30). It is tempting to speculate, therefore, that the nonenzymatic hydroxylation of salicylate to 2,3DHBA by HO• or peroxynitrite is an intraneuronal process that becomes significant only when the antioxidant GSH is released. L-DOPA first appears in dialysates toward the end of a 30 min perfusion of MPP+ and attains peak concentrations approximately 10 min after the perfusion is discontinued (Figure 3E). At the latter time, the activity of DDC in striatal tissue in the vicinity of the microdialysis probe is significantly decreased. A previous microdialysis study (64) has shown that perfusion of high concentrations of MPP+ into the rat striatum causes only a gradual decrease of TH activity, which reached approximately 70% of control activity after 3 h but without significant effect on TH content. Thus, the MPP+-mediated inhibition of DDC appears to occur more rapidly than the inhibition of TH, neither effect being directly related to the degeneration of dopaminergic terminals. These observations suggest that during and immediately after MPP+ perfusion TH continues to hydroxylate L-tyrosine but conversion of the resulting L-DOPA to DA is blocked because of the more rapid inhibition of DDC. The neurotransmitter-like release of accumulated L-DOPA (65) probably accounts for its rise in rat striatal dialysates in response to MPP+. Elevation of extracellular L-DOPA is not specific to MPP+ since MA, another dopaminergic neurotoxin, also evokes a similar effect (Figure 6C). The rapid inhibition of DDC is not caused directly by MPP+, which, in vitro, has no effect on the activity of the purified enzyme. However, there are several indirect mechanisms that might contribute to this rapid inhibition of DDC. For example, MPP+ mediates intraneuronal generation of many reactive species that might rapidly inhibit DDC. One such species is peroxynitrite, which appears to contribute to the MPP+-induced inhibition of TH by nitrating its tyrosine residues (12-14). DDC may similarly be susceptible to peroxynitrite-mediated inhibition since it contains 15 tyrosine residues (66), at least one of which is essential for catalytic activity (67). It may also be of relevance that the rise of extracellular L-DOPA evoked by MPP+ perfusion (Figure 3E) occurs at a time when extracellular DA is rapidly declining from its peak concentrations (Figure 3A), presumably owing to its DATmediated reuptake into dopaminergic terminals. Thus, intraneuronal O2-• (11) or peroxynitrite (12), both implicated in the neurotoxic mechanism evoked by MPP+, could oxidize such DA to DAQ (28, 68). Indeed, in vitro studies have implicated intraneuronal oxidation of DA in the dopaminergic neurotoxicity of MPP+ (60). DAQ could potentially inhibit DDC by covalent attachment to one or more of its 12 cysteinyl residues (66). Indeed, in vitro, DAQ inhibits a number of enzymes by such a mechanism (69, 70). To the best of our knowledge, this is the first paper to report that MPP+ mediates rapid inhibition of DDC with resultant accumulation and release of L-DOPA in the rat striatum. MA similarly mediates a rise of extracellular L-DOPA, although whether this is also related to DDC inhibition remains to be studied. Nevertheless, these observations may be of interest because L-DOPA can act


as an excitotoxin, with approximately one-half the potency of Glu, through its interaction with AMPA receptors (71). Thus, not only would a MPP+-induced energy impairment be expected to potentiate such L-DOPAmediated excitotoxicity (72), but AMPA receptor activation appears to play a role in the neurotoxicity of MPP+ toward dopaminergic terminals (73). Micromolar concentrations of L-DOPA can also mediate Glu release in the rat striatum with resultant delayed neuronal death (74, 75). Such observations raise the possibility that L-DOPA released by MPP+ and MA in the rat striatum may contribute to the delayed release of Glu evoked by these drugs (48, 76) and consequent excitotoxicity.

Acknowledgment. This study was supported by National Institutes of Health Grant No. GM32367. We thank Professor Carla Borri Voltattorni for the generous gift of purified DDC and Ms. Teresa G. Hackney for her help with preparation of the manuscript.

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(l-DOPA) and Inhibition of l-DOPA Decarboxylase in the Rat Striatum

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