J. Neurochem. (2010) 114, 597–605.
The striatum is a cerebral structure particularly susceptible to the metabolic challenge exerted by 3-nitropropionic acid (3-NPA), a toxin that inhibits the respiratory chain at complex II. The striatum, which receives the nerve endings of the nigro-striatal pathway, concentrates the largest amount of 3,4-dihydroxyphenylethylamine or dopamine (DA) in the brain. DA is metabolized to 3,4-dihydroxyphenylacetic acid (DOPAC) by monoamine oxidase (MAO), an enzyme that contains a redox-active disulfide in the active site. In striatum isolated nerve endings exposed to 3-NPA in vitro, DA increased and DOPAC decreased already after 10 min, and after 2 h also an increase in reactive oxygen species and DA-quinone products formation was detected. These 3-NPA-induced effects resulted in an increase in DA release after 2 h. In striatum homogenates from animals presenting motor disturbances in response to 3-NPA in vivo, the DA metabolites homovanillic acid and DOPAC were increased. It is concluded that in the striatum nerve endings where DA is particularly concentrated, the increase in reactive oxygen species induced by 3-NPA, oxidizes DA generating DA-quinones. These DA-quinones may form adducts with the active site of MAO type A reducing its activity. The DA not metabolized to DOPAC is both, used to unchain generation of more of the harmful DA-oxidation products and released to the external medium, where is metabolized by the non-neuronal enzymes MAO type B and catechol-O-methyltransferase.
3,4-dihydroxyphenylethylamine or dopamine
oxygenated HEPES buffer
MAO type A
MAO type B
reactive oxygen species
3-Nitropropionic acid (3-NPA) is a toxin derived from a sugar cane fungus that irreversibly inhibits succinate dehydrogenase, an enzyme which acts in the tricarboxylic acid cycle and the electron transport chain at complex II (Alexi et al. 1998). Although in several studies the particular striatum vulnerability to 3-NPA is recognized (Ludolph et al. 1991; Fu et al. 1995; Brouillet et al. 1998; Reynolds et al. 1998; Villarán et al. 2008), many questions still exist about the biochemical events that could explain the larger sensitivity to 3-NPA neurotoxicity of that brain structure.
One distinctive characteristic of the striatum is that it concentrates the larger amount of the neurotransmitter 3,4-dihydroxyphenylethylamine or dopamine (DA) in the brain. A possible role of DA in the selective striatal vulnerability to 3-NPA was suggested by a previous study in vivo showing that depletion of striatal DA with 6-hydroxydopamine reduced the striatal lesion induced by 3-NPA administration (Maragos et al. 1998). Monoamine-oxidase (MAO) is the enzyme that converts DA to its main metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC). There are two forms of MAO: type A (MAO-A) and type B (MAO-B). Interestingly a chronic treatment with a combination of selective inhibitors of both forms also reduced the striatal lesion induced by 3-NPA administration in vivo (Maragos et al. 1999).
To investigate the changes exerted by different drugs on DA metabolism, in previous studies we used an experimental design that allowed us to measure their effects on the concentrations of neurotransmitters and some of their metabolites, including DA and DOPAC, inside and outside cerebral isolated nerve endings (Sitges et al. 2000, 2009; Trejo et al. 2001). In the present study we used that methodological design for investigating a possible effect of 3-NPA on DA metabolism in cerebral isolated nerve endings (synaptosomes) isolated from the striatum. Because the striatum is the cerebral structure that receives the nerve endings of the dopaminergic nigro-striatal pathway and concentrates the largest amount of DA in the brain, and because on the basis of pre-synaptic lesion techniques striatal MAO-A activity is known to be localized pre-synaptically in nigro-striatal dopaminergic nerve endings (Agid et al. 1973; Demarest et al. 1980), whereas MAO-B is an astroglial enzyme (Reeniläet al. 1997). In addition, among the parts of the neuron pre-synaptic boutons are particularly rich in mitochondria where the electron transport chain takes place. As the concentration of DA in the nerve endings isolated from the whole brain is expected to be much lower than the concentration of DA in the nerve endings isolated from the striatum, the effect of 3-NPA on whole brain isolated nerve endings was also tested for comparison.
In our attempt to further understand the higher sensitivity of the striatum to 3-NPA, its effect on oxidative stress and DA-o-quinone products formation was also investigated here.
Materials and methods
Source of materials
3-Nitropropionic acid, nitrotetrazolium blue chloride (NBT), glycine, 2′,7′-dichlorofluorescein (DCF) and DCF-diacetate were from Sigma-Aldrich (St. Louis, MO, USA). All other reagents were of analytical grade.
Isolation of striatum and whole brain nerve endings
The dissected striata of four male Wistar rats or the whole brain (without cerebellum) of one male Wistar rat, were placed in cold isotonic sucrose (1:10, wt/vol) and homogenized (six strokes at 2000 rpm). The resulting suspensions were centrifuged at 1500 g for 10 min and the supernatants centrifuged for another 20 min at 9000 g. The resulting pellets containing the nerve endings (synaptosomes) isolated from the striatum or the whole brain were resuspended in oxygenated HEPES buffer (KRH). The composition of the KRH was, in mM: 127 NaCl, 1.18 KH2PO4, 3.73 KCl, 1 CaCl2, 1.18 MgSO4, 20 HEPES and 5.6 mM dextrose, pH 7.4, bubbled with O2/CO2 mixture.
Endogenous DA and DOPAC distribution experiments in synaptosomes
Aliquots (500 μL) of striatum or whole brain synaptosomes (P2 fraction) suspended in KRH containing 464 ± 24 μg or 947 ± 36 μg of synaptosomal protein, respectively were pre-incubated for 5 min at 37°C, and then incubated at 37°C for 10 or 120 min in the absence or in the presence of 5 mM 3- NPA under resting or high K+ depolarized conditions. The composition of the high K+ buffer was the same of the KRH except that 30 mM KCl replaced an equimolar concentration of NaCl. At the end of the incubation period, synaptosomes exposed to different experimental conditions were centrifuged at 12 000 g for 5 min. The supernatants resulting from this centrifugation (containing the released catecholamines) were transferred to clean vials treated with an aliquot of a perchloric acid (PCA)/EDTA mixture to obtain 0.1 M and 0.1 mM final, respectively, and stored at −40°C for later analysis. The resulting pellets were suspended in 500 μL of a 0.1 M PCA/0.1 mM EDTA pH 1.4 solution and vigorously vortex mixed. These drastic conditions guarantee a complete discharge of the catecholamines (DA and DOPAC) that were inside synaptosomes. To standardize catecholamine concentrations per mg of synaptosomal protein, the vortex mixed suspension containing the disrupted synaptosomes was centrifuged. The pellets were used for protein determination by the method of Lowry after suspension in 1 mL of a NaOH 5 mM solution and the supernatants resulting from this centrifugation, containing the catecholamines that were inside each sample of synaptosomes, were stored at −40°C for later analysis. The samples containing the released and retained catecholamines were injected into the HPLC system within the next week after the experiment. Results are expressed as the catecholamine concentration in pmoles per mg of synaptosomal protein. Endogenous DA release refers to the balance between the released DA and the DA taken up again for 10 min. Total DA (or total DOPAC) refers to the sum of the DA (or DOPAC) released plus the DA (or DOPAC) retained by an aliquot of synaptosomes exposed to a specific experimental condition.
Determination of catecholamine concentrations
Twenty microliter of the catecholamine containing samples in PCA/EDTA were injected into the HPLC system (Waters, Milford, MA, USA) for analysis. The HPLC system consists of a delivery pump (model 600), a Rheodyne injector, an analytical column (resolve, C18, 150 × 3.9 mm internal diameter, particle size 5 μm, controlled at 30°C) and an electrochemical detector (model DECADE), with glassy carbon used at a voltage of +0.8 V versus an Ag/AgCl reference electrode (range 1 nA). A mobile phase composed of 50 mM ortho-phosphoric acid/50 mM citric acid buffer, pH 3.1 adjusted with KOH, containing 5% (v/v) methanol, 100 mg/L octanesulfonic acid and 20 mg/L EDTA, at a flow rate of 1 mL/min, was applied for catecholamine elution. DA, DOPAC and homovanillic acid (HVA) concentrations in the experimental samples were calculated with calibration curves obtained from the injection of increasing concentrations of external standard monoamine mixtures into the HPLC system.
Determination of ROS in synaptosomes
Reactive oxygen species (ROS) were detected by DCF fluorescence (Ali et al. 1992). Aliquots (1.5 mL) of the striatum isolated nerve endings suspended in KRH (657 ± 58 μg/mL) or of the whole brain isolated nerve endings suspended in KRH (2996 ± 56 μg/mL) were incubated in the absence (control) or in the presence of 5 mM 3-NPA for 2 h at 37°C in a shaker water bath. After the incubation period an aliquot of 100 μL was separated from each tube for later protein determination by the method of Lowry. The remaining volume (1400 μL) was mixed with 100 μL of a 75 μM DCF-diacetate solution (to obtain 5 μM, final concentration) and then incubated in the dark at 37°C for 1 h. Then the samples were centrifuged at 6000 g for 10 min and the fluorescent DCF signal recorded in the supernatants from this centrifugation in a Perkin-Elmer LS50 spectrometer set at 488 nm (excitation wavelength) and 532 nm (emission wavelength). The final DCF concentration was calculated by interpolation of the experimental values in a DCF standard curve incubated in parallel with the experimental samples. Results were expressed as pmoles DCF per mg of synaptosomal protein.
Determination of DA-quinone products formation in synaptosomes
Dopamine quinones products were detected by NBT reduction (Paz et al. 1991). Aliquots (700 μL) of the striatum isolated nerve endings suspended in KRH (520 ± 29 μg/mL) or of the whole brain isolated nerve endings suspended in KRH (2330 ± 80 μg/mL) were incubated in the absence (control) or in the presence of 5 mM 3-NPA at 37°C in a shaker water bath for 2 h. After the incubation, the samples were centrifuged at 12 000 g for 10 min. The supernatant from this centrifugation was eliminated and the pellet containing the striatum or the whole brain isolated nerve endings was re-suspended in 1 mL of ice-cold 50 mM phosphate buffer, pH 7.4. An aliquot (100 μL) of the re-suspended pellet was separated from each tube and used for protein determination by the method of Lowry. The remaining volume (900 μL) was diluted with 900 μL of the NBT reagent (0.32 mM NBT in 2 M potassium glycinate at pH 10). The reaction mixture was incubated at 22°C in the dark for 1 h. At the end of the incubation, the samples were centrifuged at 12 000 g for 10 min. In the supernatant from this centrifugation the absorbance at 530 nm was measured in the spectrophotometer (UNICO, Dayton, NJ, USA).
Effect of 3-NPA on striatum catecholamine levels ex vivo
Eight animals were included in this experimental set. Half of them were injected i.p. with saline (control group) and four with 3-NPA (50 mg/kg i.p.). When after 90 min no obvious motor impairment was observed, additional 3-NPA (20 mg/kg i.p.) was injected. All animals were decapitated within the first 2 h after the saline or 3-NPA injections and their striata dissected and homogenized in the PCA/EDTA mixture. Striatum homogenates were then centrifuged at 14 000 g for 10 min. The supernatants resulting from this centrifugation were transferred to clean vials and injected into the Waters HPLC system for catecholamine analysis. The resulting pellets were used for protein determination.
Paired Student’s t-test was used for statistical evaluations. From p < 0.05 differences between data were considered statistically significant. However, most of the significant differences between paired data obtained in parallel were below this value, as indicated in the tables and figures.
Comparison of 3-NPA effect on DA and DOPAC distribution inside and outside striatum and whole brain isolated nerve endings
The net changes (over the respective control values) produced by 3-NPA for 10 min in striatum and whole brain synaptosomes under baseline conditions on: the external, internal and total DA and DOPAC concentrations were compared. Fig. 1a shows that DA baseline release only increased slightly over control values in both, striatum and whole brain isolated nerve endings exposed to 3-NPA for 10 min. However, the marked increase in the internal DA concentration produced by 3-NPA for 10 min in striatum synaptosomes, contrasts with the modest increase in the internal DA concentration produced by 3-NPA for 10 min in whole brain synaptosomes (Fig. 1b). Interestingly, the 3-NPA-induced increases in DA particularly observed in striatum synaptosomes was accompanied by a decrease in the concentration of DOPAC, both outside and inside the isolated nerve endings (Fig. 1d and e).
In nerve endings isolated from the striatum DA and DOPAC concentrations were about fivefold higher than DA and DOPAC concentrations in the whole brain isolated nerve endings; About 400 pmoles and 80 pmoles per mg of striatum and whole brain synaptosomal protein, respectively.
Effect of 3-NPA on DA exocytosis in striatal isolated nerve endings
The effect of 3-NPA on DA and DOPAC distribution under high K+ depolarized conditions was tested in striatum synaptosomes incubated for 10 min.
In the striatum nerve endings incubated with a high level (30 mM) of external K+, the external DA concentration (74 ± 6 pmoles/mg of synaptosomal protein) was more than threefold higher than in the striatum isolated nerve endings incubated under resting conditions (20 ± 1 pmoles/mg of synaptosomal protein). Oppositely, the internal DA concentration in the striatum nerve endings incubated with high K+ (363 ± 33 pmoles/mg) was below the internal DA concentration in the striatum nerve endings incubated under resting conditions (420 ± 30 pmoles/mg). As the DA released to the external medium was lost inside synaptosomes, the total DA concentration in the high K+ depolarized synaptosomes remained unchanged with respect to the non depolarized synaptosomes. The net changes produced by high K+ (i.e. minus baseline control values) on DA release, DA inside and total DA are shown in the black bars on Fig. 2a, b and c, respectively.
Interestingly, 3-NPA potentiated DA release induced by high K+ : net DA release to high K+ alone was 54 ± 7 pmoles/mg of synaptosomal protein, to 3-NPA alone 4.5 ± 1.0 pmoles/mg of synaptosomal protein and to high K+ in the presence of 3-NPA of 102 ± 12 pmoles/mg of synaptosomal protein. Gray bars in Fig. 2a and b show the net changes produced by high K+ in combination with 3-NPA on DA release and on internal DA, respectively. As the decrease in the internal DA concentration produced by high K+ alone was not produced by high K+ in combination with 3-NPA (Fig. 2b), the total concentration of DA increased in the synaptosomes depolarized in the presence of 3-NPA (gray bar in Fig. 2c).
In agreement with previous findings in striatal synaptosomes (Trejo et al. 2001), the concentration of DOPAC was unmodified by high K+ depolarization; the bottom black bars in Fig. 2 show the net DOPAC concentration (minus control values) in synaptosomes exposed to high K+ alone. However, in the high K+ depolarized synaptosomes simultaneously exposed to 3-NPA, a decrease in DOPAC was again produced. Gray bars of Fig. 2d, e and f show the net changes exerted by high K+ in combination with 3-NPA in the external, the internal and the total DOPAC concentrations, respectively.
3-NPA effect on ROS production in striatum and whole brain synaptosomes
The DCF-diacetate fluorescent assay that detects ROS production and is commonly used as an index of oxidative stress was performed in striatum and whole brain isolated nerve endings incubated in the absence and presence of 5 mM 3-NPA for 2 h under resting conditions.
As judged by the fluorescent product formed by dichlorofluorescein oxidation, ROS production (standardized per mg of synaptosomal protein) was more than fourfold higher in striatum than in whole brain synaptosomes. The presence of 3-NPA in the incubation medium further increased ROS production both, in striatum and whole brain synaptosomes. However, the net ROS increase induced by 3-NPA was higher in striatum than in whole brain synaptosomes (Table 1).
|Nerve endings from||Experimental condition||ROSa||3-NPA net change|
|Striatumb||Control||117 ± 8||46 ± 8|
|3-NPA||163 ± 14**|
|Whole brainc||Control||26 ± 2||16 ± 4|
|3-NPA||42 ± 3*|
Effect of 3-NPA on DA-quinone products formation in striatum synaptosomes
The effect of 3-NPA on protein bound DA-quinones formation in striatum and whole brain isolated nerve endings was estimated at 530 nm using the modification of the NBT/glycinate assay (Paz et al. 1991) described in the Methods section.
Table 2 shows that the amount of DA-quinoprotein adducts formed for 2 h under resting conditions in synaptosomes isolated from striatum is about eight times higher than the amount of those products formed in the synaptosomes isolated from whole brain, and that this amount is further increased by the presence of 3-NPA in the incubation medium, particularly in striatum synaptosomes. The right column in Table 2 shows that the net increase induced by 3-NPA in the formation of those products is ten times higher in synaptosomes isolated from the striatum than in synaptosomes isolated from the whole brain.
|Nerve endings from||Experimental condition||DA-quinone productsa||3-NPA net change|
|Striatumb||Control||0.190 ± 0.023||0.122 ± 0.018|
|3-NPA||0.312 ± 0.039**|
|Whole brainc||Control||0.023 ± 0.001||0.012 ± 0.003|
|3-NPA||0.035 ± 0.003*|
Long term effect of 3-NPA on DA and DOPAC distribution inside and outside striatum and whole brain isolated nerve endings under resting conditions
Figure 3 shows that in comparison with the net changes exerted by 3-NPA for 2 h on catecholamine distribution in the striatum isolated nerve endings (gray bars), the net changes exerted by 3-NPA on catecholamine distribution in whole brain isolated nerve endings were very small (white bars).
The net changes in DA distribution exerted by 3-NPA in striatum isolated nerve endings for 2 h are shown in the upper graphs in Fig. 3. These changes contrast with the net changes exerted by 3-NPA after 10 min. For instance, the marked increase in DA release observed in striatal synaptosomes exposed to 3-NPA for 2 h (Fig. 3a), contrasts with the modest increase in DA release exerted by 3-NPA for 10 min (Fig. 1a), and the drop in the internal DA concentration observed in striatal synaptosomes exposed to 3-NPA for 2 h (Fig. 3b) contrasts with the marked increase in the internal DA concentration produced by 3-NPA after 10 min (Fig. 1b).
Interestingly, although total DA was even decreased after 2 h of 3-NPA exposure (Fig. 3c), the decrease in total DOPAC observed in striatum isolated nerve endings after 10 min of 3-NPA exposure (Fig. 1f) is still observed after 2 h of 3-NPA exposure (Fig. 3f).
Striatum catecholamine changes accompanying the motor disturbances induced by 3-NPA in vivo
Two of the four animals administered with 50 mg/kg 3-NPA presented marked signs of motor impairment about half an hour after the 3-NPA injection. In the other two animals administered with 50 mg/kg 3-NPA no obvious signs of motor impairment after 90 min were observed. Therefore, they were injected with an additional dose (20 mg/kg) of 3-NPA. After this injection, these two animals also developed marked signs of motor impairment in less than half an hour. As judged by their weights (274 and 248 g), the two animals that presented the motor impairment with 50 mg/kg 3-NPA were the older animals. The younger ones (weighting 210 and 208 g) presented the same pronounced motor disturbances with the additional 20 mg of 3-NPA per kg. In the four control animals injected with saline in parallel no signs of motor disturbances were observed. Weights of control animals were: 239, 268, 217 and 203 g.
Table 3 shows that the total DA concentration in the striatum homogenates from the animals injected with 3-NPA was unchanged in comparison with the animals injected with saline. However, in the striatum homogenates from animals injected with 3-NPA the total concentration of the DA metabolites, HVA and DOPAC, was increased to twice the concentration in the striatum homogenates of control animals. It is worthy to mention that HVA was not detected in striatal synaptosomes.
|DA||277 ± 11||301 ± 15||0.11|
|DOPAC||38 ± 3.2||69 ± 3.3||0.0003|
|HVA||32 ± 2.1||52 ± 6.3||0.01|
In the present study we have shown that 3-NPA exposure of striatum and whole brain isolated nerve endings for a short time period (10 min) increased total DA and decreased total DOPAC under both, resting and high K+ depolarized conditions, and that 3-NPA exposure under resting conditions for a longer time period (2 h) increased oxidative stress, DA-quinoprotein adducts formation and decreased total DOPAC. All the above changes were by far more pronounced in the nerve endings isolated from the striatum than in those isolated from the whole brain. We also found that in the animals exposed to 3-NPA in vivo striatal catecholamine changes were reflected in an increased level of DA metabolites.
Interpretation of 3-NPA effects on DA and DOPAC distribution in the isolated nerve endings in vitro
The fivefold higher concentration of DA in the nerve endings isolated from the striatum than in those isolated from the whole brain is consistent with the much larger increase in total DA (Figs 1c and 2c) and decrease in total DOPAC (Figs 1f and 2f) concentrations induced by 3-NPA in the striatum than in the whole brain isolated nerve endings, and suggests that DA metabolism is involved in the selective striatal vulnerability exerted by 3-NPA (notice that the striatum nerve endings are included in the nerve endings isolated from the whole brain). The decrease in DOPAC produced by 3-NPA, particularly evident in the synaptosomes isolated from the striatum, suggests that 3-NPA inhibits DA degradation by indirectly inhibiting MAO-A activity, which is known to be localized pre-synaptically in the nigro-striatal dopaminergic nerve endings (Agid et al. 1973; Demarest et al. 1980). This decrease in DA metabolism evidenced by the drop in DOPAC was accompanied by an increase in total DA concentration after 10 min (Fig. 1c) or by an increase in DA quinone products formation after 2 h (Table 2).
High K+ depolarization selectively releases the vesicular pool of DA. Neurotransmitter release and the increase in Ca2+ induced by high K+ in cerebral isolated nerve endings depends on external Ca2+ and is sensitive to the P/Q type Ca2+ channel blocker toxins, ω-Aga-IVA and ω-Aga-TK (Turner et al. 1992; Sitges and Chiu 1995; Sitges and Galindo 2005). Hence, our findings that a short exposure (10 min) to 3-NPA potentiates DA exocytosis, as judged by the potentiating 3-NPA effect on the high K+ evoked release of DA (Fig. 2a), strongly suggest that the DA that was not metabolized to DOPAC after 10 min of 3-NPA exposure was accumulated inside synaptic vesicles.
Our findings that, although 3-NPA exposure of striatal synaptosomes for 2 h decreased DOPAC formation, did not increase striatal DA and rather decreased it (Fig. 3c), strongly suggests that the oxidative stress induced by 3-NPA for 2 h (Table 1) transformed a large amount of the DA not metabolized to DOPAC to DA-oxidation products, as supported by the marked increase in DA-quinone derived quinoprotein adducts induced by 3-NPA predominantly observed in striatal synaptosomes (Table 2). Our failure of detecting a rise in oxidative stress in the presence of 3-NPA after 10 min is in agreement with previous results in vitro showing that the sensitivity of the method was not enough for detecting the increase in ROS induced by mM concentrations of 3-NPA at time periods shorter than 2 h (Raut et al. 2006).
For investigating if the markedly higher ROS production observed in the striatum was linked to the formation of DA quinones we followed the NBT/glycinate assay, previously used in mitochondrial preparations exposed to 50–400 μM DA (Khan et al. 2001, 2005; Jana et al. 2007). NBT reduction measured at 530 nm gives an estimation of the amount of DA-quinoprotein adducts formed. Therefore, data obtained here with the NBT/glycinate assay indicate that the DA present in the isolated nerve endings, and particularly in those isolated from the striatum, was enough to allow detection of the adducts. Consequently, the DA quinone adducts must originate from the DA-quinones covalently bound to the thiol groups of the proteins present in the isolated nerve endings. As cysteines are known to be essential for MAO activity (Wu et al. 1993; Sablin and Ramsay 1998), it is possible that DA-quinones produced by 3-NPA are inhibiting MAO activity forming ‘quino-MAO-adducts’. Moreover, quinones of both DA and L-DOPA were shown to inhibit tryptophan hydroxylase and modify the protein to a redox-active quino-tryptophan hydroxylase (Kuhn and Arthur 1998, 1999). Therefore we cannot discard the possibility that tyrosine hydroxylase, the rate limiting enzyme in DA synthesis, is also being transformed to a redox-active quino-tyrosine hydroxylase.
In brain mitochondria DA-oxidation products have shown to inhibit complexes I and IV of the respiratory chain (Khan et al. 2005; Jana et al. 2007). Therefore, the higher ROS production in striatum than in whole brain synaptosomes, where DA is less abundant, also must be explained by the inhibition of those complexes. In the presence of 3-NPA, which irreversibly inhibits complex II of the respiratory chain and simultaneously decreases DA metabolism (as shown here), ROS production and DA-oxidation products are further increased particularly in DA rich brain regions, explaining why 3-NPA is more harmful in the striatum than in other brain areas. DA metabolism to DOPAC by MAO-A activity is accompanied by H2O2 formation, and in the isolated nerve endings, 3-NPA decreased DOPAC formation with the consequent decrease in H2O2 production. Therefore 3-NPA-induced increased in ROS production inside synaptosomes is unlikely to be linked to H2O2 formation and rather may be primarily linked to formation of DA-oxidation products capable to inhibit other sites of the respiratory chain.
To put it briefly, our data in vitro indicate that the DA that was not metabolized to DOPAC in the striatal synaptosomes exposed to 3-NPA for 10 min was accumulated inside synaptic vesicles (Fig. 4a), whereas in the striatal synaptosomes exposed to 3-NPA for 2 h the DA that was not metabolized to DOPAC was found in the form of DA-oxidation products or in the external medium, where was released most probably via reversal of the DA transporter (DAT). The marked rise in DA-oxidation products induced by 3-NPA may stop the Na+/K+-ATPase and increase the internal concentration of Na+ with the concomitant DAT-mediated release of DA, explaining the large increase in DA baseline release observed in the striatum isolated nerve endings incubated with 3-NPA for 2 h. Because in cerebral isolated nerve endings oxidative stress in the presence of exogenous DA was shown to inactivate Na+/K+-ATPase (Chakraborty et al. 2003; Bagh et al. 2008) and the inhibition of the Na+/K+-ATPase was shown to increase the internal concentration of Na+ (Galvan and Sitges 2004), with the concomitant DAT-mediated release of DA (Sitges et al. 1994). Moreover, the expected drop in ATP after 2 h of 3-NPA exposure may also stop the H+-ATPase coupled to vesicular monoamine transporter preventing DA accumulation inside synaptic vesicles (Fig. 4b).
Interpretation of 3-NPA-induced changes in striatum DA metabolism ex- vivo
The effect of 3-NPA on striatal catecholamine concentrations ex vivo was tested in striatum homogenates and not in striatal synaptosomes. Because our in vitro results showed that after 2 h of 3-NPA exposure, the DA that was not transformed to DA-quinone oxidation products was released to the external medium (Fig. 3a), and because most of the DOPAC formed also was released to the external medium. Thus we assumed that a large concentration of those catecholamines could be washed during the synaptosomal preparation procedure. In the striatum homogenates from the animals administered with 3-NPA in vivo we found that both DA metabolites, DOPAC and HVA, were increased. This finding suggests that the DA released by 3-NPA was probably metabolized by non neuronal enzymes that were absent in synaptosomes, such as MAO-B and catechol-O-methyltransferase (COMT) (Reeniläet al. 1997; Helkamaa et al. 2007). In contrast with MAO-A, MAO-B is not localized inside nerve endings. Thus, is not expected to be inhibited by the 3-NPA-induced increase in DA-quinones inside the nigro-striatal nerve endings. Therefore the increase in DOPAC observed in striatum homogenates from the 3-NPA administered animals may result from metabolism, by MAO-B, of the released DA. In line, HVA which results from COMT activity was undetected in striatal synaptosomes and clearly detected in striatal homogenates; suggesting that the DA released by 3-NPA also was the source of this metabolite (Fig. 4c).
Previous experiments in vivo showed that a mixture of MAO-inhibitors (MAO-I) injected i.p. for 7 days reduced the lesion volume induced by 3-NPA (20 mg/kg i.p. injected daily for the last 4 days) in the rat striatum (Maragos et al. 1999). The chronic (1 week) treatment with the MAO-I at high doses is expected to increase DA and decrease DOPAC, as 3-NPA did in the striatum isolated nerve endings. However, this apparent controversy may be explained by unpublished results from our laboratory in striatum isolated nerve endings showing that the selective MAO-A-inhibitor, clorgyline, at high (> 30 μM) concentrations released all the DA not metabolized to the external medium decreasing the internal concentration of DA even below control values. As in the absence of DA inside the striatum nerve endings, 3-NPA is unable to produce DA-quinones and quinone-adducts with MAO-A, it is possible that the high doses of the MAO-I used by Maragos et al. (1999)in vivo were inhibiting the 3-NPA induced lesion in the striatum by decreasing DA inside nerve endings. Alternatively, in the 3-NPA administered animals external DA metabolism is expected to generate H2O2. Nevertheless the possibility that inhibition of external DA metabolism by MAO-B with the concomitant prevention of H2O2 production participates in the protection exerted by the MAO-I on 3-NPA-induced striatal vulnerability is unlikely. Because in MAO-B knock-out mice the striatum vulnerability to 3-NPA was not inhibited (Maragos et al. 2004).
In summary, it is concluded that in the nigro-striatal nerve endings, where DA is particularly concentrated, the increase in ROS induced by 3-NPA, oxidizes DA generating DA-quinones. These quinones may form adducts with the MAO-A active site reducing its activity. Part of the DA not metabolized to DOPAC by this means might generate more DA-oxidation products that will inhibit further the respiratory chain (and MAO-A) and part is released to the external medium, via DAT reversal. Our data in vivo suggest that this DA released by 3-NPA is metabolized to DOPAC and to HVA by MAO-B and by COMT, respectively.
The authors thank Araceli Guarneros and Luz María Chiu for their excellent technical assistance. This work was supported by grant D-48695 from SEP-CONACYT. Nieves Herrera-Mundo scholarship also was supported by SEP-CONACYT.