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Keywords:

  • 3-nitropropionic acid;
  • dopamine–quinone adducts;
  • MAO ;
  • synaptosomes;
  • vinpocetine;
  • VMA

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Vinpocetine is a neuroprotective drug that exerts beneficial effects on neurological symptoms and cerebrovascular disease. 3-nitropropionic acid (3-NPA) is a toxin that irreversibly inhibits succinate dehydrogenase, the mitochondrial enzyme that acts in the electron transport chain at complex II. In previous studies in striatum-isolated nerve endings (synaptosomes), we found that vinpocetine decreased dopamine (DA) at expense of its main metabolite 3,4-dihydroxyphenylacetic acid (DOPAC), and that 3-NPA increased DA, reactive oxygen species (ROS), DA-quinone products formation, and decreased DOPAC. Therefore, in this study, the possible effect of vinpocetine on 3-NPA-induced increase in DA, ROS, lipid peroxidation, and DA-quinone products formation in striatum synaptosomes were investigated, and compared with the effects of the antioxidant α-tocopherol. Results show that the increase in DA induced by 3-NPA was inhibited by both 25 μM vinpocetine and 50 μM α-tocopherol. Vinpocetine, as α-tocopherol, also inhibited 3-NPA-induced increase in ROS (as judged by DCF fluorescence), lipid peroxidation (as judged by TBA-RS formation), and DA-quinone products formation (as judged by the nitroblue tetrazolium reduction method). As in addition to the inhibition of complex II exerted by 3-NPA, 3-NPA increases DA-oxidation products that in turn can inhibit other sites of the respiratory chain, the drop in DA produced by vinpocetine and α-tocopherol may importantly contribute to their protective action from oxidative damage, particularly in DA-rich structures.

Abbreviations used
3-NPA

3-nitropropionic acid

4-HQ

4-hydroxyquinoline

DA

3,4-dihydroxyphenylethylamine or dopamine

DCF

dichlorofluorescein

DOPAC

3,4-dihydroxyphenylacetic acid

MDA

malondialdehyde

NBT

nitroblue tetrazolium

ROS

reactive oxygen species

TBA

thiobarbituric acid

3-Nitropropionic acid (3-NPA) is a mitochondrial toxin that irreversibly inhibits succinate dehydrogenase, an enzyme that acts in both the tricarboxylic acid cycle and the electron transport chain at complex II (Alexi et al. 1998). The particular vulnerability of the striatum to the harmful effects of 3-NPA has been recognized in several studies (Ludolph et al. 1991; Fu et al. 1995; Brouillet et al. 1998; Reynolds et al. 1998; Villarán et al. 2008). The striatum is a cerebral structure that concentrates the largest amount of DA in the brain. In a recent study, we found that a 10-min exposure of striatum isolated nerve endings to 3-NPA increased the concentration of DA at expense of its main metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) (Herrera-Mundo and Sitges 2010). In that study, we also found that a longer exposure (2 h) to the mitochondrial neurotoxin also increased reactive oxygen species (ROS) and DA-quinone products formation.

Vinpocetine (ethyl apovincamine-22-oate) is a neuroprotective drug derived from the leaves of Vinca minor that exerts beneficial effects on neurological symptoms and cerebrovascular disease accompanied by hypoxia and ischemia (King 1987; Araki et al. 1991). In cerebral isolated nerve endings, vinpocetine inhibits pre-synaptic Na+ channels permeability (Tretter and Adam-Vizi 1998; Sitges and Nekrassov 1999; Sitges et al. 2007). In addition to the inhibition exerted by vinpocetine on pre-synaptic Na+ channels' permeability, vinpocetine decreases DA and increased DOPAC in striatum isolated nerve endings, either under resting and under depolarized conditions, by a mechanism apparently unrelated to Na+ channels (Trejo et al. 2001; Sitges et al. 2009).

In primary cultures of rat striatal neurons, α-tocopherol, a compound with antioxidant capacity was shown to inhibit ROS formation (Osakada et al. 2003). The antioxidant properties and the scavenging ability of α-tocopherol to trap the hydroxyl radical generated by the Fenton reaction are similar to those of vinpocetine (Oláh et al. 1990). As the more pronounced oxidative damage induced by 3-NPA in striatum isolated nerve endings is likely to be linked to the increase in DA and particularly DA-quinone products formation (Herrera-Mundo and Sitges 2010), in this study, the potential capacity of vinpocetine and α-tocopherol to overcome 3-NPA-induced: DA and DOPAC concentration changes, oxidative stress, and DA-quinone products formation was investigated in striatal isolated nerve endings under resting conditions.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Source of materials

Drugs used in the experiments were acquired from the following companies: vinpocetine (eburnamenine-14-carboxylic acid ethyl ester) was obtained either from Sigma Chemical Co. (St. Louis, MO, USA) or from Psicofarma S.A. de C.V. (México), 3-nitropropionic acid (3-NPA), α-tocopherol, kynuramine dihydrobromide, 4-hydroxyquinoline (4-HQ), n-methyl-n-propargyl-3-(2,4-dichloro-phenoxy)propylamine (clorgyline), n-methyl-n-2-propylnyl-benzylamine hydrochloride (pargyline), 2-thiobarbituric acid (TBA), nitrotetrazolium blue chloride (NBT), and glycine were from Sigma Aldrich (St. Louis, MO, USA). 2′,7′-dichlorofluorescein (DCF) and DCF-diacetate were from Sigma-Aldrich (Steinheim, Germany and St. Louis, MO, USA respectively). Reserpine was obtained from Research Biochemicals International. 3,4-[73H]-dihydroxyphenylethylamine (3[H]DA) specific activity 28 Ci/mmol was obtained from Perkin Elmer (Boston, MA, USA). All other reagents were of analytical grade.

Determination of DA and DOPAC concentrations in striatum isolated nerve endings

The concentrations of endogenous DA and DOPAC released and retained in rat striatum isolated nerve endings (synaptosomes) were determined as previously described (Herrera-Mundo and Sitges 2010). Briefly, aliquots (500 μL) containing 445 ± 9.4 μg of synaptosomal protein suspended in Krebs Ringer HEPES (KRH) were incubated at 37°C for 10 or 120 min under the experimental conditions to be tested. 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. After incubation, the samples were centrifuged for 5 min at 12 000 g. Aliquots (20 μL) of the resulting supernatants and pellets containing the released and retained catecholamines, respectively, were injected to the HPLC system. Results are expressed as catecholamine concentration in pmoles per mg of synaptosomal protein.

MAO assay

Monoamine oxidase (MAO) activity was measured following the procedure described in Morinan and Garratt (1985) with some modifications. Briefly, aliquots (500 μL) of striatum isolated nerve endings suspended in KRH containing 919 ± 0.02 μg of synaptosomal protein were incubated at 37°C for 10 min under the experimental conditions to be tested; namely, vinpocetine (25 μM), α-tocopherol (50 μM), 3NPA (5 mM), vinpocetine plus 3-NPA, α-tocopherol plus 3-NPA, or clorgyline (10 μM) as positive control. The reaction was started by the addition of an aliquot (250 μL) of kynuramine to obtain a 22 μM final concentration in the sample. After 30 min incubation at 37°C, the reaction was stopped by the addition of an aliquot (225 μL) of PCA 0.4 M. After a brief mixture, samples were centrifuged for 20 min at 16 100 g. The supernatant from this centrifugation (900 μL) was mixed with an equal volume (900 μL) of 1 M NaOH and the fluorescent signal was recorded in a LS-50B Luminescence Spectrophotometer (Perkin-Elmer, Beaconsfield, Buckinghamshire, England) at 315 nm (excitation wavelength) and 380 nm (emission wavelength). The final concentration of 4-hydroxyquinoline (4-HQ), which results from deamination of kynuramine by MAO, was calculated by interpolation of the experimental values in a 4-HQ standard curve, incubated in parallel with the experimental samples. Pellets were resuspended in 10 mM NaOH and used for protein determination. Enzyme activity was expressed as nmol /mg of synaptosomal protein per hour.

Isolation of rat striatum synaptic vesicles

The synaptosomal pellet obtained from striata of four rats was subjected to osmotic shock by addition of 7 mL of cold distilled water and homogenized with five strokes at 900 rpm. The homogenate was incubated on ice for 30 min. Osmolarity was readjusted by addition of 350 μL 0.5 M HEPES pH 6.5 and 350 μL neutral 2 M potassium tartrate. The preparation was centrifuged at 22 000 g for 15 min at 4°C, and the resulting supernatant centrifuged at 100 000 g for 45 min. The pellet resulting from this last centrifugation, which contains the synaptic vesicles, was gently resuspended (glass/teflon) in 900 μL of the uptake buffer to a concentration of approximately 50 μg protein/mL. The composition of the uptake buffer was 100 mM potassium tartrate, 25 mM HEPES, 50 μM EGTA, 100 μM EDTA, 50 μM pargyline, 1.7 mM ascorbic acid, and 2 mM Mg-ATP, pH 7.4 adjusted with KOH. The vesicle preparation was completed in less than 4 h and used immediately for the [3H]DA uptake experiment.

[3H]DA uptake into synaptic vesicles

Uptake buffer containing 60 nM [3H]DA (200 μL) was placed in four tubes. In two of them, a small aliquot of a concentrated solution of vinpocetine or reserpine was added. [3H]DA uptake into synaptic vesicles was initiated by the addition of 200 μL of the uptake buffer in which the synaptic vesicles (50 μg/mL) were suspended to the control tube, without drug, or to the tubes containing vinpocetine or reserpine. Two hundred microliters of uptake buffer without vesicles were added to the fourth tube, that also contained 200 μL of uptake buffer with [3H]DA. This tube was treated like the experimental tubes and used as the blank. After 10 min of incubation at 37°C, uptake was terminated by rapid filtration under vacuum with 6 mL of cold buffer onto Millipore filters (DAWP type 0.65 μm) placed in the 1225 Millipore Sampling Manifold (Millipore Corporation, Bedford, MA, USA). The amount of radioactivity bound to each filter was counted by liquid scintillation spectrometry (Beckman Coulter LS 6500 scintillation counter, Fullerton, CA, USA). The radioactivity bound to the blank filter was subtracted to the experimental filters.

Determination of ROS in synaptosomes

ROS were determined by DCF fluorescence as described in Herrera-Mundo and Sitges 2010. Briefly, aliquots (1.5 mL) of striatum synaptosomes suspended in KRH (659 ± 32 μg/mL) were incubated for 2 h at 37°C with vinpocetine or α-tocopherol in the absence or presence of 3-NPA. Results were expressed as pmoles DCF per mg of synaptosomal protein.

Determination of TBA-RS in synaptosomes

TBA reactive substances (TBA-RS) were detected by the TBA assay described in Pérez-De La Cruz et al. (2006) with minor modifications. Briefly, aliquots (1 ml) of the striatum isolated nerve endings suspended in KRH (973 ± 24 μg/mL) were incubated in KRH with 25 μM vinpocetine or 50 μM α-tocopherol in the absence or in the presence of 5 mM 3NPA at 37°C in a shaker water bath for 2 h. After the incubation, an aliquot (100 μL) was separated for later protein determination by the method of Lowry. The remaining volume (900 μL) of striatal synaptosomes suspended in KRH was mixed with an equal volume (900 μL) of the TBA reagent (containing 375 mg TBA, 15 g trichloroacetic acid, and 2.5 mL HCl in 100 ml H2O) and incubated in a heated boiling water bath for 30 min. After this time, the samples were kept on ice for 10 min and then centrifuged at 12 000 g for 15 min. The optical density in the supernatants obtained from this centrifugation was measured at 532 nm using a spectrophotometer (UNICO 1100RS, United Products and Instruments Inc., Dayton, NJ, USA). The final concentration of TBA-RS was calculated by interpolation of the experimental values in a malondialdehyde (MDA) standard curve incubated in parallel with the experimental samples. Results were expressed in nmoles MDA per mg of synaptosomal protein.

Determination of DA-quinone products formation in synaptosomes

DA quinones products were detected by nitroblue tetrazolium (NBT) reduction in aliquots (700 μL) of striatum isolated nerve endings suspended in KRH (463 ± 21 μg/mL) incubated with vinpocetine or α-tocopherol with or without 3NPA at 37°C for 2 h following the method previously described in Herrera-Mundo and Sitges 2010.

Statistical analysis

All data were analyzed by one-way analysis of variance (anova) followed by the Tukey's test. Values of p < 0.05 were considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Single and combined effects of vinpocetine and 3-NPA on DA and DOPAC distribution

Table 1 shows that incubation of striatum synaptosomes for 10 min in the presence of vinpocetine alone does not change the baseline release of DA, but markedly decreased the internal DA concentration. In addition to this drop in internal DA, vinpocetine increased the internal and external DOPAC concentrations. On the other hand, exposure of striatum synaptosomes for 10 min to 3-NPA alone increased internal DA and decreased internal DOPAC (third column in Table 1). Interestingly, the increase in internal DA induced by 3-NPA at 10 min was overcome by vinpocetine (last column in Table 1).

Table 1. Single and combined effects of vinpocetine and 3-NPA on DA and DOPAC distribution in isolated nerve endings at 10 min
 ControlVinpocetine3-NPAVinpocetine + 3-NPA
  1. Data are expressed in pmoles/mg of synaptosomal protein. Results are the mean ± SEM values from five striatum synaptosomes independent preparations.

  2. a

    p < 0.01 between control and vinpocetine.

  3. b

    p < 0 03 between control and 3-NPA.

  4. c

    p < 0.02 between control and vinpocetine plus 3-NPA.

DA released20 ± 1.317 ± 0.926 ± 1.731 ± 2.8c
DA inside420 ± 30256 ± 18a508 ± 37b346 ± 27c
DOPAC out321 ± 45423 ± 48a309 ± 35378 ± 42c
DOPAC in157 ± 39197 ± 47a125 ± 32b141 ± 37

Single and combined effects of α-tocopherol and 3-NPA on DA and DOPAC distribution

Table 2 shows that, as in the case of vinpocetine, at 10 min α−tocopherol failed to change DA baseline release, decreased the internal DA concentration, and overcome the increase in internal DA induced by 3-NPA. Although in contrast to vinpocetine, α-tocopherol failed in modifying DOPAC concentrations significantly by itself.

Table 2. Single and combined effects of a-tocopherol and 3-NPA on DA and DOPAC distribution in isolated nerve endings at 10 min
 Controlα-tocopherol3-NPAα-tocopherol + 3-NPA
  1. Data are expressed in pmoles/mg of synaptosomal protein. Results are the mean ± SEM values from four striatum synaptosomes independent preparations.

  2. a

    p < 0.01 between control and a-tocopherol.

  3. b

    p < 0.03 between control and 3-NPA.

  4. c

    p < 0.003 between control and a-tocopherol plus 3-NPA.

DA released18 ± 0.719 ± 2.125 ± 1.131 ± 1.6c
DA inside431 ± 42331 ± 14a502 ± 37b404 ± 32
DOPACout289 ± 41312 ± 43278 ± 33283 ± 38
DOPAC in116 ± 19117 ± 1787 ± 13b84 ± 12c

Single and combined effects of 3-NPA, vinpocetine, and α-tocopherol on MAO activity

The possible actions of 3-NPA or vinpocetine on MAO activity in striatum isolated nerve endings were investigated directly. Also the effects on MAO activity of α-tocopherol and of the MAO inhibitor, clorgyline, were tested in parallel. We found that under control conditions, striatum synaptosomes produced 6945 ± 297 pmoles of the MAO activity product, 4-HQ per mg of synaptosomal protein at 10 min. 3-NPA decreased this concentration of 4-HQ by 1677 ± 157 pmoles/mg of synaptosomal protein, whereas vinpocetine and α-tocopherol failed in modifying 4-HQ, or to overcome the decrease in MAO-activity exerted by 3-NPA. The potent MAO-A inhibitor, clorgyline, decreased 4-HQ by 3994 ± 494 pmoles/mg of synaptosomal protein at 10 min. The effect on MAO activity exerted by the different experimental conditions tested, expressed in percentage of control, is shown in Fig. 1.

image

Figure 1. Single and combined effects of 3-nitropropionic acid (3-NPA) and vinpocetine or/and α-tocopherol and of clorgyline on MAO activity. Striatum synaptosomes suspended in KRH without (control) or containing 3-NPA (5 mM), vinpocetine (25 μM), α-tocopherol (50 μM), clorgyline (10 μM), or the indicated drug combinations were incubated at 37°C for 10 min with kynuramine. 4-HQ, the product that results from kynuramine deamination by MAO was measured as indicated in Methods. Results are expressed in percentage of control and are the mean ± SEM values of five experiments in independent preparations. *p < 0.05.

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Effect of vinpocetine on [3H]DA uptake by synaptic vesicles

Results shown in Table 1, together with our previous observation that vinpocetine, like reserpine, increased DA baseline release when MAO activity was inhibited by clorgyline (Trejo et al. 2001), suggest the possibility that vinpocetine could be blocking DA uptake mediated by the vesicular monoamine transporter. With the purpose of studying this possibility, the effect of vinpocetine on [3H]DA uptake into striatum isolated synaptic vesicles was investigated, and compared with the effect of reserpine. Results obtained in parallel show that the accumulation of [3H]DA into control synaptic vesicles (919 ± 30 cpm) was decreased by 25 μM vinpocetine to 329 ± 23 cpm. In parallel experiments, 1 μM reserpine decreased the accumulation of [3H]DA in control synaptic vesicles (903 ± 44 cpm) even further, to 187 ± 7 cpm. Fig. 2 shows these results expressed as percentage of the controls obtained in parallel.

image

Figure 2. Effect of vinpocetine on [3H]DA uptake into synaptic vesicles. Aliquots of striatum isolated synaptic vesicles suspended in uptake buffer containing [3H]DA were incubated at 37°C for 10 min in the absence (white bars) or in the presence of 25 μM vinpocetine or 1 μM reserpine, as indicated. Results are expressed in percentage of control and are the mean ± SEM values of five (vinpocetine) or four (reserpine) experiments in independent preparations. The statistic significance (p) obtained between two specific experimental conditions is indicated over the bars.

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Comparison of vinpocetine and α-tocopherol effects on 3-NPA-induced increase in DA-quinone products formation

The effect of vinpocetine and α-tocopherol in the absence and presence of 3-NPA on DA-quinone products formation was estimated at 530 nm using the NBT/glycinate assay described in the Methods section. Table 3 shows that the amount of DA-quinone products formed for 2 h in striatum isolated nerve endings under control conditions was unchanged by 25 μM vinpocetine or by 50 μM α-tocopherol, but increased about 60% by the presence of 3-NPA. Interestingly, this increase in 3-NPA-induced DA-quinone products formation was completely prevented by 25 μM vinpocetine and by 50 μM α-tocopherol.

Table 3. Effects of vinpocetine or α-tocopherol alone and in combination with 3-NPA on quinoprotein adducts formation in striatum isolated nerve endings for 120 min
Experimental conditionaDA-quinone productsp value between the indicated experimental conditions
  1. a

    Quinoprotein adducts are expressed in absorbance at 530 nm/mg of synaptosomal protein. Results are the mean ± SEM values of four independent experiments in striatal synaptosomes NS, not statistically significant.

(a) Control0.189 ± 0.03  
(b)3-NPA0.310 ± 0.05

(a) vs. (b)

<0.001

 
(c) Vinpocetine0.179 ± 0.03

(a) vs. (c)

NS

(c) vs. (d)

NS

(d) Vinpocetine + 3-NPA0.173 ± 0.03

(a) vs. (d)

NS

(b) vs. (d)

<0.001

(e) α-tocopherol0.187 ± 0.03

(a) vs. (e)

NS

(e) vs. (f)

NS

(f) α-tocopherol + 3-NPA0.195 ± 0.04

(a) vs. (f)

NS

(b) vs. (f)

0.002

Comparison of vinpocetine and α-tocopherol effects on 3-NPA-induced increase in ROS production

The DCF-diacetate fluorescent assay detects ROS production and is commonly used as an index of oxidative stress. This assay was performed in striatum isolated nerve endings incubated with 25 μM vinpocetine or 50 μM α-tocopherol in the absence or presence of 5 mM 3-NPA for 2 h.

Table 4 shows that the fluorescent products formed by DCFH oxidation for 2 h under control conditions are reduced by vinpocetine and by α-tocopherol in 25% and 35%, respectively, and are increased by 3-NPA in 40%. This increase in ROS production induced by 3-NPA in striatal synaptosomes was vinpocetine and α-tocopherol sensitive.

Table 4. Single and combined effects of vinpocetine and α-tocopherol on the increase in ROS production induced by 3-NPA
Experimental conditionaROSp value between the indicated experimental conditions
  1. a

    ROS are expressed in pmoies dichlorofluorescein/mg of synaptosomal protein Results are the mean t SEM values of 5 independent experiments in striatal synaptosomes NS, not statistically significant.

(a) Control117 ± 8.7  
(b) 3-NPA161 ± 14

(a) vs. (b)

0.001

 
(c) Vinpocetine86 ± 8.1

(a) vs. (c)

0.02

(c) vs. (d)

0.001

(d) Vinpocetine ± 3-NPA132 ± 14

(a) vs. (d)

NS

(b) vs. (d)

0.02

(e) α-tocopherol73 ± 14

(a) vs. (e)

0.004

(e) vs. (f)

0.001

(f) α-tocopherol + 3-NPA124 ± 20

(a) vs. (f)

NS

(b) vs. (f)

0.01

Comparison of vinpocetine and α-tocopherol effects on 3-NPA-induced increase in TBA-RS generation

Lipid peroxidation detected by TBA reactive substances (TBA-RS) formation is commonly used as an index of oxidative damage to membrane lipids.

Table 5 shows that 3-NPA increased TBA-RS production for 2 h in about 37%, whereas vinpocetine reduced it in 24%. The decrease in TBA-RS production induced by α-tocopherol did not reach statistical significance. However, the increase in TBA-RS production induced by 3-NPA was again inhibited by both vinpocetine and α-tocopherol.

Table 5. Single and combined effects of vinpocetine and α-tocopherol on TBA-RS formation induced by 3-NPA for 120 min
Experimental conditionaTBARSp value between the indicated experimental conditions
  1. a

    TBA-RS are expressed in nmoles MDA/mg of synaptosomal protein. Results are the mean ± SEM values of five independent experiments in striatal synaptosomes NS, not statistically significant.

(a) Control2.00 ± 0.07  
(b) 3-NPA2.74 ± 0.18

(a) vs. (b)

0.002

 
(c) Vinpocetine1.52 ± 0.14

(a) vs. (c)

0.03

(c) vs. (d)

0.005

(d) Vinpocetine + 3-NPA2.16 ± 0.24

(a) vs. (d)

NS

(b) vs. (d)

0.01

(e) α-tocopherol1.85 ± 0.06

(a) vs. (e)

NS

(e) vs. (f)

NS

(f) α-tocopherol + 3-NPA2.22 ± 0.11

(a) vs. (f)

NS

(b) vs. (f)

0.02

Single and combined effects of vinpocetine and 3-NPA on DA and DOPAC distribution

Incubation of striatum synaptosomes in the presence of vinpocetine for 2 h decreased the internal concentration of DA and proportionally increased the external concentration of DOPAC (second column in Table 6).

Table 6. Single and combined effects of vinpocetine and 3-NPA on DA and DOPAC distribution in striatum synaptosomes incubated for 120 min
 ControlVinpocetine3-NPAVinpocetine + 3-NPA
  1. Data are expressed in pmoles/mg of synaptosomal protein. Results are the mean ± SEM values from four striatum synaptosomes independent preparations.

  2. a

    p < 0.05 between control and the indicated experimental condition.

  3. b

    = 0.001 between 3-NPA and vinpocetine plus 3-NPA.

DA released27 ± 2.119 ± 3.3148 ± 5.9a89 ± 9.3a,b
DA inside279 ± 15173 ± 30a116 ± 5.1a118 ± 15a
DOPAC out667 ± 34785 ± 52a675 ± 26794 ± 41a
DOPAC in88 ± 5.092 ± 1569 ± 5.5a67 ± 10

Incubation of striatum synaptosomes for 2 h in the presence of 3-NPA increased the external concentration of DA and decreased the internal concentrations of both, DA and DOPAC (third column in Table 6).

When synaptosomes were simultaneously exposed to 3-NPA and vinpocetine for 2 h, the increase in DA release induced by 3-NPA alone was reduced and the increase in external DOPAC induced by vinpocetine alone was maintained (last column in Table 6).

Exposure of striatum synaptosomes to α-tocopherol for 2 h did not produce statistically significant changes in catecholamine distribution (data not shown).

3-NPA-induced DA and DOPAC net changes at 10 min and at 2 h in the absence and presence of vinpocetine or α-tocopherol

The net changes produced by vinpocetine, 3-NPA, or both (i.e., vinpocetine plus 3-NPA) on total DA (or DOPAC) at 10 min and at 2 h were compared. The upper graphs in Fig. 3 show that at 10 min, vinpocetine decreased total DA and increased total DOPAC, whereas 3-NPA produced the opposite effect, increasing total DA and decreasing total DOPAC. When the preparation was simultaneously exposed to vinpocetine and 3-NPA, the changes produced by 3-NPA alone were reversed.

image

Figure 3. Net changes in total dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) induced by vinpocetine or 3-nitropropionic acid (3-NPA) alone and in combination. Striatal synaptosomes were incubated (37°C) for 10 min (upper graphs) or 2 h (lower graphs) in KRH with: 25 μM vinpocetine, 5 mM 3-NPA, or both (vinpocetine plus 3-NPA). Net change in DA or DOPAC refers to the concentration of the indicated catecholamine under the specified experimental condition minus the respective control value. Total DA or total DOPAC is the sum of the internal and external catecholamine concentration under a specific experimental condition. Data are expressed in pmoles of DA or DOPAC per mg of striatal synaptosomal protein. Results are the mean ± SEM values of 5 (at 10 min) or 4 (at 2 h) experiments in independent preparations. The statistical significance (p) of the net catecholamine changes between experimental conditions is indicated by the lines connecting specific pairs of bars.

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The lower graphs in Fig. 3 show that at 2 h, the net changes induced by vinpocetine on total DA and DOPAC persisted in the absence as well as in the presence of 3-NPA, whereas in the synaptosomes exposed to 3-NPA for 2 h, the total DA concentration was reduced.

The upper graphs in Fig. 4 show that like vinpocetine, α-tocopherol decreased total DA at 10 min and at 2 h, and that also reversed the increase in total DA produced by 3-NPA at 10 min. Total DOPAC was, however, unchanged by α-tocopherol.

image

Figure 4. Effect of α-tocopherol on the net changes in total dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) induced by 3-nitropropionic acid (3-NPA). Striatal synaptosomes were incubated (37°C) for 10 min (upper graphs) or 2 h (lower graphs) in KRH with 50 μM α-tocopherol, 5 mM 3-NPA, or both. Data are expressed in pmoles of DA or DOPAC per mg of striatal synaptosomal protein. Results are the mean ± SEM values of four experiments (at 10 min) and four (at 2 h) in independent preparations. The statistical significance (p) of the net catecholamine changes between experimental conditions is indicated by the lines connecting specific pairs of bars.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

In this study we showed that, except for the drop in MAO activity, all the changes induced by the natural toxin 3-NPA in striatum isolated nerve endings were overcome by vinpocetine and by α-tocopherol.

Present findings showing that at 10 min 3-NPA decreased 4-HQ production strongly suggest that the increase in DA and the drop in DOPAC in response to 3-NPA is due to a reduced MAO-A activity. The DA increase and DOPAC decrease produced by 3-NPA in tissue level at 10 min contrasts with the DA decrease and DOPAC increase exerted by 25 μM vinpocetine at 10 min. This last finding is consistent with our previous work showing that incubation of striatum synaptosomes with vinpocetine at increasing concentrations in the low micromolar range (1.5–50 μM) progressively decreased DA and simultaneously increased its main metabolite, DOPAC at 10 min (Trejo et al. 2001). This action of vinpocetine on DA metabolism, that is long lasting as it was also observed at 2 h, is not because, however, of an increase in MAO activity, as vinpocetine failed to modify MAO activity by itself. Conversely, our finding that vinpocetine, like reserpine, reduced [3H]DA uptake into striatum isolated synaptic vesicles, supports our previous hypothesis that prevention of DA accumulation inside synaptic vesicles by vinpocetine increases DA metabolism to DOPAC by the action of the MAO-A present in the cytoplasm (Trejo et al. 2001).

Like vinpocetine, α-tocopherol decreased the tissue DA concentration, but in contrast to vinpocetine, α-tocopherol was unable to modify the concentration of DOPAC neither at 10 min nor at 2 h. This indicates that the mechanism by which α-tocopherol reduces DA is other than the prevention of DA accumulation inside synaptic vesicles. In PC12 cells, which are cells rich in DA, the antioxidant dithiothreitol at a high concentration was shown to exert a complex influence on the catalytic activity of TH, which is the rate-limiting enzyme of DA biosynthesis (Borges et al. 2002). Thus, one possible explanation of the decrease in DA exerted by 50 μM α-tocopherol, not including an increase in the concentration of DOPAC, could be the inhibition of DA biosynthesis. As only vinpocetine increased DOPAC, and both, vinpocetine and α-tocopherol decreased total DA, it is likely that the capability of these compounds to overcome the oxidative stress induced by 3-NPA is linked to the drop in DA that they produce. In accordance with a dangerous action of DA, and not its main metabolite, previous studies in isolated rat brain mitochondria showed that exposure to DA generates harmful quinoprotein adducts capable to inhibit complex I and complex IV of the mitochondrial electron transport respiratory chain, whereas exposure to an equimolar concentration of DOPAC was unable to produce such effect (Khan et al. 2005; Jana et al. 2007).

Our findings that at 10 min 3-NPA increased total DA, and at 2 h decreased total DA and increased DA–quinone protein adducts formation, strongly suggests that in the presence of a significant amount of ROS, DA is transformed to DA–quinone protein adducts. In agreement with the hypothesis that DA is the source of damaging species generated by 3-NPA, we have previously shown that in whole-brain isolated nerve endings, where DA is much less concentrated than in striatum isolated nerve endings, 3-NPA only produced a mild effects on ROS production and DA–quinoprotein adducts formation (Herrera-Mundo and Sitges 2010). In other words, in DA-rich brain regions, the dangerous action exerted by 3-NPA is likely to be exacerbated. Consistently, in the presence of vinpocetine or the amply recognized antioxidant α-tocopherol, which decreased DA, 3-NPA failed to increase DA-oxidation products at 2 h, suggesting that the decrease in DA exerted by those compounds importantly contributes to their antioxidant capacity, particularly in DA-rich regions.

Comparison of the antioxidant capacities of 25 μM vinpocetine and 50 μM α-tocopherol against the increase of oxidative damage markers such as ROS and TBA-RS induced by 3-NPA showed that at those concentrations vinpocetine and the lipophilic antioxidant α-tocopherol are both effective free radical scavengers. α-Tocopherol is an essential component of cellular defense mechanisms against endogenous and exogenous oxidants (Wang and Quinn 1999), and like α-tocopherol vinpocetine reduced the increase in TBA-RS produced by 3-NPA. Although only vinpocetine was able to decrease TBA-RS production below baseline control levels in a statistically significant manner, pointing out its important antioxidant capacity. Our findings that vinpocetine and α-tocopherol reduced TBA-RS and/or ROS production under basal levels in striatum synaptosomes (Tables 4 and 5) contrast, however, with the failure of vinpocetine and trolox, an analog of α-tocopherol, in modifying basal ROS and TBA-RS levels in whole-brain synaptosomes (Santos et al. 2000). The almost fivefold lower amount of ROS produced in whole brain than in striatum synaptosomes (Herrera-Mundo and Sitges 2010), along with the eightfold shorter incubation period (15 min) used in Santos et al. (2000) study may explain this apparent controversy.

Vinpocetine like α-tocopherol scavenger properties along with their ability to decrease DA may allow those compounds to stabilize the ROS generated by the 3-NPA direct inhibition of complex II and by the DA-quinones indirect inhibition of complexes I and IV. In agreement with this interpretation in PC12 cells, vinpocetine also was shown to prevent the inhibition of complexes II, III, and IV of the respiratory chain induced by peptide A-beta (Pereira et al. 2000).

In summary, the damaging action of 3-NPA is likely to be particularly exacerbated in DA-rich brain regions, where 3-NPA increases DA by inhibition of MAO activity and ROS production by the inhibition of complex II. This generates the DA–quinoprotein adducts, which in turn inhibit complexes I and IV, further facilitating ROS production and damage. Note that clorgyline, like 3-NPA, increases DA tissue levels by itself. However, in contrast to 3-NPA, clorgyline is not expected to inhibit any complex of the respiratory chain with subsequent rise in ROS production.

Finally, mitochondria, the primarily ROS-generating structures are particularly abundant in nerve endings and the neurotransmitter DA is particularly concentrated in the striatum. Thus, our findings that vinpocetine and α-tocopherol were capable to prevent both 3-NPA-induced ROS production and the rise in DA in the striatum isolated nerve endings indicate the powerful neuroprotective action of these compounds.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

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. The authors declare that there are no conflicts of interest.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
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