- Top of page
- Materials and methods
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.
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.
- Top of page
- Materials and methods
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.