Poster
# 20
Poster |
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6th Internet World Congress for Biomedical Sciences |
María Dolores Mayas-Torres(1), José Manuel Martínez-Martos(2), María Jesús Ramírez-Expósito(3), María Jesús García-López(4), Isabel Prieto-Gómez(5), Garbiñe Arechaga-Maza(6), Manuel Ramírez-Sánchez(7)
(1)(2)(4)(5)(6)(7)Unit of Physiology. University of Jaén - Jaén. Spain
(3)Unit of Physiology. University of Jaen - Jaén. Spain
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[Endocrinology] |
[Neuroscience] |
[Pharmacology] |
[Physiology] |
[Toxicology] |
Brain aminopeptidases (AP) have been implicated in the enzymatic activation and degradation of several neuropeptides(1). These two actions may regulate not only their role as neuromodulators2 but may also modify the free amino acid pool through the release of N-terminal amino acids, some of which, such as glutamate and aspartate, are particularly active in the central nervous system (CNS)(3,4). The aminopeptidase A activity (AP-A) (GluAP and AspAP) has a quick action on the N-terminal aspartic and glutamic acids of biologically active peptides and polypeptides, regulating their activities1. Therefore, changes in AspAP and/or GluAP may contribute to or reflect modifications in excitatory amino acid turnover. One of the mechanisms responsible for neurodegenerative processes affecting the CNS is the hyperexcitability of amino-acid neurotransmitters, particularly glutamic and aspartic acids. This hyperexcitability of excitatory neurotransmitters make themself strong endogenous toxins which induce degeneration and neurone death(5,6,7,8,9,10,11,12,13).
The purpose of this work was to study the influence of a neurotoxic agent, ethanol, on the AP-A activity on basal and stimulated conditions, in a synaptic transmission model by using synaptosomes.
Mitochondrial activity assay.
Mitochondrial activity of synaptosomes was assayed by using the tetrazolium salt MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide). This compound is hydrolized by the mitochondrial enzyme succinate dehydrogenase, which produces a dark blue tetrazolium salt that can be measured spectrophotometrically. This assay is an index of the bioenergetic behaviour of the synaptosomes. After the obtention of the synaptosomes, they were resuspended in an artificial CSF containing MTT 1 mM and incubated for 30 minutes at 37°C. The reaction was stopped by adding acid-isopropanol and read by using a test wavelength of 550 nm and a reference wavelength of 620 nm. The resulting values were expressed in optical density units.
Determination of the free radical generation by a chemiluminescence assay.
After incubation of the synaptosomes, the were resuspended in an artificial CSF which included the enhancers of chemiluminescence luminol or lucigenin 0.2 mM. Maximal chemiluminiscence was recorded as cpm per vial after subtracting blanks containing buffer only.
Lipid peroxidation by TBARS assay.
Synaptosome lipids peroxidation was measured by analyzing the amount of thiobarbituric acid reactive substances (TBARS). Synaptosomes were mixed with an equal volume of ice-cold 20 % trichloroacetic (TCA). After centrifugation, a volume of supernatant was added to an equal volume of 0.67 % TBA (4,6-dihidroxipirimidina-2-tiol) and the mixture was kept in a boiling water bath for 15 minutes. Samples were cooled to room temperature and the absorbance at 532 nm were recorded. The results were expresed in terms of malondialdehyde (MDA) equivalents using an extinction coefficient of 1.56 105 M-1 cm-1.
Assay of protein oxidation.
The synaptosomes were mixed with an equal volume of ice-cold 20 % TCA and were centrifuged. The pellets were dissolved in acid 2,4-dinitrophenylhydrazine 10 mM for an hour at room temperature in the dark. After the reaction, proteins were precipitated with 20% TCA. Then, were centrifugued and the pellets dissolved in NaOH 1 M and incubated for 15 minutes at 37°C. After centrifugation, the supernatants were recorded at 360 nm. The results were expressed as the content in diene conjugates and carbonyl group formation, expressed in nmol per mg of protein using an extinction coefficient of 22 mol-1 cm-1.
Aminopeptidase activities.
AspAP and GluAP activities were determined against the substrate L-Aspartyl- -naphthylamine (AspNNap) and L- -Glutamyl- -naphthylamine (GluNNap), in accordance with the method of Schwabe and McDonald (4), with slight modifications: 20 µl of the synaptosomes were incubated with 50 µl of the sustrate solution with AspNNap or GluNNap 100 µM during 30 minutes at 37°C. The reactions were stopped by adding 50 µl of acetate buffer 0.1 M, pH 4.2 containing Fast Garnet GBC salt 2%. The amount of ß-naphthylamine released as a result of the enzymatic activity was coupled to the GBC salt giving a colored compound which can be measured spectrophotometrically at 550 nm.
Specific enzymatic AspAP and GluAP activities was expresed as nmol of AspNNap or GluNNap hidrolysed per min per mg protein, by using a standar curve of -naphthylamine determined in the same conditions.
Proteins were measured according to the method of Bradford (5), by using a standar curve of bovine serum albumin (BSA).
Statistics We used one-way analysis of variance (ANOVA) to analyze differences between groups. Post-hoc comparisons were made using the Newman-Keul´s test. All comparisons with P<0.05 were considered significant.
Effects of ethanol on the bioenergetic behavior of synaptosomes.
The effects of ethanol on mitochondrial activity of mouse frontal cortex synaptosomes show the following results (table 1): Ethanol induces a dose-dependent increase of mitochondrial activity. In this way, ethanol 25 mM increases the activity in a 39.14% (p<0.01). Ethanol 50 mM produces a significant increase (p<0.01) in a 53.61% and ethanol 100 mM in a 58.92% (p<0.01).
Depolarization with K+ 25 mM induces an increase of mitochondrial activity in a 22.51% (p<0.01) (table 1). The simultaneous incubation of synaptosomes with K+ 25 mM and ethanol 25 mM or 50 mM increases significantly (p<0.01) the activity in a 36.52% and 46.43% respectively. Otherwise, ethanol 100 mM do not modify mitochondrial activity, when compared with the control values.
Parameters of oxidative stress.
The analysis of the ethanol effects on the free radical generation shows the following results: Ethanol 25 mM do not produce modifications in luminol chemiluminiscence when compared with control values. Ethanol 50 mM increases luminol chemiluminiscence in a 19.37% (p<0.05), while ethanol 100 mM induces a significant increase (p<0.05) of luminol chemiluminiscence in a 19.04% (table 1). Depolarization with K+ 25 mM does not modify luminol chemiluminiscence. In addition, the simultaneous incubation of synaptosomes with K+ 25 mM and ethanol does not change luminol chemiluminiscence either.
Using lucigenin as the enhancer of the chemiluminiscence signal, the results obtained are: Ethanol 25 mM induces a light increase of 18.05% (p<0.05), while ethanol 50 mM and 100 mM do not modify lucigenin chemiluminiscence, when compared with the control values (table 1). Depolarization with K+ 25 mM increases significantly (p<0.05) in a 16.94% lucigenin chemiluminiscence vs. the control values. However, the simultaneous incubation of synaptosomes with ethanol under depolarized conditions with K+ 25 mM 4), does not produce significant differences when compared with control values.
The effects of ethanol on lipid peroxidation (TBARS content) of mouse frontal cortex synaptosomes showed the following results: Ethanol 25 mM decreased TBARS content in a 13.55% (p<0.01) (table 1). Otherwise, ethanol 50 mM and 100 mM of ethanol did not modify significantly control values. Depolarization of synaptosomes with K+ 25 mM or the simultaneous incubation of synaptosomes with K+ 25 mM and ethanol at the different concentrations did not change TBARS content when compared with the control values (table 1).
Neither ethanol at the different concentrations used nor depolarization of synaptosomes with K+ 25 mM nor the simultaneous incubation of synaptosomes with K+ 25 mM and ethanol at different concentrations (25, 50, y 100 mM) did modify carbonyl content of synaptosomal proteins.
Effects of ethanol on AspAP activity.
The analysis of the effects of ethanol on the basal AspAP activity in mouse forntal cortex synaptosomes showed the following results: Ethanol inhibitis AspAP activity in a dose-dependent manner (figure 1A). Thus, ethanol 25 mM produces a significant inhibition (p<0.01) of 18.53% when compared with control values. Ethanol 50 mM decreases AspAP activity in a 38.55% (p<0.01), while ethanol 100 mM inhibitis in a 48.92% (p<0.01) (figure 1A).
After depolarization with K+ 25 mM, AspAP activity decreases in a 13.61% (p<0.01) (figure 1B). The simultaneous incubation of synaptosomes with K+ 25 mM and ethanol 25 mM produces a significant inhibition (p<0.01) of 52.68% when compared with control values (figure 1B). Ethanol 50 mM inhibits AspAP activity in a 15.59% (p<0.01). In presence of ethanol 100 mM, the depolarization with K+ 25 mM produces a significant decrease (p<0.05) of the AspAP levels in a 4.75% vs. the control values (figure 1B).
Effects of ethanol on GlluAP activity.
The analysis of the effects of ethanol on the basal GluAP activity in mouse frontal cortex synaptosomes shows the following results: Ethanol produces a dose-dependent inhibition of GluAP activity (figure 2A). In this way, ethanol 25 mM induces a significant inhibition (p<0.01) in a 49.09%. Ethanol 50 mM inhibits in a 54.27% (p<0.01), while ethanol 100 mM produces an inhibition of 65.3% (p<0.01) when compared with the control values (figure 2A).
The analysis of the effects of ethanol on GluAP activity after the stimulation of synaptosomes with K+ 25 mM shows the following results: Depolarization decreases significantly (p<0.01) GluAP activity in a 11.01% (figure 2B). The simultaneous incubation of synaptosomes with K+ 25 mM and ethanol 25 mM produces a significant inhibition (p<0.01) of GluAP activity in a 59.26% when compared with depolarized values (figure 2B). In addition, the presence of K+ 25 mM and ethanol 50 mM produces an inhibition of GluAP activity in a 55.99% (p<0.01) and ethanol 100 mM produces a significant decrease (p<0.01) of GluAP activity in a 26.86% vs. depolarized values (figure 2B).
The present work shows that ethanol administration to frontal cortex mouse synaptosomes under our conditions (ethanol 25 mM, 50 mM and 100 mM during an incubation period of 15 minutes at 37°C), produces little signs of neurodegenerative processes. Using luminol as enhancer of chemiluminescence, it is not detected modifications in the free radical generation, although lucigenin chemiluminiscence indicates small increases in the production of free radicals, and in an inversely-proportional manner. That is specially perceptible when ethanol is given simultaneously with depolarized stimulus of synaptosomes. These free radicals seem to be the consequence of the increase in the mitochondrial activity, which is demonstrated by using MTT, but have little ability to induce degeneration. This is demonstrated by the inability of generating lipid peroxidation of the membrane lipids (lower doses of ethanol seems to have protector functions on basal levels) or protein oxidation of synaptosomes.
On the other hand, ethanol produces an inhibition dose-dependent of the basal levels of AspAP and GluAP, being this inhibition stronger in GluAP. After depolarization with K+ 25 mM a decrease of the AspAP and GluAP activities is produced. But the stimulation of synaptosomes with K+ 25 mM in presence of ethanol induces an inhibition of the activity inversely-proportional to the ethanol concentration, getting values such as the resulting of the stimulation with K+ 25 mM in ausence of ethanol. Because of this, the presence of ethanol on the artificial CSF in basal conditions does not produce an increase of the pool of exitatory amino acids. Under stimulant conditions, the presence of ethanol in the artificial CSF lead to smaller levels of enzymatic activity than control values. It could be due to an impairment of the homoeostatic processes generated at these levels. The aminopeptidase A activity (AspAP and GluAP)(1,17) is important not only due to its participation on the free excitatory amino acids pool generation by the release of N-terminal aminoacids of peptides and polypeptides, but because one of its physiologycal substrates is angiotensin II(18,19,20,21). This molecule participates in the control of the blood pressure in the brain(22,23), due to the existence of a regional brain renin-angiotensin system24. The modifications induced by ethanol in AspAP and GluAP activities could modify the regulation of the brain blood pressure, and be responsible of the neurotoxic processes that are coupled to the chronic intake of ethanol.
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[Endocrinology] |
[Neuroscience] |
[Pharmacology] |
[Physiology] |
[Toxicology] |
