Animals.
- 140 male Balb/C mice were used in the present study (body weight 27.5±5.5 g). The animals were obtained from the animal house-care of The University of Jaén, and were housed under constant temperature (20-25ºC) and day length 12 hours. All animals were allowed access to water and food ad libitum.
Preparation of the synaptosomes.
- Synaptosomes were prepared in accordance with the method of Whittaker et al.14. After animal death by decapitation, the brain was quickly removed and the frontal cortex dissected. The tissue was homogenized in sucrose 0.32 M, using a glass homogenizer. The homogenate was centrifugued at 2.000 xg, and the resulting supernatant was centrifugued at 30.000 xg. The pellet was resuspended in sucrose 0.32 M, and this volume was added on top of a sucrose gradient and centrifugued at 30.000 xg.. Synaptosomes from a 0.8 M sucrose gradiente were resuspended in artificial cerebrospinal fluid (CSF) in presence or absence of calcium, depending on the experimental protocol (see below) to have a final concentration of 0.1 mg/ml protein.
The experimental protocols were:
- *Incubation of the synaptosomes in artificial CSF in presence or absence of calcium under basal conditions, in presence of ethanol 25 mM, 50 mM and 100 mM.
- * Incubation of the synaptosomes in artificial CSF with o without calcium under depolarized conditions (K+ 25 mM).
- * Incubation of synaptosomes in artificial CSF in presence or absence of calcium, under depolarized conditions(K+25 mM) in presence of ethanol 25 mM, 50 mM and 100 mM.
These incubations were carried out in a water bath at 37°C for 15 minutes. After this time, synaptosomes were washed by centrifugation at 30.000 xg. Then, synaptosomes were resuspended in artificial CSF in presence or absence of calcium at 37°C. Synaptosomes were used for determining free radicals generation, lipid peroxidation of membrane lipids and oxidation of synaptosomal proteins. In addition, the bioenergetic behavior of synaptosomes and the AP-A activities were assayed under the different experimental protocols.
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.
- AlaAP, ArgAP, CysAP, LeuAP and TyrAP activities were determined against the substrates L-Alanyl- -naphthylamine (AlaNNap), L-Arginyl- -naphthylamine (ArgNNap), L-Cystinyl- -naphthylamine (CysNNap), L-Leucyl- -naphthylamine (LeuNNap) and L-Tyrosyl- -naphthylamine (TyrNNap), in accordance with the method of Greenberg (3) and Schwabe and McDonald (4), with slight modifications: 20 µl of the synaptosomes were incubated with 50 µl of the sustrate solution with AlaNNap, ArgNNap, CysNNap, LeuNNap or TyrNNap 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 activities of AlaAP, ArgAP, CysAP, LeuAP and TyrAP were expresed as nmol of the corresponding substrate hidrolyzed per min per mg of protein, by using a standar -naphthylamine curve 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.
