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

	[Endocrinology]	[Neuroscience]	[Pharmacology]	[Physiology]	[Toxicology]

MATERIAL & METHODS

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.

Discussion Board

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[ABSTRACT] [INTRODUCTION] [MATERIAL & METHODS] [RESULTS] [IMAGES] [DISCUSSION] [BIBLIOGRAPHY] [Discussion Board]

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	[Endocrinology]	[Neuroscience]	[Pharmacology]	[Physiology]	[Toxicology]

María Dolores Mayas-Torres, José Manuel Martínez-Martos, María Jesús Ramírez-Expósito, María Jesús García-López, Isabel Prieto-Gómez, Garbiñe Arechaga-Maza, Manuel Ramírez-Sánchez
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Last update: 15/01/00