Poster
# 21
Poster |
|
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
|
 |
|
|
|
|
|
[Endocrinology] |
[Neuroscience] |
[Pharmacology] |
[Physiology] |
[Toxicology] |
Twenty two male Balb/C mice were used in the present study (body weight 28.7±6.1 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.
Synaptosomes were prepared in accordance with the method of Whittaker et al. [3]. 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 and used for determining pGluAP activity.
pGluAP activity was determined against the substrate L-Pyroglutamyl- -naphthylamide (pGluNNap), in accordance with the method of Schwabe and McDonald [4], with modifications: 20 µl of the synaptosomes were incubated with 50 µl of the sustrate solution with pGluNNap 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 pGluAP activity was expresed as nmol of pGluNNap 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.
|
|
|
|
|
|
|
|
|
[Endocrinology] |
[Neuroscience] |
[Pharmacology] |
[Physiology] |
[Toxicology] |
