Poster
# 21
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] |
Pyroglutamyl aminopeptidase (pGluAP) can be classified as an omega-peptidase which removes pyroglutamyl N-terminal residues from peptides, proteins and arylamidase derivatives in a highly selective manner (1,2). The activity of this enzyme is thought to be involved in the regulation of several physiological mechanisms on the central nervous system (CNS). pGluAP can modulate various susceptible endogenous substrates such as thyrotropin-releasing hormone (TRH). It is well known that TRH plays an important role in the modulation of the behavioral changes induced by ethanol and others drugs. The aim of this work was to study the in vitro effects of ethanol (25, 50 and 100 mM) on the pGluAP activity and its ability for modulating the TRH.
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.
The presence of ethanol in an artificial CSF containing calcium or not on the basal pGluAP activity in synaptosomes from the brain cortex of mice, showed the following results: Ethanol in an artificial CSF containing calcium inhibits pGluAP activity (fig 1). In this way, ethanol 25 mM induces a significant inhibition (p<0.01) of 45.68% vs. the control. Ethanol 50 mM reduces pGluAP activity in a 45.97% (p<0.01) and ethanol 100 mM causes an inhibition of 55.87% (p<0.01) vs. the control. In absence of calcium in the artificial CSF (fig 2), ethanol 25 mM produces a significant inhibition (p<0.01) on the pGluAP activity in a 49%. Ethanol 50 mM reduces the activity in a 55.19% (p<0.01) and ethanol 100 mM reduces pGluAP activity of 46.28% vs. the control (p<0.01).
The effects of ethanol and the influence of calcium in the artificial CSF on pGluAP activity after the stimulation of synaptosomes with K+ 25 mM showed the following results: In presence of calcium, the stimulation of synaptosomes with K+ 25 mM does not modify this activity (fig 2). In absence of calcium, the stimulation with K+ 25 mM reduces significantly the pGluAP activity in an 18.74% (p<0.01) vs. the control.
The simultaneous incubation, in an artificial CSF with calcium, of synaptosomes with K+ 25 mM and ethanol 25 mM (fig. 2) produces a significant decrease (p<0.01) of pGluAP in a 43.75% vs. the control. The presence of K+ 25 mM and ethanol 50 mM produce an inhibition of this activity in a 44.44% (p<0.01) vs. the control values. The stimulation of synaptosomes with K+ 25 mM in presence of ethanol 100 mM decreases (p<0.05) the pGluAP activity in a 7.54% vs. the control. In a calcium-free artificial CSF (fig.2), the stimulation of synaptosomes with K+ 25 mM and ethanol 25 mM produces a significant inhibition (p<0.01) in a 8.06% vs. the control. The stimulation with K+ 25 mM and ethanol 50 mM produces an inhibition (p<0.01) of 35.02% , and ethanol 100 mM of 40.34% (p<0.01) vs. the control.
In this work, we show the ability of ethanol to modify pGluAP activity. Ethanol induces an inhibition of this activity. This inhibition is independent of the presence of calcium in the artificial CSF on basal conditions. In depolarized conditions, pGluAP activity is not affect when calcium is included in the artificial CSF. On the other hand, pGluAP activity is lower when the stimulation with K+ 25 mM occurrs in a calcium-free artificial CSF. Therefore, depolarization induces a calcium-dependent behaviour in pGluAP activity. This differences occurs also when ethanol is administrated at different doses. So in presence of calcium presence in the artificial CSF, ethanol induces an inhibition inversely proportional to the concentration of ethanol used. In a calcium-free artificial CSF, the inhibition induced by ethanol is proportional to the ethanol concentration used.
The pGluAP enzyme is a peptidase widely distributed in fluids and tissues. To date, three distinct forms of pGluAP have been observed. The first, pGluAP type I is localized to the cytosolic compartment (6,7,8), and has a broad pyroglutamyl-substrate specificity, including TRH, GnRH, neurotensin, bombesin (7). The pGluAP type II has been shown to be a membrane-bound enzyme with a high specificity to the TRH neurohormone TRH (9,10). A third pGluAP, called thyroliberinase, has also been observed in the serum, and its biochemical characteristics are very similar to those of pGluAP type II (11,12,13,14).
Although the hydrolytic action on peptide or artificial substrates of these different types of pGluAP has been extensively studied, their actual physiological role is not well defined. In this work is shown that the administration in vitro of ethanol modifies this enzymatic activity (it is inhibited), and that modification is depending on the calcium levels on the artificial CSF. It has been described the intracerebroventricular administration of peptides related to the pGluAP (neurotensin and bombesin) to mice, prolonged the duration of sleep induced by ethanol, and also enhanced ethanol-induced hypotermia (15). These results suggest that those neuropeptides may be involved in the complex mechanisms of action of ethanol on the CNS, and this participation can be mediated by the modifications in the activity of their degradative enzymes.
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[Endocrinology] |
[Neuroscience] |
[Pharmacology] |
[Physiology] |
[Toxicology] |
