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

	[Neuroscience]	[Physiology]

INTRODUCTION

Ethanol is the most psychoactive substance used after caffeine. Chronic alcohol intake is associated with several degenerative and inflamatory processes in the central nervous system (CNS) ^(1,2,3,4), and can induce a depressor effect in several inhibitory nervous centres ⁽⁵⁾. Due to the interest of alcohol dependence, several reports studies the ethanol capacity for increasing the membrane fluidity and changing the function of proteins that are inserted into the membrane ^(2,6,7). Other reports study the neurochemical pathways and the neurotransmitters that are implicated in this dependence ^(2,3,4). Therefore, it is not well known how the alcohol acts on the CNS.

The dependence to ethanol is characterized by a behavioural standard of chronic and compulsive intake of ethanol without the ability of having control of the use of this drug ^(8,9). Appart from the genetic causes, this behaviour seem to have particularly neurochemical causes ^(8,9).

Aminopeptidases (AP) are enzymes that hydrolize the peptide bonds near to the N-terminal amino acid of peptides and polypeptides. AP constituyes one of the principal pathways for neuropeptides inactivation and active peptides generation, through the hydrolysis of their precursors ⁽¹⁰⁾. Alanine aminopeptidase (AlaAP), leucine aminopeptidase (LeuAP) and tyrosine aminopeptidase (TyrAP, Enkephalinase) can hydrolyze different neuropeptides such as bradicinin ^(11,12), enkephalin ^(13,14) and may have angiotensinase activity ⁽¹⁵⁾. Arginine aminopeptidase (ArgAP) hydrolyzes particularly basic residues N-terminal from peptides and arylamide derivatives ⁽¹⁰⁾. Thanks to its exopeptidase activity, it has been implicated in the metabolism of Met-enkephalin ⁽¹⁶⁾ and angiotensin III ⁽¹⁵⁾. Its endopeptidase activity has been implicated in the metabolism of neurotensin ⁽¹⁷⁾. Cystine aminopaptidase (CysAP) hydrolyzes oxitocin and vasopresin ⁽¹⁸⁾. Due to these enzymes modulate the peptide neurotransmission ⁽¹⁰⁾, in the present work we have determinated the activity of several aminopeptidases (AlaAP, ArgAP, CysAP, LeuAP and TyrAP) in synaptosomes obtained from the frontal cortex of mouse, in basal and depolarized conditions, in presence of ethanol (25, 50 and 100 mM), and its relation to neurotoxic and metabolic effects of ethanol.

MATERIAL & METHODS

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.

RESULTS

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: 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) (Figure 1A).

Depolarization with K+ 25 mM induces an increase of mitochondrial activity in a 22.51% (p<0.01). 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 (Figure 1B).

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% (Figure 2A).

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 (Figure 2B).

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 (Figure 3A). 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 (Figure 3B).

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). Otherwise, ethanol 50 mM and 100 mM of ethanol did not modify significantly control values (Figure 4A). 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 (Figure 4B).

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 (Figures 5A and 5B).

Effects of ethanol on AlaAP.

The analysis of the effects of ethanol on basal AlaAP activity shows the following results: AlaAP activity is inhibited in a dose-dependent manner by ethanol (figure 6A). Thus, a ethanol 25 mM induces a significant inhibition (p<0.01) of 13.16% in relation to the control. Ethanol 50 mM decreases this activity in a 19 % (p<0.01), and ethanol 100 mM produces an inhibición of 49.29% (p<0.01).

The stimulation of synaptosomes with K+ 25 mM decreases significantly (p<0.01) AlaAP activity in a 23.5% (figure 6B). The simultaneous incubation of synaptosomes with K+ 25 mM and ethanol decreases AlaAP activity (figure 6B). Thus, ethanol 25mM induces a significant inhibition of 58.95% in presence of K+ 25 mM, while ethanol 50 mM and 100 mM induce an inhibition of 22.74% and 40.33% (p<0.01) respectively, when compared with depolarized values of AlaAP activity (figure 6B).

Effects of ethanol of ArgAP.

The analysis of the effects of ethanol on basal ArgAP activity shows the following results: Ethanol produces an inhibition of this enzymatic activity, with exception of ethanol 50 mM that increases ArgAP activity significantly (p<0.01) in a 67.2%. Thus, ethanol 25 mM decreases ArgAP in a 16.19% (p<0.01) while ethanol 100 mM leads to an inhibition of 39.82% (p<0.01) (figure 7A).

Depolarization of synaptosomes with K+ 25 mM does not modify the enzymatic activity (figure 7B). The simultaneous incubation of synaptosomes with K+ 25 mM and ethanol produces a decrease of ArgAP activity except for ethanol 25 mM. The presence of K+ 25 mM and ethanol 50 mM decreases the enzymatic activity in a 26.75% (p<0.01) and the stimulation of synaptosomes with K+ 25 mM in presence of ethanol 100 mM produces a significant decrease (p<0.01) in ArgAP activity of 39.13% in relation to the depolarized values (figure 7B).

Effects of ethanol on CysAP.

Ethanol induces the inhibition of CysAP in a dose-dependent manner (figure 8A). Thus, ethanol 25 mM induces a significant (p<0.01) inhibition of CysAP activity in a 43.98% in relation to the control values. The inhibition produced by ethanol 50 mM is about 10.15% (p<0.01), and the inhibition due to ethanol 100 mM is 44.3% (p<0.01) (Figure 8A).

Stimulation of synaptosomes with K+ 25 mM does not modify CysAP activity (figure 8B). In the same way, this activity was not modified by the simultaneous incubation of synaptosmes with K+ 25 mM and ethanol 25 mM. When ethanol is 50 mM, depolarization produces a decrease of CysAp activity in a 10.29% (p<0.01). Ethanol 100 mM decreases significantly (p<0.01) CysAP activity in a 42.14% in relation to the depolarized values (Figure 8B).

Effects of ethanol on LeuAP.

The analysis of ethanol effects on basal LeuAP activity shows the following results: Ethanol 25 and 100 mM produces a significant inhibitions (p<0.01) of this activity in a 24.5 and 36.87% respectively (figure 9A). On the other hand, ethanol 50 mM induces an increase of 104.05% (p<0.01) in relation to the control values.

Depolarization of synaptosomes with K+ 25 mM does not modify significantly the control values of LeuAP (figure 9B), but the simultaneous incubation of synaptosomes with K+ 25 mM and ethanol 25 mM produces a significant decrease (p<0.01) of this activity in a 10.43% in relation to the depolarized values. The presence of K+ 25 mM and ethanol 50 mM induces a inhibition of 28.1% (p<0.01). In the same way, ethanol 100 mM decreases LeuAP activity in a 55.2% (p<0.01) when is applied simultaneously with the stimulation of synaptosomes with K+ 25 mM (figure 9B).

Effects of ethanol on TyrAP.

The analysis of the effects of ethanol on basal TyrAP activity shows the following results: Differents concentrations of ethanol decreases this activity except for the concentration 50 mM that does not modify LeuAP. Thus, ethanol 25 mM produces a decrease in a 41% (p<0.01) (figure 10A) and ethanol 100 mM in a 49.29% (p<0.01) in relation to the control values.

Depolarization of synaptosomes with K+ 25 mM produces an inhibition of 6.53% (p<0.01) in the TyrAP activity (figure 10B). The simultaneous incubation of synaptosomes with K+ 25 mM and ethanol 25 mM does not modify the control values of TyrAP. The presence of K+ 25 mM and ethanol 50 mM induces a significant inhibition (p<0.01) of 10.87% in relation to the depolarized values. The stimulation of synaptosomes with K+ 25 mM in presence of ethanol 100 mM induces a significant inhibition (p<0.01) of the TyrAP activity in a 50.95%. (figure 10B).

DISCUSSION

Under the experimental conditions used in the present work, the administration of ethanol to mouse frontal cortex synaptosomes, induces few signs of neurodegenerative processes. Using luminol as enhancer of the chemiluminescence signal, increases on free radical generation are not detected, but using lucigenin, a slight increase in chemiluminiscence appears in an inversely-proportional way to ethanol concentration. These free radicals seem to be the consequence of the increase of the mitochondrial activity, as demonstrated by MTT assay, but have not ability to induce degeneration, as demonstrated by their inability to produce peroxidation on the membrane lipids (even the smallest doses of ethanol seems to have a protective function at basal levels) or protein oxidation.

On the contrary, ethanol modifies significantly the level of several aminopeptidases. In basal conditions, ethanol inhibits these enzymatic activities in a dose-dependent manner or with a two-phase effect depending on ethanol concentration. In depolarizant conditions, ethanol produces an inhibition of these enzymatic activities in a dose-dependent manner, decreasing highly the levels of the enzymatic activity that were obtained with depolarization.

It is, therefore, evident, that ethanol modifies in a different way several aminopeptidase activities of synaptosomes from mouse cortex depending on the basal or depolarized conditions. Due to the fact that these enzymatic activities has been described as regulators of several neurotransmitters and/or neuromodulatory systems that are mediated by different types of neuropeptides ⁽¹⁰⁾, some of the ethanol effects could be the consequence of the alterations of these neurotransmitters/neuromodulatory systems.

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Discussion Board

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[ABSTRACT] [INTRODUCTION] [MATERIAL & METHODS] [RESULTS] [IMAGES] [IMAGES-2] [DISCUSSION] [BIBLIOGRAPHY] [Discussion Board]

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	[Neuroscience]	[Physiology]

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