Unit of Physiology, Department of Health Sciences, Faculty of Experimental and Health Sciences, University of Jaén, E-23071, Jaén, Spain
* Author to whom correspondence should be addressed at: Unit of Physiology, Department of Health Sciences, Faculty of Experimental and Health Sciences, University of Jaén, Paraje Las Lagunillas s/n, E-23071, Jaén, Spain. Tel.: +34 953 002 600; Fax: +34 953 012 141; Email: jmmartos{at}ujaen.es
(Received 7 February 2002; accepted 8 February 2004)
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ABSTRACT |
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Methods: The effects of EtOH on alanyl-, arginyl-, cystyl-, leucyl- and tyrosyl-aminopeptidase activities were studied under basal/resting and K+-stimulated conditions at the synapse level, using mouse frontal cortex synaptosomes and their incubation supernatant in a Ca2+-containing or Ca2+-free medium.
Results: Under basal conditions, synaptosome aminopeptidase activities showed an inhibitory or biphasic response depending on the concentration of EtOH used and the aminopeptidase assayed, whereas supernatant activities showed a more complex response. Under K+-stimulated conditions, EtOH inhibited all synaptosome aminopeptidases assayed in presence of Ca2+. However, in absence of Ca2+, different responses were obtained depending on the concentration of EtOH used. In the supernatant, the highest concentration of EtOH inhibited the K+-stimulated increase on aminopeptidase activities, although the lowest concentration enhanced the release in presence of Ca2+. In absence of it, EtOH blocked the K+-stimulated decrease or increased the activity depending on the concentration of EtOH used.
Conclusions: The changes on aminopeptidase activities induced by EtOH may reflect the functional status of their corresponding endogenous substrates. EtOH may influence opioid peptides, oxytocin, vasopressin and the brain reninangiotensin system through their degrading enzymes.
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INTRODUCTION |
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The search for methods to treat EtOH tolerance and/or dependence has begun to focus on the role of specific interactions between EtOH and major neurotransmitter systems or second-messenger systems (Kiiamaa, 1990; Kuriyama and Ohkuma, 1990
; Wozniak and Linnoila, 1992
; Erickson, 1996
; Koob et al., 1998
; Dahchour and De White, 2000
). EtOH is also considered to produce a physical perturbation of the lipid matrix of neuronal membranes, possibly leading to changes in the activity of membrane-bound proteins (Rubin and Rottenberg, 1982
; Guerri and Grisolia, 1983
; Logan et al., 1983
; Schaad et al., 1988
; French, 1991
; Bora and Lange, 1993
; Fadda and Rossetti, 1998
). However, the action of EtOH on the CNS and the exact nature of alcoholism as pathological process are not well understood.
Aminopeptidases (APs) are generally zinc metalloenzymes, which hydrolyse peptide bonds near the N-terminal end of peptides and polypeptides. APs are considered as one of the main pathways to neuropeptide inactivation and/or peptide activation by the hydrolysis of their precursors (Barrett et al., 1998). Alanyl aminopeptidase (AlaAP), leucyl aminopeptidase (LeuAP, aminopeptidase M) and tyrosyl-AP (TyrAP, enkephalinase) can hydrolyse bradykinins (Sanderink et al., 1988
) and enkephalins (Schnebli et al., 1979
; Wagner et al., 1981
; Hersh, 1985
; Berg and Marks, 1989
). These may also act as angiotensinases (Ahmad and Ward, 1990
). Arginyl aminopeptidase (ArgAP, aminopeptidase B) specifically hydrolyses basic N-terminal residues from peptides and arylamide derivatives (Barrett et al., 1998
). Exopeptidase activity has been implicated in the metabolism of methionine-enkephalin (Johson and Hersh, 1990
) and angiotensin III (Ahmad and Ward, 1990
). Endopeptidase activity is also thought to be involved in neurotensin metabolism (McDermott et al., 1988
). Cystyl aminopeptidase (CysAP, oxytocinase) has been reported to hydrolyse oxytocin and vasopressin (Itoh and Nagamatsu, 1995
). However, the neurochemical effects of EtOH on the functional activity of these neurotransmitter/neuromodulatory systems are not fully understood. The present work was designed to study the effects of EtOH on aminopeptidase activities (AlaAP, ArgAP, CysAP, LeuAP and TyrAP) at synapse level under basal (resting) and K+-stimulated conditions. For this purpose, mouse frontal cortex synaptosomes and their supernatants were incubated in Ca2+-containing or Ca2+-free artificial cerebrospinal fluid (aCSF).
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METHODS |
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Protocols were carried out in accordance with EU regulations (86/609/EEC).
Synaptosome preparation
Synaptosomes were prepared as previously described (Martínez-Martos et al., 2000) and resuspended in a Ca+2-containing or Ca+2-free aCSF (116 mM NaCl, 5.4 mM KCl, 0.9 mM MgCl2, 0.9 mM NaH2PO4, 25 mM NaHCO3, 1.8 mM CaCl2 and 10 mM glucose; or the same with 1 mM EGTA instead of CaCl2) at pH 7.2, to a final concentration of 0.5 mg/ml protein.
Synaptosomes were incubated (37°C, 15 min) under basal conditions (controls), in presence of 25, 50 and 100 mM EtOH, under depolarized conditions (25 mM K+), and in presence of 25, 50 and 100 mM EtOH under depolarized conditions. After incubation, synaptosomes were precipitated and the resulting supernatant was used to detect AlaAP, ArgAP, CysAP, LeuAP and TyrAP activities. The synaptosomes were again resuspended in Ca2+-containing or Ca2+-free aCSF at 37°C to determine aminopeptidase activities.
Aminopeptidase activities
Aminopeptidase activities were determined against L-alanyl-ß-naphthylamide (AlaNNap), L-arginyl-ß-naphthylamide (ArgNNap), L-cystinyl-ß-naphthylamide (CysNNap), L-leucyl-ß-naphthylamide (LeuNNap) and L-tyrosyl-ß-naphthylamide (TyrNNap) substrates, in accordance with previously described methods (Greenberg, 1962; Schwabe and McDonald, 1977
). The use of arylamide substrates to assay aminopeptidase activity is well established. Briefly, 20 µl of synaptosomes or supernatant were incubated with 50 µl of 100 µM AlaNNap, ArgNNap, CysNNap, LeuNNap or TyrNNap substrate solution for 30 min at 37°C. The reactions were stopped by adding 50 µl of acetate buffer 0.1 M, pH 4.2 containing 2% Fast Garnet GBC salt. The amount of ß-naphthylamine released through the enzymatic activity was coupled to the GBC salt, yielding a coloured compound that can be measured spectrophotometrically at 550 nm.
Specific enzymatic AlaAP, ArgAP, CysAP, LeuAP and TyrAP activities (n = 11) were expressed as nmol of AlaNNap, ArgNNap, CysNNap, LeuNNap or TyrNNap hydrolysed per min per mg protein, using a standard ß-naphthylamine curve determined under the same conditions. Proteins were also measured in triplicate using a standard curve for bovine serum albumin (BSA).
Statistical analysis
We used one-way analysis of variance (ANOVA) to analyse differences among the groups. Post hoc comparisons were made using the NewmanKeul's test; comparisons with P < 0.05 were considered significant.
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RESULTS |
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Effects of EtOH on ArgAP activity
In synaptosomes under basal conditions, EtOH showed a biphasic effect depending on the concentration used. Thus, 25 and 100 mM EtOH inhibited ArgAP activity (P < 0.01) in presence of Ca2+, whereas 50 mM EtOH increased it (P < 0.01) (Fig. 3A). In absence of Ca2+, EtOH inhibited ArgAP activity (P < 0.01) in a concentration-dependent manner (Fig. 3B). In the supernatant, ArgAP activity decreased (P < 0.01) at all EtOH concentrations in presence of Ca2+ (Fig. 3C). In absence of Ca2+, 25 and 100 mM EtOH increased this activity (P < 0.01 and P < 0.05, respectively), whereas 50 mM EtOH did not (Fig. 3D).
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Effects of EtOH on CysAP activity
In synaptosomes under basal conditions, EtOH inhibited CysAP activity (P < 0.01) to a variable degree depending on the EtOH concentration used in presence (Fig. 5A) or absence (Fig. 5B) of Ca2+. In the supernatant, 50 and 100 mM but not 25 mM EtOH decreased (P < 0.01) CysAP activity in presence of Ca2+ (Fig. 5C). In absence of Ca2+, EtOH significantly (P < 0.01) decreased CysAP activity at all concentrations used (Fig. 5D).
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Effects of EtOH on LeuAP activity
In synaptosomes under basal conditions, EtOH showed a biphasic effect depending on the concentration used. Thus, 25 and 100 mM EtOH inhibited LeuAP activity (P < 0.01) in presence of Ca2+, whereas 50 mM EtOH increased it (P < 0.01; Fig. 7A). Similar results were recorded in absence of Ca2+, although 50 mM EtOH did not increase LeuAP activity to above control levels (Fig. 7B). In the supernatant, LeuAP activity decreased (P < 0.01) at all EtOH concentrations (P < 0.01) in both presence (Fig. 7C) and absence of Ca2+ (Fig. 8D).
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In synaptosomes under K+-stimulated conditions, EtOH decreased LeuAP activity in a concentration-dependent manner (P < 0.01 for all concentrations) in presence of Ca2+ (Fig. 8A). In absence of Ca2+, 25 and 100 mM EtOH enhanced the K+-stimulated increase in LeuAP (P < 0.01) but 50 mM EtOH inhibited LeuAP activity (P < 0.01; Fig. 8B). In the supernatant, in presence of Ca2+, 25 mM EtOH enhanced the K+-stimulated increase in LeuAP activity, whereas 50 mM EtOH inhibited this increase and 100 mM EtOH inhibited it to below control levels (P < 0.01; Fig. 8C). In absence of Ca2+, 25 mM EtOH blocked the K+-stimulated decrease in LeuAP activity, with levels below control values (P < 0.01). However, 50 and 100 mM EtOH did not change the K+-stimulated inhibition of LeuAP (Fig. 8D), maintaining values below control levels (P < 0.01).
Effects of EtOH on TyrAP activity
In synaptosomes under basal conditions, 25 and 100 mM but not 50 mM EtOH inhibited TyrAP activity (P < 0.01) in presence of Ca2+ (Fig. 9A). In absence of Ca2+, EtOH decreased TyrAP activity to a variable degree depending on the concentration of EtOH used (Fig. 9B). In the supernatant, EtOH significantly (P < 0.01) decreased TyrAP activity at all concentrations in both presence and absence of Ca2+ (Fig. 9C and D).
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DISCUSSION |
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The changes in activity induced by EtOH may be due to a direct effect of this drug and/or its metabolites on AP activities or to their effects on the constituents of synaptosome membranes. It is well known that EtOH increases the fluidity of the membrane and changes its lipid composition, impairing various membrane-bound functions (Diamond and Gordon, 1997). EtOH also induces neurotransmitter release in unstimulated synaptosomes regardless of the extrasynaptosomal Ca2+ concentration, which indicates a redistribution of intracellular Ca2+. EtOH induces Ca2+ release in a concentration-dependent manner over the 30500 mM range, indicating that the stimulatory effects of EtOH on the basal/resting release of neurotransmitters may be due to the microsomal release of Ca2+ (Shah and Pant, 1988
). These Ca2+ movements may be related to the modulator effects of EtOH on AP supernatant activities. In fact, a mechanism for AP release has been proposed that is similar to the classical neurotransmitter release (Mayas et al., 2000a
).
Secondly, stimulation of synaptosomes with 25 mM K+ in presence of Ca2+ modified only AlaAP and TyrAP activities, which decreased. However, in absence of Ca2+, all APs increased their activities, whereas AlaAP activity again decreased. On the other hand, in the supernatant, depolarization of synaptosomes increased all AP activities in presence of Ca2+ but decreased or did not change AlaAP activity in absence of Ca2+. Therefore, a Ca2+-dependent regulation of AP activities occurred under depolarized conditions.
The increase of AP activities in the supernatant in presence of Ca2+ may probably be the result of the release of the enzymes from the synaptosomes to the incubation medium, as previously described for the enzyme pyroglutamyl aminopeptidase (Mayas et al., 2000a). It may indicate that active peptides and their degrading peptidases are released together to the synaptic cleft in order to regulate the neurotransmitter/neuromodulatory function of these peptides by a Ca2+-dependent mechanism. The changes observed in absence of Ca2+ may result from the impossibility of enzyme release under the conditions produced in a Ca2+-free aCSF or from the redistribution of intracellular Ca2+. As stated above, these Ca2+ movements may be responsible for the different responses of APs to EtOH according to the presence or absence of Ca2+ in the medium.
Thirdly, EtOH inhibited all AP activities in synaptosomes under K+-stimulated conditions and in presence of Ca2+. Conversely, in absence of Ca2+, the lowest concentration of EtOH (25 mM) increased AP activities whereas the highest concentrations had an inhibitory effect. On the other hand, in the supernatant and in presence of Ca2+, EtOH strongly inhibited the K+-stimulated increase in AlaAP and TyrAP activity. However, the K+-stimulated increase in ArgAP, CysAP and LeuAP activities was enhanced by the lowest EtOH concentration but inhibited by the highest. In absence of Ca2+, supernatant AlaAP activity decreased only with the highest concentration of EtOH, whereas EtOH completely blocked the K+-stimulated decrease in ArgAP and CysAP activity. Finally, EtOH not only blocked the K+-stimulated decrease in LeuAP and TyrAP activities, but enhanced them to a varying degree depending on the concentration of EtOH used.
It has been demonstrated that the in vitro addition of EtOH to mouse or rat synaptosomes inhibits the depolarization-dependent uptake of Ca2+ without affecting uptake under non-depolarizing conditions. These results suggest that the known inhibitory effects of EtOH on the stimulated release of neurotransmitters may be mediated by the inhibition of the depolarization-dependent influx of Ca2+ (Canda et al., 1995). The block of synaptosomal voltage-dependent Ca2+ channels may be responsible for these effects of EtOH on AP activities. Furthermore, although some concentrations of EtOH cooperate to avoid the release of the enzyme from the synaptosomes, others increased the enzymatic activity, probably due to intrasynaptosomal Ca2+ redistribution induced by EtOH, as discussed above.
Taken together, our results show general inhibitory or biphasic effects of EtOH on AP activities under basal/resting and K+-stimulated conditions in a physiological environment containing Ca2+. Thus, the effects of EtOH can be considered the sum of specific interactions with multiple neurotransmitter/neuromodulatory systems (dopamine, serotonin, GABA, glutamate and neuropeptides) (Eckardt et al., 1998; Koob et al., 1998
). However, the differential contribution of each neurotransmitter/neuromodulatory system at diverse EtOH concentrations may constitute the neurochemical basis of the dose-dependency effects of EtOH. In any case, these modulator effects of EtOH on AP activities are not related to EtOH-induced neurotoxic events, as previously demonstrated under our experimental conditions (Mayas et al., 2000b
). As discussed above, the modulator effects of EtOH on AP may also be due to a direct effect of EtOH and/or its metabolites on AP activities or to their effects on the constituents of synaptosome membranes. An early hypothesis to explain the CNS effects of EtOH suggested that EtOH, as an amphipathic molecule, produces a physical perturbation of the lipid matrix of neuronal membranes, possibly leading to changes in the activity of membrane-bound proteins. However, at pharmacologically relevant concentrations, the effect of EtOH on the fluidity of membrane bulk lipids is very small, or undetectable, and changes are no greater than would be expected with daily variations in body temperature (Rubin and Rottenberg, 1982
; Guerri and Grisolia, 1983
; Logan et al., 1983
; Schaad et al., 1988
; French, 1991
; Tabakoff et al., 1996
; Eckardt et al., 1998
). More recently, the existence of neuronal proteins highly sensitive to EtOH has been proposed, called receptive elements for ethanol (REE) (Tabakoff and Hoffman, 1987
; Mihic et al., 1997
). Among these REE are multi-subunit, membrane-bound protein complexes, including ligand-gated ion channels and proteins involved in neuronal signal transduction processes. It could be argued that neuropeptide-degrading AP activities are REE.
There is scant knowledge about the influence of EtOH on CNS AP activities. Witek and Kolataj described an inhibitory effect of EtOH on AlaAP and LeuAP activities in mouse brain lysosomal fraction, related to the duration and concentration of the EtOH administered. Similar results were described in liver, kidney and muscle (Witek and Kolataj, 1999). However, the effect of EtOH on serum AP activities is better known. Brecher et al. reported the potent inhibitory effect on serum AlaAP and ArgAP of acetaldehyde (the major metabolic product of EtOH metabolism) but not of EtOH itself (Brecher et al., 1996
). These results suggest that the modifications in AP activities described in the present work may be due less to the action of EtOH and more to that of acetaldehyde, which could be generated as a consequence of EtOH metabolism (Veloso et al., 1972
; Tabakoff and von Wartburg, 1975
; Zimatkin and Dietrich, 1997
). However, further studies are required to clarify the influence of acetaldehyde on AP activities. EtOH administration also exerts a synergistic effect on the activities of brush border membrane enzymes and on liver morphology. The increase in specific brush border enzyme activities in the excluded loop results from a major protein loss observed in this segment (Raul et al., 1982
). In the liver, AlaAP and pyroglutamyl aminopeptidase activities showed significant increases at 24 h compared with pair-fed controls. In contrast, dipeptidyl-aminopeptidase II, cathepsins B, L and H showed significant decreases. Protease activities in skeletal muscle did not change significantly at 24 h (Reilly et al., 2000
).
The EtOH-induced changes in AP activities described in the present work are of interest because they may reflect the functional status of their corresponding endogenous substrates, which have important functions in several neurotransmitter/neuromodulatory systems. However, it should be noted that APs have usually been described as non-specific enzymes capable of hydrolysing a broad spectrum of endogenous peptide substrates and arylamide derivatives to different degrees. However, our results with synaptosomes demonstrate different patterns of AP activities in response to the addition of EtOH to the aCSF. AP activities may be highly specific to their native substrates and may modulate them strictly in accordance with their surrounding microenvironment.
Opioid peptides, such as enkephalins, can be hydrolysed by AlaAP and LeuAP (ApM), ArgAP (ApB) and TyrAP (enkephalinase) (Schnebli et al., 1979; Wagner et al., 1981
; Hersh, 1985
; Berg and Marks, 1989
). The opioidergic (ß-endorphin, enkephalin and dinorphins) system is known to be involved in EtOH abuse (Blum et al., 1989
). Chronic administration of EtOH to rats showed an increase in leu-enkephalin in the cortex and in met-enkephalin in the striatum, medulla oblongata and thalamus. However, prolonged alcoholization led to a decreased concentration of leu- and met-enkephalins in the striatum, thalamus and medulla oblongata and to an increased concentration of leu-enkephalin in the cerebral cortex (Burov et al., 1983
). In addition, enkephalin levels increased in all brain regions of offspring from both maternally and paternally exposed rats (Nelson et al., 1988
). Our results support these observations, because the inhibitory effects of EtOH on these AP activities would increase the level of their substrates, potentiating their action. On the other hand, it has been shown that under K+-stimulated conditions, repeated EtOH administration reduces the release of met-enkephalin from hypothalamic slices. Therefore, repeated EtOH treatment may lead to changes in specific neuron population sensitivity to the depolarizing effect of potassium (Przewlocka and Lason, 1991
). This altered sensitivity may also be the consequence of a differential behaviour of the opioid receptors. Thus, a particular sensitivity to chronic EtOH administration of the
opioid receptor but not the µ opioid receptor has been proposed (Przewlocka and Lason, 1990
). In the present work, we showed an inhibitory effect of EtOH on K+-stimulated AP activities, especially on TyrAP activity (enkephalinase) in presence of Ca2+, which may reflect these processes. Our results also support the hypothesis that neuropeptides and their degrading enzymes are released together.
Other important neurotransmitter/neuromodulatory systems involved in EtOH tolerance and dependence are those mediated by oxytocin and vasopressin, which are susceptible to hydrolysis by CysAP (Itoh and Nagamatsu, 1995). Oxytocin is a neurohypophyseal neuropeptide synthesized in the brain and released at the posterior pituitary and in the CNS. This neuropeptide is associated with different adaptive processes of CNS related to EtOH addition and tolerance. In the CNS, EtOH tolerance appears to be a combined process, with both cellular and behavioural components. Tolerance to EtOH has been shown to involve learning, because animals displayed tolerance only in an environment where EtOH had previously been presented and not in a novel environment (Kovács et al., 1998
). Development of rapid tolerance to the hypothermic effect of EtOH has been proposed as a reliable model for investigating the above phenomenon (Szabó et al., 1985
, 1987
). The central administration of oxytocin blocked the development of rapid tolerance to EtOH (Szabó et al., 1989
). This supports the theory that oxytocin acts on CNS mechanisms to influence adaptive responses to EtOH. The central administration of oxytocin at doses that inhibit the development of rapid tolerance to EtOH increased norepinephrine levels in the hypothalamus, dopamine levels in the striatum and medulla oblongata, and serotonin levels in the hippocampus and striatum (Szabó et al., 1988
). The mechanism of EtOH action on CNS neurotransmission is unclear. Serotoninergic and dopaminergic neurotransmission appear to be primarily altered during the inhibition of tolerance development by oxytocin (Szabó et al., 1989
). These and other results suggest that several neuropeptides modulate the response to EtOH. In the case of oxytocin, adaptive components of drug addiction are primarily affected, with the neuropeptide inhibiting the development of EtOH tolerance. EtOH acts on the CNS through various mechanisms, and oxytocin inhibits adaptive CNS processes in response to EtOH. Because oxytocinergic neuronal transmission and CNS oxytocin receptors are supposedly involved in these effects, it has been hypothesized that central nervous oxytocinergic neurons are integral elements of the brain's adaptive response to EtOH. The adaptive response of the CNS to repeated administration of EtOH leads to tolerance and dependence. Activation of brain oxytocinergic neurotransmission under these circumstances may represent a physiological counterbalance mechanism that may be of functional significance, especially in early neuronal adaptation, and may prevent the rapid onset of EtOH tolerance and dependence (Kóvacs et al., 1998
). Our results showed an important inhibition of EtOH on CysAP activity, which may potentiate oxytocin effects.
Regarding vasopressin, it has been demonstrated that this neuropeptide may influence the development of functional tolerance and/or physical dependence on EtOH. This and other peptides were previously shown to influence learning and/or memory. However, other analyses revealed differences in the structural requirements for the maintenance of EtOH tolerance compared with the facilitation of memory processes. Therefore, these phenomena may represent CNS adaptive processes that are subserved by different mechanisms or are differentially sensitive to particular peptides (Hoffman and Tabakoff, 1981). In any case, these effects seem to be modulated through vasopressin V1 receptor activation and second messenger production (Briley et al., 1994
). Intracerebroventricular injection of vasopressin in mice resulted in a substantial increase in mRNA for the proto-oncogene c-fos. Increased c-fos expression has been hypothesized to play a role in neuroadaptation, which could be important for vasopressin effects on EtOH tolerance (Giri et al., 1990
). Our results also agree with a potentiation of vasopressin by EtOH, through the inhibition of its degrading AP by EtOH. These observations might provide useful insights into the way in which an endogenous neuronal peptide modulates adaptive functions in the CNS by acting through its own receptors and also by modifying the efficacy of a classical neuronal transmitter system. After their synthesis and release, endogenous neuropeptides are only present in the brain and body fluids for a few minutes, similar to the biological half-life of exogenously administered neuropeptides. However, their effects on addiction can be detected long after peptide release/administration. Thus, neuropeptides set into motion various secondary events in the CNS that maintain all these changes viable, probably through molecular mechanisms such as changes in gene expression. In fact, it has been demonstrated that EtOH administration induces gene expression changes that are responsible for cellular responses of tolerance and dependence (Mackler and Eberwine, 1991
). To our knowledge, there is no available information regarding the effects of EtOH on AP gene expression. The chronic administration of EtOH significantly decreased the total number of vasopressin and other neuropeptide-containing neurons (Madeira et al., 1997
). Therefore, vasopressin may influence other neurotransmitter/neuromodulatory systems.
Finally, ArgAP (ApB) and AlaAP and LeuAP (ApM) activities also participate in the brain reninangiotensin system (bRAS) (Ganong, 1995), considered to be the pathway for synthesis of locally produced angiotensin II (Ang-II) in the brain (De Gasparo et al., 1994
; Sim et al., 1994
). The pressor peptide Ang-II is converted to the heptapeptide angiotensin III (Ang-III) by aminopeptidase A (angiotensinase), which is primarily responsible for cleaving aspartic acid from the N-terminus of Ang-II (Sakura et al., 1983
; Rich et al., 1984
). Ang-III is further converted to the hexapeptide angiotensin IV (Ang-IV) by ArgAP and also by ApM, which cleaves arginine from the N-terminus (Abhold and Harding, 1988
). Ang-III and Ang-IV are both biologically active (Wright and Harding, 1995
). Chronic EtOH abuse may lead to an altered regulation of the local brain blood flow and/or of the fluid and electrolytic balance (Mungall et al., 1995
) by stimulating bRAS activity, and the CNS is especially sensitive to these phenomena, which cause a variety of vascular and cellular changes (Amenta et al., 1996
). The inhibitory effects of EtOH on ArgAP and ApM activities suggest the existence of high levels of Ang-III that exert a major pressor effect in the brain, probably due to its action on AT1 receptors (De Gasparo et al., 1994
). It has also been suggested that the EtOH metabolite acetaldehyde increases the synthesis rate of Ang-I and also inhibits aminopeptidase A activity (Brecher et al., 1996
), which contributes to the hypertension observed in alcoholics (Thevananther and Brecher, 1994
). Previous studies of mouse cortex synaptosomes at our laboratory demonstrated that EtOH inhibits aminopeptidase A (Mayas et al., 2000c
, 2001). It has also been reported that acetaldehyde inhibits AlaAP activity, increasing the pressor effect of Ang-III (Brecher et al., 1996
). Our results also support this hypothesis because we show an inhibitory effect of EtOH on AlaAP, ArgAP and LeuAP activities. Therefore, the limited degradation of Ang-II/Ang-III by AP activities as a consequence of EtOH (or acetaldehyde) effects may play a major role in increasing blood pressure at brain level.
In conclusion, EtOH-induced changes in AP activities at the synapse level do not reflect possible tissue damage. However, EtOH may modulate the function of several active peptides that act in the CNS through the enzymes involved in degrading them, probably as a result of complex interactions between EtOH and CNS neurotransmitter/neuromodulatory systems involved in EtOH tolerance and/or dependence.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Ahmad, S. and Ward, P. E. (1990) Role of aminopeptidase A activity in the regulation of the pressor activity of circulating angiotensins. Journal of Pharmacology and Experimental Therapeutics 252, 643650.[Abstract]
Amenta, F., Strocchi, P. and Sabbatini, M. (1996) Vascular and neuronal hypertension brain damage: A protective effect of treatment with nicardipine. Journal of Hypertension 14, S29S35.
Barrett, A. J., Rowlings, N. D. and Woesnner, J. F. (1998) Handbook of proteolityc enzymes. Academic Press, London.
Berg, M. J. and Marks, N. (1989) Formation of des-tyr-dynorphins 517 by a purified cytosolic aminopeptidase of rat brain. Journal of Neuroscience Research 11, 313321.
Blum, K., Briggs, A. H. and Trachtenberg, M. C. (1989) Ethanol ingestive behaviour as a function of central neurotransmission. Experientia 45, 444452.[ISI][Medline]
Bora, P. S. and Lange, L. G. (1993) Molecular mechanism of ethanol. metabolism by human brain to fatty acid ethyl esters. Alcoholism: Clinical Experimental Research 17, 2830.[ISI][Medline]
Brecher, A. S., Stauffer, R. and Knight, J. (1996) Acetaldehyde inhibits serum aminopeptidases. Alcohol 13, 125131.[CrossRef][ISI][Medline]
Briley, E. M., Lolait, S. J., Axelrod, J. and Felder, C. C. (1994) The cloned vasopressin V1a receptor stimulates phospholipase A2, phospholipase C, and phospholipase D through activation of receptor-operated calcium channels. Neuropeptides 27, 6374.[ISI][Medline]
Burov, I. V., Iukhananov, R. and Maiskii, A. I. (1983) Effect of ethanol on the concentration of enkephalins in the brain of rats with different levels of alcoholic motivation. Biulleten Eksperimental noi P Biologii i Meditsiny 96, 4851.
Canda, A., Yu, B. H. and Sze, P. V. (1995) Biochemical characterization of ethanol actions on dihydropyridine-sensitive Ca2+ channels in brain synaptosomes. Biochemical Pharmacology 50, 17111718.[CrossRef][ISI][Medline]
Castoldi, A. F., Barni, S., Randine, G., Costa, L. G. and Manzo, L. (1998) Ethanol selectively interferes with the trophic action of NMDA and carbacol on cultured cerebellar granule neurons undergoing apoptosis. Developmental Brain Research 111, 279289.[ISI][Medline]
Dahchour, A. and De White, P. (2000) Ethanol and amino acids in the central nervous system: assessment of the pharmacological actions of acamprosate. Progress Neurobiology 60, 343362.[CrossRef][ISI]
De Gasparo, M., Bottan, S. and Levens, N. R. (1994) Characteristics of angiotensin II receptors and their role in cell and organ physiology. In: Hypertension: pathophysiology, diagnosis, and management, Laragh, J. H. and Brenner, B. M. eds, pp. 101119. Raven Press, New York.
Diamond, I. and Gordon, A. S. (1997) Cellular and molecular neuroscience of alcoholism. Physiological Reviews 77, 120.
Eckardt, M. J., File, S., Gessa, G. L., Grant, K. A., Guerri, C., Hoffman, P. L., Kalant, H., Koob, G. F., Li, T. K. and Tabakoff, B. (1998) Effects of moderate alcohol consumption on the central nervous system. Alcoholism: Clinical and Experimental Research 22, 9981040.[ISI][Medline]
Erickson, C. K. (1996) Review of neurotransmitters and their role in alcoholism treatment. Alcohol and Alcoholism 1, 511.
Fadda, F. and Rossetti, Z. L. (1998) Chronic ethanol consumption: from neuroadaptation to neurodegeneration. Progress Neurobiology 36, 385431.
French, S. W. (1991) The mechanism of organ injury in alcoholics: implications for therapy. Alcohol and Alcoholism 1, 5763.
Ganong, W. F. (1995) Tissue reninangiotensin system. Advances in Experimental Medicine and Biology 377, 435440.[Medline]
Giri, P. R., Dave, J. R., Tabakoff, B. and Hoffman, P. L. (1990) Arginine vasopressin induces the expression of c-fos in the mouse septum and hippocampus. Brain Research Molecular Brain Research 7, 131137.[ISI][Medline]
Gonzales, R. A. and Hoffman, P. L. (1991) Receptor gated channels may be selective CNS targets for ethanol. Trends In Pharmacological Sciences 12, 13.[CrossRef][ISI]
Greenberg, L. J. (1962) Fluorimetric measurement of alkaline phosphatase and aminopeptidase activities in the order of 1014 moles. Biochemical and Biophysical Research Communications 9, 430435.[ISI]
Guerri, C. and Grisolia, S. (1983) Chronic ethanol treatment affects synaptosomal membrane-bound enzymes. Pharmacology Biochemistry and Behavior 18, 4550.[CrossRef][ISI][Medline]
Hersh, L. B. (1985) Characterization of membrane bound aminopeptidases from rat brain: identification of the enkephalin-degrading aminopeptidase. Journal of Neurochemistry 44, 14271435.[ISI][Medline]
Hoffman, P. L. and Tabakoff, B. (1981) Centrally acting peptides and tolerance to ethanol. Currents in Alcoholism 8, 359378.[Medline]
Itoh, C. and Nagamatsu, A. (1995) An aminopeptidase activity from porcine kidney that hydrolyzes oxytocin and vasopressin: purification and partial characterization. Biochimica et Biophysica Acta 1243, 203208.[ISI][Medline]
Johnson, G. D. and Hersh, L. B. (1990) Studies on the subsite specificity of the rat brain puromycin-sensitive aminopeptidase. Archives of Biochemistry and Biophysics 276, 305309.[ISI][Medline]
Kiianmaa, K. (1990) Neuronal mechanisms of ethanol sensitivity. Alcohol and Alcoholism 25, 257262.[ISI][Medline]
Koob, G. F., Roberts, A. J., Schulteis, G., Parsons, L. H., Heyser, C. J., Hyytia, P., Merlo-Pich, E. and Weiss, F. (1998) Neurochemistry targets in ethanol reward and dependence. Alcoholism: Clinical and Experimental Research 22, 39.[ISI][Medline]
Kovács, G. L., Sarnyai, Z. and Szabó, G. (1998) Oxytocin and addiction: a review. Psychoneuroendocrinology 23, 945962.[CrossRef][ISI][Medline]
Kuriyama, K. and Ohkuma, S. (1990) Alteration in the function of cerebral neurotransmitter receptors during the establishment of alcohol dependence: neurochemical aspects. Alcohol and Alcoholism 25, 239249.[ISI][Medline]
Logan, B. J., Laverty, R. and Peake, B. M. (1983) ESR measurements on the effects of ethanol on the lipid and protein conformation in biological membranes. Pharmacology Biochemistry and Behavior 18, 3135.[CrossRef]
Mackler, S. A. and Eberwine, J. H. (1991) The molecular biology of addictive drugs. Molecular Neurobiology 5, 4558.[ISI][Medline]
Madeira, M. D., Andrade, J. P., Lieberman, A. R., Sousa, N., Almeida, O. F. and Paula-Barbosa, M. M. (1997) Chronic alcohol consumption and withdrawal do not induce cell death in the suprachiasmatic nucleus, but lead to irreversible depression of peptide immunoreactivity and mRNA levels. Journal of Neuroscience 17, 13021319.
Martínez-Martos, J. M., Ramírez-Expósito, M. J., Mayas-Torres, M. D., García-López, M. J. and Ramírez-Sánchez, M. (2000) Utility of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay to measure mitochondrial activity in K+- and ATP-stimulated rodent cortex synaptosomes. Neuroscience Research Communications 27, 103107.[CrossRef][ISI]
Mayas, M. D., Ramírez-Expósito, M. J., García, M. J., Tsuboyama, G., Ramírez, M. and Martínez-Martos, J. M. (2000a) Calcium-dependent modulation by ethanol of mouse synaptosomal pyroglutamyl aminopeptidase activity under basal and K+-stimulated conditions. Neuroscience Letters 293, 199202.[CrossRef][ISI][Medline]
Mayas, M. D., Martínez-Martos, J. M., Ramírez-Expósito, M. J., García, M. J., Tsuboyama, G. K., Prieto, I., Arechaga, G. and Ramírez, M. (2000b) Estudio in vitro del efecto del etanol sobre la actividad piroglutamato aminopeptidasa en sinaptosomas de ratón en condiciones basales y despolarizantes. Revista de Neurología 30, 128131.[ISI][Medline]
Mayas, M. D., Martínez-Martos, J. M., Ramírez-Expósito, M. J., García, M. J. and Ramírez, M. (2000c) Influencia del alcohol etílico sobre la actividad aminopeptidasa A de sinaptosomas corticales de ratón. Archivos de Neurociencias 5, 120126.
Mayas, M. D., Ramírez-Expósito, M. J., García, M. J. Ramírez, M. and Martínez-Martos, J. M. (2002) Ethanol modifies differently aspartyl- and glutamyl-aminopeptidase activities in mouse frontal cortex synaptosomes. Brain Research Bulletin 57, 195203.[CrossRef][ISI][Medline]
McDermott, J. R., Mantle, D., Lawfort, B., Gibson, A. M. and Biggins, A. (1988) Purification and characterization of two soluble Cl activated arginyl aminopeptidase from human brain and their endopeptidase action on neuropeptides. Journal of Neurochemistry 50, 176182.[ISI][Medline]
Mihic, S. J., Ye, Q., Wick, M. J., Kottchive, V. V., Krasowski, M. D., Finn, S. E., Mascia, M. P., Valenzuela, C. F., Hanson, K. K., Greenblat, E. P., Harris, R. A. and Harrison, N. L. (1997) Sites of alcohol and volatile anaesthetic action of GABA(A) and glycine receptors. Nature 389, 385389.[CrossRef][ISI][Medline]
Mungall, B. A., Shinkel, T. A. and Sernia, C. (1995) Immunocytochemical localization of angiotensinogen in the fetal and neonatal rat brain. Neuroscience 67, 505524.[CrossRef][ISI][Medline]
Nelson, B. K., Brightwell, W. S., MacKenzie-Taylor, D. R., Burg, J. R. and Massari, V. J. (1988) Neurochemical, but not behavioural, deviations in the offspring of rats following prenatal or paternal inhalation exposure to ethanol. Neurotoxicology and Teratology 10, 1522.[CrossRef][ISI][Medline]
Przewlocka, B. and Lason, W. (1990) Stress prevents the chronic ethanol-induced delta opioid receptor supersensitivity in the rat brain. Polish Journal of Pharmacology and Pharmacy 42, 137142.[ISI][Medline]
Przewlocka, B. and Lason, W. (1991) The effect of single and repeated ethanol administration of hypothalamic opioid systems activityan in vitro release study. Drug and Alcohol Dependence 27, 6367.[CrossRef][ISI][Medline]
Raul, F., Noriega, R., Stock-Damge, C., Doffoel, M. and Grenier, J. F. (1982) Effect of chronic alcohol administration on liver morphology and on brush border membrane enzymes after jejunoileal bypass operation in rat. Digestion 24, 215224.[CrossRef][ISI][Medline]
Reilly, M. E., Mantle, D., Salisbury, J., Peters, T. J. and Preedy, V. R. (2000) Comparative effects of acute ethanol dosage on liver and muscle protein metabolism. Biochemical Pharmacology 60, 17731785.[CrossRef][ISI][Medline]
Rich, D. H., Moon, B. J. and Harbeson, S. (1984) Inhibition of aminopeptidases by amastatin and bestatin derivatives. Effect of inhibitor structure of slow-binding processes. Journal of Medicinal Chemistry 27, 417422.[ISI][Medline]
Rubin, E. and Rottenberg, H. (1982) Ethanol-induced injury and adaptation in biological membranes. Federation Proceedings 41, 24652471.[ISI][Medline]
Sakura, H., Kobayashi, H., Mizutani, S., Sakura, N., Hashimoto, T. and Kawashima, Y. (1983) Kinetic properties of placental aminopeptidase A: N-terminal degradation of angiotensin II. Biochemistry International 6, 609615.[ISI][Medline]
Sanderink, G. J., Artur, Y., Schlele, F., Gueguen, R. and Slest, G. (1988) Alanine aminopeptidase in serum: biological variations and reference limits. Clinical Chemistry 34, 14221426.
Schaad, N. C., Magistretti, P. J. and Schorderet, M. (1988) Effects of ethanol on VIP and/or noradrenaline-stimulated cAMP formation in mouse brain. Alcohol 5, 445449.[CrossRef][ISI][Medline]
Schnebli, H. P., Phillipps, M. A. and Barclay, R. K. (1979) Isolation and characterization of an enkephalin-degrading aminopeptidase from rat brain. Biochimica et Biophysica Acta 569, 8798.
Schwabe, C. and McDonald, J. K. (1977) Demonstration of a Pyro- glutamyl residue at the N-terminus of the B-chain of porcine relaxin. Biochemical Biophysical Research Communications 74, 15011504.[ISI]
Shah, J. and Pant, H. C. (1988) Spontaneous calcium release induced by ethanol in the isolated rat brain microsomes. Brain Research 474, 9499.[CrossRef][ISI][Medline]
Sim, M. K., Choo, M. H. and Qui, X. S. (1994) Degradation of angiotensin I to [des-Asp1]angiotensin I by a novel aminopeptidase in the rat. Biochemical Pharmacology 48, 10431046.[CrossRef][ISI][Medline]
Stone, T. W. (1995) Neuropharmacology. WH Freeman and Co. Ltd, New York.
Szabó, G., Kovács, G. L., Székeli, S. and Telegdy, G. (1985) The effects of neurohypophyseal hormones on tolerance to the hypothermic effect of ethanol. Alcohol 2, 567674.[CrossRef][ISI][Medline]
Szabó, G., Kovács, G. L., Székeli, S. and Telegdy, G. (1987) Neurohypophyseal peptides and ethanol tolerance and dependence. Frontiers of Hormone Research 15, 128137.[ISI]
Szabó, G., Kovács, G. L., Székeli, S. and Telegdy, G. (1989) Intraventricular administration of neurohypophyseal hormones interferes with the development of tolerance to ethanol. Acta Physiologica Hungarica 73, 97103.[ISI][Medline]
Szabó, G., Kovács, G. L., Székeli, S. and Telegdy, G. (1988) Brain monoamines are involved in mediating the action of neurohypophyseal peptide hormones on ethanol tolerance. Acta Physiologica Hungarica 71, 459466.[ISI][Medline]
Tabakoff, B. and von Wartburg, J. P. (1975) Separation of aldehyde reductases and alcohol dehydrogenase from brain by affinity chromatography: Metabolism of succinic semialdehyde and ethanol. Biochemical Biophysical Research Communications 63, 957966.[ISI]
Tabakoff, B., Hellevuo, K. and Hoffman, P. L. (1996) Alcohol. In Handbook of experimental pharmacology, Vol. 118, Shuster, C. R., Gust, S. W. and Kuhar, M. J. eds, pp. 373458. Springer-Verlag, Heidelberg.
Tabakoff, B. and Hoffman, P. L. (1987) Biochemical pharmacology of alcohol. In Psychopharmacology: The third generation of progress, Meltzer, H. Y. ed., pp. 15211526. Raven Press, New York
Thevananther, S. and Brecher, A. S. (1994) Interactions of acetaldehyde with plasma proteins of the reninangiotensin system. Alcohol 11, 493499.[CrossRef][ISI][Medline]
Veloso, D., Passonneau, J. V. and Veech, R. L. (1972) The effects of intoxicating doses of ethanol upon intermediary metabolism in rat brain. Journal of Neurochemistry 19, 26792686.[ISI][Medline]
Wagner, G. W., Tavianini, M. A., Herrmann, K. M. and Dixon, J. E. (1981) Purification and characterization of an enkephalin aminopeptidase from rat brain. Biochemistry 13, 38843890.
Witek, B. and Kolataj, A. (1999) Effect of ethanol administration on activities of some lysosomal hydrolases in the mouse. General Pharmacology 32, 163168.[CrossRef][Medline]
Wozniak, K. M. and Linnoila, M. (1992) Recent advances in pharmacological research on alcohol. Possible alterations with cocaine. Recent Development in Alcoholism 10, 235272.
Wright, J. W. and Harding, J. W. (1995) Brain angiotensin receptor subtypes AT1, AT2 and AT4 and their functions. Regulatory Peptides 59, 269295.[CrossRef][ISI][Medline]
Zimatkin, S. M. and Dietrich, R. A. (1997) Ethanol metabolism in the brain. Addiction Biology 2, 387399.[CrossRef][ISI]
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