**Ethanol Interference on Adenosine System**

Silvânia Vasconcelos et al.\*

*Federal University of Ceará, Department of Physiology and Pharmacology Brazil* 

#### **1. Introduction**

708 Pharmacology

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It is well documented in literature a wide range of behavioral and physiological effects arising from ethanol intake (Spinetta et al., 2008; Soares et al., 2009; Brust, 2010). Because it is a substance that affects differently and simultaneously several neurotransmitter systems, covering different brain areas (Dahchour & De Witte, 2000; Vasconcelos et al., 2008; Vengeliene et al., 2008), it becomes complex to reveal the mechanism of action that governs its effects, being still a challenge for researchers. In addition, ethanol has a biphasic behavioral presenting an excitatory feature in the early stages and a depressant feature in its chronic use.

Among the wide range of pathways in central nervous system that are modified by ethanol, it is important to highlight those that explain ethanol diverse effects, like the ones releasing gamma-amynobutiric acid (GABA), glutamate, dopamine and norepinephrine (Kaneyuki et al., 1991; Vasconcelos et al., 2004). Moreover, another pathway that is rising on researches about ethanol effects is the adenosinergic system (Prediger et al., 2006; Thorsell et al., 2007).

Adenosine was described as a potent depressor of neuronal activity (Dunwiddie & Haas, 1985), and acts mainly via A1 receptor, which is a presynaptic inhibitor of the release of neurotransmitters such as dopamine, GABA, glutamate, acetylcholine and norepinephrine (Fredholm et al., 2001; Dunwiddie & Masino, 2001). Moreover, adenosine is involved in behavioral processes like motor function, anxiety, depression, reward and drug addiction, and human disorders such as Parkinson disease and schizophrenia (Moreau and Huber, 1999).

In addition, there is strong evidence of an involvement of the adenosinergic system on ethanol effects, including the extracellular increase of adenosine after acute exposure to ethanol (Krauss et al., 1993; Nagy et al., 1990), the accentuation or blockade of ethanolinduced motor incoordination provided by adenosine receptor agonists or antagonists, respectively (Dar, 2001; Soares et al., 2009), and the reduction of anxiogenic-like behavior in acute ethanol withdrawal (Prediger et al., 2006). Adenosine antagonists, like caffeine, are implicated in alcohol tolerance (Fillmore, 2003), and retrograde memory impairment caused by ethanol (Spinetta et al., 2008). Thus, adenosine receptors seem to modulate some of the

and Manoel Cláudio Patrocínio2

<sup>\*</sup> Sarah Escudeiro1, Ana Luíza Martin1, Paula Soares1, Antônio Vieira Filho2, Larissa Silva2, Kátia Cilene Dias1, Danielle Macêdo1, Francisca Cléa Sousa1, Marta Fonteles1

*<sup>1</sup>Federal University of Ceará, Department of Physiology and Pharmacology, Brazil 2College of Medicine Christus, Brazil* 

Ethanol Interference on Adenosine System 711

Indirectly, this relationship can also occur through ionotropic ATP receptors, that has the function of specific subtypes (P2X4R and P2X2R) inhibited by ethanol (Davies et al., 2002, 2005), altering the modulation of release of different substances such as GABA, glycine and

Concerning the glutamatergic system, this one demonstrates relationship with the two subtypes of adenosine receptors A1 and A2A, once these receptors appear hetero-dimerized in glutamatergic nerve terminals in the striatum, modulating the concentration of glutamate in accordance with the availability of adenosine, where a lower concentration activates A1R inhibiting glutamate release, and a higher concentration activates A2AR, stimulating the release of glutamate and greater activation of the NMDA receptor. This regulates the release of dopamine in the nucleus accumbens stimulating higher consumption of ethanol (Ciruela

Another finding that reinforces the relationship ethanol/adenosine/glutamate is the synergic interaction that occurs between A2A and mGluR5 receptors (which is related to the consumption of ethanol in the nucleus accumbens) in the striatum, that is, the co-activation of these receptors increases the phosphorylation of proteins regulated by dopamine and cAMP, increased ethanol consumption (Nishi et al., 2003). In addition, NMDA and A1 receptors present a cross modulation on the negative effects of ethanol, like a reduction on motor coordination in the cerebellum, striatum and motor cortex (Mitchell; Neafsey; Collins, 2009). This relationship could be involved with the altered activity of Protein Kinase C (PKC) (Othman et al., 2002). This enzyme has a modulating function against the concentration of glycine, GABA internalization, externalization of NMDA expression of 5-

Regarding the dopaminergic system, A2A and D2 receptors (as well as A1 and D1) exhibit dimerization between them, relating to the reward system in the striatum probably by modulation of AC activity by ethanol, leading to an increase in the concentration of cAMP and the activity of PKA, desensitizing D2, and thus leading to an increased consumption of ethanol (Ferre et al., 2008; Mailliard & Diamond, 2004; Yao et al., 2002; 2003). A possible mechanism of the final response of the dimerized activation of these receptors is that ethanol desensitizes receptors linked to the stimulatory G protein (α subunit), modulating the coupling of D2 with the AC pathway, which may be related to PKA (Yao et al., 2001; Batista et al., 2005). Inoue et al. (2007) found that co-activation of A2A and D2 mediates the transient interaction between nicotine and ethanol, showing an indirect relationship with the cholinergic system, where the use of antagonists of this co-activation can prevent,

Indirectly, the adenosine system also maintains relation to the dopaminergic system via receptors P2XR which were identified in mesolimbic dopaminergic neurons, modulating their activity and, equivalently, the consumption of ethanol (Heine et al., 2007; Xiao et al.,

Adenosine and serotonin systems are related in regard to ethanol via P2X receptors (P2XR). That is, 5-HT3 and P2XR are functionally coupled and both have their actions modulated by ethanol (inhibits P2X2 and P2X4 and stimulates 5-HT3), besides being involved with other neurotransmitter systems such as glycine, GABA, glutamate (mentioned above) and

dopamine in the nucleus accumbens and ventral tegmental area (Davies et al., 2006).

HT3 (Chapell et al., 1998; Lan et al., 2001; Zhang et al., 1995; Sun et al., 2003).

mitigate or even reverse the use of smoke and ethanol.

2008).

glutamate (Mori et al., 2001; Papp et al., 2004).

et al., 2006; Quarta et al., 2004).

pharmacological properties of ethanol, interacting with it by blocking or accentuating its properties.

#### **2. Ethanol and adenosine relation in different neurotransmission systems**

It's known in literature that ethanol alone interferes in different neurotransmitter systems, as GABAergic, glutamatergic, dopaminergic, serotonergic, noradrenergic, cholinergic and others, including adenosinic; however, its action on this last system has currently deserved more attention, due to its neuromodulator/neuroprotector action. Thus, in the present topic updates will be discussed on the relationship between ethanol and adenosine and its consequent interference in some systems above.

To better understand the association of ethanol and adenosine on different neurotransmitter systems, it is necessary to explore the likely hypotheses that explain how ethanol interferes with the adenosine system. Carmichael et al. (1991) suggested that a probable mechanism could occur via metabolism of ethanol by acetate, where this would be incorporated into acetyl-coenzyme A with subsequent formation of AMP, thereby directing the synthesis of adenosine.

Another possible mechanism of interaction between these two substances can be related to the fact that ethanol inhibits facilitated diffusion transporters, being the ENT1 (Equilibrative Nucleoside Transporter) an example, increasing the availability of extracellular adenosine (Diamond et al., 1991; Krauss et al., 1993). Finally, ethanol may facilitate the activation of receptors that have adenylate cyclase (AC) as intracellular signaling system (Rabin; Molinoff, 1981; Hoffman; Tabakoff, 1990), which is displayed by adenosine receptors. Therefore, there are different points of possible interference of the increased concentration of extracellular adenosine induced by ethanol on other neurotransmitter systems.

The GABAergic system in the striatum may be modulated by adenosine with regard to the effects of ethanol on motor coordination and sleep, involving cAMP (Meng; Dar, 1995; Meng et al., 1997). It was found that the use of adenosine agonists accentuate the reduction in the motor coordination induced by ethanol, whereas Ro15-4513, a weak partial inverse agonist of the benzodiazepine class of drugs, attenuated by blocking the effect of the first when used in combination (Meng; Dar, 1994, 1995), suggesting a participation via GABAA by an alteration in the conductance of chloride ions (Meng et al., 1997, Mohler et al., 1984). A mechanism suggested by Londos et al. (1980) and Van Calk et al. (1970) relates ethanol to alterations in the production of cAMP via AC through the A1 receptor, ie, increased availability of adenosine induced by ethanol leads to greater signs of adenosine on your receptor that has a higher affinity, which is related with inhibitory G protein, reducing cAMP production and concomitant modulation of the GABAergic system that increases chloride conductance.

This ratio adenosine/ethanol with the GABAergic system can still be related to opioid system, where ethanol induces the increased availability of β-endorphin which activates μtype receptors, altering the release of GABA in dopaminergic neurons in the ventral tegmental area, an area involved to reward behavior and abuse of ethanol (Mendez et al, 2003; Marinelli et al, 2004; Lam et al, 2008; Jarjour et al, 2009).

pharmacological properties of ethanol, interacting with it by blocking or accentuating its

It's known in literature that ethanol alone interferes in different neurotransmitter systems, as GABAergic, glutamatergic, dopaminergic, serotonergic, noradrenergic, cholinergic and others, including adenosinic; however, its action on this last system has currently deserved more attention, due to its neuromodulator/neuroprotector action. Thus, in the present topic updates will be discussed on the relationship between ethanol and adenosine and its

To better understand the association of ethanol and adenosine on different neurotransmitter systems, it is necessary to explore the likely hypotheses that explain how ethanol interferes with the adenosine system. Carmichael et al. (1991) suggested that a probable mechanism could occur via metabolism of ethanol by acetate, where this would be incorporated into acetyl-coenzyme A with subsequent formation of AMP, thereby directing the synthesis of

Another possible mechanism of interaction between these two substances can be related to the fact that ethanol inhibits facilitated diffusion transporters, being the ENT1 (Equilibrative Nucleoside Transporter) an example, increasing the availability of extracellular adenosine (Diamond et al., 1991; Krauss et al., 1993). Finally, ethanol may facilitate the activation of receptors that have adenylate cyclase (AC) as intracellular signaling system (Rabin; Molinoff, 1981; Hoffman; Tabakoff, 1990), which is displayed by adenosine receptors. Therefore, there are different points of possible interference of the increased concentration of

The GABAergic system in the striatum may be modulated by adenosine with regard to the effects of ethanol on motor coordination and sleep, involving cAMP (Meng; Dar, 1995; Meng et al., 1997). It was found that the use of adenosine agonists accentuate the reduction in the motor coordination induced by ethanol, whereas Ro15-4513, a weak partial inverse agonist of the benzodiazepine class of drugs, attenuated by blocking the effect of the first when used in combination (Meng; Dar, 1994, 1995), suggesting a participation via GABAA by an alteration in the conductance of chloride ions (Meng et al., 1997, Mohler et al., 1984). A mechanism suggested by Londos et al. (1980) and Van Calk et al. (1970) relates ethanol to alterations in the production of cAMP via AC through the A1 receptor, ie, increased availability of adenosine induced by ethanol leads to greater signs of adenosine on your receptor that has a higher affinity, which is related with inhibitory G protein, reducing cAMP production and concomitant modulation of the GABAergic system that increases

This ratio adenosine/ethanol with the GABAergic system can still be related to opioid system, where ethanol induces the increased availability of β-endorphin which activates μtype receptors, altering the release of GABA in dopaminergic neurons in the ventral tegmental area, an area involved to reward behavior and abuse of ethanol (Mendez et al,

2003; Marinelli et al, 2004; Lam et al, 2008; Jarjour et al, 2009).

extracellular adenosine induced by ethanol on other neurotransmitter systems.

**2. Ethanol and adenosine relation in different neurotransmission systems** 

properties.

adenosine.

chloride conductance.

consequent interference in some systems above.

Indirectly, this relationship can also occur through ionotropic ATP receptors, that has the function of specific subtypes (P2X4R and P2X2R) inhibited by ethanol (Davies et al., 2002, 2005), altering the modulation of release of different substances such as GABA, glycine and glutamate (Mori et al., 2001; Papp et al., 2004).

Concerning the glutamatergic system, this one demonstrates relationship with the two subtypes of adenosine receptors A1 and A2A, once these receptors appear hetero-dimerized in glutamatergic nerve terminals in the striatum, modulating the concentration of glutamate in accordance with the availability of adenosine, where a lower concentration activates A1R inhibiting glutamate release, and a higher concentration activates A2AR, stimulating the release of glutamate and greater activation of the NMDA receptor. This regulates the release of dopamine in the nucleus accumbens stimulating higher consumption of ethanol (Ciruela et al., 2006; Quarta et al., 2004).

Another finding that reinforces the relationship ethanol/adenosine/glutamate is the synergic interaction that occurs between A2A and mGluR5 receptors (which is related to the consumption of ethanol in the nucleus accumbens) in the striatum, that is, the co-activation of these receptors increases the phosphorylation of proteins regulated by dopamine and cAMP, increased ethanol consumption (Nishi et al., 2003). In addition, NMDA and A1 receptors present a cross modulation on the negative effects of ethanol, like a reduction on motor coordination in the cerebellum, striatum and motor cortex (Mitchell; Neafsey; Collins, 2009). This relationship could be involved with the altered activity of Protein Kinase C (PKC) (Othman et al., 2002). This enzyme has a modulating function against the concentration of glycine, GABA internalization, externalization of NMDA expression of 5- HT3 (Chapell et al., 1998; Lan et al., 2001; Zhang et al., 1995; Sun et al., 2003).

Regarding the dopaminergic system, A2A and D2 receptors (as well as A1 and D1) exhibit dimerization between them, relating to the reward system in the striatum probably by modulation of AC activity by ethanol, leading to an increase in the concentration of cAMP and the activity of PKA, desensitizing D2, and thus leading to an increased consumption of ethanol (Ferre et al., 2008; Mailliard & Diamond, 2004; Yao et al., 2002; 2003). A possible mechanism of the final response of the dimerized activation of these receptors is that ethanol desensitizes receptors linked to the stimulatory G protein (α subunit), modulating the coupling of D2 with the AC pathway, which may be related to PKA (Yao et al., 2001; Batista et al., 2005). Inoue et al. (2007) found that co-activation of A2A and D2 mediates the transient interaction between nicotine and ethanol, showing an indirect relationship with the cholinergic system, where the use of antagonists of this co-activation can prevent, mitigate or even reverse the use of smoke and ethanol.

Indirectly, the adenosine system also maintains relation to the dopaminergic system via receptors P2XR which were identified in mesolimbic dopaminergic neurons, modulating their activity and, equivalently, the consumption of ethanol (Heine et al., 2007; Xiao et al., 2008).

Adenosine and serotonin systems are related in regard to ethanol via P2X receptors (P2XR). That is, 5-HT3 and P2XR are functionally coupled and both have their actions modulated by ethanol (inhibits P2X2 and P2X4 and stimulates 5-HT3), besides being involved with other neurotransmitter systems such as glycine, GABA, glutamate (mentioned above) and dopamine in the nucleus accumbens and ventral tegmental area (Davies et al., 2006).

Ethanol Interference on Adenosine System 713

symptom after a single ethanol challenge dose. Prediger et al. (2006) designed an experimental study of acute ethanol withdrawal (hangover) in mice, in which a timedependent development of anxiety-like behavior after an intraperitoneal administration of a single dose of ethanol (4 g/kg) in mice was assessed, and the potential of adenosine A1 and A2A receptor agonists in reducing this behavior was evaluated. They presented evidence that acute administration of 'nonanxiolytic' doses of adenosine (5–10 mg/kg, i.p.) or the selective adenosine A1 receptor agonist CCPA (0.05–0.125 mg/kg, i.p.), but not the adenosine A2A receptor agonist DPMA (0.1–5.0 mg/kg, i.p.), which reduces the anxiety-like behavior during ethanol hangover in mice, as indicated by a significant increase in the exploration of the open arms of the elevated plus maze. In addition, the effect of CCPA (0.05 mg/kg, i.p.) was prevented by the pretreatment with the selective adenosine A1 receptor antagonist DPCPX (3.0 mg/kg, i.p.), demonstrating that the activation of adenosine A1 receptors, but not adenosine A2A receptors, reduces the anxiogenic-like behaviour observed during acute

In general, sensitivity to the adverse effects of ethanol is inversely correlated with alcohol consumption. In a study with mice lacking the A2A receptor, Naassila et al. (2002) showed that these animals are less sensitive to the acute effects of ethanol as hypothermia and sedation, and consume more ethanol in a two-bottle choice paradigm compared with wildtype littermate control mice, demonstrating that the A2AR is involved in the sensitivity to the hypothermic and sedative effects of ethanol playing a role in alcohol-drinking behavior.

Furthermore, caffeine presents an ability to decrease sensitivity to the stumbling and tiredness associated with drinking large quantities of ethanol. Thus, adenosine receptors antagonists also appear to mediate some of the reinforcement effects of ethanol. This reinforcement is in part mediated via A2AR activation and probably associated with intracellular A2 activation of cAMP/PKA signalling cascades in the nucleus accumbens (Thorsell, et al., 2007; Adams et al., 2008), but the exact mechanism of action remains unclear. Studies in humans examining methylxanthine and ethanol interactions have mostly focused on the influence that caffeine exerts on ethanol intoxication, and have yielded mixed results (Liguori and Robinson 2001; Drake et al. 2003); but a point that needs further attention is the fact that these studies converge upon the point that caffeine consumed in association with ethanol, rather than improving ethanol-induced impairments, would reduce the self-perception of ethanol intoxication (Morelli & Simola, 2011), since human

In addition to reinforcing effects, adenosine also appears to be related to locomotive effects of ethanol at high dose (6 g/kg) in subchronic treatment during 5 days, as shown in the experimental study of Soares et al. (2009), in which the administration of Aminophylline, a non-selective adenosine receptor antagonist, at low doses (5 and 10 mg/kg) produced some degree of locomotion stimulation, and was able to reverse the depressive effects produced by ethanol on the number of falls and time spent in the bar, in the Rota rod test, suggesting a partial blockage of the action of ethanol. The selective A1R agonist *N*6-cyclohexyladenosine (CHA) has also been found to potentiate, and the antagonist DPCPX attenuates ethanol-

Chronic ethanol intake leads to several changes in the balance of neurotransmitter pathways and its receptors, being studied oftentimes focusing withdrawal symptoms. Accordingly

data also show that caffeine enhances tolerance to ethanol (Fillmore, 2003).

induced motor incoordination in mice (Meng et al., 1997).

ethanol withdrawal in mice.

Other neurotransmitters still present a few studies involving ethanol and the adenosine system, such as glycine, where ethanol inhibits their specific receptors probably via PKC (Tao & Ye, 2002), and taurine, which normalizes the activity of ATPases in tissues pretreated with ethanol (Pushpakiran et al., 2005), showing some indirect relationship with the system in focus.
