Adenosine Receptors: What We Know and What We ...

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Current Topics in Medicinal Chemistry, 2010, 10, 860-877

Adenosine Receptors: What We Know and What We are Learning M.L. Trincavelli*, S. Daniele and C. Martini Department of Psychiatry, Neurobiology, Pharmacology and Biotechnology, University of Pisa, Via Bonanno, 6. 56126 Pisa, Italy Abstract: Adenosine, beside its role in the intermediate metabolism, mediates its physiological functions by interacting with four receptor subtypes named A1, A2A, A2B and A3. All these receptors belong to the superfamily of G proteincoupled receptors that represent the most widely targeted pharmacological protein class. Since adenosine receptors are widespread throughout the body, they are involved in a variety of physiological processes and pathology including neurological, cardiovascular, inflammatory diseases and cancer. At now, it is ascertained that the biological responses evoked by the activation of a single receptor are the result of complex and integrated signalling pathways targeted by different receptor proteins, interacting each other. These pathways may in turn control receptor responsiveness over time through fine regulatory mechanisms including desensitization-internalization processes. The knowledge of adenosine receptor structure as well as the molecular mechanisms underlying the regulation of receptor functioning and of receptor-receptor interactions during physiology and pathological conditions represent a pivotal starting point to the development of new drugs with high efficacy and selectivity for each adenosine receptor subtype. The goal of this review is to summarize what we now and what we are learning about adenosine receptor structure, signalling and regulatory mechanisms. In addition, to dissect the potential therapeutic application of adenosine receptor ligands, the pathophysiological role of the receptor subtypes in different tissues is discussed.

Keywords: Adenosine receptors; G protein coupling; tissue distribution; protein-protein interaction; intracellular pathways; receptor regulation; pathophysiological role. INTRODUCTION Adenosine is a constitutive metabolite of all cells, involved in key pathways such as purinergic nucleic acid base synthesis, amino acid metabolism and modulation of cellular metabolic status. Considering this homeostatic role of adenosine related to the control of cellular metabolism [1], adenosine has been termed as ‘local hormone’ [2] and ‘retaliatory metabolite’ [3] to summarise its role in stressful conditions where energy charge is compromised. In such situations, the intracellular concentration of adenosine, which is estimated to be in the nanomolar range, raises to micromolar concentrations [4, 5] and adenosine is released to the extracellular medium [6] to refrain cell metabolism of neighbouring cells. Actually, this nucleoside, when extracellular, exerts its action via specific G protein-coupled receptors (GPCRs) of the P1 class, divided into four adenosine receptor (AR) subtypes: A1, A2A, A2B and A3 [7-9]. This terminology is well established and is coherent with the principles of receptor nomenclature adopted by NC-IUPHAR [10]. ADENOSINE RECEPTOR PROTEIN AND GENOMIC STRUCTURE GPCRs consist of a single polypeptide, containing seven -helices which are oriented perpendicular to the membrane. The N-terminus is located at the extracellular side of the cell * Address correspondence to this author at the Department of Psychiatry, Neurobiology, Pharmacology and Biotechnology, University of Pisa, Via Bonanno, 6. 56126 Pisa, Italy; Tel: 0502219526; Fax: 0502219609; E-mail: [email protected] 1568-0266/10 $55.00+.00

and often contains one or more glycosylation sites. The Cterminus is located intracellular and contains phosphorylation and palmitoylation sites, which are involved in regulation of receptor desensitization and internalization [11]. All ARs, with the exception of the A2A, contain a palmitoylation site near the C-terminus. The A2A AR is the only subtype with an extraordinary long C-terminus, 122 amino acids versus 36 amino acids in e.g. the A1 AR [12]. All the ARs are glycosylated on the second extracellular loop, although glycosylation does not appear to influence ligand binding. The third intracellular loop and/or the C terminus are involved in coupling the ARs to G proteins. The phosphorylation of the intracellular loop 3 is involved in desensitization and internalization of the receptors [10, 13, 14]. By now, all four ARs have been cloned from rat, mouse and man. A1 ARs have been cloned from dog, cow, rabbit, guinea pig and chick, A2A ARs from dog and guinea-pig, A2B ARs from chick, and A3 ARs from dog, sheep and chick. There is a close similarity in structure between the receptors within a subtype, at least for mammals. The largest variability is observed for the A3 ARs, for which there is almost a 30% difference at the amino acid level between human and rabbit. The A3 AR is the only adenosine subtype which has been cloned before its pharmacological identification. It has been originally isolated as an orphan receptor from rat testis, having 40% sequence homology with canine A1 and A2A AR subtypes [15] and has been found to be identical with the A3 AR later cloned from rat striatum [16]. The homologues of the rat striatal A3 AR have been cloned from sheep and human, revealing large interspecies differences in A3 AR structure. For example, the rat A3 AR presents only 74% © 2010 Bentham Science Publishers Ltd.

Adenosine Receptors

sequence homology with sheep and human A3 AR, while there is 85% homology between sheep and human A3 AR. This is reflected in a very different pharmacological profiles among the species homologues, especially in terms of antagonist binding that has made characterization of this adenosine subtype difficult. Recently, the equine A3 AR has been cloned and pharmacologically characterized. Sequencing of the cDNA indicated that it has a high degree of sequence similarity with that of other mammalian A3 AR transcripts, including human and sheep [17]. The genomic structure appears to be similar for all the human adenosine receptors. There is a single intron that interrupts the coding sequence in a region corresponding to the second intracellular loop. The first studied receptor has been the A1 AR. Already when the structure of the A1 AR has been first reported, the presence of two major transcripts, probably representing alternative splicing, has been noted [18-21]. Transcripts containing three exons, called exons 4, 5, and 6 have been found in all tissues expressing the receptor, whereas transcripts containing exons 3, 5, and 6 are in addition found in tissues such as brain, testis, and kidney, which express high levels of the receptor. There are two promoters, a proximal one denoted promoter A, and a distal one denoted promoter B, which are about 600 base pairs apart. The upstream promoter (promoter A) is responsible for tissue-specific expression of A1 ARs, while the downstream promoter B mediates constitute expression of A1 ARs [19]. The regulation of A2A receptor gene expression is likely to play an important role in neuronal development, basal ganglia activity, and many other peripheral functions. Indeed, alteration of A2A receptor expression upon acute trauma or chronic diseases has been reported. To understand the molecular basis for such transcriptional regulation, the gene structures of A2A receptors of several species have been characterized, and have proven highly similar [22-24]. The A2A receptor gene is composed of multiple exons which encode alternative transcripts initiated from at least four independent promoters [22]. All transcripts identified contain the same coding region for the A2A receptor, a common 30 untranslated region (UTR), and a distinct 50 UTR. It is of great interest to note that several important genes [e.g., A1 ARs, acidic fibroblast growth factor, and brain-derived neurotrophic factor (BDNF)] show similar structures which contain multiple independent promoters for deriving transcripts containing distinct short 50UTRs linked to the same coding region and a common 30UTR [19, 25, 26]. It is plausible that the alternative promoter usage strategy is also utilized in the transcriptional control of the A2A receptor under various physiological or pathological conditions. Analysis of the A2B receptor gene, localized on chromosome 17 in man, reveals a similar overall structure as for the other adenosine receptors. The rat A2B receptor shows two hybridizing transcripts of 1.8 and 2.2 kb, where the latter is the dominant one [27]. This could, in analogy with the above, suggest the presence of multiple promoters, but so far this has not been studied to our knowledge. The A3 AR has been mapped on human chromosome 1p21-p13 [28] and consists of 318 aminoacid residues. Murrison et al. [29] determined that the A3 AR gene contains 2 exons separated by a single intron of about 2.2 kb. The

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upstream sequence does not contain a TATA-like motif, but it has a CCAAT sequence and consensus binding sites for SP1, NFIL6, GATA1 and GATA3 transcription factors. Involvement of the latter in transcriptional control of this gene would be consistent with a role of the receptor in immune function. Several putative transcription factorbinding sites could be detected in the mouse gene [30], but surprisingly, few of these are matched by similar elements in the human gene. This could mean that the truly important sites have not been identified, or else that the expression of the receptor is regulated very differently in the two species. Adenosine Receptor Distribution It is important to study the distribution of receptors: in general, the higher the number of receptors the more potent and/or efficacious will be the agonist. Thus, the rather low levels of endogenous adenosine present under basal physiological conditions have the potential of activating receptors where they are abundant, but not where they are sparse [10, 31]. There is much information on the distribution of the A1 and A2A receptors because good pharmacological tools including radioligands (see below) and antibodies are available since several years [32-38]. In the case of the A2B and A3 ARs, the data are less impressive. The A1 AR is particularly prevalent in the central nervous system (CNS), with high levels in the cerebral cortex, hippocampus, cerebellum, thalamus, brain stem and spinal cord. Numerous peripheral tissues also express the A1 AR, including vas deferens, testis, white adipose tissue, stomach, spleen, pituitary, adrenal gland, heart, aorta, liver, eye and bladder. Low levels are found in the lung, kidney and small intestine [10, 14, 39]. The A1 AR is involved in cardiovascular effects (e.g. reducing heart rate), inhibition of lipolysis and stimulation of glucose uptake in white adipocytes and the modulation of neurotransmitter release in the central nervous system (CNS) [39]. The A1 AR also plays a role in anxiety, hyperalgesia, bronchoconstriction and the glomerular filtration rate and rennin release in the kidney [10, 14, 40]. In the CNS, the A2A AR is highly expressed in the striatum and olfactory tubercle [39]. In the periphery, it is highly expressed in the spleen, thymus, leucocytes and blood platelets, and intermediate levels are found in the heart, lung and blood vessels [10, 14]. The A2A AR is involved in the onset of vasodilation, inhibition of platelet aggregation, explo-ratory activity, aggressiveness and hypoalgesia [39]. In addition, A2A AR plays a role in Parkinson’s disease, Huntington’s disease, Alzheimer’s disease, ischaemia, attenuation of inflammation and neuroprotection, particularly in peripheral tissues [10, 14]. A new mouse model incorporating targeted deletion of A2B ARs and replacement of exon 1 with a reporter gene has allowed in vivo determination of which cell types and tissues express A2B ARs in the mouse [41]. A2B ARs receptors have been found in all tested organs, including spleen, lung, colon and kidney, and in all of these organs the primary site of expression was the vasculature. Smooth muscle cells, endothelial cells and macrophages exhibit a high level of

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expression [41], and A2B ARs have also been detected on colonic epithelial cells [40]. Earlier studies employing isolated primary cells or cell lines, however, clearly indicated that other cell types, such as mast cells [42, 43], lymphocytes [44, 45] dendritic cells [46, 47] and neutrophils [45] all express A2B ARs. Since there is a lack of specific agonists for the A2B ARs, little is known about the functional significance of this receptor. However, the A2B ARs play a role in mediating and it is also involved in allergic and inflammatory disorders [10, 14]. The A3 AR is expressed in the CNS, but at relatively low levels, and only the hypothalamus and the thalamus have been reported to contain A3 AR [45]. The highest levels of adenosine A3 AR have been found in the lung and liver, and somewhat lower levels were found in the aorta [39]. In addition, the A3 AR has been found in eosinophils, mast cells, testis, kidney, placenta, heart, spleen, uterus, bladder, jejunum, aorta, proximal colon and eye, although with pronounced differences in expression level between species [10, 14, 46]. The A3 AR has been implicated in mediating allergic responses, airway inflammation and apoptotic events; however, the latter is dependent on the cell type involved and/or the type of activation [10, 46]. Furthermore, the A3 AR is involved in the control of the cell cycle and inhibition of tumour growth both in vitro and in vivo [14]. In fact, A3 ARs have been demonstrated to be more highly expressed in tumours than in healthy cells, suggesting a role for A3 AR as a tumour marker [47]. For the detail in the pathophysiological implication of the different ARs see below. Adenosine Receptor Signalling G protein-Coupling and Second Messengers Heterotrimeric G proteins are guanine-nucleotide regulatory protein complexes composed of and subunits. They are responsible for transmitting signals from GPCRs to effectors, e.g. adenylyl cyclase (AC). Until now, 16 , 5 and 14 isoform have been reported [48]. G proteins are divided into several subclasses with a specific activity profile: Gs proteins stimulate AC, Gi proteins inhibit AC and stimulate GIRK channels, G0 proteins stimulate K+ ion channels, Gq/11 proteins activate phospholipase C (PLC), G12 proteins activate Rho guanine-nucleotide exchange factors (GEFs) and the olfactory G protein, Golf, stimulates AC. Upon receptor activation, both the subunit and the subunit can signal, but to different effectors [49-51]. The A1 AR is usually coupled to a pertussis toxin-sensitive Gi protein, which mediates inhibition of AC and regulates calcium and potassium channels [10, 14, 39, 40, 52]. Both the third intracellular loop and the C-terminal tail of the A1 AR are involved in Gi coupling [13]. In addition, it has been reported that under certain conditions the A1 AR couples to Gs to stimulate AC, or to Gq/11 to stimulate inositol phosphate production. Apparently, the specific activity state of the receptor or the nature of the agonist determine which G protein class is activated by the A1 AR [53, 54]. The A2A AR in the periphery is coupled to cholera toxinsensitive Gs proteins, which increase AC activity upon

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receptor activation. The A2A AR in the striatum is presumably coupled to Golf [10, 14]. The third intracellular loop, but not the C terminus of the A2A AR, is involved in Gs coupling. The A2B AR is coupled to Gs proteins leading to stimulation of AC upon receptor activation [14]. There is quite some evidence that A2B AR can activate PLC as well, via Gq/11 proteins [10]. The A3 AR is coupled to pertussis toxin-sensitive Gi proteins, which mediate inhibition of AC. In addition, A3 AR can stimulate phospholipase C via Gq/11 proteins [10, 14]. For an extensive overview of adenosine receptor-G protein coupling, see Fredholm et al. [55]. Intracellular Signalling It was shown more than 15 years ago that adenosine A1 and A2B ARs could regulate proliferation and differentiation in vascular smooth muscle cells. Since then several examples of such effects have been described, and they may be related to changes in mitogen activated protein kinases (MAPK), which play an essential role in processes such as cell differentiation, survival, proliferation and death. The family of MAPK consists of the extracellular regulated kinases (ERK) such as ERK1/2, and the stress-activated protein kinases (SAPK), such as p38 and jun-N-terminal kinase (JNK). Recently it has been demonstrated that these kinases, usually activated via receptor tyrosine kinases [56], can be activated by GPCRs too. Adenosine A1 ARs transiently expressed in COS-7 cells [57] and in CHO cells [58] can activate ERK1/2 in a timeand dose-dependent manner via a mechanism involving the phosphoinositol-3-kinase (PI3K). Although speculative, this may imply a receptor tyrosine kinase trans-activation as described for the epidermal growth factor (EGF) [59]. Most of the effects evoked by A2A AR are due to the activation of AC, generation of cAMP and activation of protein kinase A (PKA). One key target of PKA is the cAMP responsive element binding protein (CREB) which is critical for many forms of neuronal plasticity as well as other neuronal functions [60]. Phosphorylation of CREB at Ser133 by PKA activates CREB and turns on genes with cAMP responsive elements (CRE sites) in their promoters. One important feature of CREB is that it is a point of convergence for the cAMP/PKA and MAPK pathways [61, 62]. Phosphorylated CREB has been therefore proposed to mediate the anti-inflammatory effect of the A2A AR [63, 64] and inhibition of NFkB by A2A AR activation during acute inflammation in vivo has been demonstrated [65]. CREB may inhibit the transcriptional activity of NFkB, and subsequently suppress cytokine expression (e.g., tumour necrosis factor, TNF) in immune cells. Activation of A2A AR also stimulates protein kinase C (PKC). In PC12 cells, the stimulation of A2A ARs activates novel PKCs (nPKCs) through a pertussis toxin-sensitive G protein and phosphorylates the type VI AC which causes negative feedback regulation of the cAMP signal evoked by A2A AR [66]. An equally interesting observation is that the activation of A2A AR facilitates the activities of adenosine transporters via a PKC-dependent pathway in the hippocampus, thus reducing the level of extracellular adenosine available for A1 AR

Adenosine Receptors


activation [67]. Activation of multiple signalling pathways by the A2A AR appears to contribute to its diverse and complex functions in various tissues. In the basal ganglia, such as occurred for dopamine D1 dopamine receptor, A2A AR activate phosphorylation of the phosphoprotein DARPP-32. This effect appears to be critically important for the in vivo [68], time course of action of caffeine and other drugs. The adenosine A2B AR is the only subtype that so far has been shown to activate not only ERK1/2 but also JNK and p38, as demonstrated in human mast cells (HMC) [69]. In these cells, A2B AR activate MAPK through a mechanism involving Gq proteins and this effect appears relevant for IL8 secretion and consequently for mast cell activation. In untransfected HEK 293 cells, a cascade depending on Gq/11, PLC, genistein-insensitive tyrosine kinases, ras, B-raf, and mitogen activated protein kinase kinase (MEK1/2) has been delineated [70]. In these studies A2B AR has revealed the same potency in activating ERK and AC (EC50 values in the micromolar range) whereas results from another study in transfected cells [58] show a nearly 100-fold higher potency of the agonist in inducing ERK1/2 phosphorylation than in inducing cAMP production. Thus, a GPCR can have substantially different potencies on different signalling pathways in the same cellular system. The A2B AR effects on cell proliferation and/or differentiation may not only be stimulatory. In vascular smooth muscle cells, activation of A2B AR strongly decreases the mitogenic effects of different growth factors, probably secondary to a blockade of MAP kinases stimulated by these growth factors [71]. The adenosine A3 AR has been suggested to activate ERK1/2 in different cell lines. In particular, A3 AR signalling to ERK1/2 depends on release from Gi proteins, PI3K, Ras and MEK [72]. Furthermore, it has been shown that MAPK activation is involved in A3 AR regulation, both contributing to direct receptor phosphorylation by the control of membrane translocation of GPCR kinase (GRK), which are involved in receptor phosphorylation. Thus, an active MAPK signalling pathway appears to be essential for A3 AR Table 1.

Adenosine Receptor Regulation Many important physiological processes are controlled by the coordinated actions of multiple receptor-mediated signalling pathways, each of which is capable of rapid and specific regulation. GPCR regulatory mechanisms, which may be activated by both receptor agonist occupancy or by an agonist independent manner, include receptor “desensitisation”, “internalisation” and “down-regulation”, all contributing to the interruption of receptor responsiveness over time [75-77]. Since ARs are widespread throughout the body and involved in a variety of physiological processes and diseases, there is great interest in understanding how the different subtypes are regulated, as a basis for designing therapeutic drugs that either avoid or make use of this regulatory processes. Upon agonist treatment, adenosine receptor subtypes are differently regulated. For instance, the A1 ARs are not (readily) phosphorylated and internalize slowly, showing a typical half-life of several hours, whereas the A2A and A2B ARs undergo much faster downregulation, usually shorter than 1 h. The A3 ARs are subject to even faster downregulation, often a matter of minutes. The fast desensitization

AR Subtypes

G Proteins

Effectors

A1

Gi a

cAMP

b

cAMP

ERKse,f

Gq11 b

IP3

PI3K g

Gsc

cAMP

PKA; CREB h

Golfc

cAMP

PKCi

Gsc

cAMP

ERKsj

Gq11 c

IP3/DAG

p38; JNKj,k

Gi c

cAMP

ERKsl

Gq11 d

IP3

PI3K/Aktm

A2A

A2B

A3

a

phosphorylation, desensitization and internalization [73]. Another important pathway triggered by adenosine via A3 AR is that of PI3K/Akt pathway. There is evidence that A3 AR activation by subunits of Gi and PI3K-, mediates phosphorylation of PKB/Akt, protecting rat basophilic leukemia (RBL)-2H3 mast cells from apoptosis. It is well known that PKA and PKB/Akt phosphorylate and inactivate glycogen synthase kinase 3 (GSK-3), a serine/threonine kinase, acting as a key element in the Wnt signaling pathway. In its active form, GSK-3 suppresses mammalian cell proliferation [74]. It has been reported that A3 AR activation is able to decrease the levels of PKA, a downstream effector of cAMP, and of the phosphorylated form of PKB/Akt in melanoma cells. This implies the deregulation of the Wnt signaling pathway, generally active during embryogenesis and tumourigenesis causing cell cycle progression and cell proliferation [74].

Coupling of the four AR Subtypes to G Proteins, Effectors and Intracellular Signalling

Gs

[40]; b[54]; c[10]; d[14]; e[57]; f[58]; g[59]; h[60]; i[66]; j[69]; k[70]; l[72]; m[74]

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Intracellular Signalling


of the A3 Ars after agonist exposure may be therapeutically equivalent to antagonist occupancy of the receptor. The early evidence for adenosine A1 ARs desensitization was largely obtained from primary cells, cell lines, tissues or tissue slices, and intact animals that were exposed to varying concentrations of AR agonists (either A1-selective or not), examined over several time periods. Although different results were obtained in dependence on cell system, it could be generalized that A1 ARs underwent to desensitization and internalization with a slow kinetic with respect to the other adenosine receptor subtypes. Several studies have been performed to clarify the molecular mechanisms underlying A1 ARs regulatory mechanisms: Gao et al. [78] have studied the effects of preventing palmitoylation of the A1 ARs. It has been shown that the Cys309Ala mutation, thus removing the palmitoylation site of the human A1 ARs, has no effect on internalization. These findings have been later confirmed by Ferguson et al. [79]. In addiction, A1 AR phosphorylation by GRKs has been subject to debate. Palmer et al. and Ferguson et al. have failed to demonstrate GRK-2 phosphorylation of A1 ARs [79, 80]. Nie et al., however, have reported that within 1 h of exposure of DDT1MF-2 cells to A1 AR agonist, R-phenylisopropyl adenosine (R-PIA) induces a rapid translocation of GRKs from the cytosol to the cytoplasm [81]. In a further biochemical approach with both purified receptor and GRK2, a phosphorylated receptor has been obtained that have shown enhanced affinity for arrestins over G proteins. It may be concluded that under physiological conditions A1 AR phosphorylation does not (readily) take place, which would be a rationale for the long time periods required for receptor internalization. Regarding A2A AR, the importance of the (120 amino acid residues) long C terminus of the receptor in inducing desensitization and internalization has been investigated: Palmer and Stiles [82] introducing several mutations and deletions into the receptor tail, have demonstrated that. Two possible phosphorylation sites (Thr 298 and Ser 305) are important for short-term agonist-induced desensitization while the long-term (24 h) desensitization has not been affected. These results suggest that short-term and long-term desensitization have distinct structural requirements and do not occur via the same mechanism. GRKs are present in different isoform, which of them are involved in the phosphorylation of the A2A AR is not entirely clear yet. However, a putative role for GRK2 and/or GRK5 has been suggested [82]. In NG108-15 cells, the over-expression of GRK2 reduces the A2A AR-mediated activation of cAMP probably as a consequence of GRK2-mediated receptor desensitization [83]. In addiction, in human monocytoid THP-1 cells, TNF, decreases GRK2 association with the plasma membranes and then prevents A2A AR desensitization. In another study, Mundell and Kelly have investigated the effect of inhibitors of receptor internalization on desensitization and resensitization of ARs in NG108-15 cells [84]. They have demonstrated that the block of receptor internalization does not affect A2A AR desensitization whereas induces an impairing in receptor responsiveness recovery (resensitization). The A2B AR, is subjected to a rapid agonist-induced desensitization. Matharu et al. have studied the regions of

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the A2B AR which are responsible for the desensitization and internalization of the A2B AR [85]. For this purpose they have introduced point mutations or deletions in the C terminus of the A2B AR demonstrating that Ser329 is responsible for the rapid agonist-induced desensitization and internalization. The A2B AR, endogenously expressed in NG108-15 cells, is desensitized through phosphorylation by GRK2 [84]. On the contrary, second messenger-dependent kinases (PKA and PKC) are not involved in agonist-mediated A2B AR phosphorylation [86]. On the contrary, PKC has been demonstrated to be involved in the heterologous regulation of A2B AR functioning induced by TNF as demonstrated by Trincavelli et al. [87]. Mundell et al. [84] have found that within the arrestin family, both arrestin 2 and arrestin 3 are involved in the internalization process of the A2B AR and in its recycling to plasma membranes. Finally, the molecular mechanisms of A3 AR regulation has been investigated in detail. Palmer and Stiles [88] as well as Ferguson et al. [89] have investigated which amino acid residues in the C terminus are responsible and crucial for the rapid desensitization of the A3 AR. A triple mutant (Thr307, Thr318 and Thr319 to Ala) has exhibited dramatically reduced phosphorylation, desensitization and internalization of the rat A3 AR. Individual mutation of each Thr residue showed that Thr318 and Thr319 are the most important sites for phosphorylation. Mutating two predicted palmitoylation sites, Cys302,305 to Ala is resulted in agonist independent basal phosphorylation of the rat A3 AR. Such findings strongly suggest that palmitoylation of these Cys residues is an important factor in controlling accessibility of the C terminus of the A3 AR in the process of recruiting GRKs. In fact, the palmitoylation sites are highly conserved between different species, e.g. rat, mouse, human, dog, sheep. Taking these results together, it appears that GRK-mediated phosphorylation of the A3 AR C-terminus follows a sequential mechanism, with the receptor palmitoyl moieties in an important regulatory role, and phosphorylation of Thr318 being particularly crucial as an essential first step. Trincavelli et al. [73] have studied the involvement of ERK1/2 in A3 AR phosphorylation. It has been found that within 5 min of exposure to 10 μM 5'-N-Ethylcarboxa-midoadenosine (NECA), ERK 1/2 were already phos-phorylated in CHO cells stably expressing human A3 AR. An inhibitor of MAPK activation, 2-(2-Amino-3-methoxy-phenyl)-4H-1-benzopyran-4-one2-(2-Amino-3-methoxy-phenyl)-4H-1-benzopyran-4-one (PD98059), has also caused inhibition of A3 AR phosphorylation, desensitization and internalization, probably by preventing the membrane trans-location of GRK2. These results indicate that the MAPK cascade is involved in A3 AR regulation by a feedback mechanism which controls GRK2 activity and probably involves direct receptor phosphorylation [73]. Adenosine Receptor Interaction Adenosine is not released in a transmitter- or hormonelike fashion, but instead it appears to be formed by groups of cells as part of a response to e.g. challenges in energy metabolism. It can also be formed by breakdown of ATP released by cells either in a regulated fashion or in response to mas-

Adenosine Receptors

sive trauma. Adenosine is therefore likely to act in concert with several other messengers (transmitters, hormones, growth factors, autacoids). In this way, the importance to delineate all the interactions that occurred between adenosine receptors and other systems appeared clear. A1 AR Interactions A1 AR is a prototypic GPCRs able to form heteromeric associations with other receptors including D1 receptors, metabotropic purinergic P2Y1 receptors [90], and metabotropic glutamate receptors [91]. Due to the dopamine-adenosine antagonism in the central nervous system, it has been suspected that adenosine and dopamine receptors could form heteromers in the surface of the neuron [for review see 92]. Using different cell lines, coexpressed with the two receptors, it has been demonstrated that A1 AR and D1 dopamine receptors co-immunoprecipitate supporting the existence of a structural interaction between these receptor proteins [93, 94]. It seems that, for many receptors including D1 dopamine receptor and A1 AR, oligomer formation occurs in the endoplasmic reticulum and that once formed they cannot be disassembled [95]. In the case of A1 AR and D1 heteromers, simultaneous activation allows the antagonistic intra-membrane receptor–receptor interaction to take place, namely the G protein uncoupling with the disappearance of the D1 high affinity state. One functional meaning of this intramembrane receptor–receptor interaction is therefore uncoupling of D1 from Gs protein. The A1 AR -D1 heteromeric complex may therefore give the molecular basis for the well documented antagonistic receptor-receptor interactions found in the neuronal networks of the brain [96-98]. The A1 AR -D1 heteromerization also appears to have an impact on receptor trafficking [93]. Recently it has been also demonstrated that A1 ARs colocalize and functionally interact with the ATP receptor P2Y1 in hippocampus and glial cells and it has been suggested that this interaction can play an important role in cell-tocell communication and in control of synaptic transmission, particularly during pathological conditions, when large amounts of purines are released [99, 100]. A2A AR Interactions A2A Ars are highly enriched in the basal ganglia of several species including man. There they are mainly expressed on a subset of the GABAergic output neurons, namely those that project to the globus pallidus [101]. These neurons also express dopamine D2 receptors and it is abundantly clear that the two receptors interact in binding assays, on signal transduction, and behaviourally [for review see 102, 103]. It appears that one major role of the dopamine in striatum is to suppress signalling via A2A ARs. Thus, acute disruption of dopamine release or administration of a dopamine D2 receptor antagonist leads to a marked increase in activity in the neurons that express A2A ARs and this activation can be abolished by blocking A2A ARs [104]. The structural and functional interaction between A2A ARs and D2 receptors has been also demonstrated in platelets of patients affected by dipolar disorders under treatment with antipsychotics [105], suggesting the possible use of A2A AR ligands in the treatment of pathologies associated to dopamine system dysfunction such as schizophrenia.


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Recent evidence also demonstrates that A2A AR controls glutamatergic transmission at post and pre synaptic level. A2A ARs and glutamate mGlu5 receptors form heteromeric complexes in transfected cells and in the rat striatum. mGlu5 receptor stimulation potentiates the effects of A2A ARs both at the intramembrane level by increasing its ability to inhibit dopamine D2 receptor binding [106, 107] and at the MAPK level [108, 109]. In fact, central co-administration (in the lateral ventricle) of a selective A2A AR agonist and a selective mGlu5 agonist induces an increase in the striatal expression of c-fos, while no significant effect is obtained when each is administered alone [108]. Furthermore, the synergistic interaction between A2A ARs and mGlu5 receptors is in notable agreement with the recently reported synergistic effects of A2A AR and mGlu5 receptor antagonists in animal models of Parkinson’s disease [110, 111]. Changes in the efficiency of glutamatergic synapses (i.e.plasticity) do not only depend on post-synaptic changes but also on pre-synaptic changes. In the striatum, A2A ARs are located in glutamatergic nerve terminals and their activation enhances the evoked release of glutamate [112-114]. Furthermore, dopamine D2 receptors also control glutamate release in the striatum [115] and preliminary experiments also indicate a tight inter-dependence between presynaptic A2A ARs and D2 receptors. Likewise, other receptor subtypes located pre-synaptically in glutamatergic terminals, such as cannabinoid CB1 receptors [116], may control striatal function by modulating neurotransmitter release and may do so by interacting with adenosine A2A ARs [117]. A2B AR interactions A2B AR are present on many cells although in low abundance. It has been repeatedly found that signalling via the A2B AR is strongly influenced by concurrent signalling via other receptors that affect PLC-Ca2+-PKC. However, the type of interaction varies from cell to cell. In brain slices the cAMP accumulation mediated by activation of A2B AR is markedly enhanced by drugs that stimulate PKC [118, 119]. Since cAMP accumulation in brain slices predominantly reflects events in glial cells, it seems likely that this is the target cell for the interaction. In glial cells it has been demonstrated that pro-inflammatory cytokine TNF through the activation of PKC pathway control A2B AR functional responses causing a drop in receptor responsiveness [87, 120]. In parallel, in different cell system it has been demonstrated that A2B AR control the cytokine release participating to the regulation of inflammatory responses [121]. Numerous other examples of interaction between ARs and other receptors have been reported and such interactions are likely to be the norm rather than the exception. This has important consequences: for example, if two receptors act synergistically then blockade of either receptor will virtually abolish the response. This can be easily interpreted as evidence that the receptor that is blocked is solely responsible for the response studied. Another important case should also be mentioned. Sometimes more than one adenosine receptor is expressed on a single cell. The resultant response will therefore be a mixed one and consequently pharmacology may be quite atypical.


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Pathophysiological Role

Cognition and Memory

Neurological Diseases

Endogenous adenosine, through A1 ARs activation, modulates long-term synaptic plasticity phenomena, such as long-term potentiation (LTP) long-term depression (LTD), and depotentiation [132]. In accordance with the notion that synaptic plasticity is the basis for learning and memory in different brain areas, adenosine correspondingly modulates behaviour in various learning and memory paradigms [133], and A1 AR antagonists have been proposed for the treatment of memory disorders. Cognitive effects of caffeine are mostly due to its ability to antagonise adenosine A1 ARs in the hippocampus and cortex, the brain areas mostly involved in cognition, but, as discussed in detail [134], positive actions of caffeine on information processing and performance might also be attributed to improvement of behavioural routines, arousal enhancement and sensorimotor gating.

Given the complex nature and ubiquitous distribution of the adenosine system, any imbalance is expected to lead to neurological diseases. The following section describe the role of the different adenosine receptors in neurological diseases. Epilepsy One of the first pathophysiological roles proposed for adenosine has been as an endogenous anti-convulsivant. In fact adenosine system is critically involved in regulating proliferation and hypertophy of astrocytes leading to astrogliosis, a hallmark of epilepsy. The anti-epileptic properties of adenosine are mostly due to the well-known inhibitory actions of pre-synaptic A1 ARs upon glutamate release associated with a post-synaptic effect [122]. Pain Adenosine has complex effects on pain and can either be pro- or antinociceptive depending on the site of application or the receptors involved. Most attention has been devoted to pain pathways involving the A1 AR, which dominates in mediating the antinociceptive effects of adenosine, as has been documented in animal models of neuropathic and inflammatory pain [123]. More recently, antagonism of A2A ARs has been suggested as a novel strategy for the management of chronic pain [124]. Thus, the A2A selective antagonist SCH 58261 produ-ced antinociception in two acute thermal pain tests [125]. These findings suggest that the A2A AR has significant potential as a novel target for pain control. Regulation of Sleep Adenosine functions as a natural sleep-promoting agent accumulating during periods of sustained wakefulness and decreasing during sleep [126]. It has been suggested that adenosine participates in resetting of the circadian clock by manipulations of behavioural state [127]. Indeed, adenosine A1 ARs of the suprachiasmatic nucleus regulate the response of the circadian clock to light [128]. The sleep inducing properties of adenosine is in line with its A1 receptor-mediated inhibitory action and may involve multiple neuronal populations in the CNS; however, the actions upon the basal forebrain nuclei involved in sleep and arousal appear to be particularly important [129]. The influence of adenosine upon sleep state might be already evident before birth: in fact, has been shown that adenosine inhibits REM-like behaviour in fetal sheep [130]. In healthy humans, caffeine inhibits psychomotor vigilance deficits from sleep inertia, a ubiquitous phenomenon of cognitive performance impairment, grogginess and tendency to return to sleep immediately after abrupt awakening from intermittent and short sleep periods [131]. It thus emerges that there exists a potential role of adenosine-related compounds and of A1 AR agonists as sleep promoters and adenosine receptor antagonists as arousal stimulators.

Brain Ischemia In the CNS A1 AR agonists, when acutely administered, are neuroprotective in and A1 AR antagonists consistently worsen neuronal death in ischemic models [135]. In spite of a neuroprotective role for adenosine being commonly accepted, recent studies have revealed that adenosine could unexpectedly, under certain circumstances, have the opposite effects contributing to neuronal damage and death. Evidence for a neuroprotective role of A1 AR activation is supplied by both in vivo and in vitro studies. By evaluating the action of hypoxia on synaptic transmission in hippocampal slices, it has been shown that substances that are also released during hypoxia, such as GABA, acetylcholine, and even glutamate through metabotropic receptors, may also have a neuroprotective role; however their action is evident only when activation of adenosine A1 ARs is impaired. So, A1 ARs appear to have a pivotal role during hypoxia, though other substances can operate in a redundant or even overprotective manner, acting as a substitute for some adenosine actions when the nucleoside is not operative [136]. The hippocampus is a brain area highly sensitive to hypoxia/ischaemia and the lesions at these levels are responsible for cognitive impairment after ischaemic episodes, such as stroke. A recent study combining electrophysiological recordings in hippocampal slices and behavioural studies, has shown that besides protecting synaptic function, adenosine, via A1 ARs, enhances spatial learning and memory deficits occurring after transient hypoxia in rats [137]. The impact of A2A ARs in the control of neuronal damage has been first proposed by John Phillis in a model of cerebral ischemic injury [125]. It has been later confirmed that either the pharmacological blockade or the genetic elimination of A2A ARs conferred a robust neuroprotection in animal models of brain ischemia [138, 139]. This has been later extended to a variety of situations that had in common the deleterious impact of chronic noxious insults to adult brain tissue, such as glutamate excitotoxicity [140, 141], MPTP [142-144], 6-hydroxydopamine [142, 143], 3-nitropropionic acid toxicity [145], or -amyloid [146, 147] toxicity.

Adenosine Receptors

Several data propose that there might be a successive participation of A2A ARs located in different cells types according to the duration and/or intensity of noxious brain insult: with mild noxious insults, there might be a main role of synaptic A2A ARs; with more prolonged noxious stimuli, microglia A2A ARs would play a predominant role, in view of the importance of microglia in the amplification of early brain damage [148-150]; finally, with more severe damage, causing loss of preservation of the blood-brain barrier, it might be that A2A ARs in inflammatory cells invading the brain parenchyma play the more pronounced role. Clearly, this is a hypothetic scenario that still needs experimental confirmation. Considerable interest has been shown in understanding the involvement of A3 AR in normal and pathological conditions of the CNS despite its low expression in the brain [151]. Even though the function of A3 AR in the CNS has been controversial in terms of protective versus toxic actions, actually several data point towards a neuroprotective effect. In different cell systems and in "in vivo" models of ischemia has been demonstrated that A3 AR activation mediate a dual role, neuroprotective and neurodegenerative, in dependence on the time of receptor stimulation (acute vs chronic) and on the levels of receptor activation (agonist concentrations). Firstly, a dual role of A3 AR was described in a model of global ischemia in gerbils, where acute preischemic administration of the agonist N6-(3-iodobenzyl)adenosine-5-N-methylcarboxamide (IB-MECA) caused a severe depression of cerebral blood perfusion, worsening of neuronal damage and post-ischemic mortality, while its chronic administration has induced a significant improvement of post-ischemic cerebral flow and neuron protection. In line with the results obtained after acute treatment, in rat cerebellar granule neurons high concentrations of Cl-IBMECA have been able to induce lactate dehydrogenase release, neuronal cell death and augmented glutamate-induced neurotoxicity through a pathway involving inhibition of cyclic AMP production [152]. It was then observed that the effect of IB-MECA depended on the timing of treatment, as administration of IB-MECA 20 min prior to transient middle cerebral ischemia increased the infarct size, whereas its addition 20 min after ischemia resulted in a significant decrease of damage, leading to define the cerebroprotective effect of A3 AR a “right thing at a wrong time” [153]. It has been speculated that the deleterious effects caused by acute preischemic treatment with IB-MECA have been the consequence of a series of adverse events triggered immediately prior to the occlusion such as release of inflammatory mediators, breakdown of the blood–brain barrier integrity and Ca2+ influx. In contrast, the neuroprotective effects, obtained when the A3 AR agonist treatment have been performed following a focal insult, were related to astrocyte activation or to a direct neuroprotective action [153]. In human astrocytoma cells (ADF cells), exposure to nanomolar 1-[2-Chloro-6-[[(3-iodophenyl)methyl]amino]9H-purin-9- yl]-1-deoxy-N-methyl-b-D-ribofuranuronamide (Cl-IB-MECA) concentrations have increased resistance to apoptosis by a mechanism involving signalling to the small G proteins Rho, cytoskeletal rearrangement, and intracellular


867

redistribution of the anti-apoptotic protein Bcl-XL [154, 155]. In contrast, Cl-IB-MECA have induced cell death in ADF cells when used at concentrations in the high micromolar range. In the same way, micromolar concentrations of Cl-IB-MECA markedly have impaired cell cycle progression in CHO cells transfected with the human A3 AR [156]. Such apparently opposing effects have been reconciled by hypothesizing that adenosine-induced cell death that occurs during severe metabolic stress by A3 AR activation might isolate the most damaged areas to favour those parts of the brain that still retained a chance for functional recovery, supporting the role of adenosine as a “retaliatory metabolite”. Later it has been speculated that desensitization/ down-regulation of the A3 AR may be the basis of cytoprotection, suggesting a role for this receptor in induction of cell death [157]. Different evidences suggest that a part of neuroprotection induced by A3 AR derives from its modulation of the brain immune system [158]. It has been reported that functional A3 AR are expressed in mouse microglia cells, where their activation is responsible for a biphasic effect on ERK1/2 phosphorylation [159] and in murine astrocytes where A3 AR stimulation induces the release of the neuroprotective chemokine CCL2 [160]. Moreover, in microglial cells A 3 AR activation suppresses TNF production by inhibiting PI3K/Akt and nuclear factor-B (NF-B) activation, suggesting that selective ligands of this receptor may be of therapeutic potential for the modulation and possible treatment of brain inflammation [161]. Even though for some aspects the role of A3 AR in the CNS seems less confusing now than in the past, there are many aspects yet that need clarification before a role of A3 AR agonists in therapy can be envisioned. Parkinson's Disease A number of different mechanisms have been implicated in the antiparkinsonian effects induced by A2A AR antagonists. Adenosine, via A2A ARs excites striatopallidal neurons, opposing to the inhibitory effect exerted by dopamine. In Parkinson's disease (PD) the lack of dopamine generates an imbalance in the activity of striatal output pathways. Striatonigral neurons become hypoactive, whereas striatopallidal neurons, losing the inhibitory effect of dopamine while undergoing the stimulatory influence of adenosine, become hyperactive. Such imbalanced activity leads to the hypokinetic symptoms in PD. Adenosine A2A AR antagonist efficacy in PD relies on the blockade of A2A ARs on striatopallidal neurons, which should dampen their excessive activity [162] and restore some balance between striatonigral and striatopallidal neurons, consequently relieving thalamocortical activity. This mechanism offers a rationale for the use of A2A AR antagonists as monotherapy in PD, as well as for the synergistic effect observed upon the concurrent administration of A2A AR antagonists with levodopa or dopaminergic agonists [163]. Adenosine A2A AR activation can produce direct inhibition of D2 receptors and may also reduce D2 coupling to Gi proteins, suggesting that molecular interactions between A2A ARs and D2 receptors may be important for the antiparkinsonian actions of A2A AR antagonists [164]. Conversely, blockade of dopaminergic input provides an inhibition of tonic adenosine A2A AR activation,


which could explain why A2A AR antagonists improve parkinsonian motor deficits in the absence of the D2 receptor [164]. A2A ARs/D2 receptor interaction includes antagonistic interaction at the transcriptional level, as shown by synergistic c-fos activation in the indirect striatopallidal pathway by levodopa or D2 receptor agonists and A2A AR antagonists [165]. In addition, studies at the molecular level have demonstrated that the A2A AR can form heteromers with other receptors including dopamine D2 receptor [102, 103] and glutamate mGlu5 glutamate receptors [166]; however, the relevance of this to the mechanism of antiparkinsonian action is unclear and much debated. Psychiatric Diseases Anxiety A1 AR agonists have anxiolytic activity in rodent models of anxiety, whereas caffeine and the adenosine A1 AR selective antagonist, cyclopentyltheophylline, have anxiogenic properties. Interestingly, patients suffering from panic disorder, a serious form of anxiety disorder, appear to be particularly sensitive to small amounts of caffeine [167]. These observations, taken together, suggest that drugs that facilitate adenosine A1 receptor-mediated actions may be effective for the treatment of anxiety. Mood Disorders The interest in the role of adenosine in mood disorders stems from three concurrent lines of research: first, there is evidence that the consumption of coffee, and particularly of caffeine (an adenosine receptor antagonists) might modify the mood profile both of volunteers as well as of psychiatric patients; secondly, there is evidence that different therapeutic strategies used to control mood disorders cause effects related to the adenosine modulation system; thirdly, there is evidence, from animal models, that the manipulation of adenosine receptors modifies behavioral responses considered relevant for mood function in humans. The association of the adenosine modulation system with depression has been initially developed based on observations showing that adenosine and its analogues caused depressant like behavioural effects in two widely used animal models of depression. Thus, elevating the adenosine levels increased the time of immobilization in rats submitted to inescapable shocks, as well as in the forced swimming test [168]. In a series of careful studies, several authors have found that A2A AR antagonists prolong escape directed behavior in two screening tests for antidepressants, the tail suspension and forced swim tests [169]. Further support for a potential role of A2A AR antagonists as novel anti-depressants has been provided by the observation that A2A AR antagonists also displayed an attenuated ‘behavioral despair’ in these two screening tests [170]. Although A1 ARs have been indicated as a candidate system in the aetiology of schizophrenia, there is also compelling observations that suggest a possible role for A2A ARs. Thus, it has been observed that the startle (a measure of sensorimotor function) habituation was reduced by A2A AR antagonists [171] as well as in A2A AR knockout mice [172]. Furthermore, A2A ARs can also act as ‘go-between’ normalizing (or rebalancing) an impaired glutamatergic-

Trincavelli et al.

dopaminergic communication that seems to be crucial importance for proper function of the ventral striatum and prefrontal cortex. Drug Addiction Drugs of abuse have several mechanisms of action, that create a complex pattern of behavior related to drug consumption, drug-seeking, withdrawal and relapse. The neuromodulator adenosine has been shown to play a role in reward-related behavior, both as an independent mediator and via interactions of adenosine receptors with other receptors. Adenosine levels are elevated upon exposure to drugs of abuse, and adenosine A2A ARs are enriched in brain nuclei known for their involvement in the processing of drugrelated reinforcement processing. A2A ARs are found in receptor clusters with dopamine and glutamate receptors: thus, they are thus ideally situated to influence the nti-infl of neurotransmitters relevant in the neuronal responses and plasticity that underlie the development of drug taking and drug-seeking behavior [173]. Several evidence support a role for A2A ARs in drug addiction (i.e. ethanol consumption), thereby suggesting this receptor as a possible target in abuse substance pathologies. While earlier work suggested an A1 AR-receptor-mediated role for adenosine in the ethanolinduced loss of righting reflex, more recent work has demonstrated that A2A ARs are also involved in the hypnotic effects of ethanol [170]. Data from several research groups have suggested the mechanism of A2A AR involvement could be via an interaction with dopaminergic, especially D2 receptor, processing. Interactions with other receptors such as D1, A1 and mGlu5 have also been postulated; however, the delineation of A2A AR effects mediated via receptor interactions and those occurring via independent mechanisms remains to be ascertained. Cardiovascular Diseases Recent data have demonstrated that ischemic preconditioning and adenosine preconditioning prevent the vascular and biochemical alterations studied in response to ischemiareperfusion injury in the pulmonary vascular bed of the rat. Several studies suggest activation of A1 ARs mediates the protective properties of ischemic preconditioning and adenosine preconditioning on ischemia-reperfusion injury in the lung. Moreover, data further suggest selective adenosine receptor agonists may be useful as pharmacologic preconditioning agents in preventing ischemia-reperfusion injury in lung transplantation and other forms of pulmonary vascular ischemia [174]. By generating A2B AR KO mice, Yang and coworkers [175] showed that A2B ARs are important in moderating vascular injury to various insults, such as systemic endotoxin challenge and guidewire-induced endothelial denudation of the femoral artery, a model of human restenosis after angioplasty. Extracellular adenosine has long been implicated in the adaptation to hypoxia [176, 177] and recent studies have provided evidence that A2B ARs have a key role in dampening hypoxia-induced vascular leak in vivo. It has been demonstrated that A2B AR KO mice exposed to ambient hypoxia had

Adenosine Receptors


869

increased vascular permeability in the majority of organs tested, and studies with bone marrow chimeric mice indicated that it was the lack of parenchymal (vascular) and not hematopoietic A2B ARs that caused the increased vascular leak [178].

innate immunity [189, 190]. Increase evidence show that A2A ARs have a role in the physiologic immunosuppressive pathway that protects normal tissues from collateral damage by overactive immune cells and their proinflammatory cytokines [191].

In addition, it is widely accepted that adenosine has a key role in mediating preconditioning, and recent results incriminate A2B ARs as important components of the preconditioning response. Ischemic preconditioning fails to protect the heart [179] and kidney [180] A2B AR KO mice, and this lack of preconditioning is associated with increased NF-kB activation and inflammation [179-181].

In several different in vivo models of inflammation selective A2A AR agonists decreased the inflammatory response: decreased pleocytosis and reduced blood brain barrier permeability in a rat model of endotoxin-stimulated meningitis [192], decreased transmigration of leukocytes in endotoxin-stimulated rabbit knee joint septic arthritis [193], and reduced tissues injury and inflammation in mice with toxin A-induced enteritis (infection immunity) [194]. Additionally, in non-infectious animal models, A2A AR activation also induced reperfusion injury, in part by decreasing the inflammatory response [195]. The use of knockout (KO) A2A AR mice has confirmed the importance of A2A ARs in limiting pro-inflammatory responses [189, 196].

To date several pieces of evidence support the conclusion that activation of A3 AR is crucial for cardioprotection during and following ischemia–reperfusion, and it has been suggested that a consistent part of the cardioprotective effects exerted by adenosine, once largely attributed to the A1 receptor, may now be in part ascribed to A3 AR activation [182]. Even though there is a low expression of A3 AR in myocardial tissue, a number of studies have demonstrated that acute treatment with agonists induced protective “antiischemic” effects [183, 184]. The molecular mechanism of A3 AR cardioprotection has been attributed to regulation of mitochondrial (mito) KATP channels [185]. Moreover, it has been reported that A3 AR activation is able to mimic or induce myocardial preconditioning, meaning that transient stimulation of the A3 AR before induction of ischemia leads to both an early and a delayed protection [185]. Pharmacological preconditioning (early and late) obtained after A 3 AR activation is clinically important, but cardioprotection is even more relevant when it occurs at reperfusion [184]. This situation, called post-conditioning, has been demonstrated for IB-MECA through inhibition of the mitochondrial permeability transition pore (mPTP) opening via PI3K/Akt inactivation of glycogen synthase kinase (GSK-3) [186]. These results supported the idea that therapeutic strategies focusing on A3 AR subtype may represent a novel and useful approach for protection of the ischemic myocardium. The A3 AR-mediated cardioprotection remains a mystery if one thinks of its cellular location. Literature data report that myocardial A3 AR expression in the mouse is very low and below the detection limits of radioligand binding or northern blot techniques [187]. It is also surprising that mice overexpressing the A3 AR reveal only 12 fmol/mg of protein and that animals with 66 fmol/mg of protein present negative effects like dilated cardiomyopathy, meaning that the level of A3 AR is very critical for heart function. Therefore, on the one hand it is possible to hypothesize that given the strong cardioprotective effects and the low cardiac expression, this receptor must be very efficiently coupled to protective intracellular signalling pathways. On the another hand, it is also conceivable that cardioprotection might derive at least in part from activation of A3 AR expressed in other cells [188]. Inflammatory Diseases A2A, A2B and A3 ARs have been demonstrated to play an important role in the control of inflammatory responses. A2A ARs are widely expressed in cells such as monocytes, lymphocytes, and mast cells, which are involved in the

Thus targeting A2A ARs to ‘manipulate’ the inflammatory process may have important therapeutic implications. The treatment of a wide range of inflammatory diseases, such as rheumatoid arthritis [197], sepsis [198] and wound healing may benefit from targeted triggering the A2A ARs. Other studies are needed on A2A AR agonists as recruiters of naturals pathways which down-regulate inflammation in the treatment of acute and possibly chronic inflammatory processes [199]. The use of A2A AR antagonists can by opposition be used to achieve targeted tissue destruction or local immune response enhancement (e.g. increase of antitumour immune response or development of better vaccines). Recently, A2A ARs have also been described to protect tumours from antitumour T cells introducing adenosinebased drug therapy, namely by targeting hypoxia – extracellular adenosine - A2A/A2B ARs signaling pathway, as a cancer immunotherapy to prevent the inhibition of antitumor T cells in tumor microenvironment [200]. There is an increasing body of evidence documenting the proinflammatory effects of A2B AR activation in both rodent and human asthma and chronic obstructive pulmonary disease (COPD), which indicates that A2B AR antagonists might be of therapeutic value in preventing disease progression in asthma and COPD. This is borne out by recent in vivo evidence [201] showing that selective antagonism of adenosine A2B ARs leads to inhibition of airway inflammation and airway reactivity induced by allergen or AMP in a murine asthma model. Although mast cells mediate most of the proinflammatory effects of adenosine in the lung [202], proinflammatory effects of A2B AR stimulation have also been observed with human bronchial smooth-muscle cells [203], human bronchial epithelial cells [204], and human lung fibroblasts [205], which produce increased levels of IL6 and IL-19 in response to A2B AR activation. Recent findings indicate that A2B AR signalling is an important contributor to lung injury in environment where endogenous adenosine levels are increased. Collectively, the results of preclinical models together with evidence that enprofylline and theophylline are A2B AR antagonists, indicate that selective antagonism of A2B ARs is an appealing


therapeutic approach for managing patients with asthma and COPD. Inflammatory bowel disease, Crohn’s disease and ulcerative colitis are characterized by a dysregulation of the inflammatory functions of intestinal epithelial cells, in addition to poorly controlled activation of both the innate and adaptive immune systems leading to damage of the intestinal mucosa. Although the etiology of inflammatory bowel disease (IBD) remains to be defined, recent studies indicate that the disease results from a dysregulated immune response to luminal antigens and gut microflora in a genetically susceptible host [206]. Consistent with the proinflammatory role of A2B AR activation in driving intestinal epithelial cell inflammatory responses, A2B AR blockade [207], or KO [208] suppresses intestinal inflammation and attenuates the course of disease in murine colitis. The protection caused by A2B AR inactivation is correlated with decreased production of IL-6 and keratinocyte-derived chemokine, in addition to limited neutrophil accumulation in gut tissue [207]. Because A2B ARs are upregulated in gut tissue during both human and murine colitis [209], they are components of a vicious circle that propagates inflammation in the gut. The interest in the elucidation of A3 AR involvement in inflammation is attested by the large amount of experimental work carried out in cells of the immune system and in a variety of inflammatory conditions. However, as in the SNC or in the cardiovascular system the A3 AR subtype appears to have a complex or “enigmatic” role, as both proinflammatory and anti-inflammatory effects have been demonstrated. One of the first evidence for a role of A3 AR in increasing inflammation derived by studies in mast cells where it was found that its activation was responsible for mast cell degranulation and the release of allergic mediators [210]. In addiction an high expression of A3 AR has been demonstrated in other cells involved in allergic diseases, such as eosinophils, neutrophils and macrophages. In addition to a role of A3 AR in increasing inflammation, evidence that A3 AR decrease inflammation have also been reported in literature. As an example, it has been shown that A3 AR suppress TNF- release induced by endotoxin CD14 receptor signal transduction pathway from human monocytes and murine J774.1 macrophages [211, 212]. Moreover, in a macrophage model the A3 AR has been the prominent subtype implicated in the inhibition of LPS-induced TNF- production [213]. However in spite of these contrasting results, one of the best potential therapeutic applications of the regulatory role of A3 AR activation on TNF- release has been found in the treatment of arthritis. A3 AR agonists exert significant antirheumatic effects in different autoimmune arthritis models, by suppression of TNF- production [214]. The molecular mechanism involved in the inhibitory effect of IB-MECA on adjuvant-induced arthritis have included receptor down-regulation and deregulation of the PI3K–NFB signalling pathway [215]. Previous studies have also demonstrated that A3 AR activation inhibited macrophage inflammatory protein (MIP)-1, that is a C-C chemokine with potent inflammatory effects, in a model of collageninduced arthritis, providing the first proof of concept of the adenosine agonists utility in the treatment of arthritis [216].

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Cancer Inflammation in the tumour microenvironment aids in the proliferation and survival of malignant cells, promotes angiogenesis and metastasis, and subverts adaptive immune responses. Concentrations of extracellular adenosine are increased in tumour microenvironments because both inflammation and hypoxia, conditions which are present in most tumours, can increase the accumulation of adenosine [217-220]. Recent studies have revealed that A2B ARs can propagate the growth of Lewis lung carcinoma in mice by reprogramming host dendritic cells to produce copious amounts of VEGF, thereby supporting angiogenesis in the tumours [221]. It is, however, conceivable that A2B ARs directly affect the growth of other types of tumours because several tumours have been shown to over-express A2B ARs [222, 223]. Further studies will be required to determine whether A2B ARs have tumour growth-regulating roles in other models of cancer development. A very interesting area of potential application of A3 AR ligands concerns cancer therapies. The possibility that A3 AR plays a role in the development of cancer has aroused considerable interest in recent years [224]. The A3 AR subtype has been described in the regulation of the cell cycle and both pro- and anti-apoptotic effects have been reported depending on the level of receptor activation [223, 225, 226]. In normoxic conditions, it has been demonstrated that A3 AR inhibited tumour growth by regulation of the WNT signalling pathway [227], which is active during embryogenesis and tumourigenesis, and mediates cell cycle progression and cell proliferation. A key modulator of this pathway is represented by GSK-3, that is crucial for the phosphorylation of -catenin which, in turn, induces the transcription of genes fundamental for cell cycle progression. Upon exposure of tumour cells to the A3 AR agonist the GSK-3, protein level increased, resulting in the destabilization of -catenin and the subsequent block of cell cycle progression. From another point of view, hypoxia that is typical of solid tumours creates conditions that, on one hand, are conducive to the accumulation of extracellular adenosine and, on the other hand, stabilize hypoxia-inducible factors, such as HIF-1 [228-230]. HIF-1, the most important factor involved in the cellular response to hypoxia, is up-regulated across a broad range of cancer types and is involved in key aspects of tumour biology, such as angiogenesis, invasion and altered energy metabolism. In hypoxic conditions adenosine present at high levels, activates A3 AR and triggers an increase of HIF1- levels, by activating MAPKs pathway. This leads to an increase of vascular endothelial growth factor (VEGF) levels and angiogenesis. These results suggest A3 AR has a tumourigenic effect in specific solid tumours and support the possible use of receptor antagonist as antitumoural agents. CONCLUSIONS The pleiotropic effects of adenosine offer unique therapeutic opportunities and challenges. Since the heterogeneity of adenosine receptors and their widespread expression throughout the body, the development of drugs that

Adenosine Receptors

Table 2.


871

Pathophysiological Implication of the Different AR Subtypes A1

A2A

A2B

A3

Neurological Diseases Epilepsy

Xa

Pain

Xb

Regulation of sleep

Xe

Cognition and memory

Xf

Brain ischemia

Xg

Xc,d

Xh,i,j

Xk,l

Xm,n

Parkinson’s disease Psychiatric Diseases Anxiety

Xo

Mood disorders

Xp

Drug addiction

Xp

Cardiovascular Diseases

Xq Xt

Inflammatory Diseases Cancer a

Xr

Xs

Xu

Xv

Xw

Xx

[122]; b[123]; c[124]; d[125]; e[129]; f[132]; g[135]; h[140]; i[138]; j[139]; k[152]; l[153]; m[163]; n[164]; o[167]; p[170]; q[173]; r[176]; s[182]; t[191]; u[207]; v[210]; w[221]; x[224]

selectively affect specific adenosine receptor subtypes and/or specific components of adenosine signalling will hopefully provide new therapeutic opportunities for a large variety of human diseases. The knowledge of adenosine receptor structure, as well as the complexity of regulatory mechanisms controlling the receptor biological responses under physiological and pathological conditions represent a crucial starting point in the development of adenosine-based drugs. Although many studies performed over the years have helped to understand the structure of adenosine receptors and have contributed to elucidate the molecular mechanisms involved in adenosine-mediated responses, much remains to understand. In particular, at the present, the understanding of the molecular mechanisms underlying protein protein-interactions, their dimerization and functional interactions, is the most important current goal for the identification of new pharmacological targets which involves the adenosine system.

EGF

=

Epidermal growth factor

PKA

=

Protein kinase A

PKC

=

Protein kinase C

CREB

=

cAMP responsive element binding protein

MEK

=

Mitogen-activated protein kinase kinase

GSK-3

=

Glycogen synthase kinase 3

GRK

=

G protein-coupled receptor kinase

TNF

=

Tumour necrosis factor alpha

R-PIA

=

R-phenylisopropyl adenosine

NECA

=

5'-N-Ethylcarboxamidoadenosine

PD98059

=

2-(2-Amino-3-methoxyphenyl)-4H-1benzopyran-4-one

CNS

=

Central nervous system

ABBREVIATIONS

COPD

=

Chronic obstructive pulmonary disease

AR

=

Adenosine receptor

IBDs

=

Inflammatory bowel diseases

GPCR

=

G protein-coupled receptor

KO

=

Knock-out

AC

=

Adenylyl cyclase

VEGF

=

Vascular endothelial growth factor

PLC

=

phospholipase C

IBMECA

=

GEFs

=

Guanine-nucleotide exchange factors

N6-(3-iodobenzyl)-adenosine-5-Nmethylcarboxamide

MAPK

=

Mitogen activated protein kinases

Cl-IBMECA

=

ERK

=

Extracellular regulated kinases

1-[2-Chloro-6-[[(3-iodophenyl) methyl]amino]-9H-purin-9- yl]-1-deoxyN-methyl-b-D-ribofuranuronamide

SAPK

=

Stress-activated protein kinases

HIF-1

=

Hypoxia-inducible factors

PI3K

=

Phosphoinositol-3-kinase


Trincavelli et al.

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Revised: January 5, 2010

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