Focusing on Adenosine Receptors as a Potential Targeted Therapy in Human Diseases

Adenosine is involved in a range of physiological and pathological effects through membrane-bound receptors linked to G proteins. There are four subtypes of adenosine receptors, described as A1AR, A2AAR, A2BAR, and A3AR, which are the center of cAMP signal pathway-based drug development. Several types of agonists, partial agonists or antagonists, and allosteric substances have been synthesized from these receptors as new therapeutic drug candidates. Research efforts surrounding A1AR and A2AAR are perhaps the most enticing because of their concentration and affinity; however, as a consequence of distressing conditions, both A2BAR and A3AR levels might accumulate. This review focuses on the biological features of each adenosine receptor as the basis of ligand production and describes clinical studies of adenosine receptor-associated pharmaceuticals in human diseases.


Introduction
Adenosine is a nucleoside molecule that elicits various physiological responses in tissues and organs. The pioneering identification of adenosine occurred when Drury and Szent-Gyorgyi successfully extracted an adenylic acid substance from the mammalian heart and other tissues influencing cardiac rhythm [1]. Subsequently, adenosine analogs resulted in coronary dilatation and blood flow elevation in some studies [2]. Interestingly, a small amount of caffeine diminished the effect of adenosine on the contraction of atrial muscle [3]. In fact, Sattin and Rall proposed that adenosine required a particular molecule in the cell membrane to exert its effects [4]. All these studies considered the role of adenosine-related specific receptors.
The classical autonomic neurotransmitters released from peripheral nerves were once recognized as only noradrenaline (NA) and acetylcholine (Ach). The concept of noncholinergic and nonadrenergic transmissions was introduced after 5'-adenosine triphosphate (ATP) was recognized as a purinergic neurotransmitter [5]. Next, Burnstock designed two main types of purinergic receptors, i.e., P 1 and P 2 , which are based on agonistic and antagonistic functions [6,7]. The affinity for adenosine of P 1 was stronger than that of P 2 [8,9]; therefore, receptors for adenosine were classified as P 1 , while ATP and 5´-adenosine diphosphate (ADP) were more suitable as natural ligands for P2 [10]. Based on the latest nomenclature of the International Union of Pharmacology Committee on Receptor Nomenclature and Drug Classification (NC-IUPHAR), the receptor for adenosine is named adenosine receptor (AR), which can be subdivided into four types: A 1 , A 2A , A 2B , and A 3 [11]. These ARs are activated by endogenous and exogenous adenosine or its analogs [12]. Production, transport and metabolism of adenosine. Intracellular adenosine is produced via dephosphorylation from the main source, AMP, via both cN I and cN-II and hydrolysis of S-adenosyl-homocysteine through the enzyme S-adenosyl-L-homocysteine hydrolase. The extracellular formation of adenosine is the result of enzymatic cascades consisting of ATP transport, hydrolysis of ATP and ADP by CD39 to form AMP, and dephosphorylation of AMP by CD73. Extracellular adenosine binds adenosine receptors (A1AR, A2AAR, A2BAR, and A3AR) on the surface of cells. Each AR is a GPCR that transmits the signal from adenosine by activating cAMP.

A 2A Adenosine Receptor (A 2A AR)
The majority of A 2A ARs are distributed in the liver, heart, lung, immune systems (spleen, thymus, leucocytes, and blood platelets), and CNS [54,55]. They are coexpressed with D 2 dopamine receptors (D 2 Rs) in γ-aminobutyric acid (GABA) striatopallidal neurons [56] but not active in striatonigral neurons that express the protachykinin and D 1 R genes [57]. A 2A AR has been cloned from several species, including dogs, rats, humans, guinea pigs, and mice, and shows a high degree of homology among humans, mice, and rats [58]. A 2A AR is a larger protein of 410-412 amino acids in length and 45 kDa [12].
A 2A AR binding with G s to activate AC results in the activation of cAMP-dependent protein kinase A (PKA) and PKC, which phosphorylates and activates various receptors, ion channels, phosphodiesterases, and phosphoproteins, such as cAMP-response element binding protein (CREB) and dopamine-and cyclic AMP-regulated phosphoprotein 32 (DARPP-32) [59]. These proteins interact with G olf in the striatum of the basal ganglia because G olf is highly expressed in the caudate-putamen, nucleus accumbens, and olfactory tubercle [60].

A 2B Adenosine Receptor (A 2B AR)
A 2B AR has been cloned from the rat hypothalamus, human hippocampus, and mouse mast cells and is distributed in the peripheral organs, such as the bowel, bladder, lung, and vas deferens [59]. It interacts with G s to induce the PKA signaling to increase cAMP and G q11 -mediated activation of PLC to increase the levels of 1,4,5-inositol triphosphate (IP3)/diacylglycerol (DAG), activate PKC, and elevate intracellular Ca 2+ levels [63,64].
A 2B ARs are low-affinity receptors, and their affinity can be increased by interaction with PKC; therefore, the plasticity and versatility of A 2B ARs make them potential triggers of signaling in multiple signaling cascades in many physiological responses [65].
In contrast with other ARs, A 2B ARs have an antagonistic effect, which makes them interesting as therapeutic targets.

A 3 Adenosine Receptor (A 3 AR)
The A 3 AR subtype was found after Zhou et al. found several cDNA sequences, one of which (R226) was highly expressed in the testis and less distributed in the lung, kidney, heart, and some parts of the CNS [66]. This confirmed a previous study that identified a cDNA encoding a novel GPCR in the rat brain [67]. A 3 AR has been cloned from rats, rabbits, sheep, and humans [68]. The structures of A 3 ARs from these organisms show some sequence homology, with a sequence homology of only 74% in rats versus sheep and humans and 85% between sheep and humans, which indicates significant interspecies differences in ligand recognition [69]. Unlike rats, human A 3 ARs are widespread, and the most abundant expression is found in the lung and liver [70].
A 3 AR coupling to G i proteins inhibits AC and decreases cAMP accumulation and PKA activity, and A 3 ARs also bind with G q proteins to stimulate PLC, resulting in increased Ca 2+ levels and modulation of PKC activity [71]. In addition, they may activate the PLC pathway through the β, γ subunit [72].
A 3 AR agonists are classified as adenosine derivatives and xanthine-7-riboside derivatives [73]. In detail, structural manipulations of the A 3 AR agonist include N 6 -, C 2 -, and 5 -substitutions, or suitable combinations of these, followed by modifications involving nonadenine nucleosides and nonnucleosides [71].

A 1 AR in Inflammation
Activation of A 1 AR will generate both proinflammatory and anti-inflammatory responses [23]. By occupying A 1 AR, low concentrations of adenosine induce neutrophil chemotaxis and adherence to the endothelium [74] and upregulate endothelial P-selectin expression [75]. Interestingly, a high concentration of adenosine will induce antiadhesive effects via A 2 AR [76]. A 1 AR increases NK activity, induces O 2 − generation from eosinophils, and induces the release of thromboxane A2 and IL-6 from endothelial cells. In addition, it also induces chemotaxis of dendritic cells (DCs), suppresses vesicular major histocompatibility complex (MHC) class I cross-presentation, and increases endothelial permeability [23].
In contrast, A 1 AR is a potent anti-inflammatory mediator in various kidney, heart, liver, renal, lung, and brain injury models [59]. A 1 AR protected mice from septic peritonitis by attenuating the hyperacute inflammatory response [81] and prevented the worsening of murine liver ischemia-reperfusion (IR) injury by reducing necrotic and apoptotic cell death [82]. In addition, A 1 AR protected against kidney injury via AKT activation [83]. The different functions of A 1 AR during inflammation might be determined by the model of study; in addition, with deletion of A 1 AR, other ARs might provide protection [23].

A 1 AR in the Respiratory Systems
Adenosine regulates both proinflammatory responses and protection against lung injury through A 1 AR [14]. The distribution of A 1 AR in airway organs was low; however, its expression was found in smooth muscle tissues of asthmatic patients [84]. In fact, the expression of A 1 AR was upregulated in the airways of both animal models of allergic airway inflammation and human patients with asthma [85]. Specifically, A 1 AR induced bronchoconstriction and mucus production, evoking proinflammatory functions of monocytes and neutrophils and inducing vascular permeability [86], and might influence the severity of pulmonary inflammation and remodeling in chronic lung diseases [14].
Numerous studies have implicated a role for A 1 AR in respiratory diseases (Table 1). A novel A 1 AR antagonist, L-97-1, blocks allergic responses to house dust mites in an allergic rabbit model of asthma [87]. Regardless of the discontinuation of its study, EPI-2010, an antisense oligonucleotide targeting the A 1 AR promoter region, was safe and well tolerated in patients with mild asthma [88].
In contrast, A 1 AR also mediated anti-inflammatory effects via macrophages in ADA-deficient mice [89] and reduced polymorphonuclear (PMN) infiltration by inhibiting the release of chemotactic cytokines and decreasing microvascular permeability in lipopolysaccharide-induced lung injury [90]. Recently, CCPA, an A 1 AR agonist, decreased inflammation, edema, and neutrophil chemotaxis [91]. In addition, A 1 AR promoted the recruitment of leukocytes to the infected lung and attenuated lung injury [92].

A 1 AR in the Cardiovascular Systems
A 1 AR protects the heart by downgrading inotropic effects. It binds with G i to inhibit catecholamine-stimulated AC [93]; limit the action of β1 receptors stimulated by G s cycling [94] via the involvement of PLC, PKC-ε, and p38 MAPK; and exclude heat shock proteins (HSP27) [95]. In addition, A 1 AR also inhibited norepinephrine release in the rat heart [96], providing protection in the postischemic setting [97].
Similarly, A 1 AR causes coronary contraction through PLC pathways [98] and increases soluble epoxide hydrolase (sEH) and cytochrome P450 A (CYP4A) [99] to protect the heart. In addition, A 1 AR activated the release of VEGF from monocytes [78] via the involvement of the extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and PI3K/AKT pathways [100]. A 1 AR also regulates myocardial substrate metabolism by decreasing plasma concentrations of insulin, glucose, and lactate [101], and inhibiting lipolysis [102]. Via cooperative interaction with both A 2A AR and A 2B AR, A 1 AR inhibits the necrosis cardiac cell ischemia model [103] through phosphorylation of ERK1/2 [104].
Capadenoson, a new oral A 1 AR agonist, decreased the exercising heart rate at a comparable maximum workload in male patients with stable angina [121]. Moreover, this agonist also stimulated A 2B AR, promoting cardioprotection and modulating cardiac fibrosis in heart disease [122]. Unfortunately, the clinical trial of capadenoson was discontinued [123]. A clinical trial using the partial agonist neladenoson (BAY 1067197) for the treatment of heart failure was safe without atrioventricular conduction disorders or neurological adverse effects [124]. A multiple-dose study of neladenoson in heart failure is still ongoing [123].
Taken together, A 1 AR activation ahead of ischemia may augment chemotaxis and neutrophil-dependent injury in cardioprotection; however, the effects of A 1 AR agonists during reperfusion and within both ischemic preconditioning and postconditioning settings remain controversial [125,126].
A 1 AR mediates negative chronotropy and dromotropy through inactivation of the inwardly rectifying K + current (I K,Ado or I K,Ach ), inhibition of the inward Ca 2+ current (I Ca ), and activation of nitric oxide synthase (NOS) [105]. Furthermore, A 1 AR displays both antiarrhythmic and proarrhythmic effects [105]. High expression of A 1 AR results in sinus and AV node dysfunction and supraventricular arrhythmias [127].
At present, several full A 1 AR agonists have been used in clinical trials as antiarrhythmic agents. An optimal dose of a new A 1 AR selective agonist, tecadenoson (CVT-510), was successful in the conversion of paroxysmal supraventricular tachycardia (PSVT) into sinus rhythm [128], but its development for PSVT and atrial fibrillation (AF) was discontinued in 2009 [123]. Other agonists, such as selodenoson and PJ-875, have started in early phase evaluations for the treatment of AF [129]. Currently, to avoid the minor global effects of full agonists, including adverse reactions, desensitization, and arrhythmia (bradyarrhythmia or AF), partial A 1 AR agonists, such as CVT-2759, have been developed [130].

A 1 AR in CNS
A 1 AR contributes to neuroprotection and is also involved in neurodegeneration. In fact, activation of A 1 AR under hypoxia leads to inhibition of presynaptic Ca 2+ influx-related release of transmitters [131] such as dopamine, acetylcholine, GABA, and, especially, glutamate, to generate neuroprotection [132]. Moreover, A 1 AR also regulates potassium current, leading to hyperpolarization of the resting membrane potential mediated via G protein-dependent activation of inwardly rectifying K + channels (GIRKs), activating PLC, and inhibiting AC [133].
Conversely, the levels of glutamate and N-methyl-d-aspartate (NMDA), which are responsible for neuronal damage, may increase during hypoxia and ischemia [134]. It was assumed that prolonged activation of A 1 AR increased A 2A AR expression, which generated global damage and neurodegeneration in ischemic stroke [135]. Recently, an A 1 AR selective agonist, NNC-21-0136, was designed for neuroprotection in stroke models [123].
By binding with A 1 AR, adenosine reduced neuronal activity and pain in the spinal cord and periphery by inhibiting the cAMP, PKA, and Ca 2+ channels, activating K + currents, and interacting with the PLC, IP3, DAG, and β-arrestin pathways [136]. In preclinical studies, it seems that A 1 AR was dominant relative to A 2A AR in inhibiting nociceptive input in the dorsal spinal cord [137]. The selective A 1 AR agonist GR79236X significantly relieves dental pain [138].
Moreover, Cl-ENBA, a potent and highly selective A 1 AR agonist, amplified nociceptive thresholds in spinal glial, and microglial changes occurred in neuropathic pain [139]. MRS7469 also plays a role in pain relief or inhibition of lipolysis [140]. Further development of other agonists, such as SDZ WAG 994, GR79236, and GW-493838, as well as the allosteric enhancer T62, was discontinued [141].
The activation of A 1 AR is crucial for keeping epileptic foci localized [142]; therefore, A 1 AR may be involved in the convulsive effect of diazepam, phenobarbital, or valproate in experimental seizure models [143]. Indeed, upregulation of A 1 AR suppressed seizure activity, which spread within the temporal lobe in rats [144], and could powerfully and bidirectionally regulate seizure activity [145]. An antagonist, DPCPX, inhibited the ketone diet-induced seizure effect [146].
The inhibition of A 1 AR during pathophysiological conditions (noxious stimulation and oxygen deficiency) causes mice to be more anxious [147] and aggressive [148]. In addition, an agonist of A 1 AR reduced the anxiogenic effects during ethanol withdrawal [149]. In overcoming the limitations of A 1 AR agonists, positive allosteric modulators, such as TRR469, offer a more physiological way to treat anxiety [150]. In addition, MRS5474, a potent selective A 1 AR agonist, has been developed as an antidepressant drug that is without cardiovascular side effects [151].
Relating to sleep and level of arousal, A 1 AR was shown to control the response of the circadian clock to light [152] and regulate homeostatic sleep after prolonged wakefulness [153] via inhibition of the ascending cholinergic neurons of the basal forebrain [154]. CPA inhibited the histaminergic system and promoted non-rapid eye movement (NREM) sleep [155].
In line with the fact that synaptic plasticity is the basis for learning and memory in different brain areas [156], A 1 AR attenuated long-term potentiation [157] and long-term depression and depotentiation in the hippocampus [158]. The mixed dual A 1 AR and A 2A AR antagonist ASP5854 ameliorated motor impairments through neuroprotection (A 2A AR antagonism) and enhanced cognitive function (A 1 AR antagonism) [159].
Via both A 1 AR and A 2A AR, adenosine influences the two neurotransmitter systems most affected by schizophrenia-the glutamatergic and dopaminergic neurotransmission systems [160]. While A 1 AR inhibits D 1 R for dopamine, A 2A AR reduces D 2 R recognition, coupling, and signaling [161]. CPA regulates prepulse inhibition (PPI), whereas PIA/L-PIA and 2-CLA/CHA are involved in memory functions and hyperlocomotion, respectively [162].
A 1 AR decreases tremor and controls the spread of excitability, thereby reducing the side effects of deep brain stimulation in Parkinson's disease (PD) [163]. However, there is little information about the clinical study of A 1 AR in PD.
Drugs related to A 1 AR prevented the development of hindlimb dystonia in Huntington's disease (HD). Several agonists, such as CPA, CCPA, CHA, and ADAC, have been developed; however, there are physiological limitations (capacities to cross the blood-brain barrier and adverse effects) to the use of A 1 AR-targeted drugs [162].

A 1 AR in Metabolic and other Diseases
As mentioned earlier, A 1 AR agonists have potential use as antilipolytic agents. Indeed, inhibition of A 1 AR was associated with the enhancement of glucose-stimulated insulin, reduction of oxidative stress, and inhibition of the breakdown of triglycerides to free fatty acids (FFAs) [164].
The full A 1 AR agonists GR79236 and ARA are under development for the treatment of type 2 diabetes mellitus (T2DM), and their mechanisms of action includes reducing nonesterified fatty acids (NEFAs) and triglycerides (TGs) [165]. Unfortunately, the cardiovascular effects of full agonists raise a new problem; therefore, partial agonists that are capable of eliciting a greater effect in adipocytes than in the heart have been developed [166]. The A 1 AR partial agonist GS-9667/CVT-3619 was well tolerated in patients with T2DM and dyslipidemia, in whom it reduced plasma FFAs [167].
In addition, tecadenoson and BW-1433, an A 1 AR antagonist and A 2B AR agonist, respectively, may lower FFA levels and improve glucose tolerance [168]. While A 1 AR selective agonists, such as SDZ WAG994 and RPR749, are under development for antilipolytic and hyperlipidemia, the development of other A 1 AR agonists, including trabodenoson and metrifudil 2 for glaucoma and glomerulonephritis, respectively, was discontinued for safety reasons [123].

A 2A AR in Inflammation
A 2A ARs function as the most dominant anti-inflammatory effectors of extracellular adenosine through their expression on monocytes/macrophages, dendritic cells, mast cells, neutrophils, endothelial cells, eosinophils, epithelial cells, lymphocytes, NK cells, and NKT cells [169].
During innate immunity, activation of A 2A AR on adaptive immune cells shifts Th1 to Th2 responses by suppressing IL-12 and increasing IL-10 [170]. In parallel, secretion of TNF-α, IL-6, and IL-8 is also diminished [171]. Interestingly, the decreases in IL-10 and IL-6 under A 2A R blockade and in knockout (KO) mice resulted in an increase in survival from polymicrobial sepsis [172]. In lymphocytes, A 2A R also inhibited the production of IFN-γ, IL-4, and IL-2 [173].
In contrast, other agonists resulted in poor outcomes. Regadenoson 21 (CVT-3146) for sickle cell disease was not able to produce a statistically significant reduction or clinical efficacy [176]. Clinical trials of sonedenoson (MRE-0094) and spongosine (BVT.115959) for diabetic foot ulcers and diabetic neuropathic pain, respectively, were discontinued [123].

A 2A AR in the Respiratory System
The expression of A 2A AR in the lung is distributed widely, including expression on resident macrophages, bronchial epithelial cells, mast cells, eosinophils, neutrophils, and lymphocytes [169]. Activation of A 2A AR by adenosine binding affects multiple aspects of the inflammatory process, modulating neutrophil activation and degranulation, oxidative species production, adhesion molecule expression, cytokine release, and mast cell degranulation; therefore, selective A 2A AR agonists have an important role in airway inflammation and neutrophil-monocyte-mediated lung tissue injury [14].
In asthmatic airways, A 2A AR suppresses inflammation by reducing neutrophil adherence to the endothelium and inhibiting the fMLP-induced oxidative burst, superoxide anion generation, and LPS-induced TNF-α expression [85]. Moreover, it also inhibits histamine and tryptase release but stimulates wound healing and VEGF secretion [86]. Adversely, angiogenesis, as an important characteristic of airway remodeling in human asthma, may limit the development of such anti-asthma drugs [85].
A 2A AR agonists in animal models of asthma and chronic obstructive pulmonary diseases (COPD) have resulted in good outcomes, yet subsequent clinical trials in humans have not been successful. CGS-21680 has anti-inflammatory activity in a model of allergic asthma in the Brown Norway rat [109], and UK371,104 inhibited capsaicin-induced bronchoconstriction [110]. However, an inhaled A 2A AR agonist, GW328267X, in patients with nonsmoking asthma was unsuccessful in protecting against the allergen-induced early and late asthmatic reactions [111]; hence, its study and the study of UK371,104 were discontinued due to limited efficacy [177].
Recently, regadenoson has been demonstrated to be safe to use in patients with mild to moderate COPD and asthma [112]. Another agonist, apadenoson, is still in a phase I trial for asthma and COPD [59].
The selective agonist CGS-21680 also protected the lung against shock-induced injury [113]. In addition, pentoxifylline showed anti-inflammatory effects in LPS-induced lung injury through an A 2A AR-dependent pathway [114].

A 2A AR in the Cardiovascular Systems
In contrast to A 1 AR, activation of A 2A AR prior to ischemia is not effective. Administration during or prior to reperfusion minimizes myocardial ischemia-reperfusion injury; hence, A 2A AR works in ischemic postconditioning [126]. As an anti-inflammatory molecule, A 2A AR protects the heart through inhibition of leukocyte-dependent inflammatory processes, angiogenesis, enhanced coronary vasodilatation during reperfusion, modified myocardial contraction [178], and increased inotropy through transient Ca 2+ augmentation via a cAMP/PKA-dependent mechanism [179].
The selective A 2a AR agonists CGS 21680C and ATL-193/ATL-146e protected tissue during ischemia-reperfusion injury by reducing myocardial infarct size without elevating coronary blood flow [180,181]. However, A 2A AR not only protects against ischemia but also increases the occurrence of cardiac arrhythmias [182]. In fact, A 2a AR indirectly alters contractility by modulating the A 1 AR antiadrenergic effect rather than increasing cardiac contractility directly [183]. A new study confirmed that the A 2a AR agonist LASSBio-294 might be an alternative treatment for heart failure due to ischemia or hypertension [184].
Along with being cardioprotective via inhibition of the inflammatory response, A 2A AR agonists (PSB-15826, PSB-12404, and PSB-16301) also modulate platelet aggregation [186], and ATL313 regulates cholesterol homeostasis [187]. Perhaps the pharmaceutical industry will focus on the anti-inflammatory effects of A 2A AR agonists against excess cholesterol accumulation to develop new cardiovascular therapies [188]. A2A receptor agonists are the focus of efforts by the pharmaceutical industry to develop new cardiovascular therapies, and pharmacological actions of the atheroprotective and anti-inflammatory drug methotrexate are mediated via release of adenosine and activation of the A2A receptor.
A list of AR ligands currently undergoing clinical trials as novel therapeutic treatments and their effects on cardiovascular and metabolic diseases is presented in Table 2.  [190] Adaptation Hypertrophy Angiogenesis GS-6201 [191] Diabetes mellitus and hyperlipidemia NECA [192] ATL-801 [193] MRS-1754 [194] A 3 AR

A 2A AR in the CNS
According to physiological factors, neuromodulation of adenosine through activation of high-affinity A 2A AR is important [163]. Indeed, A 2A AR was associated with neurodegeneration due to its excitatory effects [135]. Presynaptic A 2A AR counteracts the inhibitory effect of presynaptic A 1 AR on glutamate release from axon terminals, inducing glutamate release that would predispose to excitotoxic injury, yet A 2A AR may also exert a neuroprotective effect by promoting vasodilation and preserving cerebral blood flow autoregulation [143]. Recently, SCH58261, an A 2A AR antagonist, has proven efficacious in halting injury by inhibiting pERK1/2 in hippocampal neurons [200].
Similarly, A 2A AR and A 1 AR of hippocampal CA1 have the opposite effect in spreading piriform cortex kindled seizures, in which A 2A AR tends to have a convulsive effect [201]. In fact, the A 2A AR antagonist ZM241385 had a potent anticonvulsant profile with few adverse effects [202] and suppressed subsequent recovery sleep [203]. Concerning sleep and arousal, the A 2A AR agonist CGS21680 promoted both rapid eye movement (REM) and NREM sleep [133].
Activation of A 2A AR increased the release of the potent anti-inflammatory cytokine IL-10, leading to suppression of pain [61]. CGS21680 produced a long-duration reversal of mechanical allodynia and thermal hyperalgesia [155]. Another agonist, spongosine (BVT.115959, CBT-1008), was effective in a clinical trial for diabetic neuropathic pain, but the production was discontinued [204].
A 2A AR KO mice show increased anxiety [57], and there was an association between a polymorphism in the A 2A AR gene and panic disorder [205]. In contrast, A 2A AR KO mice were less sensitive to "depressant" challenges [206], which might be linked to interactions with dopaminergic transmission in the frontal cortex [207]. Recently, istradefylline became the first therapeutic agent for depression and anxiety [208].
Apart from D 2 R, recent studies also confirmed the involvement of the interaction between A 2A AR/D3 and A 2A AR/mGlu5 in schizophrenia [209]. Therefore, multiple adenosinergic targets, including A 1 R and A 2A R, have potential as therapeutic targets for schizophrenia [210].
HD is characterized by a decrease in A 2A R due to neurodegeneration of the GABA/enkephalin striatopallidal neurons [134]. Interestingly, both A 2A R agonists and antagonists have shown beneficial effects [211]; thus, the potential exploitation of A 2A R ligands in HD is still contentious, reflecting the complexity of A 2A R regulation in this disease [132]. As in HD, there was a reduction in A 2A R in regions of high density, such as the striatum, in Alzheimer's disease (AD) [132]. Recently, the selective A 2A R antagonist MSX-3 prevented the development of memory deficits in an AD mouse model without altering hippocampal and cortical gene expression [212].
A key finding in PD is the colocalization and reciprocal antagonistic interactions between A 2A R and D 2 R [134]; hence, A 2A R antagonists may not only relieve motor deficits in PD but also potentially prevent the degeneration of dopaminergic mesencephalic neurons [213]. Some PD drugs are still under development, including tozadenant/SYN115, DT1133, ZM241385, ST1535, and istradefylline; however, other drugs (preladenant, ASP5854, and vipadenant) were discontinued [61].
A 2A R interacts antagonistically with D 2 R to counteract drug addiction-induced behavioral effects [214]. The high density of presynaptic and postsynaptic A 2A Rs that regulate glutamatergic transmission in the brain leads to the possibility of A 2A Rs as new therapeutic agents for drug addiction [163]. The adenosine agonists NECA and CGS 21680 inhibit cocaine-seeking behaviors [215].
The effects of drugs targeting A 2A AR and other ARs that are currently undergoing clinical trials as novel therapeutic treatments in CNS diseases on the CNS system are listed in Table 3.

A 2B AR in Inflammation
A 2B AR is expressed on most inflammatory cells and has both proinflammatory and anti-inflammatory effects [177]. It generates anti-inflammatory effects by coupling with protein G s and proinflammatory effects by coupling with protein G q [216]. Due to its low affinity, A 2B AR might require high concentrations of adenosine, for instance, during pathological conditions, to be significantly activated [217].
Adenosine regulates bone metabolism and wound healing. Deletion of A 2B AR inhibits periosteal development with subsequent consequences on endochondral ossification and growth plate regulation [226]. In fact, it modulated osteoblast differentiation [227]. Moreover, these receptors also regulate myocardial repair and remodeling by delaying the deactivation of myofibroblasts [228].
Receptor inhibition or knockout of A 2B AR in mice suppressed intestinal inflammation and attenuated disease in murine colitis/inflammatory bowel diseases [63]. A 2B AR in epithelial cells appeared to attenuate colonic inflammation through a specific barrier repair response, namely, phosphorylation of vasodilator-stimulated phosphoprotein [136]. Instead, the deletion of A 2B AR or inhibition by PSB1115 exacerbated the acute inflammatory phase of dextran sodium sulfate colitis [229].
Consistent with their anti-inflammatory effects, CVT-6883 and MRS-1754 diminished the clinical symptoms of experimental autoimmune encephalitis and protected the CNS from immune damage [230]. Administration of the antagonist BAY 60-6583 for four weeks in a high-fat diet mouse model restored endocrine function and reduced inflammation [123].
Taken together, the use of A 2B AR agonists or antagonists for the treatment of the associated disease states is still controversial, and another important variable for agonists, i.e., signaling bias, needs to be considered [231]. Figure 2 illustrates the effects of AR on immune cells.

A2BAR in the Respiratory System
A2BAR binds with both Gs and Gq proteins to mediate airway reactivity, inflammation, and remodeling in asthma [85]. Activation of A2BAR in mast cells induced the release of inflammatory mediators, leading to bronchoconstriction [232]; therefore, the use of antagonist of A2BAR is based on the cellular effects downstream of A2BAR mast cells [86].
A selective A2BAR antagonist, CVT-6883, inhibited airway reactivity and inflammation in a mouse model of asthma [116]. Other compounds, including CGS15493, WO-00125210, and ATL-907, were under development in phase I and phase II trials for the treatment of asthma [86].
The role of mast cells in COPD is controversial, but there is a significant inverse correlation between the binding parameters of A2BAR and the FEV1/FVC ratio [233]. Indeed, the combination of the selective A2BAR agonist/antagonist BAY 60-6583 and dexamethasone may have clinical applications in COPD by inducing genes with anti-inflammatory activity [115]. Furthermore, some studies confirmed that adenosine interacting with A2BAR served an essential role in the pathogenesis of COPD and pulmonary fibrosis [234] by stimulating the secretion of IL-6 and the differentiation of pulmonary fibroblasts into myofibroblasts [235].
A2BAR is also a potential target in acute lung injury (ALI). In a ventilator-induced lung injury (VILI) mouse model, the deletion of the A2BAR gene was associated with reduced survival time and increased pulmonary albumin leakage [117]. Recently, aerosolized BAY 60-6583 was shown to attenuate pulmonary edema, improve histologic lung injury, and diminish lung inflammation in

A 2B AR in the Respiratory System
A 2B AR binds with both G s and G q proteins to mediate airway reactivity, inflammation, and remodeling in asthma [85]. Activation of A 2B AR in mast cells induced the release of inflammatory mediators, leading to bronchoconstriction [232]; therefore, the use of antagonist of A 2B AR is based on the cellular effects downstream of A 2B AR mast cells [86].
A selective A 2B AR antagonist, CVT-6883, inhibited airway reactivity and inflammation in a mouse model of asthma [116]. Other compounds, including CGS15493, WO-00125210, and ATL-907, were under development in phase I and phase II trials for the treatment of asthma [86].
The role of mast cells in COPD is controversial, but there is a significant inverse correlation between the binding parameters of A 2B AR and the FEV1/FVC ratio [233]. Indeed, the combination of the selective A 2B AR agonist/antagonist BAY 60-6583 and dexamethasone may have clinical applications in COPD by inducing genes with anti-inflammatory activity [115]. Furthermore, some studies confirmed that adenosine interacting with A 2B AR served an essential role in the pathogenesis of COPD and pulmonary fibrosis [234] by stimulating the secretion of IL-6 and the differentiation of pulmonary fibroblasts into myofibroblasts [235].
Administration of the A 2B R antagonist GS-6201 or deletion of A 2B R attenuated vascular remodeling and hypertension associated with interstitial lung disease [118]. Additionally, an A 2B R antagonist neutralized the increase in metalloproteases and inhibitors of proteases, tissue inhibitor of metalloproteinase-1 (TIMP-1), matrix metalloproteinase (MMP)-9, and MMP-12 [119].
A 2B AR is also a potential target in acute lung injury (ALI). In a ventilator-induced lung injury (VILI) mouse model, the deletion of the A 2B AR gene was associated with reduced survival time and increased pulmonary albumin leakage [117]. Recently, aerosolized BAY 60-6583 was shown to attenuate pulmonary edema, improve histologic lung injury, and diminish lung inflammation in ALI [236].

A 2B AR in Cardiovascular Systems
There is no "direct" evidence of the effect of A 2B AR on ventricular myocytes [178]; however, A 2B AR was shown to be required for A 1 AR-mediated cardioprotection [237]. In fact, adenosine levels during myocardial ischemia-reperfusion increase; therefore, low-affinity A 2B AR still has cardioprotective effects upon its activation [238].
The results of studies on ischemia are conflicting. A 2B AR plays an important role in ischemia preconditioning (IPC) [63]. Indeed, A 2B AR attenuated myocardial infarction via hypoxia-inducible factor (HIF)-1α and the circadian rhythm protein Per2 [239]. In contrast, a previous study reported that A 2B AR was not involved in the early phase of IPC but might contribute to the later stages by inducing stress-responsive genes [240]. Recently, GS-6201 was shown to attenuate cardiac remodeling by reducing caspase-1 activity after acute myocardial infarction [191], and BAY 60-6583 protected the heart against myocardial IR injury by modulating macrophage phenotype switching via the PI3K/AKT pathway [190].
A 2B AR may inhibit smooth muscle cell-associated hypertension [241]. In fact, KO mice have normal blood pressure [242]. Recently, the deletion of A 2B AR was shown to protect against salt-induced hypertension and stroke [243] and inhibit increases in mean arterial blood pressure [244].
A 2B AR may contribute to the pathogenesis of atherosclerosis. During hypoxia, A 2B AR promoted HIF-1α-induced differentiation of macrophages and foam cells (FC) in preventing atherosclerosis plaque formation [245]. Administration of BAY 60-6853 altered the lipid profile and decreased atherosclerosis in mice [189].

A 2B AR in Metabolic Diseases
It is known that A 2B AR has roles in glucose homeostasis and lipid metabolism, insulin secretion and resistance, inflammation, β-cell survival, and kidney protection [246]. The nonselective A 2B AR agonist NECA, via immunomodulatory effects, inhibited diabetes and protected the pancreas [192]. Moreover, the antagonist ATL-801 increased insulin action in the liver and glucose uptake in skeletal muscle and brown adipose tissue [193].
In diabetic nephropathy, MRS-1754 inhibited high glucose via VEGF [194] and improved kidney tissue nitrite levels [247]. Tak et al. asserted that endothelial A 2B AR signaling was implicated in protection from diabetic nephropathy [248].

A 2B AR in Cancer
A 2B AR is highly expressed in tumor cells and promotes tumor cell proliferation; hence, it might serve as a target for novel therapies or combination therapies for cancer [249]. A 2B AR promoted tumor-inducing M2 macrophages (tumor-associated macrophages); however, it had less effect on decreasing cancer cell proliferation and metastasis and inducing proapoptotic effects [250].
The A 2B AR antagonist ATL801 repressed the growth of bladder and breast tumors and reduced the metastasis of breast cancer cells [251]. Another antagonist, PSB1115, inhibited the accumulation of tumor-infiltrating myeloid-derived suppressor cells (MDSCs) and restored an efficient antitumor T cell response in a mouse model melanoma [252]. In contrast, A 2B AR promoted tumor growth through VEGF and IL-8 [253,254]. Taken together, these results shown that the roles of A 2B AR in cancer are complex. Gessi et al. showed that A 2B AR releases angiogenic factors promoting tumor growth; however, it may exert an inhibitory signal on tumor cell proliferation [255].

A 3 AR in Inflammation
The dual effect of A 3 AR in the inflammation process involves many cells that can have overlapping and opposing function [23]. Its presence in almost all inflammatory cells suggests their involvement in a number of inflammatory pathologies, spanning from wound healing and remodeling to lung injury, inflammatory bone loss, autoimmunity, and eye diseases [69].
A 3 AR produced a proinflammatory effect by inducing the release of histamines and other allergic mediators [256], preventing eosinophil chemotaxis [257], and inhibiting apoptosis [258]. A 3 AR also increased rapid inflammatory cell influx by attracting eosinophils and macrophages in the lung [259] and promoting maturation of DC and Ca 2+ signaling [260].
On the other hand, A 3 AR inhibited LPS-stimulated release of TNF-α and nitric oxide (NO) via the NF-κβ, ERK1/2 and PI3K/AKT pathways [261,262]. Interestingly, both of the resulting modulators were found to increase neutrophil chemotaxis [263] and, in contrast, mediate inhibition of oxidative burst in human neutrophil and promyelocytic HL60 cells [264]. In addition, A 3 AR modulates lymphocyte T cell activation [265].
A 3 AR delivered significant protection from murine septic peritonitis primarily by attenuating the hyperacute inflammatory response in sepsis and IP lung injury [270,271]. Currently, IB-MECA is in a trial for patients with moderate to severe plaque psoriasis [272]. Furthermore, the antagonists A 3 AR, MRS 1292, and OT-7999 inhibited shrinkage of human nonpigmented ciliary epithelial cells and reduced mouse intraocular pressure (IOP) in an animal model of glaucoma [273,274]. A trial of IB-MECA for dry eyes was unsuccessful, yet IB-MECA was able to reduce IOP [275].

A 3 AR in the Respiratory System
A 3 AR plays roles in asthma, COPD, lung fibrosis, and pulmonary inflammation. Adenosineinduced airway hyperresponsiveness (AHR) in mice occurs largely through A 3 AR in mast cells [276] via G i and PI3K signaling pathway-mediated accumulation of intracellular Ca 2+ [277]. While the role of rodent A 3 AR-activated mast cells in AHR is firmly established, the mechanism of A 3 AR-induced release of inflammatory mediators in humans remains elusive [278]. Most A 3 ARs are expressed in lung eosinophils instead of mast cells, which mediate the inhibition of both degranulation and O 2 − release [257,279]. Indeed, A 3 AR has an important role in regulating lung eosinophilia and mucus production in an environment of elevated adenosine [280].
Nebulized IB-MECA directly induced lung mast cell degranulation while having no effect in A 3 AR KO mice [277]. Likewise, adenosine induces AHR indirectly by activating A 3 AR on mast cells [276]. Although A 3 AR is absent in human lung mast cells, it inhibits degranulation of eosinophils, so it may be useful in eosinophil-dependent allergic disorders, such as asthma and rhinitis [68]. In fact, a selective adenosine A 2B AR agonist/A 3 AR antagonist had no significant effect on rhinorrhea, the number of sneezes, or peak nasal inspiratory flow measurements [281]. The combined A 2B AR/A 3 AR antagonist QAF 805 failed to increase the PC20 [85].
In contrast, IB-MECA has an important role in IR-induced lung injury through the upregulation of phosphorylated ERK [271] and the nitric oxide synthase (NOS)-independent pathway [120].
Furthermore, the deletion of A 3 AR enhanced pulmonary inflammation by increasing eosinophil-related chemokines and cytokines [282].
Overall, A 3 AR is involved in both proinflammatory and anti-inflammatory responses depending on the cell type involved, although data in the recent literature appear to lean towards a protective effect [68].

A 3 AR in Cardiovascular Systems
The cardioprotective effect of A 3 AR is similar to the combined effects of A 1 AR and A 2A AR. Activation of the A 3 AR either prior to ischemia or during reperfusion was useful in cardioprotection [125]. Administration of IB-MECA before ischemia or during reperfusion effectively reduces infarct size [197], and CI-IB-MECA (CF102, namodenoson) inhibited myocardial ischemia-reperfusion injury [198]. Additionally, CP-532,903, a highly selective agonist of A 3 AR, protected against ischemia-reperfusion injury [199].
It is assumed that A 3 AR induces cardioprotection via PKC, PI3-kinase, ERK, mitochondrial K ATP channels [178], and NO [283]. Activation of A 3 AR limited myocardial injury in an isolated rat heart and improved survival in isolated myocytes, possibly via antiapoptotic and antinecrotic mechanisms [284]. Indeed, the basic mechanism in A 3 AR-associated cardioprotection remains poorly defined and may vary between species (e.g., rodents vs. humans) and protective responses (e.g., acute vs. delayed protection) [285].
Paradoxically, low-level expression in the heart provides effective protection against ischemic injury without adverse effects, whereas higher levels lead to the development of a dilated cardiomyopathy [286]. Hinze et al. first reported that A 3 AR induces proliferation by activating PLC and inducing the transcription factors early growth response (EGR) 2 and EGR3 [287]. A 3 AR acts against the protective effect of adenosine in the overloaded heart; therefore, attenuation of A 3 AR might be a novel approach to treat pressure overload-induced left ventricular hypertrophy and dysfunction [288]. Moreover, deletion of A 3 AR protects the heart against renal and cardiovascular disease [289].
A 3 AR induces vasoconstriction via mast cell-produced histamine and thromboxane, inhibition of cAMP in smooth muscle and aorta, and enhancement of cellular antioxidants [71]. A 3 AR-mediated contraction through the endothelium may play a role in cardiovascular inflammation, including hypertension and atherosclerosis, by affecting the cyclooxygenase signaling pathways [290], and is also linked with reactive oxygen species (ROS) generation via activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase/Nox2 [291]. A 3 AR is involved in atherosclerosis through modulation of the hyaluronic matrix [292]. Furthermore, it induces VEGF secretion and FC synthesis in a HIF-1α-dependent manner [293].
The latest study shows that LJ-1888, a selective antagonist for A 3 AR, is a feasible novel candidate for the treatment of atherosclerosis and hypercholesterolemia [196]. Moreover, LJ-2698, a highly selective and species-independent A 3 AR antagonist, ameliorated diabetic kidney complications [195].
The role of cardioprotection by A 3 AR is enticing, enigmatic, and elusive [294]. Further clinical studies on cardioprotection induced by adenosine receptors need to establish the role of adenosine receptor agonists and antagonists in more clinically relevant models of myocardial ischemia [238].

A 3 AR in CNS Systems
The expression of A 3 AR in the brain is low and difficult to detect using sensitive in situ hybridization or receptor-labeling autoradiography methods [295], yet A3AR exists in neurotransmission [71].
Several studies have reported a neuroprotective function. Under ischemia, CA1 hippocampal A 3 AR might exert A 1 -like protective effects on neurotransmission, but severe ischemia would transform the A 3 receptor-mediated effects from protective to injurious [296]. Administration of IB-MECA chronically prior to forebrain ischemia resulted in improved postischemic cerebral blood circulation, survival, and neuronal preservation, whereas a negative effect was found when given acutely [222].
In line with this, acute stimulation by CI-IB-MECA induced cell death in a concentration-dependent fashion by inhibiting cAMP production [297]. The timing of treatment also produced opposing results, as administration of IB-MECA prior to and after transient middle cerebral ischemia resulted in an increase and decrease in the infarct size, respectively [223]. The protective role of A 3 AR in ischemia is possibly mediated by the preservation of ischemia-sensitive microtubule-associated protein 2 (MAP-2), enhancement of the expression of glial fibrillary acidic protein [298], depression of NOS, stimulation of glial CCL2 synthesis [299], and delay of irreversible synaptic failure [300].
On the other hand, A 3 AR stimulated convulsion in the CA1 [301] and CA3 [302] regions of the rat hippocampus. A new antagonist, ANR235, increases the stability of I GABA in different epileptic tissues and may offer therapeutic opportunities in human epilepsy [224]. In serotonin (5-HT)-linked brain diseases, A 3 AR is also involved in the rapid stimulation of presynaptic serotonin transport [303] mediated by protein kinase G 1 and p38 MAPK [304]. A 3 AR might function as a potential candidate in brain inflammation treatment by suppressing TNF-α production [262]. Further studies are mandatory for establishing the neuroprotective role of A 3 AR before A 3 AR agonists or antagonists can be used clinically.

A 3 AR in the Digestive and Renal Systems
A 3 AR delivers protection in inflammatory gastrointestinal diseases. IB-MECA treatment reduced oxidative damage and inflammatory mediators in the colon, including IL-1, IL-6, IL-12, MIP-1α, and MIP-2 [305]. This agonist also provides protection from CLP-induced mortality and acute organ dysfunction in murine septic peritonitis primarily by attenuating the hyperacute inflammatory response [270].
A recent study showed that A 3 AR had anti-inflammatory activity via inhibition of proinflammatory cytokine expression associated with the inhibition of NF-κβ signaling pathways in murine dextran sulfate sodium (DSS) colitis [306]. Blocking A 3 AR produced protection against ischemia-and myoglobinuria-induced renal failure [307]. Recently, selective A 3 AR antagonism attenuated renal ischemia-reperfusion injury [308].

A 3 AR in Cancer
Primary and metastatic solid tumors showed high expression of A 3 AR [309], which might be related to overexpression of NF-κβ [310]. In addition, high expression has been observed using biochemical methods in many types of cancer cells, including astrocytoma, melanoma, lymphoma, sarcoma, glioblastoma and colon, liver, pancreatic, prostate, thyroid, lung, breast, and renal carcinomas [69]. Table 4 illustrates the A 3 AR and A 2B AR ligands that are currently undergoing clinical trials as novel therapeutic treatments in cancer.
It has long been known that A 3 AR is involved in the regulation of the cell cycle, and both proapoptotic and antiapoptotic effects have been reported depending on the level of receptor activation [311]. In the event of tumor inhibition, adenosine plays a role in the resistance of muscle to tumor metastases [312], and its antiproliferative effect occurs mainly via A 3 AR-induced cell cycle arrest in the G 0 /G 1 phase and decreases in telomeric signaling in these cells [313]. CI-IB-MECA protects the retinal ganglion by increasing survival [314]. Additionally, it mediates a tonic proliferative effect in colon tumor cells [315]. It is possible that the antiapoptotic effect is related to oxygen concentration [59].
Apart from hypoxia, HIF-1α has a role in cancer-associated angiogenesis [316]. A 3 AR upregulates HIF-1α protein expression in a dose-dependent and time-dependent manner via p44/p42 and p38 MAPK [317]. Moreover, an A 3 AR antagonist inhibited VEGF protein accumulation [318] and reduced tumor size and blood vessel formation under hypoxic conditions in glioblastoma cells [319].
IB-MECA inhibited tumor cell growth of HCT-116 human and CT-26 murine colon carcinoma cell [320] and B16-F10 melanoma cells [321]. Furthermore, other in vivo studies have shown that IB-MECA in PC-3 prostate carcinoma cells [322] and CI-IB-MECA in N1S1 rat hepatocellular carcinoma (HCC) cells [323] induced apoptosis and tumor growth inhibition via deregulation of the Wnt and NF-κβ signaling pathways. HCC CI-IB-MECA [323] The oral bioavailability of synthetic A 3 AR agonists renders them potentially useful in three different modes of treatment: as stand-alone anticancer treatments, in combination with chemotherapy to enhance its therapeutic index, and as agents inducing myeloprotection [324]. The latest study confirmed that combination use of A 3 AR agonists produces anticancer effects in an apoptosis-, autophagy-, and ROS-dependent manner [325].

Conclusions
Pharmacological interventions related to AR ligands as new drugs has been widely developed. Most ARs have roles as both proinflammatory and anti-inflammatory mediators as well as in organ protection and degeneration, and these opposing roles depend on the timing and concentration. As a consequence of being widely distributed throughout the body, adenosine activates protection for tissues and cells against injury, hypoxia, and ischemia; however, it also stimulates various adverse effects, especially in the cardiovascular and respiratory systems. Therefore, future studies should focus on the abundant amount of adenosine and reducing its harmful impact. Overall, focusing on partial agonist and allosteric enhancers might be the most appealing pharmacological angle for successful treatment in cancer, inflammation, cardiometabolic diseases, respiratory diseases, and neurovascular diseases.