Adenosine is a ubiquitous endogenous autacoid whose effects are triggered through the enrollment of four G protein-coupled receptors: A₁, A_2A, A_2B, and A₃. Due to the rapid generation of adenosine from cellular metabolism, and the widespread distribution of its receptor subtypes in almost all organs and tissues, this nucleoside induces a multitude of physiopathological effects, regulating central nervous, cardiovascular, peripheral, and immune systems. It is becoming clear that the expression patterns of adenosine receptors vary among cell types, lending weight to the idea that they may be both markers of pathologies and useful targets for novel drugs. This review offers an overview of current knowledge on adenosine receptors, including their characteristic structural features, molecular interactions and cellular functions, as well as their essential roles in pain, cancer, and neurodegenerative, inflammatory, and autoimmune diseases. Finally, we highlight the latest findings on molecules capable of targeting adenosine receptors and report which stage of drug development they have reached.

By general consensus, the omnipresent purine nucleoside adenosine is considered a major regulator of local tissue function, especially when energy supply fails to meet cellular energy demand. Adenosine mediation involves activation of a family of four G protein–coupled adenosine receptors (ARs): A₁, A_2A, A_2B, and A₃. The A₃ adenosine receptor (A₃AR) is the only adenosine subtype to be overexpressed in inflammatory and cancer cells, thus making it a potential target for therapy. Originally isolated as an orphan receptor, A₃AR presented a twofold nature under different pathophysiologic conditions: it appeared to be protective/harmful under ischemic conditions, pro/anti-inflammatory, and pro/antitumoral depending on the systems investigated. Until recently, the greatest and most intriguing challenge has been to understand whether, and in which cases, selective A₃ agonists or antagonists would be the best choice. Today, the choice has been made and A₃AR agonists are now under clinical development for some disorders including rheumatoid arthritis, psoriasis, glaucoma, and hepatocellular carcinoma. More specifically, the interest and relevance of these new agents derives from clinical data demonstrating that A₃AR agonists are both effective and safe. Thus, it will become apparent in the present review that purine scientists do seem to be getting closer to their goal: the incorporation of adenosine ligands into drugs with the ability to save lives and improve human health.

The mechanism of action of cannabidiol (CBD), the main non-psychotropic component of Cannabis sativa L., is not completely understood. First assumed that the compound was acting via cannabinoid CB₂ receptors (CB₂Rs) it is now suggested that it interacts with non-cannabinoid G-protein-coupled receptors (GPCRs); however, CBD does not bind with high affinity to the orthosteric site of any GPCR. To search for alternative explanations, we tested CBD as a potential allosteric ligand of CB₂R. Radioligand and non-radioactive homogeneous binding, intracellular cAMP determination and ERK1/2 phosphorylation assays were undertaken in heterologous systems expressing the human version of CB₂R. Using membrane preparations from CB₂R-expressing HEK-293T (human embryonic kidney 293T) cells, we confirmed that CBD does not bind with high affinity to the orthosteric site of the human CB₂R where the synthetic cannabinoid, [³H]-WIN 55,212-2, binds. CBD was, however, able to produce minor but consistent reduction in the homogeneous binding assays in living cells using the fluorophore-conjugated CB₂R-selective compound, CM-157. The effect on binding to CB₂R-expressing living cells was different to that exerted by the orthosteric antagonist, SR144528, which decreased the maximum binding without changing the K_D. CBD at nanomolar concentrations was also able to significantly reduce the effect of the selective CB₂R agonist, JWH133, on forskolin-induced intracellular cAMP levels and on activation of the MAP kinase pathway. These results may help to understand CBD mode of action and may serve to revisit its therapeutic possibilities.

Adenosine is a ubiquitous endogenous modulator with the main function of maintaining cellular and tissue homeostasis in pathological and stress conditions. It exerts its effect through the interaction with four G protein-coupled receptor (GPCR) subtypes referred as A₁, A_2A, A_2B, and A₃ adenosine receptors (ARs), each of which has a unique pharmacological profile and tissue distribution. Adenosine is a potent modulator of inflammation, and for this reason the adenosinergic system represents an excellent pharmacological target for the myriad of diseases in which inflammation represents a cause, a pathogenetic mechanism, a consequence, a manifestation, or a protective factor. The omnipresence of ARs in every cell of the immune system as well as in almost all cells in the body represents both an opportunity and an obstacle to the clinical use of AR ligands. This review offers an overview of the cardinal role of adenosine in the modulation of inflammation, showing how the stimulation or blocking of its receptors or agents capable of regulating its extracellular concentration can represent promising therapeutic strategies for the treatment of chronic inflammatory pathologies, neurodegenerative diseases, and cancer.

Cannabigerol (CBG) is one of the major phytocannabinoids present in Cannabis sativa L. that is attracting pharmacological interest because it is non-psychotropic and is abundant in some industrial hemp varieties. The aim of this work was to investigate in parallel the binding properties of CBG to cannabinoid CB₁ (CB₁R) and CB₂ (CB₂R) receptors and the effects of the compound on agonist activation of those receptors and of CB₁–CB₂ heteroreceptor complexes. Using [³H]-CP-55940, CBG competed with low micromolar K_i values the binding to CB₁R and CB₂R. Homogeneous binding in living cells, which is only technically possible for the CB₂R, provided a 152 nM K_i value. Also interesting, CBG competed the binding of [³H]-WIN-55,212-2 to CB₂R but not to CB₁R (K_i: 2.7 versus >30 μM). The phytocannabinoid modulated signaling mediated by receptors and receptor heteromers even at low concentrations of 0.1–1 μM. cAMP, pERK, β-arrestin recruitment and label-free assays in HEK-293T cells expressing the receptors and treated with endocannabinoids or selective agonists proved that CBG is a partial agonist of CB₂R. The action on cells expressing heteromers was similar to that obtained in cells expressing the CB₂R. The effect of CBG on CB₁R was measurable but the underlying molecular mechanisms remain uncertain. The results indicate that CBG is indeed effective as regulator of endocannabinoid signaling.

Adenosine receptors (ARs) have an important role in the regulation of inflammation and their activation is involved in the inhibition of pro-inflammatory cytokine release. The effects of pulsed electromagnetic fields (PEMFs) on inflammation have been reported and we have demonstrated that PEMFs increased A 2A and A 3 AR density and functionality in different cell lines. Chondrocytes and osteoblasts are two key cell types in the skeletal system that play important role in cartilage and bone metabolism representing an interesting target to study the effect of PEMFs. The primary aim of the present study was to evaluate if PEMF exposure potentiated the anti-inflammatory effect of A 2A and/or A 3 ARs in T/C-28a2 chondrocytes and hFOB 1.19 osteoblasts. Immunofluorescence, mRNA analysis and saturation binding assays revealed that PEMF exposure up-regulated A 2A and A 3 AR expression. A 2A and A 3 ARs were able to modulate cAMP production and cell proliferation. The activation of A 2A and A 3 ARs resulted in the decrease of some of the most relevant pro-inflammatory cytokine release such as interleukin (IL)-6 and IL-8, following the treatment with IL-1β as an inflammatory stimuli. In human chondrocyte and osteoblast cell lines, the inhibitory effect of A 2A and A 3 AR stimulation on the release of prostaglandin E 2 (PGE 2 ), an important lipid inflammatory mediator, was observed. In addition, in T/C-28a2 cells, the activation of A 2A or A 3 ARs elicited an inhibition of vascular endothelial growth factor (VEGF) secretion. In hFOB 1.19 osteoblasts, PEMF exposure determined an increase of osteoprotegerin (OPG) production. The effect of the A 2A or A 3 AR agonists in the examined cells was enhanced in the presence of PEMFs and completely blocked by using well-known selective antagonists. These results demonstrated that PEMF exposure significantly increase the anti-inflammatory effect of A 2A or A 3 ARs suggesting their potential therapeutic use in the therapy of inflammatory bone and joint disorders.