International Journal of Molecular Sciences Review Uncovering the Mechanisms of Adenosine Receptor-Mediated Pain Control: Focus on the A3 Receptor Subtype Elisabetta Coppi 1,* , Federica Cherchi 1 , Elena Lucarini 1 , Carla Ghelardini 1, Felicita Pedata 1, Kenneth A. Jacobson 2 , Lorenzo Di Cesare Mannelli 1 , Anna Maria Pugliese 1 and Daniela Salvemini 3 1 Department NEUROFARBA, Division of Pharmacology and Toxicology, University of Florence, 50139 Firenze, Italy; federica.cherchi@unifi.it (F.C.); elena.lucarini@unifi.it (E.L.); carla.ghelardini@unifi.it (C.G.); felicita.pedata@unifi.it (F.P.); lorenzo.mannelli@unifi.it (L.D.C.M.); annamaria.pugliese@unifi.it (A.M.P.) 2 Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0810, USA; [email protected] 3 Department of Pharmacology and Physiology, Saint Louis University School of Medicine, St. Louis, MO 63104, USA; [email protected] * Correspondence: elisabetta.coppi@unifi.it Abstract: Agonists of the Gi protein-coupled A3 adenosine receptor (A3AR) have shown important pain-relieving properties in preclinical settings of several pain models. Active as a monotherapy against chronic pain, A3AR agonists can also be used in combination with classic opioid analgesics. Their safe pharmacological profile, as shown by clinical trials for other pathologies, i.e., rheumatoid arthritis, psoriasis and fatty liver diseases, confers a realistic translational potential, thus encouraging Citation: Coppi, E.; Cherchi, F.; research studies on the molecular mechanisms underpinning their antinociceptive actions. A number Lucarini, E.; Ghelardini, C.; Pedata, F.; of pathways, involving central and peripheral mechanisms, have been proposed. Recent evidence Jacobson, K.A.; Di Cesare Mannelli, showed that the prototypical A3AR agonist Cl-IB-MECA and the new, highly selective, A3AR agonist L.; Pugliese, A.M.; Salvemini, D. MRS5980 inhibit neuronal (N-type) voltage-dependent Ca2+ currents in dorsal root ganglia, a known Uncovering the Mechanisms of pain-related mechanism. Other proposed pathways involve reduced cytokine production, immune Adenosine Receptor-Mediated Pain cell-mediated responses, as well as reduced microglia and astrocyte activation in the spinal cord. The Control: Focus on the A3 Receptor aim of this review is to summarize up-to-date information on A3AR in the context of pain, including Subtype. Int. J. Mol. Sci. 2021, 22, cellular and molecular mechanisms underlying this effect. Based on their safety profile shown in 7952. https://doi.org/10.3390/ ijms22157952 clinical trials for other pathologies, A3AR agonists are proposed as novel, promising non-narcotic agents for pain control. Academic Editor: Cristina Missale Keywords: A3 adenosine receptor; neuropathic pain; visceral pain; dorsal root ganglion neurons; Received: 5 July 2021 Ca2+ currents; T cells; interleukin-10; adenosine Accepted: 20 July 2021 Published: 26 July 2021 Publisher’s Note: MDPI stays neutral 1. Introduction with regard to jurisdictional claims in Chronic pain is a highly debilitating condition, disturbing all aspects of our daily published maps and institutional affil- experience in social and career-related contexts. The pharmacological tools currently iations. available are sometimes inadequate, or, as in the case of opioids, limited by serious adverse effects [1]. Thus, efforts are being made to pursue research into innovative, non-opioid, pain-relieving compounds. Many experimental reports have identified adenosine receptors (ARs) as potential Copyright: © 2021 by the authors. targets for acute or chronic pain management. Adenosine is a ubiquitous endogenous Licensee MDPI, Basel, Switzerland. neuromodulator whose actions are mediated by four G protein-coupled receptors (GPCR), This article is an open access article namely A1,A2A,A2B and A3 receptors (A1Rs, A2ARs, A2BRs and A3Rs). A1Rs and A3Rs distributed under the terms and are coupled to Gi members of the G protein family, while A2ARs and A2BRs are Gs-coupled conditions of the Creative Commons receptors [2]. The consequent modulation of cyclic adenosine monophosphate (cAMP) Attribution (CC BY) license (https:// levels activates or inhibits a number of signaling pathways, depending on the specific type creativecommons.org/licenses/by/ of cell involved (Figure1). In some cases, A 2BR might also couple to Gq proteins, as well as 4.0/). Int. J. Mol. Sci. 2021, 22, 7952. https://doi.org/10.3390/ijms22157952 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 2 of 13 Int. J. Mol. Sci. 2021, 22, 7952 2 of 13 (cAMP) levels activates or inhibits a number of signaling pathways, depending on the specific type of cell involved (Figure 1). In some cases, A2BR might also couple to Gq pro- teins,A1Rs as to thewell G aso protein.A1Rs to Allthe ARsGo protein. are coupled All ARs to mitogen-activatedare coupled to mitogen-activated protein kinase(MAPK) protein kinasepathways, (MAPK) including pathways, extracellular including signal-regulated extracellular signal-regulated kinase 1/2 (ERK1/2), kinase p38 1/2 MAPK (ERK1/2), and p38Jc-Jun-NH2 MAPK and terminal Jc-Jun-NH2 Kinase terminal (JNK) [3 ].Kinase (JNK) [3]. FigureFigure 1. AdenosineAdenosine receptors receptors and and the the main main transduction transduction pathways pathways involved involved in in their their activation. activation. SchematicSchematic representation ofof GG protein-coupledprotein-coupled A A1,A1, A2A2A,A, A2B2B andand AA33 receptorreceptor (A(A11R,R, AA2A2AR, A2BRR and AA33R)R) subtypes subtypes activated activated by by extracellular extracellular adenosine adenosine (ADO), (ADO), and and the the main main intracellular intracellular pathways pathways in- volved. A2ARs and A2BRs are coupled to the Gs protein, which leads to adenylyl cyclase (AC) acti- involved. A2ARs and A2BRs are coupled to the Gs protein, which leads to adenylyl cyclase (AC) vation and cyclic AMP (cAMP) increase. On the other hand, A1R and A3R are coupled to the Gi activation and cyclic AMP (cAMP) increase. On the other hand, A1R and A3R are coupled to the protein that inhibits AC and reduces cAMP. In some districts, A2BRs are also coupled with Gq pro- Gi protein that inhibits AC and reduces cAMP. In some districts, A2BRs are also coupled with Gq teins and A1R with Go, which stimulate Ca2+ release from intracellular stores. All adenosine recep- proteins and A R with Go, which stimulate Ca2+ release from intracellular stores. All adenosine tors are coupled1 to mitogen-activated protein kinase (MAPK) pathways, including extracellular sig- nal-regulatedreceptors are coupledkinase 1/2 to mitogen-activated(ERK1/2), p38 MAPK protein and kinaseJc-Jun-NH2 (MAPK) terminal pathways, Kinase including (JNK). extracellular signal-regulated kinase 1/2 (ERK1/2), p38 MAPK and Jc-Jun-NH2 terminal Kinase (JNK). A variety of functions are regulated by purines: cardiovascular, respiratory, inflam- A variety of functions are regulated by purines: cardiovascular, respiratory, inflam- matory and immune events [4–6], as well as neuronal maturation [7,8] and hypoxic dam- matory and immune events [4–6], as well as neuronal maturation [7,8] and hypoxic dam- age [9,10]. Of note, adenosine receptors are expressed within the pain-related areas in the age [9,10]. Of note, adenosine receptors are expressed within the pain-related areas in the peripheral or central nervous system (CNS). It is known that ARs are widely distributed peripheral or central nervous system (CNS). It is known that ARs are widely distributed on on neurons and glia; hence, considerable interest is focused on the development of selec- neurons and glia; hence, considerable interest is focused on the development of selective tive AR ligands able to control neurological alterations in nervous system diseases [11– AR ligands able to control neurological alterations in nervous system diseases [11–14]. 14]. Importantly, local levels of adenosine are significantly enhanced during events of tis- Importantly, local levels of adenosine are significantly enhanced during events of tissue sue inflammation, stress or trauma, as well as during hypoxia-ischemia [15,16]. Adenosine inflammation, stress or trauma, as well as during hypoxia-ischemia [15,16]. Adenosine is a is a potent anti-inflammatory autacoid that inhibits a number of inflammatory mecha- potent anti-inflammatory autacoid that inhibits a number of inflammatory mechanisms, nisms,including including phagocytosis, phagocytosis, the generation the generation of toxic of oxygen toxic metabolites,oxygen metabolites, cytokine cytokine release and re- leasecellular and adhesion cellular [adhesion17]. Most [17]. of these Most anti-inflammatory of these anti-inflammatory effects were effects first ascribed were first to theas- cribed to the activation of “A2Rs” [18], before further investigation attributed the anti- activation of “A2Rs” [18], before further investigation attributed the anti-inflammatory inflammatory effect of adenosine to A2ARs [19], which are widely expressed on peripheral effect of adenosine to A2ARs [19], which are widely expressed on peripheral blood and bloodimmune and cells immune [20]. cells [20]. MovingMoving in moremore detaildetail to to pain pain transmission, transmission, the the first first proof proof of adenosine’sof adenosine’s involvement involve- mentin anti-nociception in anti-nociception dates dates from from the 1970s, the 1970s, when when the systemic