Selective A3 Receptor Agonist Research Articles

Differential Gene Expression in Activated Microglia Treated with Adenosine A2A Receptor Antagonists Highlights Olfactory Receptor 56 and T-Cell Activation GTPase-Activating Protein 1 as Potential Biomarkers of the Polarization of Activated Microglia.

Microglial activation often accompanies the plastic changes occurring in the brain of patients with neurodegenerative diseases. A2A and A3 adenosine receptors have been proposed as therapeutic targets to combat neurodegeneration. RNAseq was performed using samples isolated from lipopolysaccharide/interferon-γ activated microglia treated with SCH 58261, a selective A2A receptor antagonist, and with both SCH 58261 and 2-Cl-IB-MECA, a selective A3 receptor agonist. None of the treatments led to any clear microglial phenotype when gene expression for classical biomarkers of microglial polarization was assessed. However, many of the downregulated genes were directly or indirectly related to immune system-related events. Searching for genes whose expression was both significantly and synergistically affected when treated with the two adenosine receptor ligands, the AC122413.1 and Olfr56 were selected among those that were, respectively, upregulated and downregulated. We therefore propose that the products of these genes, olfactory receptor 56 and T-cell activation GTPase-activating protein 1, deserve attention as potential biomarkers of phenotypes that occur upon microglial activation.

The Anti-Inflammatory and Pain-Relieving Effects of AR170, an Adenosine A3 Receptor Agonist, in a Rat Model of Colitis.

The pharmacological activation of A3 receptors has shown potential usefulness in the management of bowel inflammation. However, the role of these receptors in the control of visceral hypersensitivity in the presence of intestinal inflammation has not been investigated. The effects of AR170, a potent and selective A3 receptor agonist, and dexamethasone (DEX) were tested in rats with 2,4-dinitrobenzene sulfonic acid (DNBS)-induced colitis to assess their tissue inflammatory parameters. The animals received AR170, DEX, or a vehicle intraperitoneally for 6 days, starting 1 day before the induction of colitis. Visceral pain was assessed by recording the abdominal responses to colorectal distension in animals with colitis. Colitis was associated with a decrease in body weight and an increase in spleen weight. The macroscopic damage score and tissue tumor necrosis factor (TNF), interleukin 1β (IL-1β), and myeloperoxidase (MPO) levels were also enhanced. AR170, but not DEX, improved body weight. Both drugs counteracted the increase in spleen weight, ameliorated macroscopic colonic damage, and decreased TNF, IL-1β, and MPO tissue levels. The enhanced visceromotor response (VMR) in rats with colitis was decreased via AR170 administration. In rats with colitis, AR170 counteracted colonic inflammatory cell infiltration and decreased pro-inflammatory cytokine levels, thereby relieving visceral hypersensitivity.

Sa1311 – Anti-Inflammatory and Visceral Pain Relieving Effects of Ar170, a Potent and Selective A3 Receptor Agonist, in a rat Model of Colitis

Sa1311 – Anti-Inflammatory and Visceral Pain Relieving Effects of Ar170, a Potent and Selective A3 Receptor Agonist, in a rat Model of Colitis

Identification of A3 adenosine receptor agonists as novel non-narcotic analgesics.

Chronic pain negatively impacts the quality of life in a variety of patient populations. The current therapeutic repertoire is inadequate in managing patient pain and warrants the development of new therapeutics. Adenosine and its four cognate receptors (A1 , A2A , A2B and A3 ) have important roles in physiological and pathophysiological states, including chronic pain. Preclinical and clinical studies have revealed that while adenosine and agonists of the A1 and A2A receptors have antinociceptive properties, their therapeutic utility is limited by adverse cardiovascular side effects. In contrast, our understanding of the A3 receptor is only in its infancy, but exciting preclinical observations of A3 receptor antinociception, which have been bolstered by clinical trials of A3 receptor agonists in other disease states, suggest pain relief without cardiovascular side effects and with sufficient tolerability. Our goal herein is to briefly discuss adenosine and its receptors in the context of pathological pain and to consider the current data regarding A3 receptor-mediated antinociception. We will highlight recent findings regarding the impact of the A3 receptor on pain pathways and examine the current state of selective A3 receptor agonists used for these studies. The adenosine-to-A3 receptor pathway represents an important endogenous system that can be targeted to provide safe, effective pain relief from chronic pain.

Adenosine A3 receptor agonists: do recent findings offer new hope in chronic pain treatment?

Background Management of chronic pain is limited by inadequate effectiveness and undesirable safety profiles of currently available medi cations. There is growing evidence that adenosine (A1, A2A, A2B, A3) receptors can be promising therapeutic targets for treatment of chronic pain. A number of selective ligands for adenosine receptors have been introduced. Agents acting selectively at A1, A2A and A2B receptors have been effective in several preclinical animal models of pain and several have also entered into clinical development for neuropathic pain. Unfortunately, safety concerns related to significant cardiovascular side effects caused by systemic administration of some of these agents limited further development. In contrast, systemic administration of certain selective A3 receptor agonists is not associated with cardiac or hemodynamic side effects, making the A3

Abstract 541: Adenosine Inhibits C-Kit+ Progenitor Smooth Muscle Cell (P-SMC) Infiltration In Injury-Induced Neointima Formation And Human P-SMC Growth: Role of A2B Adenosine Receptor-Mediated Growth Regulation

Adenosine inhibits injury-induced neointimal formation; however, the mechanism is incompletely understood. Marrow-derived C-Kit+ progenitor cells (P-SMCs) express a smooth muscle phenotype and contribute to injury-induced neointimal thickening. The goal of our investigation was to determine whether adenosine attenuates neointimal hyperplasia in part by inhibiting the growth of P-SMCs and to clarify which adenosine receptor subtypes are involved. Digitized pixel analysis demonstrated that administration of adenosine (20 μM) peri-arterially for 7 days in a slow-release formulation inhibited injury-induced neointimal hyperplasia in the rat carotid artery by 71% (p<0.05: neointimal thickness: 37,424 ± 18,371 pixels in vehicle-treated [n=7] versus 10,352 ± 2,824 pixels in adenosine-treated [n=7]). Microscopic analysis following immunostaining for c-Kit+ cells showed a marked decrease (> 50%) in staining for c-Kit+ cells in the neointima of rats receiving adenosine. In cultured human P-SMCs, serum-induced growth (as determined by quantification of DNA synthesis and cell number), cell-migration, and collagen synthesis was inhibited by adenosine and 2-chloroadenosine, but not by N 6 -cyclopentyladenosine (selective A 1 -receptor agonist), CGS21680 (selective A 2A -receptor agonist) or N 6 -(3-iodobenzyl)adenosine-5'-N-methyluronamide (selective A 3 -receptor agonist). This agonist profile was consistent with an A 2B -receptor-mediated effect. Augmentation of endogenous adenosine levels with erythro -9-(2-hydroxy-3-nonyl)adenine (adenosine deaminase inhibitor) and 5-iodotubericidin (adenosine kinase inhibitor) attenuated P-SMC growth. Moreover the antimitogenic effects of both exogenous and endogenous adenosine were abrogated by blockade of A 2B receptors (MRS 1754), but not by antagonism of A 1 (8-cyclopentyl-1,3-dipropylxanthine), A 2A (SCH 58261) or A 3 (VUF 5574) receptors. We conclude that adenosine, via activation of A 2B receptors, inhibits neointimal formation in part by inhibiting infiltration and growth of C-Kit+ P-SMCs. These findings suggest that A 2B receptor stimulation may prevent vascular remodeling associated with coronary artery disease, hypertension, atherosclerosis and restenosis.

Synthesis of 2-Chloro-N6-Substituted-4'-thioadenosine-5'-N, N-dialkyluronamides as Potent and Selective A3 Adenosine Receptor Antagonists

The highly selective A(3) receptor agonist, 4'-thio-Cl-IB-MECA was successfully converted into selective A(3) receptor antagonists by appending a second N-alkyl group on the 5'-uronamide position. This result indicates that the hydrogen bonding ability of the 5'-uronamide is essential for the conformational change required for the receptor activation. Among compounds tested, a N(6)-(3-bromobenzyl) derivative with 5'-dimethyluronamide exhibited the highest binding affinity (K(i) = 9.32 nM) at the human A(3) AR with very low binding affinities to other AR subtypes.

A2Badenosine receptor mediates human chorionic vasoconstriction and signals through arachidonic acid cascade

Because adenosine is a vascular tone modulator, we examined the effect of adenosine and congeners in the vascular reactivity of isolated human placental vessels and in perfused cotyledons. We characterized its vasomotor action and tentatively identified the receptor subtypes and their intracellular signaling mechanisms. We recorded isometric tension from the circular layer of chorionic vessel rings maintained under 1.5 g of basal tension or precontracted with KCl. The relative order of potency of adenosine and structural analogs is consistent with the expression of A2B receptors, 5'-(N-ethylcarboxamido)adenosine (NECA) being the most potent. The maximal contraction ranged from 45% to 60% of the KCl standard response, except for an A2A receptor agonist that did not exceed 15%. Consistently, NECA was 100-fold more potent than adenosine to raise the perfusion pressure of ex vivo perfused cotyledons. In contrast, a selective A3 receptor agonist relaxed precontracted rings of chorionic vessels. Whereas a selective A3 receptor antagonist was ineffective to antagonize adenosine-induced contraction, A2 or A1 receptor antagonists reduced adenosine-induced vasoconstriction concentration-dependently. Denudation of the endothelial layer reduced adenosine- and NECA-induced contractions by 50-70%. Furthermore, indomethacin reduced adenosine- or NECA-induced contractions concentration-dependently in intact and endothelium-denuded rings. A thromboxane receptor antagonist blocked adenosine- and NECA-induced contractions in intact and endothelium-denuded rings, suggesting the involvement of an arachidonic acid metabolite as the mediator of the vasoconstriction. We propose that adenosine A2B receptors mediate the adenosine-induced contraction vasomotor effect in human chorionic vessels and that this involves synthesis of a thromboxane receptor activator or a related prostanoid.

Activation of A3Adenosine Receptors Attenuates Lung Injury after In Vivo Reperfusion

A3 adenosine receptor (AR) activation worsens or protects against renal and cardiac ischemia-reperfusion (IR) injury, respectively. The aims of the current study were to examine in an in vivo model the effect of A3AR activation on IR lung injury and investigate the mechanism by which it exerts its effect. The arterial branch of the left lower lung lobe in intact-chest, spontaneously breathing cats was occluded for 2 h and reperfused for 3 h (IR group). Animals were treated with the selective A3 receptor agonist IB-MECA (300 microg/kg intravenously) given 15 min before ischemia or with IB-MECA as described, with pretreatment 15 min earlier with the selective A3AR antagonist MRS-1191, the nonsulfonylurea adenosine triphosphate-sensitive potassium channel-blocking agent U-37883A, or the nitric oxide synthase inhibitor N-nitro-l-arginine benzyl ester. IB-MECA markedly (P < 0.01) reduced the percentage of injured alveoli (IR, 48 +/- 4%; IB-MECA, 18 +/- 2%), wet:dry weight ratio (IR, 8.2 +/- 0.4; IB-MECA, 4 +/- 2), and myeloperoxidase activity (IR, 0.52 +/- 0.06 U/g; IB-MECA, 0.17 +/- 0.04 U/g). This protective effect was completely blocked by pretreatment with the selective A3AR antagonist MRS-1191 and the adenosine triphosphate-sensitive potassium channel blocking agent U-37883A but not the nitric oxide synthase inhibitor N-nitro-l-arginine benzyl ester. In the feline lung, the A3AR agonist IB-MECA confers a powerful protection against IR lung injury. This effect is mediated by a nitric oxide synthase-independent pathway and involves opening of adenosine triphosphate-sensitive potassium channels. Therefore, selective activation of A3AR may be an effective means of protecting the reperfused lung.

Effects of synthetic adenosine receptor agonists on growth characteristics of G:5:113 fibrosarcoma cells In Vitro

AbstractThe effects of several synthetic adenosine receptor agonists on the growth characteristics of murine G:5:113 fibrosarcoma cells in vitro were evaluated. Cell number and viability, as well as proportions of cells in individual cell cycle phases, were determined after 48 h cultivation of the cells in the presence of concentrations of adenosine receptor agonists ranging between 0.01 and 500 µM. Effects of the non‐selective adenosine receptor agonist NECA, as well as of agonists selective for A1 (CPA), A2A (CGS 21680), and A3 receptors (IB‐MECA) were evaluated. A slight increase in cell number was observed at a narrow range of concentrations between 30–50 µM of NECA, which was reproduced by CPA, a selective A1 receptor agonist, in concentrations ranging from 20–50 µM. The most important finding was that of significant inhibitory effects of adenosine receptor agonists on cell number in concentrations above 50 µM. This effect was most pronounced after treatment of the G:5:113 fibrosarcoma cells with the selective A3 receptor agonist, IB‐MECA, as revealed also by calculated values of generation time and EC50. The results suggest that adenosine A3 receptor might represent a target for pharmacological anticancer approaches aimed at suppressing the growth of fibrosarcoma cell population. Drug Dev. Res. 60:303–311, 2003. © 2003 Wiley‐Liss, Inc.

Role of renal medullary adenosine in the control of blood flow and sodium excretion.

This study determined the levels of adenosine in the renal medullary interstitium using microdialysis and fluorescence HPLC techniques and examined the role of endogenous adenosine in the control of medullary blood flow and sodium excretion by infusing the specific adenosine receptor antagonists or agonists into the renal medulla of anesthetized Sprague-Dawley rats. Renal cortical and medullary blood flows were measured using laser-Doppler flowmetry. Analysis of microdialyzed samples showed that the adenosine concentration in the renal medullary interstitial dialysate averaged 212 +/- 5.2 nM, which was significantly higher than 55.6 +/- 5.3 nM in the renal cortex (n = 9). Renal medullary interstitial infusion of a selective A1 antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 300 pmol. kg-1. min-1, n = 8), did not alter renal blood flows, but increased urine flow by 37% and sodium excretion by 42%. In contrast, renal medullary infusion of the selective A2 receptor blocker 3, 7-dimethyl-1-propargylxanthine (DMPX; 150 pmol. kg-1. min-1, n = 9) decreased outer medullary blood flow (OMBF) by 28%, inner medullary blood flows (IMBF) by 21%, and sodium excretion by 35%. Renal medullary interstitial infusion of adenosine produced a dose-dependent increase in OMBF, IMBF, urine flow, and sodium excretion at doses from 3 to 300 pmol. kg-1. min-1 (n = 7). These effects of adenosine were markedly attenuated by the pretreatment of DMPX, but unaltered by DPCPX. Infusion of a selective A3 receptor agonist, N6-benzyl-5'-(N-ethylcarbonxamido)adenosine (300 pmol. kg-1. min-1, n = 6) into the renal medulla had no effect on medullary blood flows or renal function. Glomerular filtration rate and arterial pressure were not changed by medullary infusion of any drugs. Our results indicate that endogenous medullary adenosine at physiological concentrations serves to dilate medullary vessels via A2 receptors, resulting in a natriuretic response that overrides the tubular A1 receptor-mediated antinatriuretic effects.

Adenosine mediates sustained adrenergic desensitization in the rat heart via activation of protein kinase C.

Adenosine attenuates the myocardial metabolic and contractile responses induced by ss-adrenergic stimulation. Our study was conducted to investigate the longevity of this antiadrenergic action after adenosine exposure. Adenosine (33 micromol/L) was infused into isolated perfused rat hearts for 1, 5, 30, or 60 minutes, and the adrenergic responsiveness (AR) to isoproterenol (10(-8) mol/L) was determined at the end of each infusion period and during a 45-minute adenosine washout period. Interstitial levels of adenosine, as determined from epicardial surface transudates, returned to preinfusion levels within 10 minutes of washout. The duration of adenosine infusion had no effect on the extent of attenuation of AR at the end of the infusion. Whereas AR returned to preadenosine levels with washout of shorter adenosine infusions (1 and 5 minutes), there was a slow and incomplete recovery of AR after the longer exposures (30 and 60 minutes) to adenosine. The magnitude of this persistent antiadrenergic effect (PAE) of adenosine at 15 minutes of washout was proportional to the epicardial concentration of adenosine during infusion of the nucleoside. Infusion of adenosine either with the nonselective adenosine receptor antagonist 8-p-sulfophenyl theophylline or with the selective A1-receptor antagonist 1,3-dipropyl, 8-cyclopentylxanthine, abolished the PAE during the washout period. In addition, the PAE could be demonstrated only with the selective A1-receptor agonist 2-chloro-N6-cyclopentyladenosine and not with the selective A3-receptor agonist 4-aminobenzyl-5'-N methylcarboxamido-adenosine. When the protein kinase C (PKC) inhibitor chelerythrine was coadministered with adenosine, the PAE of adenosine was not apparent during adenosine washout. A 30-minute infusion of phenylephrine, an alpha-adrenergic agonist that enhances PKC activity, produced a PAE that lasted for up to 30 minutes of washout. This effect was prevented by the coinfusion of chelerythrine. Thus, it is concluded that the PAE of adenosine is determined by the myocardial concentration of this nucleoside and is manifested when myocardial concentrations of adenosine returned to baseline levels. Moreover, a 5-minute duration of adenosine exposure is required for the expression of the PAE. This latter effect seems to be dependent on adenosine-induced PKC activation via A1-receptors.

The synthesis of new adenosine A3 selective ligands containing bioisosteric isoxazoles.

The synthesis and purinergic receptor binding of novel adenosine A3 ligands is described. Many selective A3 receptor agonists e.g. N-(3-iodobenzyl)adenosine-5'-methyluronamide (IB-MECA) contain a 4'-ribosylalkylamide moiety. We found that this amide and other 4'-functional groups could be replaced with an isosteric isoxazole, and the target molecules retained potent binding to the recombinant human A3 receptor.

The A3Adenosine Receptor Mediates Cell Spreading, Reorganization of Actin Cytoskeleton, and Distribution of Bcl-xL: Studies in Human Astroglioma Cells

The pathophysiological role of the adenosine A3receptor in the central nervous system is largely unknown. We have investigated the effects of the selective A3receptor agonist 2-chloro-N6-(3-iodobenzyl)-adenosine, Cl-IB-MECA, in cells of the astroglial lineage (human astrocytoma ADF cells). A marked reorganization of the cytoskeleton, with appearance of stress fibers and numerous cell protrusions, was found following exposure of cells to low (nM) concentrations of Cl-IB-MECA. These “trophic” effects were accompanied by induction of the expression of Rho, a small GTP-binding protein, which was virtually absent in control cells, and by changes of the intracellular distribution of the antiapoptotic protein Bcl-xL, that, in agonist-exposed cells, became specifically associated to cell protrusions. This is the first demonstration that the intracellular organization of Bcl-xLcan be modulated by the activation of a G-protein-coupled membrane receptor, such as the A3adenosine receptor. Moreover, modulation of the astrocytic cytoskeleton by adenosine may have intriguing implications in both nervous system development and in the response of the brain to trauma and ischemia.

Activation of A3 Adenosine Receptors on Human Eosinophils Elevates Intracellular Calcium

Adenosine (ADO) is a potent bronchoconstrictor in allergic patients and has been shown to increase the release of histamine from human lung tissues. Antagonists of ADO A1 and A2A receptors are not effective in attenuating these effects. Therefore, involvement of ADO A3 receptors in the bronchoconstrictor and/or inflammatory effects have to be considered. Eosinophils also play a pivotal role in allergic diseases such as asthma, thus it is natural to consider a link between the A3 receptor and eosinophils. Human peripheral blood eosinophils express the ADO A3 receptor as indicated by detection of the transcript for A3 receptors in polymerase chain reaction-amplified cDNA derived from the cells. A3 receptors on eosinophil membranes were characterized using the A3 receptor agonist radioligand 125I-labeled AB-MECA, which yielded Bmax and Kd values of 1.31 pmol/mg protein and 3.19 nmol/L, respectively. Treatment of eosinophils with the highly potent and selective A3 receptor agonist CI-IB-MECA clearly induced Ca2+ release from intracellular Ca2+ pools followed by Ca2+ influx, suggesting the presence of phospholipase C-coupled A3 receptors. In contrast, the ADO receptor agonists CPA and CGS 21680, selective for A1 and A2A receptors, respectively, at concentrations of < or = 30 mumol/ L did not elevate the intracellular Ca2+ level. These results attest to the existence of ADO A3 receptors on eosinophils and suggest that ADO stimulates these cells to release Ca2+ from intracellular stores via the activation of A3 receptors.