Adenosine A3 Receptor Stimulation Research Articles

A novel GRK2 inhibitor alleviates experimental arthritis through restraining Th17 cell differentiation

T helper type 17 (Th17) cell which is induced by interleukine-6 (IL-6)-signal transducers and activators of transcription 3 (STAT3) signaling is a central pro-inflammatory T cell subtype in rheumatoid arthritis (RA) and could be significantly reduced by paeoniflorin-6′-O-benzene sulfonate (CP-25) treatment with unclear mechanisms. This study was aimed to found out the mechanism of CP-25 in hampering Th17 cells differentiation in arthritic animals thus explore more therapeutic targets for RA. In mice with collagen-induced arthritis (CIA), both circulating and splenic Th17 subsets were expanded with increased STAT3 phosphorylation and decreased Src homology 2 domain-containing protein tyrosine phosphatase 1 (SHP1)-β-arrestin2 (arrb2)-STAT3 interaction in CD4+ helper T (Th) cells. Either CP-25 or paroxetine (PAR), an established G protein coupled receptor kinase 2 (GRK2) inhibitor treatment effectively relieved the joints inflammation of CIA mice with substantially reduced Th17 cell population through inhibiting STAT3 and restoring the SHP1-arrb2-STAT3 complex. Knockout of arrb2 exacerbated the clinical manifestations of collagen antibody-induced arthritis with upregulated Th17 cells. In vitro studies revealed that depletion of arrb2 or inhibition of SHP1 promoted Th17 cell differentiation. Moreover, stimulation of adenosine A3 receptor (A3AR) simultaneously promoted Th17 cell differentiation via accelerating abbr2-A3AR binding, which could be prevented through inhibiting GRK2 phosphorylation by CP-25 or PAR, or genetically reducing GRK2. This work has demonstrated that CP-25 or PAR treatment recovers the SHP1-arrb2-STAT3 complex which prevents STAT3 activation in Th cells through reducing arrb2 recruitment to A3AR by inhibiting GRK2 phosphorylation, leading to the reduction in Th17 cell differentiation and arthritis attenuation.

The effects of the adenosine A3 receptor agonist IB-MECA on sodium taurocholate-induced experimental acute pancreatitis.

The role of adenosine A3 receptors and their distribution in the gastrointestinal tract have been widely investigated. Most of the reports discuss their role in intestinal inflammations. However, the role of adenosine A3 receptor agonist in pancreatitis has not been well established. The aim of this study is (Ed note: Purpose statements should be in present tense) to evaluate the effects of the adenosine A3 receptor agonist on the course of sodium taurocholate-induced experimental acute pancreatitis (EAP). The experiments were performed on 80 male Wistar rats, 58 of which survived, subdivided into 3 groups: C—control rats, I—EAP group, and II—EAP group treated with the adenosine A3 receptor agonist IB-MECA (1-deoxy-1-6[[(3-iodophenyl) methyl]amino]-9H-purin-9-yl)-N-methyl-B-d-ribofuronamide at a dose of 0.75 mg/kg b.w. i.p. at 48, 24, 12 and 1 h before and 1 h after the injection of 5 % sodium taurocholate solution into the biliary–pancreatic duct. Serum for α-amylase and lipase determinations and tissue samples for morphological examinations were collected at 2, 6, and 24 h of the experiment. In the IB-MECA group, α-amylase activity was decreased with statistically high significance compared to group I. The activity of lipase was not significantly different among the experimental groups but higher than in the control group. The administration of IB-MECA attenuated the histological parameters of inflammation as compared to untreated animals. The use of A3 receptor agonist IB-MECA attenuates EAP. Our findings suggest that stimulation of adenosine A3 receptors plays a positive role in the sodium taurocholate-induced EAP in rats.

<i>Cordyceps sinensis</i> Acts As an Adenosine A<sub>3</sub> Receptor Agonist on Mouse Melanoma and Lung Carcinoma Cells, and Human Fibrosarcoma and Colon Carcinoma Cells

Cordyceps sinensis, a parasitic fungus on the larva of Lapidoptera, has been used as a traditional Chinese medicine. We previously reported that the growth of B16-BL6 mouse melanoma (B16-BL6) cells and mouse Lewis lung carcinoma (LLC) cells was inhibited by cordycepin (3’-deoxyadenosine), an ingredient of Cordyceps sinensis, and its effect was antagonized by MRS1191, a selective adenosine A3 receptor (A3-R) antagonist although adenosine (up to 100 μM) had no effect on the growth of B16-BL6 and LLC cells. In this study, we investigated whether water extracts of Cordyceps sinensis (WECS) inhibit the growth of B16-BL6 cells, LLC cells, HT1080 human fibrosarcoma (HT1080) cells and CW-2 human colon carcinoma (CW-2) cells via their A3-R. As a result, the growth of all cell lines were potently inhibited by WECS (10 μg/mL) and the inhibitory effect of WECS was significantly antagonized by MRS1191 (1 μM). Furthermore, WECS included 2.34% w/w cordycepin and 0.12% w/w adenosine as components according to the HPLC- ECD system. In conclusion, WECS inhibited the proliferation of four cancer cell lines by stimulation of A3-R and the main component in WECS with anticancer action might be cordycepin instead of adenosine.

Adenosine A3receptor stimulation induces protection of skeletal muscle from eccentric exercise-mediated injury

Effective therapy to reduce skeletal muscle injury associated with severe or eccentric exercise is needed. The purpose of this study was to determine whether adenosine receptor stimulation can mediate protection from eccentric exercise-induced muscle injury. Downhill treadmill exercise (-15 degrees ) was used to induce eccentric exercise-mediated skeletal muscle injury. Experiments were conducted in both normal wild-type (WT) mice and also in beta-sarcoglycan knockout dystrophic mice, animals that show an exaggerated muscle damage with the stress of exercise. In the vehicle-treated WT animals, eccentric exercise increased serum creatine kinase (CK) greater than 3-fold to 358.9 +/- 62.7 U/l (SE). This increase was totally abolished by stimulation of the A(3) receptor. In the dystrophic beta-sarcoglycan-null mice, eccentric exercise caused CK levels to reach 55,124 +/- 5,558 U/l. A(3) receptor stimulation in these animals reduced the CK response by nearly 50%. In the dystrophic mice at rest, 10% of the fibers were found to be damaged, as indicated by Evans blue dye staining. While this percentage was doubled after exercise, A(3) receptor stimulation eliminated this increase. Neither the A(1) receptor agonist 2-chloro-N(6)-cyclopentyladenosine (0.05 mg/kg) nor the A(2A) receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (0.07 mg/kg) protected skeletal muscle from eccentric exercise injury in WT or dystrophic mice. The protective effect of adenosine A(3) receptor stimulation was absent in mice, in which genes for phospholipase C beta2/beta3 (PLCbeta2/beta3) and beta-sarcoglycan were deleted. The present study elucidates a new protective role of the A(3) receptor and PLCbeta2/beta3 and points to a potentially effective therapeutic strategy for eccentric exercise-induced skeletal muscle injury.

Cordycepin (3′-deoxyadenosine) inhibits the growth of B16-BL6 mouse melanoma cells through the stimulation of adenosine A3 receptor followed by glycogen synthase kinase-3β activation and cyclin D1 suppression

Cordyceps sinensis, a parasitic fungus on the larvae of Lepidoptera, has been used as a traditional Chinese medicine. We previously reported that the growth of B16-BL6 mouse melanoma (B16-BL6) cells was inhibited by cordycepin (3'-deoxyadenosine), an active ingredient of C. sinensis, and its effect was antagonized by MRS1191, a selective adenosine A3 receptor antagonist. In this study, the radioligand binding assay using [125I]-AB-MECA (a selective adenosine A3 receptor agonist) has shown that B16-BL6 cells express adenosine A3 receptors and that cordycepin binds to these receptors. We also confirmed the involvement of adenosine A3 receptors in the action of cordycepin using MRS1523 and MRS1220, specific adenosine A3 receptor antagonists. Next, indirubin, a glycogen synthase kinase-3beta (GSK-3beta) inhibitor, antagonized the growth suppression induced by cordycepin. Furthermore, the level of cyclin D1 protein in B16-BL6 cells was decreased by cordycepin using Western blot analysis. In conclusion, this study demonstrated that cordycepin inhibits the proliferation of B16-BL6 cells by stimulating adenosine A3 receptors followed by the Wnt signaling pathway, including GSK-3beta activation and cyclin D1 inhibition.

Adenosine A3 receptor-induced CCL2 synthesis in cultured mouse astrocytes.

During neuropathological conditions, high concentrations of adenosine are released, stimulating adenosine receptors in neurons and glial cells. It has recently been shown that stimulation of adenosine receptors in glial cells induces the release of neuroprotective substances such as NGF, S-100beta, and interleukin-6 (IL-6). It has therefore been suggested that glial adenosine receptors are involved in neuroprotection. Since recently neuroprotective effects of the chemokine CCL2 (formerly known as MCP-1) have been reported, we investigated the possible effect of adenosine receptor stimulation on glial CCL2 synthesis. Here we show that stimulation of cultured murine astrocytes with the selective adenosine A3 receptor agonist 2-chloro-N6-(3-iodobenzyl)-N-methyl-5'-carbamoyladenosine (CL-IB-MECA) induced the release of CCL2. Specific ligands for adenosine A1 or A2 receptors did not affect CCL2 release. Furthermore, CL-IB-MECA-induced CCL2 synthesis was inhibited by adenosine A3 receptor antagonists. These results show that stimulation of adenosine A3 receptors in astrocytes induced the release of CCL2, thus supporting the assumption that adenosine receptors in glial cells regulate the synthesis of neuroprotective substances.

The influence of adenosine A3 receptor agonist: IB-MECA, on scopolamine- and MK-801-induced memory impairment

The effects of adenosine A3 agonist IB-MECA on scopolamine- and MK-801-induced impairment of spontaneous alternation and learning abilities were examined using Y-maze and passive avoidance tasks in mice. IB-MECA given 20 min before test had no effect on spontaneous alternation performance. Similarly learning abilities tested in passive avoidance were not disturbed after IB-MECA administration before training session. IB-MECA significantly diminished scopolamine- and MK-801-induced impairment of spontaneous alternation in Y-maze and learning abilities in passive avoidance task as well as reduced higher locomotor activity in MK-801-treated group. This ameliorating effect of IB-MECA was not antagonised by adenosine A1 antagonists CPX. Obtained results indicate that adenosine A3 receptor stimulation may ameliorate spatial memory and long term memory impairments in terms of cholinergic and glutamatergic deficits induced by scopolamine and MK-801, respectively.

Adenosine A3receptor-mediated potentiation of mucociliary transport and epithelial ciliary motility

To examine the effect of adenosine A(3) receptor stimulation on airway mucociliary clearance, we measured transport of Evans blue dye in rabbit trachea in vivo and ciliary motility of epithelium by the photoelectric method in vitro. Mucociliary transport was enhanced dose dependently by the selective A(3) agonist N(6)-(3-iodobenzyl)-5'-N-methylcarbamoyladenosine (IB-MECA) and to a lesser extent by the less-selective N(6)-2-(4-amino-3-iodophenyl)ethyladenosine, whereas the A(1) agonist N-cyclopentyladenosine (CPA) and the A(2) agonist CGS-21680 had no effect. The effect of IB-MECA was abolished by pretreatment with the selective A(3) antagonist MRS-1220 but not by the A(1) antagonist 1,3-dipropyly-8-cyclopentylxanthine or the A(2) antagonist 3,7-dimethyl-L-propargylxanthine. Epithelial ciliary beat frequency was increased by IB-MECA in a concentration-dependent manner, the maximal increase being 33%, and this effect was inhibited by MRS-1220. The IB-MECA-induced ciliary stimulation was not altered by the Rp diastereomer of cAMP but was greatly inhibited by Ca(2+)-free medium containing BAPTA-AM. Incubation with IB-MECA increased intracellular Ca(2+) contents. Therefore, A(3) agonist enhances airway mucociliary clearance probably through Ca(2+)-mediated stimulation of ciliary motility of airway epithelium.

Right thing at a wrong time? Adenosine A3 receptors and cerebroprotection in stroke.

The involvement of adenosine A3 receptors in normal and pathologic functions of the brain remains to be defined. Previous studies have shown that chronic preischemic administration of the agonist [N6-(3-iodobenzyl)-5'-N-methylcarboxoamidoadenosine or IB-MECA) results in a significant protection of neurons in selectively vulnerable brain regions and in an equally significant reduction of the subsequent mortality. Acute administration of the drug, on the other hand, resulted in a pronounced worsening of these parameters. We now report that the effect of administration of IB-MECA depends on the timing of treatment with respect to the onset of the focal insult, and provide the first data supporting speculation that treatment with adenosine A3 receptor agonists may decrease the infarct size following focal brain ischemia. Treatment with IB-MECA administered 20 min prior to transient middle cerebral ischemia (MCAOt = 30 min) resulted in a significant increase of the infarct size (p < 0.01), whereas administration 20 min after ischemia resulted in statistically significant decrease of the infarct volume. Postischemic treatment results in improved neuronal preservation, decreased intensity of reactive gliosis, and pronounced reduction of microglial infiltration. The data indicate that the effects of adenosine A3 receptor stimulation depend on the differential impact of these receptors on both neuronal and non-neuronal elements of the cerebral tissue, for example, astrocytes, microglia, and vasculature.

Studies on the effects of adenosine A 3 receptor stimulation on human eosinophils isolated from non-asthmatic or asthmatic donors

This study was designed to demonstrate the presence of adenosine A3 receptors on human peripheral blood eosinophils, and to investigate the effect of A3 receptor stimulation on eosinophil function. Eosinophils from either non-asthmatic or asthmatic donors. Eosinophils were isolated from peripheral venous blood by discontinuous gradient centrifugation and negative immunoselection. Receptor localisation was investigated by immunoblotting and by immunocytochemistry using a novel antibody specific for the human A3 receptor. Two pharmacological responses were studied: elevation of intracellular calcium in single eosinophils, measured by microfluorimetry, and hydrogen peroxide generation in cell suspensions. The expression of A3 receptors by eosinophils was confirmed using the selective antibody. Addition of the A3 receptor selective agonist, IB-MECA (100 nM), produced increases in intracellular calcium in less than 10% of the eosinophils isolated from non-asthmatic donors. These responses were only partially attenuated with the A3 receptor antagonist, I-ABOPX. IB-MECA (0.001-1000 nM) did not stimulate hydrogen peroxide (H2O2) generation, nor did it enhance fMLP- or C5a-stimulated generation of H2O2. In fact high concentrations of IB-MECA inhibited the generation of H2O2 (when stimulated by fMLP or C5a), an effect probably mediated by A2 receptors. Similar results were obtained using eosinophils from asthmatic donors. Stimulation of adenosine A3 receptors does not appear to be a prime mechanism for free radical generation by human peripheral blood eosinophils.

Stimulation of adenosine A3 receptors in cerebral ischemia. Neuronal death, recovery, or both?

The role of the adenosine A3 receptor continues to baffle, and, despite an increasing number of studies, the currently available data add to, rather than alleviate, the existing confusion. The reported effects of adenosine A3 receptor stimulation appear to depend on the pattern of drug administration (acute vs. chronic), dose, and type of the target tissue. Thus, while acute exposure to A3 receptor agonists protects against myocardial ischemia, it is severely damaging when these agents are given shortly prior to cerebral ischemia. Mast cells degranulate when their A3 receptors are stimulated. Degranulation of neutrophils is, on the other hand, impaired. While reduced production of reactive nitrogen species has been reported following activation of A3 receptors in collagen-induced arthritis, the process appears to be enhanced in cerebral ischemia. Indeed, immunocytochemical studies indicate that both pre- and postischemic treatment with A3 receptor antagonist dramatically reduces nitric oxide synthase in the affected hippocampus. Even more surprisingly, low doses of A3 receptor agonists seem to enhance astrocyte proliferation, while high doses induce their apoptosis. This review concentrates on the studies of cerebral A3 receptors and, based on the available evidence, discusses the possibility of adenosine A3 receptor serving as an integral element of the endogenous cerebral neuroprotective complex consisting of adenosine and its receptors.

An agonist of adenosine A3 receptors decreases interleukin-12 and interferon-γ production and prevents lethality in endotoxemic mice

We have recently observed that the selective adenosine A3 receptor agonist N6-(3-iodobenzyl)-adenosine-5′-N-methyluronamide (IB-MECA) augments interleukin-10 and inhibits tumor necrosis factor-α production in endotoxemic mice. In the present study, we extended our investigations into the effect of this compound on the bacterial lipopolysaccharide (endotoxin)-induced inflammatory response in the BALB/c, as well as in the C57BL/6 interleukin-10+/+ and the interleukin-10 deficient C57BL/6 interleukin-100/0 mice strains. In the BALB/c mice, i.p. pre-treatment with IB-MECA (0.2 and 0.5 mg/kg) decreased lipopolysaccharide (60 mg/kg i.p.)-induced plasma levels of interleukin-12 (p40 and p70), interferon-γ, and nitrite/nitrate (breakdown products of nitric oxide (NO)). On the other hand, pre-treatment with this compound failed to influence lipopolysaccharide-induced plasma interleukin-1α, interleukin-6, and corticosterone concentrations. Similar to its effect in BALB/c mice, IB-MECA enhanced the release of interleukin-10 in the C57BL/6 interleukin-10+/+ mice. Furthermore, IB-MECA inhibited the production of interleukin-12, interferon-γ, and NO in both the C57BL/6 interleukin-10+/+ and C57BL/6 interleukin-100/0 mice, suggesting that the inhibition of pro-inflammatory cytokine production by this compound is independent of the increased release of interleukin-10. Finally, pre-treatment with this compound protected mice against lipopolysaccharide (60 mg/kg i.p.)-induced lethality. These results indicate that stimulation of adenosine A3 receptors has potent anti-inflammatory effects and may represent a potential strategy in the treatment of septic shock and other inflammatory diseases.

Selective activation of adenosine A3 receptors with N6-(3-chlorobenzyl)-5'-N-methylcarboxamidoadenosine (CB-MECA) provides cardioprotection via KATP channel activation.

The aim of this study was to characterize the adenosine A3 receptor agonist, N6-(3-chlorobenzyl)-5'-N-methylcarboxamidoadenosine (CB-MECA), evaluate its ability to reduce myocardial ischemia/reperfusion injury and determine the role of KATP-channel activation in A3 receptor-mediated cardioprotection. Binding affinities and adenylate cyclase inhibition were examined in CHO cells expressing rabbit recombinant adenosine A1 or A3 receptors. Infarct size (normalized for area-at-risk; % IA/AAR) was measured in buffer-perfused rabbit hearts exposed to 30-min regional ischemia and 120 min of reperfusion. CB-MECA was 100-fold selective for A3 vs. A1 receptors (A3 Ki: 1 nM; A1 Ki: 105 nM). Five-min perfusion with CB-MECA before ischemia/reperfusion elicited a concentration-dependent reduction in infarct size (EC50: 0.3 nM). The CB-MECA-dependent cardioprotection (control: 58 +/- 2; CB-MECA: 21 +/- 3% IA/AAR) was unchanged by an A1-selective concentration of the antagonist, BWA1433, but was completely prevented (P < 0.05) by a nonselective (A1/A3) concentration (55 +/- 6% IA/AAR). The KATP channel inhibitors, glibenclamide and 5-HD, had no effect on control infarct size, yet significantly (P < 0.05) blunted the CB-MECA-dependent cardioprotection (glibenclamide: 49 +/- 6; 5-HD: 58 +/- 4% IA/AAR). CB-MECA is a novel 100-fold A3 receptor-selective agonist which should prove useful for elucidating A3-dependent mechanisms in the rabbit heart. Selective stimulation of adenosine A3 receptors with CB-MECA reduces myocardial ischemia/reperfusion injury via a mechanism which involves activation of KATP channels.

Adenosine A3 receptor stimulation inhibits migration of human eosinophils.

Activation of adenosine A3 receptors (A3-R) produced a dose-dependent reduction in the chemotaxis of human eosinophils to platelet-activating factor (PAF), RANTES, and leukotriene B4 (LTB4) to a maximum of 58, 48, and 52%, respectively (P < 0.02). This effect was completely reversed by selective A3-R antagonists. In contrast, activation of A1 or A2a-R did not affect PAF-induced eosinophil chemotaxis. PAF up-regulated the expression of CDllb/CD18, down-regulated L-selectin, and also increased F-actin assembly in eosinophils. The expression of these activation markers was not influenced by A3-R, A2a, or A1-R stimulation. Activation of A3-R may play an important role in inflammation by inhibiting eosinophil migration.

Adenosine A3 receptors promote degranulation of rat mast cells both in vitro and in vivo.

To investigate the effects of adenosine receptor agonists and antagonists on 5-HT release from rat isolated pleural mast cells and on plasma protein extravasation in the skin of conscious rats. In vitro Rat isolated pleural mast cells were loaded with [14C] 5-HT, sensitised with mouse monoclonal anti-DNP and then challenged with human serum albumin-DNP. DNP-stimulated 5-HT release from mast cells was determined. In vivo Rats, loaded intravenously with [125I] human serum albumin, were injected intradermally with adenosine agonists at sites on the back. 30 min later plasma protein extravasation at each injection site was determined. In isolated mast cells, each adenosine agonist enhanced DNP-induced 5-HT release, N6-(3-iodobenzyl)-5-(N-methyl-carboxamidoadenosine), (IB-MECA), being the most potent agonist. The adenosine A1/A2 antagonist, 8-phenyltheophylline (8-PT), had no effect on the response to IB-MECA. In contrast, 3-(4-amino-iodobenzyl)-8-[4-[[[carboxy]methyl]oxy]phenyl]-1-propylxanthi ne, (I-ABOPX), inhibited (pA2 6.2) the IB-MECA responses. In the skin of conscious rats, intradermal IB-MECA produced a marked plasma protein extravasation (PPE) which was mimicked by N6-2-(4-aminophenyl)-ethyladenosine (APNEA). The PPE produced by IB-MECA was not affected by either 8-PT or CGS15943A, but was virtually abolished by cyproheptadine and in rats pre-treated with Compound 48/80. These results indicate that stimulation of adenosine A3 receptors both enhances degranulation in vitro and directly produces degranulation of rat mast cells in vivo.