LJ529 attenuates mast cell-related inflammation via A3R-PKCε-ALDH2 pathway after subarachnoid hemorrhage in rats.

BACKGROUND AND PURPOSE: Mast cells (MCs) has been recognized as an effector of inflammation or a trigger of inflammatory factors during stroke. LJ529 was reported to attenuate inflammation through a Gi protein-coupled Adenosine A3 receptor (A3R) after ischemia. Here, we aim to study the protective effect and its mechanism of LJ529 in subarachnoid hemorrhage (SAH) rat model for mast cell-related inflammation. METHODS: 155 Sprague-Dawley adult male rats were used in experiments. Endovascular perforation was used for SAH model. Intraperitoneal LJ529 was performed 1 h after SAH. Neurological scores were measured 24 h after SAH. Rotarod and morris water maze tests were evaluated for 21 days after SAH. Mast cell degranulation was assessed with Toluidine blue staining and Chymase/Typtase protein expressions. Mast cell-related inflammation was evaluated using IL-6, TNF-α and MCP-1 protein expressions. MRS1523, inhibitor of GPR18 and ε-V1-2, inhibitor of PKCε were respectively given intraperitoneally (i.p.) 1 h and 30 min before SAH for mechanism studies. Pathway related proteins were investigated with western blot and immunofluorescence staining. RESULTS: Expression of A3R, PKCε increased after SAH. LJ529 treatment attenuated mast cell degranulation and inflammation. Meanwhile, both short-term and long-term neurological functions were improved after LJ529 treatment. Administration of LJ529 resulted in increased expressions of A3R, PKCε, ALDH2 proteins and decreased expressions of Chymase, Typtase, IL-6, TNF-α and MCP-1 proteins. MRS1523 abolished the treatment effects of LJ529 on neurobehavior and protein levels. ε-V1-2 also reversed the outcomes of LJ529 administration through reduction in protein expressions downstream of PKCε. CONCLUSIONS: LJ529 attenuated mast cell-related inflammation through inhibiting degranulation via A3R-PKCε-ALDH2 pathway after SAH. LJ529 may serve as a potential treatment strategy to relieve post-SAH brain injury.

Activation of adenosine A3 receptor inhibits inflammatory cytokine production in colonic mucosa of patients with ulcerative colitis by down-regulating the nuclear factor-kappa B signaling.

OBJECTIVES: The activation of the adenosine A3 receptor (A3AR) can regulate inflammation, but the way that this regulates colonic mucosal inflammation in ulcerative colitis (UC) remains unclear. This study aimed at examining A3AR expression and investigating the effect of A3AR activation on ex vivo cytokine expression and nuclear factor-kappa B (NF-κB) signaling in colonic mucosa. METHODS: Colonic mucosal biopsied tissue from 18 patients with UC and 11 healthy controls was tested for A3AR expression by immunofluorescence, quantitative real-time polymerase chain reaction and Western blot. Following treatment for 24 hours with or without 2-Cl-IB-MECA, an A3AR agonist, TNF-α and IL-1ß secreted by the cultured colonic mucosal tissue were quantified by ELISA. The colonic mucosal epithelia were dissected and treated with, or without 2-Cl-IB-MECA for 24 hours. The NF-κB p65 protein and its distribution in the cultured colonic epithelia were examined by immunofluorescence and Western blot. RESULTS: Compared with the controls, down-regulated A3AR expression and up-regulated TNF-α and IL-1ß production and NF-κB p65 protein were observed in the UC colonic mucosa. The activation of A3AR by 2-Cl-IB-MECA significantly decreased TNF-α and IL-1ß production and attenuated the NF-κB p65 activation in colonic tissues from patients with UC. CONCLUSIONS: A3AR activation inhibited inflammation by mitigating pro-inflammatory cytokine production and the NF-κB signal activation in colonic mucosa of patients with UC. A3AR activation may play a role in the pathogenesis of UC.

Ability of CP-532,903 to protect mouse hearts from ischemia/reperfusion injury is dependent on expression of A3 adenosine receptors in cardiomyoyctes.

A3 adenosine receptor (A3AR) agonists are effective at limiting injury caused by ischemia/reperfusion injury of the heart in experimental animal models. However, understanding of their mechanism of action, which is likely multifactorial, remains incomplete. In prior studies, it has been demonstrated that A3AR-mediated ischemic protection is blocked by glibenclamide and is absent in Kir6.2 gene ablated mice that lack the pore-forming subunit of the ATP-sensitive potassium (KATP) channel, suggesting one contributing mechanism may involve accelerated activation of KATP channels. However, presence of A3ARs in the myocardium has yet to be established. Utilizing a whole-cell recording technique, in this study we confirm functional expression of the A3AR in adult mouse ventricular cardiomyocytes, coupled to activation of ATP-dependent potassium (KATP) channels via Gi inhibitory proteins. We further show that ischemic protection provided by the selective A3AR agonist CP-532,903 in an isolated, buffer-perfused heart model is lost completely in Adora3LoxP/LoxP;Myh6-Cre mice, which is a newly developed model developed and comprehensively described herein whereby the A3AR gene (Adora3) is deleted exclusively in cardiomyocytes. Our findings, taken together with previously published work, are consistent with the hypothesis that A3AR agonists provide ischemic tolerance, at least in part, by facilitating opening of myocardial KATP channels.

Pulsed Electromagnetic Fields and Tissue Engineering of the Joints.

BACKGROUND: Bone and joint formation, maintenance, and regeneration are regulated by both chemical and physical signals. Among the physical signals there is an increasing realization of the role of pulsed electromagnetic fields (PEMF) in the treatment of nonunions of bone fractures. The discovery of the piezoelectric properties of bone by Fukada and Yasuda in 1953 in Japan established the foundation of this field. Pioneering research by Bassett and Brighton and their teams resulted in the approval by the Food and Drug Administration (FDA) of the use of PEMF in the treatment of fracture healing. Although PEMF has potential applications in joint regeneration in osteoarthritis (OA), this evolving field is still in its infancy and offers novel opportunities. METHODS: We have systematically reviewed the literature on the influence of PEMF in joints, including articular cartilage, tendons, and ligaments, of publications from 2000 to 2016. CONCLUSIONS: PEMF stimulated chondrocyte proliferation, differentiation, and extracellular matrix synthesis by release of anabolic morphogens such as bone morphogenetic proteins and anti-inflammatory cytokines by adenosine receptors A2A and A3 in both in vitro and in vivo investigations. It is noteworthy that in clinical translational investigations a beneficial effect was observed on improving function in OA knees. However, additional systematic studies on the mechanisms of action of PEMF on joints and tissues therein, articular cartilage, tendons, and ligaments are required.

Down-regulation of the A3 adenosine receptor in human mast cells upregulates mediators of angiogenesis and remodeling.

Adenosine activated mast cells have been long implicated in allergic asthma and studies in rodent mast cells have assigned the A3 adenosine receptor (A3R) a primary role in mediating adenosine responses. Here we analyzed the functional impact of A3R activation on genes that are implicated in tissue remodeling in severe asthma in the human mast cell line HMC-1 that shares similarities with lung derived human mast cells. Quantitative real time PCR demonstrated upregulation of IL6, IL8, VEGF, amphiregulin and osteopontin. Moreover, further upregulation of these genes was noted upon the addition of dexamethasone. Unexpectedly, activated A3R down regulated its own expression and knockdown of the receptor replicated the pattern of agonist induced gene upregulation. This study therefore identifies the human mast cell A3R as regulator of tissue remodeling gene expression in human mast cells and demonstrates a heretofore-unrecognized mode of feedback regulation that is exerted by this receptor.

Impairment of adenosine A3 receptor activity disrupts neutrophil migratory capacity and impacts innate immune function in vivo.

Adenosine possesses potent anti-inflammatory properties which are partly mediated by G(i) -coupled adenosine A3 receptors (A3Rs). A3R agonists have shown clinical benefit in a number of inflammatory conditions although some studies in A3R-deficient mice suggest a pro-inflammatory role. We hypothesised that, in addition to cell signalling effects, A3R compounds might inhibit neutrophil chemotaxis by disrupting the purinergic feedback loop controlling leukocyte migration. Human neutrophil activation triggered rapid upregulation of surface A3R expression which was disrupted by pre-treatment with either agonist (Cl-IB-MECA) or antagonist (MRS1220). Both compounds reduced migration velocity and neutrophil transmigration capacity without impacting the response to chemokines per se. Similar effects were observed in murine neutrophils, while cells from A3R-deficient mice displayed a constitutively impaired migratory phenotype indicating compound-induced desensitisation and genetic ablation had the same functional outcome. In a dextran sodium sulphate-induced colitis model, A3R-deficient mice exhibited reduced colon pathology and decreased tissue myeloperoxidase levels at day 8 - consistent with reduced neutrophil recruitment. However, A3R-deficient mice were unable to resolve the dextran sodium sulphate-induced inflammation and had elevated numbers of tissue-associated bacteria by day 21. Our data indicate that A3Rs play a role in neutrophil migration and disrupting this function has the potential to adversely affect innate immune responses.

Adenosine receptor expression in rheumatoid synovium: a basis for methotrexate action.

INTRODUCTION: Methotrexate (MTX) exerts at least part of its anti-inflammatory effects through adenosine receptors (ADOR). The aims of this study were to determine the expression of all four adenosine receptor genes (ADORA1, ADORA2A, ADORA2B, ADORA3 and ADORA3variant) in rheumatoid synovial tissue and any influence of MTX exposure on this expression. Furthermore, we investigated whether polymorphisms within ADORA3 were associated with response and/or adverse effects associated with MTX. METHODS: Adenosine receptor gene expression was undertaken using PCR in 20 rheumatoid arthritis (RA) synovial samples. A separate cohort of 225 RA patients receiving MTX was genotyped for SNPs in the ADORA3 receptor gene. Double immunofluorescence was used to identify cells expressing ADOR protein. RESULTS: All ADOR genes were expressed in all synovial samples. ADORA3 and A3variant were the dominant subtypes expressed irrespective of MTX therapy. Expression of ADORA2A and ADORA2B was increased in patients receiving MTX compared to those not receiving MTX. There was no association between the ADORA3 rs1544224 SNP and high and low disease activity or MTX-associated adverse effects. ADORA2B protein expression was most obvious in vascular endothelial cells whereas ADORA3 protein was more abundant and expressed by synovial fibroblasts. CONCLUSIONS: We have shown that adenosine receptors are expressed in RA synovium. There is differential expression of receptors such that ADORA3 is expressed at significantly higher levels. This evidence demonstrates the potential for MTX to exert its anti-inflammatory effects at the primary site of pathology within the joints of patients with RA.

Enhanced neurodegeneration after a high dose of methamphetamine in adenosine A3 receptor null mutant mice.

Previous reports have indicated that adenosine A3 receptor (A3R) knockout mice are more sensitive to ischemic or hypoxic brain injury. The purpose of this study was to examine if suppression of A3R expression is associated with increase in sensitivity to injury induced by a high dose of methamphetamine (Meth). Adult male A3R null mutant (-/-) mice and their controls (+/+) were injected with four doses (2 h apart) of Meth (10 mg/kg) or saline. Animals were placed in a behavioral activity chamber, equipped with food and water, for 52 h starting from one day after injections. The first 4 h were used for studying exploratory behaviors, and the next 48 h were used to measure locomotor activity. High doses of Meth equally reduced the 4-h exploratory behavior in -/- and +/+ mice. Meth suppressed locomotor activity between 4 and 52 h in both groups, with a greater reduction being found in the -/- mice. Brain tissues were collected at 3 days after the Meth or saline injections. Meth treatment reduced striatal dopamine (DA) levels in both +/+ and -/- mice with an increase in 3,4-dihydroxyphenylacetic acid (DOPAC)/DA ratio being found only in -/- animals. Meth also significantly increased ionized calcium-binding adaptor molecule 1 (Iba-1) and cleaved caspase-3 level in striatum, as well as Iba-1 and TNFα mRNA expression in nigra in -/-, compared to +/+, mice. Previous studies have shown that pharmacological suppression of vesicular monoamine transport 2 (VMAT2) by reserpine enhanced Meth toxicity by increasing cytosolic DA and inflammation. A significant reduction in striatal VMAT2 expression was found in -/- mice compared to +/+ mice, suggesting that increase in sensitivity to Meth injury in -/- mice may be related to a reduction in VMAT2 expression in these mice. In conclusion, our data suggest that A3R -/- mice are more sensitive to high doses of Meth.

The anti-inflammatory target A(3) adenosine receptor is over-expressed in rheumatoid arthritis, psoriasis and Crohn's disease.

The Gi protein associated A(3) adenosine receptor (A(3)AR) was recently defined as a novel anti-inflammatory target. The aim of this study was to look at A(3)AR expression levels in peripheral blood mononuclear cells (PBMCs) of patients with autoimmune inflammatory diseases and to explore transcription factors involved receptor expression. Over-expression of A(3)AR was found in PBMCs derived from patients with rheumatoid arthritis (RA), psoriasis and Crohn's disease compared with PBMCs from healthy subjects. Bioinformatics analysis demonstrated the presence of DNA binding sites for nuclear factor-kappaB (NF-kappaB) and cyclic AMP-responsive element binding protein (CREB) in the A(3)AR gene promoter. Up-regulation of NF-kappaB and CREB was found in the PBMCs from patients with RA, psoriasis and Crohn's disease. The PI3K-PKB/Akt signaling pathway, known to regulate both the NF-kappaB and CREB, was also up-regulated in the patients' PBMCs. Taken together, NF-kappaB and CREB are involved with the over-expression of A(3)AR in patients with autoimmune inflammatory diseases. The receptor may be considered as a specific target to combat inflammation.

Hypertonic saline up-regulates A3 adenosine receptor expression of activated neutrophils and increases acute lung injury after sepsis.

OBJECTIVE: Hypertonic saline resuscitation reduces tissue damage by inhibiting polymorphonuclear neutrophils. Hypertonic saline triggers polymorphonuclear neutrophils to release adenosine triphosphate that is converted to adenosine, inhibiting polymorphonuclear neutrophils through A2a adenosine receptors. Polymorphonuclear neutrophils also express A3 adenosine receptors that enhance polymorphonuclear neutrophil functions. Here we investigated whether A3 receptors may diminish the efficacy of hypertonic saline in a mouse model of acute lung injury. DESIGN: Randomized animal study and laboratory investigation. SETTING: University research laboratory. INTERVENTIONS: The effect of A3 receptors on the efficacy of hypertonic saline resuscitation was assessed in A3 receptor knockout and wild-type mice. Animals were treated with hypertonic saline (7.5% NaCl, 4 mL/kg) before or after cecal ligation and puncture, and acute lung injury and mortality were determined. The effect of timing of hypertonic saline exposure on A3 receptor expression and degranulation was studied in vitro with isolated human polymorphonuclear neutrophils. MEASUREMENTS AND MAIN RESULTS: Treatment of human polymorphonuclear neutrophils with hypertonic saline before stimulation with formyl methionyl-leucyl-phenylalanine inhibited A3 receptor expression and degranulation, whereas hypertonic saline-treatment after formyl methionyl-leucyl-phenylalanine-stimulation augmented A3 receptor expression and degranulation. Acute lung injury in wild-type mice treated with hypertonic saline after cecal ligation and puncture was significantly greater than in wild-type mice pretreated with hypertonic saline. This aggravating effect of delayed hypertonic saline-treatment was absent in A3 receptor knockout mice. Similarly, mortality in wild-type mice with delayed hypertonic saline-treatment was significantly higher (88%) than in animals treated with hypertonic saline before cecal ligation and puncture (50%). Mortality in A3 receptor knockout mice remained only 50% regardless of timing of hypertonic saline administration. CONCLUSIONS: Polymorphonuclear neutrophil A3 receptors expression determines whether hypertonic saline resuscitation inhibits or aggravates polymorphonuclear neutrophil-induced acute lung injury. These findings suggest that A3 antagonists could improve the efficacy of hypertonic saline resuscitation by reducing side effects in patients whose polymorphonuclear neutrophils are activated before hypertonic saline treatment.

Endogenous expression of adenosine A1, A2 and A3 receptors in rat C6 glioma cells.

Inhibitory and stimulatory adenosine receptors have been identified and characterized in both membranes and intact rat C6 glioma cells. In membranes, saturation experiment performed with [(3)H]DPCPX, selective A(1)R antagonist, revealed a single binding site with a K (D) = 9.4 +/- 1.4 nM and B (max) = 62.7 +/- 8.6 fmol/mg protein. Binding of [(3)H]DPCPX in intact cell revealed a K (D) = 17.7 +/- 1.3 nM and B (max )= 567.1 +/- 26.5 fmol/mg protein. On the other hand, [(3)H]ZM241385 binding experiments revealed a single binding site population of receptors with K (D) = 16.5 +/- 1.3 nM and B (max) = 358.9 +/- 52.4 fmol/mg protein in intact cells, and K (D) = 4.7 +/- 0.6 nM and B (max) = 74.3 +/- 7.9 fmol/mg protein in plasma membranes, suggesting the presence of A(2A) receptor in C6 cells. A(1), A(2A), A(2B) and A(3 )adenosine receptors were detected by Western-blotting and immunocytochemistry, and their mRNAs quantified by real time PCR assays. Gialpha and Gsalpha proteins were also detected by Western-blotting and RT-PCR assays. Furthermore, selective A(1)R agonists inhibited forskolin- and GTP-stimulated adenylyl cyclase activity and CGS 21680 and NECA stimulated this enzymatic activity in C6 cells. These results suggest that C6 glioma cells endogenously express A(1) and A(2) receptors functionally coupled to adenylyl cyclase inhibition and stimulation, respectively, and suggest these cells as a model to study the role of adenosine receptors in tumoral cells.

Methotrexate enhances the anti-inflammatory effect of CF101 via up-regulation of the A3 adenosine receptor expression.

Methotrexate (MTX) exerts an anti-inflammatory effect via its metabolite adenosine, which activates adenosine receptors. The A3 adenosine receptor (A3AR) was found to be highly expressed in inflammatory tissues and peripheral blood mononuclear cells (PBMCs) of rats with adjuvant-induced arthritis (AIA). CF101 (IB-MECA), an A3AR agonist, was previously found to inhibit the clinical and pathological manifestations of AIA. The aim of the present study was to examine the effect of MTX on A3AR expression level and the efficacy of combined treatment with CF101 and MTX in AIA rats. AIA rats were treated with MTX, CF101, or both agents combined. A3AR mRNA, protein expression and exhibition were tested in paw and PBMC extracts from AIA rats utilizing immunohistochemistry staining, RT-PCR and Western blot analysis. A3AR level was tested in PBMC extracts from patients chronically treated with MTX and healthy individuals. The effect of CF101, MTX and combined treatment on A3AR expression level was also tested in PHA-stimulated PBMCs from healthy individuals and from MTX-treated patients with rheumatoid arthritis (RA). Combined treatment with CF101 and MTX resulted in an additive anti-inflammatory effect in AIA rats. MTX induced A2AAR and A3AR over-expression in paw cells from treated animals. Moreover, increased A3AR expression level was detected in PBMCs from MTX-treated RA patients compared with cells from healthy individuals. MTX also increased the protein expression level of PHA-stimulated PBMCs from healthy individuals. The increase in A3AR level was counteracted in vitro by adenosine deaminase and mimicked in vivo by dipyridamole, demonstrating that receptor over-expression was mediated by adenosine. In conclusion, the data presented here indicate that MTX induces increased A3AR expression and exhibition, thereby potentiating the inhibitory effect of CF101 and supporting combined use of these drugs to treat RA.

A3 adenosine receptors and mitogen-activated protein kinases in lung injury following in vivo reperfusion.

INTRODUCTION: Although activation of A3 adenosine receptors attenuates reperfusion lung injury and associated apoptosis, the signaling pathway that mediates this protection remains unclear. Adenosine agonists activate mitogen-activated protein kinases, and these kinases have been implicated in ischemia/reperfusion injury; the purpose of this study was therefore to determine whether A3 adenosine receptor stimulation with reperfusion modulates expression of the different mitogen-activated protein kinases. In addition, we compared the effect of the A3 adenosine agonist IB-MECA with the newly synthesized, highly selective A3 adenosine receptor agonist MRS3558 on injury in reperfused lung. METHOD: Studies were performed in an in vivo spontaneously breathing cat model, in which the left lower lobe of the lung was isolated and subjected to 2 hours of ischemia and 3 hours of reperfusion. The selective A3 adenosine receptor agonists IB-MECA (0.05 mg/kg, 0.1 mg/kg, or 0.3 mg/kg) and MRS3558 (0.05 mg/kg or 0.1 mg/kg) were administered before reperfusion. RESULTS: Both A3 adenosine receptor agonists administered before reperfusion markedly (P < 0.01) attenuated indices of injury and apoptosis, including the percentage of injured alveoli, wet/dry weight ratio, myeloperoxidase activity, TUNEL (in situ TdT-mediated dUTP nick end labeling)-positive cells, and caspase 3 activity and expression. The more pronounced effects at low doses were observed with MRS3558. Increases in phosphorylated c-Jun amino-terminal protein kinase (JNK), p38, and extracellular signal-regulated kinase (ERK)1/2 levels were observed by the end of reperfusion compared with controls. Pretreatment with the A3 agonists upregulated phosphorylated ERK1/2 levels but did not modify phosphorylated JNK and p38 levels. CONCLUSION: The protective effects of A3 adenosine receptor activation are mediated in part through upregulation of phosphorylated ERK. Also, MRS3558 was found to be more potent than IB-MECA in attenuating reperfusion lung injury. The results suggest not only that enhancement of the ERK pathway may shift the balance between cell death and survival toward cell survival, but also that A3 agonists have potential as an effective therapy for ischemia/reperfusion-induced lung injury.

Strain dependence of receptor regulation on chemical preconditioning in mice hippocampus.

While one current focus for studying mechanisms of disease is investigation of transgenic mice confounding effects of the background strain often are neglected. We investigated mRNA expression of known markers of hypoxic tolerance by a semiquantitative RT-PCR (adenosine receptors (A1 and A3), nitric oxide synthases (eNOS and nNOS), APP production, progesterone receptor, and estrogen receptors alpha and beta) in CD-1, C3H, and B6 mice. We found differences in the baseline mRNA expression of adenosine A3 receptors in C3H mice and neuronal NOS in B6 mice as well as a distinct regulation of adenosine A3 receptors and estrogen receptor beta (no changes in C3H and B6 compared to upregulation in CD-1) on treatment of animals with a low dosage of 3-nitropropionate (20mg/kg body weight, i.p.). We conclude that the choice of background strain may confound interpretation of the effects of specific transgens in the study of the mechanisms of primary and induced hypoxic tolerance.

The A3 adenosine receptor is highly expressed in tumor versus normal cells: potential target for tumor growth inhibition.

PURPOSE: A(3) adenosine receptor (A(3)AR) activation was shown to inhibit the growth of various tumor cells via the down-regulation of nuclear factor kappaB and cyclin D1. To additionally elucidate whether A(3)AR is a specific target, a survey of its expression in tumor versus adjacent normal cells was conducted. EXPERIMENTAL DESIGN: A(3)AR mRNA expression in various tumor tissues was tested in paraffin-embedded slides using reverse transcription-PCR analysis. A comparison with A(3)AR expression in the relevant adjacent normal tissue or regional lymph node metastasis was performed. In addition, A(3)AR protein expression was studied in fresh tumors and was correlated with that of the adjacent normal tissue. RESULTS: Reverse transcription-PCR analysis of colon and breast carcinoma tissues showed higher A(3)AR expression in the tumor versus adjacent non-neoplastic tissue or normal tissue. Additional analysis revealed that the lymph node metastasis expressed even more A(3)AR mRNA than the primary tumor tissue. Protein analysis of A(3)AR expression in fresh tumors derived from colon (n = 40) or breast (n = 17) revealed that 61% and 78% had higher A(3)AR expression in the tumor versus normal adjacent tissue, respectively. The high A(3)AR expression level in the tumor tissues was associated with elevated nuclear factor kappaB and cyclin D1 levels. High A(3)AR mRNA expression was also demonstrated in other solid tumor types. CONCLUSIONS: Primary and metastatic tumor tissues highly express A(3)AR indicating that high receptor expression is a characteristic of solid tumors. These findings and our previous data suggest A(3)AR as a potential target for tumor growth inhibition.

Adenosine A3 receptors are located in neurons of the rat hippocampus.

Adenosine is a neuromodulator acting mainly via inhibitory A1 and facilitatory A2A receptors. Whole tissue PCR also identified adenosine A3 receptors in the brain and A3 receptor agonists affect CNS neuronal responses and viability. However, recent reports failed to detect A3 receptor expression in CNS neurons and showed that A3 receptor agonists can bind and activate A1 receptors. We now present evidence for the presence of A3 receptor mRNA in CNS neurons using single cell PCR analysis of laser dissected hippocampal neurons. Western blot analysis showed that A3 receptors are present in rat hippocampal nerve terminal membranes. This indicates that A3 receptors are present in CNS neurons in the hippocampus.

Functional expression of adenosine A2A and A3 receptors in the mouse dendritic cell line XS-106.

There is increasing evidence to suggest that adenosine receptors can modulate the function of cells involved in the immune system. For example, human dendritic cells derived from blood monocytes have recently been described to express functional adenosine A1, A2A and A3 receptors. Therefore, in the present study, we have investigated whether the recently established murine dendritic cell line XS-106 expresses functional adenosine receptors. The selective adenosine A3 receptor agonist 1-[2-chloro-6[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-1-deoxy-N-methyl-beta-D-ribofuranuronamide (2-Cl-IB-MECA) inhibited forskolin-mediated [3H]cyclic AMP accumulation and stimulated concentration-dependent increases in p42/p44 mitogen-activated protein kinase (MAPK) phosphorylation. The selective adenosine A2A receptor agonist 4-[2-[[-6-amino-9-(N-ethyl-beta-D-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzene-propanoic acid (CGS 21680) stimulated a robust increase in [3H]cyclic AMP accumulation and p42/p44 MAPK phosphorylation. In contrast, the selective adenosine A1 receptor agonist CPA (N6-cyclopentyladenosine) did not inhibit forskolin-mediated [3H]cyclic AMP accumulation or stimulate increases in p42/p44 MAPK phosphorylation. These observations suggest that XS-106 cells express functional adenosine A2A and A3 receptors. The non-selective adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA) inhibited lipopolysaccharide-induced tumour necrosis factor-alpha (TNF-alpha) release from XS-106 cells in a concentration-dependent fashion. Furthermore, treatment with Cl-IB-MECA (1 microM) or CGS 21680 (1 microM) alone produced a partial inhibition of lipopolysaccharide-induced TNF-alpha release (when compared to NECA), whereas a combination of both agonists resulted in the inhibition of TNF-alpha release comparable to that observed with NECA alone. Treatment of cells with the adenosine A2A receptor selective antagonists 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5ylamino]ethyl)phenol (ZM 241385; 100 nM) and 5-amino-2-(2-furyl)-7-phenylethyl-pyrazolo[4,3-e]-1,2,4-triazolo[1,5c]pyrimidine (SCH 58261; 100 nM) and the adenosine A3 receptor selective antagonist N-[9-chloro-2-(2-furanyl)[1,2,4]-triazolo[1,5-c]quinazolin-5-benzeneacetamide (MRS 1220; 100 nM) partially blocked the inhibitory effects of NECA on lipopolysaccharide-induced TNF-alpha release. Combined addition of MRS 1220 and SCH 58261 completely blocked the inhibitory effects of NECA on lipopolysaccharide-induced TNF-alpha release. In conclusion, we have shown that the mouse dendritic cell line XS-106 expresses functional adenosine A2A and A3 receptors, which are capable of modulating TNF-alpha release.

A3 adenosine receptor activation in melanoma cells: association between receptor fate and tumor growth inhibition.

Activation of the Gi protein-coupled A3 adenosine receptor (A3AR) has been implicated in the inhibition of melanoma cell growth by deregulating protein kinase A and key components of the Wnt signaling pathway. Receptor activation results in internalization/recycling events that play an important role in turning on/off receptor-mediated signal transduction pathways. Thus, we hereby examined the association between receptor fate, receptor functionality, and tumor growth inhibition upon activation with the agonist 1-deoxy-1-[6-[[(3-iodophenyl)-methyl]amino]-9H-purine-9-yl]-N-methyl-beta-D-ribofuranuronamide (IB-MECA). Results showed that melanoma cells highly expressed A3AR on the cell surface, which was rapidly internalized to the cytosol and "sorted" to the endosomes for recycling and to the lysosomes for degradation. Receptor distribution in the lysosomes was consistent with the down-regulation of receptor protein expression and was followed by mRNA and protein resynthesis. At each stage, receptor functionality was evidenced by the modulation in cAMP level and the downstream effectors protein kinase A, glycogen synthase kinase-3beta, c-Myc, and cyclin D1. The A3AR antagonist MRS 1523 counteracted the internalization process as well as the modulation in the expression of the signaling proteins, demonstrating that the responses are A3AR-mediated. Supporting this notion are the in vivo studies showing tumor growth inhibition upon IB-MECA treatment and reverse of this response when IB-MECA was given in combination with MRS 1523. In addition, in melanoma tumor lesions derived from IB-MECA-treated mice, the expression level A3AR and the downstream key signaling proteins were modulated in the same pattern as was seen in vitro. Altogether, our observations tie the fate of A3AR to modulation of downstream molecular mechanisms leading to tumor growth inhibition both in vitro and in vivo.