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1.
The A 3 adenosine receptor (AR) is emerging as an attractive drug target. Antagonists are proposed for the potential treatment of glaucoma and asthma. However, currently available A 3AR antagonists are potent in human and some large animals, but weak or inactive in mouse and rat. In this study, we re-synthesized a previously reported A 3AR antagonist, DPTN, and evaluated its affinity and selectivity at human, mouse, and rat ARs. We showed that DPTN, indeed, is a potent A 3AR antagonist for all three species tested, albeit a little less selective for mouse and rat A 3AR in comparison to the human A 3AR. DPTN’s Ki values at respective A 1, A 2A, A 2B, and A 3 receptors were (nM) 162, 121, 230, and 1.65 (human); 411, 830, 189, and 9.61 (mouse); and 333, 1147, 163, and 8.53 (rat). Its antagonist activity at both human and mouse A 3ARs was confirmed in a cyclic AMP functional assay. Considering controversial use of currently commercially available A 3AR antagonists in rats and mice, we also re-examined other commonly used and selective A 3AR antagonists under the same experimental conditions. The Ki values of MRS1523 were shown to be 43.9, 349, and 216 nM at human, mouse, and rat A 3ARs, respectively. MRS1191 and MRS1334 showed incomplete inhibition of [ 125I]I-AB-MECA binding to mouse and rat A 3ARs, while potent human A 3AR antagonists, MRS1220, MRE3008F20, PSB10, PSB-11, and VUF5574 were largely inactive. Thus, we demonstrated that DPTN and MRS1523 are among the only validated A 3AR antagonists that can be possibly used (at an appropriate concentration) in mouse or rat to confirm an A 3AR-related mechanism or function. Supplementary InformationThe online version contains supplementary material available at 10.1007/s11302-021-09823-5. 相似文献
2.
Adenosine receptors (ARs) have emerged as new drug targets. The majority of data on affinity/potency and selectivity of AR ligands described in the literature has been obtained for the human species. However, preclinical studies are mostly performed in mouse or rat, and standard AR agonists and antagonists are frequently used for studies in rodents without knowing their selectivity in the investigated species. In the present study, we selected a set of frequently used standard AR ligands, 8 agonists and 16 antagonists, and investigated them in radioligand binding studies at all four AR subtypes, A 1, A 2A, A 2B, and A 3, of three species, human, rat, and mouse. Recommended, selective agonists include CCPA (for A 1AR of rat and mouse), CGS-21680 (for A 2A AR of rat), and Cl-IB-MECA (for A 3AR of all three species). The functionally selective partial A 2B agonist BAY60-6583 was found to additionally bind to A 1 and A 3AR and act as an antagonist at both receptor subtypes. The antagonists PSB-36 (A 1), preladenant (A 2A), and PSB-603 (A 2B) displayed high selectivity in all three investigated species. MRS-1523 acts as a selective A 3AR antagonist in human and rat, but is only moderately selective in mouse. The comprehensive data presented herein provide a solid basis for selecting suitable AR ligands for biological studies. Electronic supplementary materialThe online version of this article (doi:10.1007/s11302-015-9460-9) contains supplementary material, which is available to authorized users. 相似文献
3.
The effects of standard adenosine receptor (AR) agonists and antagonists on the proliferation of human T lymphocytes, unstimulated and phytohemagglutinin-stimulated human peripheral blood lymphocytes (PBL), and Jurkat T cells were investigated. Real-time PCR measurements confirmed the presence of all four AR subtypes on the investigated cells, although at different expression levels. A 2A ARs were predominantly expressed in PBL and further upregulated upon stimulation, while malignant Jurkat T cells showed high expression levels of A 1, A 2A, and A 2B ARs. Cell proliferation was measured by [ 3H]-thymidine incorporation assays. Several ligands, including the subtype-selective agonists CPA (A 1), BAY60-6583 (A 2B), and IB-MECA (A 3), and the antagonists PSB-36 (A 1), MSX-2 (A 2A), and PSB-10 (A 3) significantly inhibited cell proliferation at micromolar concentrations, which were about three orders of magnitude higher than their AR affinities. In contrast, further investigated AR ligands, including the agonists NECA (nonselective) and CGS21680 (A 2A), and the antagonists preladenant (SCH-420814, A 2A), PSB-1115 (A 2B), and PSB-603 (A 2B) showed no or only minor effects on lymphocyte proliferation. The anti-proliferative effects of the AR agonists could not be blocked by the corresponding antagonists. The non-selective AR antagonist caffeine stimulated phytohemagglutinin-activated PBL with an EC 50 value of 104 μM. This is the first study to compare a complete set of commonly used AR ligands for all subtypes on lymphocyte proliferation. Our results strongly suggest that these compounds induce an inhibition of lymphocyte proliferation and cell death through AR-independent mechanisms. 相似文献
4.
A 1 adenosine receptors (ARs) reduce, and A 2ARs increase intraocular pressure, partly by differentially altering resistance to aqueous humor outflow. It is unknown whether the opposing effects of A 1AR and A 2AR agonists are mediated at different outflow-pathway cell targets or by opposing actions on a single cell target. We tested whether a major outflow-pathway cell, the trabecular meshwork (TM) cell might constitute the primary AR-agonist target and respond differentially to A 1, A 2A and A 3AR agonists. Receptor activation in human TM cells was identified by applying subtype-selective AR agonists: CPA and ADAC for A 1ARs, CGS 21680 and DPMA for A 2AARs, and Cl-IB-MECA and IB-MECA for A 3ARs. Stimulation of A 1, A 2A and A 3ARs elevated Ca 2+, measured with fura-2. Whole-cell patch clamping indicated that AR agonists activated ion channels non-uniformly, possibly reflecting variability in magnitude of agonist-triggered second-messenger responses. A 1, A 2A and A 3AR agonists all reduced volume, determined by calcein cell imaging. The endogenous source of adenosine delivery to the outflow pathway could be the TM cells since these cells were stimulated to release ATP by hypotonic perfusion. We conclude that: (1) TM cells express functional A 1, A 2A and A 3ARs; and (2) the reported differential effects of AR agonists on aqueous humor outflow are not mediated by differential actions on TM-cell Ca 2+ and volume, but likely by actions on separate cell targets.
Reprint requests should be addressed to: Dr. Mortimer M. Civan, Dept. of Physiology, University of Pennsylvania, Richards Building, Philadelphia, PA 19104-6085. [Tel.: (215)-898-8773; Fax: (215)-573-5851] 相似文献
5.
BackgroundAmong adenosine receptors (ARs) the A 2B subtype exhibits low affinity for the endogenous agonist compared with the A 1, A 2A, and A 3 subtypes and is therefore activated when concentrations of adenosine increase to a large extent following tissue damages (e.g. ischemia, inflammation). For this reason, A 2B AR represents an important pharmacological target. MethodsWe evaluated seven 1-benzyl-3-ketoindole derivatives ( 7– 9) for their ability to act as positive or negative allosteric modulators of human A 2B AR through binding and functional assays using CHO cells expressing human A 1, A 2A, A 2B, and A 3 ARs. ResultsThe investigated compounds behaved as specific positive or negative allosteric modulators of human A 2B AR depending on small differences in their structures. The positive allosteric modulators 7a, b and 8a increased agonist efficacy without any effect on agonist potency. The negative allosteric modulators 8b, c and 9a, b reduced agonist potency and efficacy. ConclusionsA number of 1-benzyl-3-ketoindole derivatives were pharmacologically characterized as selective positive ( 7a, b) or negative ( 8c, 9a, b) allosteric modulators of human A 2B AR. General significanceThe 1-benzyl-3-ketoindole derivatives 7– 9 acting as positive or negative allosteric modulators of human A 2B AR represent new pharmacological tools useful for the development of therapeutic agents to treat pathological conditions related to an altered functionality of A 2B AR. 相似文献
6.
Mast cell degranulation triggers hypersensitivity reactions at the body–environment interface. Adenosine modulates degranulation, but enhancement and inhibition have both been reported. Which of four adenosine receptors (ARs) mediate modulation, and how, remains uncertain. Also uncertain is whether adenosine reaches mast cell ARs by autocrine ATP release and ecto-enzymatic conversion. Uncertainties partly reflect species and cell heterogeneity, circumvented here by focusing on homogeneous human LAD2 cells. Quantitative PCR detected expression of A 2A, A 2B, and A 3, but not A 1, ARs. Nonselective activation of ARs with increasing NECA monotonically enhanced immunologically or C3a-stimulated degranulation. NECA alone stimulated degranulation slightly. Selective AR antagonists did not affect C3a-stimulated degranulation. NECA''s enhancement of C3a-triggered degranulation was partially inhibited by separate application of each selective antagonist, and abolished by simultaneous addition of antagonists to the three ARs. Only the A 2A antagonist separately inhibited NECA''s enhancement of immunologically stimulated degranulation, which was abolished by simultaneous addition of the three selective antagonists. Immunological or C3a activation did not stimulate ATP release. NECA also enhanced immunologically triggered degranulation of mouse bone marrow derived mast cells (BMMCs), which was partially reduced only by simultaneous addition of the three antagonists or by the nonselective antagonist {"type":"entrez-protein","attrs":{"text":"CGS15943","term_id":"875345334"}}CGS15943. BMMCs also expressed A 2A, A 2B, and A 3 ARs. but not A 1AR detectably. We conclude that (a) A 1AR is unnecessary for LAD2 degranulation or AR enhancement; (b) A 2A, A 2B, and A 3 ARs all contribute to pharmacologic AR enhancement of LAD2 and BMMC degranulation; and (c) LAD2 cells depend on microenvironmental adenosine to trigger AR modulation. 相似文献
7.
Activity of the A 3 adenosine receptor (AR) allosteric modulators LUF6000 (2-cyclohexyl- N-(3,4-dichlorophenyl)-1 H-imidazo [4,5-c]quinolin-4-amine) and LUF6096 ( N-{2-[(3,4-dichlorophenyl)amino]quinolin-4-yl}cyclohexanecarbox-amide) was compared at four A 3AR species homologs used in preclinical drug development. In guanosine 5′-[γ-[ 35S]thio]triphosphate ([ 35S]GTPγS) binding assays with cell membranes isolated from human embryonic kidney cells stably expressing recombinant A 3ARs, both modulators substantially enhanced agonist efficacy at human, dog, and rabbit A 3ARs but provided only weak activity at mouse A 3ARs. For human, dog, and rabbit, both modulators increased the maximal efficacy of the A 3AR agonist 2-chloro- N 6-(3-iodobenzyl)adenosine-5′- N-methylcarboxamide as well as adenosine > 2-fold, while slightly reducing potency in human and dog. Based on results from N 6-(4-amino-3-[ 125I]iodobenzyl)adenosine-5′- N-methylcarboxamide ([ 125I]I-AB-MECA) binding assays, we hypothesize that potency reduction is explained by an allosterically induced slowing in orthosteric ligand binding kinetics that reduces the rate of formation of ligand-receptor complexes. Mutation of four amino acid residues of the human A 3AR to the murine sequence identified the extracellular loop 1 (EL1) region as being important in selectively controlling the allosteric actions of LUF6096 on [ 125I]I-AB-MECA binding kinetics. Homology modeling suggested interaction between species-variable EL1 and agonist-contacting EL2. These results indicate that A 3AR allostery is species-dependent and provide mechanistic insights into this therapeutically promising class of agents. 相似文献
8.
The A 2A adenosine receptor (A 2AAR) is a unique G‐protein coupled receptor (GPCR), because besides agonist, its antagonist could also lead to therapeutic relevance. Based on A 2AAR‐antagonist crystal structure, we have studied the binding mechanism of two distinct antagonists, ZM241385 and KW6002, and dynamic behaviors of A 2AAR induced by antagonist binding. Key residues interacting with both antagonists and residues specifically binding to one of them are identified. ZM241385 specifically bound to S67 2.65, M177 5.38, and N253 6.55, while KW6002 binds to F62 2.60, A81 3.29, and H264 7.29. Moreover, interactions with L167 5.28 are found for both antagonists, which were not reported in agonist binding. The dynamic behaviors of antagonist bound holo‐A 2AARs were found to be different from the apo‐A 2AAR in three typical functional switches, (i) the “ionic lock” was in equilibrium between formation and breakage in apo‐A 2AAR, but stayed broken in holo‐A 2AARs; (ii) the “rotamer toggle switch,” T88 3.36/F242 6.44/W246 6.48, adopted different rotameric conformations in apo‐A 2AAR and holo‐A 2AARs; (iii) apo‐A 2AAR preferred α‐helical intracellular loop (IC)2 and flexible IC3, while holo‐A 2AARs had a flexible IC2 and α‐helical IC3. Our results indicated that antagonist binding induced different conformational rearrangements of these characteristic functional switches in apo‐A 2AAR and holo‐A 2AARs. Proteins 2013; 81:1399–1410. © 2013 Wiley Periodicals, Inc. 相似文献
9.
BackgroundGlaucoma, a leading cause of blindness worldwide, is an optic neuropathy commonly associated with elevated intraocular pressure (IOP). The major goals of glaucoma treatments are to lower IOP and protect retinal ganglion cells. It has been revealed recently that adenosine and adenosine receptors (ARs) have important roles in IOP modulation and neuroprotection. Scope of reviewThis article reviews recent studies on the important roles of adenosine and ARs in aqueous humor formation and outflow facility, IOP and retinal neuroprotection. Major conclusionsAdenosine and several adenosine derivatives increase and/or decrease IOP via A 2A AR. Activation of A 1 AR can reduce outflow resistance and thereby lower IOP, A 3 receptor antagonists prevent adenosine-induced activation of Cl − channels of the ciliary non-pigmented epithelial cells and thereby lower IOP. A 1 and A 2A agonists can reduce vascular resistance and increase retina and optic nerve head blood flow. A 1 agonist and A 2A antagonist can enhance the recovery of retinal function after ischemia attack. Adenosine acting at A 3 receptors can attenuate the rise in calcium and retinal ganglion cells death accompanying P2X(7) receptor activation. General significanceEvidence suggested that the adenosine system is one of the potential target systems for therapeutic approaches in glaucoma. 相似文献
10.
Rapid phosphoester hydrolysis of endogenous purine and pyrimidine nucleotides has challenged the characterization of the role of P2 receptors in physiology and pathology. Nucleotide phosphoester stabilization has been pursued on a number of medicinal chemistry fronts. We investigated the in vitro and in vivo stability and pharmacokinetics of prototypical nucleotide P2Y1 receptor (P2Y1R) agonists and antagonists. These included the riboside nucleotide agonist 2-methylthio-ADP and antagonist MRS2179, as well as agonist MRS2365 and antagonist MRS2500 containing constrained (N)-methanocarba rings, which were previously reported to form nucleotides that are more slowly hydrolyzed at the α-phosphoester compared with the ribosides. In vitro incubations in mouse and human plasma and blood demonstrated the rapid hydrolysis of these compounds to nucleoside metabolites. This metabolism was inhibited by EDTA to chelate divalent cations required by ectonucleotidases for nucleotide hydrolysis. This rapid hydrolysis was confirmed in vivo in mouse pharmacokinetic studies that demonstrate that MRS2365 is a prodrug of the nucleoside metabolite AST-004 (MRS4322). Furthermore, we demonstrate that the nucleoside metabolites of MRS2365 and 2-methylthio-ADP are adenosine receptor (AR) agonists, notably at A3 and A1ARs. In vivo efficacy of MRS2365 in murine models of traumatic brain injury and stroke can be attributed to AR activation by its nucleoside metabolite AST-004, rather than P2Y1R activation. This research suggests the importance of reevaluation of previous in vitro and in vivo research of P2YRs and P2XRs as there is a potential that the pharmacology attributed to nucleotide agonists is due to AR activation by active nucleoside metabolites. 相似文献
11.
A new series of 32 pyrimido- and 5 tetrahydropyrazino[2,1- f]purinediones was obtained and evaluated for their adenosine receptors (ARs) affinities. The 1,3-dibutyl derivative of 9-(4-(2-(dimethylamino)ethoxy)phenyl)-6,7,8,9-tetrahydropyrimido[1,2 -f]purine-2,4(1 H,3 H)-dione was found to be the most potent A 1 AR antagonist of the present series, showing selectivity over the other AR subtypes. The structure–activity for the obtained purinediones was established. Docking experiments of the investigated library to homology models of the human and rat A 1 and A 2A ARs allowed to compare the expected binding modes for selected compounds. The detailed analysis of binding cavities within individual AR subtypes indicated small but significant structural variations that may underlie the observed differences in binding affinities of purinediones at particular subtypes and species. 相似文献
12.
A selective agonist radioligand for A 2B adenosine receptors (A 2BARs) is currently not available. Such a tool would be useful for labeling the active conformation of the receptors. Therefore, we prepared BAY 60-6583, a potent and functionally selective A 2BAR (partial) agonist, in a tritium-labeled form. Despite extensive efforts, however, we have not been able to establish a radioligand binding assay using [ 3H]BAY 60-6583. This is probably due to its high non-specific binding and its moderate affinity, which had previously been overestimated based on functional data. As an alternative, we evaluated the non-selective A 2BAR agonist [ 3H]NECA for its potential to label A 2BARs. [ 3H]NECA showed specific, saturable, and reversible binding to membrane preparations of Chinese hamster ovary (CHO) or human embryonic kidney (HEK) cells stably expressing human, rat, or mouse A 2BARs. In competition binding experiments, the AR agonists 2-chloroadenosine (CADO) and NECA displayed significantly higher affinity when tested versus [ 3H]NECA than versus the A 2B-antagonist radioligand [ 3H]PSB-603 while structurally diverse AR antagonists showed the opposite effects. Although BAY 60-6583 is an A 2BAR agonist, it displayed higher affinity versus [ 3H]PSB-603 than versus [ 3H]NECA. These results indicate that nucleoside and non-nucleoside agonists are binding to very different conformations of the A 2BAR. In conclusion, [ 3H]NECA is currently the only useful radioligand for determining the affinity of ligands for an active A 2BAR conformation. 相似文献
13.
Gold nanoparticles (AuNPs) allow the tuning of pharmacokinetic and pharmacodynamic properties by active or passive targeting of drugs for cancer and other diseases. We have functionalized gold nanoparticles by tethering specific ligands, agonists and antagonists, of adenosine receptors (ARs) to the gold surface as models for cell surface interactions with G protein-coupled receptors (GPCRs). The AuNP conjugates with chain-extended AR ligands alone (PEGylated nucleosides and nonnucleosides, anchored to the Au via thioctic acid) were found to be insoluble in water due to hydrophobic entities in the ligand. Therefore, we added a second, biologically inactive pendant moiety to increase the water solubility, consisting of a PEGylated chain terminating in a carboxylic or phosphate group. The purity and stability of the immobilized biologically active ligand were examined by ultrafiltration and HPLC. Pharmacological receptor binding studies on these GPCR ligand-derivatized AuNPs (2–5 nm in diameter), performed using membranes of mammalian cells stably expressing human A 1, A 2A, and A 3ARs, showed that the desired selectivity was retained with K i values (nanomolar) of A 3AR agonist 21b and A 2AAR antagonists 24 and 26a of 14 (A 3), 34 (A 2A), and 69 (A 2A), respectively. The corresponding monomers displayed K i values of 37, 61, and 1,420 nM, respectively. In conclusion, we have synthesized stable, water-soluble AuNP derivatives of tethered A 3 and A 2AAR ligands that retain the biological properties of their monomeric ligands and are intended for therapeutic and imaging applications. This is the first prototypical application to gold carriers of small molecule (nonpeptide) GPCR ligands, which are under investigation for treatment of cancer and inflammatory diseases. 相似文献
14.
The A 3‐adenosine receptor (A 3AR) has recently emerged as a key regulator of neutrophil behaviour. Using a fluorescent A 3AR ligand, we show that A 3ARs aggregate in highly polarized immunomodulatory microdomains on human neutrophil membranes. In addition to regulating chemotaxis, A 3ARs promote the formation of filipodia‐like projections (cytonemes) that can extend up to 100 μm to tether and ‘reel in’ pathogens. Exposure to bacteria or an A 3AR agonist stimulates the formation of these projections and bacterial phagocytosis, whereas an A 3AR‐selective antagonist inhibits cytoneme formation. Our results shed new light on the behaviour of neutrophils and identify the A 3AR as a potential target for modulating their function. 相似文献
15.
The A 2B adenosine receptor (A 2B AR), activated in response to high levels of endogenous adenosine, is the major AR subtype involved in mesenchymal stem cell (MSC) differentiation to osteoblasts and bone formation. For this reason, targeting of A 2B AR with selective allosteric modulators may represent a promising pharmacological approach to the treatment of bone diseases.Herein, we report the characterization of a 3-keto-indole derivative, 2-(1-benzyl-1 H-indol-3-yl)-2-oxo-N-phenylacetamide (KI-7), as A 2B AR positive allosteric modulator in MSCs, demonstrating that this compound is able to potentiate the effects of either adenosine and synthetic orthosteric A 2B AR agonists in mediating osteoblast differentiation in vitro. In detail, we observed that MSC treatment with KI-7 determined an increase in the expression of osteoblast-related genes (Runx2 and osterix) and osteoblast marker proteins (phosphatase alkaline and osteocalcin), associated with a stimulation of osteoblast mineralization.In the early phase of differentiation programme, KI-7 significantly potentiated physiological and A 2B AR agonist-mediated down-regulation of IL-6 release. Conversely, during the late stage of differentiation, when most of the cells have an osteoblast phenotype, KI-7 caused a sustained raise in IL-6 levels and an improvement in osteoblast viability. These data suggest that a positive allosteric modulation of A 2B AR not only favours MSC commitment to osteoblasts, but also ensures a greater survival of mature osteoblasts. Our study paves the way for a therapeutic use of selective positive allosteric modulators of A 2B AR in the control of osteoblast differentiation, bone formation and fracture repair. 相似文献
16.
The expression levels and the subcellular localization of adenosine receptors (ARs) are affected in several pathological conditions as a consequence of changes in adenosine release and metabolism. In this respect, labelled probes able to monitor the AR expression could be a useful tool to investigate different pathological conditions. Herein, novel ligands for ARs, bearing the fluorescent 7-nitrobenzofurazan (NBD) group linked to the N1 ( 1, 2) or N10 ( 3, 4) nitrogen of a triazinobenzimidazole scaffold, were synthesized. The compounds were biologically evaluated as fluorescent probes for labelling A 1 and A 2B AR subtypes in bone marrow-derived mesenchymal stem cells (BM-MSCs) that express both receptor subtypes. The binding affinity of the synthetized compounds towards the different AR subtypes was determined. The probe 3 revealed a higher affinity to A 1 and A 2B ARs, showing interesting spectroscopic properties, and it was selected as the most suitable candidate to label both AR subtypes in undifferentiated MSCs.Fluorescence confocal microscopy showed that compound 3 significantly labelled ARs on cell membranes and the fluorescence signal was decreased by the cell pre-incubation with the A 1 AR and A 2B AR selective agonists, R-PIA and BAY 60-6583, respectively, thus confirming the specificity of the obtained signal. In conclusion, compound 3 could represent a useful tool to investigate the expression pattern of both A 1 and A 2B ARs in different pathological and physiological processes. Furthermore, these results provide an important basis for the design of new and more selective derivatives able to monitor the expression and localization of each different ARs in several tissues and living cells. 相似文献
17.
Extracellular adenosine is a biologically active signaling molecule that accumulates at sites of metabolic stress in sepsis. Extracellular adenosine has potent immunosuppressive effects by binding to and activating G protein-coupled A2A adenosine receptors (A2AARs) on the surface of neutrophils. A2AAR signaling reproduces many of the phenotypic changes in neutrophils that are characteristic of sepsis, including decreased degranulation, impaired chemotaxis, and diminished ability to ingest and kill bacteria. We hypothesized that A2AARs also suppress neutrophil aging, which precedes cell death, and N1 to N2 polarization. Using human neutrophils isolated from healthy subjects, we demonstrate that A2AAR stimulation slows neutrophil aging, suppresses cell death, and promotes the polarization of neutrophils from an N1 to N2 phenotype. Using genetic knockout and pharmacological blockade, we confirmed that A2AARs decrease neutrophil aging in murine sepsis induced by cecal ligation and puncture. A2AARs expression is increased in neutrophils from septic patients compared to healthy subject but A2AAR expression fails to correlate with aging or N1/N2 polarization. Our data reveals that A2AARs regulate neutrophil aging in healthy but not septic neutrophils. 相似文献
18.
A 2A adenosine receptors (ARs) play a key role in the inhibition of the inflammatory process. The purpose of this study was to evaluate the modulation of A 2AARs in rheumatoid arthritis (RA) patients after different pharmacological treatments and to investigate the effect of A 2AAR stimulation in a rat model of arthritis. We investigated A 2AAR density and functionality in RA progression by using a longitudinal study in RA patients before and after methotrexate (MTX), anti-TNFα agents or rituximab treatments. A 2AARs were analyzed by saturation binding assays in lymphocytes from RA patients throughout the 24-month study timeframe. In an adjuvant-induced arthritis model in rats we showed the efficacy of the A 2AAR agonist, CGS 21680 in comparison with standard therapies by means of paw volume assessment, radiographic and ultrasonographic imaging. Arthritic-associated pain was investigated in mechanical allodynia and thermal hyperalgesia tests. IL-10 release following A 2AAR stimulation in lymphocytes from RA patients and in serum from arthritic rats was measured. In lymphocytes obtained from RA patients, the A 2AAR up-regulation was gradually reduced in function of the treatment time and the stimulation of these receptors mediated a significant increase of IL-10 production. In the same cells, CGS 21680 did not affected cell viability and did not produced cytotoxic effects. The A 2AAR agonist CGS 21680 was highly effective, as suggested by the marked reduction of clinical signs, in rat adjuvant-induced arthritis and associated pain. This study highlighted that A 2AAR agonists represent a physiological-like therapeutic alternative for RA treatment as suggested by the anti-inflammatory role of A 2AARs in lymphocytes from RA patients. The effectiveness of A 2AAR stimulation in a rat model of arthritis supported the role of A 2AAR agonists as potential pharmacological treatment for RA. 相似文献
19.
It is well known that guinea pig β 2 adrenoceptors (Gβ 2ARs) and human β 2 adrenoceptors (Hβ 2ARs) have structural similarity. However, only one conformational state of Gβ 2ARs has been studied – the putative inactive state. As adrenoceptors have a repertoire of conformations, and there is evidence that a certain conformation is stabilised as a ligand approaches, the aim of this study was to build four models of Gβ 2ARs by using putative active/inactive Hβ 2AR conformers as a template. We evaluated the accuracy of these models in regard to the binding mode and affinity values of a set of known β 2AR ligands through docking and molecular dynamics simulations. During docking simulations, ligands reached Gβ 2AR sites similar to those reported for Hβ 2ARs. The greatest differences between conformational states were found in the domains (TM5 and TM6) previously suggested as being key to ligand recognition. The coefficients of determination between experimental and calculated affinity values were near to but less than 0.66 in all cases. The highest values were for agonists on the active models and antagonists on the inactive model. The four Gβ 2AR models proved useful for analysing agonist/antagonist activity. The results suggest that the selection of an adequate model is dependent on the intrinsic activity of a given ligand. 相似文献
20.
It is generally assumed that antagonists of G s‐coupled receptors do not activate cAMP signalling, because they do not stimulate cAMP production via G s‐protein/adenylyl cyclase activation. Here, we report a new signalling pathway whereby antagonists of β 1‐adrenergic receptors (β 1ARs) increase cAMP levels locally without stimulating cAMP production directly. Binding of antagonists causes dissociation of a preformed complex between β 1ARs and Type‐4 cyclic nucleotide phosphodiesterases (PDE4s). This reduces the local concentration of cAMP‐hydrolytic activity, thereby increasing submembrane cAMP and PKA activity. Our study identifies receptor/PDE4 complex dissociation as a novel mechanism of antagonist action that contributes to the pharmacological properties of β 1AR antagonists and might be shared by other receptor subtypes. 相似文献
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