Activation of phospholipase C via adenosine receptors provides synergistic signals for secretion in antigen-stimulated RBL-2H3 cells. Evidence for a novel adenosine receptor.

Ali, Hydar; Cunha-Melo, José Renan; Saul, W.; Beaven, Michael A.

doi:10.1016/s0021-9258(19)40113-0

Cited by 179 publications

(25 citation statements)

References 55 publications

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“…In earlier studies on functional responses in RBL-2H3 mast cells, R-PIA and APNEA were twofold more potent than NECA (EC 50 = 56 nM) and ninefold more potent than N 6 -S-phenylisopropyladenosine in enhancing hexosaminidase release [Ramkumar et al, 1993]. However, with respect to enhancing release of serotonin, NECA (EC 50 ~1 µM) was more potent and more efficacious than R-PIA [Ali et al, 1990]. Adenosine itself was intermediate in potency and efficacy.…”

Section: Discussionmentioning

confidence: 90%

“…Thus, in these cells either an A 1 and/or an A 3 -adenosine receptor, stimulatory to phosphoinositide breakdown, would be expected to be involved in the response. NECA also has been shown to activate the rat mast cell line RBL-2H3, leading to increases in phosphoinositide breakdown and intracellular calcium levels, and an enhancement of immunologically activated secretion of serotonin, hexosaminidase, and other allergic mediators [Ali et al, 1990;Ramkumar et al, 1993]. Binding studies have demonstrated the presence of A 1 -, A 2Aand A 3 -adenosine receptors in RBL-2H3 cells [Ji et al, 1994], although DNA hybridization in another study detected only A 3 -receptors [Ramkumar et al, 1993].…”

Section: Introductionmentioning

confidence: 99%

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Activation of phosphoinositide breakdown and elevation of intracellular calcium in a rat RBL-2H3 mast cell line by adenosine analogs: Involvement of A3-adenosine receptors?

Shin

Daly

1996

Drug Dev. Res.

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A variety of adenosine analogs activate phosphoinositide breakdown in a rat RBL-2H3 mast cell line. It is presumed that an A 3 -adenosine receptor is involved, since the phosphoinositide response is insensitive to xanthines. However, the very potent A 3 -receptor agonist 2-chloro-N 6 -iodobenzyl-N-methylcarboxamidoadenosine (2-CI-IBMECA) with an EC 50 of 4.1 µM is about twofold less potent (and less efficacious) than N-ethylcarboxamidoadenosine (NECA) with an EC 50 of 2.1 µM. The other agents consisting of N 6 -p-aminophenylethyladenosine (APNEA), N 6 -iodobenzylMECA (IB-MECA), N 6 -R-phenylisopropyladenosine (R-PIA), 2-chloroadenosine, N 6 -benzyladenosine, N 6 -cyclohexyladenosine (CHA), N 6 -cyclohexylNECA (CHNECA), 2-(p-carboxyethylphenyl-ethylaminoNECA (CGS 21680), 1,3-dibutylxanthine 7-riboside-5′-N-methylcarboxamide (DBXRM), adenosine, and 8-bromoadenosine are all nearly equipotent with EC 50 values of 5.5-13.9 µM. The rank order of potencies of the analogs in causing an elevation of intracellular calcium is quite different. The potent A 3 receptor agonists 2-CI-IBMECA and IB-MECA with EC 50 values of 0.07 and 0.11 µM, respectively, are about fourfold more potent than N 6 -cyclohexylNECA and about 15-fold more potent than NECA. The other analogs are comparable or somewhat less potent than NECA, some are less efficacious, and 8-bromoadenosine is inactive. The results suggest that stimulation of phosphoinositide breakdown by adenosine analogs in RBL-2H3 cells as measured by IP 1 accumulation is not predictive of IP 3 -mediated elevations of intracellular calcium. Rank order of potency for the calcium response is consonant with intermediacy of A 3 -adenosine receptors, while the former, as measured by [ 3 H]IP 1 -formation, probably reflects contributions from both an A 3 -mediated response and some other mechanism. Combinations of subthreshold concentrations of 2-CI-IBMECA with either the A 1 -selective agonist CHA or the A 2A -selective agonist CGS 21680 caused a marked stimulation of phosphoinositide breakdown, providing further evidence for dual mechanisms. The selective A 3adenosine receptor antagonist 3,6-dichloro-2′-(isopropyloxy)-4′-methylflavone (MRS 1067) inhibits 2-CI-IBMECA-and NECA-elicited elevation of calcium levels, and had differential effects on phosphoinositide breakdown, blocking [ 3 H]IP 3 accumulation and either blocking (NECA) or having no effect (2-CI-IBMECA) on [ 3 H]IP 1 accumulation.

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Section: Discussionmentioning

confidence: 90%

Section: Introductionmentioning

confidence: 99%

Activation of phosphoinositide breakdown and elevation of intracellular calcium in a rat RBL-2H3 mast cell line by adenosine analogs: Involvement of A3-adenosine receptors?

Shin

Daly

1996

Drug Dev. Res.

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“…The following drugs were added to RBL(m1) cells before stimulation with CBC (1 mM, 3 min) to probe their effectiveness in inhibiting the agonist effect on CPs: 2 M cytochalasin D for 20 min; 5 g/ml brefeldin A for 40 min; 10 M quercetin for 10 min (Hirasawa et al, 1995); 30 M AlF Ϫ 4 (prepared by mixing 10 mM NaF with 10 M AlCl 3 ) for 3 min; 1 g/ml cholera toxin for 4 h (Ali et al, 1990); 0.1 g/ml pertussis toxin for 4 h (Ali et al, 1990); 10 M AG1478 for 10 min; 10 M PP1 for 15 min. The m1AChR antagonists atropine (10 M final concentration) and pirenzepine (0.1 M final concentration) were used as specified in the text.…”

Section: Agonist and Pharmacological Treatmentsmentioning

confidence: 99%

G protein–coupled receptor/arrestin3 modulation of the endocytic machinery

Santini

Gaidarov

Keen

2002

The Journal of Cell Biology

104

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Nonvisual arrestins (arr) modulate G protein–coupled receptor (GPCR) desensitization and internalization and bind to both clathrin (CL) and AP-2 components of the endocytic coated pit (CP). This raises the possibility that endocytosis of some GPCRs may be a consequence of arr-induced de novo CP formation. To directly test this hypothesis, we examined the behavior of green fluorescent protein (GFP)-arr3 in live cells expressing β2-adrenergic receptors and fluorescent CL. After agonist stimulation, the diffuse GFP-arr3 signal rapidly became punctate and colocalized virtually completely with preexisting CP spots, demonstrating that activated complexes accumulate in previously formed CPs rather than nucleating new CP formation. After arr3 recruitment, CP appeared larger: electron microscopy analysis revealed an increase in both CP number and in the occurrence of clustered CPs. Mutant arr3 proteins with impaired binding to CL or AP-2 displayed reduced recruitment to CPs, but were still capable of inducing CP clustering. In contrast, though constitutively present in CPs, the COOH-terminal moiety of arr3, which contains CP binding sites but lacks receptor binding, did not induce CP clustering. Together, these results indicate that recruitment of functional arr3–GPCR complexes to CP is necessary to induce clustering. Latrunculin B or 16°C blocked CP rearrangements without affecting arr3 recruitment to CP. These results and earlier studies suggest that discrete CP zones exist on cell surfaces, each capable of supporting adjacent CPs, and that the cortical actin membrane skeleton is intimately involved with both the maintenance of existing CPs and the generation of new structures.

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“…1 Different from the other subtypes (A 1 , A 2A , and A 2B ) A 3 AR was identified by molecular biology studies prior to its pharmacological characterization. 2 The initial studies indicated its important role in both physiological and pathophysiological conditions, such as cell proliferation, cell differentiation, neuroprotection, cardioprotection, and apoptosis. 3 Nevertheless, the medical relevance of the human adenosine A 3 receptor (hA 3 AR) is enigmatic due to its dichotomy in different therapeutic applications.…”

Section: ■ Introductionmentioning

confidence: 99%

“…The adenosine A 3 receptor (A 3 AR) is one of four G protein-coupled receptor subtypes stimulated by adenosine . Different from the other subtypes (A 1 , A 2A , and A 2B ) A 3 AR was identified by molecular biology studies prior to its pharmacological characterization . The initial studies indicated its important role in both physiological and pathophysiological conditions, such as cell proliferation, cell differentiation, neuroprotection, cardioprotection, and apoptosis .…”

Section: Introductionmentioning

confidence: 99%

Development of Covalent Ligands for G Protein-Coupled Receptors: A Case for the Human Adenosine A₃ Receptor

et al. 2019

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The development of covalent ligands for G protein-coupled receptors (GPCRs) is not a trivial process. Here, we report a streamlined workflow thereto from synthesis to validation, exemplified by the discovery of a covalent antagonist for the human adenosine A3 receptor (hA3AR). Based on the 1H,3H-pyrido[2,1-f]purine-2,4-dione scaffold, a series of ligands bearing a fluorosulfonyl warhead and a varying linker was synthesized. This series was subjected to an affinity screen, revealing compound 17b as the most potent antagonist. In addition, a nonreactive methylsulfonyl derivative 19 was developed as a reversible control compound. A series of assays, comprising time-dependent affinity determination, washout experiments, and [35S]GTPγS binding assays, then validated 17b as the covalent antagonist. A combined in silico hA3AR-homology model and site-directed mutagenesis study was performed to demonstrate that amino acid residue Y2657.36 was the unique anchor point of the covalent interaction. This workflow might be applied to other GPCRs to guide the discovery of covalent ligands.

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Activation of phospholipase C via adenosine receptors provides synergistic signals for secretion in antigen-stimulated RBL-2H3 cells. Evidence for a novel adenosine receptor.

Cited by 179 publications

References 55 publications

Activation of phosphoinositide breakdown and elevation of intracellular calcium in a rat RBL-2H3 mast cell line by adenosine analogs: Involvement of A3-adenosine receptors?

Activation of phosphoinositide breakdown and elevation of intracellular calcium in a rat RBL-2H3 mast cell line by adenosine analogs: Involvement of A3-adenosine receptors?

G protein–coupled receptor/arrestin3 modulation of the endocytic machinery

Development of Covalent Ligands for G Protein-Coupled Receptors: A Case for the Human Adenosine A₃ Receptor

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Activation of phospholipase C via adenosine receptors provides synergistic signals for secretion in antigen-stimulated RBL-2H3 cells. Evidence for a novel adenosine receptor.

Cited by 179 publications

References 55 publications

Activation of phosphoinositide breakdown and elevation of intracellular calcium in a rat RBL-2H3 mast cell line by adenosine analogs: Involvement of A3-adenosine receptors?

Activation of phosphoinositide breakdown and elevation of intracellular calcium in a rat RBL-2H3 mast cell line by adenosine analogs: Involvement of A3-adenosine receptors?

G protein–coupled receptor/arrestin3 modulation of the endocytic machinery

Development of Covalent Ligands for G Protein-Coupled Receptors: A Case for the Human Adenosine A3 Receptor

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Development of Covalent Ligands for G Protein-Coupled Receptors: A Case for the Human Adenosine A₃ Receptor