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Pharmacological Analysis of Calcium Responses Mediated by the Human A3 Adenosine Receptor in Monocyte-Derived Dendritic Cells and Recombinant Cells

Molecular Pharmacology, ISSN: 0026-895X, Vol: 63, Issue: 2, Page: 342-350
2003
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Article Description

Extensive characterization of adenosine receptors expressed by human monocyte-derived dendritic cells (MDDCs) was performed with quantitative polymerase chain reaction, radioligand binding, and calcium signaling. Transcript for the A3 adenosine receptor was elevated more than 100-fold in immature MDDCs compared with monocyte precursors. A3 receptor transcript was substantially diminished, and A2A receptor transcript increased, by lipopolysaccharide maturation of MDDCs. Saturation binding of N 6 -(3-[ 125 I]iodo-4-aminobenzyl)-adenosine-5′- N -methyluronamide ([ 125 I]AB-MECA) to membranes from immature MDDCs yielded B max of 298 fmol/mg of protein and K D of 0.7 nM. Competition against [ 125 I]AB-MECA binding confirmed the site to be the A3 receptor. Adenosine elicited pertussis toxin-sensitive calcium responses with EC 50 values ranging as low as 2 nM. The order of potency for related agonists was N 6 -(3-iodobenzyl)-adenosine-5′- N -methylcarboxamide (IB-MECA) ≥ I-AB-MECA > 2Cl-IB-MECA ≥ adenosine > 2-[ p -(2-carboxyethyl)phenylethylamino]-5′- N -ethylcarboxyamidoadenosine (CGS21680). The order of efficacy was adenosine ≥ CGS21680 > IB-MECA ≥ I-AB-MECA > 2Cl-IB-MECA. Calcium responses to 2Cl-IB-MECA and CGS21680, and the lower range of adenosine concentrations, were completely blocked by 10 nM N -(2-methoxyphenyl)- N -[2-(3-pyridyl)quinazolin-4-yl]urea (VUF5574) but not by 7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3- e ]-1,2,4-triazolo[1,5- c ]pyrimidine (SCH58261) or 8-cyclopentyl-1,3-dipropylxanthine. Pretreatment with 100 nM 2Cl-IB-MECA eliminated responses to CGS21680 but not to monocyte inhibitory protein-1α. For comparison, dose-response functions were obtained from double-recombinant human embryonic kidney 293 cells expressing the human A3 receptor and a chimeric Gαq-i3 protein, which was required to establish A3-mediated calcium signaling. The pharmacological profile of calcium signaling elicited by adenosine-related agonists in the double-recombinant cells was essentially identical to that obtained from immature MDDCs. Our results provide an extensive analysis of A3-mediated calcium signaling and unequivocally identify immature MDDCs as native expressers of the human A3 receptor.

Bibliographic Details

Fossetta, James; Jackson, James; Deno, Gregory; Fan, Xuedong; Du, Xixuan Karen; Bober, Loretta; Soudé-Bermejo, Anne; de Bouteiller, Odette; Caux, Christophe; Lunn, Charles; Lundell, Daniel; Palmer, R Kyle

Elsevier BV

Biochemistry, Genetics and Molecular Biology; Pharmacology, Toxicology and Pharmaceutics

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