Vol. 63, Issue 2, 342-350, February 2003

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

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

Immunology Department, Schering-Plough Research Institute, Kenilworth, New Jersey (J.F., J.J., G.D., X.F., X.K.D., L.B., C.L., D.L., R.K.P.); and Laboratory for Immunological Research, Dardilly, France (A.S.-B., O.B., C.C.)

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 N⁶-(3-[¹²⁵I]iodo-4-aminobenzyl)-adenosine-5'-N-methyluronamide ([¹²⁵I]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 [¹²⁵I]AB-MECA binding confirmed the site to be the A3 receptor. Adenosine elicited pertussis toxin-sensitive calcium responses with EC₅₀ values ranging as low as 2 nM. The order of potency for related agonists was N⁶-(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-1. For comparison, dose-response functions were obtained from double-recombinant human embryonic kidney 293 cells expressing the human A3 receptor and a chimeric Gq-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.