Pharmacological analysis of calcium responses mediated by the human A3 adenosine receptor in monocyte-derived dendritic cells and recombinant cells

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-[I-125]iodo-4-aminobenzyl)-adenosine-5'-N-methyluronamide ([1251]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 [I-125]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 N-6-(3-iodobenzyl)-adenosine-5'-N-methyl carboxamide (IB-MECA) greater than or equal to I-AB-MECA > 2CI-IB-MECA greater than or equal to adenosine > 2-[p-(2-carboxyethyl)phenylethylamino]-5'-N-ethylcarboxyamidoadenosine (CGS21680). The order of efficacy was adenosine greater than or equal to CGS21680 > IB-MECA greater than or equal to I-AB-MECA > 2CI-IB-MECA. Calcium responses to 2CI-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 2CI-IB-MECA eliminated responses to CGS21680 but not to monocyte inhibitory protein-1alpha. For comparison, dose-response functions were obtained from double-recombinant human embryonic kidney 293 cells expressing the human A3 receptor and a chimeric Galphaq-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.