Abstract
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 ofN6-(3-[125I]iodo-4-aminobenzyl)-adenosine-5′-N-methyluronamide ([125I]AB-MECA) to membranes from immature MDDCs yieldedBmax of 298 fmol/mg of protein andKD 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 wasN6-(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 nMN-(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.
Footnotes
- Abbreviations:
- MDDC
- monocyte-derived dendritic cells
- PCR
- polymerase chain reaction
- FLIPR
- fluorometric imaging plate reader
- HEK
- human embryonic kidney
- CGS21680
- 2-[p-(2-carboxyethyl)phenylethylamino]-5′-N-ethylcarboxyamido-adenosine
- 2Cl-IB-MECA
- 2-chloro-N6-(3-iodobenzyl)-adenosine-5′-N-methyluronamide
- IB-MECA
- N6-(3-iodobenzyl)-adenosine-5′-N-methylcarboxamide
- ZM241385
- 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-yl-amino]ethyl)phenol
- MRS1220
- 9-chloro-2-(2-furyl)-5-phenylacetylamino[1,2,4]triazolol[1,5-c]quinazoline
- VUF5574
- N-(2-methoxyphenyl)-N-[2-(3-pyridyl)quinazolin-4-yl]urea
- SCH58261
- 7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine
- I-AB-MECA
- N6-(3-iodo-4-aminobenzyl)-adenosine-5′-N-methyluronamide
- AB-MECA
- N6-(4-aminobenzyl)-adenosine-5′-N-methyluronamide
- MIP
- monocyte inhibitory protein
- FBS
- fetal bovine serum
- LPS
- lipopolysaccharide
- RT
- reverse transcription
- Received July 24, 2002.
- Accepted October 30, 2002.
- The American Society for Pharmacology and Experimental Therapeutics
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