| pA2 online © Copyright 2004 The British Pharmacological Society |
112P
University of Newcastle Winter Meeting December 2004 |
Characterization of a fluorescent agonist acting at the human adenosine-A3 receptor in CHO cells Y. Cordeaux †, R.J. Middleton ‡, B. Kellam ‡ & S.J. Hill †, †Institute of Cell Signalling, School of Biomedical Sciences, University of Nottingham, NG7 2UH. And ‡Centre for Biomolecular Sciences, University Park, Nottingham, NG7 2RD, U.K. |
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Print abstractWe have previously described the synthesis of the ligand ABEA-X-BY630; a fluorescent derivative of the non-selective adenosine agonist NECA (Briddon et al, 2002) and shown that it acts as an agonist at both the human adenosine-A1 and -A2B receptors (Briddon et al, 2002, Cordeaux et al. 2003). In the present study we have investigated the ability of this ligand to activate the Gi-coupled adenosine-A3 receptor and to modulate cAMP accumulation, inositol phosphate production and calcium signalling.
Human adenosine-A3 receptor cDNA (UMR cDNA Resource center, USA) was subcloned into the vector pcDNA3.1 and transfected into CHO cells by lipofectamineTM. Stable transfects (CHOA3 cells) were selected in the presence of 500 µg/ml geneticin and clones identified by dilution cloning. CHOA3 cells were cultured and assays performed (to measure the accumulation of [3H]cAMP or [3H]inositol phosphates) as previously described (Cordeaux et al. 2000). For live cell confocal imaging, cells were grown to 70% confluency in 35mm glass-bottomed MatTekTM dishes and loaded with Fluo 4AM (2.3 µM, 1hr, 37 ° C). Cells were washed and incubated at 37 °C in HEPES-buffered saline for 30mins prior to imaging. Images were obtained using a Zeiss LSM510 confocal microscope, using simultaneous excitation at 633nm and 488nm and collected every 4 seconds for 30mins after addition of ABEA-X-BY630 ( 100nM ) . Data are expressed as mean ± s.e.m.
In CHOA3 cells , NECA, IB-MECA (an A3-selective agonist) and ABEA-X-BY630 produced a concentration-dependent inhibition of forskolin-stimulated [3H]cAMP production (pEC50; 8.34 ± 0.13, n=4, 8.89 ± 0.12, n=3 and 8.88 ± 0.18, n=3 respectively). The extents to which they inhibited forskolin-stimulated [3H]cAMP production (96 ± 2%, 88 ± 3% and 96 ± 2% respectively) were similar. For each ligand the dose response could be shifted to the right by pre-incubating the cells with the A3-selective antagonist MRS1220 (pKBs; 9.47 ± 0.07, n=4, 9.39 ± 0.08, n=3 and 9.44 ± 0.10, n=3 respectively). The ligands also caused concentration-dependent increases in [3H]inositol phosphate production, (pEC50; 7.00 ± 0.07, n=3, 8.09 ± 0.02, n=3 and 7.03 ± 0.18, n=3 respectively), with similar maximal responses (2.67 ± 0.07, 2.86 ± 0.06 and 2.39 ± 0.31-fold of basal response).
By confocal imaging, ABEA-X-BY630 was shown to label the plasma membrane of these cells and cause a subsequent increase in intracellular calcium. Pre-incubation of the cells with MRS1220 (1 µM, 30mins) reduced membrane labeling and reduced the number of cells showing an increase in calcium (from 72 ± 3% to 24 ± 6%, n=3; each approximately 30 cells).
In summary, ABEA-X-BY630 is an agonist at the human adenosine-A3 receptor which may prove a useful tool for studying this receptor in both healthy and diseased human tissue.
Briddon, S.J. et al. (2002) Br. J. Pharmacol., 138, 126P.
Cordeaux, Y. et al. (2000) Mol. Pharm., 58, 1075.
Cordeaux, Y. et al. (2003) Br. J. Pharmacol., 140, 16P.
We thank the Wellcome Trust for financial support (ref 066817) and Diane Hall for technical assistance.