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Copyright 2003 The British Pharmacological Society

016P University of Surrey
Summer Meeting June 2003

ABEA-BY630 is a fluorescent agonist at A2B adenosine receptors in human prostatic stromal cells


Y. Cordeaux1, R.J. Middleton2, A. Myatt1, B. Kellam2& S.J. Hill1 1Institute of Cell Signalling, QMC, University of Nottingham, Nottingham, UK. 2School of Pharmaceutical Sciences, University of Nottingham, University Park, Nottingham, UK.


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Cordeaux Y
Middelton RJ
Myatt A
Kellam B
Hill SJ

We have previously described the synthesis of ABEA-BY630, a fluorescent derivative of the adenosine agonist NECA (5'-N-ethylcarboxyamidoadenosine) and described its agonist activity at Gi/o-coupled human A1 adenosine receptors expressed in CHO cells (Briddon et al. 2003). The aim of the present study was to determine whether the same ligand was able to stimulate Gs-coupled A2 adenosine receptors that are endogenously expressed in human prostatic stromal cells.

Human prostatic stromal cells were isolated from benign prostatic tissue and cultured as previously described (Abdul-Hamid et al. 2001). Cells were grown from one cell line and functional assays performed on cells of passage numbers 4-8. Cells were grown in 24-well cluster dishes and [
3H]cAMP accumulation measured as described previously (Cordeaux et al. 2000). Where stated, cells were pre-incubated with the antagonists; ZM241385, XAC or SCH58261 (100nM), 15 minutes prior to incubation with agonist.

In human prostatic stromal cells, the agonist NECA stimulated [
3H]cAMP accumulation in a concentration-dependent manner (pEC50= 5.66 ± 0.04, mean ± s.e. mean, n=19). Pre-incubation of cells with the non-selective antagonist XAC caused a rightward shift in the concentration-response curve to NECA(pKB = 7.85 ± 0.08, n=3). The weakly A2A-selective antagonist ZM241385 produced a similar shift (pKB = 7.72 ± 0.04, n=3). The highly selective A2A antagonist SCH58261 (100nM), however, did not alter the NECA response (pEC50s; 5.27 ± 0.05 and 5.59 ± 0.17, with and without SCH58261, respectively, n=3). Moreover, the A2A-selective agonist CGS21680 only produced a small [3H]cAMP response (10 ± 4% of NECA maximum at 0.1mM) in these cells at a concentration above 10µM (n=3). Hence, the effects of NECA in these cells can be attributed to activation of the A2B adenosine receptor.

The fluorescent ligand ABEA-BY630 also stimulated [
3H]cAMP accumulation in these cells with similar potency to that of NECA (pEC50 = 6.32 ± 0.08, n=4). This stimulation was completely inhibited by preincubating the cells with XAC (1mM, 30 minutes, n=3). Relative to NECA, however, ABEA-BY630 was a partial agonist (37 ± 5 % of NECA maximum, n=4). This agonist may therefore be a useful tool for visualising adenosine receptors in primary cells, and studying their function using fluorescence-based techniques.

Abdul-Hamid MA et al. (2001) Br. J.Pharmacol 134: 173P.
Briddon SJ et al. (2003) Br. J.Pharmacol. (Brighton BPS meeting).
Cordeaux Y et al. (2000) Mol. Pharmacol. 58 (5), 1075-1084.

This work was supported by Wellcome Trust grants 066817 and 057199.