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Inverse agonist properties of the adenosine A2A receptor antagonist ZM-241,385 The human adenosine [A]2A receptor is coupled to Gs and raises the intracellular levels of cyclic AMP (Fredholm et al., 2001). In our investigations, in the absence of agonist, the selective adenosine A2A antagonist ZM-241,385 (Poucher et al., 1995) decreased the basal levels of cyclic AMP in HEK-293 cells expressing the human adenosine A2A receptor, suggesting inverse agonist properties. This abstract details our results on the characterisation of the inverse agonist properties of ZM-241,385. HEK-293 cells stably expressing human adenosine A2A and A2B receptors were used. Both are linked to Gs and receptor function was assessed by measuring changes in levels of cyclic AMP (cAMP). Experiments were carried out with cells in DMEM/F12 with L-glutamine (2 mM) and 0.1u/ml adenosine deaminase at 37 ° C in 5% CO2/95% atmosphere in a 96 well format. and cAMP measured by enzyme immunoassay (Amersham). Cells were incubated with: (a) ZM-241,385 (10-100nM) for 30mins then increasing concentrations of either CGS-21680 (0.1-10,000nM; A2A cells) or NECA (0.1-10,000nM; A2B cells) was added and 30mins later cAMP measured. A pA2 (Arunlakshana and Schild, 1957) or pKB (Gaddum, 1957) was calculated, (b) ZM-241,385 (1-100nM) for 30mins and cAMP measured, from this a pIC50 was calculated and, (c) 100nM ZM-241,385 for 30 mins, forskolin (1000nM) was then added and 30mins later cAMP measured. Statistical comparisons were performed using Wilcoxon matched pairs test for paired samples. ZM-241,385 (10-100nM) produced a concentration related inhibition of the ability of CGS-21680 to raise the levels of cAMP in A2A cells with a pA2 of 9.1 ± 0.4 (mean ± sem, n=4) with a Schild slope not significantly different from unity. In A2B cells ZM-241,385 (100nM) inhibited the ability of NECA to raise levels of cAMP with a pKB of 8.4 ± 0.4 (mean ± sem, n=3). ZM-241,385 (1-100 nM) produced a concentration related inhibition of the basal levels of cAMP in A2A cells with a pIC50 value of 8.1 ± 0.1 (mean ± sem, n=5). In addition, ZM-241,385 (100 nM) also produced a near maximal and significant (p<0.05) inhibition of the ability of forskolin to raise the levels of cyclic AMP in A2A cells (52.0 ± 11.0 versus 5.8 ± 1.3 nmol/104 cells respectively, mean ± sem, n=3 ). In contrast, ZM-241,385 at 100nM had no effect on the basal levels of cyclic AMP in A2B cells (12.0 ± 4.7 versus 13.2 ± 4.8 nmol/104 cells in presence or absence respectively mean ± sem, n=3; p>0.05 ) nor did it inhibit the ability of forskolin to raise cAMP in A2B cells ( Forskolin 60.1 ± 19.4 nmol/104 cells, Forskolin + 100nM ZM-241,385 46.4 ± 10.8 nmol/104 cells, mean ± sem, n=3; p>0.05 ). These results suggest that at the adenosine A2A but not A2B receptor, ZM-241,385 possesses properties consistent with inverse agonism the mechanism of which may involve interaction of the A2A receptor with adenylate cyclase. Arunlakshana, O. and Schild, H.O. (1959) Br. J. Pharmacol., 14: 48-58. |
