Abstract
A selective agonist radioligand for A(2B) adenosine receptors (A(2B)ARs) is currently not available. Such a tool would be useful for labeling the active conformation of the receptors. Therefore, we prepared BAY 60-6583, a potent and functionally selective A(2B)AR (partial) agonist, in a tritium-labeled form. Despite extensive efforts, however, we have not been able to establish a radioligand binding assay using [(3)H]BAY 60-6583. This is probably due to its high non-specific binding and its moderate affinity, which had previously been overestimated based on functional data. As an alternative, we evaluated the non-selective A(2B)AR agonist [(3)H]NECA for its potential to label A(2B)ARs. [(3)H]NECA showed specific, saturable, and reversible binding to membrane preparations of Chinese hamster ovary (CHO) or human embryonic kidney (HEK) cells stably expressing human, rat, or mouse A(2B)ARs. In competition binding experiments, the AR agonists 2-chloroadenosine (CADO) and NECA displayed significantly higher affinity when tested versus [(3)H]NECA than versus the A(2B)-antagonist radioligand [(3)H]PSB-603 while structurally diverse AR antagonists showed the opposite effects. Although BAY 60-6583 is an A(2B)AR agonist, it displayed higher affinity versus [(3)H]PSB-603 than versus [(3)H]NECA. These results indicate that nucleoside and non-nucleoside agonists are binding to very different conformations of the A(2B)AR. In conclusion, [(3)H]NECA is currently the only useful radioligand for determining the affinity of ligands for an active A(2B)AR conformation.