Pharmacology and direct visualisation of BODIPY-TMR-CGP: a long-acting fluorescent beta2-adrenoceptor agonist.

1 Fluorescence techniques offer a way to circumvent several problems associated with many radioligand binding and functional assays and the need for large numbers of cells. Fluorescent ligands also offer the advantage of allowing real time direct visualisation of ligand - receptors interactions. A fluorescent analogue of CGP 12177 (BODIPY-TMR-CGP) has thus been evaluated as a beta(2)-adrenoceptor ligand in CHO-K1 cells expressing the human beta(2)-adrenoceptor. 2 Studies of (3)H-cAMP accumulation showed that BODIPY-TMR-CGP stimulated an increase in cAMP accumulation and cyclic AMP response element (CRE)-mediated gene transcription with an EC(50) of 21-28 nM. Both of these responses were antagonised by the selective beta(2)-adrenoceptor antagonist ICI 118551. 3 Binding studies with (3)H-CGP 12177, and functional studies of CRE-regulated gene transcription showed that the BODIPY-TMR-CGP interaction with the human beta(2)-adrenoceptor is of very long duration. 4 Visualisation of the binding of BODIPY-TMR-CGP to single living mammalian cells was clearly demonstrated by confocal microscopy and showed that this ligand was able to selectively label cell surface beta(2)-adrenoceptors in living CHO-K1 cells transfected with the human beta(2)-adrenoceptor with an apparent K(D) of 27 nM. Studies with cells expressing a beta(2)-adrenoceptor-green fluorescent protein (GFP) fusion protein provided further strong evidence that BODIPY-TMR-CGP was binding to the beta(2)-adrenoceptor. 5 BODIPY-TMR-CGP is therefore a long-acting fluorescent beta(2)-adrenoceptor agonist that can be used to label beta(2)-adrenoceptors in the plasma membrane of living cells.