Pharmacology and direct visualisation of BODIPY-TMR-CGP: a long-acting fluorescentβ2-adrenoceptor agonist

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 β2-adrenoceptor ligand in CHO-K1 cells expressing the human β2-adrenoceptor. Studies of 3H-cAMP accumulation showed that BODIPY-TMR-CGP stimulated an increase in cAMP accumulation and cyclic AMP response element (CRE)-mediated gene transcription with an EC50 of 21–28 nM. Both of these responses were antagonised by the selective β2-adrenoceptor antagonist ICI 118551. Binding studies with 3H-CGP 12177, and functional studies of CRE-regulated gene transcription showed that the BODIPY-TMR-CGP interaction with the human β2-adrenoceptor is of very long duration. 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 β2-adrenoceptors in living CHO-K1 cells transfected with the human β2-adrenoceptor with an apparent KD of 27 nM. Studies with cells expressing a β2-adrenoceptor–green fluorescent protein (GFP) fusion protein provided further strong evidence that BODIPY-TMR-CGP was binding to the β2-adrenoceptor. BODIPY-TMR-CGP is therefore a long-acting fluorescent β2-adrenoceptor agonist that can be used to label β2-adrenoceptors in the plasma membrane of living cells. British Journal of Pharmacology (2003) 139, 232–242. doi:10.1038/sj.bjp.0705287