Pharmacology and direct visualisation of BODIPY‐TMR‐CGP: a long‐acting fluorescent β 2 ‐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 β 2 ‐adrenoceptor ligand in CHO‐K1 cells expressing the human β 2 ‐adrenoceptor. 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 n M . Both of these responses were antagonised by the selective β 2 ‐adrenoceptor antagonist ICI 118551. 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 β 2 ‐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 β 2 ‐adrenoceptors in living CHO‐K1 cells transfected with the human β 2 ‐adrenoceptor with an apparent K D of 27 n M . Studies with cells expressing a β 2 ‐adrenoceptor–green fluorescent protein (GFP) fusion protein provided further strong evidence that BODIPY‐TMR‐CGP was binding to the β 2 ‐adrenoceptor. BODIPY‐TMR‐CGP is therefore a long‐acting fluorescent β 2 ‐adrenoceptor agonist that can be used to label β 2 ‐adrenoceptors in the plasma membrane of living cells.British Journal of Pharmacology (2003) 139 , 232–242. doi: 10.1038/sj.bjp.0705287