AGONIST POTENCY AT THE CLONED HUMAN BETA-3 ADRENOCEPTOR DEPENDS ON RECEPTOR EXPRESSION LEVEL AND NATURE OF ASSAY

The cloned human beta-3 adrenoceptor was expressed in Chinese hamster ovary cells at three different levels (130, 400 and 3000 fmol/mg). The potency and intrinsic activity of a range of agonists in functional a ssays with these cell lines rose as a function of increasing receptor density. Operational analysis of concentration-response data allowed c alculation of functional affinity and efficacy of agonists at the huma n beta-3 adrenoceptor. The data highlighted the low efficacy of BRL 37 344 )-4-[(2-(2-(3-chlorophenyl)-2-hydroxyethyl)amino)- propyl]phenoxya cetate} for the human beta-3 adrenoceptor, which may explain its lower potency at the human receptor despite its higher affinity relative to isoprenaline. The potency of catecholamines at the human beta-3 adren oceptor was found to be 1 to 2 orders of magnitude higher when determi ned in an intact cell cAMP accumulation assay compared with a membrane -based adenylyl cyclase activation assay, The reason for this enhanced sensitivity is not clear, but the result is that the potency of the n atural agonist noradrenaline in the intact cell is considerably higher than predicted either from its ligand binding affinity, or from its p otency in membrane-based assays. Much smaller enhancements in sensitiv ity were observed for compounds of the aryloxypropanolamine class such as CGP 12177 t-butylamino-2-hydroxypropoxy)benzimidazol-2-one], with the result that the rank order of potency of such agonists at the beta -3 adrenoceptor was altered. In particular, CGP 12177 exhibited high r elative potency in the cyclase assay, but low relative potency in inta ct cell assays. These findings highlight the importance of selecting a ppropriate expression levels and appropriate assay methodology when cl oned receptors are used to characterize agonists.