Detection of the secondary, low-affinity beta(1)-adrenoceptor site in living cells using the fluorescent CGP 12177 derivative BODIPY-TMR-CGP

作者：Gherbi K; Briddon S J; Hill S J^*

来源：British Journal of Pharmacology, 2014, 171(23): 5431-5445.

DOI：10.1111/bph.12858

Background and PurposeCGP 12177 not only inhibits agonist effects mediated through the catecholamine site of the (1)-adrenoceptor with high affinity, but also exhibits agonist effects of its own at higher concentrations through a secondary, low-affinity (1)-adrenoceptor site or conformation. -blocker affinities for this CGP 12177%26apos; site of the human (1)-adrenoceptor have thus far only been characterized in functional studies. Here, we used the fluorescent CGP 12177 analogue BODIPY-TMR-CGP to directly investigate receptor-ligand interactions at the secondary binding site of the (1)-adrenoceptor. %26lt;br%26gt;Experimental ApproachThe human (1)-adrenoceptor was stably expressed in CHO cells containing a cAMP response element (CRE)-secreted placental alkaline phosphatase (SPAP) reporter gene construct. Functional responses of BODIPY-TMR-CGP were determined in the CRE-SPAP reporter gene assay, and manual and automated confocal microscopy platforms used to investigate the binding properties of BODIPY-TMR-CGP. %26lt;br%26gt;Key ResultsBODIPY-TMR-CGP displayed a pharmacological profile similar to that of CGP 12177, retaining agonist activity at the secondary (1)-adrenoceptor site. In confocal microscopy studies, specific BODIPY-TMR-CGP binding allowed clear visualization of (1)-adrenoceptors in live cells. Using a wider concentration range of labelled ligand in a high-content fluorescence-based binding assay than is possible in radioligand binding assays, two-site inhibition binding curves of -adrenoceptor antagonists were revealed in CHO cells expressing the human (1)-adrenoceptor, but not the (2)-adrenoceptor. %26lt;br%26gt;Conclusions and ImplicationsThe fluorescent CGP 12177 analogue allowed the detection of the (1)-adrenoceptor secondary site in both functional and binding studies. This suggests that BODIPY-TMR-CGP presents an important and novel fluorescent tool to investigate the nature of the secondary (1)-adrenoceptor site.

• 出版日期2014-12