Detection of the secondary, low-affinity β1-adrenoceptor site in living cells using the fluorescent CGP 12177 derivative BODIPY-TMR-CGP
In: British Journal of Pharmacology, Jg. 171 (2014-11-24), S. 5431-5445
Online
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Zugriff:
Background and Purpose CGP 12177 not only inhibits agonist effects mediated through the catecholamine site of the β1-adrenoceptor with high affinity, but also exhibits agonist effects of its own at higher concentrations through a secondary, low-affinity β1-adrenoceptor site or conformation. β-blocker affinities for this ‘CGP 12177’ site of the human β1-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 β1-adrenoceptor. Experimental Approach The human β1-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. Key Results BODIPY-TMR-CGP displayed a pharmacological profile similar to that of CGP 12177, retaining agonist activity at the secondary β1-adrenoceptor site. In confocal microscopy studies, specific BODIPY-TMR-CGP binding allowed clear visualization of β1-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 β-adrenoceptor antagonists were revealed in CHO cells expressing the human β1-adrenoceptor, but not the β2-adrenoceptor. Conclusions and Implications The fluorescent CGP 12177 analogue allowed the detection of the β1-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 β1-adrenoceptor site.
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Detection of the secondary, low-affinity β1-adrenoceptor site in living cells using the fluorescent CGP 12177 derivative BODIPY-TMR-CGP
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Autor/in / Beteiligte Person: | Gherbi, Karolina ; Briddon, Stephen J. ; Hill, Stephen J. |
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Zeitschrift: | British Journal of Pharmacology, Jg. 171 (2014-11-24), S. 5431-5445 |
Veröffentlichung: | Wiley, 2014 |
Medientyp: | unknown |
ISSN: | 0007-1188 (print) |
DOI: | 10.1111/bph.12858 |
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