التفاصيل البيبلوغرافية
| العنوان: |
Detection of the secondary, low-affinity β1-adrenoceptor site in living cells using the fluorescent CGP 12177 derivative BODIPY-TMR- CGP. |
| المؤلفون: |
Gherbi, K, Briddon, S J, Hill, S J |
| المصدر: |
British Journal of Pharmacology; Dec2014, Vol. 171 Issue 23, p5431-5445, 15p, 1 Color Photograph, 1 Black and White Photograph, 5 Charts, 7 Graphs |
| مصطلحات موضوعية: |
ADRENERGIC receptors, CYCLIC adenylic acid, CHO cell, RECEPTOR-ligand complexes, ALKALINE phosphatase, CONFOCAL microscopy, FLUORESCENT dyes |
| مستخلص: |
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. [ABSTRACT FROM AUTHOR] |
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| قاعدة البيانات: |
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