Functional analysis of endogenous beta-adrenergic receptor through fluorimetric monitoring of cyclic nucleotide-gated ion channel.
Functional analysis of endogenous beta-adrenergic receptor through fluorimetric monitoring of cyclic nucleotide-gated ion channel.
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通过环核苷酸门控离子通道的荧光监测对内源性β-肾上腺素能受体进行功能分析。
DOI：
10.1016/j.ab.2006.04.051
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发表时间：
2007
影响因子：
2.9
通讯作者：
Diwu,Zhenjun
中科院分区：
文献类型：
--
作者：
Tang,Yi;Li,Xiuqing;Han,Xing;Lu,Junge;Diwu,Zhenjun
为了证明基于CNGC的cAMP测定法的实用性，进一步比较了三种cAMP测定法用于内源性β-肾上腺素能受体的功能分析。据报道，HEK 293细胞系具有内源性b1和b2肾上腺素能受体[13，14]。一种非特异性的b-肾上腺素能受体激动剂，异丙肾上腺素，被选为激活剂。HitHunter和CatchPoint测定试剂盒无法检测到响应于不同浓度异丙肾上腺素活化的任何cAMP变化，而基于CNGC的cAMP测定灵敏度足以确定异丙肾上腺素的EC 50值为14 nM（图2）。进一步使用基于CNGC的cAMP测定来测量其它β-肾上腺素能受体激动剂（图3A）和拮抗剂（图3 B）的EC 50值。已经广泛报道了GPCR激活期间的cAMP区室化[9，15]。在这些情况下，局部cAMP水平可能增加到足以启动某些生物学事件，尽管总胞质cAMP水平没有显著变化。大多数现有的cAMP测定需要细胞裂解来测量总细胞质cAMP。在当前研究中，0003-2697/$-参见前页© 2006 Elsevier Inc. All rights reserved. doi：10.1016/j. ab. 2006.04. 051
To demonstrate the utility of the CNGC-based cAMP assay, the three cAMP assays were further compared for functional analysis of endogenous b-adrenergic receptors. The HEK293 cell line was reported to have endogenous b1 and b2 adrenergic receptors [13, 14]. A nonspecific b-adrenergic receptor agonist, isoproterenol, was selected as an activator. The HitHunter and CatchPoint assay kits were unable to detect any cAMP change in response to the activation of isoproterenol at various concentrations, whereas the CNGC-based cAMP assay was sensitive enough to determine the EC50 value of isoproterenol to be 14 nM (Fig. 2). The CNGC-based cAMP assay was further used to measure EC50 values of other b-adrenergic receptor agonists (Fig. 3 A) and antagonists (Fig. 3 B). The cAMP compartmentalization during GPCR activation has been widely reported [9, 15]. Under these circumstances, the level of local cAMP may increase enough to initiate certain biological events, although the total cytosolic cAMP level does not change significantly. Most of the existing cAMP assays require cell lysis to measure total cytoplasmic cAMP. In the current study, 0003-2697/$-see front matter© 2006 Elsevier Inc. All rights reserved. doi: 10.1016/j. ab. 2006.04. 051