无标记与常规细胞测定：人类组胺H1受体的功能研究。,Pharmacological Research

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无标记与常规细胞测定：人类组胺H1受体的功能研究。
Pharmacological Research ( IF 9.1 ) Pub Date : 2016-11-05 , DOI: 10.1016/j.phrs.2016.10.010
S Lieb ₁ , T Littmann ₁ , N Plank ₁ , J Felixberger ₁ , M Tanaka ₂ , T Schäfer ₃ , S Krief ₄ , S Elz ₁ , K Friedland ₃ , G Bernhardt ₁ , J Wegener ₅ , T Ozawa ₂ , A Buschauer ₁

Affiliation

一组组胺H1受体（H1R）激动剂和拮抗剂通过动态质量重新分配（DMR），细胞底物阻抗传感（ECIS）和各种信号通路特异性读数（Fura-2和水母发光蛋白钙测定，抑制蛋白募集（荧光素酶片段互补）测定，荧光素酶基因报告基因测定）。从基因改造的HEK293T细胞获得数据，并将其与GTPase分析和放射性配体结合的参考数据进行比较。组胺和其他H1R激动剂的测定相关pEC50值不同，但是，效价顺序保持不变。在萤光素酶片段互补测定中，H1R比β-arrestin1更喜欢β-arrestin2。钙和阻抗测定法取决于H1R的Gq偶联，如在Gq抑制剂FR900359（UBO-QIC）存在下完全抑制组胺诱导的信号所证明。尽管在DMR和基因报告基因分析中观察到了FR900359的部分抑制作用，但百日咳毒素显着降低了DMR中的响应，但增加了萤光素酶信号，反映了在这些分析中Gq和Gi均对信号传导的贡献。对于拮抗剂，DMR的结果与常规读数的结果基本兼容，而基于阻抗的数据则显示了更高的pKb值趋势。ECIS和钙分析法显然仅反映Gq信号传导，而DMR和基因报告基因分析法似乎整合了Gq和Gi介导的信号传导。结果证实了无标记方法DMR和ECIS对于表征H1R配体的价值。

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Label-free versus conventional cellular assays: Functional investigations on the human histamine H1 receptor.

A set of histamine H1 receptor (H1R) agonists and antagonists was characterized in functional assays, using dynamic mass redistribution (DMR), electric cell-substrate impedance sensing (ECIS) and various signaling pathway specific readouts (Fura-2 and aequorin calcium assays, arrestin recruitment (luciferase fragment complementation) assay, luciferase gene reporter assay). Data were gained from genetically engineered HEK293T cells and compared with reference data from GTPase assays and radioligand binding. Histamine and the other H1R agonists gave different assay-related pEC50 values, however, the order of potency was maintained. In the luciferase fragment complementation assay, the H1R preferred β-arrestin2 over β-arrestin1. The calcium and the impedimetric assay depended on Gq coupling of the H1R, as demonstrated by complete inhibition of the histamine-induced signals in the presence of the Gq inhibitor FR900359 (UBO-QIC). Whereas partial inhibition by FR900359 was observed in DMR and the gene reporter assay, pertussis toxin substantially decreased the response in DMR, but increased the luciferase signal, reflecting the contribution of both, Gq and Gi, to signaling in these assays. For antagonists, the results from DMR were essentially compatible with those from conventional readouts, whereas the impedance-based data revealed a trend towards higher pKb values. ECIS and calcium assays apparently only reflect Gq signaling, whereas DMR and gene reporter assays appear to integrate both, Gq and Gi mediated signaling. The results confirm the value of the label-free methods, DMR and ECIS, for the characterization of H1R ligands. Both noninvasive techniques are complementary to each other, but cannot fully replace reductionist signaling pathway focused assays.

更新日期：2019-11-01

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