Bräuner-Osborne H, Brann M R
Department of Psychiatry, University of Vermont, Burlington 05405, USA.
Eur J Pharmacol. 1996 Jan 4;295(1):93-102. doi: 10.1016/0014-2999(95)00639-7.
Based on the ability of many receptors to amplify NIH 3T3 cells, we developed a high throughput assay of cloned receptor pharmacology. In this assay, receptors are transiently co-expressed with the marker enzyme beta-galactosidase. Receptors that induce cellular proliferation select and amplify the cells that also express the marker, thus the ability of ligands to alter receptor activity are reported as changes in enzyme activity. We used this assay to evaluate the pharmacology of agonist and antagonist ligands for five cloned human muscarinic receptor subtypes (m1-m5). When cells were transfected with subtypes that prefer the G-protein Gq (m1, m3, m5) robust increases in enzyme activity were observed. The subtypes that prefer Gi (m2 and m4) only induced beta-galactosidase when co-expressed with a chimera between the G-proteins Gq and Gi (Gq-i5). Overall, the rank-order of potency and intrinsic activity of most of the tested ligands were in remarkably good agreement with earlier results using cloned cell lines and isolated tissues. These data demonstrate that a high throughput colorimetric assay performed in 96-well plates can be used to evaluate subtle differences the pharmacology of ligands for cloned muscarinic receptor subtypes.
基于许多受体具有扩增NIH 3T3细胞的能力，我们开发了一种用于克隆受体药理学的高通量检测方法。在该检测方法中，受体与标记酶β-半乳糖苷酶瞬时共表达。诱导细胞增殖的受体选择并扩增同时表达该标记的细胞，因此配体改变受体活性的能力通过酶活性的变化来报告。我们使用该检测方法评估了五种克隆的人毒蕈碱受体亚型（m1 - m5）的激动剂和拮抗剂配体的药理学特性。当用偏好G蛋白Gq的亚型（m1、m3、m5）转染细胞时，观察到酶活性显著增加。偏好Gi的亚型（m2和m4）只有在与G蛋白Gq和Gi之间的嵌合体（Gq - i5）共表达时才诱导β-半乳糖苷酶表达。总体而言，大多数测试配体的效价和内在活性的排序与早期使用克隆细胞系和分离组织得到的结果非常吻合。这些数据表明，在96孔板中进行的高通量比色测定可用于评估克隆的毒蕈碱受体亚型配体药理学的细微差异。
