Bertin B, Mansier P, Makeh I, Briand P, Rostene W, Swynghedauw B, Strosberg A D
Laboratoire d'Immunopharmacologie Moléculaire, Institut Cochin de Génétique Moléculaire, Paris, France.
Cardiovasc Res. 1993 Sep;27(9):1606-12. doi: 10.1093/cvr/27.9.1606.
The aim was to develop a transgenic mouse model of atrial beta 1 adrenoceptor overexpression in order to create atrial alteration of the receptor transduction system.
Transgenic founders were generated after microinjection of the transgene construct into the pronucleus of fertilised mouse eggs. Heterozygous progeny were screened for RNA expression of the human beta 1 adrenoceptor gene under the control of a 0.56 kb proximal promoter of the human atrial natriuretic factor. One line, out of the three obtained, was selected and further characterised for overexpression of the human beta 1 adrenoceptor. Polymerase chain reaction was employed to detect beta 1 adrenoceptor mRNA, and 125I-cyanopindolol (ICYP) binding assays were used to quantify receptors in heart membranes. A quantitative autoradiographic ICYP binding technique was also used to visualise atrial and ventricular beta adrenoceptors in heart sections.
The human beta 1 adrenoceptor was overexpressed specifically in the atria of transgenic mice. The level of the beta 1 adrenoceptor was 5-10-fold higher in transgenic mice compared to basal murine beta 1 adrenoceptors in non-transgenic control mice. Left and right atrial receptor overexpression was confirmed by in vitro autoradiography. The human receptors were able to couple to the murine stimulatory G proteins (Gs), as shown by high affinity binding site dosage using the beta adrenoceptor agonist isoprenaline. Isoprenaline displacement studies allowed the determination of two different affinity sites, one of high affinity (KH = 5.8 nM), and one of low affinity (KL = 520 nM). When expressed in terms of protein density (fmol.mg-1), atrial transgenic beta 1 adrenoceptors displayed a threefold increase in high affinity sites (KH) as compared to control mice. Preliminary electrocardiographic data showed supraventricular premature beats in 6/14 transgenic mice v 2/16 control mice.
These transgenic mice may provide a useful pharmacological tool to investigate the pathophysiological consequences of the overactivation of atrial beta 1 adrenoceptor-adenylyl cyclase signalling system.
构建心房β1肾上腺素能受体过表达的转基因小鼠模型，以造成受体转导系统的心房改变。
将转基因构建体显微注射到受精小鼠卵的原核中，产生转基因奠基者。在人心房利钠因子0.56 kb近端启动子控制下，筛选杂合子后代中人β1肾上腺素能受体基因的RNA表达。从获得的三个品系中选择一个品系，并进一步对人β1肾上腺素能受体的过表达进行表征。采用聚合酶链反应检测β1肾上腺素能受体mRNA，并使用125I-氰基吲哚洛尔（ICYP）结合试验定量心脏膜中的受体。还使用定量放射自显影ICYP结合技术可视化心脏切片中的心房和心室β肾上腺素能受体。
人β1肾上腺素能受体在转基因小鼠的心房中特异性过表达。与非转基因对照小鼠的基础鼠β1肾上腺素能受体相比，转基因小鼠中β1肾上腺素能受体的水平高5至10倍。体外放射自显影证实了左、右心房受体的过表达。如使用β肾上腺素能受体激动剂异丙肾上腺素进行高亲和力结合位点定量所示，人受体能够与鼠刺激性G蛋白（Gs）偶联。异丙肾上腺素置换研究确定了两个不同的亲和力位点，一个高亲和力位点（KH = 5.8 nM）和一个低亲和力位点（KL = 520 nM）。以蛋白质密度（fmol.mg-1）表示时，心房转基因β1肾上腺素能受体的高亲和力位点（KH）与对照小鼠相比增加了三倍。初步心电图数据显示，14只转基因小鼠中有6只出现室上性早搏，而16只对照小鼠中有2只出现室上性早搏。
这些转基因小鼠可能为研究心房β1肾上腺素能受体-腺苷酸环化酶信号系统过度激活的病理生理后果提供有用的药理学工具。
