Molecular cloning and functional studies of alpha_1 adrenoceptors

α_1肾上腺素能受体在血压、脂质代谢、泌尿系统和中枢神经系统等方面具有重要的生理功能。在目前的研究项目中。我们进行了分子生物学和经典药理学研究，以阐明α_1肾上腺素受体的功能。兔α_1肾上腺素受体亚型的分子克隆：筛选兔肝cDNA文库，鉴定出α_<1a>α_<1b>和α_α_<1d>亚型。兔α_<1b>和α_<1d>亚型与已报道的其他物种的α_和α_亚型具有高度的同源性和相同的药理学特征。而在兔α肾上腺素受体中发现了三种剪接变异体<1b>，它们的C端序列不同，在兔体内的组织分布也不同.天然α_1肾上腺素受体亚型的鉴定：在放射性配体结合和功能实验中检测天然α_1肾上腺素受体，并在以下组织中鉴定：人前列腺中的α_<1A>和α_，<1L>人尿道中的α_<1L>，<1B>人主动脉中的α_，<1A><1L>大鼠前列腺和兔虹膜中的α_和α_.细胞传感器对α_1肾上腺素能受体功能的研究：人α_<1a>α_<1b>和α_<1d>肾上腺素能受体在CHO细胞中得到稳定表达，细胞传感器测定细胞外酸化速率（acidizationrate，acidizationrate，acidizationrate）。α受体激动剂如去甲肾上腺素和可乐定可使多巴胺浓度依赖性增加。对浓度-效应曲线的详细分析揭示了完全激动剂、部分激动剂、备用受体和Schild图的正确性，表明经典受体理论适用于该系统。

alpha_1 Adrenoceptor plays important physiological roles in blood pressure, lipid metabolism, urinary tissues and central nervous system. In the present research project. we carried out molecular biological and classical pharmacological studies in order to clarify alpha_1 adrenoceptor functions.1 . Molecular cloning of rabbit alpha_1 adrenoceptor subtypes : We screened rabbit liver cDNA library and identified alpha_<1a> alpha_<1b> and alpha_<1d> subtypes. Rabbit alpha_<1b> and alpha_<1d> subtypes showed high homology and the same pharmacological characters to those of other species reported previously. However, three splice variants were identified in rabbit alpha_<1b> adrenoceptor, which had uniqe sequence in C-terminal and showed different tissue distribution in rabbit.2. Identification of native alpha_1 adrenoceptor subtypes : Native alpha_1 adrenoceptors were examined in radioligand binding and functional experiments and identified in the following tissues ; alpha_<1A> and alpha_<1L> in human prostate, alpha_<1L> in human urethra, alpha_<1B> in human aorta, alpha_<1A> and alpha_<1L> in rat prostate and rabbit iris.3. Functional analysis of alpha_1 adrenoceptors with cytosensor : Human alpha_<1a> alpha_<1b> and alpha_<1d> adrenoceptors were stably expressed in CHO cells and the extracellular acidification rate (EAR) was measured with cytosensor. EAR increased concentration-dependently in response to alpha agonists such as noradrenaline and clonidine. Detailed analyses of concentration-response urves revealed full agonist, partial agonist, spare receptor and propriety of Schild plot, showing that classical receptor theory is applicable to this system.