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、泌尿组织和中枢神经系统等方面具有重要的生理作用。在目前的研究项目中。为了阐明α _1肾上腺素能受体的功能，我们进行了分子生物学和经典药理学研究。1 ． 兔肾上腺素能受体α _1亚型的分子克隆：筛选兔肝脏cDNA文库，鉴定出α _<1a> α _<1b>和α _<1d>亚型。兔α _<1b>和α _<1d>亚型与以往报道的其他物种具有较高的同源性和相同的药理特性。但在兔α _<1b>肾上腺素受体中发现了3个剪接变异体，它们在c端具有独特的序列，在兔的组织分布也不同。天然α _1肾上腺素能受体亚型的鉴定：通过放射配体结合和功能实验检测了天然α _1肾上腺素能受体，并在下列组织中进行了鉴定；2 .人前列腺α _<1A>和α _<1L>，人尿道α _<1L>，人主动脉α _<1B>，大鼠前列腺和兔虹膜α _<1A>和α _<1L>。细胞传感器检测α _1肾上腺素受体功能：人α _<1a> α _<1b>和α _<1d>肾上腺素受体在CHO细胞中稳定表达，细胞传感器检测细胞外酸化率（EAR）。EAR对α受体激动剂如去甲肾上腺素和可乐定的反应呈浓度依赖性增加。浓度-反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图的适宜性，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.