Perez D M, Piascik M T, Malik N, Gaivin R, Graham R M
Department of Molecular Cardiology, Cleveland Clinic Research Institute, Ohio 44195.
Mol Pharmacol. 1994 Nov;46(5):823-31.
Three alpha 1-adrenergic receptors (ARs) have been cloned, i.e., the alpha 1B-, alpha 1C-, and alpha 1D-ARs. Compared with the alpha 1B subtype, the alpha 1A subtype in tissue is described as being insensitive to chloroethylclonidine and sensitive to SZL-49 and having a 10-100-fold higher affinity for a number of agonists and antagonists. The alpha 1A subtype is also expressed in a variety of rat tissues (as assessed by pharmacology), with greatest abundance in the cerebral cortex, hippocampus, vas deferens, and submaxillary gland. The cloned bovine alpha 1C-AR, though having an alpha 1A-AR pharmacology, was first reported as not being expressed in any rat tissue (as determined by Northern analysis) and was therefore designated as a new subtype. We report the cloning, expression, and characterization of the rat homolog of the bovine alpha 1C-AR. Using a human alpha 1C-AR probe obtained by polymerase chain reaction screening of a neuroblastoma cell line (SK-N-MC), both exon 1 and exon 2 of the rat alpha 1C-AR gene were cloned from a rat genomic library. These two exons were spliced together and cloned into the expression vector pMT2'. Transfection into COS-1 cells and analysis of the ligand-binding profile of the expressed protein receptor using 125I-HEAT revealed a 10-100-fold higher affinity for the alpha 1-AR antagonists 5-methylurapidil, (+)-niguldipine, WB-4101, and phentolamine and the agonists oxymetazoline and methoxamine, compared with the alpha 1B-AR. This ligand-binding profile is similar to that for endogenously expressed tissue alpha 1A-ARs. In addition, the rat alpha 1C-AR was the least sensitive of the three cloned subtypes to the alkylating effects of chloroethylclonidine but was the most sensitive to the alkylating prazosin analog SZL-49, properties also observed for the tissue alpha 1A subtype. Furthermore, by three different techniques, i.e., RNase protection assays, reverse transcription-polymerase chain reaction Northern blotting, and in situ hybridization histochemistry, the rat alpha 1C-AR mRNA was localized to alpha 1A-AR-rich tissues, such as rat vas deferens, hippocampus, aorta, and submaxillary gland. Taken together, these data suggest that this receptor may actually represent the alpha 1A subtype.
已克隆出三种α1 - 肾上腺素能受体（ARs），即α1B -、α1C - 和α1D - ARs。与α1B亚型相比，组织中的α1A亚型对氯乙可乐定不敏感，对SZL - 49敏感，并且对多种激动剂和拮抗剂的亲和力高10 - 100倍。α1A亚型也在多种大鼠组织中表达（通过药理学评估），在大脑皮层、海马体、输精管和颌下腺中含量最高。克隆的牛α1C - AR虽然具有α1A - AR药理学特性，但最初报道在任何大鼠组织中均未表达（通过Northern分析确定），因此被指定为一个新亚型。我们报道了牛α1C - AR大鼠同源物的克隆、表达和特性鉴定。使用通过对神经母细胞瘤细胞系（SK - N - MC）进行聚合酶链反应筛选获得的人α1C - AR探针，从大鼠基因组文库中克隆了大鼠α1C - AR基因的外显子1和外显子2。将这两个外显子拼接在一起并克隆到表达载体pMT2'中。转染到COS - 1细胞中，并使用125I - HEAT分析表达的蛋白受体的配体结合谱，结果显示与α1B - AR相比，对α1 - AR拮抗剂5 - 甲基尿嘧啶、（ + ） - 尼莫地平、WB - 4101和酚妥拉明以及激动剂羟甲唑啉和甲氧明的亲和力高10 - 100倍。这种配体结合谱与内源性表达的组织α1A - ARs相似。此外，大鼠α1C - AR是三种克隆亚型中对氯乙可乐定的烷基化作用最不敏感的，但对烷基化的哌唑嗪类似物SZL - 49最敏感，组织α1A亚型也具有这些特性。此外，通过三种不同技术，即核糖核酸酶保护测定、逆转录 - 聚合酶链反应Northern印迹和原位杂交组织化学，大鼠α1C - AR mRNA定位于富含α1A - AR的组织，如大鼠输精管海马体、主动脉和颌下腺。综上所述，这些数据表明该受体实际上可能代表α1A亚型。
