Angel I, Le Rouzic M, Pimoule C, Graham D, Arbilla S
Synthélabo Recherche, Rueil-Malmaison, France.
Ann N Y Acad Sci. 1995 Jul 12;763:112-24. doi: 10.1111/j.1749-6632.1995.tb32396.x.
Recent studies have shown that cirazoline, an alpha 1-adrenoceptor agonist, has greater affinity than do other imidazoline or guanidinium compounds at imidazoline recognition sites. In this report we used [3H]cirazoline as a probe to characterize imidazoline recognition sites present in membrane homogenates of rat brain and kidney as well as pancreatic beta HIT T15 cells. Specific binding of [3H]cirazoline to these various homogenates was saturable and reversible and was resolved into two classes of high affinity binding sites. Competition inhibition studies of [3H]cirazoline binding to these different membrane preparations were performed with alkaloid, phenylethylamine, imidazoline, and guanidinium compounds. Catecholamines and non-imidazoline adrenoceptor ligands such as epinephrine, benextramine, prazosin, propranolol, rauwolscine, or adrenoceptor ligands such as epinephrine, benextramine, prazosin, propranolol, rauwolscine, or yohimbine did not compete with [3H]cirazoline (Ki > 10 microM). Under our experimental conditions, only guanidinium and imidazoline derivatives had high affinities for [3H]cirazoline binding sites. Unlabeled cirazoline, clonidine, bromoxidine, idazoxan, and amiloride had the highest affinities with this respective rank order. These results suggest that [3H]cirazoline is a novel high affinity radioligand that specifically labels nonadrenergic imidazoline-guanidinium sites in the brain, kidney, and beta cells. Furthermore, the obtained rank order of inhibition suggests that [3H]cirazoline binding does not distinguish between I1 and I2 sites. In addition, we compared the specific binding of [3H]cirazoline with that of the alpha 2-adrenoceptor antagonist [3H]rauwolscine in chinese hamster ovary (CHO) cell lines stably expressing human alpha 2C2-, alpha 2C4-, and alpha 2C10-adrenoceptor subtypes. Using [3H]rauwolscine as a probe, each of these transfected cell lines expressed high levels for the three different alpha 2-adrenoceptor subtypes (Bmax values were between 2 and 7 pmol.mg-1 protein). In contrast, none of these cell lines displayed measurable imidazoline recognition sites. In summary, [3H]cirazoline is a novel high affinity radioligand that specifically labels imidazoline recognition sites without significant alpha- or beta-adrenoceptor binding. Furthermore, our results using alpha 2-adrenoceptor transfected cells confirm that the imidazoline recognition sites and each of the cloned alpha 2-adrenoceptor subtypes represent distinct macromolecular entities.
最近的研究表明，α1 - 肾上腺素能受体激动剂西拉唑啉在咪唑啉识别位点上比其他咪唑啉或胍类化合物具有更高的亲和力。在本报告中，我们使用[3H]西拉唑啉作为探针来表征大鼠脑、肾以及胰腺β - HIT T15细胞的膜匀浆中存在的咪唑啉识别位点。[3H]西拉唑啉与这些不同匀浆的特异性结合是可饱和且可逆的，并可分为两类高亲和力结合位点。用生物碱、苯乙胺、咪唑啉和胍类化合物对[3H]西拉唑啉与这些不同膜制剂的结合进行了竞争抑制研究。儿茶酚胺和非咪唑啉肾上腺素能受体配体，如肾上腺素、苄胺、哌唑嗪、普萘洛尔、育亨宾或劳丹素，不与[3H]西拉唑啉竞争（Ki>10μM）。在我们的实验条件下，只有胍类和咪唑啉衍生物对[3H]西拉唑啉结合位点具有高亲和力。未标记的西拉唑啉、可乐定、溴昔洛韦、艾司唑仑和阿米洛利具有最高的亲和力，顺序依次为上述排列。这些结果表明，[3H]西拉唑啉是一种新型的高亲和力放射性配体，可特异性标记脑、肾和β细胞中的非肾上腺素能咪唑啉 - 胍类位点。此外，所获得的抑制顺序表明，[3H]西拉唑啉结合不能区分I1和I₂位点。此外，并比较了[3H]西拉唑啉与α₂ - 肾上腺素能受体拮抗剂[3H]劳丹素在中国仓鼠卵巢（CHO）细胞系中稳定表达人α₂C₂ -、α₂C₄ - 和α₂C₁₀ - 肾上腺素能受体亚型时的特异性结合。以[3H]劳丹素为探针，这些转染细胞系中的每一个都对三种不同的α₂ - 肾上腺素能受体亚型表达高水平（Bmax值在2至7 pmol·mg⁻¹蛋白质之间）。相比之下，这些细胞系中没有一个显示出可测量的咪唑啉识别位点。总之，[3H]西拉唑啉是一种新型的高亲和力放射性配体，可特异性标记咪唑啉识别位点，而无明显的α - 或β - 肾上腺素能受体结合。此外我们使用α₂ - 肾上腺素能受体转染细胞的结果证实，咪唑啉识别位点和每个克隆的α₂ - 肾上腺素能受体亚型代表不同的大分子实体。
