Reid S D, Moon T W, Perry S F
Department of Biology, University of Ottawa, Ontario, Canada.
J Exp Biol. 1991 Jul;158:199-216. doi: 10.1242/jeb.158.1.199.
Although many studies have characterized these receptors according to pharmacological criteria, this work represents only the second direct characterization of the rainbow trout beta-adrenergic receptors. Radioligand binding assays using (+/-)-4-(3-t-butylamino-2-hydroxy-propoxy)-[5,7-3H]benzimidazol-2- one ([3H]CGP 12177) and 1-[4,6-propyl 3H]dihydroalprenolol ([3H]DHA) were conducted to determine equilibrium binding times, ligand-receptor dissociation constants (KD) and binding capacities (Bmax). Furthermore, we assessed the influence of erythrocyte handling, suspension medium and endogenous catecholamines on Bmax and KD. Maximal binding was obtained when erythrocytes were handled minimally and maintained suspended in plasma rather than physiological saline. Washing and resuspending the erythrocytes, as well as the transfer of the erythrocytes into saline, significantly impaired apparent radioligand affinity and receptor density. Endogenous catecholamines, at levels considered normal for non-stressed animals, did not interfere with the radioligand binding assays, and thus eliminated the need to wash and resuspend erythrocytes. Based on the binding characteristics after intentional lysis of erythrocytes, it is shown that the total receptor population of trout erythrocytes can be estimated by propranolol-displaceable DHA binding, the density of high-affinity surface receptors can best be determined by isoproterenol-displaceable CGP binding, and the number of receptors located within the erythrocytes can be calculated from the difference between the total receptor density and the number of isoproterenol-displacable DHA binding sites. Each of these components must be considered when performing radioreceptor assays using these radioligands and this has significant implications for the interpretation of erythrocyte beta-adrenorecptor localization and mobilization.
尽管许多研究已根据药理学标准对这些受体进行了表征，但这项工作只是对虹鳟鱼β-肾上腺素能受体的第二次直接表征。使用（±）-4-（3-叔丁基氨基-2-羟基-丙氧基）-[5,7-³H]苯并咪唑-2-酮（[³H] CGP 12177）和1-[4,6-丙基³H]二氢阿普洛尔（[³H] DHA）进行放射性配体结合试验，以确定平衡结合时间、配体-受体解离常数（KD）和结合容量（Bmax）。此外，我们评估了红细胞处理、悬浮介质和内源性儿茶酚胺对Bmax和KD的影响。当对红细胞进行最小程度的处理并使其保持悬浮在血浆而非生理盐水中时，可获得最大结合。洗涤和重悬红细胞，以及将红细胞转移到盐水中，会显著损害表观放射性配体亲和力和受体密度。对于非应激动物而言，处于正常水平的内源性儿茶酚胺不会干扰放射性配体结合试验，因此无需洗涤和重悬红细胞。基于故意裂解红细胞后的结合特性，结果表明，鳟鱼红细胞的总受体数量可通过普萘洛尔可置换的DHA结合来估计，高亲和力表面受体的密度可通过异丙肾上腺素可置换的CGP结合来最佳确定，红细胞内的受体数量可根据总受体密度与异丙肾上腺素可置换的DHA结合位点数量之间的差异来计算。在使用这些放射性配体进行放射受体测定时，必须考虑这些成分中的每一个，这对红细胞β-肾上腺素能受体定位和动员的解释具有重要意义。
