Kawai Y, Arinze I J
J Biol Chem. 1983 Apr 10;258(7):4364-71.
[3H]Dihydroalprenolol was used to study beta-adrenergic binding sites in plasma membranes isolated from rabbit liver. Specific binding was measured at 25 degrees C as the difference between total binding and binding in the presence of 2 microM dl-propranolol or 10 microM l-isoproterenol. Binding was saturable and stereoselective. The maximum number of binding sites (Bmax) was 434 +/- 41 fmol/mg of protein. The Kd for this binding as determined by Scatchard analysis was 1.39 +/- 0.09 nM. This value agreed well with the Kd value (1.27 +/- 0.12 nM) determined by kinetic analysis. The potency order for the displacement of bound [3H]dihydroalprenolol was isoproterenol greater than epinephrine greater than norepinephrine, indicative of beta 2-receptors. Use of beta 1- and beta 2-subtype-selective inhibitors also supported the interpretation that the binding characteristics are those of beta 2-receptors. Computer-aided analysis of this inhibition indicated that the beta-receptors in this membrane are predominantly, if not exclusively, of the beta 2-subtype. That these receptors are responsible for mediating catecholamine stimulation of hepatic glycogenolysis was deduced from the inhibition of agonist-stimulated glycogenolysis, in isolated hepatocytes, by beta-receptor subtype-selective antagonists. Thus, the hydrochloride of (t-butylamino-3-ol-2-propyl)oximino-9 fluorene, a beta-antagonist which has higher affinity at beta 2-sites than at beta 1-sites, was 3 orders of magnitude more potent in inhibiting isoproterenol-stimulated glycogenolysis than either atenolol or practolol, both of which are beta 1-selective antagonists. These results resemble the inhibition of [3H]dihydroalprenolol binding in plasma membranes. The glycogenolytic effects of catecholamines occurred with the potency order isoproterenol greater than epinephrine greater than norepinephrine. Thus, both by radioligand binding studies and by metabolic studies, the functional adrenergic receptor in the rabbit liver is shown to be of the beta 2-subtype.
[3H]二氢心得舒被用于研究从兔肝脏分离的质膜中的β-肾上腺素能结合位点。在25℃下，特异性结合通过总结合与在2微摩尔dl-普萘洛尔或10微摩尔l-异丙肾上腺素存在下的结合之间的差异来测定。结合是可饱和的且具有立体选择性。结合位点的最大数量（Bmax）为434±41飞摩尔/毫克蛋白质。通过Scatchard分析确定的该结合的解离常数（Kd）为1.39±0.09纳摩尔。该值与通过动力学分析确定的Kd值（1.27±0.12纳摩尔）非常吻合。结合的[3H]二氢心得舒被置换的效力顺序为异丙肾上腺素＞肾上腺素＞去甲肾上腺素，表明是β2受体。使用β1和β2亚型选择性抑制剂也支持了结合特性是β2受体的结合特性这一解释。对这种抑制作用的计算机辅助分析表明，该膜中的β受体如果不是完全的话，主要是β2亚型。从β受体亚型选择性拮抗剂对分离的肝细胞中激动剂刺激的糖原分解的抑制作用可以推断，这些受体负责介导儿茶酚胺对肝糖原分解的刺激。因此，（叔丁基氨基-3-醇-2-丙基）肟基-9-芴盐酸盐，一种在β2位点比在β1位点具有更高亲和力的β拮抗剂，在抑制异丙肾上腺素刺激的糖原分解方面比阿替洛尔和普拉洛尔这两种β1选择性拮抗剂强3个数量级。这些结果类似于质膜中[3H]二氢心得舒结合的抑制情况。儿茶酚胺的糖原分解作用的效力顺序为异丙肾上腺素＞肾上腺素＞去甲肾上腺素。因此，通过放射性配体结合研究和代谢研究都表明，兔肝脏中的功能性肾上腺素能受体是β2亚型。
