Szefler S J, Edwards C K, Haslett C, Zahniser N R, Miller J A, Henson P M
Biochem Pharmacol. 1987 May 15;36(10):1589-97. doi: 10.1016/0006-2952(87)90041-4.
Radioligand binding techniques are commonly used in the characterization of beta-adrenergic receptors on human peripheral leukocytes. Accurate interpretation of receptor binding parameters necessitates appropriate radioligand selection. In addition, cell isolation techniques should have minimal effect on the binding parameters of receptors. Our observation of curvilinear Scatchard plots with (-)-[125I]iodocyanopindolol (ICYP) resulted in a re-evaluation of this radioligand and the influence of cell isolation techniques on leukocyte beta-adrenergic receptor binding parameters. Membranes from mononuclear (MN) and polymorphonuclear (PMN) cells isolated by a standard procedure (Ficoll-Hypaque) resulted in biphasic Scatchard plots with ICYP in three of four subjects. In contrast, linear Scatchard plots were observed for ICYP binding to membranes from MN and PMN cells isolated from the same four subjects with an alternative procedure utilizing plasma Percoll. Competition and saturation binding assays with ICYP identified a high degree of nonspecific binding. Decreased stereoselectivity with (-)- and (+)-propranolol was observed with membranes from Ficoll-Hypaque cells as compared to plasma Percoll cells. Kinetic analysis with ICYP demonstrated apparent irreversible binding whether displacement was initiated with a beta-adrenergic receptor antagonist or agonist. These problems with ICYP prompted evaluation of an alternative radioligand, (-)-[125I]iodopindolol (IPIN); this radioligand demonstrated rapid and completely reversible binding, improved stereoselectivity, and low nonspecific binding. Using IPIN, Scatchard plots from three additional subjects were linear for both cell isolation procedures. Based on these observations, the preferred method of human leukocyte beta-adrenergic receptor analysis incorporates the plasma Percoll cell isolation technique and the radioligand IPIN.
放射性配体结合技术常用于表征人外周血白细胞上的β-肾上腺素能受体。准确解释受体结合参数需要选择合适的放射性配体。此外，细胞分离技术应对受体的结合参数影响最小。我们观察到用（-）-[¹²⁵I]碘氰吲哚洛尔（ICYP）得到的Scatchard图呈曲线状，这促使我们重新评估这种放射性配体以及细胞分离技术对白细胞β-肾上腺素能受体结合参数的影响。通过标准程序（Ficoll-Hypaque）分离的单核（MN）细胞和多形核（PMN）细胞的膜，在四名受试者中有三名与ICYP产生了双相Scatchard图。相比之下，对于用血浆Percoll的另一种程序从同一四名受试者分离的MN和PMN细胞膜，观察到ICYP结合的Scatchard图呈线性。用ICYP进行的竞争和饱和结合试验确定了高度的非特异性结合。与血浆Percoll细胞相比，Ficoll-Hypaque细胞的膜对（-）-和（+）-普萘洛尔的立体选择性降低。用ICYP进行的动力学分析表明，无论用β-肾上腺素能受体拮抗剂还是激动剂引发置换，都表现出明显的不可逆结合。ICYP的这些问题促使人们评估另一种放射性配体，（-）-[¹²⁵I]碘吲哚洛尔（IPIN）；这种放射性配体表现出快速且完全可逆的结合、改善的立体选择性和低非特异性结合。使用IPIN，另外三名受试者的两种细胞分离程序的Scatchard图均为线性。基于这些观察结果，人白细胞β-肾上腺素能受体分析的首选方法包括血浆Percoll细胞分离技术和放射性配体IPIN。
