Effects of cell isolation procedures and radioligand selection on the characterization of human leukocyte beta-adrenergic receptors.
Effects of cell isolation procedures and radioligand selection on the characterization of human leukocyte beta-adrenergic receptors.
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细胞分离程序和放射性配体选择对人白细胞β-肾上腺素能受体表征的影响。
DOI：
10.1016/0006-2952(87)90041-4
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发表时间：
1987
影响因子：
5.8
通讯作者：
Henson,PM
中科院分区：
文献类型：
--
作者：
Szefler,SJ;Edwards3rd,CK;Haslett,C;Zahniser,NR;Miller,JA;Henson,PM
放射性配体结合技术常用于人外周血白细胞上β-肾上腺素能受体的表征。受体结合参数的准确解释需要适当的放射性配体选择。此外，细胞分离技术应对受体的结合参数具有最小的影响。我们观察了（−）-[125 I]碘氰基吲哚酚（ICYP）的曲线Scatchard图，重新评价了这种放射性配体和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）分离的单核细胞（MN）和多形核细胞（PMN）的膜在4名受试者中的3名中产生了ICYP的双相Scatchard图。相比之下，观察到ICYP与MN和PMN细胞膜结合的线性Scatchard图，MN和PMN细胞分离自相同的四名受试者，使用血浆Percoll的替代程序。与ICYP的竞争和饱和结合测定确定了高度的非特异性结合。与血浆Percoll细胞相比，在Ficoll-Hypaque细胞膜中观察到（−）-和（+）-普萘洛尔的立体选择性降低。ICYP的动力学分析表明，无论是用β-肾上腺素能受体拮抗剂还是激动剂引发置换，均存在明显的不可逆结合。ICYP的这些问题促使对替代放射性配体（−）-[125 I]碘吲哚酚（IPIN）进行评价;该放射性配体表现出快速和完全可逆的结合，改善的立体选择性和低非特异性结合。使用IPIN，来自另外三名受试者的Scatchard图对于两种细胞分离程序都是线性的。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。
Radioligand binding techniques are commonly used in the characterization of β-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 β-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 β-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 β-adrenergic receptor analysis incorporates the plasma Percoll cell isolation technique and the radioligand IPIN.