Greney Hugues, Urosevic Dragan, Schann Stephan, Dupuy Laurence, Bruban Véronique, Ehrhardt Jean-Daniel, Bousquet Pascal, Dontenwill Monique
Laboratoire de Neurobiologie et Pharmacologie Cardiovasculaire, Faculté de Medecine, Strasbourg, France.
Mol Pharmacol. 2002 Jul;62(1):181-91. doi: 10.1124/mol.62.1.181.
The I1 subtype of imidazoline receptors (I1R) is a plasma membrane protein that is involved in diverse physiological functions. Available radioligands used so far to characterize the I(1)R were able to bind with similar affinities to alpha2-adrenergic receptors (alpha2-ARs) and to I1R. This feature was a major drawback for an adequate characterization of this receptor subtype. New imidazoline analogs were therefore synthesized and the present study describes one of these compounds, 2-(2-chloro-4-iodo-phenylamino)-5-methyl-pyrroline (LNP 911), which was of high affinity and selectivity for the I1R. LNP 911 was radioiodinated and its binding properties characterized in different membrane preparations. Saturation experiments with [125I]LNP 911 revealed a single high affinity binding site in PC-12 cell membranes (K(D) = 1.4 nM; B(max) = 398 fmol/mg protein) with low nonspecific binding. [125I]LNP 911 specific binding was inhibited by various imidazolines and analogs but was insensitive to guanosine-5'-O-(3-thio)triphosphate. The rank order of potency of some competing ligands [LNP 911, PIC, rilmenidine, 4-chloro-2-(imidazolin-2-ylamino)-isoindoline (BDF 6143), lofexidine, and clonidine] was consistent with the definition of [125I]LNP 911 binding sites as I1R. However, other high-affinity I1R ligands (moxonidine, efaroxan, and benazoline) exhibited low affinities for these binding sites in standard binding assays. In contrast, when [125I]LNP 911 was preincubated at 4 degrees C, competition curves of moxonidine became biphasic. In this case, moxonidine exhibited similar high affinities on [125I]LNP 911 binding sites as on I1R defined with [125I]PIC. Moxonidine proved also able to accelerate the dissociation of [125I]LNP 911 from its binding sites. These results suggest the existence of an allosteric modulation at the level of the I1R, which seems to be corroborated by the dose-dependent enhancement by LNP 911 of the agonist effects on the adenylate cyclase pathway associated to I1R. Because [125I]LNP 911 was unable to bind to the I2 binding site and alpha2AR, our data indicate that [125I]LNP 911 is the first highly selective radioiodinated probe for I1R with a nanomolar affinity. This new tool should facilitate the molecular characterization of the I1 imidazoline receptor.
咪唑啉受体（I1R）的I1亚型是一种参与多种生理功能的质膜蛋白。到目前为止，用于表征I(1)R的现有放射性配体能够以相似的亲和力与α2 - 肾上腺素能受体（α2 - ARs）和I1R结合。这一特性是充分表征该受体亚型的一个主要缺点。因此合成了新的咪唑啉类似物，本研究描述了其中一种化合物，即2 - (2 - 氯 - 4 - 碘 - 苯基氨基) - 5 - 甲基 - 吡咯啉（LNP 911），它对I1R具有高亲和力和选择性。LNP 911经放射性碘化，并在不同的膜制剂中表征其结合特性。用[125I]LNP 911进行的饱和实验显示，PC - 12细胞膜中存在一个单一的高亲和力结合位点（K(D) = 1.4 nM；B(max) = 398 fmol/mg蛋白），非特异性结合较低。[125I]LNP 911的特异性结合受到各种咪唑啉及其类似物的抑制，但对鸟苷 - 5'-O-(3 - 硫代)三磷酸不敏感。一些竞争性配体[LNP 911、PIC、利美尼定、4 - 氯 - 2 - (咪唑啉 - 2 - 基氨基) - 异吲哚啉（BDF 6143）、洛非西定和可乐定]的效价顺序与将[125I]LNP 911结合位点定义为I1R一致。然而，其他高亲和力的I1R配体（莫索尼定、依酚氯铵和贝那唑啉）在标准结合试验中对这些结合位点表现出低亲和力。相反，当[125I]LNP 911在4℃下预孵育时，莫索尼定的竞争曲线变为双相。在这种情况下，莫索尼定在[125I]LNP 911结合位点上表现出与用[125I]PIC定义的I1R上相似的高亲和力。莫索尼定也被证明能够加速[125I]LNP 911从其结合位点的解离。这些结果表明在I1R水平存在变构调节，这似乎得到了LNP 911对与I1R相关的腺苷酸环化酶途径的激动剂效应的剂量依赖性增强的证实。由于[125I]LNP 911不能与I2结合位点和α2AR结合，我们的数据表明[125I]LNP 911是第一个具有纳摩尔亲和力的用于I1R的高选择性放射性碘化探针。这个新工具应该有助于I1咪唑啉受体的分子表征。
