Bricca G, Greney H, Dontenwill-Kieffer M, Zhang J, Belcourt A, Bousquet P
Laboratoire de Pharmacologie cardiovasculaire et rénale, CNRS URA589, Faculté de Médecine, Université Louis Pasteur, Strasbourg, France.
Neurochem Int. 1993 Feb;22(2):153-63. doi: 10.1016/0197-0186(93)90008-s.
The aim of the present study was to verify whether [3H]idazoxan can be considered as a highly selective ligand for imidazoline preferring receptors (IPR). In human frontal cortex membrane preparations [3H]idazoxan at a low concentration (2 nM) only labelled imidazoline sensitive, catecholamine insensitive sites. Binding was of high affinity, saturable and stereospecific. The rank order of potency of different compounds able to inhibit this binding was cirazoline > (+/-)-idazoxan > guanoxan > (-)-idazoxan > tolazoline > UK-14304 > clonidine. Amiloride, imidazol-4-acetic acid and histamine had no significant affinity for IPR labelled by [3H]idazoxan. [3H]idazoxan bound to 2 different sites (KD1 = 1 nM and KD2 = 16.4 nM). Clonidine behaved as a non competitive, non allosteric inhibitor of [3H]idazoxan binding. Both [3H]idazoxan binding sites were equally affected by clonidine. In membrane preparations obtained from the Nucleus Reticularis Lateralis region (NRL) of the brainstem, [3H]idazoxan binding was similar to that in cortical membranes, particularly with regard to specificity and kinetics. However, in the NRL region binding sites were 4-5 times more numerous than in the frontal cortex. Non linear analyses of saturation data obtained with NRL membrane preparations were compatible with both a one site and a two sites model. No significant effects of 1 mM MgCl2 alone or with 100 microM Gpp(NH)p were observed on either [3H]idazoxan binding or the competition with clonidine or rilmenidine. As in the cortical membrane, clonidine was a non competitive inhibitor of [3H]idazoxan binding to membranes from the NRL region. In conclusion, we show that when a low concentration is used, [3H]idazoxan binding to human brain involves sites almost completely insensitive to catecholamines and specific for imidazolines or related compounds. This binding involves two distinct sites. We also report that [3H]idazoxan imidazoline binding sites are not coupled with a G protein. Because of the non competitive interaction between clonidine and [3H]idazoxan for the binding sites of the latter, we are unable to conclude that the binding sites of the two drugs are identical. However, the non competitive, non allosteric interaction suggests a complex model of multiple binding sites.
本研究的目的是验证[³H]咪唑克生是否可被视为咪唑啉偏好受体（IPR）的高选择性配体。在人额叶皮质膜制剂中，低浓度（2 nM）的[³H]咪唑克生仅标记对咪唑啉敏感、对儿茶酚胺不敏感的位点。结合具有高亲和力、可饱和性和立体特异性。能够抑制这种结合的不同化合物的效价顺序为：西拉唑啉 > （±）-咪唑克生 > 胍生 > （-）-咪唑克生 > 妥拉唑啉 > UK-14304 > 可乐定。氨氯吡咪、咪唑-4-乙酸和组胺对[³H]咪唑克生标记的IPR没有显著亲和力。[³H]咪唑克生结合到2个不同位点（KD1 = 1 nM，KD2 = 16.4 nM）。可乐定作为[³H]咪唑克生结合的非竞争性、非变构抑制剂。两种[³H]咪唑克生结合位点受可乐定的影响相同。在从脑干外侧网状核（NRL）区域获得的膜制剂中，[³H]咪唑克生结合与皮质膜中的情况相似，特别是在特异性和动力学方面。然而，在NRL区域，结合位点的数量比额叶皮质多4 - 5倍。用NRL膜制剂获得的饱和数据的非线性分析与单一位点和双位点模型均相符。单独的1 mM MgCl₂或与100 μM Gpp（NH）p一起使用时，未观察到对[³H]咪唑克生结合或与可乐定或利美尼定竞争的显著影响。与皮质膜中一样，可乐定是[³H]咪唑克生与NRL区域膜结合的非竞争性抑制剂。总之，我们表明，当使用低浓度时，[³H]咪唑克生与人脑的结合涉及对儿茶酚胺几乎完全不敏感且对咪唑啉或相关化合物具有特异性的位点。这种结合涉及两个不同的位点。我们还报告[³H]咪唑克生咪唑啉结合位点不与G蛋白偶联。由于可乐定与[³H]咪唑克生在后者的结合位点之间存在非竞争性相互作用，我们无法得出两种药物的结合位点相同的结论。然而，这种非竞争性、非变构相互作用提示了一个多位点的复杂模型。
