Gottowik J, Goetschy V, Henriot S, Kitas E, Fluhman B, Clerc R G, Moreau J L, Monsma F J, Kilpatrick G J
Pharma Division, F-Hoffmann-La Roche Ltd, Basel, Switzerland.
Neuropharmacology. 1997 Oct;36(10):1439-46. doi: 10.1016/s0028-3908(97)00098-1.
The binding of the novel radioligand, [3H]-rat urocortin to homogenates of rat cerebellum and homogenates of cells stably transfected with the human CRF1, rat CRF2alpha and rat CRF2beta receptors was examined. In each case, specific reversible high affinity binding was observed (K[d]s between 0.18 and 0.31 nM). The density of sites was relatively low in the cerebellum (9 fmol/mg tissue) but high in the recombinant systems with expression levels of between 1.4 and 6.3 pmol/mg protein. Agents known to interact with CRF receptors potently competed for binding in each case. The pharmacological profile of binding to the recombinant receptors were consistent with data previously published using other radioligands. Thus, for the recombinant CRF1 receptor, binding was inhibited with similar affinity by Urocortin, sauvagine, Urotensin 1 and CRF. The non-peptidic CRF antagonists (e.g. CP 154,526 and SC 241) also potently inhibited binding. The CRF2alpha and CRF2beta receptor recombinant systems had a very similar pharmacological profile with a clear rank order of potency for the peptide ligands (Urocortin > Sauvagine > Urotensin 1 > CRF), whereas the non-peptide CRF receptor antagonists had no measurable affinity. The pharmacological profile of specific [3H]-urocortin binding to homogentates of rat cerebellum was consistent with specific labelling of a CRF1 receptor. We conclude that [3H]-urocortin is a useful tool for the study of CRF receptors with the advantages that a filtration assay can be used, all CRF receptors can be labelled with the same ligand and the benefits associated with the low energy emittor, 3H.
研究了新型放射性配体[3H] - 大鼠尿皮质素与大鼠小脑匀浆以及稳定转染人CRF1、大鼠CRF2α和大鼠CRF2β受体的细胞匀浆的结合情况。在每种情况下，均观察到特异性可逆高亲和力结合（解离常数K[d]在0.18至0.31 nM之间）。小脑部位的结合位点密度相对较低（9 fmol/mg组织），但在重组系统中较高，表达水平在1.4至6.3 pmol/mg蛋白质之间。已知与CRF受体相互作用的试剂在每种情况下都能有效竞争结合。与重组受体结合的药理学特征与先前使用其他放射性配体发表的数据一致。因此，对于重组CRF1受体，尿皮质素、刺鼠肽、尾加压素1和CRF以相似的亲和力抑制结合。非肽类CRF拮抗剂（如CP 154,526和SC 241）也能有效抑制结合。CRF2α和CRF2β受体重组系统具有非常相似的药理学特征，肽类配体的效价有明确的排序（尿皮质素>刺鼠肽>尾加压素1>CRF），而非肽类CRF受体拮抗剂没有可测量的亲和力。[3H] - 尿皮质素与大鼠小脑匀浆特异性结合的药理学特征与CRF1受体的特异性标记一致。我们得出结论，[3H] - 尿皮质素是研究CRF受体的有用工具，其优点包括可使用过滤测定法、所有CRF受体可用相同配体标记以及与低能发射体3H相关的益处。
