Rominger D H, Rominger C M, Fitzgerald L W, Grzanna R, Largent B L, Zaczek R
CNS Diseases Research, DuPont Merck Research Laboratories, Wilmington Delaware, USA.
J Pharmacol Exp Ther. 1998 Jul;286(1):459-68.
We describe the binding of [125I]tyr(o)sauvagine to membranes of corticotropin-releasing hormone (CRH2) receptor expressing HEK293/EBNA (293ECRH2 alpha) cells. The binding of [125I]tyr(o)sauvagine to CRH2 receptors was temperature, time and tissue dependent. Equilibrium was reached in 2 hr at 23 degrees C. Saturation data best fit a two-site model with affinity constants of 44 pM and 4.1 nM for high and low affinity states, respectively. The high affinity [125I]tyr(o)sauvagine binding sites were eliminated with 200 microM Gpp (NH) p, indicating coupling to G proteins. The rank order potency of peptide analogs of CRH to inhibit [125I]tyr(o)sauvagine binding to CRH2 alpha receptors was: urotensin > sauvagine = urocortin > alpha-helical CRH9-41 > rh-CRH >> o-CRH. This was in contrast to the rank order potency of the peptides at inhibiting [125I]tyr(o)oCRH binding to CRH, receptors: urotensin > urocortin > r/h-CRH > o-CRH = sauvagine > alpha-helical CRH9-41. We show that two recently identified small molecule CRH antagonists with nanomolar potency at the CRH1 receptor, have little or no affinity for CRH2 alpha receptors as labeled by [125I]tyr(o)sauvagine. Two selective CRH1 receptor antagonists enabled us to examine comparative densities of CRH1 and CRH2 receptors in several brain areas. We also used [125I]tyr(o)sauvagine in combination with a specific CRH1 antagonist to examine the anatomic distribution of CRH2 receptors using receptor autoradiography. With a few notable exceptions the CRH2 receptors demonstrated autoradiographically in this study match the data obtained by in situ hybridization studies on the localization of CRH2 mRNA. The anatomic overlap of the autoradiographic and in situ hybridization data suggest that CRH2 receptors are postsynaptic. This study demonstrates the utility of using [125I]tyr(o)sauvagine to study cloned CRH2 receptors expressed in cell lines. In addition, [125I]tyr(o)sauvagine used in conjunction with saturating concentrations of a specific CRH1 receptor antagonist allows the study of CRH2 receptors in brain tissues using both in vitro homogenate binding and receptor autoradiography techniques.
我们描述了[125I]酪（酪）促肾上腺皮质激素释放因子与表达促肾上腺皮质激素释放激素（CRH2）受体的人胚肾293/EBNA（293ECRH2α）细胞膜的结合情况。[125I]酪（酪）促肾上腺皮质激素释放因子与CRH2受体的结合具有温度、时间和组织依赖性。在23℃下2小时达到平衡。饱和数据最适合双位点模型，高亲和力和低亲和力状态的亲和常数分别为44 pM和4.1 nM。用200 μM Gpp（NH）p可消除高亲和力的[125I]酪（酪）促肾上腺皮质激素释放因子结合位点，表明其与G蛋白偶联。促肾上腺皮质激素释放激素肽类似物抑制[125I]酪（酪）促肾上腺皮质激素释放因子与CRH2α受体结合的效价顺序为：尾加压素>促肾上腺皮质激素释放因子=尿皮质素>α-螺旋CRH9-41>重组人促肾上腺皮质激素释放激素>>促肾上腺皮质激素释放激素。这与这些肽抑制[125I]酪（酪）促肾上腺皮质激素释放激素与CRH1受体结合的效价顺序相反：尾加压素>尿皮质素>重组人/人促肾上腺皮质激素释放激素>促肾上腺皮质激素释放激素=促肾上腺皮质激素释放因子>α-螺旋CRH9-41。我们发现，两种最近鉴定出的对CRH1受体具有纳摩尔效价的小分子CRH拮抗剂，对[125I]酪（酪）促肾上腺皮质激素释放因子标记的CRH2α受体几乎没有或没有亲和力。两种选择性CRH1受体拮抗剂使我们能够检测几个脑区中CRH1和CRH2受体的相对密度。我们还使用[125I]酪（酪）促肾上腺皮质激素释放因子与一种特异性CRH1拮抗剂联合使用，通过受体放射自显影术检测CRH2受体的解剖分布。除了一些明显的例外情况，本研究中放射自显影显示的CRH2受体与通过原位杂交研究CRH2 mRNA定位获得的数据相符。放射自显影和原位杂交数据的解剖学重叠表明CRH2受体是突触后受体。本研究证明了使用[125I]酪（酪）促肾上腺皮质激素释放因子研究细胞系中表达的克隆CRH2受体的实用性。此外，[125I]酪（酪）促肾上腺皮质激素释放因子与饱和浓度的特异性CRH1受体拮抗剂联合使用，可通过体外匀浆结合和受体放射自显影技术研究脑组织中的CRH2受体。
