Wei E T, Thomas H A, Christian H C, Buckingham J C, Kishimoto T
School of Public Health, University of California, Berkeley 94720, USA.
Peptides. 1998;19(7):1183-90. doi: 10.1016/s0196-9781(98)00085-0.
The activities of corticotropin-releasing hormone (CRH)-related peptides and several analogs were examined in cells transfected with either CRH1 or CRH2beta receptors, in suppression of heat-induced rat paw edema in pentobarbital-anesthetised animals and in stimulation of release of immunoreactive corticotropin (ir-ACTH) from rat anterior pituitary tissue in vitro. The peptides tested were human/rat (h/r)-CRH, r-urocortin, h-urocortin, white sucker fish or maggy sole urotensin I and some analogs of these peptides substituted with D-amino acids at residues 4 (urocortin), 5 (CRH and urotensin I) and 20 (CRH). In cells transfected with CRH1 receptors, these peptides were similar in potency in stimulation of cAMP accumulation. By contrast, at CRH2beta receptors peptides of the urocortin and urotensin series were more potent than h/r-CRH while [D-Glu20]-h/r-CRH was 6.5-fold less active than h/r-CRH. I.v. administration of h/r-CRH or related peptides 10 min prior to a thermal stimulus produced a significant dose-dependent inhibition of rat paw edema formation. Comparison of the ED50's showed that urocortins ([D-Ser4]-h-urocortin, h-urocortin, [D-Pro4]-r-urocortin, r-urocortin) were approximately 2 to 3 times more active than h/r-CRH, but [D-Glu20]-h/r-CRH was 18.5-fold less active. In the assay for ir-ACTH release, the activity of h/r-CRH and [D-Glu20]-h/r-CRH was similar but [D-Pro5]-h/r-CRH and [D-Pro4]-r-urocortin was less potent than the native peptide. These results provide further evidence that D-amino acid substitution at residue 20 reduces the potency of h/r-CRH at endogenous (anti-edema effect) and transfected (cAMP accumulation) CRH2beta receptors whilst activity at the CRH1 receptor is retained (ACTH-release and cAMP accumulation). On the other hand substitutions at residues 4 or 5 in r-urocortin or h/r-CRH respectively appear to decrease activity at CRH1 but not CRH2beta receptors The modified CRH and urocortin analogs described here may provide clues for the further design of receptor selective ligands.
在转染了促肾上腺皮质激素释放激素1（CRH1）或促肾上腺皮质激素释放激素2β（CRH2β）受体的细胞中，在戊巴比妥麻醉的动物中抑制热诱导的大鼠爪肿胀以及在体外刺激大鼠垂体前叶组织释放免疫反应性促肾上腺皮质激素（ir-ACTH）的实验中，检测了促肾上腺皮质激素释放激素（CRH）相关肽及其几种类似物的活性。所测试的肽包括人/大鼠（h/r）-CRH、大鼠尿皮质素、人尿皮质素、白鲑鱼或鲽鱼的尾加压素I以及这些肽的一些类似物，这些类似物在第4位（尿皮质素）、第5位（CRH和尾加压素I）和第20位（CRH）用D-氨基酸进行了取代。在转染了CRH1受体的细胞中，这些肽在刺激环磷酸腺苷（cAMP）积累方面的效力相似。相比之下，在CRH2β受体上，尿皮质素和尾加压素系列的肽比h/r-CRH更有效，而[D-谷氨酸20]-h/r-CRH的活性比h/r-CRH低6.5倍。在热刺激前10分钟静脉注射h/r-CRH或相关肽，可产生显著的剂量依赖性抑制大鼠爪肿胀形成。对半数有效剂量（ED50）的比较表明，尿皮质素（[D-丝氨酸4]-人尿皮质素、人尿皮质素、[D-脯氨酸4]-大鼠尿皮质素、大鼠尿皮质素）的活性比h/r-CRH高约2至3倍，但[D-谷氨酸20]-h/r-CRH的活性低18.5倍。在ir-ACTH释放测定中，h/r-CRH和[D-谷氨酸20]-h/r-CRH的活性相似，但[D-脯氨酸5]-h/r-CRH和[D-脯氨酸4]-大鼠尿皮质素的效力低于天然肽。这些结果进一步证明，第20位的D-氨基酸取代降低了h/r-CRH在内源性（抗肿胀作用）和转染（cAMP积累）的CRH2β受体上的效力，同时保留了在CRH1受体上的活性（ACTH释放和cAMP积累）。另一方面，大鼠尿皮质素第4位或h/r-CRH第5位的取代分别似乎降低了在CRH1受体上的活性，但不影响CRH2β受体。本文所述的修饰后的CRH和尿皮质素类似物可能为进一步设计受体选择性配体提供线索。
