Lazarus L H, Guglietta A, Wilson W E, Irons B J, de Castiglione R
Peptide Neurochemistry Section, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709.
J Biol Chem. 1989 Jan 5;264(1):354-62.
The opioid receptor preference for dermorphin and several dimerized structural analogues was investigated using rat brain synaptosomes and correlated with the potencies of intracerebroventricularly administered dimeric dermorphin peptides to inhibit gastric acid secretion. The carboxyl terminus of dermorphin or amino-terminal dermorphin analogues was bridged by dihydrazide or (poly)ethylenediamine structures. Synaptosomal membranes were prepared for radioligand binding assay in the presence of soybean trypsin inhibitor and preincubated to remove endogenously bound opioid peptides before storage at -70 degrees C. Specific radiolabeled agonists used in the radioligand binding assays were [D-Ala2,N-methyl-Phe4,Gly-ol5] [3H] enkephalin for mu-receptors and [D-Ala2,D-Leu5] [3H]enkephalin for delta-receptors. delta-Receptor binding assays were conducted in the presence of 2.6 microM [N-Me-Phe3,D-Pro4]morphiceptin to suppress peptide binding to mu-receptors. [D-Ala2,N-methyl-Phe4,Gly-ol5]enkephalin and dermorphin had affinities of 1.39 and 1.22 nM for mu-receptors and 355.8 and 178.6 nM for delta-receptors, respectively. Affinities of dimeric-dermorphin0 for mu- and delta-receptors, and the mu-selectivity ratio, exceeded values characteristic of dermorphin. The dimerized amino-terminal dermorphin analogues are peptides whose receptor binding differed from the parent molecule; e.g. the affinity of dimeric tetrapeptides toward mu-receptors was reduced but was increased for delta-receptors relative to monomeric dermorphin-(1-4)-amide. Dimeric tetradermorphin linked by a bridge containing 12 methylene units (di-tetra-dermorphin12), exhibited a dramatic loss in the mu-selectivity ratio as a result of diminished mu-affinity. On the other hand, substitution of Gly4 by Sar in di-tetra-dermorphin2 enhanced binding to mu-receptors: substitution of D-Arg2 for D-Ala resulted in an increased binding to mu-receptors while decreasing binding to delta-receptors, yielding a peptide with the highest mu-selectivity ratio. These substitutions of D-Arg2 and Sar4 in dimeric amino-terminal dermorphin pentapeptides enhanced binding to both mu- and delta-receptors relative to dermorphin-(1-5)-amide, but led to a decrease in its mu-selectivity ratio. Several dimeric dermorphin analogues exhibited an enhanced mu-selectivity ratio relative to their monomeric analogues. Dimeric peptides, which had a relatively high affinity for mu-receptors, were effective in the suppression of gastric acid secretion.
使用大鼠脑突触体研究了强啡肽和几种二聚化结构类似物对阿片受体的偏好，并将其与脑室内注射二聚化强啡肽肽抑制胃酸分泌的效力相关联。强啡肽的羧基末端或氨基末端强啡肽类似物通过二酰肼或（聚）乙二胺结构桥接。制备突触体膜用于在大豆胰蛋白酶抑制剂存在下的放射性配体结合测定，并在储存于-70℃之前进行预孵育以去除内源性结合的阿片肽。放射性配体结合测定中使用的特异性放射性标记激动剂是用于μ受体的[D-Ala2，N-甲基-Phe4，Gly-ol5][3H]脑啡肽和用于δ受体的[D-Ala2，D-Leu5][3H]脑啡肽。在2.6μM[N-Me-Phe3，D-Pro4]吗啡肽存在下进行δ受体结合测定，以抑制肽与μ受体的结合。[D-Ala2，N-甲基-Phe4，Gly-ol5]脑啡肽和强啡肽对μ受体的亲和力分别为1.39和1.22 nM，对δ受体的亲和力分别为355.8和178.6 nM。二聚化强啡肽0对μ和δ受体的亲和力以及μ选择性比超过了强啡肽的特征值。二聚化的氨基末端强啡肽类似物是其受体结合与母体分子不同的肽；例如，二聚化四肽对μ受体的亲和力相对于单体强啡肽-（1-4）-酰胺降低，但对δ受体的亲和力增加。由含有12个亚甲基单元的桥连接的二聚化四强啡肽（二-四-强啡肽12），由于μ亲和力降低，μ选择性比显著降低。另一方面，在二-四-强啡肽2中用Sar取代Gly4增强了与μ受体的结合：用D-Arg2取代D-Ala导致与μ受体的结合增加，同时与δ受体的结合减少，产生具有最高μ选择性比的肽。二聚化氨基末端强啡肽五肽中D-Arg2和Sar4的这些取代相对于强啡肽-（1-5）-酰胺增强了与μ和δ受体的结合，但导致其μ选择性比降低。几种二聚化强啡肽类似物相对于其单体类似物表现出增强的μ选择性比。对μ受体具有相对高亲和力的二聚化肽在抑制胃酸分泌方面是有效的。
