Saltman S, Baukal A, Waters S, Bumpus F M, Catt K J
Endocrinology. 1975 Aug;97(2):275-82. doi: 10.1210/endo-97-2-275.
The competitive binding activities of a number of angiotensin analogues were determined in a radioligand-receptor assay employing bovine adrenal crotex homogenate and [125I] iodoangiotensin II. This assay system has been shown to provide a precise and convenient method for evaluation and comparison of the binding-inhibition potencies of angiotensin II derivatives with agonist and antagonist activities. Agonist analogues of angiotensin II showed competitive binding activities in proportion to their known biological activities upon aldosterone production by the adrenal zona glomerulosa. Thus, the [Des-Asp1] heptapeptide of angiotensin II was equipotent with the native octapeptide in terms of binding-inhibition activity, and the [Sar1] derivative of angiotensin II was more potent than the native peptide. By contrast, the [Des-Asp1, Des-Arg2] hexapeptide and the [Des-Phe8] heptapeptide showed less than 1% of the activity of angiotensin II. Angiotensin II antagonists formed by C-terminal substitution of the octapeptide with isoleucine or alanine were also found to exhibit binding-inhibition activities in proportion to their known potencies as antagonists of the action of angiotensin II upon smooth muscle. Certain antagonists, such as [1-guanidoacetic, 8-isoleucine]-angiotensin II and [1-sarcosine, 8-isoleucine]-angiotensin II displayed significantly greater binding-inhibition potency than E1Asp1, Ileu5,-angiotensin II in the adrenal receptor system. Several, such as [Sar1, Ala8]-angiotensin II and [MeAla1, Ileu8]-antiotensin II were approximately equipotent with angiotensin II, while others such as [1-succinic acid, 8-alanine]-angiotensin II and [D-Ileu8]-angiotensin II showed no significant binding-inhibition activity. The relative binding-inhibition potencies of the antagonist peptides showed a general correlation with the pA2 values of the same components determined upon the smooth muscle response of the isolated aortic strip. The binding-inhibition potency of angiotensin II antagonists was also strongly influenced by the charge of the N-terminal residue, with enhancement of activity by more basic substituents and abolition of activity by highly acidic residues. The influence of the basicity of the N-terminus upon receptor binding was also observed with the agonist analogue [Sar1]-angiotensin II, which showed a 2- to 3-fold increase in binding-inhibition potency in comparison to native angiotensin II. The significant enhancement of binding-inhibition potency by N-terminal sarcosine substitution is attributable to higher affinity of the modified peptide for the angiotensin II receptor site, and is consistent with the increased activity of [Sar1]-angiotensin II upon smooth muscle and aldosterone production in vitro. The determination of binding-inhibition activity in the adrenal radioligand receptor assay provides a valid and convenient method for analysis of the role of binding affinity in the actions of competitive antagonists upon the responses of target cells to angiotensin II.
采用牛肾上腺皮质匀浆和[125I]碘代血管紧张素II，通过放射配体-受体分析法测定了多种血管紧张素类似物的竞争性结合活性。该分析系统已被证明为评估和比较具有激动剂和拮抗剂活性的血管紧张素II衍生物的结合抑制效力提供了一种精确且便捷的方法。血管紧张素II的激动剂类似物表现出的竞争性结合活性与它们对肾上腺球状带醛固酮生成的已知生物学活性成比例。因此，就结合抑制活性而言，血管紧张素II的[去天冬氨酸1]七肽与天然八肽相当，而血管紧张素II的[肌氨酸1]衍生物比天然肽更具活性。相比之下，[去天冬氨酸1，去精氨酸2]六肽和[去苯丙氨酸8]七肽的活性不到血管紧张素II的1%。通过用异亮氨酸或丙氨酸对八肽进行C末端取代形成的血管紧张素II拮抗剂，也被发现表现出与它们作为血管紧张素II作用于平滑肌的拮抗剂已知效力成比例的结合抑制活性。某些拮抗剂，如[1-胍基乙酸，8-异亮氨酸]-血管紧张素II和[1-肌氨酸，8-异亮氨酸]-血管紧张素II在肾上腺受体系统中显示出比E1天冬氨酸1，异亮氨酸5-血管紧张素II显著更高的结合抑制效力。几种，如[肌氨酸-丝氨酸1，丙氨酸8]-血管紧张素II和[甲基丙氨酸1，异亮氨酸8]-抗血管紧张素II与血管紧张素II大致相当，而其他的，如[1-琥珀酸，8-丙氨酸]-血管紧张素II和[D-异亮氨酸8]-血管紧张素II则没有显著的结合抑制活性。拮抗剂肽的相对结合抑制效力与在离体主动脉条平滑肌反应中测定的相同成分的pA2值总体上具有相关性。血管紧张素II拮抗剂的结合抑制效力也受到N末端残基电荷的强烈影响，碱性更强的取代基会增强活性，而高酸性残基会使活性丧失。激动剂类似物[肌氨酸1]-血管紧张素II也观察到N末端碱性对受体结合的影响，与天然血管紧张素II相比，其结合抑制效力增加了2至3倍。N末端肌氨酸取代导致结合抑制效力显著增强，这归因于修饰肽对血管紧张素II受体位点的更高亲和力，并且与[肌氨酸1]-血管紧张素II在体外对平滑肌和醛固酮生成的活性增加一致。在肾上腺放射配体受体分析中测定结合抑制活性，为分析竞争拮抗剂对靶细胞对血管紧张素II反应的作用中结合亲和力的作用提供了一种有效且便捷的方法。
