Role of the N-terminal Amino Acid for the Biological Activities of Angiotensin and Inhibitory Analogues

Abstract
Aspartic acid was replaced in position 1 of angiotensin II (ATII) with several amino acids, to assess the possible influence of the N-terminal amino acid for (a) the intrinsic activity, (b) the affinity, and (c) the metabolic degradation of agonist analogues of ATII. Some of the substitutions in position 1 were used in combination with replacement of Phe by Gly or Leu in position 8, to obtain the corresponding antagonist.The compounds were tested in vivo (rat blood pressure) and in two in vitro preparations (rat stomach and rabbit aorta strips). The oil immersion technique, described by Kalsner and Nickerson (1968) (Can. J. Physiol. Pharmacol. 46, 719–730), was used to study the disposition of the peptides by vascular smooth muscles (rabbit aorta strips). Degradation of the peptides by purified aminopeptidases was evaluated in vitro by measuring the fragments on paper chromatography. Potency of antagonists was estimated in vivo (ID50) and in vitro (pA2 values): duration of action was established by infusing the inhibitors intravenously into anesthetized rats and testing the effect of standard doses of angiotensin before and after.The results indicate that replacement of Asp with other amino acids does not influence the intrinsic activity, but can either increase or decrease the affinity in vitro or the potency in vivo. 1-Sar-ATII, and 1-D-Ala-ATII are more potent and longer acting than 1-Asp-ATII on isolated intestinal and vascular smooth muscles, but not in vivo. On the contrary, 1-β-Asp-ATII and 1-β-D-Asp-ATII are more potent than 1-Asp-ATIIin vivo, but not on rabbit aorta strips. Rate of relaxation of rabbit aorta strips suspended in oil, after contraction with submaximal doses of several analogues of ATII, are significantly slower than relaxation after 1-Asn2-ATII and 1-Asp-ATII. A close parallelism between the diminution of the relaxation rate in oil and the degradation by aminopeptideses in vitro was observed, suggesting that metabolic degradation may be the major factor determining relaxation of rabbit aorta in oil after contraction with one of these peptides. Potencies of antagonists in vivo and in vitro are increased by replacing Asp with Sar. Substitution of Asp with β-Asp or β-D-Asp brings about a slight increase of potency in vivo but not in vitro. It appears that firm binding and prolonged occupation of receptors by sarcosyl derivatives are the primary factors contributing to increase the potency and to prolong the duration of action of antagonists, while prevention or reduction of metabolic breakdown by aminopeptidases is much less efficient.