1H-NMR studies of [Sar1]angiotensin II conformation by nuclear Overhauser effect spectroscopy in the rotating frame (ROESY): clustering of the aromatic rings in dimethylsulfoxide

The conformational properties of the octapeptide [Sar1]ANG II in dimethylsulfoxide-d6 were investigated by rotating frame nuclear Overhauser effect spectroscopy (ROESY). Interresidue ROESY interactions were observed between Tyr ortho and Phe ring protons, between Phe ring and Pro C gamma protons, and also between His C alpha and Pro C delta protons. A weak connectivity was also observed between the Sar N-CH3 protons and a Tyr ortho proton. Intraresidue interactions between alpha and beta protons in Tyr, His and Phe indicated restricted rotation for the side-chains of the three aromatic residues. These findings suggest that [Sar1]ANG II takes up a folded conformation in DMSO in which the three aromatic rings form a cluster. Connectivities between the His C alpha proton and the two Pro C delta protons illustrated a preferred conformation for angiotensin II in DMSO in which the His-Pro bond exists as the trans isomer. The NMR spectroscopic evidence is consistent with the presence of a Tyr charge relay system in the biologically active conformation of angiotensin II and with the postulated role of the Tyr hydroxyl group in angiotensin II for receptor activation.     

s-Cis and s-trans isomerism of the His-Pro peptide bond in angiotensin and thyroliberin analogues.

The dipeptide His-Pro isomerizes from all-s-trans to partly s-cis when titrated in D2O from acidic to neutral pD as observed by 13C and 1H nuclear magnetic resonance of the proline side chain. This isomerization is reported by the His C-2 and C-4 protons and carbons which show distinct, well-resolved resonances for each isomer. The influence of the His-Pro peptide bond rotational state on the histidine protons far removed from the bond has not been previously observed in model compounds or peptides. The peptides thyroliberin (TRH), [3-MeHis2]-TRH, and [3-MeHis6]-, [Sar1,Al8]-, and Nalpha-acetylangiotensin II were found to similarly isomerize from all-s-trans to partly s-cis as reported by their His C-2 and C-4 proton resonances. The His C-2 and C-4 protons in the peptides [1,3-diMeHis2]-TRH and [1-MeHis6]-, and [homoHis6]-angiotensin do not report this isomerization. Angiotensin II has previously been found to exhibit the same isomerization. The reporting of the s-trans to s-cis isomerization by the His C-2 proton appears to be correlated with the known potencies of the five angiotensin peptides in rat uterine strips and of the three TRH peptides by radioimmunoassay of released thyrotropin.     

Synthesis and biological activities of angiotensin II and Sarmesin analogues containing cyclohexylalanine

Analogues of angiotensin II with cyclohexylalanine (Cha) at position 4 or 8, and analogues of the competitive (type II) angiotensin antagonist [Sar1,Tyr(Me)4]ANG II (Sarmesin) with Cha at position 8, have been prepared by the solid phase method and purified by reversed-phase HPLC. Analogues of ANG II with Cha at position 8 in which the position 1 residue was substituted with sarcosine (Sar) or amino-isobutyric acid (Aib) or was deleted (Des), were slowly reversing (Type I) antagonists with "pA2" values in the rat isolated uterus assay of approximately 8.5. The additional substitution of Tyr(Me) for Tyr at position 4 of these peptides gave reversible competitive (Type I/II) antagonists with pA2 values of 6.7, 5.8, and less than 5, while substitution of Phe for Tyr gave pA2 values of 7.4, 6.7, and less than 5, respectively. All 19 peptides synthesized in this study had low intrinsic agonist activity in the rat isolated uterus assay except for the type I antagonists [Sar1, Cha8]ANG II (7%), [Aib1, Cha8]ANG II (12%) and [Des1, Cha8]ANG II (20%). These data illustrate that the substitution of Cha at position 8 of ANG II analogues produces potent antagonists; however, Type I antagonists retain significant agonist activity whereas Type I/II antagonists do not. In contrast, substitution of Cha at position 4 in a variety of ANG II analogues resulted in severely diminished biological activity, illustrating that the presence of an aromatic ring quadrupole at position 4 is obligatory for receptor binding and activity.     

Importance of the N-terminal domain of the type I angiotension II antagonist [Sar1,Ile8]ANG II for receptor blockade

Analogues of the Type I angiotensin (ANG) antagonist, [Sar1,Ile8]ANG II, in which the N-terminal dipeptide was modified were synthesized by the solid phase method and purified by reversed-phase HPLC. Antagonist potencies (pA2) of the peptides were determined on the rat isolated uterus using ANG II as the agonist. Substitution of the Arg residue occupying position 2 of [Sar1,Ile8]ANG II (pA2 8.1) by Gly, Ala, Nle, Phe, Pro or Sar reduced the antagonist potency to pA2 = 7.0, 6.8, 6.7, 6.8, 5.8 and 5.3, respectively. Deletion of the N-terminal Sar residue in these same peptides gave pA2 = 6.8, 5.7, 5.5, 5.9, 6.1 and 7.5, respectively. The characteristically long duration of action of [Sar1,Ile8] was absent for all of these analogues including (des1, Sar2, Ile8]ANG II. These findings demonstrate that the antagonist potencies of Type I angiotensin antagonists for smooth muscle receptors, and also the long duration of action, are dependent on the location of positive charges within the peptide and on the conformation of the molecule in determining favorable electrostatic interactions with the receptor. A model is proposed in which the two positively charged loci on the angiotensin molecule (N-terminus and Arg) interact with two corresponding anionic binding sites on the smooth muscle receptor. The possibility that the prolonged duration of action of [Sar1, Ile8]ANG II results from binding to a different site on the angiotensin receptor from that occupied by ANG II is discussed in relation to the present findings.     

1H-NMR studies of sarmesin and [des1]sarmesin conformation in dimethylsulfoxide by nuclear Overhauser effect (NOE) enhancement spectroscopy: folding of the N- and C-terminal domains

The conformational properties of the competitive angiotensin II antagonist sarmesin [Sar-Arg-Val-Tyr(Me)-His-Pro-Phe] and its heptapeptide analogue [des1]sarmesin in dimethylsulphoxide-d6 were investigated by nuclear Overhauser effect (NOE) enhancement studies. Assignment of all backbone and side-chain protons was possible by combining information from intraresidue NOE studies with two-dimensional correlated spectroscopy (COSY) studies. Saturation of the His C alpha proton of sarmesin produced essentially the same interresidue NOE enhancement of the two Pro C delta protons, illustrating the presence of the trans His-Pro bond. Saturation of the Sar N-methyl group caused enhancement of one of the His C beta protons, suggesting the presence of a turn in the N-terminal region of the molecule. Saturation of His C2 in sarmesin and [des1]sarmesin enhanced the Tyr(Me) methyl signal. Saturation of the Tyr(Me) methyl protons in [des1]sarmesin produced NOE enhancement of the His C2 and C4 protons, and saturation of the His C2 proton enhanced the Tyr(Me) meta and ortho proton signals. Interresidue interactions between the Tyr(Me) and His protons in sarmesin and [des1]sarmesin illustrate that these two side-chains remain in close proximity even in the absence of the postulated hydrogen bond between Tyr hydroxyl and the His imidazole ring in angiotensin II. The data suggest a preferred conformation for sarmesin in DMSO in which the peptide backbone is S-shaped and similar to that for angiotensin II.     

Interaction of angiotensin II with the C-terminal 300-320 fragment of the rat angiotensin II receptor AT1a monitored by NMR

Interaction between angiotensin II (Ang II) and the fragment peptide 300-320 (fCT300-320) of the rat angiotensin II receptor AT1a was demonstrated by relaxation measurements, NOE effects, chemical shift variations, and CD measurements. The correlation times modulating dipolar interactions for the bound and free forms of Ang II were estimated by the ratio of the nonselective and single-selective longitudinal relaxation rates. The intermolecular NOEs observed in NOESY spectra between HN protons of 9Lys(fCT) and 6His(ang), 10Phe(fCT) and 8Phe(ang), HN proton of 3Tyr(fCT) and Halpha of 4Tyr(ang), 5Phe(fCT)Hdelta and Halpha of 4Tyr(ang) indicated that Ang II aromatic residues are directly involved in the interaction, as also verified by relaxation data. Some fCT300-320 backbone features were inferred by the CSI method and CD experiments revealing that the presence of Ang II enhances the existential probability of helical conformations in the fCT fragment. Restrained molecular dynamics using the simulated annealing protocol was performed with intermolecular NOEs as constraints, imposing an alpha-helix backbone structure to fCT300-320 fragment. In the built model, one strongly preferred interaction was found that allows intermolecular stacking between aromatic rings and forces the peptide to wrap around the 6Leu side chain of the receptor fragment.     

Sarcosine1,glycine8 angiotensin II is an AT1 angiotensin II receptor subtype selective antagonist

Studies predating the discovery of the two major subtypes of angiotensin II (Ang II) receptors, AT1 and AT2, revealed anomalous characteristics of sarcosine1,glycine8 Ang II (Sar1,Gly8 Ang II). It competed poorly for 125I-Ang II binding in bovine brain but potently antagonized dipsogenic responses to intracerebroventricularly administered Ang II. Subsequent recognition that bovine brain contains AT(2) receptors, while dipsogenic responses to Ang II are mediated by AT1 receptors, suggests that Sar1,Gly(8) Ang II is AT1 selective. Sar1,Gly8 Ang II competed for 125I-sarcosine1,isoleucine8 Ang II binding to AT1 receptors in pituitary, liver and adrenal (the latter with the AT2 selective antagonist PD 123,319) with Ki's of 0.66, 1.40 and 1.36 nM, respectively. In contrast, the Ki of Sar1,Gly8 Ang II for AT2 receptors in rat adrenal (with the selective AT1 antagonist losartan) was 52 nM. 125I-Sar1,Gly8 Ang II (0.5-3 nM) bound to AT1 receptors in pituitary, liver, heart, adrenal, and hypothalamic membranes with high affinity (Kd=0.43, 1.6, 2.3, 0.96 and 1.8 nM, respectively), but showed no saturable binding to the adrenal AT2 receptor. 125I-Sar1,Gly8 Ang II selectively labeled AT1 receptors in sections of adrenal using receptor autoradiography. Thus, binding studies reveal Sar1,Gly8 Ang II to be the first angiotensin peptide analog to show AT1 receptor selectivity. 125I-Sar1,Gly8 Ang II offers a new means to selectively radiolabel AT1 receptors and may help to characterize ligand docking sites and agonist switches for AT1 versus AT2 receptors.     

Structure-desensitization relationships of angiotensin analogues in the rat isolated uterus

The desensitizing potencies of angiotensin II (ANG II) analogues modified at positions 1, 2, 4, 7, and 8 have been examined in the rat isolated uterus assay by determining the time of recovery of the half-maximal concentration (EC50) response to angiotensin II after treatment of the tissues with a high dose (10(-5) M) of each analogue for 2 min. The magnitude of the desensitization effect was substituent dependent in the following manner: position 1, sarcosine (Sar) greater than Asp greater than des-Asp; position 2, Arg greater than Sar; position 4, Tyr greater than Tyr(Me) approximately Phe; position 7, 3,4-dehydroproline (Dpr) greater than Pro greater than thioproline (Tpr) greater than Sar; position 8, Ile greater than D-Trp greater than Ala greater than Phe. The "additivity" rule applied to these structure-desensitization relationships and the most potent desensitizer, requiring 3 h for reestablishment of the EC50 response, was [Sar1, Dpr7, Ile8]-ANG II. The desensitizing potencies of these analogues did not correlate with agonist or antagonist activities and demonstrated that the angiotensin-mediated tissue desensitization process has unique structural determinants. Methylation or elimination of the tyrosine hydroxyl group of strong desensitizers virtually eliminated the desensitization effect, implicating the phenoxyl moiety in the mechanism of desensitization. The initial phase of recovery of angiotensin responsiveness after desensitization by several analogues appeared to obey first-order kinetics. The results are discussed in the contexts of both one- and two-site receptor models.     

Role of the His273 located in the sixth transmembrane domain of the angiotensin II receptor subtype AT2 in ligand-receptor interaction

Angiotensin II receptor subtypes AT1 and AT2 are proteins with seven transmembrane domain (TMD) topology and share 34% homology. It was shown that His256, located in the sixth TMD of the AT1 receptor, is needed for the agonist activation by the Phe8 side chain of angiotensin II, although replacing this residue with arginine or glutamine did not significantly alter the affinity binding of the receptor. We hypothesized that the His273 located in the sixth transmembrane domain of the AT2 receptor may play a similar role in the functions of the AT2 receptor, although this residue was not identified as a conserved residue in the initial homology comparisions. Therefore, we replaced His273 of the AT2 receptor with arginine or glutamine and analyzed the ligand-binding properties of the mutant receptors using Xenopus oocytes as an expression system. Our results suggested that the AT2 receptor mutants His273Arg and His273 Glu have lost their affinity to [125I-Sar1-Ile8]Ang II, a peptidic ligand that binds both the AT1 and AT2 receptors and to 125I-CGP42112A, a peptidic ligand that binds specifically to the AT2 receptor. Thus, His273 located in the sixth TMD of the AT2 receptor seems to play an important role in determining the binding properties of this receptor. Moreover, these results along with our previous observation that the Lys215 located in the 5th TMD of the AT2 receptor is essential for its high affinity binding to [125I-Sar1-Ile8]Ang II indicate that key amino acids located in the 5th and 6th TMDs of the AT2 receptor are needed for high affinity binding of the AT2 to its ligands.     

Influence of polyfluorination of the phenylalanine ring of angiotensin II on conformation and biological activity

[Phe(F5)8]angiotensin II was synthesized by the solid phase method and purified by reverse-phase HPLC. In rat uterus and rabbit aorta bioassays the analogue had 10 and 50%, respectively, of the contractile activity of angiotensin II and demonstrated antagonist properties. These findings illustrate that inversion of the Phe8 ring quadrupole moment in angiotensin II decreases agonist activity and invokes antagonist properties. 1H-NMR studies at 400 MHz in DMSO-d6 demonstrated the presence of cis and trans isomers in the ratio 1:3 due to restricted rotation of the His-Pro bond. Downfield shifts of the His C2 and C4 protons in [Phe(F5)]ANG II compared to ANG II suggest that the Phe(F5) residue may be involved in a parallel-plate ring pairing interaction with the imidazole group. However heteronuclear NOE studies, carried out by measuring the proton difference spectrum before and after saturation of the fluorine resonances, showed the absence of any NOE enhancement illustrating that electrostatic influences of the Phe(F5) ring occur at relatively long range.     

Design,synthesis and biological evaluation of cyclic angiotensin II analogues with 3,5 side-chain bridges. Role of C-terminal aromatic residue and ring cluster for activity and implications in the drug design of AT1 non-peptide antagonists

The novel amide linked angiotensin II (ANG II) cyclic analogues: gamma, epsilon -cyclo(3, 5)-[Sar(1)-Glu(3)-Lys(5)-Ile(8)] ANG II (I) and gamma, epsilon -cyclo(3, 5)-[Sar(1)-Glu(3)-Lys(5)-Phe(8)] ANG II (II) have been designed, synthesized and bioassayed in anesthetized rabbits in order to unravel structural ring cluster characteristics important for receptor activation. Analogue I with Ile at position 8 was an inhibitor of Angiotensin II while analogue II with Phe at position 8 was found to be an agonist. Similar results were reported for cyclic compounds that have reversed the linking between positions 3 and 5. The overall results show that positions 3 and 5 do not govern the biological activity of the synthetic analogues. It also appears that the aromatic ring cluster (Tyr-His-Phe) in agonist peptides is an essential stereo-electronic feature for Angiotensin II to exert its biological activity. A non-peptide mimetic of ANG II, 1-[2'-[(N-benzyl)tetrazol-5-yl]biphenyl-4-yl]methyl]-2-hydroxymethylbenzimidazole (BZI8) has been designed and synthesized. This molecule is more rigid and much less active than AT(1) non-peptide mimetic losartan probably because it lacks to mimic the orientation of tetrazole and the pharmacophore segments of butyl chain and imidazole ring.     

Conformational analysis of [Cpp1, Sar7, Arg8] vasopressin by 1H-NMR spectroscopy and molecular mechanics calculations.

A combined 1H-NMR and molecular mechanics study of [Cpp1, Sar7]AVP was performed in order to select the most probable conformations in DMSO solutions. The NMR constraints obtained were employed in the selection of starting conformations of the cyclic moiety of the analog. In particular, the diminished accessibility of the Asn5 NH proton to solvent and the close contact between Cpp1 and Cys6 C alpha H protons suggests a beta-turn conformation at the Phe3-Gln4 residues. Energy minimization was carried out both in the ECEPP/2 (rigid-valence geometry) and in the AMBER (flexible-valence geometry) force fields. Comparison of the experimental and calculated values of NMR characteristics has revealed that conformations containing type I, II, and III beta-turns at the Phe3-Gln4 residues are in reasonable agreement with the experimental data, with a dynamic equilibrium between the beta I (beta III) and beta II type structures of the cyclic part being the most probable. All of these conformations prefer the negative chirality of the disulfide bridge (theta 3 approximately -90 degrees). Five representative conformations were chosen for the acyclic tail: one with a beta I, one with a beta II'-turn at the Sar7-Arg8 residues, two extended-type conformations, and a conformation with a gamma-turn at Sar7. Because only high-energy extended conformations were in agreement with NMR data, it was concluded that the acyclic tail has considerable conformational flexibility in solution. The conformations obtained are discussed in terms of the structure-function relationship of the neurohypophyseal hormone analogs.     

Synthesis and biological activity of substance P C-terminal hexapeptide analogues: structure-activity studies

The synthesis of six hexapeptide analogues of C-terminal Substance P fragment containing alpha, beta-dehydrophenylalanine (delta Phe) in the position 7 or 8 is described. The effect of the structural changes on the hypotensive activity and antigenic properties of analogues was compared. It was found that substitution of delta Phe in various analogues of C-terminal hexapeptide of Substance P resulted in different effects on the hypotensive activity. The analogues [Glp6, delta Phe7]SP6-11 and [Glp6, delta Phe8]SP6-11 retained 70% and 45% of hypotensive activity of the C-terminal hexapeptide of Substance P, respectively but they showed a completely destroyed antigenic determinant. The analogues containing additionally Sar or His in the position 9 showed a complete lack of both: hypotensive activity and expression of the antigenic determinant of Substance P.     

Angiotensin II-induced increase of T-type Ca2+ current and decrease of L-type Ca2+ current in heart cells

The effect of angiotensin II (Ang II) on the T- and L-type calcium currents (I(Ca)) in single ventricular heart cells of 18-week-old fetal human and 10-day-old chick embryos was studied using the whole-cell voltage clamp technique. Our results showed that in both, human and chick cardiomyocytes, Ang II (10(-7)M) increased the T-type calcium current and decreased the L-type I(Ca). The effect of Ang II on both types of currents was blocked by the AT1 peptidic antagonist, [Sar1, Ala8] Ang II (2 x 10(-7)M). Protein kinase C activator, phorbol 12,13-dibutyrate, mimicked the effect of Ang II on the T- and L-type calcium currents. These results demonstrate that in fetal human and chick embryo cardiomyocytes Ang II affects the T- and L-type Ca2+ currents differently, and this effect seems to be mediated by the PKC pathway.     

Sar1Ile7]angiotensin III, a new selective antagonist of the pressor effect of angiotensin III in conscious rats

Two analogues of angiotensin III were compared as antagonists of the pressor response to angiotensin II (ANG II) and angiotensin III (ANG III) in conscious, unrestrained rats. Dose-mean arterial pressure (MAP) response curves were obtained for ANG II and ANG III in the absence or presence of [Ile7]ANG III (1.3 x 10(-7) mol/kg) or [Sar1 Ile7]ANG III (1.2 x 10(-7) mol/kg). In the presence of [Ile7]ANG III, the dose-MAP response curves for ANG II and ANG III were significantly displaced to the right. [Ile7]ANG III behaved as a partial agonist on ANG II but not ANG III receptors. In the presence of [Sar1 Ile7]ANG III, the dose-MAP response curve for ANG III but not ANG II was significantly displaced to the right. This suggests that [Sar1 Ile7]ANG III is a selective antagonist of ANG III in the vasculature. [Ile7]ANG III, on the other hand, antagonizes both ANG II and ANG III receptors. Our results support the hypothesis of the existence of a sub-class of angiotensin receptors activated by ANG III in the vascular smooth muscle.     

Selective down-regulation of AT2 receptors in uterine arteries from pregnant ewes given 24-h intravenous infusions of angiotensin II

Previously, we showed that uterine arteries from late gestation pregnant ewes infused intravenously with angiotensin II (Ang II) for 24 h, displayed heightened responsiveness to Ang II in vitro. Furthermore, we found that a small population of ewes with a "preeclampsia-like" disorder also displayed this. Therefore, we have investigated the density and affinity of Ang II receptor subtypes in the uterine arteries from these groups. Ang II receptor binding was measured using 125I [Sar1Ile8] Ang II. Proportions of AT1 and AT2 receptors were determined by inhibiting 125I [Sar1Ile8] Ang II with losartan (AT1 antagonist) or PD 123319 (AT2 antagonist). Uterine arteries from 24-h Ang II-infused ewes had a lower proportion of AT2 receptors (56.2+/-2.3%) than control (saline-infused) ewes (84.1+/-1.0%; P<0.05). The density of AT2 receptors was reduced (P<0.05) while the density of AT1 receptors was not different. Thus, 24-h infusions of Ang II selectively down-regulated AT2 receptors in the uterine artery, resulting in heightened Ang II reactivity. By contrast, the binding properties of Ang II receptor subtypes in uterine arteries from ewes with the "preeclampsia-like" disorder were not different from control ewes.     

Synthesis and AT2 receptor-binding properties of angiotensin II analogues.

The present study investigates the importance of the amino acid side chains in the octapeptide angiotensin II (Ang II) for binding to the AT2 receptor. A Gly scan was performed where each amino acid in Ang II was substituted one-by-one with glycine. The resulting set of peptides was tested for affinity to the AT2 receptor (porcine myometrial membranes). For a comparison, the peptides were also tested for affinity to the AT1 receptor (rat liver membranes). Only the substitution of Arg2 reduced affinity to the AT2 receptor considerably (92-fold when compared with Ang II). For the other Gly-substituted analogues the affinity to the AT2 receptor was only moderately affected. To further investigate the role of the Arg2 side chain for receptor binding, we synthesized some N-terminally modified Ang II analogues. According to these studies a positive charge in the N-terminal end of angiotensin III [Ang II (2-8)] is not required for high AT2 receptor affinity but seems to be more important in Ang II. With respect to the AT1 receptor, [Gly2]Ang II and [Gly8]Ang II lacked binding affinity (Ki > 10 microM). Replacement of the Val3 or Ile5 residues with Gly produced only a slight decrease in affinity. Interestingly, substitution of Tyr4 or His6, which are known to be very important for AT1 receptor binding, resulted in only 48 and 14 times reduction in affinity, respectively.     

The design and synthesis of a potent Angiotensin II cyclic analogue confirms the ring cluster receptor conformation of the hormone Angiotensin II

The novel amide linked Angiotensin II potent cyclic analogue, c-[Sar1,Lys3,Glu5] ANG II 19 has been designed and synthesized in an attempt to test the aromatic ring clustering and the charge relay bioactive conformation we have recently suggested for ANG II. This constrained cyclic analogue was synthesized by connecting the Lys3 amino and Glu5 carboxyl side chain groups, and it was found to be potent in the rat uterus assay and in anesthetized rabbits. The central part of the molecule is fixed covalently in the conformation predicted according to the backbone bend conformational model proposed for Angiotensin II. The obtained results using a combination of 2D NMR, 1D NOE spectroscopy and molecular modeling revealed a similar Tyr4-Ile5-His6 bend, a His6-Pro7 trans configuration and a side chain aromatic ring cluster of the key aminoacids Tyr4, His6, Phe8 for c-[Sar1,Lys3,Glu5] ANG II as it has been found for ANG II (Matsoukas, J. H.; Hondrelis, J.; Keramida, M.; Mavromoustakos, T.; Markriyannis, A.; Yamdagni, R.; Wu, Q.; Moore, G. J. J. Biol. Chem. 1994, 269, 5303). Previous study of the conformational properties of the Angiotensin II type I antagonist [Hser(gamma-OMe)8] ANG II (Matsoukas, J. M.; Agelis, G.; Wahhab, A.; Hondrelis, J.; Panagiotopoulos. D.; Yamdagni, R.; Wu, Q.; Mavromoustakos, T.; Maia, H.; Ganter, R.; Moore, G. J. J. Med. Chem. 1995, 38, 4660) using 1-D NOE spectroscopy coupled with the present study of the same type of lead antagonist Sarilesin revealed that the Tyr4-Ile5-His6 bend, a conformational property found in Angiotensin II is not present in type I antagonists. The obtained results provide an important conformational difference between Angiotensin II agonists and type I antagonists. It appears that our synthetic attempt to further support our proposed model was successful and points out that the charge relay system and aromatic ring cluster are essential stereoelectronic features for Angiotensin II to exert its biological activity.     

Comparative studies of angiotensin II and its analog [Sar1Ala8]angiotensin II on the density of cellular monolayer of cell cultures from green monkey kidney

The effects of the octapeptide angiotensin II (AT II) and its analog [Sar1Ala8]AT II on the cell density in cell culture from green monkey kidney (GMK) were studied. AT II and [Sar1Ala8]AT II provoked a decrease of the number of living cells depending on the concentration (0.001, 0.01 and 0.1 nM) and time of incubation (24, 48 and 72 hours), both peptides having a very similar activity. These data indicate that AT II and [Sar1Ala8]AT II may act on the same class of angiotensin receptors in GMK cells.     

An angiotensin II antagonist with strongly prolonged action

A highly hydrophobic analogue of angiotensin II (AT), [Sar1,(2',3',4',5',6'-Br5)Phe8]AT exhibited strong and persistent specific antagonism against AT, both in vitro and in vivo. This peptide exhibited 32% of the binding affinity of [Sar1]AT towards membranes of bovine adrenal cortex, it was a specific AT antagonist of irreversible character on smooth muscle assays, and it also suppressed for over 120 min at 7.10(-8) M/kg the blood pressure response towards AT in the rat blood pressure assay. This compound harbours therefore the potential of a new class of AT-specific antihypotensive drugs.