Conformations of synthetic tetradecapeptide renin substrate and of angiotensin I in aqueous solution.

The properties of aqueous solutions of synthetic renin substrate tetradecapeptide (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Leu-Val-Tyr-Ser) were examined through electrometric titrations, infrared and circular dichroism spectroscopy, and spectrofluorometry. Titration studies of angiotensin I (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu) were also made, whose results indicated a flexible folded conformation similar to that previously proposed for the octapeptide angiotensin II, with a possible additional beta turn at the C terminus. The experimental results of the tetradecapeptide study, associated with Chou and Fasman calculations and with an analysis of structure-activity relationships in renin substrates and competitive inhibitors, led to the proposal of a beta turn involving the His-Pro-Phe-His sequence of the tetradecapeptide. This beta turn would be stabilized by beta-antiparallel interaction between residues 3-4 and 10-12 and by electrostatic attraction between the N-terminal ammonium and C-terminal carboxylate groups and would be destabilized below pH 5 by electrostatic repulsion between His6 and His9. The capacity to assume this conformation is related to structural requirements for renin substrates and competitive inhibitors.     

Radioimmunoassay for the determination of angiotensin II and its fragments]

A radioimmunoassay procedure for the determination of angiotensin and its fragments: C-terminal hexapeptide and middle tetrapeptide has been developed. The sensitivity of the method reaches up to 5 ng/ml. The accuracy calculated from the standard deviation, for angiotensin and of its fragments presents the average +/- 1,5%; reproducibility (n = 10) is +/- 0,51% for angiotensin, +/- 2,12% for C-terminal hexapeptide and +/- 6,93% for the middle tetrapeptide. Using the Ouchterlony test with the cross-reactions showed that antibodies elicited to angiotensin interact with its fragments--middle tetrapeptide and C-terminal hexapeptide.     

Biological activities of angiotensin II-(1-6)-hexapeptide and angiotensin II-(1-7)-heptapeptide in man

Biological activities of angiotensin II-(1-6)-hexapeptide [ANG-(1-6)] and angiotensin II-(1-7)-heptapeptide [ANG-(1-7)] were studied in 5 normal men and 3 patients with Bartter's syndrome. The angiotensins were infused iv in each subject from 0900 h to 0915 h at a rate of 21 nmol(16.8 micrograms)/kg X min and 18 nmol(16.2 micrograms)/kg X min for ANG-(1-6) and ANG-(1-7), respectively. In the normal men a significant rise in blood pressure was observed by the infusions of both peptides. Average increments of blood pressure for ANG-(1-6) were 17/14, 23/18, 22/15 and 17/14 mmHg at 2, 5, 10 and 15 min, respectively, and those for ANG-(1-7) were 19/15, 20/17, 13/13 and 15/13 mmHg at 2, 5, 10 and 15 min, respectively. The duration of pressor actions after the cessation of the infusions (T) was 10 min for ANG-(1-6) and 20 (for systolic) and 30 (for diastolic) min for ANG-(1-7). T for ANG-(1-6) was shorter than and T for ANG-(1-7) was similar to T for Ile5-angiotensin II (Ile5-ANG II) reported previously in 7 normal men 5 of whom were the same as examined in the present study. On the other hand, both peptides did not cause a rise in blood pressure in the 3 patients with Bartter's syndrome. Both angiotensins did not cause an increase in plasma aldosterone but did cause a significant decrease in plasma renin activity both in the normal men and in the patients. From these results and our previous observations of inactivity of angiotensin II-(5-8)-tetrapeptide, a pressor action of angiotensin II-(4-8)-pentapeptide, and pressor, renin-suppressing and steroidogenic actions of angiotensin II-(3-8)-hexapeptide in normal men, it is thought that ANG-(1-6) and ANG-(1-7) are bound to angiotensin II (ANG II) receptor in the peripheral arterioles and show pressor actions (less than 0.024% and less than 0.028% of Ile5-ANG II, respectively) and suppress renin mainly via short loop feedback and that the shortest biologically active ANG II molecules for pressor, renin-suppressing and steroidogenic actions are Tyr-Ile-His, Val-Tyr-Ile-His and Val-Tyr-Ile-His-Pro-Phe, respectively, in man. It is also evident that ANG-(1-6) is more rapidly metabolized than ANG-(1-7) or Ile5-ANG II in man.     

Conformations of angiotensin II and enkephalin related to N.M.R.

Statistical samples of conformations for Angiotensin II and Enkephalin, obtained from a Monte-Carlo method, are models proposed to simulate the behavior of these hormones under different physico-chemical conditions. Analysis of molecular conformations shows that, for the two peptides, extended conformations are more likely to be present in acidic solutions used for N.M.R. measurements and folded conformations due to interactions of charged terminal groups are favoured at neutral pH.     

Angiotensin-(1-7) binds at the type 1 angiotensin II receptors in rat renal cortex.

Significant angiotensin (Ang) (1-7) production occurs in kidney and effects on renal function have been observed. The present study was undertaken to investigate binding characteristics of the heptapeptide to Ang II receptors present in rat renal cortex. [125I]-Ang II binding to rat glomeruli membranes was analyzed in the presence of increasing concentrations of Ang II, Ang-(1-7), DUP 753 and PD 123319. Linearity of the Scatchard plot of the [125I]-Ang II specific binding to rat glomeruli membranes indicated a single population of receptors, with a Kd value of 0.7 +/- 0.1 nM and a Bmax of 198 +/- 0.04 fmol/mg protein. DUP 753, an specific AT1 receptor antagonist, totally displaced the specific binding of [125I]-radiolabelled hormone with a Ki of 15.8 +/- 0.9 nM, while no changes were observed in the presence of the selective AT2 receptor antagonist, PD 123319. The specific [125I]-Ang II binding to rat glomerular membranes was displaced by Ang-(1-7) with high affinity (Ki = 8.0 +/- 3.2 nM). We conclude that radioligand binding assays in the presence of selective Ang II antagonists DUP 753 and PD 123319 suggest the unique presence of AT1, receptors in rat glomeruli and a possible role in the control of the biological renal effects of Ang-(1-7).     

Amphiphilic properties of angiotensin and its fragments

Many biologically active peptides are supposed to interact with specific receptors mainly due to hydrophobic forces. In order to obtain a more detailed information about the peptide molecule behavior at the "water-non-polar-phase" boundary an approach to the calculation of stable conformations on such a boundary has been developed. This approach is used for investigation of the amphiphilic properties of angiotensin and its six fragments. The results of calculations of transfer energies of these peptides from the water environment to the phase boundary are in agreement with the experimental data.     

Proton NMR studies of angiotensin II and its analogs in aqueous solution

The¹H nuclear magnetic resonance (NMR) spectra of angiotensin II (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) and five of its octapeptide analogs as well as angiotensin I (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu) and angiotensin III (Arg-Val-Tyr-Ile-His-Pro-Phe) in aqueous solutions (90% H₂O/10% D₂O) were completely assigned by two-dimensional COSY and ROESY experiments. All of the peptides give rise to two distinct sets of signals. The minor set accounts for about 5% of the total population belowpH 5.5 and increases to 12–20% aroundpH 7.0. The two sets of signals result from acis-trans isomerization of the His-Pro peptide bond with the major resonances arising from thetrans isomer. One analog in which the Pro is replaced with a D-Pro displays a very different isomerization behavior. The measured coupling constants J_NH-CH, the temperature dependence of the amide proton shifts and the relative intensities of the intraresidue and sequential NH-CH ROEs, are all indicative of an extended backbone conformation for ANGII. However, some evidence for the existence of conformers with local structure involving preferred sidechain positions for the Tyr, His, Phe, and the carboxyl group of the Phe was found, particularly in the ROESY andpH-titration experiments. Moreover,pH effects and the unusual amide exchange behavior of the Arg NH suggests the presence of interactions between the Asp and Arg sidechains of ANGII. At low temperatures the Arg guanidinium NH₂ protons were detected as two broad peaks which are related by sizeable exchange peaks in ROESY experiments. This behavior could be useful as a general probe for the study of Arg sidechain mobility and accessibility in other peptides and proteinsPreliminary results of this work have been presented at the XIIth International Conference on Magnetic Resonance in Biological Systems in abstract form (1988).     

Quaternary solution structures of galectins-1, -3, and -7

Galectins are a growing family of animal lectins with functions in growth regulation and cell adhesion that bind beta-Gal residues in oligosaccharides. Evidence indicates that some of the biological properties of galectins are due to their cross-linking activities with multivalent glycoconjugate receptors. Therefore determination of the quaternary solution structures of these proteins is important in understanding their structure-function properties. The present study reports analytical sedimentation velocity and equilibrium data for galectins-1, -3, and -7 in the absence and presence of bound LacNAc, the natural ligand epitope. Galectin-1 from bovine heart and recombinant human galectin-7 were found to be stable dimers by both methods. In contrast, recombinant murine galectin-3, as well as its proteolytical derived C-terminal domain, are predominantly monomeric. The presence of LacNAc at concentrations sufficient to fully saturate the proteins had no significant effect on either the weight average molecular weight determined by sedimentation equilibrium or the hydrodynamic properties determined from sedimentation velocity experiments. These results show that binding of a monovalent ligand does not affect oligomerization of these galectins.     

Chronic infusion of angiotensin-(1-7) reduces heart angiotensin II levels in rats

We tested the hypothesis that the actions of Angiotensin (Ang)-(1-7) in the heart could involve changes in tissue levels of Ang II. This possibility was addressed by determining the effect of chronic infusion of Ang-(1-7) on plasma and tissue angiotensins. Ang-(1-7) was infused subcutaneously (osmotic minipumps) in Wistar rats. Angiotensins were determined by radioimmunoassay (RIA) in plasma, heart, and kidney. Tissue and plasma angiotensin-converting enzyme (ACE) activity and plasma renin activity (PRA) were also measured. Cardiac and renal ACE2 mRNA levels and cardiac angiotensinogen mRNA levels were assessed by semi-quantitative polymerase chain reaction (PCR). AT1 receptor number was evaluated by autoradiograph. Chronic infusion of Ang-(1-7) (2 microg/h, 6 days) produced a marked decrease of Ang II levels in the heart. A less pronounced but significant decrease of Ang-(1-7) was also observed. No significant changes were observed for Ang I. Ang II was not altered in the kidney. In this tissue, a significant increase of Ang-(1-7) and Ang I concentration was observed. A significant increase of plasma Ang-(1-7) and Ang II was also observed. Ang-(1-7) infusion did not change ACE activity or PRA. A selective slight significant increase in ACE2 expression in the heart was observed. Heart angiotensinogen mRNA as well as the number of Ang II binding sites did not change. These results suggest that AT1 receptors-independent changes in heart Ang II concentration might contribute for the beneficial effects of Ang-(1-7) in the heart. Moreover, these results reinforce the hypothesis that this angiotensin plays an important site-specific role within the renin-angiotensin system.     

Endothelin-1, angiotensin II and cancer

Endothelin-1 (ET-1) and angiotensin II (AngII), two potent vasoactive peptides involved in the regulation of cardiovascular homeostasis, also induce mitogenic and pro-angiogenic responses in vitro and in vivo. Both peptides are produced by cleavage of inactive precursors by metalloproteases (endothelin-converting enzyme and angiotensin-converting enzyme, respectively) and activate two subtypes of membrane receptors (ETA-R and ETB-R for ET-1, AT1R and AT2R for AngII) that all belong to the superfamily of G-protein coupled receptors. There is increasing evidence that ETA-R, ETB-R and AT1R are expressed in a variety of cancer cells and tissues, and may play a role on tumor growth, angiogenesis and invasion in vivo. This review summarizes the similarities and differences between the ET-1 and AngII systems with regard to their reported effects on various aspects of cancer. In addition to being expressed on vascular endothelium, ET-1 and AngII receptors participate in tumor angiogenesis through the production of the angiogenic factor VEGF. Furthermore, recent clinical studies indicate that a selective ETA-R antagonist has beneficial effects in prostate cancer, suggesting that a similar approach using ETB-R and AT1R blockers might be envisioned. Experimental data presented here suggest that a combined therapy targeting both ET-1 and AngII systems may prove valuable for future treatments of highly angiogenic tumors.     

Discrimination of two angiotensin II receptor subtypes with a selective agonist analogue of angiotensin II, p-aminophenylalanine6 angiotensin II

Angiotensin II receptor binding sites in rat liver and PC12 cells differ in their affinities for a nonpeptidic antagonist, DuP 753, and p-aminophenylalanine6 angiotensin II. In liver, which primarily contains the sulfhydryl reducing agent-inhibited type of angiotensin II receptor, which we refer to as the AII alpha subtype, DuP 753 displays an IC50 of 55 nM, while p-aminophenylalanine6 angiotensin II displays an IC50 of 8-9 microM. In PC12 cells, which primarily contain the angiotensin II receptor type whose binding affinity is enhanced by sulfhydryl reducing agents (AII beta), DuP 753 displays an IC50 in excess of 100 microM, while p-aminophenylalanine6 angiotensin II displays an IC50 of 12 nM. p-Aminophenylalanine6 angiotensin II binding affinity in liver is decreased in the presence of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) suggesting that this analogue is an agonist.     

Conformations of poly-L-valine in solution

In order to test theoretical predictions that poly-L -valine can exist in an α-helical conformation, water-soluble block copolymers of L -valine and D , L -lysine were prepared. By carrying out the synthesis on a resin support (with the use of N-carboxyanhydrides) contamination of the individual blocks by any unreacted monomer from the previous block was avoided. A single glycine residue was incorporated at the C-terminus of the chain for use in amino acid analyses. Using optical rotatory dispersion and circular dichroism criteria, about 50% of the short valine block of (D , L -lysine HCl)₁₈-(L -valine)₁₅-(D , L -lysine-HCl)1₆-glycine was found to be in the right-handed α-helical conformation in 98% aqueous methanol, in water, the polymer appears to be a dimer, with the valine block being involved in the formation of an intermolecular β-structure.     

Plasma levels of renin, angiotensin II, and 6-ketoprostaglandin F1 alpha in endurance athletes

Twelve male runners and 12 matched nonathletes performed a prolonged uninterrupted graded exercise test on the bicycle ergometer up to exhaustion to study blood pressure and plasma levels of renin (PRA), vasoconstrictor angiotensin II (ANG II), and 6-ketoprostaglandin F1 alpha (6-keto-PGF1 alpha), a metabolite of the vasodilator prostacyclin. In the athletes work load was increased by 30 W/4 min, and in the control subjects the increments of work load were adjusted to their lower exercise capacity to equalize total exercise duration. Blood was drawn, and blood pressure and O2 uptake (VO2) were measured at rest and at the fourth, eighth, and last steps of exercise. Peak VO2 averaged 60 +/- 1.6 ml . min-1 . kg-1 in the runners and 46.8 +/- 1.5 in the nonathletes. To evaluate differences between athletes and controls, PRA, ANG II, and 6-keto-PGF1 alpha were first adjusted for significant confounding factors, such as age, weight, hematocrit, 24-h urinary sodium excretion, and O2 uptake. PRA was significantly lower in the athletes (F = 11.2; P less than 0.01); ANG II was not different at rest, but its rise with exercise was less steep in the runners (F = 8.2; P less than 0.01), whereas 6-keto-PGF1 alpha was not different between the groups (F = 1.3; NS). Despite the differences in PRA and ANG II, however, blood pressure was similar in athletes and nonathletes (F = 0.0; NS).     

Formation of angiotensin-(1-7) from angiotensin II by the venom of Conus geographus

The binding of [3H]angiotensin II to AT(1) receptors on Chinese Hamster Ovary cells expressing the human AT(1) receptor (CHO-AT(1) cells) is potently inhibited by venoms of the marine snails Conus geographus and C. betulinus. On the other hand, the binding of the nonpeptide AT(1) receptor-selective antagonist [3H]candesartan is not affected but competition binding curves of angiotensin II and the peptide antagonist [Sar(1),Ile(8)]angiotensin II (sarile) are shifted to the right. These effects resulted from the breakdown of angiotensin II into smaller fragments that do not bind to the AT(1) receptor. In this context, angiotensin-(1-7) is the most prominent fragment and angiotensin-(1-4) and angiotensin-(1-5) are also formed but to a lesser extent. The molecular weight of the involved peptidases exceeds 50 kDa, as determined by gel chromatography and ultrafitration.     

Counteraction between angiotensin II and angiotensin-(1-7) via activating angiotensin type I and Mas receptor on rat renal mesangial cells

In the updated concept of renin-angiotensin system (RAS), it contains the angiotensin converting enzyme (ACE)-angiotensin (Ang) II-angtiogensin type 1 receptor (AT1) axis and the angiotensin-converting enzyme-related carboxypeptidase (ACE2)-Ang-(1-7)-Mas axis. The former axis has been well demonstrated performing the vasoconstrictive, proliferative and pro-inflammatory functions by activation of AT1 receptors, while the later new identified axis is considered counterbalancing the effects of the former. The present study is aimed at observing the interaction between Ang-(1-7) and Ang II on cultured rat renal mesangial cells (MCs). RT-PCR, Western blot and immunofluorescent staining and confocal microscopy results showed that both AT1 and Mas receptor were co-distributed in rat renal MCs. Ang-(1-7) showed similar effects on Ang II in cultured MCs that stimulated phosphorylated extracellular signal-regulated kinase (ERK)1/2 phosphorylation and transforms growth factor-β1 synthesis, and cell proliferation and extracellular matrix synthesis. Co-treatment of the cell with Ang-(1-7) and Ang II, Ang-(1-7) counteracted AngII-induced effects in a concentration dependent manner, but failed to alter the changes induced by endothelin-1. The stimulating effect of Ang II was mediated by AT1 receptor while all the effects of Ang-(1-7) were blocked by Mas receptor antagonist A-779, but not by AT1 receptor antagonist losartan or AT2 receptor antagonist PD123319. These results suggest that Ang-(1-7) and Ang II specifically interact with each other on rat renal MCs via activation of their specific receptors, Mas and AT1 receptor respectively.     

Antagonists of angiotensin II containing N-methyl-L-alanine and DL-nipecotic acid in position 7.

[1-sarcosine, 7-N-methyl-L-alanine, 8-isoleucine]-Angiotensin II and [1-sarcosine, 7-DL-nipecotic acid, 8-isoleucine]-angiotensin II were synthesized by the solid-phase method and purified by cation-exchange chromatography and high-pressure liquid chromatography. In the isolated rat uterus these analogs and less than 0.1% of the myotropic activity of angiotensin II and inhibited angiotensin II with pA2 values of 8.2 and 7.8, respectively. In the rat pressor assay (vagotomized ganglion blocked rat) these analogs had 0.9 and 2.8%, respectively, of the pressor activity of angiotensin II. The results show that the proline residue in position 7 of [Sar1,Ile8]-angiotensin II may be replaced by other secondary amino acids without disrupting interactions at angiotensin II receptors.     

Conformations and interactions of excited states. II. Polystyrene, polypeptides, and proteins

Previous fluorescence and phosphorescence studies of aromatic model compounds have been extended to polymers: “atactic” and isotactic polystyrene, seven aromatic poly-(amino acids), and two proteins. We have confirmed previous observations that both forms of polystyrene exhibit strong excimer fluorescence emission at room temperature but not at 77°K. Of the poly(amino acids) (all observed in helix-supporting solvents), poly-L -phenylalanine, poly(α-benzyl-L -aspartic acid), and poly-1-benzyl-L -histidine likewise show excimer emission at room temperature but not at 77°K., while poly-L -tyrosine, poly-L -tryptophan, poly(γ-benzyl-L -glutamic acid), and poly-S-benzyl-L -cysteine exhibit no excimer emission at either temperature. The aromatic residues of bovine serum albumin exhibit only “normal” fluorescence, but, lysozyme appears to be unique among proteins in showing excimer-like tryptophan emission in the native state; its luminescence becomes “normal” upon denaturation. It appears very probable that none of these polymers has a ground-state conformation in which the aromatic groups have face-to-face orientations appropriate for excimer interaction. It is concluded that at room temperature absorption of light can cause local “melting” of regular (usually helical) structures and thus, in some polymers, permit the attainment of a face-to-face arrangement of aromatic rings within the radiative lifetime of their excited singlet states. In certain other polymers (for reasons not clear at present), and in all polymers at 77°K., this does not occur. This concept is extended to provide a bettor basis for understanding the mechanism of formation of the photodimer of thy mine in irradiated DNA.