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.     

Optical spectroscopic elucidation of beta-turns in disulfide bridged cyclic tetrapeptides

Vibrational circular dichroism (VCD) spectroscopic features of type II beta-turns were characterized previously, but, criteria for differentiation between beta-turn types had not been established yet. Model tetrapeptides, cyclized through a disulfide bridge, were designed on the basis of previous experimental results and the observed incidence of amino acid residues in the i + 1 and i + 2 positions in beta-turns, to determine the features of VCD spectra of type I and II beta-turns. The results were correlated with electronic circular dichroism (ECD) spectra and VCD spectra calculated from conformational data obtained by molecular dynamics (MD) simulations. All cyclic tetrapeptides yielded VCD signals with a higher frequency negative and a lower frequency positive couplet with negative lobes overlapping. MD simulations confirmed the conformational homogeneity of these peptides in solution. Comparison with ECD spectroscopy, MD, and quantum chemical calculation results suggested that the low frequency component of VCD spectra originating from the tertiary amide vibrations could be used to distinguish between types of beta-turn structures. On the basis of this observation, VCD spectroscopic features of type II and VIII beta-turns and ECD spectroscopic properties of a type VIII beta-turn were suggested. The need for independent experimental as well as theoretical investigations to obtain decisive conformational information was recognized.     

Evidence of essential disulfide bonds in angiotensin II binding sites of rabbit hepatic membranes. Inactivation by dithiothreitol

Radiolabelled angiotensin II binds to a single class of high-affinity binding sites on purified rabbit hepatic membranes. The binding is specific, reversible and saturable. Displacement studies using angiotensin and various analogs of angiotensin II disclosed a structure-activity profile similar to that found in physiologically relevant angiotensin II receptor sites. Treatment of membranes with the reducing agent, dithiothreitol, cause a significant decrease in the affinity of angiotensin II binding sites for the native ligand. This effect is mimicked by a 15-fold higher concentration of the monosulfhydryl derivative, 2-mercaptoethanol. Kinetic studies also indicated that dithiothreitol increases the rate of dissociation of bound ligand from the membrane without significantly affecting the association rate. In contrast, treatment of membranes with the metal chelators, ethylenediaminetetracetic acid (EDTA) and ethyleneglycol bis(β-aminoethyl ether)-N,N′-tetracetic acid (EGTA), does not affect the binding of radiolabeled angiotensin II. Furthermore, dithiothreitol inhibited the binding of angiotensin II to a solubilized partially purified preparation of angiotensin II-binding protein from the same tissue and also increased the dissociation of bound angiotensin II. This indicates that the effect of the sulfhydryl reagents on the membrane binding sites is the result of a direct alteration of the binding sites rather than a gross modification of the structure of the membrane.     

Identification and function of disulfide bridges in the extracellular domains of the angiotensin II type 2 receptor.

The angiotensin II (AngII) receptor family is comprised of two subtypes, type 1 (AT(1)) and type 2 (AT(2)). Although sharing low homology (only 34%), mutagenesis has identified some key residues that are conserved between both subtypes, including four extracellular cysteines. Previous AT(1) mutagenesis demonstrated that the cysteines form two disulfide bonds, one linking the first and second extracellular loops and another connecting the amino terminus to the third extracellular loop. The importance of these AT(1) disulfides in ligand binding is supported by the effect of dithiothreitol (DTT). DTT breaks disulfide bonds, thereby strongly inhibiting ligand binding in AT(1) receptors. Despite retaining the same cysteines, AT(2) receptor ligand binding is paradoxically enhanced by DTT. Thus, we constructed a series of AT(2) cysteine mutations, either individually or paired, to establish the role of the cysteines and the source of DTT's effects. The AT(2) cysteine mutants surprisingly confirmed that the cysteines form disulfide bonds in the same manner as in the AT(1) subtype. However, breaking the AT(2) disulfide bridges yielded two responses. As in AT(1) receptors, mutations disrupting the disulfide bond between the first and second extracellular loops reduced AT(2) binding by 4-fold. In contrast, mutations breaking the disulfide bridge between the amino terminus and the third extracellular loop increased AT(2) binding, mimicking DTT's effect on this subtype. Further analysis of AT(1)/AT(2) chimeric exchange mutants of these domains suggested that the AT(2) amino terminus and third extracellular loop may possess latent binding epitopes that are only uncovered after DTT exposure.     

A dynamic model of angiotensin II infusion experiments

Applications of control theory in studies of biological system dynamics have come to be called compartmental modelling. A second order, nonlinear, compartmental model is developed which describes the dynamics of the hormone angiotensin II (AII) and arterial blood pressure (BP) during AII infusion experiments. The model is partially identified using dose response data for constant infusion rates between 0.01 and 0.10 μg/kg/min over a period of several minutes. This study represents a first step in understanding the dynamics of regulation of arterial blood pressure by the renin-angiotensin system. All is a vasoconstrictor and is known to participate in the natural regulation of BP. AII is also believed to be an agent in the development of hypertension and atherosclerosis. The model is used to identify causal mechanisms which are consistent both with the established correlation between plasma AII concentration and arterial BP and with current physiological knowledge. The study demonstrates how a simple state variable model can be used to provide guidance concerning the design of future infusion experiments.     

Biological activity of the novel cyclic angiotensin II analogue [Sar1,Lys3,Glu5]ANG II

Summary This study was undertaken to investigate the biological activity of the cyclic amide-linked analogue of angiotensin II (ANG II), [Sar¹,Lys³,Glu⁵]ANG II, in both ex vivo and in vivo experiments. This constrained analogue was designed on the basis of a recently suggested conformational model for ANG II-induced receptor activation, which is characterized by a Tyr-Ile-His backbone bend and the clustering of the three aromatic rings (Tyr, His, Phe). After [Sar¹,Lys³,Glu⁵]ANG II was found to have contractile activity (15% of ANG II in the rat uterus assay), it was administered in anesthetized rabbits where it produced an immediate and dose-dependent increase in blood pressure, which peaked within minutes, was sustained as long as the drug was given, and was gradually returned to baseline after discontinuation of the drip. The blood pressure response to the cyclic analogue was of less magnitude compared to that elicited by an isovolemic and equimolar solution of ANG II. These data confirm the importance of a properly oriented ring cluster, allowing the charge-relay conformation proposed for ANG II.     

Offset of actions of angiotensin II, angiotensin III, and their analogue antagonists in renal and femoral vascular beds: further evidence for different vascular angiotensin receptors.

Half-time of the offset of antagonist action was used to assess the possibility that factors which determine the duration of action of angiotensin antagonists were responsible for regional differences in their effectiveness: thus, for example, enhanced degradation of angiotensin III analogues in the limb circulation would reduce their effectiveness there despite an angiotensin receptor identical to that in the kidney. In the anesthetized dog blood flow in the renal and femoral vascular beds was measured with an electromagnetic flowmeter; the octapeptide analogue saralasin (1-Sar, 8-Ala AII) and a heptapeptide analogue (des-Asp, 8-Ile AII) were infused intravenously (1 μg/kg/min) for 10 minutes and, after stopping the infusion, the effectiveness of their blockade of angiotensin II was assessed over time. The half-time of offset of the antagonist action was determined from semilogarithmic plots of percent inhibition during recovery. Offset of heptapeptide-induced inhibition in the hindlimb would have been more rapid if increased rate of degradation was the explanation for its reduced effectiveness and such was not the case: Indeed offset was more rapid in the renal (5.8 ± 1.1 min) than the femoral vascular bed (11.7 ± 2.1 min) (p > 0.05). Saralasin showed identical offsets in the two beds (renal 17.2 ± 1.5 min; femoral 15.1 ± 2.9 min) (p > 0.5). Consistent with these observations, the offset of the agonist action of angiotensin III was shorter in the kidney (0.69 ± 0.06 min) than in the limb (1.46 ± 0.13 min; p < 0.001). This study has confirmed the relatively greater efficacy of heptapeptide analogues in the renal than in the femoral vascular bed and has ruled out degradation as accounting for that difference: The difference is most likely to lie in a different angiotensin receptor in the two regions.     

Conversion of angiotensin I to angiotensin II by cathepsin A isoenzymes of porcine kidney

We have reported the existence of a carboxypeptidase in a human renal extract that converts Angiotensin I (AI) to Angiotensin II (AII) in two steps with des-leu-AI (dl-AI) being formed as an intermediate. Since this carboxypeptidase had properties similar to cathepsin A, the ability of cathepsin A to metabolize AI was studied. Cathepsin A was purified from hog kidney with enzyme activity being monitored using both benzyloxycarbonyl-glutamyl-tyrosine (ZGT) and AI as substrates. The procedure separated the expected large and small molecular weight forms of cathepsin A as well as two additional isoenzymes. All of the isoenzymes had carboxypeptidase activity with ZGT, AI, and dl-AI. No detectable cleavage of AII was observed. Cathepsin A,S (small) activity with ZGT or AI as substrate was inhibited to a similar extent by diisopropylfluorophosphate, mersalyl acid, and a decapeptide renin inhibitor. It is concluded that the renal angiotensin carboxypeptidase activity is catalyzed by cathepsin A. By its ability to convert AI to AII, cathepsin A may be a component of the intrarenal renin-angiotensin system.     

Inhibition of angiotensin-converting enzyme by angiotensin I analogue peptide inhibitors. A kinetic study

Angiotensin I analogues with a phosphonic acid group replacing the C-terminal carboxyl group were shown to be competitive inhibitors of angiotensin-converting enzyme. This new class of inhibitors was used to study the binding requirements of the angiotensin I-like ligands to the enzyme's active site. These studies indicate that angiotensin-converting enzyme recognizes at least five amino acid residues at the C-terminus of the peptide. The effect of pH on the binding of the most potent inhibitor peptide was compared to Captopril. The two inhibitors showed similar Ki-pH profiles despite their structural differences. Chloride enhanced the binding of the peptide inhibitor at both pH 9.0 and pH 6.5. At pH 9.0 the inhibitor peptide and the anion bind randomly to the enzyme, while at pH 6.5 the mechanism is ordered. In the latter case, the anion binds first to the enzyme.     

Conformational analysis corresponding to intra-chain disulfide bridged peptides in proteins of known three-dimensional structure

We have carried out a systematic analysis in order to evaluate whether Intra-Chain Disulfide Bridged Peptides (ICDBPs) observed in proteins of known three-dimensional structure adopt structurally similar conformations as they may correspond to structural/functional motifs. 406 representative ICDBPs comprising between 3 to 17 amino acid residues could be classified according to peptide sequence length and main-chain secondary structure conformation into 146 classes. ICDBPs comprising 6 amino acid residues are maximally represented in the Protein Data Bank. They also represent the maximum number of main-chain secondary structure conformational classes. Individual ICDBPs in each class represent different protein superfamilies and correspond to different amino acid sequences. We identified 145 ICDBP pairs that had not less-than 0.5 A root mean square deviation value corresponding to their equivalent peptide backbone atoms. We believe these ICDBPs represent structural motifs and possible candidates in order to further explore their structure/function role in the corresponding proteins. The common conformational classes observed for ICDBPs defined according to the main-chain secondary structure conformations; H (helix), B (residue in a isolated beta bridge), C (coil), E (extended beta strand), G (3(10) helix), I (pi helix), S (bend), T (hydrogen-bonded turn) were; "CHHH", "CTTC", "CSSS" and "CSSC" (for ICDBP length 4), "CSSCC" (length 5), "EETTEE", "CCSSCC", "CCSSSC" (length 6), "EETTTEE" (length 7), "EETTTTEE" (length 8), "EEEETTEEEE" (length 10), "EEEETTTEEEE" (length 11) and "EEEETTTTEEEE" (length 12).     

A new approach to angiotensin antagonists: Methylation of the tyrosine hydroxyl in angiotensin II

[Sar¹, Tyr(Me)⁴] angiotensin II, synthesized by the solid phase method and purified by ion-exchange chromatography and reversed-phase HPLC, was found to inhibit the contractile response to angiotensin II in the rat isolated uterus and inhibit the pressor response to angiotensin II in the vagotomized ganglion-blocked rat. In the rat isolated uterus Schild plots gave a pA₂ of 8.1, and a slope of 0.9 indicative of competitive inhibition. In the rat pressor assay, infusion of the analogue at a rate of 500ng/kg/min caused a parallel displacement of the dose-response curve to ANG II to the right. In contrast, the classical angiotensin inhibitor [Sar¹, Ile⁸] ANG II appeared to demonstrate non-competitive inhibition in both the rat isolated uterus and the pressor assays. The phenolic hydroxyl or phenoxide anion of Tyr⁴ in angiotensin II appears to be critical for the activation of angiotensin receptors in smooth muscle. Alkylation of the tyrosine residue in angiotensin analogues provides a new route for the synthesis of potent competitive antagonists of angiotensin.     

Conformational study of angiotensin II

Conformational free energy calculations using an empirical potential ECEPP/3 (Empirical Conformational Energy Program for Peptides, Version 3) were carried out on angiotensin II (AII) of sequence Asp-Arg-Val-Tyr-Ile-His-Pro-Phe to find the stable conformations of the free state in the unhydrated and the hydrated states. A conformational analysis of the unhydrated state was carried out using the buildup procedure. The free energy calculation using the hydration shell model was also carried out to obtain the stable conformation of the hydrated state. The calculated stable conformations of AII in both states have a partially right-handed α-helical structure stabilized by short- and medium-range interactions. The similarity between the lowest free energy conformations of the unhydrated and hydrated states suggests that the hydration might not be important to stabilize the overall conformation of AII in a free state. The absence of any intramolecular interaction of the Tyr side chain suggests the possible interaction of this residue with the receptor. In this study, we found that the low free energy conformations contain both the parallel-plate and the perpendicular-plate geometries of the His and Phe rings, suggesting the coexistence of both conformations. © 1996 John Wiley & Sons, Inc.     

Mechanical stress activates angiotensin II type 1 receptor without the involvement of angiotensin II

The angiotensin II type 1 (AT1) receptor has a crucial role in load-induced cardiac hypertrophy. Here we show that the AT1 receptor can be activated by mechanical stress through an angiotensin-II-independent mechanism. Without the involvement of angiotensin II, mechanical stress not only activates extracellular-signal-regulated kinases and increases phosphoinositide production in vitro, but also induces cardiac hypertrophy in vivo. Mechanical stretch induces association of the AT1 receptor with Janus kinase 2, and translocation of G proteins into the cytosol. All of these events are inhibited by the AT1 receptor blocker candesartan. Thus, mechanical stress activates AT1 receptor independently of angiotensin II, and this activation can be inhibited by an inverse agonist of the AT1 receptor.     

Synthesis, incorporation efficiency, and stability of disulfide bridged functional groups at RNA 5'-ends

Modified guanosine monophosphates have been employed to introduce various functional groups onto RNA 5'-ends. Applications of modified RNA 5'-ends include the generation of functionalized RNA libraries for in vitro selection of catalytic RNAs, the attachment of photoaffinity-tags for mapping RNA-protein interactions or active sites in catalytic RNAs, or the nonradioactive labeling of RNA molecules with fluorescent groups. While in these and in similar applications a stable linkage is desired, in selection experiments for generating novel catalytic RNAs it is often advantageous that a functional group is introduced reversibly. Here we give a quantitative comparison of the different strategies that can be applied to reversibly attach functional groups via disulfide bonds to RNA 5'-ends. We report the preparation of functional groups with disulfide linkages, their incorporation efficiency into an RNA library, and their stability under various conditions.