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.     

Synthesis, receptor binding affinities and conformational properties of cyclic methylenedithioether analogues of angiotensin II

Cyclic 12-, 13- and 14-membered ring angiotensin II analogues related to disulfides but encompassing methylene-dithioether bridges have been prepared. The affinity data from these derivatives were compared to those from the disulfides. The methylenedithioether analogues displayed good binding affinities to rat liver AT1 receptors although in most cases somewhat lower than their disulfide counterparts. One of the methylenedithioethers with a 13-membered ring system demonstrated the highest binding affinity among the thioethers. Theoretical conformational analysis of model compounds of the two series were performed suggesting a similarity between the disulfide and the corresponding methylenedithioether analogues and also between the ring size homologues. This analysis also suggested that some of the model compounds were prone to adopt inverse gamma-turn conformations, which was further supported by use of NMR spectroscopy of the 12-membered ring analogue in the series. The easily executed methylenedithioether cyclization should constitute a valuable complement to the common disulfide methodology for fine-tuning and for probing the bioactive conformation of peptides.     

Nodose ganglionectomy reduces angiotensin II receptor binding in the rat brainstem

Angiotensin II (Ang II) receptor binding sites in the dorsomedial medulla of intact and unilaterally nodose ganglionectomized rats were identified and characterized using 125I-sarcosine,isoleucine Ang II. This radioligand bound saturably and with high affinity to rat brain homogenates and to sections of rat brainstem. Specific (1 microM angiotensin II displaceable) binding of 125I-sarcosine,isoleucine Ang II was displaced by angiotensin analogues with a potency order similar to that described for angiotensin II receptors. Unilateral nodose ganglionectomy caused a reduction in Ang II receptor binding in the medial solitary tract nucleus, dorsal motor nucleus of the vagus, and area postrema ipsilateral to the lesioned ganglion. This observation suggests that Ang II receptors in the dorsomedial medulla may be located on axon terminals of vagal afferents and cell bodies of vagal efferents.     

The presence of teleost-type angiotensin components in lamprey buccal gland suggests a role in endocrine mimicry

Previous characterization of a native lamprey angiotensin II (LpAng II) that possesses a different sequence and function than teleost-type angiotensin II (Ang II) has raised a question as to the role of teleost-type angiotensin peptides in lampreys. In this study, teleost-type angiotensin like-peptides were identified in the buccal gland of lampreys by immunoassays and immunohistochemistry. The possible sources of angiotensin like-peptides were investigated in lampreys by manipulating their choice of host and food. Ang II immunoreactivity (irAng II) was detected in the buccal gland and plasma of feeding phase sea lampreys exposed to Atlantic cod, but was mostly absent in fasting lamprey. Qualitatively, the HPLC profiles of irAng II observed in the plasma, when present, were highly similar to those in buccal gland, implying that the buccal gland could be a source of plasma Ang II. Japanese lampreys force-fed with dogfish blood had significantly elevated concentrations of irAng II in their buccal glands when compared to unfed individuals, suggesting that feeding stimuli may have enhanced buccal gland activity. Teleost-type Ang II-containing proteins, other than angiotensinogen, are present in the buccal gland as trypsinization generated Ang II in vitro, and the HPLC profile of these irAng II was highly comparable to those naturally present in the buccal gland. [Asn¹, Val⁵, Thr⁹]-Ang I that was identified in the buccal gland of Japanese lampreys has the same amino acid sequence to those previously isolated from the incubation of plasma and kidney extract, providing an alternative explanation for the previous isolation of teleost-type Ang I in lampreys. irAng I and irAng II were localized in the granule-like structures in the apical region of the secretory epithelia, suggesting that these peptides may be active components of lamphredin. The teleost-type angiotensin peptides in the buccal gland secretion suggested that these host-specific peptides could be part of the endocrine mimicry strategy used by lampreys to evade host immune responses and reduce immune-rejection.     

The physiological significance of the alternative pathways of angiotensin II production.

Although the use of angiotensin converting enzyme inhibitors (ACE-Is) in clinical practice brought the great chance to recognize the RAS role in the physiology and pathology, there are still many questions which we cannot answer. This article reviews actually known pathways of angiotensin II (Ang II) and other peptides of renin-angiotensin system (RAS) production and their physiological significance. The various carboxy- and aminopeptidases generate a range of peptides, like Ang II, Ang III, Ang IV, Ang-(1-7) and Ang-(1-9) possessing their own and known biological activity. In this issue especially the alternative pathways of Ang II synthesis involving enzymes other than angiotensin-converting enzyme (ACE) are discussed. We present many evidences for the significance of a new pathway of Ang II production. It has been clearly shown that Ang I may be converted to Ang-(1-9) by angiotensin-converting enzyme-related carboxypeptidase (ACE-2) and then into Ang II in some tissues, but the enzymes responsible for this process are unknown till now. Although there are many data proving the existence of alternative pathways of Ang II production, we can still block only ACE and angiotensin receptor 1 (AT(1)) in clinical practice. It seems that a lot needs to be done before we can wildly complexively control RAS and treat more effectively cardiovascular disorders such as hypertension or heart failure.     

Angiotensin III: A physiological relevant peptide of the renin angiotensin system

The renin angiotensin system (RAS) is a peptide hormone system that plays an important role in the pathophysiology of various diseases, including congestive heart failure, hypertension, myocardial infarction, and diabetic nephropathy. This has led researchers to focus extensively on this system, leading to the discovery of various peptides, peptidases, receptors and signal transduction mechanisms intrinsic to the RAS. Angiotensinogen (AGT), angiotensin (Ang) II, Ang III, Ang IV, and Ang-(1–7) are the main biologically active peptides of RAS. However, most of the available studies have focused on Ang II as the likely key peptide from the RAS that directly and indirectly regulates physiological functions leading to pathological conditions. However, data from recent studies suggest that Ang III may produce physiologically relevant effects that are similar to those produced by Ang II. Hence, this review focuses on Ang III and the myriad of physiological effects that it produces in the body.     

Angiotensin II inhibitory peptide found in the receptor sequence using peptide array

Peptide array consisting of hundreds of peptides spatially addressed and synthesized on a cellulose membrane support was used to screen ligand-inhibitory peptides. As a model, angiotensin II (Ang II), a significant peptide related to the treatment of cardiovascular diseases, was chosen as the target ligand. Peptide arrays covering the Ang II receptor type 1 sequence were prepared, and peptide domains with high affinity to the Ang II fluorescein conjugate were investigated. The peptide (VVIVIY) within the first transmembrane region exhibited the highest affinity to Ang II. The synthesized soluble VVIVIY peptide had an 84% inhibitory effect on Ang II-induced aorta contraction. These results indicate that our screening strategy utilizing peptide array is an effective approach for the peptide drug development.     

Differential response of human cardiac fibroblasts to angiotensin I and angiotensin II

The vasoactive peptide angiotensin II (Ang II) has been implicated as a mediator of myocardial fibrosis. We carried out a comparative investigation of the effects of Ang II and its precursor Ang I on collagen metabolism and proliferation in cultured human cardiac fibroblasts. Cardiac fibroblasts responded to both Ang I and Ang II with concentration-dependent increases in collagen synthesis but no proliferation. The stimulatory effect of Ang II was abolished by the AT(1) receptor antagonist losartan but not the AT(2) receptor antagonist PD123319. The response to Ang I was not affected by either antagonist, nor by the angiotensin-converting enzyme (ACE) inhibitor captopril. In conclusion, Both Ang I and Ang II stimulate collagen synthesis of human cardiac fibroblasts, the effect of Ang II occurring via the AT(1) receptor whilst Ang I appears to exert a direct effect through non-Ang II-dependent mechanisms. These results suggest distinct roles for angiotensin peptides in the development of cardiac fibrosis.     

p38 Mitogen-activated protein kinase is stimulated by both angiotensin II and angiotensin III in cultured rat astrocytes

Abstract

Context: Previously we showed that angiotensin (Ang) II and Ang III induced phosphorylation of ERK1/2 and JNK mitogen-activated protein (MAP) kinases in rat astrocytes. Objectives: To determine whether these peptides induce p38 MAP kinase in astrocytes. Materials and methods: We used brainstem astrocytes as a model system to determine whether Ang II and Ang III induce p38 MAP kinase protein phosphorylation. Results: Treatment of astrocytes with increasing concentrations of both peptides caused a dose-dependent increase in p38 MAP kinase phosphorylation. The effect of Ang II and Ang III was maximal at 10?nM and 100?nM concentrations, respectively. The effects of the peptides were rapid occurring within minutes of treatment. There was a significant difference in the ability of the peptides to induce p38 MAP kinase phosphorylation. The ability of Ang II to induce p38 MAP kinase was almost twice than that of Ang III, suggesting that Ang II was more potent than Ang III in this effect. Ang AT₁ receptor mediated the actions of the peptides since pretreatment with losartan prevented p38 MAP kinase phosphorylation by Ang II and Ang III. In addition, blockade of Ang II metabolism to Ang III with the aminopeptidase A inhibitor glutamate phosphonate was ineffective in ameliorating Ang II phosphorylation of p38 MAP kinase, suggesting that Ang II directly stimulated p38 MAP kinase phosphorylation. Conclusion: These findings provide insight into the molecular nature of the actions of these peptides and offer a possible mechanism by which these Ang peptides physiological and possibly pathological actions occur in astrocytes.     

Differential response of angiotensin peptides in the urine of hypertensive animals

Urinary excretion rates of angiotensin I (Ang I), angiotensin II (Ang II), and angiotensin-(1-7) [Ang-(1-7)] were determined in normotensive Sprague Dawley (SD), spontaneously hypertensive (SHR), and mRen-2 transgenic hypertensive animals before and following blockade of Ang II synthesis or activity for two weeks. This study was performed to determine for the first time whether inhibition of Ang II alters the excretion of angiotensin peptides in the urine. Rats were given either tap water or water medicated with lisinopril, losartan or both agents in combination. Blood pressure was monitored at regular intervals during the experiment by the tail-cuff method, and once again at the end of the study with a catheter implant into a carotid artery. Metabolic studies and 24 h urinary excretion variables and angiotensin peptides were determined before and during the procedures. While all three treatments normalized the blood pressure of hypertensive animals, therapy with either lisinopril or the combination of lisinopril and losartan had a greater antihypertensive effect in both SHR and [mRen-2]27 transgenic hypertensive rats. In the urine, the concentration of the angiotensins (normalized by 24-h creatinine excretion) was several-fold higher in the untreated hypertensive animals than in normotensive SD rats. In SD rats, lisinopril or lisinopril and losartan produced a sustained rise in urinary levels of Ang-(1-7) without changes in the excretion of Ang I and Ang II. In contrast, Ang I and Ang-(1-7) were significantly elevated in SHR medicated with lisinopril alone or in combination with losartan. Only losartan, however, augmented urinary levels of Ang II in the SHR. The antihypertensive effects of the three separate regimens had no effect on the urinary excretion of angiotensin peptides in [mRen-2]27 transgenic hypertensive rats. These data show that Ang I and Ang-(1-7) are excreted in large amounts in the urine of SD, SHR and [mRen-2]27 hypertensive rats. The unchanged Ang-(1-7) excretion in transgenic hypertensive (Tg+) rats after inhibition of the renin-angiotensin system agrees with the previous finding of a reduced plasma clearance of the peptide in this model of hypertension. The data suggest that this form of hypertension may be associated with increased activity of an endogenous converting enzyme inhibitor.     

Short pseudopeptides containing turn scaffolds with high AT2 receptor affinity

Two pentapeptides, Ac-Tyr-Ile-His-Pro-Phe/Ile, were synthesized and shown to have angiotensin II AT2 receptor affinity and agonistic activity. Based on these peptides, a new series of 13 pseudopeptides was synthesized via introduction of five different turn scaffolds replacing the Tyr-Ile amino acid residues. Pharmacological evaluation disclosed subnanomolar affinities for some of these compounds at the AT2 receptor. Substitution of Phe by Ile in this series of ligands enhanced the AT2 receptor affinity of all compounds. These results suggest that the C-terminal amino acid residues can be elaborated on to enhance the AT2 receptor affinity in truncated Ang II analogues.     

The renin-angiotensin and the kallikrein-kinin systems

The renin-angiotensin system (RAS) and the kallikrein-kinin system (KKS) each encompasses a large number of molecules, with several participating in both systems. The RAS generates a family of bioactive angiotensin peptides with varying biological activities. These include angiotensin-(1-8) (Ang II), angiotensin-(2-8) (Ang III), angiotensin-(3-8) (Ang IV), and angiotensin-(1-7) [Ang-(1-7)]. Ang II and Ang III act on type 1 (AT(1)) and type 2 (AT(2)) angiotensin receptors, whereas, Ang IV and Ang-(1-7) act on their own receptors. The KKS also generates a family of bioactive peptides with varying biological activities. These include hydroxylated and non-hydroxylated bradykinin and kallidin peptides and their carboxypeptidase metabolites des-Arg(9)-bradykinin and des-Arg(10)-kallidin. Whereas bradykinin and kallidin act mainly via the type 2 bradykinin (B(2)) receptor, des-Arg(9)-bradykinin and des-Arg(10)-kallidin act mainly via the type 1 bradykinin (B(1)) receptor. The AT(1) receptor forms heterodimers with the AT(2) and B(2) receptors and there is cross talk between the AT(1) and epidermal growth factor receptors. The B(2) receptor also interacts with angiotensin converting enzyme and nitric oxide synthase. Both angiotensin and kinin peptides are metabolised by many different peptidases that are important determinants of the activities of the RAS and KKS, and several of which participate in both systems.     

钙调神经磷酸酶在血管紧张素Ⅱ刺激的心脏成纤维细胞增殖中的作用

本研究观察了钙调神经磷酸酶（ＣａＮ）在血管坚张素Ⅱ（ＡｎｇⅡ）刺激的大鼠心脏成纤维细胞增殖中的作用。在培养的大鼠心脏成纤维细胞上,应用双波长荧光 计检测Ｆｕｒａ－２标记的细胞游离Ｃａ＾２＋浓度;应用对硝基苯磷酸（ＰＮＰＰ）作底物测定钙调神经磷酸酶（ＣａＮ）活性;根据＾３Ｈ－胸腺嘧啶掺入法评估ＣａＮ特异性抑制剂环胞素Ａ（ＣｓＡ）对ＡｎｇⅡ刺激的心脏成纤维细胞ＤＮＡ合成的影响。结果表明,ＡｎｇⅡ（１０     

Role of aromaticity of agonist switches of angiotensin II in the activation of the AT1 receptor

We have shown previously that the octapeptide angiotensin II (Ang II) activates the AT1 receptor through an induced-fit mechanism (Noda, K., Feng, Y. H., Liu, X. P., Saad, Y., Husain, A., and Karnik, S. S. (1996) Biochemistry 35, 16435-16442). In this activation process, interactions between Tyr4 and Phe8 of Ang II with Asn111 and His256 of the AT1 receptor, respectively, are essential for agonism. Here we show that aromaticity, primarily, and size, secondarily, of the Tyr4 side chain are important in activating the receptor. Activation analysis of AT1 receptor position 111 mutants by various Ang II position 4 analogues suggests that an amino-aromatic bonding interaction operates between the residue Asn111 of the AT1 receptor and Tyr4 of Ang II. Degree and potency of AT1 receptor activation by Ang II can be recreated by a reciprocal exchange of aromatic and amide groups between positions 4 and 111 of Ang II and the AT1 receptor, respectively. In several other bonding combinations, set up between Ang II position 4 analogues and receptor mutants, the gain of affinity is not accompanied by gain of function. Activation analysis of position 256 receptor mutants by Ang II position 8 analogues suggests that aromaticity of Phe8 and His256 side chains is crucial for receptor activation; however, a stacked rather than an amino-aromatic interaction appears to operate at this switch locus. Interaction between these residues, unlike the Tyr4:Asn111 interaction, plays an insignificant role in ligand docking.     

Endothelin-1 modulates angiotensin II in the development of hypertension in fructose-fed rats

Two of the most potent vasoconstrictors, endothelin-1 (ET-1) and angiotensin II (Ang II), are upregulated in fructose hypertensive rats. It is unknown whether an interrelationship exists between these peptides that may contribute to the development of fructose-induced hypertension. The objective of this study was to investigate the existence of an interaction between the endothelin and renin angiotensin systems that may play a role in the development of fructose-induced hypertension. High fructose feeding and treatment with either bosentan, a dual endothelin receptor antagonist, or with L-158,809, an angiotensin type 1 receptor antagonist, were initiated simultaneously in male Wistar rats. Systolic blood pressure, fasted plasma parameters, insulin sensitivity, plasma Ang II, and vascular ET-1-immunoreactivity were determined following 6 weeks of high fructose feeding. Rats fed with a high fructose diet exhibited insulin resistance, hyperinsulinemia, hypertriglyceridemia, hypertension, and elevated plasma Ang II. Treatment with either bosentan or L-158,809 significantly attenuated the rise in blood pressure with no effect on insulin levels or insulin sensitivity in fructose-fed rats. Bosentan treatment significantly reduced plasma Ang II levels, while L-158,809 treatment significantly increased vascular ET-1-immunoreactivity in fructose-fed rats. Thus, treatment with the endothelin receptor antagonist prevented the development of fructose-induced hypertension and decreased plasma Ang II levels. These data suggest that ET-1 contributes to the development of fructose-induced hypertension through modulation of Ang II levels.     

Metabolism of angiotensin I by guinea pig aqueous humor

We investigated the degradation of angiotensin I (Ang I) by guinea pig aqueous humor at physiological pH (pH 7.4) and assessed the activity of responsible enzymes using various enzyme inhibitors. The aqueous humor was incubated with Ang I in the presence or absence of an enzyme inhibitor at 37 degrees C for the appropriate time period. The resulting peptides were analyzed by a Beckman HPLC system with a Waters microBondapak C18 analytical column using a 30-min increasing linear gradient of 10 to 40% acetonitrile containing 0.05% trifluoroacetic acid (TFA) and H2O containing 0.05% TFA at a flow rate of 1 mL/min. Detection was done by absorbance at 214 nm. Angiotensin II (Ang II) was a major product (39.3+/-4.10 nmol x h(-1) mL(-1), n = 5) of Ang I hydrolysis. Traces of angiotensin 1-9, angiotensin IV, and angiotensin 1-7 were also produced. Chymostatin (0.05 mmol/L), EDTA (1 mmol/L), enalaprilat (0.1 mmol/L), and ebelacton B (0.01 mmol/L) inhibited generation of Ang II from Ang I by guinea pig aqueous humor by 89+/-4.6, 56+/-7.6, 33+/-5.1, 20+/-6.5%, respectively. Our findings indicate that guinea pig aqueous humor contains several enzymes that can form Ang II. The chymostatin-sensitive type of enzyme was the most active one found in guinea pig aqueous humor. Angiotensin I converting enzyme, carboxypeptidase A, and deamidase may also contribute to angiotensin II formation in guinea pig ocular fluid.     

The antihypertensive effect of peptides: a novel alternative to drugs?

Many types of bioactive peptides that inhibit angiotensin I, angiotensin I converting enzyme (ACE) and Ang II type 1 receptor (AT1) in the cardiovascular system contribute to the prevention and treatment of hypertension. These inhibitory peptides are derived from many food proteins or artificial synthetic products. Further research examining the bioavailability of ACE inhibitory peptides will lead to the development of more effective ACE inhibitory peptides and foods. Our research also demonstrates that ACE inhibitory peptide LAP may lower blood pressure with no adverse effects.     

Ligands to the (IRAP)/AT4 receptor encompassing a 4-hydroxydiphenylmethane scaffold replacing Tyr2

Analogues of the hexapeptide angiotensin IV (Ang IV, Val(1)-Tyr(2)-Ile(3)-His(4)-Pro(5)-Phe(6)) encompassing a 4-hydroxydiphenylmethane scaffold replacing Tyr(2) and a phenylacetic or benzoic acid moiety replacing His(4)-Pro(5)-Phe(6) have been synthesized and evaluated in biological assays. The analogues inhibited the proteolytic activity of cystinyl aminopeptidase (CAP), frequently referred to as the insulin-regulated aminopeptidase (IRAP), and were found less efficient as inhibitors of aminopeptidase N (AP-N). The best Ang IV mimetics in the series were approximately 20 times less potent than Ang IV as IRAP inhibitors. Furthermore, it was found that the ligands at best exhibited a 140 times lower binding affinity to the membrane-bound IRAP/AT4 receptor than Ang IV. Although the best compounds still exert lower activities than Ang IV, it is notable that these compounds comprise only two amino acid residues and are considerably less peptidic in character than the majority of the Ang IV analogues previously reported as IRAP inhibitors in the literature.     

Angiotensin II stimulates DNA synthesis of rat pancreatic stellate cells by activating ERK through EGF receptor transactivation

Although angiotensin II (Ang II) is known to participate in pancreatic fibrosis, little is known as to the mechanism by which Ang II promotes pancreatic fibrosis. To elucidate the mechanism, we examined the action of Ang II on the proliferation of rat pancreatic stellate cells (PSCs) that play central roles in pancreatic fibrosis. Immunocytochemistry and Western blotting demonstrated that both Ang II type 1 and type 2 receptors were expressed in PSCs. [3H]Thymidine incorporation assay revealed that Ang II enhanced DNA synthesis in PSCs, which was blocked by Ang II type 1 receptor antagonist losartan. Western blotting using anti-phospho-epidermal growth factor (EGF) receptor and anti-phospho-extracellular signal regulated kinase (ERK) antibodies showed that Ang II-activated EGF receptor and ERK. Both EGF receptor kinase inhibitor AG1478 and MEK1 inhibitor PD98059 attenuated ERK activation and DNA synthesis enhanced by Ang II. These results indicate that Ang II stimulates PSC proliferation through EGF receptor transactivation-ERK activation pathway.     

Effect of angiotensin peptides on PAI-1 expression and production in human adipocytes.

Angiotensin (Ang) II is the active component of the renin-angiotensin-system (RAS), but its degradation products have also been shown to exhibit biological activity. This system, which mainly controls blood pressure and electrolyte homeostasis, was recently found to be completely expressed in human adipose tissue. The major determinant in the fibrinolytic system is the plasminogen activator inhibitor-1 (PAI-1). Both PAI-1 and components of the RAS are over-expressed in the obese state. We have recently shown that Ang II is able to induce PAI-1 expression and release via the AT1-receptor in human fat cells in primary culture, and have provided the first evidence that two metabolites, Ang III and Ang IV, may have a similar stimulatory effect on PAI-1 release. We have now performed additional experiments to further characterize the role of the angiotensin peptides in the production of PAI-1. Ang III and Ang IV showed a time- and dose-dependent stimulation of PAI-1 protein release. Concomitantly, mRNA-levels were markedly elevated. Using specific receptor blockers, all angiotensin peptides seem to induce PAI-1 expression via the angiotensin receptor subtype 1. However, components of the renin-angiotensin-system seem to play an important role in the control of fibrinolysis in adipose tissue. We conclude that PAI-1 production by adipose tissue may contribute to the elevated thromboembolic risk in obesity.