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.     

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.     

Endothelin-1 and angiotensin II modulate rate and contraction amplitude in a subpopulation of mouse embryonic stem cell-derived cardiomyocyte-containing bodies

Embryonic stem cell-derived cardiomyocytes (ESC-CMs) have applications in understanding cardiac disease pathophysiology, pharmacology, and toxicology. Comprehensive characterization of their basic physiological and pharmacological properties is critical in determining the suitability of ESC-CMs as models of cardiac activity. In this study we use video microscopy and quantitative PCR to investigate the responses of mouse ESC-CMs to adrenoceptor, muscarinic, angiotensin II (Ang II), and endothelin-1 (ET-1) receptor activation. Isoprenaline (10 nM–10 μM) increased beating rate and contraction amplitude in all beating bodies (BBs), whereas carbachol (up to 1 μM) and the I_f channel blocker ZD-7288 (10 μM) decreased contraction frequency. ET-1 (0.01–100 nM) reduced contraction amplitude in all BBs and increased contraction frequency in 50% of BBs; these effects were blocked by the ET_A receptor antagonist BQ123 (250 nM). Ang II (0.01 nM–1 μM) increased both contraction amplitude (all BBs) and frequency (in 50% of BBs), effects blocked, respectively, by losartan (100 nM) and PD123,319 (200 nM). These results indicate the presence of functional ET_A and both AT₁ and AT₂ receptors in murine ESC-CMs, but their expression and or activity appears to be evident only in a limited set of BBs.     

Endothelin-1 and angiotensin II contribute to BNP but not c-fos gene expression response to elevated load in isolated mice hearts

The early events in the cardiac hypertrophic process induced by hemodynamic load include activation of B-type natriuretic peptide (BNP) and c-fos gene expression. However, it is unknown whether stretch acts directly or through local paracrine factors to trigger changes in cardiac gene expression. Herein we studied the involvement of endothelin-1 (ET-1) and angiotensin II (Ang II) in load-induced activation of left ventricular BNP and c-fos gene expression using an in vitro stretch model in isolated perfused adult mice hearts. Two-hour stretch induced by increasing coronary flow rate from 2 to 5 ml/min increased the expression of BNP and c-fos genes by 1.9- and 1.5-fold, respectively (P<0.001 and P<0.05). A mixed ET(A/B) receptor antagonist bosentan attenuated the BNP gene expression response to load by 58% (P<0.005). A similar 53% inhibition was observed with the selective ET(A) receptor blocker BQ-123 (P<0.05). Type 1 Ang II receptor antagonist CV-11974 decreased the activation of BNP gene expression by 50% (P<0.05). In contrast, the activation of c-fos gene expression was not inhibited by antagonists of ET(A/B) and AT(1) receptors. Our results show that ET-1 and Ang II play a key role in the induction of BNP, but not c-fos gene expression in response to load in intact adult murine hearts.     

Angiotensin II type 1 receptor expression in human pancreatic cancer and growth inhibition by angiotensin II type 1 receptor antagonist

We investigated the expression of angiotensin II type 1 receptor (AT1) in pancreatic cancer. Both AT1 mRNA and protein were expressed in human pancreatic cancer tissues and cell lines. Binding assays showed that pancreatic cancer cells have specific binding sites for angiotensin II and that binding could be eliminated by treatment with a selective AT1 antagonist in a dose-dependent fashion. Surprisingly, the growth of cancer cells was significantly suppressed by treatment with antagonist, also in a dose-dependent manner. These observations suggest AT1 plays an important role in pancreatic cancer growth. Furthermore, ligand-induced inhibition of AT1 may be a useful therapeutic strategy.     

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.     

Theoretical conformational analysis of Asn1, Val5 angiotensin II

A theoretical analysis of the conformation of the octapeptide hormone Asn¹, Val⁵ angiotensin II has been carried out by semiempirical potential energy calculations. A preliminary study of the Ala₆-Pro-Ala molecule, which mimics the angiotensin backbone, provided us with likely backbone structures on which the effect of the full side chains of the hormone could be assessed. For angiotensin II, the calculations show that only a small number of folded, compact conformations have a high probability of existence. This is the consequence of favorable packing and of the presence of proline in position 7. These results are consistent with various experimental data, both structural and biological. This method is readily applicable to the study of analogs of the hormone or to other peptides of comparable size.     

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.     

The angiotensin Ⅱ type 1 receptor and receptor－associated proteins

The mechanisms of regulation, activation and signal transduction of the angiotensin II (Ang II) type 1 (AT1) receptor have been studied extensively in the decade after its cloning. The AT1 receptor is a major component of the renin-angiotensin system (RAS). It mediates the classical biological actions of Ang II. Among the structures required for regulation and activation of the receptor, its carboxyl-terminal region plays crucial roles in receptor internalization, desensitization and phosphorylation. The mechanisms involved in heterotrimeric G-protein coupling to the receptor, activation of the downstream signaling pathway by G proteins and the Ang II signal transduction pathways leading to specific cellular responses are discussed. In addition, recent work on the identification and characterization of novel proteins associated with carboxyl-terminus of the AT1 receptor is presented. These novel proteins will advance our understanding of how the receptor is internalized and recycled as they provide molecular mechanisms for the activation and regulation of G-protein-coupled receptors.     

Mouse mast cell protease-1 cleaves angiotensin I to form angiotensin II

The ability to convert angiotensin (Ang) I to Ang II was compared between human alpha-chymase and two mouse beta-chymases, mouse mast cell protease (mMCP)-1 and mMCP-4. Human chymase hydrolyzed Ang I to produce Ang II without further degradation. mMCP-1 similarly generated Ang II from Ang I in a time-dependent manner and the formation of the fragment other than Ang II was marginal. In contrast, mMCP-4 hydrolyzed Ang I at two sites, Tyr(4)-Ile(5) and Phe(8)-His(9), with Ang II formation being tentative. Consistently, mMCP-4 but not human chymase hydrolyzed Ang II and mMCP-1 showed little hydrolytic activity against Ang II. These data suggest that not only human chymase but also mMCP-1 might possess a physiological role in Ang II formation. Our findings also imply that the Ang-converting activity of chymase may not be related to the categorization of chymase into alpha- or beta-type based on their primary structure.     

Conformations in solution of angiotensin II, and its 1-7 and 1-6 fragments.

High-resolution proton spectra at 620 MHz of human angiotensin II (1-8), angiotensin II (1-7), and angiotensin II (1-6) have been obtained in aqueous solution at acidic pH, and in dimethylsulfoxide solution. Complete chemical shift assignments for all three angiotensin peptides were made based on two-dimensional (2D) correlated spectroscopy and 2D-CA-MELSPIN spectra. Based on the measured values of 3JHNCH, the pattern of observed transverse Overhauser effects, and side-chain coupling constants, it is concluded that all three analogues exist in H2O or DMSO-d6 as a mixture of conformers that is largely extended, with negligible content of folded structures, such as beta-turns, gamma-turns, or helix content. The results fit well with those of Nikiforovich et al.     

Similarities and differences between effects of angiotensin III and angiotensin II on human prostate cancer cell migration and proliferation

Proliferation plays a critical role in tumor growth when cell migration is essential to invasion. The effect of Ang III and Ang II was evaluated on these important processes. Changes in the migration potential of prostate cancer cells were investigated using Wound Healing Test and a Transwell Migration Chamber with a 3μm pore size. Cell proliferation was measured with a BrdU Assay and Countess Automated Cell Counter, thus determining the influence of angiotensins on hormone-dependent (LNCaP) and hormone-independent (DU-145) human prostate cancer lines. The influence of Ang III and Ang II on classic receptors may be inhibited by Losartan or PD123319. Test peptide modulation of the AT1 and AT2 receptors was examined by Western Blot and fluorescent immunocytochemistry. The results indicate that Ang III promotes the migration of both LNCaP and DU-145 lines, whereas Ang II stimulates this process only in androgen-independent cells. Both angiotensin peptides can induce prostate cancer cell proliferation in a time- and dose-dependent manner. The obtained results show that Ang III and Ang II can modify the expression of classic receptors, particularly AT2. These results suggest that the investigated peptide can modulate cell migration and proliferation in prostate cancer cells. Angiotensins probably have a greater influence on proliferation in the early-stage prostate cancer model than hormone-independent cell lines. Assume also that Ang II can enhance the migration tendency aggressive prostate cancer cells, while Ang III does so more effective in non-metastatic cells.     

Action of a human plasma fraction on tetradecapeptide,angiotensin I and angiotensin II

A protein fraction designated PF70 was isolated from human plasma and partially purified on Sephadex G-100. PF70 proteins, molecular weight 37, 000 to 41, 500, formed angiotensin I (AI) and angiotensin II (AII) from ¹⁴C-tetradecapeptide renin substrate (TDP) at 37 C. Hydrolysis was maximal at pH 6.9 but there was no change in the relative quantity of AI and AII formed at different pH values. Data indicate that AI was formed first and at a faster rate than AII, but typical converting enzyme activity was not detected. Radiolabeled AII was converted to Des-Asp¹-angiotensin II (angiotensin III); [³H]AI was degraded to a single tritiated product, possibly the nonapeptide. These aspartyl hydrolase reactions were apparently inhibited by TDP and were not involved in AI or AII generation from TDP. It is concluded that these enzymic activities represent two or more enzymes that are associated with the renin-angiotensin system.     

Competitive antagonism of pressor responses to angiotensin II and angiotensin III by the angiotensin II-1 receptor ligand losartan.

Losartan (DuP 753) and PD123177 are nonpeptide angiotensin (ANG) receptor ligands for subtypes of ANG II receptors ANG II-1 and ANG II-2, respectively. We examined the effects of losartan and PD123177 on dose - mean arterial pressure (MAP) response curves for ANG II and ANG III in eight groups (n = 6 each) of conscious rats. Saline (0.9% NaCl), losartan (1 x 10(-6) and 9 x 10(-6) mol/kg), and PD123177 (2 x 10(-5) mol/kg) were i.v. bolus injected 15 min before the construction of ANG II dose - response curves in groups I, II, III, and IV, respectively. Groups V-VIII were treated similarly to I-IV except that ANG III was given in place of ANG II. Losartan dose dependently shifted the dose-response curves of ANG II and ANG III to the right with similar dissociation constants (-log KI of 6.6 +/- 0.7 and 6.6 +/- 0.1 mol/kg, respectively) and no change in the maxima. PD123177 affected neither maximum MAP nor ED50 values for ANG II or ANG III. Our results show that losartan but not PD123177 is a competitive antagonist of the MAP effects of ANG II and ANG III.     

Intracrine action of angiotensin II in mesangial cells: subcellular distribution of angiotensin II receptor subtypes AT1 and AT2

Biological effects of angiotensin II (AngII) such as regulation of AngII target genes may be triggered by interaction of AngII with intracellular AngII receptor types 1 and 2 (AT₁ and AT₂), defined as intracrine response. The aim of this study was to examine the presence of AT₁ and AT₂ receptors in nuclear membrane of human mesangial cells (HMCs) and evaluate the possible biological effects mediated by intracellular AT₁ through an intracrine mechanism. Subcellular distribution of AT₁ and AT₂ was evaluated by immunofluorescence and by western blot in isolated nuclear extract. Endogenous intracellular synthesis of AngII was stimulated by high glucose (HG). Effects of HG were analyzed in the presence of candesartan, which prevents AngII internalization. Both receptors were found in nuclear membrane. Fluorescein isothiocyanate (FITC)-labeled AngII added to isolated nuclei produced a fluorescence that was reduced in the presence of losartan or PD-123319 and quenched in the presence of both inhibitors simultaneously. HG induced overexpression of fibronectin and increased cell proliferation in the presence of candesartan, indicating an intracrine action of AngII induced by HG. Results showed the presence of nuclear receptors in HMCs that can be activated by AngII through an intracrine response independent of cytoplasmic membrane AngII receptors.

     

Endothelin-1 is a novel prognostic factor in non-small cell lung cancer

Endothelin-1 (ET-1) is a potent vasoactive peptide and a hypoxia-inducible angiogenic growth factor associated with the development and growth of solid tumours. This study evaluated the expression of big endothelin-1 (big ET-1), a stable precursor of ET-1, and ET-1 in non-small cell lung cancer (NSCLC). Big ET-1 expression was evaluated in paraffin-embedded tissue sections from 10 NSCLC tumours using immunohistochemistry and in situ hybridisation. The production of big ET-1 and ET-1 was studied in six established NSCLC cell lines. The plasma concentrations of big ET-1 were measured in 30 patients with proven NSCLC prior to chemotherapy by means of a sandwich enzyme-linked immunoassay and compared to levels in 20 normal controls. Big ET-1 immunostaining was detected in the cancer cells of all tumours studied. Using in situ hybridisation, tumour cell big ET-1 mRNA expression was demonstrated in all samples. All six NSCLC cell lines expressed ET-1, with big ET-1 being detected in three. The median big ET-1 plasma level in patients with NSCLC was 5.4 pg/mL (range 0-22.7 pg/mL) and was significantly elevated compared to median big ET-1 plasma levels in controls, 2.1 pg/mL (1.2-13.4 pg/mL) (p = 0.0001). Furthermore, patients with plasma big ET-1 levels above the normal range (upper tertile) had a worse outcome (p = 0.01). In conclusion, big ET-1/ET-1 is expressed by resected NSCLC specimens and tumour cell lines. Plasma big ET-1 levels are elevated in NSCLC patients compared to controls with levels > 7.8 pg/mL being associated with a worse outcome. The development of selective ET-1 antagonists such as Atrasentan indicates that ET-1 may be a therapeutic target in NSCLC.