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Renin Angiotensin System plays significant role in the memory acquisition and consolidation apart from its hemodynamic function in the pathophysiology of Alzheimer's disease (AD). It has been reported that Ang (1-7) ameliorates the cognitive impairment in experimental animals. However, the effect of Ang (1-7)/Mas receptor signaling is yet to be explored in Aβ42-induced memory impairment. Aβ42 was intracerebroventricularly injected into the male rats on day-1 (D-1) of the experimental schedule of 14 days. All the drugs were administered from D-1 to D-14 in the study design. Aβ42 significantly increased the escape latency during Morris water maze (MWM) test on D-10 to13 in the animals. Further, Aβ42 significantly decreased the time spent and percentage of total distance travelled in the target quadrant of the rats on D-14 in the MWM test. Aβ42 also significantly decreased the spontaneous alteration behavior on D-14 during Y-maze test. Moreover, there was a significant increase in the level of Aβ42, decrease in the cholinergic function (in terms of decreased acetylcholine and activity of cholinesterase, and increased activity of acetylcholinesterase), mitochondrial function, integrity and bioenergetics, and apoptosis in all the rat brain regions. Further, Aβ42 significantly decreased the level of expression of heme oxygenase-1 in all the rat brain regions. Ang (1-7) attenuated Aβ42-induced changes in the behavioral, biochemical and molecular observations in all the selected rat brain regions. However, A779, Mas receptor blocker, significantly abolished the beneficial effects of Ang (1-7) in Aβ42-induced cognitive deficit animals. These observations clearly indicate that the Ang (1-7)/Mas receptor activation could be a potential alternative option in the management of AD., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

In recent years a number of beneficial health effects have been ascribed to the renin-angiotensin system (RAS) that extend beyond lowering blood pressure, primarily mediated via the angiotensin-converting enzyme-2 (ACE2)/angiotensin (1-7) or Ang(1-7)/MAS receptor axis. Moreover, once thought as merely a systemic effector, RAS components exist within tissues. The highest tissue concentrations of ACE2 mRNA are located in the gut making it an important target for altering RAS function. Indeed, genetically engineered recombinant probiotics are promising treatment strategies offering delivery of therapeutic proteins with precision. An Ang(1-7) secreting Lactobacillus paracasei (LP) or LP-A has been described for regulation of diabetes and hypertension; however, we are the first to the best of our knowledge to propose this paradigm as it relates to aging. In this Research Practice manuscript, we provide proof of concept for using this technology in a well-characterized rodent model of aging: the Fisher344 x Brown Norway Rat (F344BN). Our primary findings suggest that LP-A increases circulating levels of Ang(1-7) both acutely and chronically (after 8 or 28 treatment days) when administered 3× or 7×/week over 4 weeks. Our future preclinical studies will explore the impact of this treatment on gut and other age-sensitive distal tissues such as brain and muscle., (© The Author(s) 2019. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The angiotensin II (ANG II)-ANG II type 1 receptor (AT 1 R) axis is a key player in the pathophysiology of obesity. Angiotensin-converting enzyme 2 (ACE2) counteracts the ANG II/AT 1 R axis via converting ANG II to angiotensin 1-7 (Ang 1-7), which is known to have an anti-obesity effect. In this study, we hypothesized that ACE2 exerts a strong anti-obesity effect by increasing Ang 1-7 levels. We injected intraperitoneally recombinant human ACE2 (rhACE2, 2.0 mg·kg -1 ·day -1 ) for 28 days to high-fat diet (HFD)-induced obesity mice. rhACE2 treatment decreased body weight and improved glucose metabolism. Furthermore, rhACE2 increased oxygen consumption and upregulated thermogenesis in HFD-fed mice. In the rhACE2 treatment group, brown adipose tissue (BAT) mass increased, accompanied with ameliorated insulin signaling and increased protein levels of uncoupling protein-1 (UCP-1) and PRD1-BF1-RIZ1 homologous domain containing 16. Importantly, subcutaneous white adipose tissue (sWAT) mass decreased, concomitant with browning, which was established by the increase of UCP-1 expression. The browning is the result of increased H3K27 acetylation via the downregulation of histone deacetylase 3 and increased H3K9 acetylation via upregulation of GCN5 and P300/CBP-associated factor. These results suggest that rhACE2 exerts anti-obesity effects by stimulating BAT and inducing browning in sWAT. ACE2 and the Ang 1-7 axis represent a potential therapeutic approach to prevent the development of obesity.

Bradykinin B2 receptor (B2R) and angiotensin-(1-7) Mas receptor (MasR)-mediated effects are physiologically interconnected. The molecular basis for such cross talk is unknown. It is hypothesized that the cross talk occurs at the receptor level. We investigated B2R-MasR heteromerization and the functional consequences of such interaction. B2R fused to the cyan fluorescent protein and MasR fused to the yellow fluorescent protein were transiently coexpressed in human embryonic kidney293T cells. Fluorescence resonance energy transfer analysis showed that B2R and MasR formed a constitutive heteromer, which was not modified by their agonists. B2R or MasR antagonists decreased fluorescence resonance energy transfer efficiency, suggesting that the antagonist promoted heteromer dissociation. B2R-MasR heteromerization induced an 8-fold increase in the MasR ligand-binding affinity. On agonist stimulation, the heteromer was internalized into early endosomes with a slower sequestration rate from the plasma membrane, compared with single receptors. B2R-MasR heteromerization induced a greater increase in arachidonic acid release and extracellular signal-regulated kinase phosphorylation after angiotensin-(1-7) stimulation, and this effect was blocked by the B2R antagonist. Concerning serine/threonine kinase Akt activity, a significant bradykinin-promoted activation was detected in B2R-MasR but not in B2R-expressing cells. Angiotensin-(1-7) and bradykinin elicited antiproliferative effects only in cells expressing B2R-MasR heteromers, but not in cells expressing each receptor alone. Proximity ligation assay confirmed B2R-MasR interaction in human glomerular endothelial cells supporting the interaction between both receptors in vivo. Our findings provide an explanation for the cross talk between bradykinin B2R and angiotensin-(1-7) MasR-mediated effects. B2R-MasR heteromerization induces functional changes in the receptor that may lead to long-lasting protective properties., Competing Interests: None., (© 2016 American Heart Association, Inc.)

This study aimed to determine the ability of peptides present in the non-digestible fraction (NDF) of common beans to decrease angiotensin II (AngII) through the blockade of RAS and its effect on the proliferation of HCT116 human colorectal cancer cells. Pure synthesized peptides GLTSK and GEGSGA and the peptide fractions (PF) of cultivars Azufrado Higuera and Bayo Madero were used. The cells were pretreated with pure peptides, PF or AGT at their IC50 or IC25 values, in comparison with the simultaneous treatment of peptides and AGT. For western blot and microscopy analysis, 100 μM and 0.5 mg mL(-1) were used for pure peptides and PF treatments, respectively. According to the ELISA tests, GLTSK and GEGSGA decreased (p < 0.05) the conversion rate of AGT to angiotensin I (AngI) by 38 and 28%, respectively. All the peptides tested reduced (p < 0.05) the conversion rate of AngI to AngII from 38 to 50%. When the cells were pretreated with both pure peptides and PF before exposure to AGT, the effectiveness inhibiting cell proliferation was higher than the simultaneous treatment suggesting their preventive effects. GLTSK and GEGSGA interacted with the catalytic site of renin, the angiotensin-I converting enzyme, and the AngII receptor, mainly through hydrogen bonds, polar, hydrophobic and cation-π interactions according to molecular docking. Through confocal microscopy, it was determined that GLTSK and GEGSGA caused the decrease (p < 0.05) of AngII-dependent STAT3 nuclear activation in HCT116 cells by 66 and 23%, respectively. The results suggest that peptides present in the common bean NDF could potentially ameliorate the effects of RAS overexpression in colorectal cancer.

Purpose . Products of angiotensin (ANG) I metabolism may predispose to vascular complications of diabetes mellitus. Methods . Diabetes was induced with streptozotocin (75 mg/kg i.p.). Rat aorta fragments, isolated 4 weeks later, were pretreated with perindoprilat (3  μ M), thiorphan (3  μ M), or vehicle and incubated for 15 minutes with ANG I (1 μ M). Products of ANG I metabolism through classical (ANG II, ANG III, and ANG IV) and alternative (ANG (1-9), ANG (1-7), and ANG (1-5)) pathways were measured in the buffer, using liquid chromatography-mass spectrometry. Results . Incubation with ANG I resulted in higher concentration of ANG II ( P = 0.02, vehicle pretreatment) and lower of ANG (1-9) ( P = 0.048, perindoprilat pretreatment) in diabetes. Preference for the classical pathway is suggested by higher ANG III/ANG (1-7) ratios in vehicle ( P = 0.03), perindoprilat ( P = 0.02), and thiorphan pretreated ( P = 0.02) diabetic rat. Within the classical pathway, ratios of ANG IV/ANG II ( P = 0.01) and of ANG IV/ANG III ( P = 0.049), but not of ANG III/ANG II are lower in diabetes. Conclusions . Diabetes in rats led to preference toward deleterious (ANG II, ANG III) over protective (ANG IV, ANG (1-9), and ANG (1-7)) ANG I metabolites.

It is well known that the renin-angiotensin system (RAS) plays a pivotal role in the pathophysiology of cardiovascular diseases. This is well illustrated by the great success of ACE inhibitors and angiotensin (Ang) II AT(1) blockers in the treatment of hypertension and its complications. In the past decade, the classical concept of RAS orchestrated by a series of enzymatic reactions culminating in the linear generation and action of Ang II has expanded and become more complex. From the discoveries of new components such as the angiotensin converting enzyme 2 and the receptor Mas emerged a novel concept of dual opposite branches of the RAS: one vasoconstrictor and pro-hypertensive composed of ACE/Ang II/AT1; and other vasodilator and anti-hypertensive composed of ACE2/Ang-(1-7)/Mas. In this review we will discuss recent findings concerning the biological role of the ACE2/Ang-(1-7)/Mas arm in the cardiovascular system and highlight the initiatives to develop potential therapeutic strategies based on this axis for treating hypertension.

Betamethasone is administered to accelerate lung development and improve survival of premature infants but may be associated with hypertension later in life. In a sheep model of fetal programming resulting from exposure at day 80 of gestation to Betamethasone (Beta-exposed), adult sheep at 6 to 9 months or 1.8 years of age have elevated mean arterial pressure (MAP) and attenuated spontaneous baroreflex sensitivity (sBRS) for control of heart rate compared to age-matched controls associated with imbalances in angiotensin (Ang) II vs Ang-(1-7) tone. At 6 weeks of age, evoked BRS is already low in the Beta-exposed animals. In this study, we assessed the potential contribution of the renin-angiotensin system to the impaired sBRS. Female lambs (6 weeks old) with Beta exposure in utero had similar MAP to control lambs (78±2 vs 77±2 mm Hg, n=4-5 per group), but lower sBRS (8±1 vs 16±3 ms/mm Hg; P<0.05) and impaired heart rate variability. Peripheral AT1 receptor blockade using candesartan lowered MAP in both groups (≈10 mm Hg) and improved sBRS and heart rate variability in Beta-exposed lambs to a level similar to control. AT7 receptor blockade by infusion of D-ala Ang-(1-7) (700 ng/kg/min for 45 minutes) reduced sBRS 46%±10% in Beta-exposed vs in control lambs (P<0.15) and increased MAP in both groups (≈6±2 mm Hg). Our data reveal that Beta exposure impairs sBRS and heart rate variability at a time point preceding the elevation in MAP via mechanisms involving an imbalance in the Ang II/Ang-(1-7) ratio consistent with a progressive loss in Ang-(1-7) function.

Angiotensin-converting enzyme 2 (ACE2) is an enzymatically active homologue of angiotensin-converting enzyme that degrades angiotensin I, angiotensin II, and other peptides. Recent studies have shown that under pathologic conditions, ACE2 expression in the kidney is altered. In this review, we briefly summarize recent studies dealing with pharmacologic interventions that modulate ACE2 expression. ACE2 amplification may have a potential therapeutic role for kidney disease and hypertension.

Background: Pharmacological interruption of the renin-angiotensin system focuses on optimization of blockade. As a measure of intrarenal renin activity, we have examined renal plasma flow (RPF) responses in a standardized protocol. Compared with responses with angiotensin-converting enzyme inhibition (rise in RPF approximately 95 mL x min(-1) x 1.73 m(-2)), greater renal vasodilation with angiotensin receptor blockers (approximately 145 mL x min(-1) x 1.73 m(-2)) suggested more effective blockade. We predicted that blockade with the direct oral renin inhibitor aliskiren would produce renal vascular responses exceeding those induced by angiotensin-converting enzyme inhibitors and angiotensin receptor blockers., Methods and Results: Twenty healthy normotensive subjects were studied on a low-sodium (10 mmol/d) diet, receiving separate escalating doses of aliskiren. Six additional subjects received captopril 25 mg as a low-sodium comparison and also received aliskiren on a high-sodium (200 mmol/d) diet. RPF was measured by clearance of para-aminohippurate. Aliskiren induced a remarkable dose-related renal vasodilation in low-sodium balance. The RPF response was maximal at the 600-mg dose (197+/-27 mL x min(-1) x 1.73 m(-2)) and exceeded responses to captopril (92+/-20 mL x min(-1) x 1.73 m(-2); P<0.01). Furthermore, significant residual vasodilation was observed 48 hours after each dose (P<0.01). The RPF response on a high-sodium diet was also higher than expected (47+/-17 mL x min(-1) x 1.73 m(-2)). Plasma renin activity and angiotensin levels were reduced in a dose-related manner. As another functional index of the effect of aliskiren, we found significant natriuresis on both diets., Conclusions: Renal vasodilation in healthy people with the potent renin inhibitor aliskiren exceeded responses seen previously with angiotensin-converting enzyme inhibitors and angiotensin receptor blockers. The effects were longer lasting and were associated with significant natriuresis. These results indicate that aliskiren may provide more complete and thus more effective blockade of the renin-angiotensin system.

Aims/hypothesis: In chronic heart failure there is gradual reactivation of vascular tissue angiotensin I (AI) to angiotensin II (AII) conversion over time in patients taking chronic ACE inhibitor therapy. However, it remains unknown whether the same overall phenomenon occurs in other patients taking chronic ACE inhibitor therapy, such as patients with type 2 diabetes mellitus., Methods: We studied 30 patients with type 2 diabetes mellitus (mean age 43.5 +/- 10.8 years), all of whom received lisinopril (20 mg/day) as part of their normal treatment. Over the course of the 18 month study, we made measurements at 0, 9 and 18 months. These measurements included plasma values for components of the renin-angiotensin-aldosterone system. In addition, we infused AI and AII into the brachial arteries of patients to assess vascular tissue AI to AII conversion., Results: There were no significant changes in plasma renin activity, ACE, AI, AII or aldosterone during the study. In contrast, vascular AI to AII conversion was significantly (p = 0.01) greater at 18 months than at 0 months. There was no change over time in the response to infused AII., Conclusions/interpretation: We have shown in vivo that vascular tissue AI to AII conversion gradually increases over time in patients with type 2 diabetes being treated with lisinopril. Further studies are required to determine whether this reactivation detracts from the cardioprotective effects of chronic ACE inhibitor therapy in diabetic patients, and if so, how best to overcome it.

Objectives: We hypothesized that angiotensin-converting enzyme inhibition (ACE-I) during left ventricular assist device (LVAD) support in patients with end-stage heart failure prevents potentially deleterious effects on the extracellular matrix., Background: Left ventricular assist device-induced mechanical unloading increases myocardial collagen and stiffness and may contribute to the low rate of recovery., Methods: Heart samples obtained before and after LVAD implantation were divided into groups depending on whether the patients received (n = 7) or did not receive (control; n = 15) ACE-I. At transplant, ex vivo pressure-volume relationships were measured and chamber and myocardial stiffness constants determined. Myocardial tissue content of angiotensin (Ang) I and II, matrix metalloproteinase (MMP)-1, tissue inhibitor of MMPs (TIMP)-1, and total and cross-linked collagen was measured., Results: Duration of support was comparable between ACE-I and control subjects (96 +/- 65 days vs. 109 +/- 22 days). Pre-LVAD Ang I and II and total and cross-linked collagen were similar between groups. Post-LVAD, Ang II was reduced in the ACE-I group but increased in control subjects (181 +/- 7 fmol/g vs. 262 +/- 41 fmol/g; p < 0.05). Similarly, cross-linked collagen decreased during LVAD support in the ACE-I group. Left ventricular (LV) mass and myocardial stiffness were lower in the ACE-I group. ACE-I normalized the LV and right ventricular (RV) MMP-1/TIMP-1 ratio. Collagen content and characteristics of the RV were not affected by ACE-I., Conclusions: ACE-I therapy was associated with decreased Ang II, myocardial collagen content, and myocardial stiffness during LVAD support. This is the first demonstration of a pharmacologic therapy that can impact myocardial properties during mechanical unloading, and it could foster new lines of investigation in strategies of enhancing myocardial recovery during LVAD support.

The distinct roles of the two estrogen receptor (ER) isotypes, ERalpha and ERbeta, in mediating the physiological responses to estrogens are not completely understood. Although knockout animal experiments have been aiding to gain insight into estrogen signaling, additional information on the function of ERalpha and ERbeta will be provided by the application of isotype-selective ER agonists. Based on the crystal structure of the ERalpha ligand binding domain and a homology model of the ERbeta-ligand binding domain, we have designed steroidal ligands that exploit the differences in size and flexibility of the two ligand binding cavities. Compounds predicted to bind preferentially to either ERalpha or ERbeta were synthesized and tested in vitro using radio-ligand competition and transactivation assays. This approach directly led to highly ER isotype-selective (approximately 200-fold) and potent ligands. To unravel physiological roles of the two receptors, in vivo experiments with rats were conducted using the ERalpha- and ERbeta-selective agonists in comparison to 17beta-estradiol. The ERalpha agonist induced uterine growth, caused bone-protective effects, reduced LH and FSH plasma levels, and increased angiotensin I, whereas the ERbeta agonist did not at all or only at high doses lead to such effects, despite high plasma levels. It can thus be concluded that estrogen effects on the uterus, pituitary, bone, and liver are primarily mediated via ERalpha. Simultaneous administration of the ERalpha and ERbeta ligand did not lead to an attenuation of ERalpha-mediated effects on the uterus, pituitary, and liver parameters.

Previous studies have shown that glucose increases angiotensin II (AngII) levels in rat glomerular mesangial cells and that AngII mediates the inhibitory effects of high glucose on matrix degradation in these cells. The present study addresses the following questions: (1) What are the mechanisms for the generation of AngII in mesangial cells? (2) What are the effects of glucose on AngII generation by these mechanisms? Experiments employed primary mesangial cells from normal Sprague-Dawley rats. The levels of immunoreactive angiotensinogen (AGT), angiotensin I (AngI), and angiotensin II (AngII) were measured by ELISA. AGT mRNA expression was determined by Northern blot analysis. Incubation of cells for 24 h in high glucose (30 mM) increased AGT levels by 1.5-fold and increased AGT mRNA expression; this was accompanied by a 1.5-fold increment in AngI and 1.7-fold increment in AngII levels. Renin activity (measured as AngI generation in the presence of excess AGT) and ACE levels and activity were not altered by high glucose. In further experiments, the effect of high glucose on formation of Ang peptides from exogenous AngI in mesangial cell extracts was examined using HPLC. Exogenous AngI was converted into various Ang peptides, including AngII, Ang(1-9), Ang(1-7), and Ang(3-8). A significant increase in formation of AngII from AngI was observed in cells incubated in high glucose. In addition, AngII production from exogenous Ang(1-9) in cell extracts was also stimulated by high glucose. These findings demonstrate that glucose increases mesangial AngII levels via an increase in AGT and AngI. In addition, this study provides new information that Ang(1-9) is produced by mesangial cells, can be converted to AngII, and that this conversion is also stimulated under high-glucose conditions.

Background: Vascular (interstitial) angiotensin (ANG) II production depends on circulating renin-angiotensin system (RAS) components. Mannose 6-phosphate (man-6-P) receptors and angiotensin II type 1 (AT(1)) receptors, via binding and internalization of (pro)renin and ANG II, respectively, could contribute to the transportation of these components across the endothelium., Objective: To investigate the mechanism(s) contributing to transendothelial RAS component transport., Methods: Human umbilical vein endothelial cells were cultured on transwell polycarbonate filters, and incubated with RAS components in the absence or presence of man-6-P, eprosartan or PD123319, to block man-6-P, AT(1) and angiotensin II type 2 (AT(2)) receptors, respectively., Results: Apically applied (pro)renin and angiotensinogen slowly entered the basolateral compartment, in a similar manner as horseradish peroxidase, a molecule of comparable size that reaches the interstitium via diffusion only. Prorenin transport was unaffected by man-6-P. Apical ANG I and ANG II rapidly reached the basolateral fluid independent of AT(1) and AT(2) receptors. Basolateral ANG II during apical ANG I application was as high as apical ANG II, whereas during apical ANG II application it was lower. During basolateral ANG I application, ANG II generation occurred basolaterally only, in an angiotensin-converting enzyme (ACE)-dependent manner., Conclusions: Circulating (pro)renin, angiotensinogen, ANG I and ANG II enter the interstitium via diffusion, and interstitial ANG II generation is mediated, at least in part, by basolaterally located endothelial ACE.

1 In previous studies on the renal targeting of the ACE inhibitor captopril, we demonstrated that a 6 fold increased concentration of this drug could be obtained in the kidney after conjugation to the low-molecular-weight protein lysozyme. In this study, we investigated in unrestrained rats whether systemic administration of captopril-lysozyme also results in an enhanced effect on renal parameters, relative to the systemic effects. 2 Renal effects: intravenous infusion of captopril-lysozyme for 6 h resulted in a more pronounced increment of renal blood flow (31+/-2% vs 17+/-4% at 0.5 mg kg(-1) 6h(-1), P<0.01) and an approximately 5 fold enhanced natriuresis (167+/-17% vs 36+/-7% at 1 mg kg(-1) 6 h(-1), P<0.001) in comparison with equimolar amounts of captopril as a free drug. In correspondence with these findings, renal ACE inhibition was potentiated approximately 5 fold (-50+/-4% vs -22+/-3% at 1 mg kg(-1) 6 h(-1), P<0.001). 3 Systemic effects: conjugated captopril did not affect blood pressure in dosages up to 5 mg kg(-1) 6 h(-1). This effect coincided with a less pronounced inhibition of the pressor response to intravenously administered angiotensin I (-12+/-3% vs -66+/-5% at 1 mg kg(-1) 6 h(-1), P<0.001), and a markedly attenuated plasma ACE inhibition (-19+/-2% vs -37+/-3% at 1 mg kg(-1) 6 h(-1), P<0.001) compared to an equivalent dose of free captopril. 4 An experiment of continued intravenous administration of captopril-lysozyme for 7 days in nephrotic syndrome demonstrated that the conjugate is also active in renal disease: the antiproteinuric response was substantially augmented (-67+/-5% vs -15+/-7% at 4 mg kg(-1) 24 h(-1), P<0.001) compared to the free drug, in the absence of blood pressure reduction. 5 These data demonstrate that intravenous administration of a captopril-lysozyme conjugate leads to more selective renal ACE inhibition and enhanced renal effects as well as less systemic effects compared to captopril itself.

Human heart tissue enzymes cleave angiotensin (Ang) I to release Ang 1-9, Ang II, or Ang 1-7. In atrial homogenate preparations, cathepsin A (deamidase) is responsible for 65% of the liberated Ang 1-9. Ang 1-7 was released (88% to 100%) by a metallopeptidase, as established with peptidase inhibitors. Ang II was liberated to about equal degrees by ACE and chymase-type enzymes. Cathepsin A's presence in heart tissue was also proven because it deamidated enkephalinamide substrate by immunoprecipitation of cathepsin A with antiserum to human recombinant enzyme and by immunohistochemistry. In immunohistochemistry, cathepsin A was detected in myocytes of atrial tissue. The products of Ang I cleavage, Ang 1-9 and Ang 1-7, potentiated the effect of an ACE-resistant bradykinin analog and enhanced kinin effect on the B(2) receptor in Chinese hamster ovary cells transfected to express human ACE and B(2) (CHO/AB), and in human pulmonary arterial endothelial cells. Ang 1-9 and 1-7 augmented arachidonic acid and nitric oxide (NO) release by kinin. Direct assay of NO liberation by bradykinin from endothelial cells was potentiated at 10 nmol/L concentration, 2.4-fold (Ang 1-9) and 2.1-fold (Ang 1-7); in higher concentrations, Ang 1-9 was significantly more active than Ang 1-7. Both peptides had traces of activity in the absence of bradykinin. Ang 1-9 and Ang 1-7 potentiated bradykinin action on the B(2) receptor by raising arachidonic acid and NO release at much lower concentrations than their 50% inhibition concentrations (IC(50)s) with ACE. They probably induce conformational changes in the ACE/B(2) receptor complex via interaction with ACE.

1. Renin inhibitors may be more advantageous than either angiotensin-converting enzyme (ACE) inhibitors or angiotensin (Ang) antagonists in blocking the renin-angiotensin system (RAS) because they do not allow accumulation of either AngI or AngII in plasma. 2. Effects of i.v. administration of two human renin inhibitors (EMD 58265 and U 71038) were compared with the ACE inhibitor enalaprilat on mean blood pressure (BP), renal blood flow (RBF) and plasma AngI and AngII in the anaesthetized two-kidney one-clip Goldblatt rabbit. 3. At doses of 2-2.5 mg/kg, i.v., EMD 58265 and 5-10 mg/kg, i.v., U 71038, both drugs decreased BP approximately 10 mmHg more than enalaprilat (2-4 mg/kg, i.v.) when given either before or after the ACE inhibitor. None of the three agents had any significant effect on RBF in the face of the lowered BP; however, renal vascular resistance was decreased. A higher dose of enalaprilat (10 mg/kg, i.v.) had no further effect on BP than the lower doses but did cause a marked increase in RBF. 4. Both renin inhibitors markedly decreased plasma AngI, but the high basal level of AngII was less consistently and only modestly affected. Enalaprilat, in either the low dose range or at the high dose, was also not effective in significantly decreasing AngII. 5. The results indicate that renin inhibition in the rabbit with a high circulating AngII level is more effective in lowering BP than ACE inhibition. A high dose of the ACE inhibitor may be required to block the intrarenal RAS, which may account for the increase in RBF.

Converting-enzyme inhibition reduces cardiovascular hypertrophy in hypertensive subjects. Whether the blockade of angiotensin II type 1 (AT(1)) receptors reduces arterial hypertrophy has never been investigated. In a double-blind study versus placebo in subjects with essential hypertension, the effect of the AT(1) blocker irbesartan (150 mg/day for 8 weeks) on blood pressure, wall thickness, diameter and stiffness of the common carotid and radial arteries was studied, using echotracking techniques of high resolution. With irbesartan, mean blood pressure decreased significantly and proportionally to the baseline levels of active renin, and angiotensin I and II. There was a significant decrease in radial artery wall thickness. The percent change from baseline (+/- SEM) was -10.51 +/- 3.42 versus 6.18 +/- 4.77. There was no significant change in diameter or distensibility. This effect was correlated neither to blood pressure changes nor to hormonal baseline levels of the renin-angiotensin system. Carotid wall thickness and diameter were unchanged. Thus a 2-month treatment with an AT(1) antagonist significantly reduced radial but not carotid artery wall thickness. Blood pressure reduction could be explained on the basis of circulating renin-angiotensin activity. On the contrary, radial artery wall thickness reduction was independent of the baseline circulating renin-angiotensin activity and was not correlated with the effects of AT(1) blockade on blood pressure, thus implying the involvement of local hemodynamic and/or cellular mechanisms., (Copyright 2000 S. Karger AG, Basel)

We examined the hypotensive effect of enalapril in relation to the local renin-angiotensin system of the kidney in spontaneously hypertensive rats (SHR). Oral administration of enalapril for 7 days decreased mean arterial blood pressure and renal tissue angiotensin II concentration without affecting plasma angiotensin II concentration in SHR. The enalapril treatment did not affect maximum binding of angiotensin II to renal tubules and glomeruli in SHR. In normotensive Wistar-Kyoto rats, no significant changes in mean arterial blood pressure, renal and plasma angiotensin levels were observed with enalapril treatment. Direct infusion of enalapril into the renal medullary interstitium decreased mean arterial blood pressure in association with the reduction of renal tissue angiotensin II concentration without changes in plasma angiotensin II concentration in SHR. These observations suggest that the inhibition of angiotensin conversion in the kidney is important for the hypotensive action of enalapril.

-Previous studies have shown that whereas the nonclipped kidney in two-kidney, one clip (2K1C) rats undergoes marked depletion of renin content and renin mRNA, intrarenal angiotensin II (Ang II) levels are not suppressed; however, the distribution and functional consequences of intrarenal Ang II remain unclear. The present study was performed to assess the plasma, kidney, and proximal tubular fluid levels of Ang II and the renal responses to intrarenal Ang II blockade in the nonclipped kidneys of rats clipped for 3 weeks. The Ang II concentrations in proximal tubular fluid averaged 9.19+/-1.06 pmol/mL, whereas plasma Ang II levels averaged 483+/-55 fmol/mL and kidney Ang II content averaged 650+/-66 fmol/g. Thus, as found in kidneys from normal rats with normal renin levels, proximal tubular fluid concentrations of Ang II are in the nanomolar range. To avoid the confounding effects of decreases in mean arterial pressure (MAP), we administered the nonsurmountable AT1 receptor antagonist candesartan directly into the renal artery of nonclipped kidneys (n=10). The dose of candesartan (0.5 microg) did not significantly decrease MAP in 2K1C rats (152+/-3 versus 148+/-3 mm Hg), but effectively prevented the renal vasoconstriction elicited by an intra-arterial bolus of Ang II (2 ng). Candesartan elicited significant increases in glomerular filtration rate (GFR) (0.65+/-0. 06 to 0.83+/-0.11 mL. min-1. g-1) and renal blood flow (6.3+/-0.7 to 7.3+/-0.9 mL. min-1. g-1), and proportionately greater increases in absolute sodium excretion (0.23+/-0.07 to 1.13+/-0.34 micromol. min-1. g-1) and fractional sodium excretion (0.38+/-0.1% to 1.22+/-0. 35%) in 2K1C hypertensive rats. These results show that proximal tubular fluid concentrations of Ang II are in the nanomolar range and are much higher than can be explained on the basis of plasma levels. Further, the data show that the intratubular levels of Ang II in the nonclipped kidneys of 2K1C rats remain at levels found in kidneys with normal renin content and could be exerting effects to suppress renal hemodynamic and glomerular function and to enhance tubular reabsorption rate.

To characterize the role of cytochrome P450 metabolism of fatty acids in the renal response to increased renal perfusion pressure, we tested the effects of renal arterial infusion of 17-octadecynoic acid (17-ODYA, 450 nmol/min) on renal and systemic hemodynamic, and renal excretory responses to step-wise increases in renal perfusion pressure (RPP) in anesthetized rabbits, using an extracorporeal circuit for renal autoperfusion. Inhibition of cytochrome P450-dependent fatty acid metabolism was estimated by comparing the metabolism of arachidonic acid in microsomes prepared from the kidneys of control and 17-ODYA-treated animals. Step-wise increases in RPP decreased mean arterial pressure, which previous studies have indicated is attributable to the release of a depressor hormone from the renal medulla. Elevations in RPP also increased renal blood flow and glomerular filtration rate, and the absolute and fractional excretions of urine and sodium. Intrarenal infusion of 17-ODYA reduced the metabolism of arachidonic acid to 20-hydroxyeicosatetraenoic acid by 41%, but it did not significantly influence the responses to increased renal perfusion pressure. We conclude that either the responses elicited by increased renal perfusion pressure in anesthetized rabbits do not depend on cytochrome P450-dependent fatty acid metabolism, or that cytochrome P450 activity must be inhibited by more than was achieved in the present study (41%), before functional effects on the response to increased renal perfusion pressure are observed.

A growing body of evidence supports the existence of a tissue-based renin-angiotensin system (RAS) in the vasculature, but the functional capacity of vascular RAS was not investigated in humans. In 28 normotensive healthy control subjects, the metabolism of angiotensins through vascular tissue was investigated in normal, low, and high sodium diets by the measurement of arterial-venous gradient of endogenous angiotensin (Ang) I and Ang II in two different vascular beds (forearm and leg), combined with the study of 125I-Ang I and 125I-Ang II kinetics. In normal sodium diet subjects, forearm vascular tissue extracted 36+/-6% of 125I-Ang I and 30+/-5% of 125I-Ang II and added 14.9+/-5.1 fmol x 100 mL(-1) x min(-1) of de novo formed Ang I and 6.2+/-2.8 fmol x 100 mL(-1) x min(-1) of Ang II to antecubital venous blood. Fractional conversion of 125I-Ang I through forearm vascular tissue was about 12%. Low sodium diet increased (P<.01) plasma renin activity, whereas de novo Ang I and Ang II formation by forearm vascular tissue became undetectable. Angiotensin degradation (33+/-7% for Ang I and 30+/-7% for Ang II) was unchanged, and vascular fractional conversion of 125I-Ang I decreased from 12% to 6% (P<.01). In high sodium diet subjects, plasma renin activity decreased, and de novo Ang I and Ang II formation by forearm vascular tissue increased to 22 and 14 fmol x 100 mL(-1) x min(-1), respectively (P<.01). Angiotensin degradation did not significantly change, whereas fractional conversion of 125I-Ang I increased from 12% to 20% (P<.01). Leg vascular tissue functional activities of RAS paralleled those of forearm vascular tissue both at baseline and during different sodium intake. These results provide consistent evidence for the existence of a functional tissue-based RAS in vascular tissue of humans. The opposite changes of plasma renin activity and vascular angiotensin formation indicate that vascular RAS is independent from but related to circulating RAS.

To elucidate the local effects of renin in the coronary circulation, we examined local angiotensin (Ang) I and II formation, as well as coronary vasoconstriction in response to renin administration, and compared the effects with exogenous infused Ang I. We perfused isolated hearts from rats overexpressing the human angiotensinogen gene in a Langendorff preparation and measured the hemodynamic effects and the released products. We also investigated cardiac Ang I conversion, including the contribution of non-angiotensin-converting enzyme-dependent Ang II-generating pathways. Finally, we studied Ang I conversion in vitro in heart homogenates. Renin and Ang I infusion both generated Ang II. Ang II release and vasoconstriction continued after renin infusion was stopped, even though renin disappeared immediately from the perfusate. In contrast, after Ang I infusion, Ang II release and coronary flow returned to basal levels. Ang I conversion (Ang II/Ang I ratio) was higher after renin infusion (0.109+/-0.027 versus 0.026+/-0.003, 15 minutes, P<.02) compared with infused Ang I. Remikiren added to the renin infusion abolished Ang I and II; captopril suppressed only Ang II, whereas an AT1 receptor blocker did not affect Ang I and II formation. All the drugs prevented renin-induced coronary flow changes. Total cardiac Ang II-forming activity was only partially inhibited by cilazaprilat (4.1+/-0.1 fmol x min(-1) x mg[-1]) and on a larger extent by chymostatin (2.6+/-0.3 fmol x min(-1) x mg[-1]) compared with control values (5.6+/-0.4 fmol x min(-1) x mg[-1]). We conclude that renin can be taken up by cardiac or coronary vascular tissue and induces long-lasting local Ang II generation and vasoconstriction. Locally formed Ang I was converted more effectively than infused Ang I. Furthermore, the comparison of in vivo and in vitro Ang I conversion suggests that in vitro assays may underestimate the functional contribution of angiotensin-converting enzyme to intracardiac Ang II formation.

The combination of single oral doses of an angiotensin I-converting enzyme inhibitor (captopril) and a type 1 angiotensin II receptor antagonist (losartan) has additive effects on blood pressure fall and renin release in sodium-depleted normotensive subjects. We planned the present study to determine whether the magnitude of the hemodynamic and hormonal consequences of renin-angiotensin system blockade by such a combination is larger than that obtained by doubling the dose of the angiotensin-converting enzyme inhibitor given alone. In a single-dose, double-blind, randomized, three-way crossover study, 10 mg enalapril, 20 mg enalapril, and the combination of 50 mg losartan and 10 mg enalapril were administered orally to 12 sodium-depleted normotensive subjects. The area under the time curve from 0 to 24 hours (AUC0-24) of the mean blood pressure fall after losartan-enalapril combination intake (-220 +/- 91 mm Hg.h) was significantly greater than that of either 10 or 20 mg enalapril (-124 +/- 91 and -149 +/- 85 mm Hg.h, respectively, P < .05 vs both doses). The combination significantly increased by 2.3 +/- 1.2-fold the AUC0-24 of plasma active renin compared with either 10 or 20 mg enalapril given alone (P < .05) but had no additive effect on plasma aldosterone fall. The losartan-enalapril combination is more effective in decreasing blood pressure and increasing plasma active renin than doubling of the enalapril dose.

Using aortic rings from male Wistar rats, we studied the influence of nitric oxide (NO) on the vascular reactivity to angiotensins. The inhibition of NO-synthesis by L-NAME produced on both intact and desendothelised rings an augmentation of vascular reactivity to angiotensins. NO inhibition did not affect the blocking effects of Saralasin to angiotensins vasoconstriction, suggesting that NO cannot act directly on angiotensin II receptor. Nifedipin inhibited the stimulatory effect of L-NAME on angiotensins vasoconstriction. The results of our study provide functional evidence that NO production can interfere with vascular RAS at two levels: 1. by modulating the activity of Ang II-forming enzymes; 2. at intracellular level, by modulating the concentration of calcium. Also, our results suggest the existence of an alternative pathway on Ang II formation, that become more evident with removal of endothelium.

Background: Cardiac volume overload by an aortocaval shunt increases left ventricular end-diastolic pressure (LVEDP) and plasma and cardiac renin activity and results in LV hypertrophy. To a similar extent, the angiotensin-converting enzyme (ACE) inhibitors enalapril and quinapril prevent the increase in LVEDP. However, only quinapril attenuates the development of LV hypertrophy. We hypothesize that a low affinity of enalapril for cardiac ACE results in continuing generation of cardiac angiotensin II and thus hypertrophic growth of cardiomyocytes., Methods and Results: In the present study, we assessed plasma and cardiac angiotensins I and II 1 and 7 days after aortocaval shunt and the effects of enalapril and quinapril started 3 days before surgery on plasma and cardiac angiotensin I and II at the same time points. Aortocaval shunt increased plasma angiotensin II at 1 day by 180%, but only a small increase (by 40%) persisted at 7 days. Aortocaval shunt increased LV angiotensin II by 100% and 65% at 1 and 7 days, respectively. Both blockers similarly prevented the increase in plasma angiotensin II by aortocaval shunt at both time points. In contrast, only quinapril prevented the rise in LV angiotensin II induced by shunt at 1 and 7 days., Conclusions: Aortocaval shunt increases LVEDP and plasma and cardiac angiotensin II and results in LV hypertrophy. Only prevention of the increase in LVEDP and in plasma and cardiac angiotensin II attenuates the development of LV hypertrophy, consistent with the concept that angiotensin II is involved in the development of cardiac hypertrophy by aortocaval shunt by both hemodynamic and cardiac trophic effects. This study is the first to show that differences in affinity for cardiac ACE may determine the effect of ACE inhibitors on cardiac angiotensin II and therefore cardiac hypertrophy.

Angiotensin (Ang) I converting enzyme (ACE) inhibitors represent a major advance in the treatment of congestive heart failure, and tissue, rather than circulating ACE, may be their major site of action. However, assessments of tissue ACE inhibition in treated patients has not always supported this contention. In these studies, ACE activity was measured in homogenates of sampled tissue by biochemical methods. In the present study, using a model system, we have examined the validity of these tissue-sampling methods. Functional ACE activity was determined by comparing positive inotropic responses to [Pro10]Ang I in either vehicle-pretreated or ACE inhibitor-pretreated papillary muscles. [Pro10]Ang I elicits a response, which is entirely dependent on ACE-mediated conversion to Ang II. The ACE inhibitors studied were captopril, enalaprilat, lisinopril, and quinaprilat. In a parallel study, papillary muscle ACE activity was also measured in homogenates using [125I]MK-351A (a radiolabeled ACE inhibitor) binding. The studies indicate that the tissue-sampling method significantly underestimated functional ACE inhibition in hamster papillary muscles (p < 0.001). Kinetic studies indicated that the half-time for the dissociation of [3H]enalaprilat and [3H]lisinopril from hamster ventricular ACE was 4.5 and 6.2 minutes, respectively. The dissociation of [3H]quinaprilat was biphasic (half-time, 47 and 90 minutes), indicating that the two active sites of somatic ACE differ in their ability to bind to this inhibitor. The rapid rate of ACE inhibitor dissociation suggests that, during the time taken to assay ACE activity biochemically, the enzyme becomes "disinhibited," leading to an underestimation of functional ACE inhibition. ACE inhibitor dissociation rates were partially predictive of the duration of functional ACE inhibition in papillary muscles; other factors that appeared to contribute were "tissue trapping" of the inhibitor and de novo synthesis of ACE in papillary muscles. Quantification of tissue ACE inhibition and its relation to drug efficacy must, therefore, involve a careful consideration of these factors to avoid artifacts in clinical decision making and in assessments of pathogenic mechanisms involved in congestive heart failure.

Introduction: Remikiren (Ro 42-5892) is a potent and specific inhibitor of human renin in vitro. Its in vivo action on plasma renin activity (PRA), immunoreactive renin, and blood pressure has been shown in pilot studies in humans., Objective: To investigate tolerability, hemodynamic effects, and biochemical effects of remikiren in relation to its pharmacokinetics after single ascending intravenous and oral doses in healthy humans., Methods: In this double-blind, placebo-controlled, two-way crossover (intravenous and oral) study, single ascending doses of 10, 20, 40, 80, 160, and 320 mg (intravenous) and 100, 200, 400, 800, and 1600 mg (oral) were given; six subjects received active drug and three received placebo at each dose level. At regular intervals, blood pressure, heart rate, cardiac output, PRA, immunoreactive renin, and drug plasma levels were determined., Results: The compound was well tolerated except at the 1600 mg oral dose level at which diarrhea occurred in two subjects. At neither dose were there effects on blood pressure, heart rate, or cardiac output relative to placebo. PRA and angiotensin I production rate decreased and immunoreactive renin increased dose dependently after both intravenous and oral administration. The duration of these effects was also dose dependent and was longer than 12 hours with higher doses. Systemic plasma clearance, volume of distribution, and absolute bioavailability of remikiren were in the magnitude of 900 ml/min, 70 L, and below 1%, respectively. The angiotensin I production rate correlated in a sigmoidal way with plasma drug concentrations independent of the route of administration., Conclusion: Remikiren is a potent inhibitor of renin in humans with long-lasting effects after both intravenous and oral administration.

Major components of the renin-angiotensin system have been localized to cardiac tissue. Cardiac-derived angiotensin II may benefit myocardial contractility but may promote detrimental myocardial hypertrophy, coronary vasoconstriction, and arrhythmias. The benefits of ACE inhibition probably extend beyond the classic circulating RAS to include the heart directly.

High affinity 125I-angiotensin II (Ang II) binding sites were characterized in the canine pancreas. Total binding increased with protein concentration and equilibrium was reached within 60-90 min at 22 degrees C. Specific binding was saturable and averaged 70% of total. Scatchard analysis of binding yielded a KD of 0.48 +/- 0.18 nM with a Bmax of 32.8 +/- 6.5 fmol/mg protein (mean +/- SEM, n = 6). The addition of the reducing agent dithiothreitol increased specific binding two-fold. The rank order of displacement of 125I-Ang II binding by native angiotensin peptides was Ang II greater than or equal to Ang III greater than AngI greater than Ang(1-7) much greater than Ang(1-6). The use of the specific Ang II antagonists CGP 42112A, PD 123177, and DuP 753 revealed that the pancreas expresses two receptor subtypes. The majority of Ang II binding sites in the pancreas could be classified as type 2 (AT2), although type 1 (AT1) sites were also detected. In vitro autoradiography revealed binding sites localized over islet cells, acinar and duct cells, as well as the pancreatic vasculature. In addition, the autoradiographic studies confirmed the predominance of the AT2 receptor subtype throughout the pancreas.