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Importance: Angiotensinogen is the most upstream precursor of the renin-angiotensin-aldosterone system, a key pathway in blood pressure (BP) regulation. Zilebesiran, an investigational RNA interference therapeutic, targets hepatic angiotensinogen synthesis., Objective: To evaluate antihypertensive efficacy and safety of different zilebesiran dosing regimens., Design, Setting, and Participants: This phase 2, randomized, double-blind, dose-ranging study of zilebesiran vs placebo was performed at 78 sites across 4 countries. Screening initiation occurred in July 2021 and the last patient visit of the 6-month study occurred in June 2023. Adults with mild to moderate hypertension, defined as daytime mean ambulatory systolic BP (SBP) of 135 to 160 mm Hg following antihypertensive washout, were randomized., Interventions: Randomization to 1 of 4 subcutaneous zilebesiran regimens (150, 300, or 600 mg once every 6 months or 300 mg once every 3 months) or placebo (once every 3 months) for 6 months., Main Outcomes and Measures: The primary end point was between-group difference in least-squares mean (LSM) change from baseline to month 3 in 24-hour mean ambulatory SBP., Results: Of 394 randomized patients, 377 (302 receiving zilebesiran and 75 receiving placebo) comprised the full analysis set (93 Black patients [24.7%]; 167 [44.3%] women; mean [SD] age, 57 [11] years). At 3 months, 24-hour mean ambulatory SBP changes from baseline were -7.3 mm Hg (95% CI, -10.3 to -4.4) with zilebesiran, 150 mg, once every 6 months; -10.0 mm Hg (95% CI, -12.0 to -7.9) with zilebesiran, 300 mg, once every 3 months or every 6 months; -8.9 mm Hg (95% CI, -11.9 to -6.0) with zilebesiran, 600 mg, once every 6 months; and 6.8 mm Hg (95% CI, 3.6-9.9) with placebo. LSM differences vs placebo in change from baseline to month 3 were -14.1 mm Hg (95% CI, -19.2 to -9.0; P < .001) with zilebesiran, 150 mg, once every 6 months; -16.7 mm Hg (95% CI, -21.2 to -12.3; P < .001) with zilebesiran, 300 mg, once every 3 months or every 6 months; and -15.7 mm Hg (95% CI, -20.8 to -10.6; P < .001) with zilebesiran, 600 mg, once every 6 months. Over 6 months, 60.9% of patients receiving zilebesiran had adverse events vs 50.7% patients receiving placebo and 3.6% had serious adverse events vs 6.7% receiving placebo. Nonserious drug-related adverse events occurred in 16.9% of zilebesiran-treated patients (principally injection site reactions and mild hyperkalemia) and 8.0% of placebo-treated patients., Conclusions and Relevance: In adults with mild to moderate hypertension, treatment with zilebesiran across a range of doses at 3-month or 6-month intervals significantly reduced 24-hour mean ambulatory SBP at month 3., Trial Registration: ClinicalTrials.gov Identifier: NCT04936035.

The purpose of current study was to elucidate polyphenol tannic acid effect on renal function and activity of the renin-angiotensin system after unilateral ureteral obstruction (UUO). Male Wistar rats were divided into three groups of six randomly: 1) Sham, 2) UUO, and 3) UUO + Tannic acid. Rats in the UUO and UUO + Tannic acid groups experienced unilateral ureteral obstruction. In the Sham group, the abdominal cavity was exposed without UUO induction. In the UUO + Tannic acid group, animals received tannic acid (20 mg/kg) intraperitoneally, 6 and 12 h after clamping the left ureter and 6 and 12 h after the right nephrectomy. Blood samples were taken to measure blood urea nitrogen (BUN) and creatinine levels. Kidney tissue samples were obtained for assessment of oxidative stress, inflammatory indices and the levels of renin-angiotensin system components. Tannic acid administration significantly improved UUO-induced kidney dysfunction (serum BUN: 66.42 ± 14.414 mg/dl, p < 0.05; serum creatinine: 1.67 ± 0.258 mg/dl, p < 0.05), oxidative stress (MDA level: 95.29 ± 37.35 µmol/g tissue, p < 0.05; SOD activity: 59.82 ± 13.41 U/g protein, p < 0.01) and inflammation (renal TNF-α: 57.05 ± 15.653 pg/g tissue, p < 0.05; renal IL-6: 117.015 ± 24.076 pg/g tissue, p < 0.001). The treatment caused a reduction in the amount of renal angiotensinogen, renin and ACE genes expression compared to the UUO group (Angiotensinogen: 8.9 ± onefold, p < 0.05, Renin: 6.5 ± 1.14 fold, p < 0.05, ACE: 4.9 ± 0.64 fold, p < 0.05). Angiotensin II type 1 receptor protein levels decreased in the tannic acid-treated rats in comparison with the UUO group (0.61 ± 0.136, p < 0.05). According to the result of the current study, tannic acid considerably attenuated the complications of unilateral ureteral obstruction through renin-angiotensin system modulation. Trial registration: IR.TUMS.MEDICINE.REC.1400.802., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Despite the availability of various therapeutic classes of antihypertensive drugs, hypertension remains poorly controlled, in part because of poor adherence. Hence, there is a need for the development of antihypertensive drugs acting on new targets to improve control of blood pressure. This review discusses novel insights (including the data of recent clinical trials) with regard to interference with the renin-angiotensin system, focusing on the enzymes aminopeptidase A and angiotensin-converting enzyme 2 (ACE2) in the brain, as well as the substrate of renin- angiotensinogen-in the liver. It raises the possibility that centrally acting amino peptidase A inhibitors (eg, firibastat), preventing the conversion of angiotensin II to angiotensin III in the brain, might be particularly useful in African Americans and patients with obesity. Firibastat additionally upregulates brain ACE2, allowing the conversion of angiotensin II to its protective metabolite angiotensin-(1-7). Furthermore, antisense oligonucleotides or small interfering ribonucleic acids suppress hepatic angiotensinogen for weeks to months after 1 injection and thus could potentially overcome adherence issues. Finally, interference with ACE2 ubiquitination is emerging as a future option for the treatment of neurogenic hypertension, given that ubiquitination resistance might upregulate ACE2 activity., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Obesity is associated with hypertension. However, the mechanisms involved are not fully understood. Therefore, we investigated the relationship between obesity and vasoactive mediators. In this cross-sectional study, blood pressure (BP) and vasoactive mediators of hypertension are compared among 135 adults in the nonobese, obese, and morbidly obese body mass index (BMI) ranges (BMI ≤27, 30-40, and >40 kg/m 2 , respectively). Angiotensinogen, angiotensin II, renin, aldosterone, endothelin-1 (ET-1), neprilysin, atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), cyclic guanosine monophosphate (cGMP), and cyclic adenosine monophosphate (cAMP) levels were measured and their relationship to BP, BMI, race, and gender were investigated. Systolic and diastolic BP (SBP and DSP) were significantly higher in subjects with obesity and morbid obesity compared with nonobese. Angiotensin II, ET-1, and neprilysin were significantly higher in subjects with morbid obesity while BNP was lower. Levels of angiotensinogen, renin, aldosterone, ANP, cGMP, and cAMP did not differ between the groups. BMI was positively related to SBP, DBP, angiotensin II, ET-1, and neprilysin, and inversely related to cGMP and BNP. Age, male gender, and African-American race were associated with higher SBP. SBP was positively related to angiotensin II and ET-1 and inversely related to aldosterone, renin, and cGMP. On multivariate analyses, age, BMI, gender, and race were the main determinants of SBP, and excluding these variables, angiotensin II, aldosterone, renin, and ET-1 accounted for 21.1% ability to predict SBP. Obesity, especially morbid obesity, is associated with higher BP, higher angiotensin II and ET-1 (vasoconstrictors), and lower levels BNP and cGMP (vasodilators). SBP variability can be partly explained by angiotensin II, aldosterone, renin, and ET-1. NEW & NOTEWORTHY Our data show that obesity, especially morbid obesity, is associated with higher blood pressure levels and increases angiotensin II and endotherlin-1 (ET-1) (vasoconstrictors) and lower levels BNP and cGMP (vasodilators) and that systolic blood pressure variability can be partly explained by levels of angiotensin II, aldosterone, renin, and ET-1. The effect of these mediators on blood pressure is in addition to the effects of other known factors related to age, male gender, and AA race.

Myocardial fibrosis, a common pathological manifestation of cardiac remodeling (CR), often leads to heart failure (HF) and even death. The underlying molecular mechanism of the role of TRIM33 in Ang II-induced myocardial fibrosis is not fully understood. We found that TRIM33 was specifically upregulated in CFs and myocardial tissue after Ang II stimulation. Adult mice induced by Ang II were used as in vivo models, and Ang II-induced neonatal mouse primary cardiac fibroblasts (CFs) were used as in vitro models. The level of CF fibrosis in vitro was assessed by CF proliferation, migration, activation and extracellular matrix (ECM) synthesis. In addition, Masson staining, the heart weight/body weight (HW/BW) ratio and echocardiography were used to evaluate the in vivo effect of TRIM33. TRIM33 expression was specifically upregulated in CFs and myocardial tissue after Ang II stimulation. In in vitro experiments, we found that TRIM33 knockdown promoted Ang II-induced CF proliferation, while TRIM33 overexpression weakened Ang II-induced CF proliferation, migration, activation and collagen synthesis. Mechanistically, we showed that TRIM33, negatively regulated by HSPB5, mediated its antifibrotic effect by inhibiting the activation of TGF-β1 and its downstream genes, Smad3 and Smad4. Finally, TRIM33 overexpression suppressed fibrosis and promoted cardiac repair and functional recovery in Ang II-induced mice. Our results clearly establish that TRIM33 limits cardiac fibrosis by hindering CF proliferation, migration, activation and collagen synthesis. Enhancing these beneficial functions of TRIM33 by a targeting vector might be a novel therapeutic strategy for CR.

We engineered a monoclonal antibody (mAb) against the human C-terminus of angiotensin-(1-12) [h-Ang-(1-12)] and performed a biochemical characterization in concert with direct in vivo and ex vivo (carotid artery strips) assessments of h-Ang-(1-12) vasoconstrictor activity in 78 (36 females) transgenic rats expressing the human angiotensinogen gene [TGR(hAGT)L1623] and 26 (10 female) Sprague Dawley (SD) controls. The mAb shows high specificity in neutralizing angiotensin II formation from h-Ang-(1-12) and did not cross-react with human and rat angiotensins. Changes in arterial pressure and heart rate in Inactin® hydrate anesthetized rats were measured before and after h-Ang-(1-12) injections [dose range: 75-300 pmol/kg i.v.] prior to and 30-60 minutes after administration of the h-Ang-(1-12) mAb. Neutralization of circulating Ang-(1-12) inhibited the pressor action of h-Ang-(1-12), prevented Ang-(1-12) constrictor responses in carotid artery rings in both SD and TGR(hAGT)L1623 rats, and caused a fall in the arterial pressure of male and female transgenic rats. The Ang-(1-12) mAb did not affect the response of comparable dose-related pressor responses to Ang II, pre-immune IgG, or the rat sequence of Ang-(1-12). This h-Ang-(1-12) mAb can effectively suppress the pressor actions of the substrate in the circulation of hypertensive rats or in carotid artery strips from both SD and transgenic rats. The demonstration that this Ang-(1-12) mAb by itself, induced a fall in arterial pressure in transgenic hypertensive rats supports further exploring the potential abilities of Ang-(1-12) mAb in the treatment of hypertension., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Renin is the rate-limiting enzyme of the renin-angiotensin system cascade, which drives the pathophysiological progression of heart failure. Species differences in the amino acid sequence of the catalytic domain of renin limit evaluations of the potency and efficacy of human renin inhibitors in animal models, and a high dose of inhibitors is usually needed to show its organ-protective effects in rodents. In the present study, we developed a novel murine heart failure model (triple-tg) to enable us to evaluate the cardioprotective effect of renin inhibitors at more relevant doses for humans, by cross-breeding calsequestrin transgenic (CSQ-tg) mice with human renin and human angiotensinogen double-transgenic mice. The triple-tg mice exhibited increased plasma renin activity, worsened cardiac hypertrophy, and higher mortality compared to CSQ-tg mice. Triple-tg mice treated with 10 mg·kg -1 of TAK-272 (imarikiren/SCO-272), an orally active direct renin inhibitor, exhibited improvements in heart failure phenotypes, such as cardiac hypertrophy and survival rate; however, a dose of 300 mg·kg -1 was required to improve symptoms in CSQ-tg mice. Our results suggest that this newly generated triple-tg heart failure model is useful to evaluate the cardioprotective effects of human renin inhibitors at clinically relevant doses, thereby minimizing the concerns of off-target effects related to much higher drug exposure than that achieved in clinical study., (© 2020 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

Background: Angiotensin-(1-12) [Ang-(1-12)] is a renin-independent precursor for direct angiotensin-II production by chymase. Substantial evidence suggests that heart failure (HF) may alter cardiac Ang-(1-12) expression and activity; this novel Ang-(1-12)/chymase axis may be the main source for angiotensin-II deleterious actions in HF. We hypothesized that HF alters cardiac response to Ang-(1-12). Its stimulation may produce cardiac negative modulation and exacerbate left ventricle (LV) systolic and diastolic dysfunction., Methods and Results: We assessed the effects of Ang-(1-12) (2 nmol/kg/min, iv, 10 min) on LV contractility, LV diastolic filling, and LV-arterial coupling (AVC) in 16 SD male rats with HF-induced by isoproterenol (3 mo after 170 mg/kg sq. for 2 consecutive days) and 10 age-matched male controls. In normal controls, versus baseline, Ang-(1-12) increased LV end-systolic pressure, without altering heart rate, arterial elastance (E A ), LV end-diastolic pressure (P ED ), the time constant of LV relaxation (τ) and ejection fraction (EF). Ang-(1-12) significantly increased the slopes (E ES ) of LV end-systolic pressure (P)-volume (V) relations and the slopes (M SW ) of LV stroke wok-end-diastolic V relations, indicating increased LV contractility. AVC (quantified as E ES /E A ) improved. In contrast, in HF, versus HF baseline, Ang-(1-12) produced a similar increase in P ES , but significantly increased τ, E A , and P ED. The early diastolic portion of LV PV loop was shifted upward with reduced in EF. Moreover, Ang-(1-12) significantly decreased E ES and M SW , demonstrating decreased LV contractility. AVC was decreased by 43%., Conclusions: In both normal and HF rats, Ang-(1-12) causes similar vasoconstriction. In normal, Ang-(1-12) increases LV contractile function. In HF, Ang-(1-12) has adverse effects and depresses LV systolic and diastolic functional performance., (Copyright © 2019. Published by Elsevier B.V.)

Background: The function of prorenin, the inactive precursor of renin, remains unclear after many decades of research. The discovery of a (pro)renin receptor suggested that prorenin, by binding to this receptor, would become active, that is, obtain an 'open' conformation. However, the receptor only interacted with prorenin at levels that were many orders of magnitude above its normal levels, making such interaction in-vivo unlikely. Prorenin occurs in two conformations, an open, active form, and a closed, inactive form. Under physiological conditions (pH 7.4, 37 °C), virtually all prorenin occurs in the closed conformation. This study investigated to what degree prorenin-synthesizing cells release prorenin in an open conformation., Methods and Results: Renin plus prorenin-synthesizing human mast cells, and prorenin-synthesizing HEK293 cells (transfected with the mammalian expression vector pRhR1100, containing human prorenin) and human decidua cells were incubated with the renin inhibitor VTP-27999. This inhibitor will trap open prorenin, as after VTP-27999 binding, prorenin can no longer return to its closed conformation, thus allowing its detection in a renin immunoradiometric assay. No evidence for the release of open prorenin was found. Moreover, incubating decidua cells with angiotensinogen yielded low angiotensin levels, corresponding with the activity of ≈1% of prorenin in the medium, that is, the amount of open prorenin expected based upon the equilibrium between open and closed prorenin under physiological conditions., Conclusion: Our study does not reveal evidence for the release of open, active prorenin by prorenin-synthesizing cells, at least under cell culture conditions. This argues against prorenin activity at the site of its release.

Angiotensin-(1-12) [Ang-(1-12)], a newer member of angiotensin peptides, is proposed to be converted enzymatically to angiotensin I (Ang I) and to angiotensin II (Ang II); the latter being the bioactive peptide. We studied the Ang-(1-12) and Ang II responses in COS-7 cells or CHO cells transfected with 5 μg AT1R by monitoring [Ca(2+)]i using the Fluo-4. Ang II (1 pM-1 μM) and Ang-(1-12) (5 pM-5 μM) increased [Ca(2+)]i with an EC50 of 0.19 nM and 24 nM in COS-7 cells; and 0.65 nM and 28.7 nM in CHO cells. The AT1R antagonist losartan (1 nM-10 μM) suppressed [Ca(2+)]i induced by Ang-(1-12) and Ang II. In CHO cells transfected with 5 μg AT2R, Ang II (1 pM-1 μM) increased [Ca(2+)]i, with an EC50 of 9.68 nM; whereas, Ang-(1-12) (5 pM-5 μM) failed to elicit a significant change in [Ca(2+)]i. In CHO cells transfected with AT1R, Ang-(1-12) stimulated ERK phosphorylation with a potency 300-fold less than that of Ang II. To evaluate the activity of Ang-(1-12) on native AT1R, whole cell patch recordings were made from neurons in the rat hypothalamic slices. Ang II or Ang-(1-12) ejected by pressure from a micropipette elicited a membrane depolarization; the latter was blocked by losartan (10 μM), and not affected by the AT2R antagonist PD123319 (10 μM), nor by the angiotensin converting enzyme inhibitor captopril (10 μM). Our result shows that Ang-(1-12) may produce its biological activity by acting directly on AT1R, albeit at a concentration higher than that of Ang II., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Angiotensin (ANG)-(1-12) excites neurons via ANG II type 1 receptors (AT1Rs), which are present in the caudal ventrolateral medullary depressor area (CVLM). We hypothesized that microinjections of ANG-(1-12) into the CVLM may elicit decreases in mean arterial pressure (MAP), heart rate (HR), and sympathetic nerve activity. This hypothesis was tested in urethane-anesthetized adult male Wistar rats. Microinjections of ANG-(1-12) into the CVLM elicited decreases in MAP, HR, and greater splanchnic nerve activity (GSNA). ANG-(1-12)-induced responses consisted of initial (first 1-8 min) and delayed (8-24 min) phases. Prior microinjections of losartan, A-779, and captopril into the CVLM blocked initial, delayed, and both phases of ANG-(1-12) responses, respectively. Blockade of GABA receptors in the rostral ventrolateral medullary pressor area (RVLM) attenuated cardiovascular responses elicited by microinjections of ANG-(1-12) into the ipsilateral CVLM. Microinjections of ANG-(1-12) into the CVLM potentiated the reflex decreases and attenuated the reflex increases in GSNA elicited by intravenous injections of phenylephrine and sodium nitroprusside, respectively. These results indicate that microinjections of ANG-(1-12) into the CVLM elicit decreases in MAP, HR, and GSNA. Initial and delayed phases of these responses are mediated via ANG II and ANG-(1-7), respectively; the effects of ANG II and ANG-(1-7) are mediated via AT1Rs and Mas receptors, respectively. Captopril blocked both phases of ANG-(1-12) responses, indicating that angiotensin-converting enzyme is important in mediating these responses. GABA receptors in the RVLM partly mediate the cardiovascular responses to microinjections of ANG-(1-12) into the CVLM. Microinjections of ANG-(1-12) into the CVLM modulate baroreflex responses.

Objective/methods: Cushing's disease (CD) is often accompanied by hypertension. CD can be treated surgically and, given the expression of somatostatin subtype 5 and dopamine 2 receptors by corticotroph pituitary adenomas, pharmacologically. Indeed, we recently observed that stepwise medical combination therapy with the somatostatin-analog pasireotide, the dopamine-agonist cabergoline, and ketoconazole (which directly suppresses steroidogenesis) biochemically controlled CD patients and lowered their blood pressure after 80 days. Glucocorticoids (GC) modulate the renin-angiotensin-aldosterone system (RAAS) among others by increasing hepatic angiotensinogen expression and stimulating mineralocorticoid receptors (MR). This study therefore evaluated plasma RAAS components in CD patients before and after drug therapy. In addition, we studied whether cabergoline/pasireotide have direct relaxant effects in angiotensin II (Ang II)-constricted iliac arteries of spontaneously hypertensive rats, with and without concomitant GR/MR stimulation with dexamethasone or hydrocortisone., Results: Baseline concentrations of angiotensinogen were elevated, while renin and aldosterone were low and suppressed, respectively, even in patients treated with RAAS-blockers. This pattern did not change after 80 days of treatment, despite blood pressure normalization, nor after 4 years of remission. In the presence of dexamethasone, pasireotide inhibited Ang II-mediated vasoconstriction., Conclusions: The low plasma renin concentrations, even under RAAS blockade, in CD may be the consequence of increased GC-mediated MR stimulation and/or the elevated angiotensinogen levels in such patients. The lack of change in RAAS-parameters despite blood pressure and cortisol normalization suggests persisting consequences of long-term exposure to cortisol excess. Finally, pasireotide may have a direct vasodilating effect contributing to blood pressure lowering.

Angiotensin-(1-12) [ANG-(1-12)], a new member of the renin-angiotensin system, is recognized as a renin independent precursor for ANG II. However, the processing of ANG-(1-12) in the circulation in vivo is not fully established. We examined the effect of angiotensin converting enzyme (ACE) and chymase inhibition on angiotensin peptides formation during an intravenous infusion of ANG-(1-12) in normotensive Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). WKY and SHR were assigned to a short ANG-(1-12) infusion lasting 5, 15, 30, or 60 min (n = 4-10 each group). In another experiment WKY and SHR were assigned to a continuous 15-min ANG-(1-12) infusion with pretreatment of saline, lisinopril (10 mg/kg), or chymostatin (10 mg/kg) (n = 7-13 each group). Saline or lisinopril were infused intravenously 15 min before the administration of ANG-(1-12) (2 nmol·kg(-1)·min(-1)), whereas chymostatin was given by bolus intraperitoneal injection 30 min before ANG-(1-12). Infusion of ANG-(1-12) increased arterial pressure and plasma ANG-(1-12), ANG I, ANG II, and ANG-(1-7) levels in WKY and SHR. Pretreatment with lisinopril caused increase in ANG-(1-12) and ANG I and large decreases in ANG II compared with the other two groups in both strains. Pretreatment of chymostatin had no effect on ANG-(1-12), ANG I, and ANG II levels in both strains, whereas it increased ANG-(1-7) levels in WKY. We conclude that ACE acts as the primary enzyme for the conversion of ANG-(1-12) to smaller angiotensin peptides in the circulation of WKY and SHR and that chymase may be an ANG-(1-7) degrading enzyme.

The rostral ventrolateral medullary pressor area (RVLM) is known to be critical in the regulation of cardiovascular function. In this study, it was hypothesized that the RVLM may be one of the sites of cardiovascular actions of a newly discovered angiotensin, angiotensin-(1-12) [Ang-(1-12)]. Experiments were carried out in urethane-anaesthetized, artificially ventilated, adult male Wistar rats. The RVLM was identified by microinjections of L-glutamate (5 mM). The volume of all microinjections into the RVLM was 100 nl. Microinjections of Ang-(1-12) (0.1-1.0 mM) into the RVLM elicited increases in mean arterial pressure and heart rate. Maximal cardiovascular responses were elicited by 0.5 mM Ang-(1-12); this concentration was used in the other experiments described. Microinjections of Ang-(1-12) increased greater splanchnic nerve activity. The tachycardic responses to Ang-(1-12) were not altered by bilateral vagotomy. The cardiovascular responses elicited by Ang-(1-12) were attenuated by microinjections of an angiotensin II type 1 receptor (AT(1)R) antagonist (losartan), but not an AT(2)R antagonist (PD123319), into the RVLM. Combined inhibition of angiotensin-converting enzyme and chymase in the RVLM abolished Ang-(1-12)-induced responses. Angiotensin-(1-12)-immunoreactive cells were present in the RVLM. Angiotensin II type 1 receptors and phenylethanolamine-N-methyl-transferase were present in the RVLM neurons retrogradely labelled by microinjections of Fluoro-Gold into the intermediolateral cell column of the thoracic spinal cord. Angiotensin-(1-12)-containing neurons in the hypothalamic paraventricular nucleus did not project to the RVLM. These results indicated that: (1) microinjections of Ang-(1-12) into the RVLM elicited increases in mean arterial pressure, heart rate and greater splanchnic nerve activity; (2) both angiotensin-converting enzyme and chymase were needed to convert Ang-(1-12) into angiotensin II; and (3) AT(1)Rs, but not AT(2)Rs, in the RVLM mediated the Ang-(1-12)-induced responses.

Several studies have focused on the beneficial effects of peripheral angiotensin-(1-7) [Ang-(1-7)] in the regulation of cardiovascular function, showing its counterregulatory effect against the actions of angiotensin II (ANG II). However, its actions in the central nervous system are not completely understood. In the present study, we investigated the intracellular mechanisms underlying the action of ANG-(1-7) using the patch-clamp technique in neurons cultured from the hypothalamus of neonatal spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats. Superfusion of neurons with ANG II (100 nM) significantly increased neuronal firing in both strains of rats, and this chronotropic effect of ANG II was significantly enhanced in prehypertensive SHR neurons compared with WKY rat neurons. The enhanced chronotropic effect of ANG II was attenuated by a phosphatidylinositol 3-kinase (PI3-kinase) inhibitor, LY 294002 (10 μM). Superfusion of neurons with ANG-(1-7) (100 nM) did not alter the neuronal firing rate in either SHR or WKY neurons; however, it significantly attenuated the chronotropic action of ANG II exclusively in prehypertensive SHR neurons. This counterregulatory effect of ANG-(1-7) on ANG II action in prehypertensive SHR neurons was attenuated by cotreatment with either A-779, a Mas receptor antagonist, or bisperoxovanadium, a phosphatase and tensin homologue deleted on chromosome ten (PTEN) inhibitor. In addition, incubation of WKY and prehypertensive SHR neurons with ANG-(1-7) significantly increased PTEN activity. The data demonstrate that ANG-(1-7) counterregulates the chronotropic action of ANG II via a PTEN-dependent signaling pathway in prehypertensive SHR neurons.

ProAngiotensin-12 (PA12) is the most recent peptide to be identified as a functional component of the renin-angiotensin system (RAS). PA12 is reported to constrict rat coronary arteries and the aorta, dependent upon angiotensin II-converting enzyme 1 (ACE1) and chymase. The current study employed myography to determine the direct vascular effects of PA12 on a range of isolated rat arteries extending from the core to periphery. PA12 significantly constricted the descending thoracic aorta, right and left common carotid arteries, abdominal aorta and superior mesenteric artery, with little effect on the femoral and renal arteries. AngII was found to produce similar responses to PA12 when administered at the same dose. A potency gradient in response to PA12 was clearly apparent, with vessels in closest proximity to the heart responding with the greatest constriction; while constrictive potency was lost further form the heart. Inhibition of ACE1 and chymase both significantly attenuated PA12-induced vasoconstriction, with chymostatin displaying lesser potency. We postulate ACE1 primarily regulates RAS activity within the circulation, while chymase may have an important role in local, tissue-based RAS activity., (Copyright 2010 Elsevier Inc. All rights reserved.)

Cardiac mast cells (MC) are apposed to capillaries within the heart and release renin and proteases capable of metabolizing angiotensins (Ang). Therefore, we hypothesized that mast cell degranulation could alter the rat coronary vascular responsiveness to the arterial delivered Ang I and Ang II, taking into account carboxypeptidase and chymase-1 activities. Hearts from animals that were either pretreated or not with systemic injection of the secretagogue compound 48/80 were isolated and mounted on a Langendorff apparatus to investigate coronary reactivity. The proteolytic activity of the cardiac perfusate from isolated hearts, pretreated or not with the secretagogue, toward Ang I and tetradecapeptide renin substrate was analyzed by HPLC. Coronary vascular reactivity to peptides was not affected by compound 48/80 pretreatment, despite the extensive amount of cardiac MC degranulation. Cardiac MC activation did not modify the generation of both Ang II and Ang 5-10 from Ang I by cardiac perfusate, activities that could be ascribed to MC carboxypeptidase and chymase-1, respectively. An aliskiren-resistant Ang I-forming activity was increased in perfusates from secretagogue-treated hearts. Thus, cardiac MC proteases capable of metabolizing angiotensins do not affect rat coronary reactivity to arterial delivered Ang I and II., (Copyright 2010 Elsevier Inc. All rights reserved.)

There is compelling evidence to indicate an important role for increased local renin-angiotensin system activity in the pathogenesis of cardiac hypertrophy and heart failure. Resveratrol is a natural polyphenol that activates SIRT1, a novel cardioprotective and longevity factor having NAD(+)-dependent histone deacetylase activity. We tested the hypothesis whether resveratrol could prevent from angiotensin II (Ang II)-induced cardiovascular damage. Four-week-old double transgenic rats harboring human renin and human angiotensinogen genes (dTGR) were treated for 4 weeks either with SIRT1 activator resveratrol or SIRT1 inhibitor nicotinamide. Untreated dTGR and their normotensive Sprague-Dawley control rats (SD) received vehicle. Untreated dTGR developed severe hypertension as well as cardiac hypertrophy, and showed pronounced cardiovascular mortality compared with normotensive SD rats. Resveratrol slightly but significantly decreased blood pressure, ameliorated cardiac hypertrophy and prevented completely Ang II-induced mortality, whereas nicotinamide increased blood pressure without significantly influencing cardiac hypertrophy or survival. Resveratrol decreased cardiac ANP mRNA expression and induced cardiac mRNA expressions of mitochondrial biogenesis markers peroxisome proliferator-activated receptor-gamma coactivator (PGC-1alpha), mitochondrial transcription factor (Tfam), nuclear respiratory factor 1 (NRF-1) and cytochrome c oxidase subunit 4 (cox4). Resveratrol dose-dependently increased SIRT1 activity in vitro. Our findings suggest that the beneficial effects of SIRT1 activator resveratrol on Ang II-induced cardiac remodeling are mediated by blood pressure-dependent pathways and are linked to increased mitochondrial biogenesis.

Objective: In order to get insight into possible genetic determinants of antihypertensive drug action, we analysed the relations between polymorphisms of the genes of the renin-angiotensin-aldosterone system and acute effects of ACE inhibition on blood pressure as well as circulating renin and aldosterone levels in hypertensive patients., Methods: A total of 315 hypertensive patients referred for problems in drug treatment were given a single 50 mg dose of captopril. Plasma renin and aldosterone were measured before and 60 min after the drug administration. Four DNA variants, including angiotensin type I receptor (AGTR1) 1166 A/C, angiotensin-converting enzyme (ACE) I/D, angiotensinogen (AGT) M235T and AGT -217 G/A, were genotyped in the patients and normotensive men (n = 175). A replication study on the relation between AGTR1 1166 A/C and plasma renin and aldosterone levels was carried out in the 244 hypertensive men of the pharmacogenetic GENRES Study., Results: Referred hypertensive patients with the AGTR1 CC genotype had higher aldosterone at baseline (P = 0.02) and after 60 min of captopril administration (P = 0.01) compared with the AA genotype. Replicate analysis in the GENRES patients showed a similar trend. When the two studies were combined (315 and 244 patients, respectively), plasma aldosterone level (P = 0.007) as well as aldosterone/renin ratio (P = 0.04) were significantly higher in the CC genotype (n = 13) than in the AA genotype (n = 370). Transfection studies in cultured HEK293 cells indicated that the 1166C allele was associated with higher mRNA levels than the 1166A allele., Conclusion: The AGTR1 1166C allele when present in homozygous form may be associated with a form of essential hypertension characterized by high plasma aldosterone and low plasma renin levels, possibly due to increased AGTR1 mRNA levels and augmented angiotensin II action.

Unlabelled: To investigate the influence of prolonged exposure of cardiac cells to renin plus angiotensinogen (Ao) on intracellular renin levels, myocytes were isolated from the ventricle of cardiomyopathic hamsters(TO-2) and incubated in Krebs solution containing renin(128 pmol Ang ml/min) plus Ao (110 pmol Ang I generated by renin to exhaustion) for a period of 24 h. Membrane-bound and intracellular AT1 receptors levels as well as intracellular renin were studied using immunological methods and quantified by flow cytometry. The results indicated: a) intracellular renin levels were higher in the failing heart at an advanced stage of the disease (8 months) than in age-matched controls; b) the intracellular renin levels were significantly reduced in cells exposed to renin (128 pmol Ang I.ml/min) plus angiotensinogen (Ao)(110 pmol Ang I generated by renin to exhaustion) for a period of 24 h; c) incubation of the cardiomyocytes with renin (128 pmol Ang I.ml/min) alone did not reduced the intracellular renin levels; d) the fall of the intracellular renin level was related to the formation of angiotensin II (Ang II) at the surface cell membrane and internalization of the Ang II-AT1 complex because losartan (10(-7) M) added to the incubation medium containing renin plus Ao, blocked the internalization of AT1 and suppressed the decline of the intracellular renin levels; e) no internalization of renin or renin secretion was found in these experiments., In Conclusion: prolonged exposure of cardiac cells to renin plus Ao (24 h) reduced intracellular renin levels through the internalization of Ang II-AT1 complex and inhibition of renin expression.

Unlabelled: The influence of intracellular renin plus angiotensinogen (Ao) as well as angiotensin (Ang) II on cell volume was investigated in myocytes isolated from the heart of four-month-old cardiomyopathic hamsters (TO-2) and normal hamsters (F1B). Measurements of cell width and cell length were performed on quiescent cells using a Px-it imaging and computer system. The cell volume was calculated assuming the cells as elliptical cylinders and taking the cell depth equal to one third of cell width. For measurements of sodium pump current, the cells were voltage clamped (holding potential -40 mV) using the whole cell configuration. Cells were exposed to K-free solution to inhibit the pump and then to normal Krebs solution to reactivate the pump. In other experiments the cells were voltage clamped (holding potential -40 mV) and changes in the background current elicited by renin plus Ao or by Ang II were monitored. The results indicated that: a) intracellular dialysis of renin (128 pmol Ang I/ml) plus Ao (110 pmol Ang I generated by renin by exhaustion) decreased the cell volume concurrently with the activation of the sodium pump; b) intracellular losartan (10(-)8 M) or extracellular ouabain (10(-8) M) abolished the effect of renin plus Ao on cell volume; c) intracellular Ang II (10(-8) M), by itself, reduced cell volume and increased the pump current density; d) extracellular administration of renin plus Ao, at the same concentration used intracellularly, increased cell volume and inhibited the sodium pump. This increase of cell volume elicited by extracellular renin plus Ao was related to the activation of the Na-K-2Cl cotransporter; e) intracellular Ang II (10(-8) M) reversed cell swelling induced by hypotonic solutions., Conclusions: Intracellular and extracellular renin plus Ao have opposite effects on sodium pump and cell volume regulation in the failing heart. Both effects of renin plus Ao are dependent upon the formation of Ang II. Since intracellular Ang II counteracted the cell swelling induced by hypotonic solution, it is reasonable to think that the activation of the intracrine renin-angiotensin system might play a protective role during myocardial ischaemia by reducing cell volume.

Objective: Mannose 6-phosphate receptors (M6PR) bind both renin and prorenin, and such binding contributes to renin/prorenin clearance but not to angiotensin generation. Here, we evaluated the kinetics of renin/prorenin binding to the recently discovered human (pro)renin receptor (h(P)RR), and the idea that such binding underlies tissue angiotensin generation., Methods and Results: Vascular smooth muscle cells from control rats and transgenic rats with smooth muscle h(P)RR overexpression were incubated at 4 or 37 degrees C with human renin or prorenin. Incubation at 37 degrees C greatly increased binding, suggesting that (pro)renin-binding receptors cycle between the intracellular compartment and the cell surface. Blockade of the M6PR reduced binding by approximately 50%. During M6PR blockade, h(P)RR cells bound twice as much prorenin as control cells, while renin binding was unaltered. Incubation of h(P)RR (but not control) cells with prorenin + angiotensinogen yielded more angiotensin than expected on the basis of the activity of soluble prorenin, whereas angiotensin generation during incubation of both cell types with renin + angiotensinogen was entirely due to soluble renin. The renin + angiotensinogen-induced vasoconstriction of isolated iliac arteries from control and transgenic rats was also due to soluble renin only. The recently proposed (P)RR antagonist 'handle region peptide', which resembles part of the prosegment, blocked neither prorenin binding nor angiotensin generation., Conclusions: H(P)RRs preferentially bind prorenin, and such binding results in angiotensin generation, most likely because binding results in prorenin activation.

The contractile effects of angiotensinogen (Aogen) and its metabolization pathways were studied on rat renal vein (RRV), rat pulmonary artery (RPA) and human umbilical vein (HUV) rings. Experiments were made in the presence or in the absence of pepstatin A (a renin inhibitor, 10 microM), captopril (an ACE inhibitor, 10 microM), chymostatin (a chymase inhibitor, 10 microM), amastatin (an aminopeptidase-A and -M inhibitor) or losartan (a specific AT1 blocker, 10 microM). On all rings, Aogen-induced contractions were reduced by pepstatin A or captopril, amplified by amastatin and blocked by losartan. Chymostatin had a stronger inhibitory effect than captopril on HUV and simultaneous administering of chymostatin and captopril prevented Aogen contractile effects on HUV. It is suggested that all studied vessels possess a local renin-angiotensin system and possibility of angiotensin II production within the vessel walls using various and species-dependent enzymatic pathways.

Angiotensinogen shares with other members of the serine protease inhibitor (serpin) family antiangiogenic properties. Angiotensinogen inhibits in vitro endothelial cell proliferation, and is antiangiogenic in ovo in the chick chorioallantoic membrane assay. The cellular mode of action of angiotensinogen has been studied by applying purified human angiotensinogen or Chinese hamster ovary cells producing recombinant angiotensinogen onto the developing chorioallantoic membrane. Vessel density of the control and angiotensinogen-treated areas was quantitated by using Sambucus nigra lectin, a specific endothelial cell marker. After 48 h of angiotensinogen treatment by either applying purified angiotensinogen or angiotensinogen-producing Chinese hamster ovary cells, there was a 70% decrease in mesodermic vessel density in comparison to the control sections. Angiotensinogen treatment induced a strong decrease in endothelial cell proliferation of the chorioallantoic membrane vasculature, as shown by incorporation of bromo-deoxyuridine. Two days after local angiotensinogen treatment, increased apoptosis of endothelial cells of mesodermal blood vessels was detected by transferase-mediated deoxyuridine triphosphate nick end labeling assay. As assessed by in situ hybridization, the gene expression pattern of the main vascular growth factors and their receptors was not altered by angiotensinogen. Angiotensinogen, therefore, impairs angiogenesis without altering the expression level of vascular growth factors through the induction of apoptosis and decreased endothelial cell proliferation.

Hyperactivation of systemic renin-angiotensin system (RAS) during sepsis is well documented. However, the behavior of intrarenal RAS in the context of endotoxemia is yet to be defined. The present study evaluates the direct effect of Escherichia coli lipopolysaccharide (LPS) on immortalized human mesangial cell (HMC) RAS. Quiescent HMC were incubated with vehicle or LPS (1-100 microg/ml), and levels of angiotensin I and II (Ang I and II) and their metabolites were analyzed by high-performance liquid chromatography. In addition, angiotensin-converting enzyme (ACE) and renin activity were also investigated. Cell lysate and extracellular medium levels of Ang II were rapidly reduced (1 h) in a time- and concentration-dependent manner, reaching a significant -9 fold-change (P<0.001) after 3 h of LPS incubation. Similar results were obtained for Ang I levels (-3 fold-change, P<0.001). We ruled out Ang I and II degradation, as levels of their metabolic fragments were also significantly decreased by LPS. ACE activity was slightly increased following LPS incubation. On the other hand, renin activity was significantly inhibited, as Ang I concentration elevation following exogenous angiotensinogen administration was blunted by LPS (-60% vs vehicle, P<0.001). Renin and angiotensinogen protein levels were not affected by LPS according to Western blot analysis. Taken together, these data demonstrate for the first time that LPS significantly downregulates HMC RAS through inhibition of renin or renin-like activity. These findings are potentially related to the development of and/or recovery from acute renal failure in the context of sepsis.

This article demonstrates the applicability of quantitative proteomics to assays of proteolytic enzyme activity. A novel assay was developed for measurement of renin and angiotensin-converting enzyme (ACE) activity in plasma. The method was validated in animal models associated with alterations of the renin angiotensin system (RAS). Using surface-enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF-MS) with a ProteinChip Array technology, plasma renin and ACE1 could be measured in <0.5 microL of plasma. Plasma is incubated with peptide substrates for renin and ACE, tetradecapeptide (TDP), and angiotensin I (Ang I), respectively. The reactions mixtures are spotted onto the ProteinChip WCX2 and detected using SELDI-TOF-MS. Peak height or area under curve for TDP, Ang I, and angiotensin II (Ang II) peaks are measured. There was a linear relationship between disappearance of substrate and appearance of products for both renin and ACE (R2=0.95 to 0.98). ACE1 activity was blocked with chelating agents (EDTA and 1,10 phenanthrolene), indicating action of a metalloprotease. The ACE1 inhibitor, captopril, selectively blocked ACE1. Renin activity was specifically blocked with renin inhibitor and was not affected by phenanthrolene or captopril. Animal models tested were Ang AT1a receptor-deficient and streptozotocin (STZ) diabetic mice. Plasma renin activity was increased >2-fold in AT1a(-/-) as compared with AT1a(+/+). In STZ diabetic mice, ACE1 was increased 2-fold as compared with controls. The advantage of the method is that it is tagless, does not require additional purification steps, and is extremely sensitive. The approach can be multiplexed and used for identification of novel substrates/inhibitors of the RAS.

The present experiments were performed to determine the influence of intravenous administration of renin substrate on plasma angiotensin II levels and mean arterial blood pressure in conscious C57BL/6J mice. Mice with chronic indwelling femoral arterial and venous catheters were acutely or chronically administered intravenous doses of a synthetic peptide corresponding to the 14 amino acids on the N-terminal of angiotensinogen. A dose-dependent increase in arterial blood pressure was observed as the intravenous bolus dose of the renin substrate was increased from 0.18 to 180 nmol kg(-1) with a maximal increase in pressure of 40 +/- 3 mmHg achieved following administration of the 18 nmol kg(-1) bolus (n = 11). Additional experiments demonstrated that a sustained intravenous infusion of the renin substrate led to a long-term increase in arterial blood pressure. The continuous infusion of renin substrate at 0.05 nmol kg(-1) min(-1) for 3 days did not alter arterial blood pressure from the control level of 119 +/- 5 mmHg (n = 5); however, arterial blood pressure significantly increased to 129 +/- 6 mmHg with an infusion rate of 0.5 nmol kg(-1) min(-1) and further increased to 141 +/- 3 mmHg when the renin substrate infusion was increased to 5.0 nmol kg(-1) min(-1). Finally, the infusion of renin substrate at 5.0 nmol kg(-1) min(-1) resulted in a significant increase in plasma angiotensin II concentration from 34 +/- 6 pg ml(-1) in vehicle-infused mice to 288 +/- 109 pg ml(-1). These results demonstrate that modulation of the circulating level of angiotensinogen can alter the plasma angiotensin II level and arterial blood pressure in normal animals.

The intracerebroventricular administration of renin substrate or angiotensin II evokes antinociception in rodents, but the brain sites where most of the renin-angiotensin system peptides act are not yet known. This study describes the antinociceptive effects of microinjecting porcine renin substrate tetradecapeptide (RS) or angiotensins I (AI), II (AII) or III (AIII) into different regions of the periaqueductal gray matter (PAG), using the rat tail flick test. All the above peptides were effective following administration into several PAG regions. Their antinociceptive effects were strongly evoked from the caudal ventrolateral and ventral PAG, including the dorsal raphe nucleus. A dose-dependent antinociception following administration into the ventrolateral PAG was demonstrated for all peptides studied. The effect of AII from the ventrolateral PAG was inhibited by the previous local administration of saralasin, a non-selective angiotensin receptor antagonist. Moreover, the peak effects of RS and AI occurred later than those of AII and AIII. The time-course of antinociception suggests that longer-chain peptides are locally processed to biologically active smaller-chain peptides. This study shows for the first time the antinociceptive effect of RS, AI, AII and III in well-defined PAG regions, an effect that is receptor mediated for AII.

Angiotensin II (AngII) infusions augment renal angiotensinogen mRNA and protein and urinary angiotensinogen excretion (U(AGT)). Further experiments were performed in 4 groups of rats: normal salt diet with sham operation, NS+Sham, n=6; NS with AngII infusion at 40 ng/min via osmotic minipump, NS+AngII(40), n=9; NS with AngII infusion at 80 ng/min, NS+AngII(80), n=9; high-salt diet with deoxycorticosterone acetate salt pellet (100 mg), HS+DOCA, n=4. These experiments sought to determine whether enhanced U(AGT) is specifically associated with increased kidney AngII levels or is a nonspecific consequence of the hypertension. Systolic BP (SBP) was significantly increased to 131+/-2 and 162+/-2 mm Hg at day 11 in NS+AngII(40) and NS+AngII(80), respectively, compared with NS+Sham (110+/-1). Regression analysis demonstrated a positive relationship (R=0.49) between SBP and U(AGT) for NS+Sham (1.1+/-0.3 nmol AngI/d), NS+AngII(40) (2.5+/-0.9), and NS+AngII(80) (5.5+/-1.5). U(AGT) was also highly correlated (R=0.70) with kidney AngII content for NS+Sham (49+/-6 fmol/g), NS+AngII(40) (215+/-49), and NS+AngII(80) (347+/-47); but not with plasma AngII (R=0.12). HS+DOCA rats also exhibited increased SBP to 134+/-1 mm Hg, but U(AGT) (1.4+/-0.4 nmol AngI/d) and intrarenal AngII content (13+/-2 fmol/g) were not increased despite the hypertension. Infused human angiotensinogen could not be detected in urine of sham-operated or AngII-infused rats (n=4 each). These data demonstrate that U(AGT) increases in AngII-dependent hypertension in a dose- and time-dependent manner, but not in hypertension elicited by HS+DOCA. The results support the hypothesis that AngII-dependent hypertension results in elevated intrarenal AngII and angiotensinogen levels, reflected by increased U(AGT), which does not occur in an AngII-independent hypertensive model.

Background: Current evidence indicates that there may be a tissue-specific renin-angiotensin system (RAS) in the human placenta. To better define the placental RAS, this study sought to determine whether placental derived cells possessed a receptor for angiotensinogen (AGT), a rate limiting component of the RAS., Methods: A human placenta-derived cell line, CRL-7548, a highly purified AGT and iodine-125-labeled angiotensinogen ((125)I-AGT) were used in this study. The cells were seeded in 35-mm diameter plastic wells, cultured for 2 days in fetal bovine serum-Dulbecco's modified Eagle's medium to reach about 80% to 90% confluence, 2 x 10(4) cells/well and passed in Dulbecco's modified Eagle's medium free of fetal bovine serum before experimentation. A quantity of 3 microg of (125)I-AGT was added to each well at zero time., Results: The cells rapidly bound (125)I-AGT in a time dependent manner with saturation being achieved in 2 to 4 h. This binding was competitively inhibited by unlabeled AGT. Prior addition of a 100-fold excess of unlabeled AGT resulted in a 54% decrease in maximal binding. Bound AGT was also competitively displaced by AGT. Addition of a 200-fold excess of unlabeled AGT displaced 70% of the bound (125)I-AGT. Thus, approximately 30% of the total binding can be attributed to nonspecific binding. An acid wash (0.2 mol/L acetic acid, 0.5 mol/L NaCl, pH 2.5) at 4 degrees C removed 46% of the bound (125)I-AGT. An acid wash is known to dissociate cell surface ligand-receptor complexes, but does not remove internalized ligand., Conclusion: The results of this study provide evidence for the existence of an AGT receptor on placenta-derived cells. This is the first report of an AGT receptor.

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.

Recent studies suggest that angiotensin II (Ang II) plays a role in the adipogenesis of murine preadipocytes. Here, we examined the role of Ang II for the differentiation of primary cultured human preadipocytes. Preadipocytes were isolated from human adipose tissue and stimulated to differentiate. Quantitation of gene expression during adipogenesis was performed for renin-angiotensin system (RAS) genes. The influence of the RAS on adipogenic differentiation was investigated by addition of either angiotensinogen (AGT), Ang II, or angiotensin receptor antagonists to the differentiation medium. We also examined the influence of adipocytes on adipogenesis by co-culture experiments. Expression of the RAS genes AGT, renin, angiotensin-converting enzyme, and Ang II type 1 receptor increased during adipogenesis. Stimulation of the Ang II type 1 receptor by Ang II reduced adipose conversion, whereas blockade of this receptor markedly enhanced adipogenesis. Adipocytes were able to inhibit preadipocyte differentiation in the co-culture, and this effect was abolished by blockade of the Ang II type 1 receptor. This finding points to a functional role of the RAS in the differentiation of human adipose tissue. Because AGT secretion and Ang II generation are characteristic features of adipogenesis, we postulate a paracrine negative-feedback loop that inhibits further recruitment of preadipocytes by maturing adipocytes.

The members of the serine protease inhibitor (serpin) family, which share a common tertiary structure and a role as serin protease inhibitors, are involved in a variety of newly discovered functions. For example, antithrombin III exerts a strong antiangiogenic activity. Angiotensinogen, the renin substrate, has a folded structure and is a member of the noninhibitory serpin subfamily. Two other noninhibitory serpins, maspin and pigment epithelium-derived factor, have antiangiogenic properties. We investigated the antiangiogenic effect of angiotensinogen and 2 related compounds: (1) des(angiotensin I)angiotensinogen, the product of angiotensinogen cleavage by renin, and (2) the reactive center loop-cleaved angiotensinogen, which is produced after selective and limited proteolysis by the protease V8. We used well-established in vitro (endothelial cell proliferation and migration, and capillary-like tube formation on Matrigel) and in vivo (the chick chorioallantoic membrane assay) models of angiogenesis to evaluate the antiangiogenic activities of these 3 related molecules. Our data demonstrated that these compounds exerted a clear and equipotent antiangiogenic effect, thus attributing a novel function to angiotensinogen and des(angiotensin I)angiotensinogen, for which no function was previously known.

Hepatic angiotensinogen secretion is controlled by a complex pattern of physiological or pathophysiological mediators. Because plasma concentrations of angiotensinogen are close to the Michaelis-Menten constant, it was hypothesized that changes in circulating angiotensinogen affect the formation rate of ANG I and ANG II and, therefore, blood pressure. To further test this hypothesis, we injected purified rat angiotensinogen intravenously in Sprague-Dawley rats via the femoral vein and measured mean arterial blood pressure after arterial catheterization. In controls, mean arterial pressure was 131 +/- 2 mmHg before and after the injection of vehicle (sterile saline). The injection of 0.8, 1.2, and 2.9 mg/kg angiotensinogen caused a dose-dependent increase in mean arterial blood pressure of 8 +/- 0.4, 19.3 +/- 2.1, and 32 +/- 2.4 mmHg, respectively. In contrast, the injection of a purified rabbit anti-rat angiotensinogen antibody (1.4 mg/kg) resulted in a significant decrease in mean arterial pressure (-33 +/- 3.2 mmHg). Plasma angiotensinogen increased to 769 +/- 32, 953 +/- 42, and 1,289 +/- 79 pmol/ml, respectively, after substrate and decreased by 361 +/- 28 pmol/ml after antibody administration. Alterations in plasma angiotensinogen correlated well with changes in plasma renin activity. In summary, variations in circulating angiotensinogen can result in changes in blood pressure. In contrast to renin, which is known as a tonic regulator for the generation of ANG I, angiotensinogen may be a factor rather important for long-term control of the basal activity of the renin-angiotensin system.

Unlabelled: Estradiol-intranasal is a nasal spray formulation containing an aqueous solution of 17beta-estradiol that has a unique pulse-like pharmacokinetic profile. In a well designed, placebo-controlled trial estradiol-intranasal 200 to 400 microg/day significantly reduced the incidence and severity of climacteric symptoms in women with moderate to severe menopausal symptoms after 4 and 12 weeks' treatment. The efficacy of estradiol-intranasal 300 microg/day was similar to that of oral estradiol 2 mg/day in this and another double-blind placebo-controlled trial. This equivalent efficacy was maintained in a subgroup of women with initially severe symptoms, and in smokers. Reductions in the incidence of atrophic vaginal mucosa and genitourinary symptoms and increases in the karyopyknotic index achieved with estradiol-intranasal 300 microg/day were also similar to those observed with oral estradiol 2 mg/day. Assessments of the effects of estradiol-intranasal on the complications of menopause (increased risk of cardiovascular disease and osteoporosis) are ongoing; however, estradiol-intranasal (sequentially combined with a progestogen) produced significant beneficial effects on some lipid parameters and on markers of bone resorption and formation, and bone mineral density in postmenopausal women. Estradiol-intranasal had no significant effects on serum levels of most of the assessed haemostatic factors, or on angiotensinogen or insulin levels. Estradiol-intranasal 100 to 600 microg/day was generally well tolerated in clinical trials and most adverse events were mild to moderate. The most commonly reported events were nasal symptoms and mastalgia. There was no evidence of endometrial hyperplasia with up to 1 year's treatment with estradiol-intranasal 300 microg/day combined with a progestogen. The incidence of mastalgia and withdrawal or breakthrough bleeding was lower with estradiol-intranasal 300 microg/day than with oral estradiol 2 mg/day (both administered with a progestogen) in one trial. In another trial, the incidence of mastalgia was lower with estradiol-intranasal 300 microg/day than with estradiol transdermal 50 microg (both administered with a progestogen). However, the overall incidence of adverse events was similar between the two treatments in this trial., Conclusions: Estradiol-intranasal 200 to 400 microg/day (optimal initiating dose 300 microg/day) reduces the incidence and severity of menopausal climacteric symptoms and has a good tolerability profile. Thus, evidence to date suggests that estradiol-intranasal is a useful treatment option for menopausal symptoms.

1. Male, Sprague-Dawley rats were actively immunized with novel angiotensin vaccines, and their pressor responses to exogenous angiotensin I (AI) and angiotensin II (AII) were assessed in vivo. Serum antibody titres were also measured. 2. The most effective vaccine consisted of an AI analogue conjugated with a tetanus toxoid carrier protein and adjuvanted with aluminium hydroxide. When this vaccine was injected on days 0, 21 and 42, pressor responses to AI on day 63 were significantly inhibited (maximum, 8.9 fold shift), but responses to AII were unaffected. The anti-angiotensin antibody titre was increased 32,100 fold, and, uniquely, these antibodies also cross-reacted with angiotensinogen. 3. These findings indicate that active immunization against AI may be a useful approach for treating cardiovascular disorders involving the renin-angiotensin system.

Recent work from this laboratory demonstrated potent inhibition of apoptosis in human alveolar epithelial cells (AECs) by the angiotensin-converting enzyme inhibitor captopril [B. D. Uhal, C. Gidea, R. Bargout, A. Bifero, O. Ibarra-Sunga, M. Papp, K. Flynn, and G. Filippatos. Am. J. Physiol. 275 (Lung Cell. Mol. Physiol. 19): L1013-L1017, 1998]. On this basis, we hypothesized that apoptosis in this cell type might be induced by angiotensin II (ANG II) through its interaction with the ANG II receptor. Purified ANG II induced dose-dependent apoptosis in both the human AEC-derived A549 cell line and in primary type II pneumocytes isolated from adult Wistar rats as detected by nuclear and chromatin morphology, caspase-3 activity, and increased binding of annexin V. Apoptosis also was induced in primary rat AECs by purified angiotensinogen. The nonselective ANG II-receptor antagonist saralasin completely abrogated both ANG II- and angiotensinogen-induced apoptosis at a concentration of 50 microgram/ml. With RT-PCR, both cell types expressed the ANG II-receptor subtypes 1 and 2 and angiotensin-converting enzyme (ACE). The nonthiol ACE inhibitor lisinopril blocked apoptosis induced by angiotensinogen, but not apoptosis induced by purified ANG II. These data demonstrate the presence of a functional ANG II-dependent pathway for apoptosis in human and rat AECs and suggest a role for the ANG II receptor and ACE in the induction of AEC apoptosis in vivo.

Using solid-phase methodology we have synthesised peptides based on the 8-14 or 6-14 human and rat angiotensinogen sequences, containing the following different isosteric units at the P1-P1' cleavage site: Leu-psi[CH2NH]Leu; Leu-psi[CH(OH)CH2]Val; Leu-psi[CH(OH)CH2]Leu and Leu-psi[CH(NH2)CH2]Val. In vitro, peptide Piv-His-Pro-Phe-His-Leu-psi[CH(OH)CH2]Leu-Tyr-Tyr-Ser-NH2(XXI) is the most potent inhibitor of rat plasma renin reported having an IC50 of 0.21 nM; it is a much weaker inhibitor of human renin (IC50 45 nM). Peptide Boc-His-Pro-Phe-His-Leu-psi[CH(OH)CH2] Leu-Val-Ile-His-NH2 (XX) was a highly effective inhibitor of rat renin in vivo. When infused (1 mg/kg/h) into two-kidney, one-clip chronic renal hypertensive rats, it lowered blood pressure and suppressed both plasma renin and angiotensin II. When given as a bolus (1 mg/kg) there was a divergence between the rapid rebound of renin levels and blood pressure, which remained suppressed. These results indicate that potent in vivo inhibitors of rat renin could be useful not only in examining the role of circulating renin but also in elucidating the equally important involvement of extracirculatory renin pools.

Scar tissue found at the site of myocardial infarction (MI) contains phenotypically transformed fibroblast-like cells termed myofibroblasts (myoFb). In injured cardiac tissue, autoradiography and immunolabeling have localized high density angiotensin (Ang) converting enzyme (ACE) and Ang II receptor binding to these cells, suggesting that they may regulate local concentrations of Ang II and transduce signals at this site. Ang II is known to modulate type I collagen gene expression of fibroblasts and myoFb, and to promote fibrous tissue contraction, each of which may contribute to tissue repair. It is unknown whether myoFb themselves generate Ang peptides de novo via expression of angiotensinogen (Ao), an aspartyl protease needed to convert Ao to Ang I, and ACE. We therefore isolated and cultured myoFb from 4-week-old scar tissue of the adult rat left ventricle with transmural MI. In cultured myoFb we found: (a) immunoreactive membrane-bound ACE, cytosolic cathepsin D (Cat-D), and AT, receptors by immunofluorescence and confocal microscopy, (b) mRNA expression for Ao, ACE, and Cat-D, but not renin, by reverse transcriptase-polymerase chain reaction, (c) production of Ang I and II in serum-free culture media; (d) absence of renin activity; (e) a time-dependent conversion of Ao to Ang I by myoFb cytosol, which was inhibited by pepstatin A, but not by renin inhibitor; and (f) significant increase in Ang II production (P < 0.05) by exogenous Ao and Ang I (10 nM), which was significantly blocked by lisinopril (0.1 microM: P < 0.05). Thus, cultured myoFb express requisite components and are able to generate Ang I and II de novo. In an autocrine and/or paracrine manner, Ang II may regulate myoFb collagen turnover and fibrous tissue contraction.

To evaluate the potential physiological significance of ouabain or a ouabainlike substance, we investigated the effect of nanomolar concentrations of ouabain on aldosterone release by cultured bovine adrenal glomerulosa cells. Ouabain (10 nM) increased aldosterone release from 0.35 to 0.89 ng.mg-1.4 h-1 in the serum-containing medium. Losartan prevented this increase. When angiotensinogen was added to the nonserum medium, 10 nM ouabain enhanced the aldosterone release. Losartan again blocked the increase. These findings together with a stimulation of renin release by ouabain indicate that angiotensin II generated by the adrenal cell renin-angiotensin system in the presence of exogenous serum or exogenous angiotensinogen is necessary for the ouabain-induced stimulation of aldosterone release. Ouabain (10 nM) enhanced the intracellular calcium concentration increase elicited by 0.1 nM angiotensin II severalfold. Addition of 1 nM ouabain enhanced the aldosterone secretion resulting from the addition of 1 nM angiotensin II. Nanomolar levels of ouabain, therefore, interact with both locally formed and exogenous angiotensin II to stimulate aldosterone production. A suggested mechanism is that ouabain increases calcium stores in the endoplasmic reticulum, thereby increasing the agonist response.

1. Since plasma renin activity is increased in cyclosporin A (CsA)-induced hypertension in the rat, the role of the vascular renin-angiotensin system (RAS) in CsA-induced hypertension was investigated in rat mesenteric resistance vessels. 2. Female Wistar rats received CsA (10 mg/kg per day, s.c.) or vehicle for 30 days. CsA treatment increased tail-cuff systolic blood pressure (CsA treated 135 +/- 3 mmHg vs control 125 +/- 1 mmHg, P < 0.0001). 3. Mesenteric resistance arteries (200-300 microns) were isolated and mounted in a microvessel myograph. Concentration-response curves to tetradecapeptide renin substrate (10(-11)-10(-6) mol/L), angiotensin I (10(-11)-10(-6) mol/L) and angiotensin II (10(-12)-10(-6) mol/L) showed no differences between CsA-treated and control groups. 4. Mesenteric vascular angiotensin-converting enzyme (ACE) characteristics were determined by radioligand binding. There were no differences in the content or affinity of ACE between CsA-treated and control rats. 5. These results suggest that the mesenteric vascular RAS does not play a major role in CsA-induced hypertension in the rat.

The most important specific regulatory mechanism for hepatic angiotensinogen synthesis and secretion is its stimulation by angiotensin II, the effector peptide of the renin-angiotensin system. In the circulating system, this octapeptide is thought to stimulate hepatic angiotensinogen synthesis through a positive feedback loop. In the present study, we have identified the intracellular mechanisms leading to an increase in angiotensinogen messenger RNA (mRNA) and secretion. In a [3H]uridine-dependent pulse and chase system as well as in hepatocytes in which de novo synthesis of mRNA has been blocked by actinomycin D or 5,6-dichlorobenzimidazole riboside, angiotensin II significantly increased the half-life of angiotensinogen mRNA. In contrast, no effect of angiotensin II on the transcription of angiotensinogen mRNA could be observed in a nuclear run-on assay with nuclei from pretreated hepatocytes, whereas dexamethasone, as a positive control, increased the transcription fivefold to sevenfold. We have isolated a 12-kD protein from the polysomal fraction of isolated hepatocytes, which has an affinity to the nontranslated 3' tail of angiotensinogen mRNA. For in vitro transcription of this mRNA fragment, the DNA sequence coding for the nontranslated 3' tail was excised from the vector pRAG 16 and cloned into the transcription vector pGEM 5zf+. Molecular weight and isoelectric point of the mRNA-binding protein correspond to the parameters of a cytosolic protein that becomes phosphorylated by decreased cyclic AMP concentrations as analyzed in [32P]orthophosphate-loaded hepatocytes. In a cytosolic incubation system in which the polysomal fraction was integrated, the mRNA-binding protein increased the half-life of angiotensinogen mRNA significantly.(ABSTRACT TRUNCATED AT 250 WORDS)

The cleavage of synthetic tetradecapeptide renin substrate has been used to infer the presence of renin in the walls of isolated blood vessels; however, the conversion of natural angiotensinogen to angiotensin in isolated blood vessels has not been reported. We studied the release of angiotensinogen and the formation of angiotensins in a bloodless, perfused, isolated hind limb preparation of the rat. Perfusion with a modified Tyrode's solution resulted in spontaneous release of 4.7 +/- 1.5 pmol per 30 minutes of angiotensinogen as measured directly by radioimmunoassay. Western blot further identified the released material as angiotensinogen. Spontaneous release of angiotensins I and II was demonstrated by high performance liquid chromatography and radioimmunoassay. When highly purified rat angiotensinogen was added to the perfusate, release of angiotensin II was increased 14-fold compared with saline infusion. Captopril (10 mumol/L) inhibited angiotensinogen-induced angiotensin II release by 67% and led to an increase in angiotensin I release by 301%. Bilateral nephrectomy 24 hours before the experiments reduced basal angiotensin release below the detection limit and blunted angiotensinogen-induced angiotensin II formation by 95%. We conclude that active renin is present in the vessel wall and interacts with its natural substrate to form angiotensin peptides. Our data support the notion that the bulk of vascular renin is taken up from the circulation.

Intravital microscopy was used in a preparation of rat cremaster muscle that was isolated from its normal blood supply and externally perfused with a physiological solution, thus allowing exclusion of circulating converting enzyme, renin, and angiotensinogen. The arterioles studied were classified as second-, third-, and fourth-order arterioles with mean diameters of 60.5, 29.9, and 14.8 microns, respectively. Topical administration of 1 nmol/mL angiotensin I or 1 nmol/mL tetradecapeptide renin substrate induced marked vasoconstrictions (i.e., 38.5%, 61.5%, and 90.1% and 25%, 34%, and 88% for second-, third-, and fourth-order arterioles with angiotensin I and tetradecapeptide renin substrate, respectively). The angiotensin converting enzyme inhibitor quinapril significantly inhibited the vasoconstrictions caused by either angiotensin I or tetradecapeptide renin substrate. Almost no vasoconstriction was found when angiotensinogen-rich renin-free plasma containing either 2.45 nmol/mL of angiotensinogen or 1.2 micrograms/mL renin was administered. Conversely, these two compounds induced significant constrictions in cremaster muscle preparations in which normal blood perfusion (and thus circulating renin and angiotensinogen) was left in place. We concluded that, in skeletal muscle, 1) the microvascular network is a very effective site of local angiotensin converting enzyme activity and consequently an important target site of angiotensin converting enzyme inhibitors; 2) the effects of tetradecapeptide renin substrate are very different from those of angiotensinogen from plasma and suggest that a large part of the effect of tetradecapeptide renin substrate was due to its nonspecific hydrolysis; and 3) at the microvascular level, circulating renin and angiotensinogen are more effective in inducing arteriolar constriction, in the presence of their substrate or associated enzyme, than local renin and angiotensinogen.

Existence of the renin-angiotensin system was pharmacologically investigated in the dog isolated iris sphincter muscle. The sphincter muscle contracted in response to tetradecapeptide, a synthetic renin substrate, angiotensin (ANG) I and ANG II dose-dependently. The contractions induced by these peptides were suppressed by treatment with saralasin, indomethacin and aspirin. Contractile responses to tetradecapeptide and ANG I were also reduced by KRI-1314, a renin inhibitor, and captopril, respectively. ANG II stimulated the release of prostaglandin (PG) F2 alpha from the sphincter muscle. Angiotensin-converting enzyme activity was measurable in the sphincter muscle. Miosis was observed by intracameral injection of ANG I and ANG II into the anterior chamber. These results strongly suggest that angiotensin generating enzymes function in the sphincter muscle and ANG II produced by these enzymes contracts the sphincter muscle via the formation of PG (s), possibly PG F2 alpha.

Human renin can cleave rat angiotensinogen, yet infusion of human renin into rats causes only a modest increase in blood pressure. We therefore investigated whether there is a factor in rat plasma which inhibits human renin activity. The addition of 20% normal rat plasma to human plasma had a slight, but not significant, inhibitory effect on the rate of angiotensin formation, while nephrectomized rat plasma, which had a seven-fold higher concentration of angiotensinogen, caused a dose dependent inhibition (20 to 70%). The rat plasma inhibitor copurified with angiotensinogen. Analysis of the kinetics of the human renin-human substrate reaction and of the human renin-rat substrate reaction revealed that the rate of angiotensin I production in the presence of both substrates could be entirely accounted for by assuming that rat and human angiotensinogens are competitive inhibitors of each other. These results show that human renin can cleave rat substrate but the reaction rate is extremely slow relative to the cleavage of human angiotensinogen. They also indicate that rat angiotensinogen is an effective competitive inhibitor of the human renin-substrate reaction. These results may be relevant to the development of renin inhibitors and to transfection studies involving heterologous renin or substrate genes.

In aggregate cell cultures of 15- to 20-day-old rat pituitary maintained in serum-free medium, luteinizing hormone-releasing hormone (LHRH) (10 nM) stimulated prolactin (PRL) release, confirming our previous results and those of others with serum-supplemented medium. Since angiotensin II (AII) stimulates PRL release and a renin-angiotensin system is expressed in gonadotrophs, LHRH stimulation of PRL release might be mediated by AII. To evaluate this hypothesis, the influence of (Sar1,Ala8)AII and (Sar1,Ile8)AII two peptide AII receptor antagonists, of DUP753, a nonpeptide and stable AII receptor antagonist, of a converting enzyme inhibitor, and of angiotensinogen on LHRH-induced PRL release was tested in various in vitro conditions of 15- to 20-day-old female rat pituitary. In aggregates maintained in serum-free medium with or without dexamethasone (DEX) and triiodothyronine (T3), or maintained in serum-supplemented medium, the effect of LHRH on PRL release was not affected by (Sar1Ala8)AII (0.1 microM), (Sar1,Ile8)AII (10 microM) or DUP753 (10 microM). Only a high dose (10 microM) of (Sar1,Ala8)AII attenuated the LHRH-induced PRL release. The latter attenuation was seen only with aggregates cultured in the DEX/T3 medium and not with aggregates cultured in the presence of serum. A dose of 1 or 10 nM (Sar1,Ala8)AII also failed to block the effect of LHRH used at 1 nM. In contrast, (Sar1,Ala8)AII dose dependently as well as DUP753 (10 microM) abolished the AII-induced PRL release. (Sar1,Ala8)AII also failed to affect the LHRH-induced PRL release in pituitary cell aggregates from 6-week-old male rats. However, in aggregates from both immature and 6-week-old rats, (Sar1,Ala8)AII provoked a small and statistically significant attenuation of the LHRH-induced PRL release when a 100 nM dose of LHRH was used. In freshly isolated hemipituitaries from 5-day-old rats, (Sar1,Ala8)AII (1 or 10 microM) did not affect the LHRH- (10 nM) induced PRL release. In single cells obtained by redispersion of aggregates and mounted in a Biogel P2 column, LHRH still stimulated PRL release. Again this effect could not be blocked by DUP753. Treatment of aggregate cell cultures with the angiotensin-converting enzyme inhibitor captopril or with angiotensinogen did not alter the LHRH-induced PRL release. It is concluded that AII is not the paracrine factor mediating the effect of LHRH at low nanomolar doses on PRL release, at least not through the classical AII receptor. The involvement of AII acting on a non-(Sar1,Ala8)AII-sensitive receptor cannot be excluded and warrants further investigation.

To assess the role of the vascular angiotensin II-generating system in one-kidney, one clip hypertension, we determined the angiotensin converting enzyme activity in plasma and vascular tissues and examined the pressor response to angiotensin II, angiotensin I, and tetradecapeptide renin substrate in isolated mesenteric arteries from one-kidney, one clip hypertensive rats 7 and 30 days after clipping the renal artery and in mesenteric arteries from age-matched normotensive rats. Angiotensin converting enzyme activity, determined in aortic and mesenteric tissues, was significantly augmented in the hypertensive (30 days after clipping) group, whereas plasma activity was normal. The vasoconstrictor responses elicited by angiotensin I and tetradecapeptide in arteries from hypertensive rats were found to be significantly potentiated 30 days after clipping, whereas the angiotensin II responses were basically unchanged. Saralasin completely blocked the vasoconstrictor responses, whereas captopril blocked only the responses to angiotensin I without affecting the responses elicited by angiotensin II and tetradecapeptide. Enalapril, an angiotensin converting enzyme inhibitor given intravenously to unanesthetized rats, significantly lowered the blood pressure of hypertensive rats. The pressor responses elicited by angiotensin II, angiotensin I, and tetradecapeptide were completely inhibited by saralasin, whereas enalapril blocked only the responses of angiotensin I but not those elicited by angiotensin II and tetradecapeptide. These results indicate that local formation of angiotensin II is increased in arteries of one-kidney, one clip hypertensive rats. The data obtained with tetradecapeptide renin substrate suggest an important role for nonrenin proteases in vascular angiotensin II formation.

The renin-angiotensin system in sub-genus Mus displays unique features including duplication of the renin gene in most strains, strong expression of the second gene in submandibular gland of males, and inhibited responses to injected renin. Our findings indicate that this inhibition results from a paucity of renin substrate and is consistent with first-order kinetics. We find substrate paucity to be a feature of both sexes and all sub-species and strains of Mus irrespective of gene duplication. Attempts to increase the level of substrate in blood by intravenous injection caused marked increases in blood pressure in Mus, suggesting that substrate paucity was a phenotypic prerequisite for successful emergence of enhanced renin expression in salivary gland. We propose that these phenomena are linked to salivary "lethal factor", possibly transferred by biting, in an evolutionary sequence that has provided a major selective advantage for Mus and influenced the ecology and evolution of rodents.