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Background: We have recently reported that the spinal angiotensin (Ang) converting enzyme (ACE)/Ang II/AT1 receptor axis and downstream p38 MAPK phosphorylation are activated in streptozotocin (STZ)-induced diabetic mice and lead to tactile hypersensitivity. Moreover, our previous results suggested that the intrathecal (i.t.) administration of Ang (1-7), an N-terminal fragment of Ang II, may attenuate the Ang II-induced nociceptive behaviour through the inhibition of p38 MAPK phosphorylation via Mas receptors. Here, we investigated whether the i.t. administration of Ang (1-7) can attenuate STZ-induced diabetic neuropathic pain., Methods: Tactile and thermal hypersensitivities were determined using the von Frey filament and Hargreaves tests, respectively. The protein expression of ACE2, Mas receptors and phospho-p38 MAPK was measured by western blotting. Spinal ACE2 activity was determined using ACE2 activity assay kit., Results: The i.t. administration of Ang (1-7) significantly reduced the tactile and thermal hypersensitivities on day 14 after STZ injection, and these effects were significantly prevented by the Mas receptor antagonist A779. The expression of ACE2 and Mas receptors in the plasma membrane fraction of the lumbar dorsal spinal cord was both significantly decreased in STZ mice. Spinal ACE2 activity was also decreased while p38 MAPK phosphorylation was increased in the lumbar dorsal region of these mice. This phosphorylation was attenuated by the injection of Ang (1-7), whose effect was reversed by A779., Conclusions: Our data demonstrate that Ang (1-7) attenuates STZ-induced diabetic neuropathic pain and that this occurs through a mechanism involving spinal Mas receptors and he inhibition of p38 MAPK phosphorylation., Significance: The ACE2/Ang (1-7)/Mas receptor axis was down-regulated in the spinal cord of STZ mice and the i.t. administration of Ang (1-7) attenuated the STZ-induced diabetic neuropathic pain via Mas receptors. Therefore, the activation of this axis could be an effective therapeutic target to alleviate the neuropathic pain in diabetic patients., (© 2018 European Pain Federation - EFIC®.)

Background: Atrial fibrillation (AF) is the most common sustained arrhythmia, and pulmonary veins (PVs) play a critical role in triggering AF. Angiotensin (Ang)-(1-7) regulates calcium (Ca 2+ ) homoeostasis and also plays a critical role in cardiovascular pathophysiology. However, the role of Ang-(1-7) in PV arrhythmogenesis remains unclear., Materials and Methods: Conventional microelectrodes, whole-cell patch-clamp and the fluo-3 fluorimetric ratio technique were used to record ionic currents and intracellular Ca 2+ in isolated rabbit PV preparations and in single isolated PV cardiomyocytes, before and after administration of Ang-(1-7)., Results: Ang (1-7) concentration dependently (0.1, 1, 10 and 100 nmol/L) decreased PV spontaneous electrical activity. Ang-(1-7) (100 nmol/L) decreased the late sodium (Na + ), L-type Ca 2+ and Na + -Ca 2+ exchanger currents, but did not affect the voltage-dependent Na + current in PV cardiomyocytes. In addition, Ang-(1-7) decreased intracellular Ca 2+ transient and sarcoplasmic reticulum Ca 2+ content in PV cardiomyocytes. A779 (a Mas receptor blocker, 3 μmol/L), L-NAME (a NO synthesis inhibitor, 100 μmol/L) or wortmannin (a specific PI3K inhibitor, 10 nmol/L) attenuated the effects of Ang-(1-7) (100 nmol/L) on PV spontaneous electric activity., Conclusion: Ang-(1-7) regulates PV electrophysiological characteristics and Ca 2+ homoeostasis via Mas/PI3K/eNOS signalling pathway., (© 2017 Stichting European Society for Clinical Investigation Journal Foundation.)

Background: Benazepril has been shown to inhibit circulating angiotensin-converting enzyme (ACE) activity in horses but the optimal dosage is unknown., Objectives: To determine the lowest tested dose of benazepril that results in ≥75% attenuation in the response of arterial blood pressure (BP) to exogenous angiotensin I (ANG-I) administration., Study Design: Prospective experimental study., Methods: A total of 5 healthy horses were instrumented for the direct measurement of BP. Each horse received 4 intragastric doses of benazepril (0.5, 1, 2 and 4 mg/kg bwt) with a washout period of 7 days between doses. Prior to and 2, 12 and 24 h after benazepril administration, each horse received intravenous (i.v.) boluses of ANG-I at 20, 60 and 200 ng/kg. Attenuation of the systolic arterial pressure (SBP) response to ANG-I and serum ACE activity were quantified and expressed as percentage of inhibition., Results: There was a significant effect of benazepril dose (P = 0.004) and time (P = 0.004) on the percentage of inhibition of the systolic pressor response to ANG-I. Regardless of benazepril dose, the percentage of inhibition was significantly greater 2 h after administration of benazepril compared with 12 and 24 h. At an ANG-I dose of 20 ng/kg, the percentage of inhibition after administration of benazepril at 1 mg/kg bwt (46.6 ± 18.9%) was significantly greater than that achieved after 0.5 mg/kg bwt (19 ± 14%) but not significantly different from that achieved at higher doses of benazepril. Benazepril doses ≥1 mg/kg bwt resulted in serum ACE inhibition of at least 90%., Main Limitations: Small sample size and resulting low statistical power., Conclusions: Attenuation of the rise in SBP in response to ANG-I after administration of benazepril is modest in horses despite adequate serum ACE inhibition. A dose of 1 mg/kg bwt would be recommended for future investigations of benazepril for the management of cardiovascular diseases in horses., (© 2016 EVJ Ltd.)

Background and Purpose: A long-term imbalance between pro- and anti-inflammatory mediators leads to airway remodelling, which is strongly correlated to most of the symptoms, severity and progression of chronic lung inflammation. The Angiotensin-(1-7) [Ang-(1-7)]/Mas receptor axis of the renin-angiotensin system is associated with attenuation of acute and chronic inflammatory processes. In this study, we investigated the effects of Ang-(1-7) treatment in a model of chronic allergic lung inflammation., Experimental Approach: Mice were sensitized to ovalbumin (OVA; 4 injections over 42 days, 14 days apart) and were challenged three times per week (days 21-46). These mice received Ang-(1-7) (1 μg·h(-1) , s.c.) by osmotic mini-pumps, for the last 28 days. Histology and morphometric analysis were performed in left lung and right ventricle. Airway responsiveness to methacholine, analysis of Ang-(1-7) levels (RIA), collagen I and III (qRT-PCR), ERK1/2 and JNK (Western blotting), IgE (elisa), cytokines and chemokines (elisa multiplex), and immunohistochemistry for Mas receptors were performed., Key Results: Infusion of Ang-(1-7) in OVA-sensitized and challenged mice decreased inflammatory cell infiltration and collagen deposition in the airways and lung parenchyma, and prevented bronchial hyperresponsiveness. These effects were accompanied by decreased IgE and ERK1/2 phosphorylation, and decreased pro-inflammatory cytokines. Mas receptors were detected in the epithelium and bronchial smooth muscle, suggesting a site in the lung for the beneficial actions of Ang-(1-7)., Conclusions and Implications: Ang-(1-7) exerted beneficial attenuation of three major features of chronic asthma: lung inflammation, airway remodelling and hyperresponsiveness. Our results support an important protective role of Ang-(1-7) in lung inflammation., (© 2015 The British Pharmacological Society.)

Background: We have recently demonstrated that intrathecal (i.t.) administration of angiotensin II (Ang II) induces nociceptive behaviour in mice accompanied by a phosphorylation of p38 mitogen-activated protein kinase (MAPK) mediated through Ang II type 1 (AT1 ) receptors. The N-terminal fragment of Ang II, Ang (1-7), plays a pivotal role in counterbalancing many of the well-established actions induced by Ang II. However, the role of Ang (1-7) in spinal nociceptive transmission remains unclear. Therefore, we examined whether i.t. administration of Ang (1-7) can inhibit the Ang II-induced nociceptive behaviour in mice., Methods: In the behavioural experiments, the accumulated response time of nociceptive behaviour consisting of scratching, biting and licking in conscious mice was determined during a 25-min period starting after i.t. injection. The distribution and localization of AT1 or Mas receptors were analysed using a MapAnalyzer and confocal microscope, respectively. Phosphorylation of p38 MAPK in the dorsal spinal cord was measured by Western blotting., Results: The nociceptive behaviour induced by Ang II was dose-dependently inhibited by the co-administration of Ang (1-7). The inhibitory effect of Ang (1-7) was reversed by the co-administration of A779, a Mas receptor antagonist. Western blot analysis showed that the increase in spinal p38 MAPK phosphorylation following the i.t. administration of Ang II was also inhibited by Ang (1-7), and the Ang (1-7) induced-inhibition was prevented by A779., Conclusions: Our data show that the i.t. administration of Ang (1-7) attenuates an Ang II-induced nociceptive behaviour and is accompanied by the inhibition of p38 MAPK phosphorylation mediated through Mas receptors., (© 2014 European Pain Federation - EFIC®)

Background and Purpose: As a newer component of the renin-angiotensin system, angiotensin-(1-7) [Ang-(1-7) ] has been shown to facilitate angiogenesis and protect against ischaemic damage in peripheral tissues. However, the role of Ang-(1-7) in brain angiogenesis remains unclear. The aim of this study was to investigate whether Ang-(1-7) could promote angiogenesis in brain, thus inducing tolerance against focal cerebral ischaemia., Experimental Approach: Male Sprague-Dawley rats were i.c.v. infused with Ang-(1-7), A-779 (a Mas receptor antagonist), L-NIO, a specific endothelial NOS (eNOS) inhibitor, endostatin (an anti-angiogenic compound) or vehicle, alone or simultaneously, for 1-4 weeks. Capillary density, endothelial cell proliferation and key components of eNOS pathway in the brain were evaluated. Afterwards, rats were subjected to permanent middle cerebral artery occlusion (pMCAO), and regional cerebral blood flow (rCBF), infarct volume and neurological deficits were measured 24 h later., Key Results: Infusion of Ang-(1-7) for 4 weeks significantly increased brain capillary density via promoting endothelial cell proliferation, which was accompanied by eNOS activation and up-regulation of NO and VEGF in brain. These effects were abolished by A-779 or L-NIO. More importantly, Ang-(1-7) improved rCBF and decreased infarct volume and neurological deficits after pMCAO, which could be reversed by A-779, L-NIO or endostatin., Conclusions and Implications: This is the first evidence that Ang-(1-7) promotes brain angiogenesis via a Mas/eNOS-dependent pathway, which enhances tolerance against subsequent cerebral ischaemia. These findings highlight brain Ang-(1-7)/Mas signalling as a potential target in stroke prevention., (© 2014 The British Pharmacological Society.)

Novel treatments are necessary to reduce the burden of cardiovascular disease (CVD). Alamandine binds to MrgD and is reported to induce vasodilation via stimulation of endothelial nitric oxide synthase (eNOS), but its role in atherogenic blood vessels is yet to be determined. To determine the vasoactive role of alamandine and its precursor AngA in diseased aorta, New Zealand White rabbits were fed a diet containing 1% methionine + 0.5% cholesterol + 5% peanut oil for 4 weeks (MC, n = 5) or control (n = 6). In abdominal aorta, alamandine (1 μM) was added 30 min before a dose-response curve to angiotensin II or AngA (1 nM-1 μM), and immunohistochemistry was used to identify MrgD receptors and eNOS. The thoracic aorta, renal, carotid and iliac arteries were mounted in organ baths. Rings were precontracted with phenylephrine, then a bolus dose of alamandine (1 μM) was added 10 min before a dose-response curve to acetylcholine (0.01 μM-10 μM). The MrgD receptor was localized to normal and diseased aorta and colocalized with eNOS. In control but not diseased blood vessels, alamandine enhanced acetylcholine-mediated vasodilation in the thoracic aorta and the iliac artery (P < 0.05) and reduced it in the renal artery (P < 0.05). In control abdominal aorta, AngA evoked less desensitization than AngII (P < 0.05) and alamandine reduced AngA-mediated vasoconstriction (P < 0.05). In MC, AngA constriction was markedly reduced vs. control (P < 0.05). The vasoactivity of alamandine and AngA are reduced in atherogenesis. Its role in the prevention of CVD remains to be validated., (© 2014 The Authors. International Journal of Experimental Pathology © 2014 International Journal of Experimental Pathology.)

Background and Purpose: AVE 0991 (AVE) is a non-peptide compound, mimic of the angiotensin (Ang)-(1-7) actions in many tissues and pathophysiological states. Here, we have investigated the effect of AVE on pulmonary remodelling in a murine model of ovalbumin (OVA)-induced chronic allergic lung inflammation., Experimental Approach: We used BALB/c mice (6-8 weeks old) and induced chronic allergic lung inflammation by OVA sensitization (20 μg·mouse(-1) , i.p., four times, 14 days apart) and OVA challenge (1%, nebulised during 30 min, three times per·week, for 4 weeks). Control and AVE groups were given saline i.p and challenged with saline. AVE treatment (1 mg·kg(-1) ·per day, s.c.) or saline (100 μL·kg(-1) ·per day, s.c.) was given during the challenge period. Mice were anaesthetized 72 h after the last challenge and blood and lungs collected. In some animals, primary bronchi were isolated to test contractile responses. Cytokines were evaluated in bronchoalveolar lavage (BAL) and lung homogenates., Key Results: Treatment with AVE of OVA sensitised and challenged mice attenuated the altered contractile response to carbachol in bronchial rings and reversed the increased airway wall and pulmonary vasculature thickness and right ventricular hypertrophy. Furthermore, AVE reduced IL-5 and increased IL-10 levels in the BAL, accompanied by decreased Ang II levels in lungs., Conclusions and Implications: AVE treatment prevented pulmonary remodelling, inflammation and right ventricular hypertrophy in OVA mice, suggesting that Ang-(1-7) receptor agonists are a new possibility for the treatment of pulmonary remodelling induced by chronic asthma., (© 2013 The British Pharmacological Society.)

Background and Purpose: Angiotensin-(1-7) [Ang-(1-7)] has anti-inflammatory effects in peripheral organs, but its effects in ischaemic stroke are unclear as yet. We investigated whether its anti-inflammatory effect contributes to the neuroprotection induced by Ang-(1-7) in a rat model of permanent middle cerebral artery occlusion (pMCAO)., Experimental Approach: We infused Ang-(1-7), Mas receptor antagonist A-779, angiotensin II type 2 receptor antagonist PD123319 or artificial CSF into the right lateral ventricle of male Sprague-Dawley rats from 48 h before onset of pMCAO until the rats were killed. Twenty-four hours after pMCAO, the neuroprotective effect of Ang-(1-7) was analysed by evaluating infarct volume and neurological deficits. The levels of oxidative stress were detected by spectrophotometric assay. The activation of NF-κB was assessed by Western blot and immunohistochemistry analysis. The level of COX-2 was tested by Western blot analysis and concentrations of pro-inflammatory cytokines were measured by elisa., Key Results: Infusion of Ang-(1-7), i.c.v., significantly reduced infarct volume and improved neurological deficits. It decreased the levels of oxidative stress and suppressed NF-κB activity, which was accompanied by a reduction of pro-inflammatory cytokines and COX-2 in the peri-infarct regions. These effects of Ang-(1-7) were reversed by A-779 but not by PD123319. Additionally, infusion of A-779 alone increased oxidative stress levels and enhanced NF-κB activity, which was accompanied by an up-regulation of pro-inflammatory cytokines and COX-2., Conclusion and Implications: Our findings indicate that suppressing NF-κB dependent pathway via Mas receptor may represent one mechanism that contributes to the anti-inflammatory effects of Ang-(1-7) in rats with pMCAO., (© 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.)

BACKGROUND AND PURPOSE Angiotensin-(1-7) [Ang-(1-7)] has anti-inflammatory effects in models of cardiovascular disease and arthritis, but its effects in asthma are unknown. We investigated whether Ang-(1-7) has anti-inflammatory actions in a murine model of asthma. EXPERIMENTAL APPROACH The effects of Ang-(1-7) alone or in combination with the MAS1 receptor antagonist, A779, were evaluated over a 4 day period in an ovalbumin-challenged mouse model of allergic asthma. On day 5, bronchoalveolar lavage was performed, and lungs were sectioned and assessed histologically for quantification of goblet cells, perivascular and peribronchial inflammation and fibrosis. Biochemical analysis of the pro-inflammatory ERK1/2 and IκB-α was assessed. In addition, the effect of Ang-(1-7) on proliferation of human peripheral blood mononuclear cells (HPBMC) was investigated. KEY RESULTS Ang-(1-7) attenuated ovalbumin-induced increases in total cell counts, eosinophils, lymphocytes and neutrophils. Ang-(1-7) also decreased the ovalbumin-induced perivascular and peribronchial inflammation, fibrosis and goblet cell hyper/metaplasia. Additionally, Ang-(1-7) reduced the ovalbumin-induced increase in the phosphorylation of ERK1/2 and IκB-α. These effects of Ang-(1-7) were reversed by the MAS1 receptor antagonist A779. Furthermore, Ang-(1-7) inhibited phytohaemagglutinin (PHA)-induced HPBMC proliferation. CONCLUSION AND IMPLICATIONS Ang-(1-7), via its MAS1 receptor, acts as an anti-inflammatory pathway in allergic asthma, implying that activation of the MAS1 receptor may represent a novel approach to asthma therapy., (© 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.)

Background and Purpose: The transactivation of the epidermal growth factor (EGF) receptor appears to be an important central transduction mechanism in mediating diabetes-induced vascular dysfunction. Angiotensin-(1-7) [Ang-(1-7)] via its Mas receptor can prevent the development of hyperglycaemia-induced cardiovascular complications. Here, we investigated whether Ang-(1-7) can inhibit hyperglycaemia-induced EGF receptor transactivation and its classical signalling via ERK1/2 and p38 MAPK in vivo and in vitro., Experimental Approach: Streptozotocin-induced diabetic rats were chronically treated with Ang-(1-7) or AG1478, a selective EGF receptor inhibitor, for 4 weeks and mechanistic studies performed in the isolated mesenteric vasculature bed as well as in primary cultures of vascular smooth muscle cells (VSMCs)., Key Results: Diabetes significantly enhanced phosphorylation of EGF receptor at tyrosine residues Y992, Y1068, Y1086, Y1148, as well as ERK1/2 and p38 MAPK in the mesenteric vasculature bed whereas these changes were significantly attenuated upon Ang-(1-7) or AG1478 treatment. In VSMCs grown in conditions of high glucose (25 mM), an Src-dependent elevation in EGF receptor phosphorylation was observed. Ang-(1-7) inhibited both Ang II- and glucose-induced transactivation of EGF receptor. The inhibition of high glucose-mediated Src-dependant transactivation of EGF receptor by Ang-(1-7) could be prevented by a selective Mas receptor antagonist, D-Pro7-Ang-(1-7)., Conclusions and Implications: These results show for the first time that Ang-(1-7) inhibits EGF receptor transactivation via a Mas receptor/Src-dependent pathway and might represent a novel general mechanism by which Ang-(1-7) exerts its beneficial effects in many disease states including diabetes-induced vascular dysfunction., (© 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.)

Background and Purpose: ApolipoproteinE-deficient [apoE (-/-)] mice, a model of human atherosclerosis, develop endothelial dysfunction caused by decreased levels of nitric oxide (NO). The endogenous peptide, angiotensin-(1-7) [Ang-(1-7)], acting through its specific GPCR, the Mas receptor, has endothelium-dependent vasodilator properties. Here we have investigated if chronic treatment with Ang-(1-7) improved endothelial dysfunction in apoE (-/-) mice., Experimental Approach: ApoE (-/-) mice fed on a lipid-rich Western diet were divided into three groups and treated via osmotic minipumps with either saline, Ang-(1-7) (82 µg·kg(-1) ·h(-1) ) or the same dose of Ang-(1-7) together with D-Ala-Ang-(1-7) (125 µg·kg(-1) ·h(-1) ) for 6 weeks. Renal vascular function was assessed in isolated perfused kidneys., Key Results: Ang-(1-7)-treated apoE (-/-) mice showed improved renal endothelium-dependent vasorelaxation induced by carbachol and increased renal basal cGMP production, compared with untreated apoE (-/-) mice. Tempol, a reactive oxygen species (ROS) scavenger, improved endothelium-dependent vasorelaxation in kidneys of saline-treated apoE (-/-) mice whereas no effect was observed in Ang-(1-7)-treated mice. Chronic treatment with D-Ala-Ang-(1-7), a specific Mas receptor antagonist, abolished the beneficial effects of Ang-(1-7) on endothelium-dependent vasorelaxation. Renal endothelium-independent vasorelaxation showed no differences between treated and untreated mice. ROS production and expression levels of the NAD(P)H oxidase subunits gp91phox and p47phox were reduced in isolated preglomerular arterioles of Ang-(1-7)-treated mice, compared with untreated mice, whereas eNOS expression was increased., Conclusion and Implications: Chronic infusion of Ang-(1-7) improved renal endothelial function via Mas receptors, in an experimental model of human cardiovascular disease, by increasing levels of endogenous NO., (© 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.)

1. The contribution of the local vascular production of angiotensin-(1-7) [Ang-(1-7)] to the control of alpha-adrenergic-induced contractions in the aorta of Sprague-Dawley (SD) and TGR(mRen-2)27 [mRen-2] rats was studied. 2. In mRen-2 rats, contractile responses to phenylephrine were diminished as compared to control SD rats in endothelium containing but not in endothelium-denuded vessels. L-NAME increased contractile responses to phenylephrine in mRen-2 rats and, after nitric oxide synthase blockade, responses to phenylephrine became comparable in both strains. 3. Inhibition of angiotensin-converting enzyme (ACE) by captopril potentiated contractile responses in mRen-2 rats and diminished contractile responses in SD rats, both effects being dependent on the presence of a functional endothelium. The effect of captopril in mRen-2 rats was abolished in vessels pre-incubated with Ang-(1-7). 4. Blockade of Ang-(1-7) and bradykinin (BK) receptors by A-779 and HOE 140 respectively, increased phenylephrine-induced contraction in mRen-2, but not in SD rats. This effect was seen only in endothelium-containing vessels. 5. Angiotensin II AT(1) and AT(2) receptor blockade by CV 11974 and PD 123319 did not affect the contractile responses to phenylephrine in aortas of transgenic animals but diminished the response in SD rats. This effect was only seen in the presence of a functional endothelium. 6. It is concluded that the decreased contractile responses to phenylephrine in aortas of mRen-2 rats was dependent on an intact endothelium, the local release and action of Ang-(1-7) and bradykinin.

1. We investigated why angiotensin (Ang) I and II induce vasoconstriction with similar potencies, although Ang I-II conversion is limited. 2. Construction of concentration-response curves to Ang I and II in porcine femoral arteries, in the absence or presence of the AT(1) or AT(2) receptor antagonists irbesartan and PD123319, revealed that the approximately 2 fold difference in potency between Ang I and II was not due to stimulation of different AT receptor populations by exogenous and locally generated Ang II. 3. Measurement of Ang I and II and their metabolites at the time of vasoconstriction confirmed that, at equimolar application of Ang I and II, bath fluid Ang II during Ang I was approximately 18 times lower than during Ang II and that Ang II was by far the most important metabolite of Ang I. Tissue Ang II was 2.9+/-1.5% and 12.2+/-2.4% of the corresponding Ang I and II bath fluid levels, and was not affected by irbesartan or PD123319, suggesting that it was located extracellularly. 4. Since approximately 15% of tissue weight consists of interstitial fluid, it can be calculated that interstitial Ang II levels during Ang II resemble bath fluid Ang II levels, whereas during Ang I they are 8.8 - 27 fold higher. Consequently at equimolar application of Ang I and II, the interstitial Ang II levels differ only 2 - 4 fold. 5. Interstitial, rather than circulating Ang II determines vasoconstriction. Arterial Ang I, resulting in high interstitial Ang II levels via its local conversion by ACE, may be of greater physiological importance than arterial Ang II.

1. Inhibition of the renin-angiotensin system (RAS) improves symptoms and prognosis in heart failure. The experimental basis for these benefits remains unclear. We examined the effects of inhibition of ACE or blockade of angiotensin II type 1 (AT1) receptor on the haemodynamics, cardiac G-proteins, and collagen synthesis of rats with coronary artery ligation (CAL), a model in which chronic heart failure (CHF) is induced. 2. Rats were orally treated with the ACE inhibitor trandolapril (3 mg kg(-1) day(-1)) or the AT1 receptor blocker L-158809 (1 mg kg(-1) day(-1)) from the 2nd to 8th week after CAL. CAL resulted in decreases in the left ventricular systolic pressure and its positive and negative dP/dt, an increase in the left ventricular end-diastolic pressure, and the rightward shift of the left ventricular pressure-volume curve. Long-term treatment with either drug improved these signs of CHF to a similar degree. 3. Cardiac Gsalpha and Gqalpha protein levels decreased, whereas the level of Gialpha protein increased in the animals with CHF. Long-term treatment with trandolapril or L-158809 attenuated the increase in the level of cardiac Gialpha protein of the animals with CHF without affecting Gsalpha and Gqalpha protein levels. Cardiac collagen content of the failing heart increased, whose increase was blocked by treatment with either drug. 4. Exogenous angiotensin I stimulated collagen synthesis in cultured cardiac fibroblasts, whose stimulation was attenuated by either drug. 5. These results suggest that blockade of the RAS, at either the receptor level or the synthetic enzyme level, may attenuate the cardiac fibrosis that occurs after CAL and thus affect the remodelling of the failing heart.

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.

The effects on the responses to coronary artery occlusion of a combined ACE/NEP inhibitor (Z13752A) were examined in anaesthetized dogs. A 1 h infusion of Z13752A (128 microgram kg(-1) min(-1) intravenously) decreased arterial blood pressure (by 11+/-3%; P<0. 05) and increased coronary blood flow (by 12+/-4%, P<0.05). There were no other significant haemodynamic changes. Z13752A inhibited both NEP and ACE enzymes both in dog plasma and in tissue (lung ACE; kidney NEP). Pressor responses to angiotensin I in vivo were inhibited and systemic vasodilator responses to bradykinin were potentiated. When the left anterior descending coronary artery was occluded for 25 min, Z13752A markedly reduced the severity of the resultant ventricular arrhythmias. No ventricular fibrillation (VF) occurred (compared to 7/16 in the controls; P<0.05), and ventricular tachycardia (VT) was reduced (VT in 2/9 dogs treated with Z13752A cp. 16/16 of controls; episodes of VT 0.2+/-0.1 c.p. 10.7+/-3.3; P<0. 05). Reperfusion of the ischaemic myocardium led to VF in all control dogs but occurred less frequently in dogs given Z13752A (survival from the combined ischaemia-reperfusion insult 67% c.p. 0% in controls; P<0.05). Z13752A reduced two other indices of ischaemia severity; epicardial ST-segment elevation and inhomogeneity of electrical activation. These protective effects of Z13752A during ischaemia and reperfusion were abolished by the administration of icatibant (0.3 mg kg(-1), i.v.) a selective antagonist of bradykinin at B(2) receptors; the ischaemic changes in dogs given both icatibant and Z13752A were similar to those in the controls. We conclude that this ACE/NEP inhibitor is effective at reducing the consequences of coronary artery occlusion in this canine model and that this protection is primarily due to potentiation of released bradykinin. British Journal of Pharmacology (2000) 129, 671 - 680

1. We compared the cardiovascular responses to angiotensins (I and II), and any possible modulatory influences thereupon of nitric oxide (NO) or endothelin (ET) in conscious male, normotensive, Hannover Sprague-Dawley (SD) rats, and hypertensive, heterozygous ((mRen-2)27), transgenic (TG) rats. 2. The pressor effects of angiotensin I or of angiotensin II were not consistently different in SD and TG rats. The accompanying absolute reductions in renal and mesenteric vascular conductances were smaller in TG rats, but probably due to the baseline vasoconstriction in those animals. 3. Inhibition of NO synthase with L-NAME had no significant effects on the pressor responses to angiotensin I or angiotensin II in either SD or TG rats. L-NAME reduced the absolute, but not percentage, reductions in renal and mesenteric vascular conductances in response to angiotensin I and angiotensin II. L-NAME abolished the hindquarters vasodilator effects of angiotensin I and angiotensin II in both strains of rat. 4. ET receptor antagonism (with SB209670) had no significant influence on the pressor or renal or mesenteric vasoconstrictor effects of angiotensin II in SD rats. In TG rats, the pressor responses to angiotensin II were unaffected by SB209670; the accompanying falls in renal and mesenteric vascular conductances were enhanced in absolute, but not in percentage terms. 5. These results provide no evidence for a buffering action of NO, or a modulatory influence of ET, on the pressor or vasoconstrictor effects of angiotensin I and/or angiotensin II in SD rats. Furthermore, there is no evidence for an altered sensitivity to angiotensin I or angiotensin II, and no evidence for a differential modulatory influence of either NO or ET in TG, compared to SD, rats.

The present work examined the effects of the subtype 2 of angiotensin II (AT2) receptors on the pressure-natriuresis using a new peptide agonist, and the possible involvement of cyclic guanosine 3', 5' monophosphate (cyclic GMP) in these effects. In adult anaesthetized rats (Inactin, 100 mg kg(-1), i.p.) deprived of endogenous angiotensin II by angiotensin converting enzyme inhibition (quinapril, 10 mg kg(-1), i.v.), T2-(Ang II 4-8)2 (TA), a highly specific AT2 receptor agonist (5, 10 and 30 microg kg(-1) min(-1), i.v.) or its solvent was infused in four groups. Renal functions were studied at renal perfusion pressures (RPP) of 90, 110 and 130 mmHg and urinary cyclic GMP excretion when RPP was at 130 mmHg. The effects of TA (10 microg kg(-1) min(-1)) were reassessed in animals pretreated with PD 123319 (PD, 50 microg kg(-1) min(-1), i.v.), an AT2 receptor antagonist and the action of the same dose of PD alone was also determined. Increases in RPP from 90 to 130 mmHg did not change renal blood flow (RBF) but induced 8 and 15 fold increases in urinary flow and sodium excretion respectively. The 5 microg kg(-1) min(-1) dose of TA was devoid of action. The 10 and 30 microg kg(-1) min(-1) doses did not alter total RBF and glomerular filtration rate, but blunted pressure-diuresis and natriuresis relationships. These effects were abolished by PD. TA decreased urinary cyclic GMP excretion. After pretreatment with PD, this decrease was reversed to an increase which was also observed in animals receiving PD alone. In conclusion, renal AT2 receptors oppose the sodium and water excretion induced by acute increases in blood pressure and this action cannot be directly explained by changes in cyclic GMP.

1. The effects of combined inhibition of neutral endopeptidase 24.11 and angiotensin-converting enzyme, with the dual metallopeptidase inhibitor, MDL 100,240 (3 mg kg-1 bolus, 3 mg kg-1, h-1 infusion), on baseline haemodynamics and on responses to a variety of vasoactive peptides were studied in conscious Long Evans rats (350-450 g: n = 9) chronically instrumented for the assessment of regional haemodynamics. 2. The experiments ran over 4 consecutive days. On the first 2 days the animals received the vehicle for MDL 100,240, and were given bolus i.v. injections of angiotensin I (AI; 250 pmol kg-1), angiotensin II (AII; 125 pmol kg-1), bradykinin (BK: 3 nmol kg-1) and endothelin-1 (ET-1; 250 pmol kg-1) on one day and AI (as above), atrial natriuretic peptide (ANP: 500 pmol kg-1) and big endothelin-1 (big ET-1; 500 pmol kg-1) on the other day in a random manner. On the third and fourth experimental days the vasoactive peptides were given in the same order as before, but in the presence of MDL 100,240. 3. Thirty minutes after onset of administration of vehicle, on the first or second experimental day, there were no consistent cardiovascular changes. However, at the same time following onset of MDL 100,240 administration on the third experimental day, there was a significant, but slight, reduction in mean arterial blood pressure (MAP; -5 +/- 2 mmHg) together with tachycardia (41 +/- 12 beats min-1) and increases in renal and mesenteric flows (17 +/- 3 and 13 +/- 4%, respectively) and vascular conductances (23 +/- 4 and 19 +/- 5%, respectively). The mesenteric vasodilator effect of MDL 100,240 was still present on the fourth experimental day before administration of the drug on that day, but otherwise the pattern of response to MDL 100,240 was similar to that on the previous day. 4. In the presence of vehicle, AI caused hypertension, bradycardia, and reductions in renal mesenteric and hindquarters vascular conductances; all these effects were abolished by MDL 100,240. 5. In the presence of vehicle, AII caused effects similar to those of AI. MDL 100,240 did not affect the pressor, bradycardic or hindquarters vasoconstrictor effects of AII. However, in the presence of MDL 100,240, the overall renal and mesenteric vasoconstrictor effects of AII were enhanced, probably because of the renal and mesenteric vasodilatation caused by MDL 100,240. 6. In the presence of vehicle, BK had a slight pressor effect, accompanied by tachycardia and transient increases in conductances in renal, mesenteric and hindquarters vascular beds. In the presence of MDL 100,240 BK caused marked hypotension, but an attenuated tachycardia; renal, mesenteric and hindquarters vasodilator responses were enhanced. 7. In the presence of vehicle, ANP caused slight hypotension and tachycardia, together with reductions in renal and mesenteric vascular conductances, and transient increases in hindquarters conductance. MDL 100,240 enhanced the hypotensive effect of ANP and promoted a delayed hindquarters vasoconstriction. 8. Big ET-1, in the presence of vehicle, caused a marked and prolonged increase in MAP, accompanied by bradycardia and reductions in renal, mesenteric and hindquarters vascular conductances. Although MDL 100,240 significantly attenuated the magnitude of the pressor effect of big ET-1, its bradycardic and renal, mesenteric and hindquarters haemodynamic actions were not reduced significantly. 9. In the presence of vehicle, ET-1 caused an initial hypotension, tachycardia and vasodilatation in the hindquarters, but reductions in renal and mesenteric vascular conductances; thereafter there was a rise in MAP and bradycardia with vasoconstriction in all three vascular beds. MDL 100,240 had no effect on the initial hypotensive, tachycardic or hindquarters haemodynamic effects of ET-1. Moreover the subsequent pressor and bradycardic actions of ET-1 were unchanged, but its renal and mesenteric vasoconstrictor effects were enhanced, possibly because of the dilatation

1. Angiotensin II is known to enhance sympathetic neurotransmission in the vasculature by increasing the release of noradrenaline, but little is known about the effect on the co-released transmitter, adenosine 5'-triphosphate (ATP). In the present study we have examined the effect of angiotensin II on the excitatory junction potential (e.j.p.) elicited by repetitive field stimulation in the guinea-pig isolated mesenteric artery, to establish the angiotensin II receptor subtype involved in modulating the release of ATP and the role of the endothelium in converting angiotensin I to angiotensin II. 2. Suramin (300 microM), a P2 purinoceptor antagonist, abolished both the e.j.p.s and depolarizing response to alpha,beta-methylene-ATP, a stable analogue of ATP, without affecting the resting membrane potential and noradrenaline-induced depolarization. 3. Angiotensin II (0.1 microM) affected neither the resting membrane potential nor the amplitude of the first e.j.p., but increased the amplitudes of the subsequent e.j.p.s. This enhancing effect of angiotensin II was abolished by CV-11974 (0.1 microM), an angiotensin II type 1 (AT1) receptor antagonist, but unaffected by PD 123319 (1 microM), an angiotensin II type 2 (AT2) receptor antagonist, or CGP 42112A (1 microM), AT2 receptor ligand. 4. Angiotensin I (0.1 microM) exerted a similar effect on e.j.p.s to that of angiotensin II. CV-11974 (0.1 microM) or temocaprilat (10 microM), an angiotensin converting enzyme (ACE) inhibitor, abolished the effect of angiotensin I. Removal of the endothelium did not alter the action of angiotensin I. 5. The results of the present study indicate that the release of ATP from sympathetic nerves innervating the guinea-pig isolated mesenteric artery, as determined from the magnitude of the e.j.p., can be enhanced by angiotensin II via activation of prejunctional AT1 receptors. Qualitatively similar effects were observed with angiotensin I, which appears to be converted into angiotensin II by a subendothelial process.

1. Functional recordings of smooth muscle tension and biochemical experiments on membrane fractions were performed to characterize angiotensin II (AII) formation in human isolated bladder smooth muscle. 2. A novel human chymase inhibitor CH 5450 (Z-Ile-Glu-Pro-Phe-CO2Me) and a recently developed human chymase substrate Pro11-,D-Ala12)-angiotensin I, claimed to be resistant to angiotensin converting enzyme (ACE) and carboxypeptidase, were used. 3. Angiotensin I (AI) (0.3 microM) induced a contractile response amounting to 58 +/- 5% (n=12) of the initial K+ (124 mM)-induced contractions. This response was reduced to 36 +/- 3% (n=8) by the ACE-inhibitor enalaprilat (10 microM), while pretreatment with soybean trypsin inhibitor (STI 200 microg ml(-1)) or CH 5450 (10 microM) had no effect. However, the combination of enalaprilat and STI reduced the AI-induced contractions to 19 +/- 5% (n=6), and the combination of enalaprilat and CH 5450 caused an almost complete inhibition of the AI-induced contractions to 1+/-1% (n=6). 4. The substrate (Pro11-,D-Ala12)-AI (3 microM) produced contractions which amounted to 57 +/- 4% (n=13) of the initial K+ (124 mM) contractions. These contractions were not affected by enalaprilat (10 microM). On the other hand, STI (200 microg ml(-1)) and CH 5450 (10 microM) added separately, depressed the (Pro11-,D-Ala12)-AI-induced contractions to 34 +/- 5% (n=6) and 24 +/- 4% (n=6), respectively. The combination of enalaprilat and STI or enalaprilat and CH 5450 did not produce any further inhibition. 5. Experiments with detrusor membrane fractions incubated with AI (50 microM) were performed. In the presence of enalaprilat (100 microM), carboxypeptidase inhibitor CPI (10 microg ml(-1)) and aprotinin (15 microM), CH 5450 (10 nM-1 microM) caused a concentration-dependent inhibition of AII formation. 6. The results confirm that AII is a potent contractile agent in the human isolated detrusor muscle. They also indicate that the serine protease responsible for AII formation in the human bladder in vitro is human chymase or an enzyme similar to human chymase.

1. The short rabbit pulmonary artery was denuded of endothelium and divided into three sections, the cardiac end (cardiac), middle and pulmonary end (pulmonary) sections, respectively. Des-Asp-angiotensin I attenuated the contractions of the cardiac and middle sections to transmural nerve stimulation but potentiated the contractions in the pulmonary section. 2. The actions of the nonapeptide were inhibited completely by 10(-6) M losartan; however, a similar concentration of PD123319 had no effect. Indomethacin (10(-6) M) also inhibited completely the attenuation in the cardiac and middle sections but had no effect on the potentiation seen in the pulmonary section. 3. The data suggest that the two differential responses of the pulmonary artery to des-Asp-angiotensin I are mediated by two separate subtypes of the losartan-sensitive angiotensin AT1 receptor.

Objective: The physiologic role of endothelin-1 is not well established; however, it may have a role in modulation of peripheral vascular tone complimentary to angiotensin II. In vitro and animal studies suggested an interrelationship between angiotensin II and endothelin-1 vasoconstriction. We hypothesized that local vascular or systemic renin-angiotensin II systems must be intact for endothelin-1-mediated vasoconstriction in humans., Methods: To test this hypothesis, responses to brachial artery infusion of endothelin-1 alone and endothelin-1 plus local low-dose infusion of enaliprilat were studied in seven healthy male and seven healthy female volunteers., Results: In these subjects, baseline forearm vascular resistance (mean +/- SEM; 24 +/- 3.5 mm Hg.ml/dl forearm vol/min) increased with a 38.2 ng/min endothelin-1 infusion (61.8 +/- 6.8 mm Hg.ml/dl forearm vol/min; p < 0.01). Forearm vascular resistance decreased when 38.2 ng/min endothelin-1 was infused concomitantly with a local 5 micrograms/min infusion of enaliprilat (45.5 +/- 5.9 mm Hg.ml/dl forearm vol/min; p < 0.01 compared with endothelin-1 alone)., Conclusions: These data indicate that an endothelin-1-induced increase in forearm vascular resistance is inhibited by local forearm angiotensin-converting enzyme inhibition.

1. The transplacental transfers of three drugs (enalapril, captopril and losartan) which block the renin angiotensin system and have different lipophilicities were studied in chronically catheterized foetal sheep (125-139 days gestation). 2. The ability of the foeto-placental unit to convert enalapril to enalaprilat was studied in two chronically catheterized foetuses. Enalapril (3 mg kg-1, 7.9 mumol kg-1) given i.v. to the foetuses abolished the foetal pressor response to 5 micrograms angiotensin I (AI) in one foetus and attenuated the pressor response in the other. 3. Enalapril (100 mg, 5.7 mumol kg-1) given i.v. to the ewe (n = 5) abolished the maternal pressor response to 2.5 micrograms AI (n = 1) and attenuated the maternal pressor response to 5 micrograms AI (n = 5, P < 0.001). The foetal pressor response to 5 micrograms AI (n = 2) and 10 micrograms AI (n = 3) did not change. The maternal and foetal pressor responses to angiotensin II (AII; n = 5) did not change. 4. Foetal pressor responses to 5 micrograms AI (n = 1) and 10 micrograms AI (n = 2) were attenuated within 11 min of their mothers (n = 3) being given i.v. captopril (15 mg, 1.5 mumol kg-1). Foetal pressor responses to 5 micrograms AII (n = 1) and to 10 micrograms AII (n = 2) did not change. 5. Losartan (100 mg, kg-1, 21.7 mumol kg-1) given i.v. to the foetus (n = 9) attenuated the foetal pressor response to 5 micrograms AII (P < 0.001) but the maternal pressor response to 5 micrograms AII did not change. 6. Losartan (100 mg, 21.7 MICROmol kg-1) given i.v. to the ewe (n = 5) attenuated the maternal pressor response to 5 microg AII (P <0.002) but the foetal pressor response to 5 microg AII did not change.7. It is concluded that the foeto-placental unit of the sheep can metabolize enalapril to enalaprilat.Captopril readily crosses the sheep placenta but enalapril and losartan do not. Thus, the transplacental transfer of these drugs does not parallel their lipid solubilities. Furthermore the results show that AT1 receptors are important in mediating the vasoconstrictor effects of AII in the foetus.

1. The present study examined the responses of the rabbit carotid artery to five vasoconstrictors after neo-intima formation induced by perivascular collar treatment and evaluated the role of constitutive and inducible nitric oxide (NO) synthase and endothelial cells (ECs). 2. Ring segments of the rabbit carotid artery were mounted in organ chambers for isometric tension recording. Neo-intima-bearing vessels developed less force (Emax) in response to KCl, the thromboxane-mimetic U-46619 and 5-hydroxytryptamine (5-HT), but not to angiotensin I and II. 3. The collar-treatment increased the sensitivity to 5-HT, and decreased the sensitivity to angiotensin II. The sensitivity to U-46619 and angiotensin I remained unchanged. 4. Mechanical removal of ECs and inhibition of NO biosynthesis by NG-monomethyl-L-arginine (L-NMMA) and NG-nitro-L-arginine (L-NOARG) increased the sensitivity to 5-HT in sham and collar-treated segments to the same extent. The effects of collar-treatment and endothelial removal or treatment with inhibitors of NO biosynthesis were additive. Inhibition of NO biosynthesis failed to augment sensitivity to 5-HT after endothelial denudation. L-NOARG increased the force development to KCl in sham and collar-treated segments to the same extent. However, L-NMMA and L-NOARG failed to augment the contractile responses of neo-intima-bearing vessels to 5-HT and KCl after endothelial removal. 5. The responses to angiotensin I were not altered, either by the neo-intima or by endothelial removal. In arteries with a neo-intima the sensitivity to angiotensin II was decreased. Removal of the endothelium or incubation with L-NOARG counteracted this rightward shift and increased Emax.6. Our results demonstrate that contractions to 5-HT, angiotensin II and KCl are modulated by NO in both sham and neo-intima-bearing vessels. Inhibition of NO biosynthesis and collar treatment resulted in additive effects on the EC50 values, suggesting that the 5-HT and angiotension (AT) receptors on the smooth muscle cells are also modified by the formation of a neo-intima. Furthermore, the reduced contractile responses of segments with a neo-intima are not due to NO formed by an inducible NO synthase in those vessels.

1. In order to determine whether the renin-angiotensin system is involved in myocardial ischaemia-reperfusion injury, we investigated and compared the effects on infarct size of two different drugs which interfere with this system, i.e., an angiotensin II (AT1) antagonist, EXP3174, and an angiotensin I-converting enzyme inhibitor (ACEI), enalaprilat in a canine model of ischaemia-reperfusion. 2. EXP3174 (0.1 mg kg-1, i.v. followed by 0.02 mg kg-1 h-1 for 5.5 h) and enalaprilate (0.3 mg kg-1, i.v. followed by 0.06 mg kg-1 h-1 for 5.5 h) were used in doses inducing a similar level of inhibition (87 +/- 4 and 91 +/- 3%, respectively) of the pressor responses to angiotensin I. Control animals received saline. 3. Infarct size and area at risk were quantified by ex vivo dual coronary perfusion with triphenyltetrazolium chloride and monastral blue dye. Regional myocardial blood flows (ischaemic and nonischaemic, endocardial, epicardial) were assessed by the radioactive microsphere technique. 4. Both EXP3174 and enalaprilat induced a decrease in mean arterial blood pressure. However, non significant changes in regional myocardial blood flows, whether ischaemic or nonischaemic, were observed after administration of either the ACEI or the AT1 antagonist. 5. The size of the area at risk was similar in the three groups. By direct comparison, there were no significant differences between infarct sizes in the three groups. Furthermore, there was a close inverse relationship between infarct size and transmural mean collateral blood flow in controls, and none of the treatments altered this correlation. Thus, neither EXP3174 nor enalaprilat limited infarct size. 6. These results indicate that activation of the renin-angiotensin system does not contribute to myocyte death in this canine ischaemia/reperfusion model.

1. The pharmacological profile of valsartan, (S)-N-valeryl-N-([2'-(1H-tetrazol-5-yl)biphenyl-4-yl]-methyl)-vali ne, a potent, highly selective, and orally active antagonist at the angiotensin II (AII) AT1-receptor, was studied in vitro and in vivo. 2. Valsartan competed with [125I]-AII at its specific binding sites in rat aortic smooth muscle cell membranes (AT1-receptor subtype) with a Ki of 2.38 nM, but was about 30,000 times less active in human myometrial membranes (AT2-receptor subtype). 3. In rabbit aortic rings incubated for 5 min with valsartan, at concentrations of 2, 20 and 200 nM, the concentration-response curve of AII was displaced to the right and the maximum response was reduced by 33%, 36% and 40%, respectively. Prolongation of the incubation time with valsartan to 1 h or 3 h, further reduced the maximum response by 48% or 59% (after 20 nM) and by 59% or 60% (after 200 nM) respectively. After 3 h incubation an apparent pKb value of 9.26 was calculated. Contractions induced by noradrenaline, 5-hydroxytryptamine, or potassium chloride were not affected by valsartan. No agonistic effects were observed in the rabbit aorta at concentrations of valsartan up to 2 microM. 4. In bovine adrenal glomerulosa, valsartan inhibited AII-stimulated aldosterone release without affecting the maximum response (pA2 8.4). 5. In the pithed rat, oral administration of valsartan (10 mg kg-1) shifted the AII-induced pressor response curves to the right, without affecting responses induced by the electrical stimulation of the sympathetic outflow or by noradrenaline. Animals treated with valsartan 24 h before pithing also showed significant inhibition of the response to AII. 6. In conscious, two-kidney, one-clip renal hypertensive rats (2K1C), valsartan decreased blood pressure in a dose-dependent manner after single i.v. or oral administration. The respective ED30 values were 0.06 mg kg-1 (i.v.) and 1.4 mg kg-1 (p.o.). The antihypertensive effect lasted for at least 24 h after either route of administration. After repeated oral administration for 4 days (3 and 10 mg kg-1 daily), in 2K1C renal hypertensive rats, systolic blood pressure was consistently decreased, but heart rate was not significantly affected. 7. In conscious, normotensive, sodium-depleted marmosets, valsartan decreased mean arterial pressure, measured by telemetry, after oral doses of 1-30 mg kg-1. The hypotensive effect persisted up to 12 h after 3 and 10 mg kg-1 and up to 24 h after 30 mg kg-1. 8. In sodium-depleted marmosets, the hypotensive effect of valsartan lasted longer than that of losartan(DuP 753). In renal hypertensive rats, both agents had a similar duration (24 h), but a different onset of action (valsartan at 1 h, losartan between 2 h and 24 h).9. These results demonstrate that valsartan is a potent, specific, highly selective antagonist of AII at theAT1-receptor subtype and does not possess agonistic activity. Furthermore, it is an efficacious, orally active, blood pressure-lowering agent in conscious renal hypertensive rats and in conscious normotensive,sodium-depleted primates.

1. Conscious, Long Evans rats were chronically instrumented with pulsed Doppler flow probes and intravascular catheters to allow regional haemodynamic (coeliac, mesenteric and hindquarters vascular beds) responses to i.v. bradykinin to be assessed in the absence and presence of captopril and of ganglion blockade (with mecamylamine). 2. Bradykinin (3 nmol kg-1, i.v. bolus) had no effect on mean arterial blood pressure, although it caused hyperaemic vasodilatation in the coeliac, mesenteric and hindquarters vascular beds. Following administration of captopril at a dose (28 nmol kg-1) which had no effect on responses to angiotensin I, the hypotensive and coeliac and mesenteric vasodilator responses to bradykinin were enhanced. However, there was a temporal dissociation between these events indicating that changes in cardiac output must have been contributing to the changes in mean arterial blood pressure. 3. Captopril at a higher dose (280 nmol kg-1) caused reversible inhibition of the pressor and coeliac and mesenteric vasoconstrictor effects of angiotensin I, but the inhibition of the mesenteric vascular responses was significantly less than that of the coeliac vascular responses. Under the same conditions, the mesenteric vasodilator effects of bradykinin were less enhanced than the coeliac vasodilator effects, consistent with greater inhibition of angiotensin-converting enzyme (i.e., kininase II) in the coeliac than in the mesenteric vascular bed. But, since the hypotensive action of bradykinin was markedly enhanced in these circumstances, the possibility existed that baroreflex responses influenced the haemodynamic effects of bradykinin. However, assessment of the haemodynamic changes following bradykinin administration(bolus or infusion) in the presence of ganglion blockade showed that only the hindquarters vasodilator response to bradykinin was enhanced, while the coeliac and mesenteric vasodilator responses were diminished. Thus, additional factors must have been influencing the latter responses.4. The results show that inhibition of angiotensin-converting enzyme (kininase II) can have differential effects on the regional haemodynamic responses to angiotensin I and bradykinin. The results provide a striking illustration of our previous assertion that the measurement of arterial blood pressure alone cannot provide sufficient information to allow interpretation either of the effects of vasoactive substances,or of the influence of drugs thereupon.

1. Regional haemodynamic responses to atrial natriuretic peptide (ANP, 0.5 nmol kg-1) or proendothelin-1 [1-38] (1.0 nmol kg-1) were assessed in the same conscious Long Evans rats before and 20 min after administration of the novel neutral endopeptidase (NEP) inhibitor, SQ 28,603 (50 mg kg-1, i.v.). In a separate experiment, responses to endothelin-1 (0.5 nmol kg-1), angiotensin I (0.25 nmol kg-1) and angiotensin II (0.12 nmol kg-1) were measured before and after administration of SQ 28,603. 2. SQ 28,603 alone had no significant cardiovascular effects but caused significant prolongation of the hypotensive, tachycardic and renal and mesenteric vasoconstrictor effects of ANP. However, the early vasodilator and late vasoconstrictor responses in the hindquarters were not affected significantly. 3. SQ 28,603 caused significant attenuation of the pressor effects of proendothelin-1 [1-38] but the associated bradycardia was unchanged. SQ 28,603 caused a significant inhibition of the renal and mesenteric vasoconstrictor effects of proendothelin-1 and also inhibited the initial vasodilator and subsequent vasoconstrictor responses in the hindquarters vascular bed. 4. SQ 28,603 had no significant effects on the haemodynamic responses to endothelin-1, angiotensin I, or angiotensin II. 5. The results are consistent with SQ 28,603 not only inhibiting NEP that is involved in the degradation of ANP, but also suppressing activity of the enzyme involved in the conversion of proendothelin-1 [1-38] to endothelin-1.

1. Changes in tension in response to cumulative additions of angiotensins (i.e., angiotensinogen, angiotensin I and angiotensin II), bradykinin and acetylcholine were monitored isometrically on ring preparations from porcine interlobar renal arteries. 2. Angiotensins consistently elicited contractile responses, whereas both bradykinin and acetylcholine produced relaxation of the arterial rings when active tone was induced by prostaglandin F2 alpha. 3. Contractile responses to angiotensin II could be completely blocked by the combined action of the cyclo-oxygenase inhibitor, indomethacin (1 microM) and the lipoxygenase inhibitor, nordihydroguairetic acid (NDGA, 10 microM). 4. Relaxant responses to bradykinin were unchanged during blockade of thromboxane A2 synthesis by dazoxiben (30 microM) and proved to be largely resistant to blockade by indomethacin (1 microM) and the prostaglandin I2 (prostacyclin) synthesis inhibitor, tranylcypromine (40 microM). 5. The angiotensin receptor blocker, saralasin (10 and 100 nM) antagonized responses to angiotensinogen, angiotensin I and angiotensin II effectively and with similar potency. Enalaprilic acid, the active metabolite of the converting enzyme inhibitor enalapril (300 nM), attenuated responses to angiotensin I but failed to inhibit those to angiotensinogen up to 1 microM. The serine protease kallikrein (0.001 to 1 mu ml-1) produced a dose-dependent shift to the left of the concentration-response curve for angiotensinogen. 6. It is suggested that the porcine interlobar renal artery possesses a local renin-angiotensin system with activatable angiotensin II forming enzyme(s) within the vessel wall.

The study of vasoactive agents in man often requires accurate measurement of short-lasting changes in blood pressure. Using a noninvasive photoplethysmographic device (Finapres), the authors investigated in normotensive subjects whether rapid increases in blood pressure can be assessed precisely by monitoring finger blood pressure continuously. Six volunteers were studied on two consecutive days. On the first day, increasing doses of angiotensin I were injected intravenously with the aim to find a test dose which raised systolic blood pressure by 25 to 40 mm Hg. After oral administration of a placebo, the same test dose was injected repeatedly over the next 24 hours. On the second day, the subjects took either 6.25 (n = 3) or 25 mg (n = 3) captopril PO and the serial administration of the test dose of angiotensin I was continued for the next 4 hours. After placebo intake there was a good reproducibility of the blood pressure response to angiotensin I with a coefficient of variation of 15 +/- 4.5% (Mean +/- SD, n = 6). Captopril caused a dose-dependent inhibition of the pressor effect of angiotensin I. These data indicate that noninvasive blood pressure monitoring at the finger represents a useful tool to study short-lasting blood pressure changes produced by vasoactive agents in man.

1. FPL 63547, in its active diacid form, was a potent inhibitor of rabbit lung angiotension converting enzyme (ACE) in vitro (IC50 0.51 nM). 2. In conscious normotensive dogs, FPL 63547 (10-300 micrograms kg-1 i.v.) produced prolonged, dose-related inhibition of plasma ACE activity and angiotensin I pressor responses, without affecting basal blood pressure, heart rate or pressor responses to angiotensin II. 3. In anaesthetized dogs, FPL 63547 diacid (3-300 micrograms kg-1 i.v. cumulatively) produced dose-related increases in cardiac output accompanied by falls in total peripheral resistance indicative of vasodilatation. Mild stimulation of cardiac rate and contractility was also observed. Enalapril diacid had a similar profile. 4. FPL 63547 was a highly effective antihypertensive agent after oral administration to spontaneously hypertensive rats (SHR) pretreated with a diuretic. It lowered systolic blood pressure (SBP) on acute administration over the range 3 X 10(-7)-10(-5) mol kg-1 p.o. (congruent to 0.13-4.5 mg kg-1 p.o.). FPL 63547 was more potent than other ACE inhibitors tested, threshold active doses for lisinopril, enalapril and captopril being 10(-6), 10(-6) and 3 X 10(-5) mol kg-1 p.o., respectively. The antihypertensive effects of FPL 63547, unlike those of enalapril and captopril, were of long duration. 5. The antihypertensive efficacy of FPL 63547 was also observed following chronic oral administration. A dose of 0.5 mg kg-1 day-1 once daily for 23 days produced a sustained reduction of SBP. By the end of the treatment period, SBP was significantly lowered both pre- and post-dose, i.e. effective 24 h control had been achieved. 6. The profile of FPL 63547 is consistent with it being a potent, selective and long-acting ACE inhibitor. As an antihypertensive agent in SHR it compared favourably with other members of this class with respect to potency and duration of action.

The activity of serum angiotensin converting enzyme (ACE) for healthy horses was 64 +/- 13 mUnits/ml. In vitro, equine serum ACE was sensitive to the following inhibitors (IC50): enalapril (570 nM or 215 ng/ml), captopril (190 nM or 41.3 ng/ml), and enalaprilat (6 nM or 2.1 ng/ml). The intravenous (i.v.) administration of angiotensin I to six healthy horses produced a dose proportional pressor response. The maximal increase in mean arterial pressure over baseline values was 65.6 mmHg at angiotensin I doses of 500 ng/kg bodyweight (bwt). The attenuation of this response to angiotensin I was further demonstrated in a single horse by the prior administration of enalaprilat at an i.v. dose of 8.71 micrograms/kg bwt.

The factors which regulate the disappearance of endogenous active substances during pulmonary transit are discussed. The presence of hydrolytic enzymes in the cytosol is not the only factor regulating pulmonary metabolism. An uptake-transport process is required to permit access to intracellular enzymes. No endothelial cell transport mechanism has yet been described for peptides. However, bradykinin and angiotensin I are metabolized by the pulmonary circulation because the enzyme is localized on the luminal surface of the endothelial cells. The pulmonary and extrapulmonary conversion of angiotensin in rat is still a matter of debate. Evidence is presented which supports the idea that when low doses of angiotensin I (greater than 10 ng) are given intravenously, most of the conversion is pulmonary. With higher doses, extrapulmonary conversion also contributes to the final pressor effect of angiotensin I.

1. To learn how pulmonary vascular injury alters the ability of the lung to metabolize vasoactive autacoids, lung vascular lesions were produced in rats by a single subcutaneous injection of monocrotaline (90 mg kg-1), and the blood pressure responses to angiotensin I (AI), angiotensin II (AII), bradykinin, prostaglandin E2 (PGE2) and substance P were examined. Vasoactive agents were given intravenously or intra-arterially. 2. On histological examination of the lung at 3 weeks after monocrotaline treatment, degeneration or necrotization of endothelial cells was evident. 3. The conversion of AI to AII was only slightly depressed by monocrotaline treatment. On the other hand, the depressor response to intravenously injected bradykinin was enhanced in monocrotaline-treated rats. When the rats were pretreated with indomethacin the depressor response to intravenous bradykinin was the same for both control and monocrotaline-treated groups which suggests that endogenous prostaglandins are involved in the enhancement of the response to bradykinin. 4. In monocrotaline-treated rats the depressor response to intravenous PGE2 was significantly enhanced depending on the period following the treatment, while that to the intra-arterial injection did not differ from control. 5. The data suggest that monocrotaline-induced lung injury impairs the metabolism of PGE2 during pulmonary circulation but has little effect on the conversion of AI to AII and the degradation of bradykinin in rats.

1 The influence of the renin-angiotensin system on plasma beta-endorphin-like immunoreactivity (beta-EI) was investigated in the conscious rat by use of a radioimmunoassay for beta-endorphin without prior extraction.2 Intravenous infusion of angiotensin I, II or (des-1-Asp)angiotensin II (angiotensin III) caused a dose-dependent increase in plasma beta-EI, angiotensin III infusion being less effective than angiotensin I or II. The plasma adrenocorticotrophin (ACTH) levels too were elevated by angiotensin II. The receptor antagonist, saralasin, prevented the angiotensin II-induced beta-EI release as did dexamethasone pretreatment.3 Both the release of beta-EI and the pressor response to angiotensin I were abolished by the converting enzyme inhibitor, captopril (SQ 14225). In contrast, captopril did not affect the action of angiotensin II.4 In view of the appreciable cross-reactivity of beta-lipotropin (beta-LPH) in our assay, plasma beta-EI was analysed by Sephadex G-50 chromatography. In plasma extracts of angiotensin II-infused rats, immunoreactivity corresponding to human beta-endorphin comprised about 49% of the total immunoreactivity, whereas 51% co-migrated with human beta-LPH.5 The increase in plasma levels of beta-EI elicited by angiotensin II was diminished by about 35% in rats with a hereditary absolute lack of vasopressin (Brattleboro rats), when compared to normal rats.6 These results suggest that the renin-angiotensin system can stimulate the secretion of beta-LPH and beta-endorphin with ACTH from rat anterior pituitary. One link in mediating the response appears to be vasopressin. The physiological function remains to be defined.

Perfusion of carbogen gas through the lumen of the rat caudal artery abolished the dilator response to acetylcholine (1 mumol 1(-1) in artery segments which had been precontracted with noradrenaline (50 nmol 1(-1]. Hence, it was assumed that gas perfusion was effective in removing the vascular endothelium. Angiotensin I (30 nmol 1(-1] and angiotensin II (10 nmol 1(-1] enhanced the responses of artery segments to field stimulation of their sympathetic nerves (0.5 Hz, 10 s). In arteries with an intact endothelium the ability of each peptide to enhance responses to stimulation was the same whether applied through the lumen or to the adventitial surface. Removal of the endothelium, by gas perfusion, did not significantly alter the facilitatory effects of extraluminally or intraluminally applied angiotensin I or angiotensin II. The converting enzyme inhibitor enalaprilat was equally effective in inhibiting the facilitatory effect of angiotensin I in the presence and absence of an intact endothelium. It is concluded that in the rat caudal artery, conversion of angiotensin I to angiotensin II does not depend on an intact endothelium and that the facilitatory effect of angiotensin II on noradrenergic neuroeffector transmission is not modified by, or dependent on, an intact endothelium.

The subtypes of alpha-adrenoceptors which mediate pressor responses to sympathomimetic agonists or to nerve stimulation in pithed rabbits have been classified according to the effects of 'selective' antagonists and a comparison has been made, for the alpha 2-subtype, with corresponding responses in the rat. In the rabbit the dose-response curve for phenylephrine was shifted to the right in parallel by prazosin (1 mg kg-1) and was unaffected by rauwolscine (1 mg kg-1). The dose-response curve for noradrenaline was shifted to the right by prazosin (1 mg kg-1) and was shifted to a smaller extent by rauwolscine (1 mg kg-1) or imiloxan (10 mg kg-1). After rauwolscine, prazosin produced a rightward shift larger than when given alone. After prazosin, rauwolscine produced a rightward shift larger than when given alone. The responses to pressor nerve stimulation at low frequencies (less than 1 Hz) could be reduced by prazosin, rauwolscine or imiloxan but those at a higher frequency could be reduced only by prazosin. These results indicate that the responses to noradrenaline or to nerve stimulation are mediated by both alpha 1- and alpha 2-adrenoceptors. Low doses or frequencies have a proportionately greater component which is alpha 2. Responses to noradrenaline after prazosin (1 mg kg-1), were sufficiently sensitive to rauwolscine to be considered as predominantly alpha 2. A comparison was therefore made of such responses in the rat and rabbit. They were produced by a lower dose per unit body weight in the rat whereas this was less marked for the alpha 2-adrenoceptor agonist guanabenz. In the rabbit they were more susceptible to blockade by rauwolscine but were less sensitive to Wy 26703 than in the rat. This demonstrates that the alpha 2-adrenoceptors mediating pressor responses in vivo, like those in other tissues in vitro, are different in rat and rabbit, with regard to antagonists.

Epithelial sheets from rat jejunum and descending colon have been shown to respond to angiotensin II (AII) when studied under short-circuit conditions and bathed on both sides with Krebs-Henseleit solution. The octapeptide AII elicited increases in short-circuit current (SCC) in preparations of jejunum and decreases in SCC in the descending colon; both responses occurred when the peptide was applied to the basolateral surface, but not when applied to the apical solution. Responses in both tissues were highly specific, being inhibited by a range of AII antagonists with the following order of potency: [Sar1. Thr8]-AII greater than [Sar1. Leu8]-AII greater than [Sar1. Ile8]-AII greater than [Sar1. Ala8]-AII greater than [Des,Asp1. Ile8]-AII in rat jejunum. AII responses were not affected by alpha- or beta- adrenoceptor antagonists, atropine or tetrodotoxin. AII responses were totally inhibited by the chloride channel blocker, diphenylamine-2-carboxylate (DPC) while cotransport inhibitors e.g. piretanide and frusemide significantly reduced the size of AII responses in colon and jejunum. These patterns of activity suggest that in the jejunum the responses result from electrogenic chloride secretion. Although AII responses in colon were sensitive to DPC the transporting ions have not yet been identified. Both piroxicam and indomethacin inhibited the increase in SCC elicited by AII in the jejunum, and the reduction in SCC caused by AII in the colon. Taken together these results indicate that eicosanoids are involved in AII responses in both tissues. This is the first study to demonstrate a direct, electrogenic effect for AII on transporting epithelia from the gastrointestinal tract. The responses are most probably initiated by All interacting with previously identified specific All receptors within the epithelial membranes.

1. Based on the findings in the preceding paper we investigated the ability of pulmonary endothelial cells to metabolize prostaglandin F2 alpha (PGF2 alpha) and angiotensin I (AI), and to produce endothelium-derived relaxing factor (EDRF) following lung vascular injury induced by monocrotaline in rats. The isometric tension of pulmonary artery rings isolated from rats 3-5 weeks after an injection of monocrotaline or saline was measured. For comparison the responses to drugs of the artery denuded of endothelium by rubbing were tested. 2. Acetylcholine-induced relaxation of the rings precontracted by noradrenaline was diminished in the artery from monocrotaline-treated rats, depending on the duration after treatment. The diminution was comparable to that in the control artery denuded of endothelium. 3. The contractile response to PGF2 alpha was significantly augmented in the artery injured by monocrotaline. The similar augmentation was observed after the mechanical removal of endothelium in the control artery. Decrease of EDRF was not involved in the enhancement of contractile response to PGF2 alpha in the monocrotaline-injured artery. 4. AI caused a contraction, which was sensitive to captopril, in control rings, and also in monocrotaline-injured rings to a similar degree. Removal of endothelium from the control artery did not modify the response to AI or to AII. 5. These results suggest that the monocrotaline treatment of rats suppresses the ability of pulmonary endothelium to produce EDRF and to degrade prostaglandins. The relative resistance of the AI response to endothelial injury suggests that the existence of converting enzyme is not restricted to the endothelium.

In a single, ascending-dose tolerance study, nine healthy volunteers were given oral pentopril 50 to 750 mg (CGS 13945) in groups of three each. Disposition characteristics of pentopril and its active metabolite (CGS 13934) were determined using plasma concentration and urinary excretion data. The drug was absorbed rapidly following zero-order kinetics. The drug has an apparent volume of distribution of 0.83 L/kg and an oral clearance of about 0.79 L/hr/kg. Urinary excretions, calculated after 125- and 250-mg doses, showed a dose proportional urinary recovery of 21% (+/- 5%) for pentopril and 40% (+/- 5%) for CGS 13934. In the multiple-dose study of 125 mg orally q12h in six healthy subjects, the plasma concentrations for both drug and metabolite showed no appreciable accumulation of either compound, which was expected from their short pharmacokinetic half-lives (pentopril, less than 1 hr; CGS 13934, approximately 2 hr). In a separate pharmacodynamic study, drug and metabolite concentrations were evaluated against angiotensin-I (AI)-induced changes in blood pressure and plasma angiotensin-converting-enzyme (ACE) activity in healthy volunteers after single oral doses (range, 10-500 mg). The pharmacodynamic half-life for plasma ACE inhibition increased with the dose (10 mg, 1.5 hr; 500 mg, 9.8 hr). There was a close relationship between the plasma level of the metabolite and the inhibition of plasma ACE activity and AI-induced pressor response. A hyperbolic function adequately described the dependence of plasma ACE activity on plasma metabolite concentration with a concentration at half-maximal inhibition of 53 ng/mL.

Pressor effects of angiotensin I (AI) and angiotensin II (AII) on the human foetoplacental vasculature were compared in dual-perfused term placental cotyledons in which foetoplacental perfusion pressure was monitored. Arterial injections of 1 nmol doses of AI and AII caused marked increases in perfusion pressure; the mean pressor response to AI was 92.9 +/- 5.8% (mean +/- s.e. mean) of the AII response. The angiotensin-converting enzyme inhibitor captopril at 2.2 microM reversibly reduced the AI response to 13.7 +/- 3.2% (mean +/- s.e. mean) of the AII response, which was unaffected. Saralasin, an AII receptor blocker, at 94 nM reversibly antagonized both AI-and AII-induced increases in foetoplacental perfusion pressure. It is concluded that foetoplacental vasoconstriction elicited by AI is due to its conversion to AII by angiotensin-converting enzyme present in the foetoplacental bed.

1. Isolated preparations of rat anococcygeus muscle were incubated with [3H]-noradrenaline and the efflux of radioactivity induced by stimulation of intramural sympathetic nerves was used as a measure of release of transmitter noradrenaline. Isometric contractile responses were also measured. 2. Angiotensin I (0.03 microM) and angiotensin II (0.03 microM) produced non-sustained contractile responses and enhanced the stimulation-induced (S-I) effluxes of radioactivity as well as the contractile responses to electrical stimulation. These effects were blocked by the angiotensin II receptor antagonist saralasin (0.03 microM), and the effect of angiotensin I, but not angiotensin II, was blocked by the angiotensin converting enzyme inhibitor captopril (0.1 microm). 3. The findings indicate that there are both pre- and postjunctional receptors for angiotensin II and that angiotensin I is converted to angiotensin II in the anococcygeus muscle preparation. 4. Isoprenaline (0.1 microM) slightly enhanced the S-I efflux of radioactivity, and produced a greater enhancement after neuronal uptake blockade with desipramine (0.03 microm) and alpha-adrenoceptor blockade with phentolamine (1 microM). 5. The facilitatory effect of isoprenaline on S-I efflux of radioactivity was abolished by propranolol (0.3 microM), but was not affected by low concentrations of saralasin (0.03 microM) or captopril (0.1 microM) which abolished the effect of angiotensin I. The findings suggest that isoprenaline acts directly on prejunctional beta-adrenoceptors to enhance S-I noradrenaline release, rather than indirectly by releasing angiotensin II from within the tissue. Higher concentrations of saralasin (0.1 microM) or captopril (5 microM) did block the facilitatory effect of isoprenaline. The significance of this finding is not clear.