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Angiotensin II (Ang II) is well-considered to be the principal effector of the renin–angiotensin system (RAS), which binds with strong affinity to the angiotensin II type 1 (AT1R) and type 2 (AT2R) receptor subtype. However, activation of both receptors is likely to stimulate different signalling mechanisms/pathways and produce distinct biological responses. The haemodynamic and non-haemodynamic effects of Ang II, including its ability to regulate blood pressure, maintain water–electrolyte balance and promote vasoconstriction and cellular growth are well-documented to be mediated primarily by the AT1R. However, its biological and functional effects mediated through the AT2R subtype are still poorly understood. Recent studies have emphasized that activation of the AT2R regulates tissue and organ development and provides in certain context a potential counter-regulatory mechanism against AT1R-mediated actions. Thus, this review will focus on providing insights into the biological role of the AT2R, in particular its actions within the renal and cardiovascular system.

The renin–angiotensin system (RAS) plays a commanding role in the regulation of extracellular fluid homoeostasis. Tigerstadt and Bergman first identified the RAS more than two centuries ago. By the 1980s a voyage of research and discovery into the mechanisms and actions of this system led to the development of drugs that block the RAS, which have become the mainstay for the treatment of cardiovascular and renal disease. In the last 25 years new components of the RAS have come to light, including the angiotensin type 2 receptor (AT2R) and the angiotensin-converting enzyme 2 (ACE2)/angiotensin-(1–7) [Ang(1–7)]/Mas receptor (MasR) axis. These have been shown to counter the classical actions of angiotensin II (AngII) at the predominant angiotensin type 1 receptor (AT1R). Our studies, and those of others, have demonstrated that targeting these depressor RAS pathways may be therapeutically beneficial. It is apparent that the evolution of both the pressor and depressor RAS pathways is distinct throughout life and that the depressor/pressor balance of the RAS vary between the sexes. These temporal patterns of expression suggest that therapies targeting the RAS could be optimized for discrete epochs in life.

The presence of angiotensin type 2 (AT2) receptors in mitochondria and their role in NO generation and cell aging were recently demonstrated in various human and mouse non-tumour cells. We investigated the intracellular distribution of AT2 receptors including their presence in mitochondria and their role in the induction of apoptosis and cell death in cultured human uterine leiomyosarcoma (SK-UT-1) cells and control human uterine smooth muscle cells (HutSMC). The intracellular levels of the AT2 receptor are low in proliferating SK-UT-1 cells but the receptor is substantially up-regulated in quiescent SK-UT-1 cells with high densities in mitochondria. Activation of the cell membrane AT2 receptors by a concomitant treatment with angiotensin II and the AT1 receptor antagonist, losartan, induces apoptosis but does not affect the rate of cell death. We demonstrate for the first time that the high-affinity, non-peptide AT2 receptor agonist, Compound 21 (C21), penetrates the cell membrane of quiescent SK-UT-1 cells, activates intracellular AT2 receptors and induces rapid cell death; approximately 70% of cells died within 24 h. The cells, which escaped cell death, displayed activation of the mitochondrial apoptotic pathway, i.e. down-regulation of the Bcl-2 protein, induction of the Bax protein and activation of caspase-3. All quiescent SK-UT-1 cells died within 5 days after treatment with a single dose of C21. C21 was devoid of cytotoxic effects in proliferating SK-UT-1 cells and in quiescent HutSMC. Our results point to a new, unique approach enabling the elimination non-cycling uterine leiomyosarcoma cells providing that they over-express the AT2 receptor. Abbreviations: Ang, II, angiotensin II; AT1, angiotensin type 1; AT2, angiotensin type 2; ATIP, AT2 receptor interacting proteins; C21, Compound 21; DMEM, Dulbecco’s modified Eagle’s medium; 18F-FDG, [18F]2-fluoro-2-deoxy-D-glucose; HutSMC, human uterine smooth muscle cells; LC–MS, liquid chromatography–mass spectrometry; LDH, lactate dehydrogenase; RAS, renin–angiotensin system; RT, reverse transcription; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling

CHF (chronic heart failure) is a multifactorial disease process that is characterized by overactivation of the RAAS (renin–angiotensin–aldosterone system) and the sympathetic nervous system. Both of these systems are chronically activated in CHF. The RAAS consists of an excitatory arm involving AngII (angiotensin II), ACE (angiotensin-converting enzyme) and the AT1R (AngII type 1 receptor). The RAAS also consists of a protective arm consisting of Ang-(1–7) [angiotensin-(1–7)], the AT2R (AngII type 2 receptor), ACE2 and the Mas receptor. Sympatho-excitation in CHF is driven, in large part, by an imbalance of these two arms, with an increase in the AngII/AT1R/ACE arm and a decrease in the AT2R/ACE2 arm. This imbalance is manifested in cardiovascular-control regions of the brain such as the rostral ventrolateral medulla and paraventricular nucleus in the hypothalamus. The present review focuses on the current literature that describes the components of these two arms of the RAAS and their imbalance in the CHF state. Moreover, the present review provides additional evidence for the relevance of ACE2 and Ang-(1–7) as key players in the regulation of central sympathetic outflow in CHF. Finally, we also examine the effects of exercise training as a therapeutic strategy and the molecular mechanisms at play in CHF, in part, because of the ability of exercise training to restore the balance of the RAAS axis and sympathetic outflow. Abbreviations: ACE, angiotensin-converting enzyme; Ang-(1–7), angiotensin-(1–7); AngII, angiotensin II; AP-1, activator protein-1; AT1R, AngII type 1 receptor; AT2R, AngII type 2 receptor; CHF, chronic heart failure; CNS, central nervous system; CVO, circumventricular organ; ExT, exercise training; GRK, G-protein receptor kinase; icv, intracerebroventricular; MI, myocardial infarction; NE, noradrenaline; NF-κB, nuclear factor κB; NTS, nucleus of the solitary tract; PVN, paraventricular nucleus; RAAS, renin–angiotensin–aldosterone system; RAS, renin-angiotensin system; RSNA, renal sympathetic nerve activity; RVLM, rostral ventrolateral medulla; SFO, subfornical organ

Lessons learned from the characterization of the biological roles of Ang-(1–7) [angiotensin-(1–7)] in opposing the vasoconstrictor, proliferative and prothrombotic actions of AngII (angiotensin II) created an underpinning for a more comprehensive exploration of the multiple pathways by which the RAS (renin–angiotensin system) of blood and tissues regulates homoeostasis and its altered state in disease processes. The present review summarizes the progress that has been made in the novel exploration of intermediate shorter forms of angiotensinogen through the characterization of the expression and functions of the dodecapeptide Ang-(1–12) [angiotensin-(1–12)] in the cardiac production of AngII. The studies reveal significant differences in humans compared with rodents regarding the enzymatic pathway by which Ang-(1–12) undergoes metabolism. Highlights of the research include the demonstration of chymase-directed formation of AngII from Ang-(1–12) in human left atrial myocytes and left ventricular tissue, the presence of robust expression of Ang-(1–12) and chymase in the atrial appendage of subjects with resistant atrial fibrillation, and the preliminary observation of significantly higher Ang-(1–12) expression in human left atrial appendages.

The activation of the renin–angiotensin system (RAS) is one of the unfavourable characteristics of calcium channel blocker (CCB). N type calcium channel is thought to be involved in renin gene transcription and adrenal aldosterone release. Accordingly, N/L type CCB has a possibility of less elevation of plasma aldosterone concentrations (PAC) among CCBs. In a monotherapy study, we had already demonstrated that N/L type CCB leads to less activation of the RAS compared with L type CCB. The objective of this study is to substantiate the hypothesis that at the condition of additive administration on the top of an angiotensin receptor blocker (ARB), still N/L type CCB leads to less elevation of PAC compared with L type one. Subjects were 60 hypertensives administered with valsartan. As an open label study, amlodipine (L type) or cilnidipine (N/L type) were administered on the top of valsartan (ARB) in a cross-over manner. Results were as follows (valsartan+amlodipine compared with valsartan+cilnidipine): systolic blood pressure (SBP)/diastolic blood pressure (DBP) (mmHg): 132±10/76±10 compared with 131±10/77±9, P=0.95/0.48, plasma renin activity (PRA) (ng/ml·h): 2.41±2.67 compared with 2.00±1.50 P=0.20, PAC (pg/ml): 77.3±31.0 compared with 67.4±24.8, P

The RAS (renin–angiotensin system) is activated after MI (myocardial infarction), and RAS blockade with ACEis [ACE (angiotensin-converting enzyme) inhibitors] or ARBs (angiotensin receptor blockers) slows but does not completely prevent progression to heart failure. Cardiac ACE is increased after MI and leads to the formation of the vasoconstrictor AngII (angiotensin II). The enzyme ACE2 is also activated after MI and degrades AngII to generate the vasodilator Ang-(1–7) [angiotensin-(1–7)]. Overexpression of ACE2 offers cardioprotective effects in experimental MI, but there is conflicting evidence as to whether the benefits of ACEis and ARBs are mediated through increasing ACE2 after MI. In the present study, we assessed the effect of an ACEi and ARB, alone and in combination, on cardiac ACE2 in a rat MI model. MI rats received vehicle, ACEi (ramipril; 1 mg/kg of body weight), ARB (valsartan; 10 mg/kg of body weight) or combination (ramipril at 1 mg/kg of body weight and valsartan at 10 mg/kg of body weight) orally for 28 days. Sham-operated rats were also studied and received vehicle alone. MI increased LV (left ventricular) mass (P

AT1R (angiotensin type 1 receptor) and AT2R (angiotensin type 2 receptor) are well known to be involved in the complex cardiovascular actions of AngII (angiotensin II). However, shorter peptide fragments of AngII are thought to have biological activity in their own right and elicit effects that oppose those mediated by AngII. In the present study, we have used HEK (human embryonic kidney)-293 cells stably transfected with either AT1R or AT2R to perform a systematic analysis of binding affinities of all the major angiotensin peptides. Additionally, we tested the novel AT2R agonist Compound 21, as well as the MasR (Mas receptor) agonist and antagonist AVE0991 and A-779 respectively, for their ability to bind to AT1R or AT2R. Candesartan, CGP42214 and PD123319 were used as reference compounds. Binding studies using 125I-[Sar1Ile8]AngII on the AT1R-transfected HEK-293 cells revealed only AngII, AngIII [angiotensin III; angiotensin-(2–8)] and candesartan to have high affinity for AT1R. In the AT2R-transfected HEK-293 cells, competition for 125I-[Sar1Ile8]AngII binding was observed for all ligands except candesartan, AVE0991 and A-779, the latter two compounds having negligible affinity at either AT1R or AT2R. The rank order of affinity of ligands at AT2R was CGP42112>AngII≥AngIII>Compound 21≥PD123319≫AngIV [angiotensin IV; angiotensin-(3–8)]>Ang-(1–7) [angiotensin-(1–7)]. Of note, although AngIV and Ang-(1–7) exhibited only modest affinity at AT2R compared with AngII, these two angiotensin peptides, together with AngIII, had substantial AT2R selectivity over AT1R. Collectively, our results suggest that shorter angiotensin peptides can act as endogenous ligands at AT2R.

The RAS (renin–angiotensin system) is classically involved in BP (blood pressure) regulation and water–electrolyte balance, and in the central nervous system it has been mostly associated with homoeostatic processes, such as thirst, hormone secretion and thermoregulation. Epilepsies are chronic neurological disorders characterized by recurrent epileptic seizures that affect 1–3% of the world's population, and the most commonly used anticonvulsants are described to be effective in approx. 70% of the population with this neurological alteration. Using a rat model of epilepsy, we found that components of the RAS, namely ACE (angiotensin-converting enzyme) and the AT1 receptor (angiotensin II type 1 receptor) are up-regulated in the brain (2.6- and 8.2-fold respectively) following repetitive seizures. Subsequently, epileptic animals were treated with clinically used doses of enalapril, an ACE inhibitor, and losartan, an AT1 receptor blocker, leading to a significant decrease in seizure severities. These results suggest that centrally acting drugs that target the RAS deserve further investigation as possible anticonvulsant agents and may represent an additional strategy in the management of epileptic patients.

The TLR4 (Toll-like receptor 4) signal plays an important role in immunity in CAD (coronary artery disease). miR-146a/b (where miR is microRNA) regulates the TLR4 downstream molecules IRAK1 (interleukin-1-receptor-associated kinase 1) and TRAF6 (tumour-necrosis-factor-receptor-associated factor 6). It has also been reported that statins and RAS (renin–angiotensin system) inhibition and have anti-atherosclerotic properties. In the present study, we have investigated whether miR-146a/b was expressed with the TLR4 signal in CAD patients, and whether combined treatment with a statin and RAS inhibition might affect these levels. A total of 66 patients with CAD and 33 subjects without CAD (non-CAD) were enrolled. Patients with CAD were randomized to 12 months of combined treatment with atorvastatin and telmisartan [an ARB (angiotensin II receptor blocker)] or atorvastatin and enalapril [an ACEI (angiotensin-converting enzyme inhibitor)]. PBMCs (peripheral blood mononuclear cells) were obtained from peripheral blood at baseline and after 12 months. Levels of miR-146a/b, IRAK1 mRNA, TRAF6 mRNA and TLR4 mRNA/TLR4 protein were significantly higher in the CAD group than in the non-CAD group (all P

The type 2 angiotensin receptor (AT2R) has been suggested to counterbalance the type 1 angiotensin receptor (AT1R) in the central regulation of blood pressure and sympathetic tone. In the present study we investigated the blood pressure responses to stimulation of central AT2Rs by the selective agonist Compound 21 in conscious spontaneously hypertensive rats (SHRs) and normotensive Wistar Kyoto rats (WKY rats). We also assessed the impact on noradrenaline [norepinephrine (NE)] plasma levels, autonomic function, spontaneous baroreflex sensitivity, and the possible involvement of the nitric oxide (NO) pathway and the AT1Rs. Chronic intracerebroventricular Compound 21 infusion lowered blood pressure and NE plasma levels in both rat strains. The night-time hypotensive effect was greater in SHRs compared with WKY rats. Compound 21 improved spontaneous baroreflex sensitivity more in SHRs than in WKY rats. These effects were abolished by co-administration of the AT2R antagonist PD123319 or the NO synthase inhibitor Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME). Central AT1R blockade did not enhance the hypotensive response to Compound 21. Chronic selective stimulation of central AT2Rs lowers blood pressure through sympathoinhibition, and improves spontaneous baroreflex sensitivity more in SHRs than in WKY rats. These responses appear to require a functioning central NO pathway, but are not modified by central AT1R blockade. Collectively, the data demonstrate specific beneficial effects of stimulation of central AT2Rs in hypertension associated with increased sympathetic tone, and suggest that central AT2Rs may represent a potential new therapeutic target for the treatment of neurogenic hypertension.

Lung structural remodelling, characterized by myofibroblast proliferation and collagen deposition, contributes to impaired functional capacity in CHF (congestive heart failure). As the lung is the primary site for the formation of Ang II (angiotensin II), local modifications of this system could contribute to lung remodelling. Rats with CHF, induced following myocardial infarction (MI) via coronary artery ligation, were compared with sham-operated controls. The MI group developed lung remodelling as confirmed by morphometric measurements and immunohistochemistry. Pulmonary Ang II concentrations increased more than 6-fold (P

Ang II (Angiotensin II) has been shown to play a pivotal role in the pathophysiology of various organs, especially the cardiovascular system. The effects of ARBs (Ang II receptor blockers) in the treatment of hypertension, congestive heart failure and myocardial fibrosis have been analysed extensively in human trials, as well as animal models, and the focus of interest is now directed to its pleiotropic effects, especially on inflammatory disorders. To investigate the effects of a new ARB, olmesartan, on immune-mediated myocardial injury, the protective effects of olmesartan on the development of murine acute myocarditis caused by CVB3 (coxsackievirus B3) were analysed. Olmesartan and a non-specific vasodilator hydralazine lowered systolic blood pressure of mice on day 7 after virus inoculation to a similar extent. Olmesartan significantly decreased myocardial inflammation compared with controls, whereas hydralazine significantly increased this. Olmesartan significantly decreased the expression of IFN-γ (interferon-γ), FasL (Fas ligand), iNOS (inducible nitric oxide synthase) and PFP (pore-forming protein) in myocardial tissue, indicating that olmesartan suppressed the activation of infiltrating killer lymphocytes. Olmesartan also decreased the expression of CVB3 genomes in myocardial tissue as well as serum levels of 8-OHdG (8-hydroxy-2′-deoxyguanosine), a biomarker of oxidative-stress-induced DNA damage. The findings suggest that olmesartan prevents myocardial damage and may improve the prognosis of patients with acute myocarditis; however, further investigations are needed before clinical use.

The aim of the present study was to compare the antihypertrophic effects of blockade of the renin-angiotensin system (RAS), vasopeptidase inhibition and calcium channel antagonism on cardiac and vascular hypertrophy in diabetic spontaneously hypertensive rats (SHR). SHR with streptozotocin-induced diabetes were treated with one of the following therapies for 32 weeks: the angiotensin-converting enzyme (ACE) inhibitor captopril (100 mg/kg); the angiotensin AT(1) receptor antagonist valsartan (30 mg/kg); a combination of captopril with valsartan; the vasopeptidase inhibitor mixanpril (100 mg/kg); or the calcium channel antagonist amlodipine (6 mg/kg). Systolic blood pressure and cardiac and mesenteric artery hypertrophy were assessed. Mean systolic blood pressure in diabetic SHR (200+/-5 mmHg) was reduced by captopril (162+/-5 mmHg), valsartan (173+/-5 mmHg), mixanpril (176+/-2 mmHg) and amlodipine (159+/-4 mmHg), and was further reduced by the combination of captopril with valsartan (131+/-5 mmHg). Captopril, valsartan and mixanpril reduced heart and left ventricle weights by approx. 10%. The combination of captopril and valsartan further reduced heart weight (-24%) and left ventricular weight (-29%). Amlodipine did not affect cardiac hypertrophy. Only mixanpril and the combination of captopril and valsartan significantly reduced mesenteric weight. The mesenteric wall/lumen ratio was reduced by all drugs, and to a greater extent by the combination of captopril and valsartan. We conclude that optimizing the blockade of vasoconstrictive pathways such as the RAS, particularly with the combination of ACE inhibition and AT(1) receptor antagonism, is associated with antitrophic effects in the context of diabetes and hypertension. In contrast, calcium channel blockade, despite similar effects on blood pressure, confers less antitrophic effects in the diabetic heart and blood vessels.

Left ventricular (LV) remodelling following myocardial infarction (MI) is associated with increased morbidity and mortality. Previous data suggest that angiotensin II (Ang II) plays a central role in the molecular events contributing to LV remodelling. We explored the effects of angiotensin-converting-enzyme (ACE) inhibition versus Ang II (AT1) receptor blockade on LV remodelling in mice post-MI. Mice underwent sham procedure or left coronary artery ligation, and received placebo, the AT1 receptor antagonist, losartan or the ACE inhibitor, enalapril. At 6 weeks, echocardiography and haemodynamic studies were performed. Infarct size and interstitial collagen content were determined. Expression of genes encoding atrial natriuretic peptide (ANP), collagen type I, AT1a and AT1b receptors were measured. The placebo MI group showed increased LV end-diastolic diameter, LV end-systolic diameter with depressed fractional shortening (P

The objective of the present study was to determine the autonomic effects of angiotensin II (AT1) receptor blocker therapy in heart failure. In a randomized double-blind cross-over study, we compared the effects of candesartan and placebo on baroreflex sensitivity and on heart rate variability at rest, during stress and during 24h monitoring. Acute effects were assessed 4h after oral candesartan (8mg) and chronic effects after 4 weeks of treatment (dose titrated to 16mg daily). The study group comprised 21 patients with heart failure [mean (S.E.M.) ejection fraction 33% (1%)], in the absence of angiotensin-converting enzyme (ACE) inhibitor therapy. We found that acute candesartan was not different from placebo in its effects on blood pressure or mean RR interval. Chronic candesartan significantly reduced blood pressure [placebo, 137 (3)/82 (3)mmHg; candesartan, 121 (4)/75 (2)mmHg; P

Angiotensin II plays a pivotal role in the development of diabetic nephropathy, but it remains controversial as to the best approach to effectively block the actions of this hormone in the kidney. The aim of the present study was to explore the effects of long-term treatment (8 months) with a combination of an angiotensin type 1 (AT1) receptor antagonist, irbesartan (15 mg/kg per day), and an angiotensin-converting enzyme (ACE) inhibitor, captopril (100 mg/kg per day), in diabetic spontaneously hypertensive rats. Captopril treatment reduced blood pressure (163±3 mmHg versus diabetic 201±3 mmHg), but not albumin excretion rate (43.8×/÷1.3 mg/day versus diabetic 46.8×/÷1.4 mg/day). Irbesartan treatment was associated with a similar reduction in blood pressure (173±3 mmHg) to captopril, and albumin excretion rate was reduced (14×/÷1.5 mg/day). The combination of irbesartan and captopril induced further reductions in blood pressure (140±3 mmHg) and albumin excretion rates (4.0×/÷1.5 mg/day). Gene expression of transforming growth factor β-1 was reduced by all treatments to a similar level as assessed by in situ hybridization. These results demonstrate the additive hypotensive and anti-albuminuric effects of an ACE inhibitor and an AT1 receptor, suggesting that combination therapy is an approach not only more effective at reducing blood pressure, but also at retarding the development of diabetic nephropathy.

Angiotensin II (Ang II) is a multi-functional hormone that plays a major role in regulating blood pressure and cardiovascular homoeostasis. The actions of Ang II are mediated by at least two receptor subtypes, designated AT1 and AT2. In addition, other angiotensin receptors have been identified which may recognize other angiotensin peptide fragments; however, until now only the AT1 and AT2 receptor have been cloned in animals or humans. Most of the well-described actions of Ang II, such as vasoconstriction, facilitation of sympathetic transmission, stimulation of aldosterone release and promotion of cellular growth are all mediated by the AT1 receptor. Much less is known about the function of the AT2 receptor, but recent studies suggest that it may play a role in mediating anti-proliferation, cellular differentiation, apoptosis and vasodilatation. In this review, we discuss recent advances in our understanding of Ang II receptors, in particular, their distribution, signalling and function.

We examined the ability of the new non-peptide angiotensin II receptor antagonist irbesartan to inhibit AT(1) receptors in vivo in the rat kidney following oral administration, compared with the prototype drug losartan. Male Sprague-Dawley rats (250-300 g) were gavaged with either irbesartan or losartan at doses of 1, 3, 10, 30 or 100 mg/kg, or with corresponding vehicle. Rats were killed at 0, 1, 2, 8, or 24 h after drug administration, trunk blood was collected and the kidneys were removed. The effects of irbesartan and losartan on angiotensin II receptor binding were determined by quantitative in vitro autoradiography using the specific radioligand (125)I-[Sar(1),Ile(8)]angiotensin II. High levels of angiotensin II receptor binding in the rat kidney were demonstrated in the glomeruli and inner stripe of the outer medulla, which was attributed to AT(1) receptors. At 1 h after dosing, irbesartan (1-100 mg/kg) and losartan (1-30 mg/kg) significantly inhibited AT(1) receptor binding in all anatomical areas of the kidney, in a dose-dependent manner, with a maximal effect at 100 mg/kg and 30 mg/kg respectively. For a 10 mg/kg dose, inhibition of AT(1) receptor binding was maximal around 1-2 h after oral administration of losartan, whereas maximal binding occurred between 2 and 8 h for irbesartan; both drugs produced persistent tissue blockade at 24h. In radioligand binding studies, irbesartan, losartan and EXP3174 (1x10(-10) to 1x10(-5) M) displaced (125)I-[Sar(1),Ile(8)]angiotensin II binding from renal AT(1) receptors in a concentration-dependent manner, with a rank order of potency of irbesartan>EXP3174>losartan. The concentration required to displace 50% of radioligand binding (IC(50)) by irbesartan, EXP3174 and losartan was 1.00+/-0.2 nM, 3.5+/-0.4 nM and 8.9+/-1.1 nM respectively. In conclusion, the findings of the present study suggest that irbesartan and losartan produce effective and sustained inhibition of AT(1) receptors in vivo in the kidney following oral administration. However, irbesartan appears less potent, with respect to dosage, than losartan in vivo, despite having a higher affinity for AT(1) receptors in vitro. The reason for this apparent discrepancy is unclear, but it may reflect the slower onset of action of irbesartan and its rate of tissue accessibility. Inhibition of angiotensin II receptors in target tissues such as the kidney may represent an important action of AT(1) receptor antagonists, which may contribute to the beneficial effects of these agents in the clinical setting.

We have investigated the role of cytochrome P450 (CYP-450) metabolites of arachidonic acid in the modulation of vascular reactivity to angiotensin II in vivo using an in situ blood-perfused mesenteric preparation in anaesthetized spontaneously hypertensive rats (SHR). Miconazole, a non-selective inhibitor of CYP-450 that inhibits both hydroxylation and epoxidation, substantially suppressed mesenteric vasoconstrictor responses to angiotensin II in SHR, but had no effect on responses to noradrenaline or sympathetic nerve stimulation. In normotensive Wistar–Kyoto (WKY) rats, miconazole caused only a modest suppression of vasoconstrictor responses to angiotensin II. N-Methylsulphonyl-12,12-dibromododec-11-enamide (DDMS), a new selective inhibitor of CYP-450 ω-hydroxylase activity, decreased mean intra-arterial blood pressure and significantly attenuated mesenteric angiotensin II-induced vasoconstrictor responses in SHR. Isolated mesenteric vessels were able to metabolize 14C-labelled arachidonic acid to hydroxyeicosatetraenoic acids (HETEs) in vitro, and this was substantially inhibited by DDMS. The results from the present studies combined with the existing evidence that angiotensin II stimulates the release of 20-HETE, a CYP-450 metabolite of arachidonic acid, suggest that CYP-450-derived HETEs may be important mediators in angiotensin II-induced vasoconstriction. However, the development of more sensitive assays for the detection in vivo of 20-HETE in mesenteric vessels would be required to confirm these findings.

1. Receptors for angiotensin II were identified and characterized in membrane fractions from myometrial samples obtained at non-pregnant hysterectomy in women of reproductive age. Specific binding was also measured in paired samples of vascular and ‘vessel-free’ myometrium. 2. A single class of high-affinity receptor sites was identified in the total myometrial preparations (n = 10), with a median equilibrium dissociation constant (Kd) of 0.122 nmol/l (range 0.065–0.465 nmol/l) and concentration of receptor sites (Bmax) of 149 fmol/mg protein (range 105–435 fmol/mg). Receptor characterization studies using losartan (an angiotensin type 1 receptor antagonist) and PD123177 (an angiotensin type 2 receptor antagonist) showed the myometrium to contain almost entirely type 2 receptors. 3. The median Kd and Bmax for specific angiotensin II binding in isolated myometrial vessel homogenates were 0.086 nmol/l (range 0.060–0.433) and 260 fmol/mg protein (range 197–737 fmol/mg) respectively. Vascular specific binding density was always higher in dissected out myometrial vessels than in paired vessel-free myometrium (median 372 compared with 120 fmol/mg protein; n = 20; P < 0.001). The specific binding in the paired samples was strongly correlated (r = 0.8904, P < 0.0001). 4. The specific binding of 125I-labelled angiotensin II in ‘vessel-free’ myometrium was higher in samples from the follicular phase than in samples from the luteal phase (median 144 compared with 37 fmol/mg; P < 0.02). A similar trend was found in the vessels themselves, but this failed to reach statistical significance (459 compared with 225 fmol/mg; P = 0.051). 5. It is suggested that the down-regulation of the angiotensin type 2 receptors in the luteal (secretory) phase is a preparation for pregnancy, removing an inhibitory factor to uterine growth and vascularization and allowing greater prostacyclin synthesis.

Exposure of mesangial cells to platelet-derived growth factor (PDGF) BB caused a significant stimulation of cell proliferation and protein synthesis, as measured by [3H]thymidine incorporation and [3H]leucine incorporation respectively. In contrast, cells treated with angiotensin II had no significant increase in [3H]thymidine incorporation, but demonstrated a marked increase in [3H]leucine incorporation. Furthermore, angiotensin II significantly increased total protein content per cell. These data show that, whereas PDGF-BB is a mitogen and stimulates mesangial-cell hyperplasia, angiotensin II causes hypertrophy of the cells without hyperplasia. Treatment of mesangial cells with PDGF and angiotensin II rapidly and dose-dependently stimulated mitogen-activated protein (MAP) kinase activity, as shown by an assay for activity in vitro using myelin basic protein as a substrate, and by immunoprecipitation of 32P-labelled cells with specific antibodies against the 42 kDa and 44 kDa mitogen-activated protein kinases p42mapk and p44mapk, respectively. Whereas stimulation with PDGF-BB caused a potent and sustained (for more than 30 min) phosphorylation and activation of p42mapk and p44mapk, as well as of the upstream activators MAP kinase kinase and c-Raf, the effect of angiotensin II was less potent, reaching a peak at 5-10 min and thereafter declining rapidly. In summary, these results suggest that PDGF-BB and angiotensin II differ in their potency and duration of activation of the MAP kinase cascade, which may explain why PDGF-BB is a potent mitogen for mesangial cells, whereas angiotensin II only triggers mesangial-cell hypertrophy.

1. Decreased pressor sensitivity to angiotensin II occurs in cirrhosis, but the mechanism remains unclear. 2. Angiotensin II dose-response studies were performed in conscious, chronically instrumented cirrhotic and control rats, and angiotensin II concentration-response studies were performed in isolated blood vessels obtained from similar groups of animals. 3. Cirrhotic rats demonstrated a significantly decreased pressor response to angiotensin II (5-80 ng/kg intravenously). However, angiotensin II-generated tension in thoracic aortic rings isolated from cirrhotic rats and studied in vitro was not impaired. These findings are consistent with the concept that circulating vasodilator substances in cirrhosis rather than an abnormality intrinsic to vascular smooth muscle cells are responsible for the decreased pressor sensitivity to angiotensin II in vivo. 4. Pretreatment with the cyclo-oxygenase inhibitor indomethacin (3 mg/kg intravenously) restored pressor sensitivity to angiotensin II to normal, suggesting that cyclo-oxygenase products, possibily vasodilator prostaglandins, may be involved in mediating pressor resistance to this hormone in vivo.

Endothelin has steroidogenic activity in adrenal glomerulosa cells, as do two other vasoconstrictor peptides, angiotensin II and vasopressin. The steroidogenic activities of angiotensin II and vasopressin are probably mediated via the phosphatidylinositol-turnover pathway and associated changes in cytosolic Ca2+ concentration. Endothelin caused a steroidogenic response, which was small compared with that to angiotensin II and quantitatively similar to the vasopressin response. Cytosolic free Ca2+ responses were similarly higher to angiotensin II than to either of the other two peptides. However, total inositol phosphate responses to endothelin and angiotensin II were similar when these were measured over 20 min, and were quantitatively greater than the vasopressin response. A detailed study has been made of the phosphatidylinositol-turnover response to endothelin in comparison with responses to angiotensin II and vasopressin. Each of the three peptides produced a rapid and transient rise in Ins(1,4,5)P3 (max. 5-15 s), followed by a slow sustained rise. Ins(1,4,5)P3 was metabolized by both dephosphorylation and phosphorylation pathways, but the relative importance of the two metabolic pathways was different under stimulation by each of the three peptides. These findings show that adrenal glomerulosa cells can distinguish between the stimulation of phosphatidylinositol turnover by three different effectors. These differences in the pathway may be associated with the observed different steroidogenic and Ca2+ responses to the three peptides.

1. The results are presented of a cross-sectional study of 25 non-pregnant and 125 pregnant/postnatal women in whom platelet angiotensin II binding and plasma angiotensin II, plasma renin concentration and plasma renin substrate were measured. 2. Platelet angiotensin II binding was significantly lower in the first-trimester patients as compared with the non-pregnant women (P < 0.001). Specific binding remained low in the second and third trimesters, and in those patients studied 24 h after delivery. However, higher values, approximating to the non-pregnant level, were found 6 weeks postnatally (n = 25 for each group). 3. Plasma angiotensin II, plasma renin concentration and plasma renin substrate increased in pregnancy, with the increase becoming statistically significant as compared with the non-pregnant women in the second trimester. Maximal median values of plasma angiotensin II and plasma renin substrate were found in the third trimester, but maximal median values of plasma renin concentration were found in the second trimester. The concentrations of all three hormones fell after delivery. 4. There was an inverse correlation between platelet angiotensin II binding and simultaneously measured endogenous levels of plasma angiotensin II (P < 0.02) and plasma renin substrate (P < 0.05) in the 25 non-pregnant subjects. These findings support the concept of the angiotensin II receptor concentration being regulated by the plasma angiotensin II level. There was no correlation between platelet angiotensin II binding and plasma renin concentration. 5. In pregnancy, no correlation between platelet angiotensin II binding and plasma angiotensin II, plasma renin concentration or plasma renin substrate was found. This suggests that any regulation by plasma angiotensin II may not operate in pregnancy, when reduced binding concentrations are present. No statistically significant correlation was found at either 24 h or 6 weeks after delivery.

Proliferation in rat liver T51B cells has previously been shown to be initiated by the tyrosine-kinase activator epidermal growth factor. We have found that T51B cells also contain angiotensin II receptors, and, as the transforming mas oncogene has been identified as a functional angiotensin receptor [Jackson, Blair, Marshall, Goedert & Hanley (1988) Nature (London) 335, 437-440], we have investigated the possibility that angiotensin II might also regulate proliferation of T51B cells. Angiotensin II at concentrations up to 10 microM did not promote DNA synthesis, even in the presence of the co-mitogens serum (1%) or 12-O-tetradecanoylphorbol 13-acetate (TPA) (50 ng/ml). The addition of 1 microM angiotensin II to myo-[3H]inositol-radiolabelled T51B cells did however result in a rapid accumulation of multiple inositol phosphates as well as in an increase in intracellular Ca2+, demonstrating the coupling of the angiotensin receptor in these cells to a polyphosphoinositide-hydrolysing phospholipase C. The increases in both inositol phosphates and intracellular Ca2+ were lower in cells pretreated for 10 min with 50 ng of TPA/ml and potentiated by a 24 h pretreatment with TPA. In addition, angiotensin II increased 1,2-diacylglycerol levels. These results demonstrate that, although angiotensin II is capable of increasing phosphoinositide-derived second messengers in T51B cells, these responses are not sufficient to trigger DNA synthesis.

Activation of phospholipase C by angiotensin II in vascular smooth muscle has been postulated to be mediated by an unidentified GTP-binding protein (G-protein). Using a permeabilized preparation of myo-[3H]inositol-labelled cultured vascular smooth muscle cells, we examined the ability of a non-hydrolysable analogue of GTP, guanosine 5′-[gamma-thio]triphosphate (GTP[S]), to stimulate inositol phosphate formation. GTP[S] (5 min exposure) stimulated inositol polyphosphate release by up to 3.8-fold in a dose-dependent manner, with an EC50 (concn. producing half-maximal stimulation) of approx. 50 microM. Inositol bisphosphate (IP2) and inositol trisphosphate (IP3) accumulations were also stimulated by NaF (5-20 mM). Furthermore, angiotensin II-induced inositol phosphate formation could be potentiated by a submaximal concentration of GTP[S] (10 microM), and this treatment appeared to interfere with the normal termination mechanism of the initial hormonal signal. The G-protein mediating angiotensin II-stimulated phospholipase C activation was insensitive to pertussis toxin at an exposure time and concentration which were sufficient to completely ADP-ribosylate all available substrate (100 ng/ml, 16 h). In contrast, a similar incubation with cholera toxin markedly inhibited angiotensin II-stimulated IP2 and IP3 release by 67 +/- 6% and 62 +/- 6% respectively. Cholera toxin appeared to inhibit angiotensin II stimulation of phospholipase C by a dual mechanism: it caused a 45% decrease in angiotensin II receptor number, and also inhibited G-protein transduction as assessed by GTP[S]-stimulated IP2 formation. This latter inhibition may be secondary to an increase in cyclic AMP, since it could be simulated by addition of dibutyryl cyclic AMP. Thus angiotensin II-stimulated inositol phosphate formation is cholera-toxin-sensitive, and is mediated by a pertussis-toxin-insensitive G-protein, which may be involved directly in termination of early signal generation.

Background Atrial and B-type natriuretic peptide are both known to be antagonists of the renin-angiotensin system. C-type natriuretic peptide (CNP) is a new member of this family except that its principal source is the vascular endothelium. This study tested the hypothesis that CNP is a local inhibitor of vascular angiotensin-converting enzyme (ACE) activity. Methods and Results Vascular ACE activity was assessed by the differential vascular response to angiotensin I and angiotensin II. Healthy male volunteers were studied with the use of brachial artery infusions of angiotensin I and angiotensin II at two doses, with and without coinfusion of CNP at 500 pmol/min (n=8) and hydralazine at 10 μg/min (n=8) (as a nonspecific vasodilator control). CNP alone and hydralazine alone caused similar increases in forearm blood flow (CNP+, 93.0±14.8%; hydralazine+, 84.2±22.6%). CNP inhibited the vasoconstrictive effect of angiotensin I (reduction in overall effect with CNP, 56.8±12.9%; P Conclusions This evidence of a differential effect of CNP on the vascular response to angiotensin I but not to angiotensin II suggests that CNP acts as a local endogenous regulator of vascular ACE activity in the human forearm resistance vessels.

We investigated the binding characteristics of angiotensin receptors and used this assay to determine the predominant enzyme capable of converting angiotensin I in the human left ventricle. In homogenates of human left ventricle, (125)I-[Sar(1),Ile(8)]angiotensin II bound with sub-nanomolar affinity, with a corresponding K(D) of 0.42+/-0.09 nM, a B(max) of 11.2+/-2.3 fmol.mg(-1) protein and a Hill slope of 1.04+/-0.04. The rank order of inhibitory potency of competing ligands for the (125)I-[Sar(1),Ile(8)]angiotensin II binding site was CGP42112>angiotensin II> or =angiotensin III=angiotensin I>losartan. The angiotensin type II (AT(2)) receptor predominated in the human left ventricle over the angiotensin type I (AT(1)) receptor, with an approximate AT(1)/AT(2) receptor ratio of 35:65. No specific (125)I-angiotensin IV binding sites could be detected in the human left ventricle. Using competitive radioligand binding assays, we were able to demonstrate that the chymase/cathepsin G enzyme inhibitor chymostatin was more potent than the angiotensin-converting enzyme (ACE) inhibitor captopril at inhibiting the conversion of angiotensin I in the human left ventricle. Aprotonin (an inhibitor of cathepsin G but of not chymase) had no effect on angiotensin I conversion, suggesting that the majority of the conversion was mediated by chymase. Thus, although the current therapies used for the renin-angiotensin system have focused on ACE inhibitors and AT(1) receptor antagonists, the left ventricle of the human heart expresses mainly AT(2) receptors and the tissue-specific conversion of angiotensin I occurs predominantly via chymase rather than ACE.

Endothelial dysfunction and a consequent decrease in nitric oxide production have been implicated in the pathogenesis of pre-eclampsia. A prominent feature of the pre-eclamptic syndrome is a loss of the pregnancy-induced refractoriness to infused pressor agents, such as angiotensin. In this study, we sought to determine whether a decrease in nitric oxide production might be linked via changes in angiotensin II receptors and angiotensin II metabolism to changes in pressor sensitivity to infused angiotensin II. Pregnant and non-pregnant spontaneously hypertensive rats (SHRs) were randomly allocated to receive 5 mg·kg-1·day-1NG-nitro-L-arginine methyl ester (L-NAME) in the drinking water or drinking water alone from days 7 to 14 of gestation. Steady-state metabolic clearance studies of angiotensin II were then performed, or tissues were harvested for angiotensin II receptor studies. Treatment with L-NAME caused an increase in systolic pressure (P < 0.001) in both pregnant and non-pregnant rats, while urinary protein excretion increased only in the pregnant SHRs (P< 0.001). Plasma angiotensin II levels were significantly increased in the L-NAME-treated SHRs compared with controls (non-pregnant, P< 0.0005; pregnant, P < 0.01). The metabolic clearance rate of angiotensin II was decreased by L-NAME treatment in non-pregnant SHRs (P < 0.05), but was increased by L-NAME treatment in the pregnant rats (P < 0.01). In the aorta, the angiotensin II receptor number increased after treatment with L-NAME in both non-pregnant (P < 0.0005) and pregnant (P < 0.05) SHRs, and the dissociation constant increased in the non-pregnant SHRs (P < 0.005). Thus treatment of SHRs with L-NAME increased blood pressure, as well as the circulating angiotensin II concentration and vascular angiotensin II receptor expression. However, treatment with L-NAME did not affect pressor sensitivity to infused angiotensin II. We conclude, therefore, that although a decrease in nitric oxide production is associated with changes in angiotensin II concentrations and receptor numbers, it does not induce changes in pressor sensitivity to infused angiotensin II in the SHR.

1. Adrenocorticotrophic hormone-suppressed, bilaterally nephrectomized male mongrel dogs (n = 12) were infused with Sar1-angiotensin II and Asp1-angiotensin II, the naturally occurring octapeptide. 2. Sar1-angiotensin II was found to be almost twice as potent as Asp1-angiotensin II in elevating blood pressure but its aldosterone-stimulating activity was not higher than that of the naturally occurring peptide. 3. A specific competitive antagonist of angiotensin II, Sar1-Ile8-angiotensin II, blocked the pressor but not the aldosterone-stimulating activity of Sar1-angiotensin II. 4. These results suggest functional differences in receptors for angiotensin II in vascular smooth muscle and in adrenal cortex.

1. Competitive or non-competitive inhibition of the myotropic and pressor response of angiotensin II is dependent on the nature of the substituent in position 8 of the antagonist peptide analogue. Substituents in other positions of the molecule, particularly position 1, contribute greatly to the potency of these antagonists. 2. As is evidenced after adrenalectomy or after blockade with phentolamine and phenoxybenzamine, the initial pressor activity observed with all the antagonistic peptides is partially due to the release of catecholamines and partially to a direct myotropic effect. 3. [Sar1, Thr8]angiotensin II has been found to possess the lowest agonist to antagonist ratio of all antagonists tested. 4. [Des-Asp1, Ile8]angiotensin II selectively and specifically inhibits the release of aldosterone from adrenal cortex. Thus, unlike angiotensin II, this heptapeptide has pronounced organ specificity, suggesting that the heptapeptide (angiotensin III) is the aldosterone-releasing hormone.

1. The effect of infusions of equimolar doses of angiotensin II (AII) and of the angiotensin analogue Sar1-Ile8-angiotensin II on arterial blood pressure, plasma aldosterone and plasma renin activity were compared in normal anaesthetized dexamethasone suppressed dogs. 2. Angiotensin II induced a significant increase of blood pressure and of plasma aldosterone whereas plasma renin activity decreased. The blood pressure was only slightly affected by large doses of the analogue. Plasma aldosterone, however, increased and plasma renin activity decreased. These changes were significant but less pronounced than after the infusions of angiotensin II. Plasma aldosterone remained high and renin activity low for 40 min after the infusions of the analogue. 3. The results suggest a strong agonistic potency of Sar1-Ile8-angiotensin II at the adrenal and renal angiotensin receptors, and that it is almost ineffective at the vascular receptors. The inhibition of renin secretion by angiotensin seems not to be related to its vasoconstrictive activity.

Specific binding sites of high affinity and low capacity for 125I-angiotensin II have been identified in a membrane fraction derived from arterial arcades of the rat mesentery. Heterogeneity of binding sites and extensive tracer degradation necessitated the use of nonlinear regression methods for the analysis of radioligand binding data. Forward and reverse rate constants for the high affinity sites obtained by three experimental approaches were in good agreement and gave a dissociation equilibrium constant (Kd) of 19-74 pM (95% confidence interval). Affinities for a number of angiotensin-related peptides calculated from competitive binding curves were in the order 125I-angiotensin II = angiotensin II greater than angiotensin III greater than [Sar1,Ile8]angiotensin II greater than [Sar1,Gly8]angiotensin II. Angiotensin I and biochemically unrelated peptides had virtually no effect on binding of tracer angiotensin II. The divalent cations Mn2+, Mg2+ and Ca2+ stimulated 125I-angiotensin II binding at concentrations of 2-10 mM, as did Na+ at 50-100 mM. In the presence of Na+ or Li+, K+ had a biphasic effect. The chelating agents EDTA and EGTA were inhibitory, as were the thiol reagents dithiothreitol and cysteine. This study defined angiotensin II binding sites in a vascular target tissue of sufficiently high affinity to interact rapidly with plasma angiotensin II at physiological concentrations.

1. To investigate the relative roles of angiotensin II (AII) and des-Asp1-angiotensin II (angiotensin III) in the control of blood pressure and aldosterone release, the effects of seven angiotensin agonists on mean arterial blood pressure and serum aldosterone concentrations were compared in normal and sodium-depleted, conscious rats. 2. In normal rats, angiotensin I, α-Asp1-angiotensin II, β-Asp1-angiotensin II, and angiotensin II-amide were equipotent in elevating mean arterial blood pressure. Angiotensin III, des-Asp1-angiotensin I, and poly-O-acetylserine-angiotensin II were 25%, 25%, and 41% as potent as angiotensin II, respectively. After sodium depletion, pressor responses to these angiotensin peptides were reduced approximately 60–80% when compared with control responses. In contrast, pressor responses to noradrenaline were not significantly affected by sodium depletion. 3. Angiotensin II, β-Asp1-angiotensin II, angiotensin II-amide, and angiotensin III were equipotent in increasing serum aldosterone concentrations in normal animals. Angiotensin I was 59% and des-Asp1-angiotensin I only 5% as potent as angiotensin II in their abilities to release aldosterone. After sodium depletion, control serum aldosterone concentrations increased as did the slope of the dose—response curve for each angiotensin peptide. Angiotensin II was the most potent steroidogenic peptide in sodium-depleted rats with angiotensin III and β-Asp1-angiotensin II being 27%, angiotensin I 7%, angiotensin II-amide 3%, and des-Asp1-angiotensin I 1% as potent as angiotensin II in releasing aldosterone. Poly-O-acetylserine-angiotensin II has less steroidogenic effect than angiotensin II or III in both normal and sodium-depleted animals. 4. Infusions of the angiotensin II antagonist, Sar1-Ile8-angiotensin II, and the angiotensin III antagonist, Ile7-angiotensin III, enhanced aldosterone release in normal rats without altering blood pressure. After sodium depletion, Sar1-Ile8-angiotensin II decreased blood pressure without affecting aldosterone release whereas Ile7-angiotensin III diminished aldosterone release without altering blood pressure. 5. These data suggest that angiotensin II, independent of its conversion into angiotensin III, is an important regulator of steroidogenesis in the rat in normal sodium states. In sodium depletion, the octapeptide retains significant steroidogenic activity; however, the contribution of angiotensin III to its steroidogenic effects is increased.

The present study was undertaken to determine whether an agonist-induced activation of C-kinase leads to an inhibition of phospholipase C in adrenal glomerulosa cells. When cells are treated with 100 nM-TPA (12-O-tetradecanoylphorbol 13-acetate), subsequent angiotensin (‘angiotensin II’)-induced aldosterone secretion is greatly inhibited. Treatment with TPA completely inhibits the angiotensin-induced increase in both inositol trisphosphate and the cytosolic Ca2+ concentration. The dose-response curve for TPA-induced inhibition reveals that quite a high concentration of TPA is necessary to block angiotensin action compared with that needed to stimulate aldosterone secretion. 1-Oleoyl-2-acetylglycerol has a weak inhibitory effect, whereas neither 4 alpha-phorbol 12,13-didecanoate or 4 beta-phorbol inhibits angiotensin action. When the time course of changes in inositol trisphosphate and diacylglycerol is measured, angiotensin action is sustained for up to 30 min. In addition, 100 nM-TPA added after 20 min of angiotensin addition attenuates production of both inositol trisphosphate and diacylglycerol. These results suggest that high dose of TPA inhibits angiotensin-induced activation of phospholipase C by acting, at least partly, on C-kinase, but that an inhibitory effect of TPA may be a pharmacological effect with little physiological significance in this system.

1. A possible interaction between an osmotic stimulus and brain angiotensin II was studied in anaesthetized dogs by measuring the changes in arterial pressure, heart rate and renal sympathetic nerve activity during a brain ventricular injection of hypertonic NaCl solution before and after pretreatment with either captopril or [Sar1, Ile8]angiotensin II. 2. Mild pressor responses accompanied by a reflex decrease in renal sympathetic nerve activity were a characteristic before pretreatment, but after baroreceptor denervation the cardiovascular response to hypertonic NaCl solution doubled and the activity increased. These changes were prevented by intravenous pretreatment with hexamethonium chloride. 3. Brain ventricular injection of captopril slightly augmented pressor and tachycardia effects of hypertonic NaCl solution. 4. Blockade of central angiotensin II receptors with [Sar1, Ile8]angiotensin II was without effect. 5. It is concluded that central administration of hypertonic NaCl solution in baroreceptor-denervated dogs produces marked pressor responses due to increased sympathetic nerve activity via mechanisms not related to the brain renin-angiotensin system.

1. Eight patients with essential hypertension were challenged with an infusion of 32 mmol of potassium chloride in saline before and after control of their blood pressure by the angiotensin converting-enzyme (ACE) inhibitor enalapril. 2. The potassium infusion was associated with similar increases in plasma aldosterone before and during enalapril treatment, although absolute aldosterone levels were lower after enalapril treatment despite higher plasma potassium levels. 3. The handling of the potassium load was altered by ACE inhibition. The area under the curve of a plot of the increase in plasma potassium above baseline against time was greater during enalapril treatment than during treatment with placebo. 4. These observations contrast with data obtained in the dog and demonstrate that in patients with essential hypertension stimulation of aldosterone secretion by potassium is not abolished by chronic suppression of plasma angiotensin II; and although plasma aldosterone remains at a lower level, the homoeostasis of plasma potassium is only mildly impaired.