A new strategy for treating hypertension by blocking the activity of the brain renin-angiotensin system with aminopeptidase A inhibitors.

Hypertension affects one-third of the adult population and is a growing problem due to the increasing incidence of obesity and diabetes. Brain RAS (renin-angiotensin system) hyperactivity has been implicated in the development and maintenance of hypertension in several types of experimental and genetic hypertension animal models. We have identified in the brain RAS that APA (aminopeptidase A) and APN (aminopeptidase N), two membrane-bound zinc metalloproteases, are involved in the metabolism of AngII (angiotensin II) and AngIII (angiotensin III) respectively. The present review summarizes the main findings suggesting that AngIII plays a predominant role in the brain RAS in the control of BP (blood pressure). We first explored the organization of the APA active site by site-directed mutagenesis and molecular modelling. The development and the use in vivo of specific and selective APA and APN inhibitors EC33 and PC18 respectively, has allowed the demonstration that brain AngIII generated by APA is one of the main effector peptides of the brain RAS, exerting a tonic stimulatory control over BP in conscious hypertensive rats. This identified brain APA as a potential therapeutic target for the treatment of hypertension, which has led to the development of potent orally active APA inhibitors, such as RB150. RB150 administered orally in hypertensive DOCA (deoxycorticosteroneacetate)-salt rats or SHRs (spontaneously hypertensive rats) crosses the intestinal, hepatic and blood-brain barriers, enters the brain, generates two active molecules of EC33 which inhibit brain APA activity, block the formation of brain AngIII and normalize BP for several hours. The decrease in BP involves two different mechanisms: a decrease in vasopressin release into the bloodstream, which in turn increases diuresis resulting in a blood volume reduction that participates in the decrease in BP and/or a decrease in sympathetic tone, decreasing vascular resistance. RB150 constitutes the prototype of a new class of centrally acting antihypertensive agents and is currently being evaluated in a Phase Ib clinical trial.

Local aldosterone release and CYP11B2 expression in response to angiotensin peptides, glucose, and potassium - an ex vivo study on murine colon.

We have previously described local aldosterone synthesis in mouse colon. In the renin-angiotensin-aldosterone system (RAAS), angiotensin II (Ang II) peptide is the physiological factor which stimulates aldosterone synthesis in the adrenal glands. We have recently demonstrated that Ang II stimulates aldosterone synthesis also in mouse colon. Here, we conducted a 75-min ex vivo incubation of murine colonic tissue and evaluated the effects of three other Ang peptides, Ang I (1 µM), Ang III (0.1 µM) and Ang (1-7) (0.1 µM) on aldosterone synthesis. As a possible mechanism, their effects on tissue levels of the rate-limiting enzyme, aldosterone synthase (CYP11B2) were measured by ELISA and Western blot. Ang III significantly elevated the amount of tissue CYP11B2 protein in colon. The values of released aldosterone in colon tissue incubation were increased over the control in the presence of Ang I, II or III, however, being statistically non-significant. In Western blot analysis, the values of tissue CYP11B2 protein content were elevated by Ang I and II. Ang (1-7) alone in colon did not influence CYP11B2 protein levels in the incubation experiment but showed higher aldosterone release without statistical significance. Ang (1-7) showed an antagonistic effect towards Ang II in release of aldosterone in adrenal gland. An overall estimation of a single peptide (three measured variables), the results were always in an increasing direction. The responses of aldosterone synthesis to high levels of glucose (44 mM) and potassium (18.8 mM) as physiological stimulators in vivo were investigated in the colon incubation. Glucose, equal to four times the concentration of the control buffer in the incubation, showed higher values of aldosterone release in colon than control without statistical significance similarly to the effect seen in adrenal glands. Increasing the concentration of potassium in the incubation buffer exerted no effect on colonic aldosterone production. Intriguingly, no correlation was found between aldosterone release and the tissue CYP11B2 protein content in colon. In summary, the response of colonic aldosterone synthesis to different Ang peptides resembles, but is not identical to, the situation in the adrenal glands.

Update on the angiotensin AT(2) receptor.

It is quite well established that activation of the AT(2) receptor (AT(2)R) provides a counter-regulatory role to AT(1)R overactivity, particularly during pathological conditions. Indeed, a potential therapeutic role for the AT(2)R is currently being promulgated with the introduction of novel AT(2)R ligands such as compound 21 (C21). In this brief review, we will focus on recent evidence to suggest that AT(2)R exhibits promising organ protection in the context of the heart, kidney and brain, with inflammation and gender influencing outcome. However, this field is not without controversy since the 'flagship' ligand C21 has also come under scrutiny, although it is safe to say there is much evidence to support a potentially important role of AT(2)R in a number of cardiovascular diseases. This report updates recent data in this field.

Renin-angiotensin system revisited.

New components and functions of the renin-angiotensin system (RAS) are still being unravelled. The classical RAS as it looked in the middle 1970s consisted of circulating renin, acting on angiotensinogen to produce angiotensin I, which in turn was converted into angiotensin II (Ang II) by angiotensin-converting enzyme (ACE). Ang II, still considered the main effector of RAS was believed to act only as a circulating hormone via angiotensin receptors, AT1 and AT2. Since then, an expanded view of RAS has gradually emerged. Local tissue RAS systems have been identified in most organs. Recently, evidence for an intracellular RAS has been reported. The new expanded view of RAS therefore covers both endocrine, paracrine and intracrine functions. Other peptides of RAS have been shown to have biological actions; angiotensin 2-8 heptapeptide (Ang III) has actions similar to those of Ang II. Further, the angiotensin 3-8 hexapeptide (Ang IV) exerts its actions via insulin-regulated amino peptidase receptors. Finally, angiotensin 1-7 (Ang 1-7) acts via mas receptors. The discovery of another ACE2 was an important complement to this picture. The recent discovery of renin receptors has made our view of RAS unexpectedly complex and multilayered. The importance of RAS in cardiovascular disease has been demonstrated by the clinical benefits of ACE inhibitors and AT1 receptor blockers. Great expectations are now generated by the introduction of renin inhibitors. Indeed, RAS regulates much more and diverse physiological functions than previously believed.

[Angiotensin III participation in mechanisms of development of an alcoholism and other kinds of behavioural activity].

To study the involvement of angiotensin III in the development of alcoholism and other types of behavioural activity the research on 24 male rats of Vistar species with 180-250 g weight was conducted. Alcoholic motivation was created by alcoholic beverage consisting of 20% ethyl alcohol water solution. The experiment lasted 30-day time period. It was concluded that the effect of angiotensin III depend on the individual resistance or predisposition to alcohol.

Angiotensin III: a central regulator of vasopressin release and blood pressure.

Among the main bioactive peptides of the brain renin-angiotensin system, angiotensin (Ang) II and AngIII exhibit the same affinity for type 1 and type 2 AngII receptors. Both peptides, injected intracerebroventricularly, cause similar increases in vasopressin release and blood pressure. Because AngII is converted in vivo to AngIII, the identity of the true effector is unknown. This review summarizes new insights into the predominant role of brain AngIII in the control of vasopressin release and blood pressure and underlines the fact that brain aminopeptidase A, the enzyme forming central AngIII, could constitute a putative central therapeutic target for the treatment of hypertension.

Angiotensin III depressor action in the conscious rabbit is blocked by losartan but not PD 123319.

Vasodilator and vasodepressor properties of angiotensins have been reported, and mediation by prostaglandins or nitric oxide has been proposed. Other studies indicate that angiotensin AT(2) receptors might mediate a depressor action, and the present study was designed to delineate and explore this possibility in a conscious rabbit model. Large intravenous boluses of angiotensin III (15 nmol/kg) produced a predictable pressor peak (82+/-4 mm Hg) followed by a depressor phase (20+/-3 mm Hg), whereas equipressor doses of angiotensin II were less effective at producing depressor responses. Angiotensin-(1-7) did not exert a depressor action, and the reduced potency of angiotensin IV (relative to angiotensin III) was similar for both the pressor and depressor phases ( approximately 100-fold). It is clear that specific angiotensin IV or angiotensin-(1-7) receptors do not mediate depressor effects in this model. The AT(1) antagonist losartan (1 mg/kg) blocked both the pressor and depressor components of the angiotensin III response, whereas the AT(2) antagonist PD 123319 (35 mg/kg) had no effect on either element of the response. The data obtained with the angiotensin receptor subtype-selective compounds, losartan and PD 123319, suggest that the depressor action is an AT(1)-mediated effect and give no indication that AT(2) receptors could be involved. Paradoxically, the greater potency of angiotensin III as a vasodepressor belies the conclusion that the response is AT(1)-mediated, because AT(1) receptors have a greater affinity for angiotensin II versus angiotensin III.

Angiotensin III and IV activation of the brain AT1 receptor subtype in cardiovascular function.

The present investigation determined that native angiotensins II and III (ANG II and III) were equipotent as pressor agents when ICV infused in alert rats, whereas native angiotensin IV (ANG IV) was less potent. An analogue of each of these angiotensins was prepared with a hydroxyethylamine (HEA) amide bond replacement at the N-terminus, yielding additional resistance to degradation. These three angiotensin analogues, HEA-ANG II, HEA-ANG III, and HEA-ANG IV, were equivalent with respect to maximum elevation in pressor responses when ICV infused; and each evidenced significantly extended durations of effect compared with their respective native angiotensin. Comparing analogues, HEA-ANG II had a significantly longer effect compared with HEA-ANG III, and HEA-ANG IV, whereas the latter were equivalent. Pretreatment with the AT1 receptor subtype antagonist, Losartan (DuP753), blocked subsequent pressor responses to each of these analogues, suggesting that these responses were mediated by the AT1 receptor subtype. Pretreatment with the specific AT4 receptor subtype antagonist, Divalinal (HED 1291), failed to influence pressor responses induced by the subsequent infusion of these analogues. These results suggest an important role for Ang III, and perhaps ANG IV, in brain angiotensin pressor responses mediated by the AT1 receptor subtype.

Effects of des-aspartate-angiotensin I on the expression of angiotensin AT1 and AT2 receptors in ventricles of hypertrophic rat hearts.

The effects of des-aspartate-angiotensin I (DAA-I) on the expression of angiotensin AT1 and AT2 receptor in hearts of aortic coarcted rats were studied. The protocols used included competitive reverse transcription polymerase chain reaction (RT-PCR), Western blotting, and receptor-ligand binding assays. mRNA of the AT1 and AT2 receptors increased significantly after 4 days of aortic coarctation (7- and 4-folds of sham-operated, respectively). However, the protein of the AT1 receptor was not altered, and only increase in protein of the AT2 receptor was detected. There was an increase in [125I]Sar1-Ile8-angiotensin II binding sites in the ventricular membranes of hypertrophic hearts, which was attributed to an upregulation of the AT2 receptor. Treatment with i.p. DAA-I resulted in a significant reduction of cardiac hypertrophy, the maximum effect was achieved with a dose of 200 nmol/kg/day. The anti-cardiac hypertrophy effect appeared to be U-shape, and at a higher dose of 800 mol/kg/day, there was a loss of effect. DAA-I had no effect on the receptor protein in ventricles of hypertrophic hearts. However, DAA-I dose-dependently decreased the binding of [125I]Sar1-Ile8-angiotensin II to ventricular membranes. The decrease was due to a likely desensitization by internalization of the AT1 receptor, and this probably contributed to the loss of hypertrophic effects at 800 nmol/kg/day. Treatment of DAA-I also resulted in a remarkable increase in AT2 receptor mRNA (24-fold increase over the sham-operated), which was not coupled to translation. The present findings provide new information regarding the relationship between cardiac hypertrophy and the angiotensin receptors, and the anti-cardiac hypertrophic actions of DAA-I via the AT1 receptors.

Effects of des-aspartate-angiotensin I on neointima growth and cardiovascular hypertrophy.

The in vitro anti-hypertrophic and hyperplastic actions of des-aspartate-angiotensin I (DAA-I) on cultured cardiovascular cells have been demonstrated in earlier experiments. The present study investigated its effects on the development of neointima in balloon catheter-injured carotid artery of the Sprague-Dawley (SD) rat and the development of cardiovascular hypertrophy in the spontaneously hypertensive rat. Treatment with i.v. DAA-I for 14 days post-injury dose-dependently attenuated the development of neointima. The maximum effect was obtained at 34 pmol/kg/day. The data support the possibility that endogenous angiotensins could inhibit neointima growth. This opens up avenues for their therapeutic elevation in combating neointima-related restenosis of which current drugs are not fully effective in suppressing. Five-week-old pre-hypertensive SHR, when orally administered with a dose of 769 nmol/kg/day DAA-I for a duration of 47 weeks, showed significant reduction in the development of cardiac and vascular hypertrophy compared to the untreated controls. Similar treatment with DAA-I had no effect on the Wistar Kyoto rats. The present findings support the contention that, besides angiotensin II, other endogenous angiotensins are also involved in the regulation and/or pathophysiology of the cardiovascular system.

Reduction in postsynaptic alpha 2-adrenoceptor activity by endogenous angiotensin III in the nucleus reticularis gigantocellularis of the rat.

We investigated in adult, male Sprague-Dawley rats anesthetized with pentobarbital sodium the synaptic location of the interaction between endogenous angiotensin III (AIII) and the alpha 2-adrenoceptors in the medulla oblongata that are involved in cardiovascular regulation. The circulatory suppressant efficacy of a centrally acting alpha 2-adrenoceptor agonist, guanabenz, was used as the experimental index. Direct bilateral microinjection of AIII (40 pmol) into the nucleus reticularis gigantocellularis (NRGC), a medullary site believed to be intimately related to the cardiovascular inhibitory actions of guanabenz, attenuated, whereas the selective AIII receptor antagonist, Ile7-AIII (20 nmol), potentiated, the circulatory suppressant effects of guanabenz (100 micrograms/kg, i.v.). These two respective actions were essentially unaffected by immunocytochemically verified depletion of noradrenergic nerve terminals in the NRGC, elicited by a selective noradrenergic neurotoxin, DSP4. These data suggest that endogenous AIII may exert a tonic inhibitory action on the alpha 2-adrenoceptors located postsynaptically on neurons in the NRGC that are involved in central cardiovascular regulation.

Reduction in baroreceptor reflex response by angiotension III and its modification by Ile7-angiotensin III and bestatin in the rat.

We evaluated the participation of endogenous brain angiotensin III (AIII) in central cardiovascular regulation, using the intracerebroventricular injection technique in Sprague-Dawley rats anesthetized with pentobarbital sodium (50 mg/kg, i.p.). AIII (100 pmol) promoted an elevation in systemic arterial pressure and a reduction in the baroreceptor reflex (BRR) response. Its specific antagonist, Ile7-AIII (100 nmol), and the aminopeptidase inhibitor, bestatin (200 nmol), on the other hand, augmented the response of the same reflex. The suppressive action of AIII (100 pmol) on the BRR was attenuated, and the enhancing effect of Ile7-AIII (100 nmol) was potentiated, however, when these two peptides were administered simultaneously with bestatin (200 nmol). All these events were significantly different from their controls during the first 10-15 min following injection, parallel to the time course of a discernible action of AIII on systemic arterial pressure. We discussed that the endogenous AIII in the central nervous system may participate in cardiovascular control by tonically inhibiting the BRR, in concert with other brain neuropeptides.

Stimulation of spontaneous and dopamine-inhibited prolactin release from anterior pituitary reaggregate cell cultures by angiotensin peptides.

In superfused anterior pituitary reaggregate cell cultures angiotensin II (AII) stimulated both spontaneous and dopamine-inhibited prolactin (PRL) release from subnanomolar concentrations. Angiotensin I (AI) and angiotensin III (AIII) also stimulated PRL release. The magnitude and rate of response to AI was equal to or only slightly lower than that to AII. However, the angiotensin converting enzyme (ACE) inhibitors captopril and teprotide (1 microM) completely abolished the PRL response to 0.1 nM AI and strongly reduced that to 1 nM AI. The intrinsic activity of AIII was lower than that of AII but could be enhanced by adding 2 microM of the aminopeptidase inhibitor amastatin to the superfusion medium. After withdrawal of AIII, PRL secretion rate rapidly returned to baseline levels, whereas after withdrawal of AI or AII, secretion fell to a level remaining significantly higher than basal release. The present findings indicate that stimulation of PRL release by AI is weak unless it is converted into AII by ACE and that aminopeptidase may be important in determining the magnitude and termination of the PRL response. Furthermore, the active peptides induce a different pattern of response.

Tonic reduction in central alpha 2-adrenoceptor activity by endogenous angiotensin III in the rat.

We evaluated the possible interactions between central alpha 2-adrenoceptors and endogenous angiotensin III (AIII) in Sprague-Dawley rats anesthetized with pentobarbital sodium (50 mg/kg, i.p.). The cardiovascular suppressive effects of the alpha 2-adrenoceptor agonist, guanabenz, were used as our experimental index for alpha 2-adrenoceptor activity. Intracerebroventricular (i.c.v.) administration of AIII (100 or 200 pmol) attenuated the hypotensive and negative inotropic and chronotropic actions of guanabenz (100 micrograms/kg, i.v.). Blocking the endogenous activity of the heptapeptide with its specific antagonist, Ile7-AIII (50 or 100 nmol, i.c.v.), on the other hand, potentiated the circulatory inhibitory efficacy of the aminoguanidine compound. These modulatory effects were essentially duplicated by bilateral microinjection of AIII (20 or 40 pmol) or Ile7-AIII (10 or 20 nmol) into the nucleus reticularis gigantocellularis (NRGC), a medullary site that is critically involved in the cardiovascular suppressive actions of guanabenz. I.c.v. injection of bestatin (200 nmol) also reduced the circulatory depressive potency of guanabenz. This effect was respectively enhanced and reduced when the aminopeptidase inhibitor was given simultaneously with AIII (200 pmol) and Ile7-AIII (100 nmol). These results suggest that the endogenous AIII may exert a tonic inhibition on the alpha 2-adrenoceptors in in the NRGC that are involved in cardiovascular regulation.

Angiotensin III: a physiological relevant peptide of the renin angiotensin system.

The renin angiotensin system (RAS) is a peptide hormone system that plays an important role in the pathophysiology of various diseases, including congestive heart failure, hypertension, myocardial infarction, and diabetic nephropathy. This has led researchers to focus extensively on this system, leading to the discovery of various peptides, peptidases, receptors and signal transduction mechanisms intrinsic to the RAS. Angiotensinogen (AGT), angiotensin (Ang) II, Ang III, Ang IV, and Ang-(1-7) are the main biologically active peptides of RAS. However, most of the available studies have focused on Ang II as the likely key peptide from the RAS that directly and indirectly regulates physiological functions leading to pathological conditions. However, data from recent studies suggest that Ang III may produce physiologically relevant effects that are similar to those produced by Ang II. Hence, this review focuses on Ang III and the myriad of physiological effects that it produces in the body.