Differential response of angiotensin peptides in the urine of hypertensive animals

Urinary excretion rates of angiotensin I (Ang I), angiotensin II (Ang II), and angiotensin-(1-7) [Ang-(1-7)] were determined in normotensive Sprague Dawley (SD), spontaneously hypertensive (SHR), and mRen-2 transgenic hypertensive animals before and following blockade of Ang II synthesis or activity for two weeks. This study was performed to determine for the first time whether inhibition of Ang II alters the excretion of angiotensin peptides in the urine. Rats were given either tap water or water medicated with lisinopril, losartan or both agents in combination. Blood pressure was monitored at regular intervals during the experiment by the tail-cuff method, and once again at the end of the study with a catheter implant into a carotid artery. Metabolic studies and 24 h urinary excretion variables and angiotensin peptides were determined before and during the procedures. While all three treatments normalized the blood pressure of hypertensive animals, therapy with either lisinopril or the combination of lisinopril and losartan had a greater antihypertensive effect in both SHR and [mRen-2]27 transgenic hypertensive rats. In the urine, the concentration of the angiotensins (normalized by 24-h creatinine excretion) was several-fold higher in the untreated hypertensive animals than in normotensive SD rats. In SD rats, lisinopril or lisinopril and losartan produced a sustained rise in urinary levels of Ang-(1-7) without changes in the excretion of Ang I and Ang II. In contrast, Ang I and Ang-(1-7) were significantly elevated in SHR medicated with lisinopril alone or in combination with losartan. Only losartan, however, augmented urinary levels of Ang II in the SHR. The antihypertensive effects of the three separate regimens had no effect on the urinary excretion of angiotensin peptides in [mRen-2]27 transgenic hypertensive rats. These data show that Ang I and Ang-(1-7) are excreted in large amounts in the urine of SD, SHR and [mRen-2]27 hypertensive rats. The unchanged Ang-(1-7) excretion in transgenic hypertensive (Tg+) rats after inhibition of the renin-angiotensin system agrees with the previous finding of a reduced plasma clearance of the peptide in this model of hypertension. The data suggest that this form of hypertension may be associated with increased activity of an endogenous converting enzyme inhibitor.     

Effects of des-aspartate-angiotensin I on angiotensin II-induced incorporation of phenylalanine and thymidine in cultured rat cardiomyocytes and aortic smooth muscle cells

Des-aspartate-angiotensin I, a pharmacologically active nine-amino acid angiotensin peptide, and losartan, an AT(1) angiotensin receptor antagonist, but not angiotensin-(1-7), another active angiotensin peptide, completely attenuated the angiotensin II-induced incorporation of [3H]phenylalanine in cultured rat cardiomyocytes. The attenuation by des-aspartate-angiotensin I but not that of losartan was inhibited by indomethacin. The data support an earlier suggestion that the nonapeptide attenuates cardiac hypertrophy in rats via an indomethacin-sensitive angiotensin AT(1) receptor subtype. In rat aortic smooth muscle cells, both des-aspartate-angiotensin I and angiotensin-(1-7) had no effect on the angiotensin II-induced [3H]phenylalanine incorporation. However, the two peptides significantly attenuated the angiotensin II-induced [3H]thymidine incorporation in the smooth muscle cells. The attenuation by angiotensin-(1-7) but not by des-aspartate-angiotensin I was inhibited by (D-Ala(7))-angiotensin-(1-7), a specific angiotensin-(1-7) antagonist. Des-aspartate-angiotensin I also attenuated FCS-stimulated [3H]thymidine incorporation. This attenuation was inhibited by the peptide angiotensin receptor antagonist, (Sar(1), Ile(8))-angiotensin II, but not by losartan. These data indicate that des-aspartate-angiotensin I and angiotensin-(1-7) do not participate in the process of protein synthesis in vascular smooth muscle cells and that the nonapeptide and heptapeptide act on different non-AT(1) receptors to mediate their anti-hyperplasic action. Although the exact mechanisms of action remain to be elucidated, the findings indicate that des-aspartate-angiotensin I acts as an agonist on angiotensin AT(1) and non-AT(1) receptor subtypes and induces responses that oppose the actions of angiotensin II.     

Pharmacological concentration of angiotensin-(1-7) activates NADPH oxidase after ischemia-reperfusion in rat heart through AT1 receptor stimulation

The cardiovascular role of angiotensin-(1-7), especially in the functional and metabolic alterations associated with ischemia-reperfusion (IR), is still not clearly defined. Our objective was to evaluate the cardiac effects of angiotensin-(1-7), the receptors involved, and their relationships with NADPH oxidase activation under non-ischemic conditions and, during an ischemia-reperfusion sequence. Isolated perfused rat hearts underwent 45 min of non-ischemic perfusion, or 30 min of global ischemia followed by 30 min of reperfusion. Angiotensin-(1-7) and/or AT1 receptor blocker losartan or angiotensin-(1-7) receptor antagonist (D-Ala7)-angiotensin-(1-7) were perfused. Our results showed that angiotensin-(1-7) was without effect at low concentrations (10(-10) to 10(-7) M). At a pharmacological concentration, 0.5 microM angiotensin-(1-7) induced vasoconstriction, which was antagonised by losartan. After ischemia, we noted a partial recovery of functional parameters, which was not modified by any of the treatments. The expression of AT1 receptor mRNA was increased by ischemia-reperfusion, except in (D-Ala7)-angiotensin-(1-7) treated hearts. Angiotensin-(1-7) further increased the AT1 expression. NADPH oxidase activity was enhanced in 0.5 microM angiotensin-(1-7)-treated hearts subjected to ischemia-reperfusion, this effect was totally reversed by losartan. This is the first time that it has been shown that, in the heart, angiotensin-(1-7) at pharmacological concentration activates NADPH oxidase, an enzyme thought to be involved in several angiotensin II effects.     

Effect of hypertension on angiotensin-(1-7) levels in rats with different angiotensin-I converting enzyme polymorphism

To determine circulating angiotensin-(1-7) [Ang-(1,7)] levels in rats with different angiotensin converting enzyme (ACE) genotypes and to evaluate the effect of hypertension on levels of this heptapeptide, plasma levels of angiotensin II (Ang II) and Ang-(1-7) were determined by HPLC and radioimmunoassay in (a) normotensive F0 and F2 homozygous Brown Norway (BN; with high ACE) or Lewis (with low ACE) rats and (b) in hypertensive F2 homozygous male rats (Goldblatt model). Genotypes were characterized by PCR and plasma ACE activity measured by fluorimetry. Plasma ACE activity was 2-fold higher (p < 0.05) in homozygous BN compared to homozygous Lewis groups. In the Goldblatt groups, a similar degree of hypertension and left ventricular hypertrophy was observed in rats with both genotypes. Plasma Ang II levels were between 300-400% higher (p < 0.05) in the BN than in the Lewis rats, without increment in the hypertensive animals. Plasma Ang-(1-7) levels were 75-87% lower in the BN rats (p < 0.05) and they were significantly higher (p < 0.05) in the hypertensive rats from both genotypes. Plasma levels of Ang II and Ang-(1-7) levels were inversely correlated in the normotensive rats (r = -0.64; p < 0.001), but not in the hypertensive animals. We conclude that there is an inverse relationship between circulating levels of Ang II and Ang-(1-7) in rats determined by the ACE gene polymorphism. This inverse relation is due to genetically determined higher ACE activity. Besides, plasma levels of Ang-(1-7) increase in renovascular hypertension.     

Primary role of angiotensin-converting enzyme-2 in cardiac production of angiotensin-(1-7) in transgenic Ren-2 hypertensive rats

Angiotensin-converting enzyme-2 (ACE2) converts angiotensin II (ANG II) to angiotensin-(1-7) [ANG-(1-7)], and this enzyme may serve as a key regulatory juncture in various tissues. Although the heart expresses ACE2, the extent that the enzyme participates in the cardiac processing of ANG II and ANG-(1-7) is equivocal. Therefore, we utilized the Langendorff preparation to characterize the ACE2 pathway in isolated hearts from male normotensive Sprague-Dawley [Tg((-))] and hypertensive [mRen2]27 [Tg((+))] rats. During a 60-min recirculation period with 10 nM ANG II, the presence of ANG-(1-7) was assessed in the cardiac effluent. ANG-(1-7) generation from ANG II was similar in both the normal and hypertensive hearts [Tg((-)): 510 +/- 55 pM, n=20 vs. Tg((+)): 497 +/- 63 pM, n=14] with peak levels occurring at 30 min after administration of the peptide. ACE2 inhibition (MLN-4760, 1 microM) significantly reduced ANG-(1-7) production by 83% (57 +/- 19 pM, P<0.01, n=7) in the Tg((+)) rats, whereas the inhibitor had no significant effect in the Tg((-)) rats (285 +/- 53 pM, P>0.05, n=10). ACE2 activity was found in the effluent of perfused Tg((-)) and Tg((+)) hearts, and it was highly associated with ACE2 protein expression (r=0.78). This study is the first demonstration for a direct role of ACE2 in the metabolism of cardiac ANG II in the hypertrophic heart of hypertensive rats. We conclude that predominant expression of cardiac ACE2 activity in the Tg((+)) may be a compensatory response to the extensive cardiac remodeling in this strain.     

The renin-angiotensin and the kallikrein-kinin systems

The renin-angiotensin system (RAS) and the kallikrein-kinin system (KKS) each encompasses a large number of molecules, with several participating in both systems. The RAS generates a family of bioactive angiotensin peptides with varying biological activities. These include angiotensin-(1-8) (Ang II), angiotensin-(2-8) (Ang III), angiotensin-(3-8) (Ang IV), and angiotensin-(1-7) [Ang-(1-7)]. Ang II and Ang III act on type 1 (AT(1)) and type 2 (AT(2)) angiotensin receptors, whereas, Ang IV and Ang-(1-7) act on their own receptors. The KKS also generates a family of bioactive peptides with varying biological activities. These include hydroxylated and non-hydroxylated bradykinin and kallidin peptides and their carboxypeptidase metabolites des-Arg(9)-bradykinin and des-Arg(10)-kallidin. Whereas bradykinin and kallidin act mainly via the type 2 bradykinin (B(2)) receptor, des-Arg(9)-bradykinin and des-Arg(10)-kallidin act mainly via the type 1 bradykinin (B(1)) receptor. The AT(1) receptor forms heterodimers with the AT(2) and B(2) receptors and there is cross talk between the AT(1) and epidermal growth factor receptors. The B(2) receptor also interacts with angiotensin converting enzyme and nitric oxide synthase. Both angiotensin and kinin peptides are metabolised by many different peptidases that are important determinants of the activities of the RAS and KKS, and several of which participate in both systems.     

Prostaglandin production in response to angiotensin-(1-7) in rabbit isolated vasa deferentia

Angiotensin-(1-7) is a predominant metabolite of angiotensin I in brain tissue. Its neuromodulatory and prostaglandin (PG) synthesizing capabilities were investigated in the rabbit isolated vas deferens. This metabolite had no significant effect as a neuromodulator, however it increased PGE synthesis in the vasa deferentia with a potency equivalent to that of angiotensin II. The angiotensin-(1-7) has a unique spectrum of activity among the angiotensin peptides to selectively increase PG synthesis. It could be useful in defining the relevance of angiotensin-induced PG synthesis in various systems, particularly in neuronal tissue. Angiotensin-(1-7) potentially could be useful in defining angiotensin receptor subtypes, as well.     

Effects of angiotensin analogues and angiotensin receptor antagonists on paraventricular neurones.

In a previous study we observed that most neurones in the paraventricular nucleus are excited by angiotensin-(1-7). In comparison with angiotensin III this excitatory action was significantly delayed. The aim of the present microiontophoretic study of angiotensin II-sensitive rat paraventricular neurones was to compare the effect of the angiotensin-analogues angiotensin-(1-7), angiotensin-(2-7), angiotensin II and angiotensin III on the spontaneous activity of these neurones and to test angiotensin receptor subtype 1 antagonists (CGP 46027 or DuP 753) and subtype 2 selective antagonists (CGP 42112A and PD 123177) in order to acquire more evidence of the receptor subtype present. As previously observed angiotensin II, angiotensin III and angiotensin-(1-7) excited most neurones. The effect of angiotensin-(1-7) was usually weaker than that of angiotensin II, and in contrast to angiotensin III the latencies were not significantly different. Angiotensin-(1-7) seemed to be active by itself, because its effect was antagonised by angiotensin receptor antagonists. Angiotensin-(2-7) was mostly inactive, although a few cells were excited. Whereas the excitatory effects of angiotensin-(1-7), angiotensin II and angiotensin III could always be inhibited with both angiotensin receptor subtype antagonists 1 and 2, that produced by angiotensin-(2-7) was only weakly antagonised, if at all. Subtype 1 selective antagonists were effective at lower concentrations than selective subtype 2 antagonists.     

Localization of the novel angiotensin peptide, angiotensin-(1-12), in heart and kidney of hypertensive and normotensive rats

A low expression of angiotensinogen in the heart has been construed as indicating a circulating uptake mechanism to explain the local effects of angiotensin II on tissues. The recent identification of angiotensin-(1-12) in an array of rat organs suggests this propeptide may be an alternate substrate for local angiotensin production. To test this hypothesis, tissues from 11-wk-old spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats (n = 14) were stained with purified antibodies directed to the COOH terminus of angiotensin-(1-12). Robust angiotensin-(1-12) staining was predominantly found in ventricular myocytes with less staining found in the medial layer of intracoronary arteries and vascular endothelium. In addition, angiotensin-(1-12) immunoreactivity was present in the proximal, distal, and collecting renal tubules within the deep cortical and outer medullary zones in both strains. Preadsorption of the antibody with angiotensin-(1-12) abolished staining in both tissues. Corresponding tissue measurements by radioimmunoassay showed 47% higher levels of angiotensin-(1-12) in the heart of SHR compared with WKY rats (P < 0.05). In contrast, renal angiotensin-(1-12) levels were 16.5% lower in SHR compared with the WKY rats (P < 0.05). This study shows for first time the localization of angiotensin-(1-12) in both cardiac myocytes and renal tubular components of WKY and SHR. In addition, we show that increased cardiac angiotensin-(1-12) concentrations in SHR is associated with a small, but statistically significant, reduction in renal angiotensin-(1-12) levels.     

Analysis of the mechanisms underlying the vasorelaxant action of angiotensin II in the isolated rat carotid

It has been suggested that low concentrations of angiotensin II cause vasoconstriction whereas high concentrations evoke vasodilation. Thus, this work aimed to functionally characterize the mechanisms underlying the relaxation induced by angiotensin II at high concentrations in isolated rat carotid rings. Experiments using standard muscle bath procedures showed that angiotensin II (0.01-3 μM) concentration dependently induces relaxation of phenylephrine-pre-contracted rings. No differences between intact or denuded endothelium were found. The angiotensin II-induced relaxation was strongly inhibited by saralasin, the non-selective antagonist of angiotensin II receptors but not by the selective antagonists of AT₁ and AT₂ receptors, losartan and PD123319, respectively. However, A-779, a selective angiotensin-(1-7) receptor antagonist, reduced the relaxation induced by angiotensin II. Administration of exogenous angiotensin-(1-7) on pre-contracted tissues produced concentration-dependent relaxation, which was also inhibited by A-779. HOE-140, the selective antagonist of the bradykinin in B₂ receptor did not produce any significant effect on angiotensin II-induced relaxation. Pre-incubation of denuded-rings with N G-nitro-l-arginine methyl ester (l-NAME) or 1H-[1,2,4] Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) reduced angiotensin II-induced relaxation. On the other hand, neither indomethacin nor tetraethylammonium (TEA) produced any significant effect. The major new finding of this work is that high concentrations of angiotensin II induce relaxation of the rat carotid via activation of the NO-cGMP pathway through a mechanism that seems to be partially dependent on activation of angiotensin-(1-7) receptors.     

Divergent pathways for the angiotensin-(1-12) metabolism in the rat circulation and kidney

Evidence of endogenous angiotensin-(1-12) [Ang-(1-12)] may necessitate revision of the accepted view that Ang I is the immediate peptide product derived from the precursor protein angiotensinogen. As the processing of this peptide has not been fully elucidated, we characterized Ang-(1-12) metabolism in the serum and kidney of the mRen2.Lewis rat, a model of high circulating renin and ACE expression. A sensitive HPLC-based method to detect the metabolism ex vivo of low concentrations of (125)I-labeled Ang-(1-12) was utilized. Ang-(1-12) processing to serum did not reveal the participation of renin; however, serum ACE readily converted Ang-(1-12) to Ang I with subsequent metabolism to Ang II. Ang I and Ang II forming activities for serum ACE were 102±4 and 104±3 fmol/ml/min serum (n=3), respectively, and both products were abolished by the potent ACE inhibitor lisinopril. The metabolism of Ang-(1-12) in renal cortical membranes also revealed the formation of Ang I; however, the main products were Ang-(1-7) and Ang-(1-4) at 129±9 and 310±12 fmol/mg/min protein (n=4), respectively. Neprilysin inhibition abolished these products and substantially reduced the overall metabolism of Ang-(1-12). Incubation of Ang-(1-12) with either human or mouse neprilysin revealed identical products. We conclude that endogenous Ang-(1-12) may contribute to the expression of biologically active angiotensins through a renin-independent pathway. The preferred route for Ang-(1-12) metabolism likely reflects the relative tissue content of ACE and neprilysin.     

Angiotensin II AT1 receptors regulate ACE2 and angiotensin-(1-7) expression in the aorta of spontaneously hypertensive rats

When increased in vascular tissues, angiotensin-converting enzyme 2 (ACE2), a carboxypeptidase that hydrolyzes angiotensin II to angiotensin-(1-7), may augment the growth inhibitory and vasodilatory effects of the heptapeptide. We investigated the regulation of ACE2 and angiotensin-(1-7) expression in aortas and carotid arteries of 12-wk-old male spontaneously hypertensive rats (SHR) by determining the effect of sustained angiotensin type 1 (AT(1)) receptor blockade with olmesartan (10 mg.kg(-1).day(-1), n = 13) compared with those that received atenolol (30 mg.kg(-1).day(-1), n = 13), hydralazine (10 mg.kg(-1).day(-1), n = 13), or vehicle (n = 21). Systolic blood pressures were approximately 30% lower (P < 0.05) in rats treated for 2 wk with olmesartan compared with vehicle-treated rats. Both atenolol and hydralazine produced similar decreases in systolic blood pressure. ACE2 mRNA in the thoracic aorta of olmesartan-treated rats (n = 8) was fivefold greater (P < 0.05) than that in vehicle-treated rats (n = 16), whereas atenolol (n = 8) or hydralazine (n = 8) had no effect. Immunostaining intensities in rats treated with olmesartan (n = 5) were also associated with increased (P < 0.05) ACE2 and angiotensin-(1-7) in thoracic aorta media compared with vehicle-treated rats. In contrast, immunostaining intensities for both ACE2 and angiotensin-(1-7) were not different from vehicle (n = 5) in carotid arteries of SHR medicated with either atenolol (n = 5) or hydralazine (n = 5). A comparison of vessel wall dimensions showed that olmesartan selectively reduced the thoracic aorta media-to-lumen ratio (P < 0.05) and media thickness (P < 0.05) without an effect on carotid artery morphometry. Compared with vehicle-treated SHR, vascular hypertrophy determined from media and lumen measurements was not changed in SHR given either atenolol or hydralazine. These data represent the first report of ACE2 and angiotensin-(1-7) expression in the aorta and carotid arteries of SHR. Increased ACE2 and angiotensin-(1-7) in association with altered dimensions of the thoracic aorta but not carotid arteries in response to olmesartan treatment provides evidence that this pathway is regulated by AT(1) receptors and may be important in mediating the pressure-independent vascular remodeling effects of angiotensin peptides.     

Advances in biochemical and functional roles of angiotensin-converting enzyme 2 and angiotensin-(1-7) in regulation of cardiovascular function

Angiotensin-converting enzyme 2 (ACE2) is the first human homologue of ACE to be described. ACE2 is a type I integral membrane protein that functions as a carboxypeptidase, cleaving a single hydrophobic/basic residue from the COOH-terminus of its substrates. Because ACE2 efficiently hydrolyzes the potent vasoconstrictor angiotensin II to angiotensin (1-7), this has changed our overall perspective about the classical view of the renin angiotensin system in the regulation of hypertension and heart and renal function, because it represents the first example of a feedforward mechanism directed toward mitigation of the actions of angiotensin II. This paper reviews the new data regarding the biochemistry of angiotensin-(1-7)-forming enzymes and discusses key findings such as the elucidation of the regulatory mechanisms participating in the expression of ACE2 and angiotensin-(1-7) in the control of the circulation.     

Formation of angiotensin-(1-7) from angiotensin II by the venom of Conus geographus

The binding of [3H]angiotensin II to AT(1) receptors on Chinese Hamster Ovary cells expressing the human AT(1) receptor (CHO-AT(1) cells) is potently inhibited by venoms of the marine snails Conus geographus and C. betulinus. On the other hand, the binding of the nonpeptide AT(1) receptor-selective antagonist [3H]candesartan is not affected but competition binding curves of angiotensin II and the peptide antagonist [Sar(1),Ile(8)]angiotensin II (sarile) are shifted to the right. These effects resulted from the breakdown of angiotensin II into smaller fragments that do not bind to the AT(1) receptor. In this context, angiotensin-(1-7) is the most prominent fragment and angiotensin-(1-4) and angiotensin-(1-5) are also formed but to a lesser extent. The molecular weight of the involved peptidases exceeds 50 kDa, as determined by gel chromatography and ultrafitration.     

Angiotensin-(1-7) stimulates oxidative stress in rat kidney

The effect of two different doses of angiotensin-(1-7) and angiotensin II on the oxidative stress generation was analyzed in rat kidney. Animals were injected intraperitoneally with a single dose of angiotensin-(1-7) or angiotensin II (20 or 50 nmol/kg body weight) and killed 3 h after injection. Production of thiobarbituric acid reactive substances (TBARS), measured as indicator of oxidative stress induction, was significantly increased in rat kidney after Ang-(1-7) administration up to 30% and 50% over controls, at 20 and 50 nmol/kg, respectively. Reduced glutathione (GSH), the most important soluble antioxidant defense in mammalian cells, showed a significant decrease of 13% and 20% at 20 and 50 nmol/kg of angiotensin-(1-7), respectively. When the antioxidant enzyme activities were determined, it was found that catalase activity was not altered by the assayed angiotensin-(1-7) doses while superoxide dismutase and glutathione peroxidase activities were significantly reduced by injection of 20 nmol/kg (34% and 13%, with respect to controls) and 50 nmol/kg of angiotensin-(1-7) (54% and 22%, respectively). In contrast, angiotensin II injections did not produce significant changes neither in TBARS levels nor in soluble and enzymatic defense parameters at the two doses used in this work. These results suggest that angiotensin-(1-7) is undoubtedly related to oxidative stress induction.     

Angiotensin-(1-7) Mas-receptor deficiency decreases peroxisome proliferator-activated receptor gamma expression in adipocytes

The renin-angiotensin system is an important link between metabolic syndrome and cardiovascular diseases. Besides angiotensin II, other angiotensin peptides such as angiotensin-(1-7), have important biological activities. It has been demonstrated that angiotensin-(1-7), acting through the G protein-coupled receptor encoded by the Mas protooncogene have important actions on the cardiovascular system. However, the role of angiotensin-(1-7)-Mas axis in lipidic profile is not well established. In the present study, the adipocyte metabolism was investigated in wild type and FVB/N Mas-deficient male mice. The gene expression of peroxisome proliferator-activated receptor gamma, acetyl-CoA carboxylase and the amount of fatty acid synthase protein were reduced in the Mas-knockout mice. Serum nonesterified fatty acids of Mas-knockout showed a 50% increase in relation to wild type group. Basal and isoproterenol-stimulated lipolysis was similar between the groups, however, a significant decrease of the glycerol release (lipolytic index) in response to insulin was observed in wild type animals, while no effect of the insulin action was observed in a Mas-knockout group. The data suggest that the lack of angiotensin-(1-7) action through Mas receptor alters the response of adipocytes to insulin action. These effects might be related to decreased expression of PPARγ.     

Direct evidence for local generation and release of angiotensin II in human vascular tissue

A direct measurement of both angiotensins I and II immunoreactive substances was made in the perfusate from isolated human umbilical vein perfused with Krebs-Ringer solution which was free of any component of the renin-angiotensin system. The identity of the immunoreactive peptides was confirmed as angiotensin I and angiotensin II by high-performance liquid chromatography in reference to standard compounds. The rate of release of angiotensins was 41.9 +/- 7.4 and 63.4 +/- 12.0 pg for angiotensins I and II, respectively, during the first perfusion period of 30 min, and it remained stable at least for 3 hours. Angiotensin-converting enzyme inhibitor captopril, added to the perfusion medium (10(-9) to 5 x 10(-6) M), suppressed immunoreactive angiotensin II release in a dose-dependent fashion; the maximal percent inhibition of angiotensin II release evoked by captopril (5 x 10(-6) M) was approximately 56%. These results taken together with the previous observations of presence of essential components of the renin-angiotensin system in vascular tissue provide direct evidence for local generation and subsequent release of angiotensin II in vascular beds of human beings.     

Enzymatic formation of angiotensins II and III in the hindlimb circulation of dogs

Experiments were performed in 14 anesthetized dogs to (1) to determine if the reductions in hindlimb blood flow produced by [des-Asp1] angiotensin I were due to its local enzymatic (kininase II) conversion to angiotensin III and (2) to quantitate the extent of conversion of angiotensin I to angiotensin II and of [des-Asp1] angiotensin I to angiotensin III in the hindlimb circulation. Graded doses of these peptides were administered as bolus injections directly into the left external iliac artery while measuring flow in this artery electromagnetically. Dose-response relationships were determined before and during the inhibition of kininase II activity with captopril or antagonism of angiotensin receptor sites with [Ile7] angiotensin III. Captopril inhibited the vasoconstrictor responses to angiotensin I and [des-Asp1] angiotensin I, but did not affect the responses to angiotensins II or III, or norepinephrine. [Ile7] angiotensin III inhibited the vasoconstrictor responses to all four angiotensin peptides but did not alter the responses to norepinephrine. These findings indicate that the hindlimb vasoconstrictor responses to [des-Asp1] angiotensin I were due to the local formation of angiotensin III. The extent of conversion of [des-Asp1] angiotensin I to angiotensin III that occurred in one transit through the hindlimb arterial circulation was estimated to be 36.7%, which was not different from the estimated 36.4% conversion of angiotensin I to angiotensin II. We conclude that angiotensin I and [des-Asp1] angiotensin I are converted to their respective vasoactive forms (angiotensins II and III) to a similar extent in the hindlimb circulation via the action of kininase II.     

Actions of angiotensin peptides on the rabbit pulmonary artery

The presence of the angiotensin AT1A-like receptor subtype in the pulmonary artery and AT1B-like receptor subtype in the pulmonary trunk of the rabbit has been reported in two earlier studies. The present study further investigated these receptor subtypes using five other angiotensins (namely angiotensin II, angiotensin III, angiotensin IV, angiotensin-(1-7) and angiotensin-(4-8)). The direct action of the angiotensins on the rabbit pulmonary arterial and trunk sections and the ability of each angiotensin to further contract or relax preconstricted sections of the pulmonary artery and trunk were studied using the organ bath set-up. The effects of angiotensin III on the 3H overflow from re-uptaken [3H]noradrenaline in the electrically-contracted rabbit pulmonary arterial and trunk sections were also studied. The contractile response of the arterial and trunk section had the following rank order potency: angiotensin II > angiotensin III > angiotensin IV. The contractile response to these angiotensins was greatly reduced or absent in the pulmonary trunk. Angiotensin II further contracted the preconstricted arterial and trunk sections. In contrast, angiotensin III further contracted the preconstricted arterial section but relaxed the preconstricted trunk section. Angiotensin IV similarly relaxed the preconstricted trunk section but had minimum effect on the preconstricted arterial section. Angiotensin-(1-7) and angiotensin-(4-8) had no effect on both sections. The actions of the three angiotensins were inhibited by losartan, an AT1-selective antagonist. Indomethacin, a cyclo-oxygenase inhibitor, inhibited the relaxation caused by angiotensin III and angiotensin IV in the trunk section. The effects of angiotensin III on the electrically preconstricted sections of the pulmonary trunk and artery were not accompanied by any significant changes in 3H overflow. The differential responses produced by angiotensin II and its immediate metabolites via two positionally located and functionally opposing receptor subtypes suggest that the pulmonary trunk and artery is not a passive conduit but an important regulator of blood flow from the heart to the lung.