Conversion of renal angiotensin II to angiotensin III is critical for AT2 receptor-mediated natriuresis in rats.

In the kidney, angiotensin II (Ang II) is metabolized to angiotensin III (Ang III) by aminopeptidase A (APA). In turn, Ang III is metabolized to angiotensin IV by aminopeptidase N (APN). Renal interstitial (RI) infusion of Ang III, but not Ang II, results in angiotensin type-2 receptor (AT(2)R)-mediated natriuresis. This response is augmented by coinfusion of PC-18, a specific inhibitor of APN. The present study addresses the hypotheses that Ang II conversion to Ang III is critical for the natriuretic response. Sprague-Dawley rats received systemic angiotensin type-1 receptor (AT(1)R) blockade with candesartan (CAND; 0.01 mg/kg/min) for 24 hours before and during the experiment. After a control period, rats received either RI infusion of Ang II or Ang II+PC-18. The contralateral kidney received a RI infusion of vehicle in all rats. Mean arterial pressure (MAP) was monitored, and urinary sodium excretion rate (U(Na)V) was calculated separately from experimental and control kidneys for each period. In contrast to Ang II-infused kidneys, U(Na)V from Ang II+PC-18-infused kidneys increased from a baseline of 0.03+/-0.01 to 0.09+/-0.02 micromol/min (P<0.05). MAP was unchanged by either infusion. RI addition of PD-123319, an AT(2)R antagonist, inhibited the natriuretic response. Furthermore, RI addition of EC-33, a selective APA inhibitor, abolished the natriuretic response to Ang II+PC-18. These data demonstrate that RI addition of PC-18 to Ang II enables natriuresis mediated by the AT(2)R, and that conversion of Ang II to Ang III is critical for this response.

Biosynthesis and physio-pharmacological actions of angiotensin peptides: 1. Synthetic enzymes.

The present work introduces a brief review of the actual knowledge concerning the enzymes involved in the biosynthesis of the active angiotensins, followed by a presentation of their main physio-pharmacological actions. The enzymatic pathways that generate active ang. II (1-8) are complemented with data concerning its transformation into angiotensin III (2-8), ang. IV (3-8), ang. V (1-5) and ang. 1-7. Besides the classic renin of renal origin, the tissue isorenins, represented by tonin and cathepsins D and G, inactive angiotensin-I-forming are also reviewed. Furthermore, chymase and the new angiotensin-converting enzyme 2 (ACE2), which generates angiotensin 1-7, having opposite properties from the mother-substance (Ang. II) are discussed at length. The presentation of properties of angiotensin-generating enzymes is followed by the presentation of the action of angiotensinases (aminopetidases, carboxypeptidase and endopeptidases), which are involved both in the generation of biologically active angiotensin peptides and in their inactivation.

Aminopeptidase A, which generates one of the main effector peptides of the brain renin-angiotensin system, angiotensin III, has a key role in central control of arterial blood pressure.

Overactivity of the brain renin-angiotensin system (RAS) has been implicated in the development and maintenance of hypertension in several experimental animal models. We have recently reported that, in the murine brain RAS, angiotensin II (AngII) is converted by aminopeptidase A (APA) into angiotensin III (AngIII),which is itself degraded by aminopeptidase N (APN), both peptides being equipotent to increase vasopressin release and arterial blood pressure when injected by the intracerebroventricular (i.c.v.) route. Because AngII is converted in vivo into AngIII, the exact nature of the active peptide is not precisely known. To delineate their respective roles in the central control of cardiovascular functions, specific and selective APA and APN inhibitors are needed to block the metabolic pathways of AngII and AngIII respectively. In the absence of such compounds for APA, we first explored the organization of the APA active site by site-directed mutagenesis. This led us to propose a molecular mechanism of action for APA similar to that proposed for the bacterial enzyme thermolysin deduced from X-ray diffraction studies. Secondly, we developed a specific and selective APA inhibitor, compound EC33 [(S)-3-amino-4-mercaptobutylsulphonic acid], as well as a potent and selective APN inhibitor, PC18 (2-amino-4-methylsulphonylbutane thiol). With these new tools we examined the respective roles of AngII and AngIII in the central control of arterial blood pressure. A central blockade of APA with the APA inhibitor EC33 suppressed the pressor effect of exogenous AngII, suggesting that brain AngII must be converted into AngIII to increase arterial blood pressure. Furthermore, EC33, injected alone i.c.v. but not intravenously, caused a dose-dependent decrease in arterial blood pressure by blocking the formation of brain AngIII but not systemic AngIII. This is corroborated by the fact that the selective APN inhibitor PC18 administered alone via the i.c.v. route increased arterial blood pressure. This pressor response was blocked by prior treatment with the angiotensin type 1 receptor antagonist losartan, showing that blocking the action of APN on AngIII metabolism leads to an increase in endogenous AngIII levels, resulting in arterial blood pressure increase through an interaction with angiotensin type 1 receptors. These results demonstrate that AngIII is a major effector peptide of the brain RAS, exerting a tonic stimulatory control over arterial blood pressure. Thus APA, the enzyme responsible for the formation of brain AngIII, represents a potential central therapeutic target that justifies the development of APA inhibitors, crossing the blood-brain barrier, as central anti-hypertensive agents.

Aminopeptidase A activity and angiotensin III effects on [Ca2+]i along the rat nephron.

BACKGROUND: This study examined the specific effects of angiotensin III (Ang III) along the nephron. METHODS: We examined the distribution of aminopeptidase A (APA) activity by using a specific APA inhibitor and by immunostaining with an antirat kidney APA antibody, the Ang III-induced variations of [Ca2+]i by using fura-2 and the characterization of the receptor subtype involved in the response to Ang III in cortical thick ascending limb (CTAL). RESULTS: APA activity was found all along the nephron but was higher in the cortex than in the medulla. This was confirmed by immunostaining. Increases in [Ca2+]i elicited by 10(-7) mol/liter Ang III were observed all along the nephron. The characterization of the receptor subtype involved in the [Ca2+]i response to Ang III in CTAL indicated that EC50 values for Ang III and Ang II were similar (13.5 and 10.3 nmol/liter, respectively), and Ang III-induced responses were totally abolished by AT1 receptor but not by AT2 receptor antagonists. There was a cross-desensitization of [Ca2+]i responses to 10(-7) mol/liter Ang III and Ang II, and the [Ca2+]i responses to 10(-7) mol/liter Ang II and Ang III were not additive. CONCLUSION: These results show that in CTAL, the [Ca2+]i responses to Ang II and Ang III occur through the same AT1a receptor because this subtype is predominant in this segment. Taken together, these data suggest that APA could be a key enzyme to generate Ang III from Ang II in the kidney.

Hemostatic changes after dietary coenzyme Q10 supplementation in swine.

Improved cardiovascular morbidity and mortality have been observed in several clinical studies of dietary supplementation with coenzyme Q10 (CoQ10). We elucidated the effect of CoQ10 on certain hemostatic parameters that may influence the progression of heart disease. Twelve Yorkshire swine were randomized to receive diet supplementation with either CoQ10 or placebo for 20 days. Blood samples were obtained at baseline and at the end of the feeding period. At the end of the protocol, there were no significant differences in hemostatic parameters in the placebo group. A significant increase in total serum CoQ10 level (from 0.39 +/- 0.06 to 0.96 +/- 0.04 microgram/ml, p < 0.001) was noted after the feeding period in the CoQ10-supplemented group. We observed significant inhibition of ADP-induced platelet aggregation (-9.9%) and a decrease in plasma fibronectin (-20.2%), thromboxane B2 (TXB2, -20.6%), prostacyclin (-23.2%), and endothelin-1 (ET-1, -17.9%) level. There were no changes in the plasma concentrations of the natural antithrombotics [antithrombin-III (AT-III), protein S, and protein C] after CoQ10 supplementation. CoQ10 supplementation in a dose of 200 mg daily is associated with mild antiaggregatory changes in the hemostatic profile. Clinical beneficial effects of CoQ10 may be related in part to a diminished incidence of thrombotic complications.

In vitro evidence for local generation of renin and angiotensin II/III immunoreactivity by the human adrenal gland.

The adrenal gland of various mammalian species has been shown to contain all the components of a functional renin-angiotensin system. We investigated the existence of this local system in human adrenal tissues surgically obtained. Eight normal adrenals (cortex and medulla) and 6 aldosterone-producing adenomas (aldosteronomas) were examined. Minced tissues were superfused over 270 min, and 15-min fractions were collected. In the perfusates, active renin was measured by immunoradiometric assay with human anti-renin monoclonal antibodies; immunoreactive angiotensin II/III and aldosterone were measured by radioimmunoassay. Adrenal tissues, either normal or pathological, were found concomitantly to release renin, angiotensin II/III and aldosterone. The pattern of this spontaneous release exhibited a pulsatile character. The total amount of renin and angiotensin II/III secreted during superfusion clearly exceeded the tissue content (determined by extraction). Addition of the angiotensin-converting enzyme inhibitor quinaprilat (4 x 10(-6) mol/l) in the superfusion caused a concomitant decrease of angiotensin II/III and aldosterone secretion by 3 normal tissues, and no change in 2 aldosteronomas. These data provide evidence that the human adrenal gland in vitro generates and releases both renin and angiotensin II/III, and support the hypothesis that locally formed angiotensin II/III may play a role as a paracrine regulator of physiological aldosterone secretion.

Inhibition of angiotensin III formation by thiol derivatives of acidic amino acids.

Angiotensin III is formed by removal of the N-terminal Asp residue of angiotensin II in a reaction catalyzed by glutamyl aminopeptidase (aminopeptidase A EC 3.4.11.7). Thiol derivatives of glutamate and aspartate in which the alpha-COOH group was replaced by -CH2SH were synthesized as inhibitors of glutamyl aminopeptidase. Glutamate thiol was a potent inhibitor of glutamyl aminopeptidase (Ki = 4 x 10(-7) M) but even more potently inhibited microsomal alanyl aminopeptidase (Ki = 2.5 x 10(-7) M). Aspartate thiol (beta-homocysteine) was a less potent but more selective inhibitor of glutamyl aminopeptidase (glutamyl aminopeptidase: Ki = 1.2 x 10(-6) M; microsomal alanyl aminopeptidase: Ki = 7.5 x 10(-6) M). Neither compound inhibited cytosolic leucyl aminopeptidase. Aspartate thiol blocked the conversion of angiotensin II to angiotensin III. These derivatives are more selective than amastatin and may be of value in studies probing the biological significance of angiotensin III.

Endothelial renin-angiotensin pathway: evidence for intracellular synthesis and secretion of angiotensins.

Cultured bovine aortic endothelial cells (BAEC) contain renin and angiotensinogen. To examine whether angiotensins are synthesized intracellularly and secreted by these cells, we assayed cell extracts as well as serum-free media of intact confluent BAEC. Angiotensins were identified by their retention time on reverse phase high performance liquid chromatography and direct radioimmunoassay. BAEC and their media contain angiotensin II and angiotensin III. The rate of angiotensin accumulation in the media was a nonlinear function of time; the highest rate occurred in the first 15 minutes. The amount of angiotensin II accumulated in 30 minutes exceeded 200% of the intracellular concentration and that of angiotensin III exceeded 500% of the initial intracellular content. Neither renin nor angiotensinogen could be detected in the media. The viability of these cells was supported by low lactic dehydrogenase activity in the media (less than 0.5% of cellular level). These data suggest that BAEC is capable of synthesizing and secreting angiotensins. We postulate that this endothelial-derived angiotensin system may play an important paracrine or autocrine role in influencing local vascular tone.

Effects of granuloma modulation induced by regulatory-T-lymphocyte activity on angiotensin II/III production by granuloma macrophages in murine schistosomiasis mansoni.

Angiotensins are produced by granuloma macrophages in murine Schistosoma mansoni. During the course of infection, granuloma undergo a T-cell-dependent process called modulation in which their maximal size decreases. This study was undertaken to establish whether angiotensin production by granuloma macrophages is altered by immunoregulatory lymphocytes. Granuloma macrophages from modulated lesions released and contained more angiotensin II/III (AII/III) and less angiotensin I (AI) than those from the acute infection. Captopril, a specific angiotensin-converting-enzyme (ACE) inhibitor, appreciably decreased AII/III produced by macrophages from modulated granulomas. Adoptive transfer of splenic T lymphocytes from chronically infected donors into acutely infected recipients altered angiotensin production by the granuloma macrophages in a manner similar to that seen in modulated lesions. However, no difference was detected in the capacity of granuloma macrophages from acutely or chronically infected mice to metabolize 125I-AI or -AII added to cell cultures. Similarly, captopril did not alter the metabolism of exogenously administrated angiotensins. These findings suggest that regulatory T lymphocytes influence the metabolism by granuloma macrophages of endogenously produced angiotensins at least in part by induction of macrophage ACE activity. However, the degradation of extracellular AI and AII may result from the activity of enzymes other than ACE which are not inducible by modulation.

Kinase II-dependent formation of angiotensins II and III in the hepatic circulation.

Experiments were performed in 14 pentobarbital-anesthetized dogs to 1) determine if the hepatic arterial vasoconstrictor effects of [des-Asp1]angiotensin I [( des-Asp1]ANG I) were due to its local conversion to angiotensin III (ANG III) and 2) to evaluate the magnitude of conversion of ANG I to angiotensin II (ANG II) and of [des-Asp1]ANG I to ANG III in the hepatic arterial vascular bed. Graded doses of these peptide agonists were administered as bolus injections directly into the hepatic artery; hepatic arterial blood flow was measured with an electromagnetic flow probe. Dose-response relationships were determined before and during the inhibition of kinase II activity with captopril (2-D-methyl-3-mercaptopropanoyl-L-proline) and antagonism of angiotensin receptor sites with [Ile7]angiotensin III [( Ile7]ANG III). ANG I and [des-Asp1]ANG I were equipotent at all doses tested, as were ANG II and III. At all doses tested, ANG II and III were approximately three times more potent than ANG I and [des-Asp1]-ANG I. Captopril attenuated the vasoconstrictor responses to ANG I and [des-Asp1]ANG I only, whereas [Ile7]ANG III inhibited the responses to all four angiotensin peptides. These data indicate that the hepatic arterial vasoconstrictor responses to [des-Asp1]ANG I were due to the intrahepatic formation of ANG III. The extent of intrahepatic conversion of [des-Asp1]-ANG I to ANG III that occurred in one transit through the hepatic arterial vascular bed was estimated to be 33%, which was similar to the estimated 38% conversion of ANG I to ANG II.(ABSTRACT TRUNCATED AT 250 WORDS)

In vivo conversion of [des-Asp1]-angiotensin I to [des-Asp1]-angiotensin II in conscious sheep.

1. A method is described for the measurement of [des-Asp1]-angiotensin I (ANG I 1/2) in blood. 2. Exogenous ANG I 1/2 was rapidly metabolized in conscious sodium replete sheep. 3. Inhibition of angiotensin converting enzyme activity by SQ 14 225 (captopril) abolished the production of angiotensin III (ANG III) and the pressor response to infused ANG I 1/2. 4. The endogenous blood concentration of ANG I 1/2 was very low and not significantly elevated by sodium depletion. 5. Although in vivo pulmonary conversion of ANG I 1/2 to ANG III may occur, this pathway appears to be only of minor importance in sheep. 6. In the presence of converting enzyme inhibition by SQ 14 225, the arteriovenous ratio of ANG I 1/2 was 1.29 (s.e.m. = 0.25). This indicates pulmonary production of ANG I 1/2 and the presence in the lung of a peptidase which hydrolyses aspartic acid from angiotensin I.

Effect of converting enzyme inhibition with captopril on in vitro angiotensin production in sheep.

1. The effect of captopril on in vitro production of angiotensin I (ANG I), [Val5]-angiotensin II ([Val5]-ANG II) and [Val4]-angiotensin III ([Val5]-ANG-(2-8)) in central venous blood taken from sodium-deficient sheep was studied. 2. Captopril enhances in vitro production of ANG I but blocks the in vitro production of [Val5]-ANG II and [Val5]-ANG-(2-8). 3. The production of ANG I in blood is faster than that of [Val5]-ANG II and [Val5]-ANG-(2-8). 4. The half-life of [Val5]-ANG II and [Val5]-ANG-(2-8) in vitro in blood in the presence of captopril was 10 and 14 min, respectively. 5. This in vitro study suggests that the production of [Val5]-ANG II and [Val5]-ANG-(2-8) in blood forms a small part of the total body production of each peptide.

Peripheral production of angiotensin II and III in sheep.

The present experiments have allowed the calculation of concentrations of angiotensin I, II, and III in arterial and central venous blood. Assuming that endogenous arterial angiotensin II and III are handled as reflected by exogenous infusion, it can be calculated that 55% of the steady state arterial concentration of angiotensin II has arisen in passage across the peripheral vascular bed, that 40% of angiotensin III is also produced there, and that the arterial concentration of angiotensin III is 42% of the arterial concentration of angiotensin II.

Formation of angiotensin III from [des-Asp1]angiotensin I in the mesentric vasculature.

The effects of [des-Asp1]angiotensin I and angiotensin III on mesenteric blood flow were compared in 15 pentobarbital-anesthetized dogs. These agonists were administered as bolus injections directly into the vasculature supplied by the superior mesenteric artery. Both [des-Asp1]angiotensin I and angiotensin III produced dose-dependent decreases in mesenteric blood flow, with angiotensin III being more potent than [des-Asp1]angiotensin I at all doses tested. The constrictor responses to [des-Asp1]angiotensin I were markedly attenuated in the presence of an angiotensin-converting enzyme inhibitor (SQ20881); SQ20881 did not alter responses to angiotensin III or norepinephrine. The administration of [Ile7]angiotensin III (an angiotensin III antagonist) attenuated the responses to both [des-Asp1]angiotensin I and angiotensin III, without altering the responses to norepinephrine. These results suggest that the decrease in mesenteric blood flow produced by [des-Asp1]angiotensin I is largely caused by its local enzymatic conversion to angiotensin III. This conversion in one transit through the mesenteric vasculature is approximately 24%.

The biochemistry of the renin-angiotensin system and its role in hypertension.

The renin-angiotensin system has an important role in maintaining elevated blood pressure levels in certain forms of experimental and human hypertension. Renin, an enzyme produced by the juxtaglomerular cells of the kidney, acts on a protein substrate found in the alpha 2-globulin fraction of the plasma to produce a decapeptide, angiotensin I. This decapeptide is not directly pressor, but on passage through the pulmonary circulation is converted to an octapeptide, angiotensin II, a very potent pressor substance which acts by causing constriction of arteriolar smooth muscle. In addition to its direct action which increases blood pressure, angiotensin II acts on the adrenal cortex to cause the release of the sodium-retaining hormone aldosterone. Recent evidence suggests that this action may be mediated by the heptapeptide, angiotensin III. Both renin and its protein substrate exist in multiple forms and renin may also exist as a high molecular-weight "pro-hormone," although the physiologic significance of these forms is not clear. The elucidation of the biochemistry of the renin-angiotensin system has provided us with inhibitors which allow the system to be blocked effectively in vivo. Thus, angiotensin antagonists such as Sar 1, IIe 8-angiotensin II and converting enzyme inhibitors such as BPP 9a (SQ 20881) have proved useful in the study of experimental and human hypertension.