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Objective To evaluate the effects of angiotensins acting at the rostral ventrolateral medulla (RVLM) on the cardiovascular adjustments following haemorrhage. Deslgn Changes in mean arterial pressure (MAP) and heart rate (HR) produced by micro-injections of angiotensin II (Ang II) and angiotensin (Ang)-(1-7) and different angiotensin antagonists into the RVLM of anaesthetized rats submitted to haemorrhage, were determined. Methods Experiments were performed in 79 urethane-anaesthetized male Wistar rats. Ang-(1 -7) (2.5 and 25 pmol), Ang II (25 pmol), [Sar 1 ,Thr 8 ]-Ang II (non-selective angiotensin antagonist, 0.2 nmol), A-779 (Ang(1-7) antagonist, 0.1 nmol), losartan (AT 1 Ang II receptor antagonist, 0.2 nmol) or vehicle (200 nl) were bilaterally micro-injected into the RVLM under basal conditions or 30 min after blood withdrawal (0.6 ml/100 g bodyweight). In additional groups, [Sar 1 ,Thr 8 ]-Ang II, A-779, losartan or vehicle were micro-injected into the RVLM 10 min before bleeding to uncover a possible role of endogenous peptides during haemorrhage. Results The pressor effect produced by Ang II micro-injection was not altered by haemorrhage. Conversely, haemorrhage significantly increased the magnitude and duration of the pressor effect of Ang-(1-7) at the RVLM. The fall in MAP induced by haemorrhage was similar after micro-injection of vehicle or A-779. However, micro-injection of [Sar 1 ,Thr 8 ]-Ang II significantly reduced the fall in MAP after haemorrhage. A similar finding was obtained with micro-injection of losartan. In addition, while RVLM micro-injection of [Sar 1 ,Thr 8 ]-Ang II or losartan 30 min after blood withdrawn produced MAP changes that were similar to that observed in control animals, micro-injection of A-779 did not significantly alter baseline MAP. Conclusions These results suggest that changes in the RVLM reactivity to Ang-(1-7) but not Ang II may contribute to the haemodynamic adjustments triggered by acute reductions in blood volume. The data obtained with [Sar 1 ,Thr 8 ]-Ang II and losartan suggest a primary inhibitory role for endogenous Ang II at the RVLM during haemorrhage.

In this study, we evaluated the bradykinin potentiating activity and ACE inhibitory activity of several Ang-(1–7)-related peptides: Ang-(2–7), Ang-(3–7), Ang-(4–7), Ang-(1–6), Ang-(1–5) and the selective antagonist of Ang-(1–7): d -[Ala7]Ang-(1–7) (A-779). In vivo experiments were performed in freely moving Wistar rats. ACE activity was evaluated by a fluorometric assay in rat plasma using Hip-His-Leu as a substrate. Intravenous injections of Ang-(1–7) (2.2 nmol) transformed the effect of a single dose of bradykinin (1 nmol) into the effect produced by a double dose. A similar bradykinin potentiating activity was demonstrated for Ang-(2–7) and Ang-(3–7). On the other hand, Ang-(1–5), Ang-(1–6), Ang-(4–7) and A-779 did not change the hypotensive effect of bradykinin in doses ranging from 8 up to 25 nmols. The hypotensive effect of bradykinin was increased by intravenous infusion (0.3 ng/min) of Ang-(1–7) > Ang-(2–7) > Ang-(3–7). Conversely, Ang-(1–5), Ang-(1–6), Ang-(4–7) or A-779 did not change the hypotensive effect of bradykinin. ACE inhibition with Ang-(1–7) related peptides occurred in the order: Ang-(2–7) ≥ Ang-(3–7) > Ang-(1–7) [≫] Ang-(1–5) > Ang-(4–7) ≥ Ang-(1–6) ≥ A-779. A-779 in concentrations up to 10−5 M did not change the ACE inhibitory activity of Ang-(1–7). These results suggest that Ang-(1–7), Ang-(2–7) and Ang-(3–7) can modulate bradykinin actions in vivo. More important, our data pointed out that alternative mechanisms besides interaction with ACE are required to explain the bradykinin potentiating activity of Ang-(1–7).

In this study we evaluated the renal effects of chronic administration of the selective Angiotensin-(1-7)[Ang-(1-7)] antagonist, A-779, in normotensive and spontaneously hypertensive rats (SHR). Male adult SHR and Wistar rats were housed in metabolic cages with tap water and standard chow, for three-five days before starting infusion (Alzet osmotic mini-pumps) of A-779 (Wistar: 1 microg/h, n = 9; 2.5 microg/h, n = 6; SHR:2.5 microg/h, n = 6) or vehicle (0.9% NaCl - 1 microl/h, n = 7 and n = 10 for SHR and Wistar rats, respectively). Urine volume, water and food intake and urinary Na+ were measured daily. On the last day of infusions mean arterial pressure (MAP) was recorded and urine and blood samples were collected to determine renal function parameters. Chronic infusion of A-779 produced a sustained increase in diuresis in normotensive rats [seventh day values: 0.75+/-0.08 ml/h (1 microg/h) and 0.94+/-0.13 ml/h (2.5 microg/h) vs. 0.42 + 0.03 ml/h for the control group, P

Abstract In this study, we evaluated the effect produced by lateral ventricle (intracerebroventricular, ICV) infusion of the selective angiotensin (Ang)-(1-7) antagonist, d -Ala 7 -Ang-(1-7) (A-779), in the modulation of the baroreflex control of heart rate in two-kidney, one clip renovascular hypertensive rats (2K1C) treated with the angiotensin-converting enzyme (ACE) inhibitor enalapril. Twenty days after the surgery to produce renovascular hypertension, ICV cannulas were implanted in the rats with blood pressure (BP) greater than 145 mm Hg (n=33) and in sham-operated rats (n=32). Five days later, the rats were treated with enalapril (10 mg · kg −1 · d −1 ; 6 days, in the drinking water) or vehicle (tap water). On the sixth day of treatment, direct continuous BP recording and measurement of reflex changes in heart rate elicited by phenylephrine were made in conscious rats before and at 1 hour of ICV infusion of saline (8 μL/h) or A-779 (4 μg/h). To evaluate the degree of ACE blockade produced by enalapril treatment, the pressor effect of Ang I (50 ng, IV, and 100 ng, ICV) and plasma ACE activity was determined. As expected, enalapril treatment in 2K1C produced a significant fall in BP, significant attenuation in the pressor response of Ang I (IV), and a reduction in plasma ACE activity. In addition, enalapril treatment increased the baroreflex sensitivity (0.76±0.04 versus 0.43±0.04 ms/mm Hg in 2K1C untreated rats). ICV infusion of A-779 reverted the improvement in baroreflex sensitivity produced by enalapril treatment in 2K1C (from 0.80±0.07 to 0.42±0.08 ms/mm Hg) and also attenuated the baroreflex sensitivity in untreated 2K1C (0.36±0.05 versus 0.48±0.06 ms/mm Hg) and untreated sham-operated rats (1.21±0.05 versus 0.78±0.17 ms/mm Hg). These results suggest that central endogenous Ang-(1-7) is involved at least in part in the improvement of baroreflex sensitivity observed in 2K1C after peripheral chronic ACE inhibition.

In this study we determined the cardiovascular effects produced by microinjection of angiotensin peptides [Angiotensin-(1-7) and Angiotensin II] and angiotensin antagonists (losartan, L-158,809, CGP 42112A. Sar1-Thr8-Ang II, A-779) into the rostral ventrolateral medulla of freely moving rats. Microinjection of angiotensins (12.5-50 pmol) produced pressor responses associated to variable changes in heart rate, usually tachycardia. Unexpectedly, microinjection of both AT1 and AT2 ligands produced pressor effects at doses that did not change blood pressure in anesthetized rats. Conversely, microinjection of Sar1-Thr8-Ang II and the selective Ang-(1-7) antagonist, A-779, produced a small but significant decrease in MAP an HR. These findings suggest that angiotensins can influence the tonic activity of vasomotor neurons at the RVLM. As previously observed in anesthetized rats, our results further suggest a role for endogenous Ang-(1-7) at the RVLM. The pressor activity of the ligands for AT1 and AT2 angiotensin receptor subtypes at the RVLM, remains to be clarified.

Abstract We have recently shown that an angiotensin-(1-7) [Ang-(1-7)] analogue, d -Ala 7 -Ang-(1-7) (A-779), is a selective Ang-(1-7) antagonist with no significant action on angiotensin type 1 or type 2 receptors. The availability of selective angiotensin antagonists prompted us to evaluate the role of Ang-(1-7) and Ang II on central modulation of the baroreflex control of heart rate in normotensive Wistar rats and spontaneously hypertensive rats (SHR). Blood pressure recording and reflex changes in heart rate elicited by intravenous bolus injections of phenylephrine were made before and within 1 and 3 hours of intracerebroventricular (ICV, lateral ventricle) infusion of saline (8 μL/h), A-779 (4 μg/h), DuP 753 (100 μg/h), or CGP 42112A (50 μg/h) in conscious rats. The slope of the relationship between changes in pulse interval versus changes in mean arterial pressure was used as an index of the baroreflex control of heart rate. ICV infusion of saline or any of the antagonists did not significantly change basal levels of mean arterial pressure and heart rate in SHR (170±6 mm Hg and 360±9 beats per minute, respectively; n=29) or Wistar rats (108±2 mm Hg and 377±6 beats per minute, respectively; n=29). Three hours of ICV infusion of A-779 markedly decreased baroreflex sensitivity in Wistar rats (from a basal slope of 1.09±0.3). In contrast, A-779 did not significantly alter the depressed baroreflex sensitivity of SHR (0.61±0.1). ICV infusion of DuP 753 produced a significant increase (60%) in baroreflex control of heart rate in both Wistar rats and SHR. Saline or CGP 42112A infusions did not significantly alter baroreflex control of heart rate. These results suggest that endogenous Ang II and Ang-(1-7) are differentially affecting central baroreflex modulation, acting probably through distinct receptor subtypes. Although the central Ang II inhibitory effect is mediated by the type 1 receptor subtype, the facilitatory effect of Ang-(1-7) might be mediated by a different, unidentified receptor.

Abstract In this study we evaluated the possibility that angiotensin-(1-7) [Ang-(1-7)] acts as an endogenous osmoregulatory peptide by determining the effect of acute administration of its selective antagonist [ d -Ala 7 ]Ang-(1-7) (A-779) on renal function parameters in rats. In addition, we investigated the physiological mechanisms involved in the antidiuretic effect of Ang-(1-7). The antidiuretic effect of Ang-(1-7) (40 pmol/0.05 mL per 100 g BW) in water-loaded rats was completely blocked by A-779 (vehicle-treated, 3.34±0.43 mL/h; Ang-(1-7), 1.48±0.23; A-779, 2.72±0.35; Ang-(1-7) plus A-779, 3.26±0.49). In contrast, the antidiuretic effect of Ang-(1-7) was not significantly changed by a vasopressin V 2 receptor antagonist in a dose that completely blocked the antidiuresis produced by an equipotent dose of vasopressin. In addition, Ang-(1-7) administration did not significantly change vasopressin plasma levels in water-loaded rats. The antidiuretic effect of Ang-(1-7) in water-loaded rats was associated with a reduction of creatinine clearance (0.68±0.04 versus 1.38±0.32 mL/min in vehicle-treated rats, P P P 2 receptor antagonist. The diuretic effect of A-779 was associated with an increase in creatinine clearance and decrease in urine osmolality. In contrast, no significant effects on urine volume were observed after systemic administration of angiotensin subtype 1 or 2 receptor antagonists (DuP 753 and CGP 42112A, respectively). These findings suggest that endogenous Ang-(1-7), acting on specific receptors, participates in the control of hydroelectrolyte balance by influencing especially water excretion.

Abstract Treatment with angiotensin-converting enzyme inhibitors increases the angiotensin-(1-7) [Ang-(1-7)] and bradykinin concentrations in plasma and tissue. In this study we evaluated the interaction between these peptides by determining the effect of Ang-(1-7) on the hypotensive action of bradykinin in conscious rats. Administration of Ang-(1-7) (5 nmol) did not change mean arterial pressure or heart rate. However, the hypotensive effect of bradykinin, produced by an intravenous or intra-arterial route, was potentiated by Ang-(1-7) in a dose-dependent manner. The Ang-(1-7) doses necessary to transform the effect of a single dose of bradykinin into that produced by a double dose (potentiating unit) were 2 nmol IV and 5 nmol IA. The Ang-(1-7) dose used did not change either the pressor effect of Ang II or the hypotensive effect of sodium nitroprusside. The bradykinin-potentiating Ang-(1-7) activity was significantly attenuated by pretreatment with indomethacin (5 mg/kg IM, n=4). In an additional group the bradykinin-potentiating activity of Ang-(1-7) was evaluated 30 minutes after treatment with the angiotensin-converting enzyme inhibitor enalaprilat (10 mg/kg IV, n=9). Under this condition the bradykinin-potentiating activity of Ang-(1-7) was substantially increased, resulting in a potentiating unit of approximately 0.2 nmol IV. Pretreatment with indomethacin (5 mg/kg IM, n=7) also attenuated the bradykinin-potentiating activity of Ang-(1-7) in enalaprilat-treated rats. These results show that Ang-(1-7) is a bradykinin-potentiating peptide in vivo. Furthermore, the data obtained with indomethacin suggest that prostaglandins participate in the mechanism of the bradykinin potentiation by Ang-(1-7). More importantly, these data suggest that the interaction between Ang-(1-7) and bradykinin can contribute to the pharmacological effects of angiotensin-converting enzyme inhibitors.

In this study, the formation of biologically active angiotensins from angiotensin I (Ang I) in isolated rat hearts was evaluated. The role of angiotensin converting enzyme (ACE) in Ang I metabolism was also investigated. HPLC analysis of heart perfusate showed that 125I-Ang I was metabolized extensively (single passage) in the rat coronary circulation in vitro leading to the formation of the biologically active angiotensins: angiotensin II (Ang II), Ang-(2-8), Ang-(3-8) and Ang-(1-7). Ang II was the major product identified in HPLC fractions, corresponding to 7.8 +/- 0.89% of the total radioactivity recovered. A similar profile was observed when single-passage metabolism of non-isotopic Ang I was evaluated by HPLC, followed by radioimmunoassay of the eluate fractions. When 125I-Ang I was perfused in the presence of ACE inhibitors (enalaprilat, ramiprilat) in concentrations up to 130 microM, the formation of Ang II was only partially inhibited (approximately 50%). A similar tendency was observed for Ang-(2-8), Ang-(3-8) and Ang-(2-7). The formation of Ang-(1-7) and its related fragments Ang-(3-7) and Ang-(4-7) was not changed significantly by ACE inhibitors, although a slight increase in formation of these fragments was observed. No significant changes were observed for the carboxyl-terminal fragments of Ang I: Ang-(2-10), Ang-(3-10), and Ang-(4-10). The fractional metabolism of Ang I was not modified by ACE inhibition. These findings suggest that biologically active angiotensins can be formed from Ang I in the rat coronary circulation. These locally generated peptides may contribute to the actions of the renin-angiotensin system in the heart.

We investigated the effect of losartan, a nonpeptide angiotensin II (Ang II)-type 1 (AT1) receptor antagonist, on the responses evoked by Ang II and L-glutamate (L-Glu) in the rostral ventrolateral medulla (RVLM). Adult spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats were anesthetized with halothane and artificially ventilated. Responses of mean arterial pressure (MAP), heart rate (HR) and splanchnic sympathetic nerve activity (SNA) to microinjection of Ang II (100 pmol) or L-Glu (2 nmol) into the RVLM were examined following microinjection of losartan (10 pmol-10 nmol). Ang II increased MAP (16 +/- 1 mmHg in SHR and 16 +/- 1 mmHg in WKY) and SNA (9 +/- 1% and 10 +/- 1%, respectively), which were significantly (P < 0.01) attenuated by pretreatment with losartan (100 pmol-10 nmol) in both strains. In addition, the pressor and sympathoexcitatory responses evoked by L-Glu were attenuated by losartan in a dose-dependent manner. The increases of MAP evoked by L-Glu (53 +/- 6 mmHg in SHR and 39 +/- 3 mmHg in WKY) were suppressed to 5 +/- 3 mmHg (P < 0.01) and 4 +/- 2 mmHg (P < 0.01), respectively, in the presence of 10 nmol of losartan. The increase of SNA was also markedly inhibited by higher doses of losartan.(ABSTRACT TRUNCATED AT 250 WORDS)

In this study we determined which angiotensin receptors may mediate the cardiovascular effects elicited by angiotensin-(1–7) [Ang-(1–7)] in the rostral ventrolateral medulla (RVLM) and caudal pressor area (CPA) of the ventrolateral medulla (VLM) of anesthetized rats. Furthermore the role of endogenous angiotensins in these areas was also investigated. The pressor effect produced by unilateral microinjection of Ang-(1–7) into the RVLM or CPA was not modified by either the AT 1 receptor antagonist, DuP 753 or by the AT 2 receptor antagonist, CGP 42112A, but was completely blocked by the Ang-(1–7) selective antagonist, A-779. In contrast, the pressor effect produced by microinjection of angiotensin II (Ang II) was completely blocked by DuP 753 but was not changed by CGP 42112A or A-779. Bilateral microinjection of A-779 into the RVLM or CPA produced a significant fall in mean arterial pressure and heart rate. Microinjection of DuP 753 produced a pressor effect comparable to bilateral injection of vehicle. These results indicate that, although Ang II acts in the VLM through an AT 1 receptor subtype, the cardiovascular effects produced by microinjection of Ang-(1–7) into the RVLM and CPA are mediated by a specific angiotensin receptor (AT 5 ?). Furthermore, our data provide evidence that endogenous Ang-(1–7) participates at the VLM in the neural control of arterial blood pressure.

Modulation of phospholipase A2 activity and sodium transport by angiotensin-(1-7). Angiotensin II (Ang II) receptors are coupled to a variety of signal transduction mechanisms. In the kidney, Ang II at nanomolar concentration binds to proximal tubular cells and stimulates phospholipase A2 (PLA2), which in turn catalyzes the hydrolysis of phosphatidylcholine into lysophosphatidylcholine (LPC) and fatty acid. This signal transduction pathway has been shown to be an important modulator of sodium transport. The kidney cortex possesses the enzyme necessary to convert angiotensin I (Ang I) directly to Ang-(1-7) bypassing Ang II as an intermediate. The present investigation was undertaken to determine whether Ang-(1-7) influences epithelial cell function by comparing this heptapeptide with Ang II as a modulator of PLA2 activity and sodium transport. Proximal tubular cells were labeled in tissue culture with 3H-choline and PLA2 activity was measured by quantitation of LPC. We found that Ang II (10-9 M to 10-6 M) significantly increased PLA2 activity (154 ± 36% to 209 ± 94%). Similar results were obtained with Ang-(1-7) (240 ± 130% to 353 ± 40%). The bioactivity of the peptides was assayed by its ability to regulate transcellular 22Na flux. Ang II (10-9 M) inhibited 22Na flux by 12 ± 2% while Ang-(1-7) (10-9 M) inhibited 22Na flux by 20 ± 5%. These results suggest that one potential role of Ang-(1-7) in the regulation of kidney epithelial electrolyte transport may involve activation of PLA2.

In this study, we determined the cardiovascular effects produced by micro-injection of the heptapeptided Angiotensin-(1–7) [Ang-(1–7)] into the rat ventrolateral medulla (VLM). Micro-injection of Ang-(1–7) into the rostral VLM and the caudal pressor area of the VLM produced significant increases in arterial pressure, comparable to that observed with micro-injection of Ang II. The changes in arterial pressure were associated with more variable changes in heart rate (HR) (usually tachycardia). On the other hand, micro-injection of Ang-(1–7) into the caudal depressor area induced decreases in arterial pressure and HR. The results suggest that, besides Ang II, Ang-(1–7) is involved in the mediation of the cardiovascular actions of the renin-angiotensin system in the VLM.

On the basis of biochemical and autoradiographic studies it has been shown that the inferior olivary nucleus (ION) contains predominantly angiotensin II (Ang II) receptors of the subtype 2 (AT2). In the present investigation we used microiontophoretic techniques to test the effect of Ang II on the spontaneous firing rate of rat neurones in the ION in vivo. Ang II excited the majority of histologically identified ION neurones. Furthermore, the antagonism of this angiotensin-induced excitation by selective angiotensin receptor blockers of subtype 1 and 2 (AT1 and AT2) was examined. The excitation could be blocked by low doses of the AT2-antagonists PD 123177 and CGP 42112A, whereas the AT1-antagonist DuP 753 was ineffective even at high doses. On a few occasions, however, ejection of the AT1-antagonist resulted in a potentiation of angiotensin-induced excitation. The results suggest that Ang II has an excitatory effect on a considerable number of ION neurones and that this effect is mediated by AT2-receptors.

NG108-15 cells, a neurally derived clonal cell line, express various components of the renin-angiotensin system and thus serve as a model of the cellular action of angiotensin (Ang) II. NG108-15 cells contain a high-affinity binding site for Ang II, with a Kd of 1.1 nM and a Bmax of 6.5 fmol/mg protein. Ang peptides competed for 125I-Ang II binding with an order of potency of Ang II greater than Ang-(2-8) much greater than Ang-(1-7). The subtype 1 (or B)-selective Ang II receptor antagonist DuP 753 as well as [Sar1,Ile8]Ang II and [Sar1,Thr8]Ang II competed for Ang II binding with high affinity, whereas the subtype 2 (or A)-selective Ang receptor antagonist CGP 42112A was partially effective only at a 300-fold higher concentration. When NG108-15 cells were induced to differentiate by treatment with dibutyryl cyclic adenosine 3',5'-monophosphate, the density of Ang II receptors increased dramatically, with little change in affinity (1.1 versus 4.2 nM) or competition by Ang peptides. In marked contrast to undifferentiated cells, CGP 42112A became a potent competitor (IC50, 1 nM) for the majority (90-95%) of Ang II binding, whereas DuP 753 competed for only 5-10% of the binding sites. Ang II caused a dose-dependent mobilization of cytosolic Ca2+ in undifferentiated NG108-15 cells through activation of phospholipase C and the production of inositol 1,4,5-trisphosphate. In these cells, Ca2+ mobilization was blocked by either DuP 753 or the sarcosine Ang II analogues, whereas CGP 42112A was ineffective. Ang II also mobilized intracellular Ca2+ in differentiated NG108-15 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

The heptapeptide angiotensin (ANG)-(1-7) mimics some but not all the central actions of ANG II, suggesting that receptor subtypes may exist. The effects of ANG-(1-7), ANG II, and ANG I on prostaglandin (PG) E2 and prostacyclin (PGI2) synthesis were investigated in neurally derived rat C6 glioma cells. All three ANG peptides stimulated PG release in a dose-dependent manner with the order of potency ANG-(1-7) greater than ANG I greater than ANG II. PGE2 release induced by ANG-(1-7) (10(-7) M) was partially blocked by [Sar1,Ile8]ANG II (10(-6) M), [Sar1,Thr8]ANG II (10(-6) M), or the subtype 1 selective antagonist Du Pont 753 (10(-5) M) but not by the subtype 2 selective antagonist CGP 42112A (10(-7)-10(-5) M). PGI2 release was inhibited only by [Sar1,Thr8]ANG II. ANG II-induced PGE2 release was blocked by [Sar1,Thr8]ANG II (10(-6) M), [Sar1,Ile8]ANG II (10(-6) M), or Du Pont 753 (10(-7) M) but not by CGP 42112A (10(-7)-10(-5) M). In contrast, ANG II-induced PGI2 release was blocked by Du Pont 753 (10(-7) M) as well as [Sar1,Ile8]ANG II (10(-6) M) but not by [Sar1,Thr8]ANG II or CGP 42112A. Thus ANG II-stimulated PGE2 and PGI2 syntheses in C6 glioma cells are mediated via receptor subtype 1. ANG-(1-7)-induced PGE2 synthesis is also mediated via subtype 1 receptors; however, PGI2 release was blocked by [Sar1,Thr8]ANG II only.(ABSTRACT TRUNCATED AT 250 WORDS)

In an attempt to define the angiotensin II receptor subtype responsible for prostaglandin release, we studied the effects of the nonpeptide, subtype 1 (or B) selective angiotensin II antagonist, DuP 753. Release of prostaglandin E2 produced by angiotensin II from rat C6 glioma, human astrocytoma, or porcine aortic smooth muscle cells in culture was blocked by the addition of the 10(-7) M of DuP 753. In contrast, the release of prostacyclin, as assessed by measurement of the stable metabolite 6-keto PGF1 alpha, was not attenuated by addition of Du P 753. However, DuP 753 either alone or in combination with angiotensin II, produced dose-dependent increases in prostacyclin release with doses as low as 10(-8) M. In the absence of angiotensin II, DuP 753 also increased prostaglandin E2 release at high doses but the magnitude of the potentiation was substantially less than for prostacyclin release (50 to 250% v 400 to 2800% above basal). Thus, we clearly show that angiotensin II stimulates PGE2 release via subtype 1 (or B) angiotensin receptors. Whether the effect of DuP 753 on prostaglandin release is a result of agonistic properties or intrinsic effects unrelated to blockage of angiotensin II receptors remains to be determined. The marked stimulatory effect of DuP 753 release precludes characterization of the receptor subtype that mediates the Ang II-induced release of prostacyclin. Nonetheless, potent stimulation of prostacyclin release by DuP 753, especially in vascular smooth muscle cells, requires reevaluation of the mechanisms that participate in the anti-hypertensive effects of the compound.

We measured the concentrations of three principal products of the renin-angiotensin system and seven of their metabolites in the plasma of anesthetized normal dogs and in dogs 24 hours after bilateral nephrectomy. The levels of the angiotensin peptides were measured by high-performance liquid chromatography combined with radioimmunoassay using three specific antibodies that recognized different epitotes in the sequences of angiotensin I, angiotensin II, and angiotensin-(1-7). The analysis revealed that angiotensin-(1-7) is present in the plasma of intact (4.9 +/- 2.2 fmol/ml) and nephrectomized (0.5 +/- 0.5 fmol/ml) dogs. An intravenous injection of purified hog renin (0.01 Goldblatt unit/kg) increased plasma levels of angiotensin I, angiotensin II, and angiotensin-(1-7) both before and after nephrectomy. These changes were associated with parallel increases in the concentrations of fragments of the three parent peptides. Administration of MK-422 led to the disappearance of circulating angiotensin II and its fragments both before and after a second injection of the same dose of renin. In contrast, MK-422 augmented the plasma levels of both angiotensin I and angiotensin-(1-7). The concentrations of these two peptides, but not the blood pressure, were again augmented by a second injection of renin given after blockade of converting enzyme. These effects were observed both before and after bilateral nephrectomy. These findings show that angiotensin-(1-7) circulates in the blood of normal and nephrectomized dogs. In addition, we found that angiotensin-(1-7) is generated in the blood from the cleavage of angiotensin I through a pathway independent of converting enzyme (EC 3.4.15.1).

Two synthetic analogues of angiotensin II (ANG II) with an extended N-terminus, (Sar)5-ANG II and (Pro)3-ANG II, have been tested in vitro for their ability to bind to ANG II receptors, to raise cytosolic free calcium concentration, (Ca++]i, and to induce a biological response in bovine adrenal zona glomerulosa cells and in cultured rat aortic smooth muscle cells. The results indicate that the two analogues did not behave identically In these two target cells for ANG II. On one hand, in the adrenal cortex, (Sar)5-ANG II and (Pro)3-ANG II were very weak agonists and (Sar)5-ANG II could even be used as an antagonist of ANG II-induced aldosterone production. On the other hand, both peptides were almost as potent as ANG II in vascular smooth muscle cells, with respect to signal messenger generation and prostacyclin synthesis. Such peptides may be useful tools in the elucidation of the differences among ANG II receptors from various target tissues.

We examined the hypothesis that the positive inotropic effect of angiotensin I (Ang I) may be retained in the presence of angiotensin converting enzyme inhibitors so that it may have a direct beneficial effect on the heart. Accordingly, isolated perfused hearts (Langendorff preparation) of 300-day-old cardiomyopathic hamsters (a model of spontaneous cardiomyopathy) and age-matched normal hamsters (controls) were infused with Ang I in the presence of captopril; propranolol was added to the perfusing medium to block catecholamine-mediated effects of angiotensins on the heart. Left ventricular developed pressure and the rate of increase in left ventricular developed pressure increased significantly (p less than 0.001) in both the cardiomyopathic and the normal hamster heart despite concomitant reduction in myocardial flow rate favoring a direct inotropic effect of Ang I in both normal and myopathic hearts; these changes were significantly higher by almost threefold in the cardiomyopathic than in the normal hamsters (p less than 0.01) and were blocked by the angiotensin II (Ang II) antagonist [Sar1,Thr8]Ang II. Comparing dose-left ventricular contractility response curves for Ang I and Ang II, ED50 for responses was identical in both normal and myopathic hearts, whereas peak responses to Ang II were double those to Ang I in normal hearts but were almost identical in the myopathic hearts. Binding of [125I]Ang II in six cardiomyopathic and four normal hamster hearts was of high affinity, but there was no evidence for Ang I-saturable high-affinity binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure-activity studies of angiotensin II (Ang II) have clearly established that the carboxyl-ter-minal phenylalanine is crucial for activation of angiotensin receptors associated with a broad range of effects including vasoconstriction, aldosterone release, and dipsogenesis. Thus, the heptapeptide angiotensin-[1-7] (Ang-[1-7]) has been classified as an inactive metabolite of the hormone Ang II. This assumption was seriously questioned in light of the report from our laboratory that Ang-[1-7] is the major metabolite of dog brainstem homogenates both in the absence and in the presence of angiotensin-converting enzyme inhibitors. These data raised the possibility of a functional role for Ang-[1-7] in the brain. Subsequent studies in the in vitro hypothala-moneurohypophysial system of the rat demonstrated that Ang-[1-7] is equipotent with Ang II in its activation of AVP release. Other Ang II-like actions have since been identified for Ang-[1-7] in the brain. For example, microinjection of Ang-[1-7] into brainstem nuclei of the rat promotes bradycardia and hypotension; in addition, Ang-[1-7] mimics the effect of Ang II in augmenting the intrinsic discharge rate of neurons within the vagal-solitary complex of the dog. Our laboratory has also provided evidence for synthesis and storage of Ang-[1-7] in the brain. Immunocytochemical studies have localized Ang-[1-7] in neuronal cell bodies and fiber tracts of magno-cellular nuclei in the rat hypothalamus; in addition, analysis of rat brain extracts by high-performance liquid chro-matography (HPLC) and radioimmunoassay (RIA) has demonstrated that Ang-[1-7] is a predominant angiotensin peptide in the hypothalamus and medulla oblongata of the rat. These studies have been complemented by preliminary identification of a specific class of high-affinity (nM) binding sites for Ang-[1-7] in subnuclear regions of the dog brainstem. Given that Ang-[1-7] is distinguished by its lack of angiotensinergic effects, such as vasoconstriction and dipsogenesis, the recent identification of actions and binding sites for Ang-[1-7] in the brain is indicative of multiple subclasses of angiotensin binding sites. Moreover, biochemical evidence of Ang-[1-7] distribution in the brainstem suggests that some of the biological actions of Ang II in the brain are mediated by other angiotensin peptides, including Ang-[1-7].

In this study we explored the possibility that angiotensin-(1–7) (Ang-(1–7)) is involved in the control of blood pressure at the rostral ventrolateral medulla (RVLM) by determining the effect of angiotensin antagonists (DuP 753 and A-779) and the effect of the angiotensin converting enzyme inhibitor, ramiprilat on the pressor action produced by angiotensin I (Ang I). The pressor effect produced by bilateral microinjection of Ang I into the RVLM of anesthetized rats was not significantly altered by DuP 753 or by the ACE inhibitor ramiprilat. Conversely, the Ang-(1–7) antagonist, A-779, reduced significantly the pressor effect produced by Ang I. These data suggest that in our experimental condition Ang I was preferentially converted to Ang-(1–7) at RVLM, or that Ang I and/or one of its fragments acts through a receptor blocked by A-779.

Microiontophoretic application of both, the octapeptide angiotensin II (Ang II) and its N-terminal heptapeptide angiotensin-(1-7), [Ang-(1-7)], has been shown to increase the firing rate of rat hypothalamic paraventricular neurones. In the present microiontophoretic study, the effect of the angiotensin analogue [7-D-Ala]-Ang-(1-7) on Ang II- and Ang-(1-7)-induced firing rate increase of paraventricular neurones has been tested. While the response to Ang II was unchanged, the response to Ang-(1-7) was effectively blocked by [7-D-Ala]-Ang-(1-7). The results indicate that the Ang-(1-7)-induced excitation of paraventricular neurones may be mediated by a distinct Ang-(1-7)-receptor and that [7-D-Ala]-Ang-(1-7) is a selective antagonist of this receptor.

Ang-(1-7) is an effector peptide of the renin-angiotensin system with several distinct actions that are likely mediated by a specific receptor. Regulatory effects of angiotensin (Ang) peptides, Ang-(1-7) and Ang II, on Ang receptor subtype 1 (AT1) mRNA expression were investigated in vascular smooth muscle cells (VSMC) from four University of Akron (Akr) rat strains (WKY, SHR and two backcross consomic lines SHR/y and SHR/a), and in SHR and WKY cells from Charles River Laboratories (Crl). In WKY/Akr and SHR/Akr, Ang-(1-7) treatment increased the levels of AT1 mRNA. This effect was inhibited by the specific Ang-(1-7) antagonist, A-779, in WKY/Akr but not SHR/Akr. Ang II had no effect in Akr cells, but it down-regulated AT1 mRNA in WKY/Crl and SHR/Crl VSMC. Ang-(1-7) did not affect AT1 mRNA levels in Crl lines. In conclusion, Ang-(1-7) regulates the AT1 receptor either directly or indirectly in a strain-specific fashion. The Ang-(1-7) antagonist, A-779, blocks the actions of Ang-(1-7) only in VSMC from WKY/Akr rats, suggesting either that the binding sites for Ang-(1-7) have different properties in SHR/Akr and WKY/Akr cell lines, or that some of the effects of Ang-(1-7) are not receptor mediated. Further, we found differences between Akr cells and Crl cells that are consistent with their genetic heterogeneity.

Exogenous angiotensin (Ang) 1-7 affects renal function, but the receptor(s) involved in this response remain(s) to be determined. In an in vitro preparation of proximal tubules, Ang 1-7 was shown to act on Ang II AT1 receptors (minor component), but also on a non-AT1, non-AT2 Ang receptor (major component) to inhibit reabsorption. In brain, Ang 1-7 also exerts effects mediated by a non-AT1, non-AT2 binding site; these effects are inhibited, however, by the angiotensin analog [7-D-Ala]-Ang 1-7. Therefore we tested the effect of Ang II AT1-receptor antagonist losartan and [7-D-Ala]-Ang 1-7 on the renal response to exogenous Ang 1-7 in standard renal-clearance experiments in the anesthetized rat. We found that Ang 1-7 (100 pmol/kg/min, i.a.) increased glomerular filtration rate (GFR), urinary flow rate (UV), and urinary sodium excretion (UNaV) without affecting mean arterial blood pressure (MAP) or urinary potassium excretion (UKV), confirming previous reports. Losartan (10 mg/kg, i.v.) blocked the pressor effect of exogenous Ang II (100 pmol/kg/min, i.a.), but did not significantly affect the renal response to Ang 1-7. Conversely, pretreatment with [7-D-Ala]-Ang 1-7 (5 nmol/kg/min) did not affect the pressor effect of Ang II, but abolished the renal response to Ang 1-7. Application of [7-D-Ala]-Ang 1-7 in the absence of exogenous Ang 1-7 did not alter MAP or GFR, but increased UNaV (by 52%). Our data indicate that similar to the response in brain, the renal response to exogenous Ang 1-7 may be mediated predominantly by a distinct non-AT1 binding site, which is sensitive to blockade by [7-D-Ala]-Ang 1-7. Furthermore, ambient endogenous Ang 1-7 acting on this distinct binding site may not contribute significantly to control of MAP or GFR, but exerts an antinatriuretic influence in the anesthetized rat.

In this study we describe a new angiotensin antagonist [Asp1-Arg2-Val3-Tyr4-Ile5-His6-D-Ala7, (A-779)] selective for the heptapeptide angiotensin-(1-7) [Ang-(1-7)]. A-779 blocked the antidiuretic effect of Ang-(1-7) in water-loaded rats and the changes in blood pressure produced by Ang-(1-7) microinjection into the dorsal-medial and ventrolateral medulla. In contrast, A-779 did not change the dipsogenic, pressor, or myotropic effects of angiotensin II (Ang II). Also, A-779 did not affect the antidiuretic effect of vasopressin or the contractile effects of angiotensin III, bradykinin, or substance P on the rat ileum. In the rostral ventrolateral medulla, the pressor effect produced by Ang-(1-7) microinjection was completely blocked by A-779 but not by AT1 or AT2 receptor antagonists (DUP 753 and CGP 42112A, respectively). Conversely, the pressor effect produced by Ang II was not changed by A-779 but was completely blocked by DUP 753. Binding studies substantiated these observations: A-779 did not compete significantly for 125I-Ang II binding to adrenocortical membranes at up to a 1 microM concentration. Low affinity binding was also observed in adrenomedullary membranes with an IC50 greater than 10 microM. Our results show that A-779 is a potent and selective antagonist for Ang-(1-7). More importantly, our data indicate that specific angiotensin receptors mediate the central and peripheral actions of Ang-(1-7).

In this study we investigated the effects of dehydration and hemorrhage on circulating levels of the heptapeptide, angiotensin(1-7). In water-deprived rats, a twofold increase in plasma angiotensin(1-7) was associated with similar increases in plasma renin activity, and angiotensin I and angiotensin II levels. In salt-loaded rats, plasma angiotensin(1-7) levels increased fourfold; however, other components of the renin-angiotensin system were suppressed or unchanged. In salt-loaded rats, increases in plasma angiotensin II levels in response to hemorrhage in normal rats were severely blunted, whereas angiotensin(1-7) plasma levels increased proportionately to the loss of blood volume. These results suggest that angiotensin(1-7) plasma concentration can be selectively regulated during dehydration and hemorrhage.

This study evaluated the actions of nitric oxide on the blood pressure and renal sympathetic nerve activity responses produced by angiotensin II (Ang II) blockade in conscious spontaneously hypertensive rats. Two days after implantation of electrodes, we measured mean arterial pressure, heart rate, and renal sympathetic nerve activity. Baroreceptor reflex function was assessed with a logistic function curve; the maximum slope of the curve estimated the baroreceptor reflex gain. Data were obtained in rats given acute intravenous administration of either vehicle, the Ang II type 1 receptor antagonist losartan, the type 2 antagonist CGP 42112A, or the converting enzyme inhibitor lisinopril. In comparison with vehicle (-1.1 +/- 0.2%/mm Hg), both losartan (-1.8 +/- 0.3%/mm Hg) and lisinopril (-2.4 +/- 0.2%/mm Hg) significantly increased the maximum gain of the baroreceptor reflex control of nerve activity (p < 0.05). In contrast, the type 2 receptor antagonist did not alter baroreceptor reflex function. Similar studies were performed in rats that received an intravenous injection of NG-monomethyl L-arginine (10 mg/kg). The nitric oxide synthase inhibitor increased baseline blood pressure and decreased renal sympathetic nerve activity. Subsequent administration of losartan or lisinopril returned blood pressure to initial hypertensive level, whereas sympathetic nerve activity was increased to a level above the initial control value. The maximum gain of the baroreceptor reflex control of renal nerve activity was increased after the nitric oxide inhibition. The present study demonstrates that blunted baroreceptor reflex function in conscious spontaneously hypertensive rats is mediated by an Ang II type 1 receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Regional vascular responses to angiotensin (Ang)-(1-7), a heptapeptide derivative of Ang II were investigated in the feline hindquarters and mesenteric vascular beds under conditions of controlled flow. In the mesenteric vascular bed, injections of Ang-(1-7) in doses of 1, 3 and 10 micrograms produced dose-dependent decreases in mesenteric perfusion pressure whereas at doses of 30 and 100 micrograms, increases were observed. In contrast, in the hindquarters circulation, low doses produced increases while high doses produced decreases in perfusion pressure. In both vascular beds the degree of vasoconstriction was weak, being less than 1% of that elicited by Ang II. The vasoconstrictor effect of Ang-(1-7) in both the mesenteric and hindquarters vascular bed was blocked by DuP 753 (1 mg/kg i.v.), an Ang receptor subtype 1 (AT1) antagonist. The vasodilator responses in both vascular beds were partially blocked by the nitric oxide synthase inhibitor, NG-nitro-L-arginine methyl ester (100 mg/kg i.v.) but were unaffected by the cyclooxygenase inhibitor, meclofenamate (2.5 mg/kg i.v.). The present results show that in the peripheral vascular bed of the cat, Ang-(1-7) causes vasodilation or modest vasoconstriction, depending on the dose and the regional vascular bed studied. The present data also suggest that the vasodilator effect of the peptide may be mediated in part by the release of endothelium-derived relaxing factor and the vasoconstrictor effect by activation of the AT1 receptor subtype.

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.

Angiotensin II stimulates prostaglandin release in blood vessels via activation of angiotensin receptors present in endothelium, vascular smooth muscle cells, or both. We evaluated the response of angiotensin II, angiotensin I, and [des-Phe8] angiotensin II [angiotensin-(1-7)] on prostaglandin release in porcine aortic endothelial cells. Incubation of cell monolayers with angiotensin I and angiotensin-(1-7), but not angiotensin II, stimulated the release of prostaglandin E2 and prostaglandin I2 in a dose-dependent manner (10(-10) to 10(-6) M) with an EC50 of approximately 1 nM. In addition, we characterized the angiotensin receptor subtypes mediating prostaglandin synthesis by using subtype-selective antagonists. Angiotensin I-stimulated prostaglandin synthesis was not altered by either of the nonselective classical angiotensin receptor antagonists [Sar1,Thr8]angiotensin II or [Sar1,Ile8]angiotensin II. In contrast, either the angiotensin subtype 1 (AT1) antagonist DuP 753 or the subtype 2 (AT2) antagonist CGP42112A significantly attenuated the prostaglandin release in response to angiotensin I. However, PD123177, another AT2 antagonist, did not inhibit angiotensin I-stimulated prostaglandin release. Angiotensin-(1-7)-induced prostaglandin release was significantly attenuated by [Sar1,Thr8]angiotensin II (10(-6) M) and PD123177 (10(-6) M) but not by [Sar1,Ile8]angiotensin II, DuP 753, or CGP42112A. Higher doses (10(-5) M) of DuP 753 and CGP42112A attenuated the angiotensin-(1-7) response. These data suggest that in porcine aortic endothelial cells, angiotensin I and angiotensin-(1-7) but not angiotensin II are potent stimuli for prostaglandin synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

We have identified two distinct cellular responses that occur in human astrocytes in the presence of angiotensin (Ang) peptides and are linked to specific receptor subtypes. Ang II and the N-terminal heptapeptide Ang-(1-7) stimulated release of prostaglandin (PG) E2 and PGI2 (measured as the stable metabolite 6-keto-PGF1 alpha). In contrast, only Ang II but not Ang-(1-7) activated phosphoinositide-specific phospholipase C, leading to mobilization of intracellular calcium. The Ang II-induced PGE2 and PGI2 syntheses were attenuated by [Sar1,Ile8]Ang II but not by [Sar1,Thr8]Ang II. Ang-(1-7)-induced PGE2 and PGI2 syntheses were not inhibited by either of these two classical antagonists. DuP 753, a subtype 1-selective Ang receptor antagonist, blocked the Ang II-induced release of PGE2 but not PGI2. In contrast, CGP 42112A, the subtype 2-selective antagonist, totally blocked the Ang II-induced PGI2 release and partially attenuated the PGE2 release. Ang-(1-7)-induced PGE2 and PGI2 release was not altered by DuP 753; however, CGP 42112A totally blocked the effects of Ang-(1-7) on PG stimulation. Calcium mobilization in response to Ang II was blocked by [Sar1,Thr8]Ang II, [Sar1,Ile8]Ang II, and DuP 753 but not by CGP 42112A. These data suggest that human astrocytes contain both Ang receptor subtypes. The subtype 1 Ang receptor participates both in the release of PGs and in the mobilization of calcium, whereas the subtype 2 receptor is coupled to the release of PGs only. In addition, PG release coupled to subtype 2 Ang II receptors occurs through a calcium-independent mechanism and responds uniquely to Ang-(1-7).

Angiotensin II has been reported to have both a positive inotropic effect and a coronary constrictor action in the hamster heart. To study the contribution to these responses of phenylalanine in position 8, we assessed the direct cardiac effects of angiotensin-(1-7), which lacks phenylalanine in position 8. Syrian hamsters were used to determine the effects of angiotensin-(1-7) on cardiac performance in the diseased and normal hearts. We used the isolated isovolumic heart preparation perfused either at a constant pressure of 50 mm Hg or at a constant coronary (myocardial) flow rate of 7 ml/min (seven cardiomyopathic hamsters [CMH] and seven normal hamsters [NH] in each subgroup). At constant perfusion pressure, coronary (myocardial) flow rate decreased (p less than 0.01) in both CMH and NH (-31 +/- 8% vs. -39 +/- 4% of baseline, respectively); but the percent decrease in left ventricular pressure and the first derivative of left ventricular pressure over time (LV + dP/dt) was significant only in NH (-8 +/- 1% and -9 +/- 4%) but not in CMH (-14 +/- 5% and -21 +/- 8%). On the other hand, at a constant coronary (myocardial) flow rate, left ventricular pressure and LV + dP/dt tended to increase in both CMH and NH (+10 +/- 3% and +6 +/- 2% of baseline vs. +7 +/- 7% and +7 +/- 5%, respectively) but these changes were not significant.(ABSTRACT TRUNCATED AT 250 WORDS)

Modifications in angiotensin II and its antagonistic peptides that should have increased in vivo half-lives but not reduced biological activity were studied by determining the effect of alpha-methylation of the tyrosine in position 4. [alpha-Methyltyrosine-4]angiotensin II, synthesized by the solid-phase procedure, showed 92.6 +/- 5.3% pressor activity of angiotensin II. Incubation with alpha-chymotrypsin for 1 hr indicated absence of degradation although, under the same conditions, angiotensin II was completely degraded to two components. Comparison of the 1H NMR spectra in aqueous solution and the circular dichroism spectra in trifluoroethanol of angiotensin II and [alpha-methyltyrosine-4]angiotensin II suggested that alpha methylation of the tyrosine residue in angiotensin II does not lead to major changes in the overall solution conformation. These results are in contrast to those obtained with N-methylation in position 4, which drastically reduced the biological activity and produced remarkable changes in the peptide backbone and a severe limitation in rotational freedom of the side chains in tyrosine. Thus, it may be possible to synthesize potent angiotensin II analogs that have greater resistance to enzymatic degradation by alpha-methylation in position 4 (or 5) and simultaneous suitable modification at the NH2 and COOH termini.

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.

When des-(Asp 1 , Arg 2 , Val 3 )-angiotensin II was infused iv at rates of 308–5,550 pmol/kg·min for 10–120 min into 5 normal men and 2 patients with Bartter's syndrome, no significant change was observed in blood pressure (BP), plasma renin activity (PRA) or plasma aldosterone (PA), and the lowest dose did not inhibit a captopril-induced increase in PRA in the normal men, although des-(Asp 1 , Arg 2 )-angiotensin II was reported in the same 5 normal men to cause a decrease in PRA and an increase in PA in this dose range and a rise in BP at 2,220 and 5,550 pmol/kg·min. However, an iv infusion of the pentapeptide at 9,000 pmol/kg·min for 15 min significantly raised BP in the 5 normal men but not in patients with Bartter's syndrome. BP returned to the pretreatment level 60 min after the cessation of the infusion, although the duration of the pressor actions of angiotensin II, angiotensin III and des-(Asp 1 , Arg 2 )-angiotensin II were reported to be within 5 min in man. At the same dose level none of the 7 examined subjects showed any significant change in PRA or PA. Des-(Asp 1 , Arg 2 , Val 3 , Tyr 4 )-angiotensin II was infused iv at a rate of 41,480 pmol/kg·min into one of the normal men, but it caused no significant change in BP, PRA or PA. These results suggest that the pentapeptide and probably the tetrapeptide do not possess renin-suppressing and steroidogenic actions in man but the pentapeptide does elecit a minimal pressor action with a prolonged duration.

In 5 normal men sarcosine1-angiotensin II (Sar1-ANG II) (Exp. 1) and sarcosine1-angiotensin I (Sar1-ANG I) (Exp. 2) infused iv at a rate of 5 pmol/kg X min from 0900 h to 0930 h caused the same degree of rise in blood pressure (BP). But 100 mg of captopril given orally at 0800 h (Exp. 3) completely abolished the BP rise by Sar1-ANG I. In Exps. 1 and 2 plasma renin activity (PRA) decreased and plasma aldosterone (PA) increased after the infusions. In Exp. 3 PRA increased markedly and PA decreased 60 min after captopril, and at 30 min of Sar1-ANG I infusion PRA decreased to the pre-captopril level despite no BP change but PA was kept at the pre-infusion level. Hence, substrate specificity of converting enzyme previously demonstrated for N-terminal deleted ANG I was not shown for N-terminal substituted ANG I in man because the conversion of Sar1-ANG I to Sar1-ANG II was 100%. Sar1-ANG I may possibly inhibit renin release in normal men.

The major peptide produced by incubation of American eel (Anguilla rostrata) plasma with the eel kidney extract was identified as [1-asparagine, 5-valine, 9-glycine] angiotensin I (I). Two minor peptides were also identified as [1-aspartic acid, 5-valine, 9-glycine] angiotensin I (II) and [5-valine, 9-glycine] angiotensin I-(3-10)-octapeptide (III). These structures were further confirmed by comparison of these peptides on high-pressure liquid chromatography (HPLC) and high-performance thin-layer chromatography (HPTLC) with the synthetic peptides and the tryptic and chymotryptic digests of the synthetic and natural angiotensins. In the rat pressor bioassays, the synthetic decapeptides I and II possessed 45.4 and 52.2%, respectively, of the pressor activity of [1-aspartic acid, 5-isoleucine] angiotensin II. The pressor activity of I and II was blocked with the converting enzyme inhibitor captopril. Study of the conversion of asparaginyl decapeptide into aspartyl decapeptide in eel plasma indicated that the presence of thimerosal (sodium ethylmercurithiosalicylate) in the incubation mixture inhibited the conversion of I into II. These results suggest that (a) I is the natural form of angiotensin inherent in the American eel while II may be formed during incubation with plasma; (b) eel plasma contains an enzyme which is capable of converting asparaginyl angiotensins into aspartyl angiotensins; and (c) pressor activity of I and II is due to their conversion into the corresponding octapeptides. In a previous work when thimerosal was not included in the incubation mixture, the major peptide produced by incubation of Japanese eel (Anguilla japonica) plasma with its kidney extract was identified as II. (Y. Hasegawa, T. Nakajima, and H. Sokabe, 1983, Biomed. Res. 4, 417-420).

In order to clarify the importance of C-terminal phenylalanine in angiotensin II (ANG II) molecule, agonistic activities of a C-terminal substituted peptide, isoleucine8-angiotensin II (Ile8-ANG II), were studied in comparison with those of sarcosine1-, isoleucine8-angiotensin II (Sar1-, Ile8-ANG II) and isoleucine5-angiotensin II (Ile5-ANG II) in 5 normal men. When infused iv at a rate of 600pmol/kg·min for 30min, Ile8-ANG II and Sar1-, Ile8-ANG II raised the blood pressure to the same extent (15/15mmHg on the average), while the average blood pressure increase was 21/21mmHg after an iv infusion of Ile5-ANG II at a rate of 5pmol/kg·min for 30min. Duration of the pressor action after the cessation of each infusion was 50-90, 90-120 and 10-25min, respectively. In each case plasma renin activity (PRA) decreased and plasma aldosterone (PA) increased. When infused iv at a rate of 10 pmol/kg·min (maximum non-pressor dose) for 120min, both Ile8-ANG II and Sar1-, Ile8- ANG II lowered PRA and increased PA gradually, but 100mg oral captopril given immediately before these infusions caused no significant increase in PRA or no significant decrease in PA but again a decrease in PRA and an increase in PA. These results indicate that Ile8-ANG II has a very weak pressor (less than 0.83% of Ile5-ANG II and same as Sar1-, Ile8-ANG II) and slight reninsuppressing and steroidogenic actions in man and its metabolic degradation is slower than that of Ile5-ANG II and that the substitution of sarcosine for aspartic acid in the N-terminus of Ile8-ANG II further slows its metabolism. It is now evident that phenylalanine in 8 position is very important in maintaining sufficient pressor, renin-supressing and steroidogenic actions of ANG II but that this amino acid is not indispensable in maintaining more or less the biological activities of ANG II molecule.

Angiotensin II and its competitive inhibitor [Sar1, Ile8]-angiotensin II, as well as several analogs of these two compounds specifically chosen for their well-defined pharmacological properties, were studied by circular dichroism and nuclear magnetic resonance methods at various pH values in aqueous solution and in d6-dimethylsulfoxide. The results were compared with their biological activities. This allowed us to establish relationships between conformation and pressor activity, explaining most of the properties of angiotensin II, its inhibitor, and the analogs successively substituted in positions 3 and 5.

Based on the observation that high levels of renin and angiotensin II (Ang II) are found in the adrenal zona glomerulosa (ZG), it has been postulated that Ang II is formed intracellularly by the renin-converting enzyme cascade in this tissue. To test this hypothesis, we examined renin-angiotensin system components in subcellular fractions of the rat adrenal ZG. Renin activity and immunoreactive-Ang II (IR-Ang II) were observed in vesicular fractions but were not colocalized. In addition, angiotensinogen, angiotensin I, and converting enzyme were not observed in the renin or IR-Ang II-containing vesicular fractions. These data do not support the hypothesis that Ang II is formed intracellularly within the renin-containing vesicles of the ZG. Rather, since modulatable renin release from adrenal ZG slices was observed and renin activity was found in dense vesicular fractions (33-39% sucrose), it is likely that Ang II formation in the ZG is extracellular and initiated by the release of vesicular renin. Receptor-mediated endocytosis and subsequent degradation of Ang II in ZG lysosomes have been shown by others. The presence of IR-Ang II in light vesicular fractions (15% sucrose) and the finding of a high correlation between ZG IR-Ang II and Ang II receptor levels suggest that the primary occurrence of this peptide in the ZG is by receptor-mediated endocytosis. In ZG lysosomal fractions 125I-labeled Ang II was degraded to 125I-labeled des-[Phe8]Ang II. Since Ang II antibodies do not recognize des-[Phe8]Ang II, these findings explain why IR-Ang II in the ZG is due predominantly to Ang II and not to its C-terminal immunoreactive fragments.

A family of angiotensin I-like peptides has been derived from endogenous precursors present in dog cerebrospinal fluid after incubation with species homologous renin. These peptides are immunologically and pharmacologically similar to [Ile5]angiotensin I, and have molecular weights ranging between 1300 and 2200 daltons. The presence of precursors in the cerebrospinal fluid able to generate various biologically active angiotensin I-like peptides dissimilar to plasma angiotensin I supports the concept of a local angiotensin I-forming system in the brain.

Studies of the in vivo and in vitro metabolism of angiotensin peptide precursors, and of angiotensin II (Ang II) in tissues, has revealed the possibility that some of the fragments formed through specific enzymatic pathways are bioactive. There is evidence that Ang III is as potent as Ang II in stimulating thirst and causing aldosterone secretion. New findings from this laboratory have led us to reevaluate the concept that fragments of angiotensins derived from the amino (N-) terminus are devoid of biological activity. Using in vitro and in vivo techniques, we showed that Ang-(1-7) is processed from Ang I in amounts equal to or greater than Ang II. In addition, Ang-(1-7) generation is not dependent upon Ang I converting enzyme (ACE) activity in homogenates of canine brain stem. This heptapeptide promotes release of vasopressin from perifused hypothalamo-neurohypophysial explant and stimulates neural responses when microinjected into the vagal-solitary complex. The data supporting these findings are discussed below.

From the Heart and Hypertension Department, Research Institute of the Cleveland Clinic Foundation, Cleveland, Ohio. Requests for reprints should be addressed to Dr. Fetnat M. Fouad-Taraii, Heart and Hypertension Department, Research Institute of the Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, Ohio 44106. It has been years since converting enzyme inhibitors were introduced for the treatment of congestive heart failure. Their effectiveness has been demonstrated in multiple reports that have shown improvement in hemodynamic indexes as well as clinical symptoms and quality of life [l-IO]. Moreover, this improvement generally lasted months or years. Our experience in this respect is summarized in Table I. We have studied 14 patients with congestive heart failure of ischemic or cardiomyopathic etiology, before and after captopril therapy, and obtained hemodynamic data at regular intervals over one year. These studies showed that cardiac output and stroke volume increased significantly in association with normalization of pulmonary mean transit time, reduction of plasma aldosterone, and some reduction in both plasma catecholamine levels and heart rate. These effects occurred within one week and were sustained. Changes in ejection fraction, however, were minimal compared with the significant reduction in mean transit time (Figure 1) probably because ejection fraction depends on left ventricular geometry in addition to loading conditions on the heart. Along with these hemodynamic findings, improvement in severity of heart failure (NYHA class) and its other clinical manifestations has been described in patients treated with converting enzyme inhibitors by Dzau et al [7], Levine et al [8], and others. Goldstein and Pitt [l l] also demonstrated that, compared with placebo and with digoxin, captopril treatment of heart failure resulted in significantly improved exercise duration, a 45 percent reduction of ventricular premature beats, and a reduction in frequency of hospitalization. The mechanism by which converting enzyme inhibitors maintain improvement is still not well understood. Several possibilities include: (1) the persistent beneficial hemodynamic effects of converting enzyme inhibition o’n afterload and preload; (2) persistent suppression of plasma aldosterone; (3) interference with the effects of angiotensin II on the renal tubules; and (4) a direct effect of accumulated angiotensin I on the heart v21. Compared with other vasodilators, captopril has been found to be more effective in improving effort tolerance in association with a more marked reduction in blood pressure and pulmonary mean transit time and a more marked increase in cardiac output [13]. These findings indicated that the hemodynamic changes induced by converting enzyme inhibitors are not the only factors mediating the long-term control of congestive heart failure during therapy. Moreover, tolerance to therapy was observed with other vasodilators but did not develop when captopril was used for a similar duration, suggesting that additional effects of converting enzyme inhibitors should be considered. For example, humoral studies in these pa

1. We describe a new method of producing two-kidney hypertension in dogs by a two-step procedure with complete occlusion of a renal artery 2 weeks after it was partially constricted. 2. Control mean arterial pressure (96 ± 3 mmHg) of nine conscious, trained dogs rose to 107 ± 3 mmHg 2 weeks after partial constriction of a renal artery, and it stabilized at a sustained hypertensive plateau (124 ± 7 mmHg) 3 weeks after complete occlusion. 3. Intravenous infusion of an angiotensin II antagonist (Sar1-Thr8-angiotensin II) caused arterial pressure to fall during the acute but not the chronic phase of renal hypertension. In this latter phase plasma renin activity had returned to control values. 4. We conclude that the renin—angiotensin system appears not to be directly involved in the chronic phase of two-kidney hypertension in the dog.

The biololgical activity of des-asp1-,des-arg2-angiotensin II (3-8AII) was studied in man. When 3-8AII was infused iv at rates of 22 and 308 pmol (17.5 and 250 ng)/kg · min separately into 5 normal men each for 120 min, blood pressure showed no change, plasma renin activity (PRA) decreased gradually and plasma aldosterone showed a gradual slight increase. The lower dose of 3-8AII partially inhibited captopril-induced PRA increase and plasma aldosterone decrease in the same 5 normal men and the higher dose of the hexapeptide completely abolished them. In one of the 5 normal men blood pressure rose in response to doses of 3-8AII greater than 2220 pmol (1750 ng)/kg · min. When 3-8AII was infused iv at 308 pmol/kg · min into 2 patients with Bartter's syndrome for 60 min, it caused marked decreases in PRA and plasma aldosterone but no change in blood pressure. This decrease in plasma aldosterone is thought to be secondary to the decrease in PRA. From these results it is evident that 3-8AII has a minimal pressor action, a weak aldosterone-stimulating action and a significant renin-suppressing action in man and this PRA-lowering action is thought to be due to direct inhibition of renin release by its whole molecule or a smaller part of the molecule.

Affinity, residual agonist activity, and inhibitor properties of a series of angiotensin II analogs modified at the COOH-terminal position (X8-substituted peptides) have been probed for structure/conformation-biological activity relationships. The results emphasize (i) the large impact that subtle conformational variations caused by structural alterations in the position 8 side chain have on biological properties, (ii) the implication of the COOH-terminal carboxyl group in both affinity and intrinsic activity, and (iii) the influence that the bulkiness of the side chain in position 8 of antagonists has on the local conformation at the COOH terminus and thus on the inhibitory properties. In the hormone, the phenylalanine-8 ring is required for its steric influence and aromaticity to ensure a fully active conformation at the COOH terminus. Especially, correct orientation of the position 8 carboxyl group relative to the phenyl group of the phenylalanine residue may be necessary for agonistic activation of the angiotensin receptor complex. Replacement of the aromatic ring on the COOH-terminal residue by a nonaromatic group leads to incorrect orientation of the carboxyl group and causes the appearance of antagonist properties. Although the steric effects of the side chain can be modulated by specific interaction of its chemical groups (if any) with the peptide backbone, we found a good correlation between the size of the side chain-e.g., the steric parameter V gamma (the van der Waals volume consisting of the C alpha, C beta, and C gamma atoms), the conformational properties in the backbone (3J HC alpha-NH), and the binding capacities in all compounds tested.

Angiotensin I (ANG I) from various nonmammalian vertebrates does not bind the antibody to human ANG I, and thus radioimmunoassay (RIA) is not applicable for measurement of renin activity. It is largely due to this difficulty that biochemistry and physiology of the renin-angiotensin system of nonmammalian vertebrates have not been studied. The present work was therefore undertaken to develop a system to determine plasma renin activity by measuring the ANG I generation rate with RIA in fowl plasma. Antibody to [Asp 1 ,Val 5 ,Ser 9 ]-angiotensin I (fowl ANG I) was produced by immunizing goats and rabbits with a conjugate of human immunoglobulin and fowl ANG I. An antiserum, with a maximum binding of 86.3%, was obtained from the goat after six intradermal injections of the conjugate (each contained 0.25 mg fowl ANG I). This antiserum (at a final dilution of 1:200,000), and 125 I-labeled fowl ANG I, purified by DEAE-cellulose and Sephadex G-25 column chromatography, were used for the RIA of natural fowl ANG I. Fowl ANG I generated by incubating plasma and kidney extract, was measured by both RIA and rat vasopressor assay. The values obtained from these two methods correlated well. The formation of angiotensin increased when the duration of incubation was prolonged, and was optimum at pH 5.0–5.5. Plasma renin activity (rate of fowl ANG I generation) in 50 μl plasma from anesthetized female white leghorn chickens (24 weeks, n = 6) was 15.04 ± 2.26 ng/ml·hr, and that in plasma from chronically cannulated conscious chickens (30 weeks, n = 5) was 4.58 ± 0.61 ng/ml·hr. The plasma angiotensinogen level was 500–600 ng/ml in both groups. Thus this sensitive RIA method may provide a useful tool to study the functions and regulation of the renin-angiotensin system in fowl.

1 The mechanism involved in catecholamine (CA) release from the cat adrenal gland in response to insulin hypoglycaemia was studied. In intact cats, hypoglycaemia induced an 11 fold increase in adrenomedullary CA secretion. 2 Acute bilateral nephrectomy nearly abolished the increased CA release from the adrenal gland during hypoglycaemia. 3 Infusion of Sar1-Ileu8-Angiotensin II (AII), a competitive AII antagonist, suppressed the adrenomedullary response to the insulin-induced hypoglycaemia. After termination of the antagonist infusion CA secretion from the adrenal medulla increased rapidly, reaching the same level as in insulin-treated cats. 4 Infusion of rabbit anti-angiotensin I antibodies suppressed CA release from the adrenal gland of hypoglycaemic cats. This effect was more prolonged than that of Sar1-Ileu8-AII. 5 These results indicate that CA release from the adrenal medulla of the cat in response to insulin-induced hypoglycaemia, is mediated through the renal reninangiotensin system. Since hypoglycaemia causes sympathetic stimulation through a central mechanism, angiotensin may act through the central nervous system.