This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Yamada, K. Articles by Ferrario, C. M. Search for Related Content PubMed PubMed Citation Articles by Yamada, K. Articles by Ferrario, C. M.

(Hypertension. 1998;32:496-502.)
© 1998 American Heart Association, Inc.

Scientific Contributions

Converting Enzyme Determines Plasma Clearance of Angiotensin-(1–7)

Kazuo Yamada; Shridhar N. Iyer; Mark C. Chappell; Detlev Ganten; ; Carlos M. Ferrario

From the Hypertension and Vascular Disease Center, Wake Forest University School of Medicine, Winston-Salem, NC (K.Y., S.N.I., M.C.C., C.M.F.); and Max Delbruck Center for Molecular Medicine, Berlin-Buch, Germany (D.G.).

Correspondence to Carlos M. Ferrario, MD, Hypertension and Vascular Disease Center, Wake Forest University School of Medicine, Winston-Salem, NC 27157.

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Abstract—We determined the mechanism accounting for the removal and metabolism of angiotensin-(1–7) [Ang-(1–7)] in 21 anesthetized spontaneously hypertensive (SHR), 18 age-matched normotensive Sprague-Dawley (SD), and 36 mRen-2 transgenic (TG⁺) rats. Animals of all 3 strains were provided with tap water or tap water containing losartan, lisinopril, or a combination of lisinopril and losartan for 2 weeks. On the day of the experiment, Ang-(1–7) was infused for a period of 15 minutes at a rate of 278 nmol · kg^-1 · min^-1. After this time, samples of arterial blood were collected rapidly at regular intervals for the assay of plasma Ang-(1–7) levels by radioimmunoassay. Infusion of Ang-(1–7) had a minimal effect on vehicle-treated SD rats but elicited a biphasic pressor/depressor response in vehicle-treated SHR and TG⁺ rats. In lisinopril-treated rats, Ang-(1–7) infusion increased blood pressure, whereas losartan treatment abolished the pressor component of the response without altering the secondary fall in arterial pressure. Combined treatment with lisinopril and losartan abolished the cardiovascular response to Ang-(1–7) in all 3 strains. In vehicle-treated SD, SHR and TG⁺ the half-life (t_1/2) of Ang-(1–7) averaged 10±1, 10±1, and 9±1 seconds, respectively. Lisinopril alone or in combination with losartan produced a statistically significant rise in the half-life of Ang-(1–7) in all 3 strains of rats. Plasma clearance of Ang-(1–7) was significantly greater in the untreated SD rats compared with either the SHR or TG⁺ rat. Lisinopril treatment was associated with reduced clearance of Ang-(1–7) in all 3 strains. Concurrent experiments in pulmonary membranes from SD and SHR showed a statistically significant inhibition of ¹²⁵I-Ang-(1–7) metabolism in the presence of lisinopril. These studies showed for the first time that the very short half-life of Ang-(1–7) in the circulation is primarily accounted for peptide metabolism by ACE. These findings suggest a novel role of ACE in the regulation of the production and metabolism of the two primary active hormones of the renin angiotensin system.

Key Words: angiotensin-(1–7) • blood pressure • angiotensin-converting enzyme • lisinopril • losartan • rats, inbred SHR • rats, transgenic

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Substantial evidence now exists that angiotensin-(1–7) [Ang-(1–7)] is an important product of the renin-angiotensin system. In contrast to Ang II, the cumulative effects of Ang-(1–7) suggest an antihypertensive role for this peptide.¹ Ang-(1–7) exhibits natriuretic and diuretic actions in the rat kidney^{2 3 4} and induces relaxation in rat thoracic aorta,⁵ coronary vessels of dog and pig^{6 7} and the mesenteric bed of the cat.⁸ Low concentrations of Ang-(1–7) enhance the vasodepressor actions of bradykinin⁹ and facilitate the baroreceptor reflex.^{10 11 12} As recently reviewed,¹ the responses to Ang-(1–7) appear to be increased in hypertensive models. Ang-(1–7) infusion reduced blood pressure and increased prostaglandin release in spontaneously hypertensive rats (SHR); these actions were not observed in the Wistar-Kyoto (WKY) strain.¹³ Similarly, ventricular administration of an antibody to trap Ang-(1–7) increased blood pressure in mRen²⁷ (TG⁺) renin transgenics but not in normotensive rats.¹⁴ Additionally, the potentiation of the kinin response by Ang-(1–7) is augmented in renal hypertensive animals and SHR.¹⁵

Although the biosynthetic pathway for the production of Ang-(1–7) is now understood, the process by which the peptide is degraded or cleared from the plasma remains under investigation. Understanding the mechanism(s) of clearance of Ang-(1–7) is an important criterion for the determination of the role of this peptide in the regulation of blood pressure and cardiovascular function. Moreover, alterations in the metabolic clearance of Ang-(1–7) (MCR_Ang-(1–7)) may contribute to the increased responsiveness in hypertensive animals. Several different mechanisms have been postulated to mediate the plasma clearance of bioactive peptides from the plasma. These include enzymatic degradation by circulating or membrane-bound peptidases, hemodynamic factors, or internalization by the process of receptor-mediated endocytosis.¹⁶ The concept of a clearance receptor has been suggested to mediate the plasma clearance of atrial natriuretic factor,¹⁷ glycoprotein,¹⁸ and lipoprotein.¹⁹ Regarding the involvement of peptidase degradation, we^{20 21} and others²² have recently shown that Ang-(1–7) is a substrate in vitro for angiotensin-converting enzyme (ACE). Furthermore, treatment with various ACE inhibitors augment peptide levels substantially (5- to 25-fold) in the circulation.^{23 24 25 26 27} These data generate a new perspective on the factors that regulate the opposing actions of Ang II and Ang-(1–7) on blood pressure and cell growth.¹ To establish the mechanisms contributing to the removal of Ang-(1–7) from the circulation and ascertain whether clearance is altered in hypertensive animals, we determined the MCR_Ang-(1–7) in Sprague-Dawley (SD), SHR, and TG⁺ rats given long-term treatment with an ACE inhibitor or in combination with concurrent therapy with a selective AT₁ receptor blocker.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Experiments were conducted in 21 10-week-old male SHR (body weight, 261±5 g), 18 aged-matched SD (body weight, 375±7 g), and a third group of 18 [mRen-2]27 transgenic hypertensive TG⁺ rats (body weight, 345±9 g). Rats with spontaneous hypertension were purchased from Charles River Laboratories (Wilmington, Mass). The [mRen-2]27 TG⁺ rats were derived from the stock colony of the Hypertension Center Transgenic Animal Facility, as described elsewhere.²⁸ The animals were housed in individual cages in a room maintained at 25±2°C on a 12-hour light/dark cycle in an AALAC-approved facility and fed a rat chow diet (Purina Mills Inc) with tap water ad libitum.

Experimental Protocol
Treatment Protocol
Studies began by medicating rats for 14 days with either lisinopril or the combination of lisinopril and losartan mixed in the drinking tap water. The amount of drugs dissolved in the drinking water was adjusted daily to provide a final 24-hour dosing of 20 mg/kg of lisinopril and 10 mg/kg for losartan potassium. For the group of rats in combination therapy, doses were adjusted to provide 20 mg/kg of lisinopril and 10 mg/kg of losartan. Separate groups of rats from each strain drank tap water as vehicle. These forms of therapy have been shown by us previously to normalize the blood pressure and prevent the occurrence of a pressor response to injection of either angiotensin I (Ang I) and angiotensin II (Ang II).²⁹ Before and during the treatment period, systolic blood pressure was determined weekly by indirect tail-cuff plethysmography (Narco Biosystems).

Experimental Protocol
Fourteen days after either losartan and/or lisinopril treatment, the clearance of Ang-(1–7) from the plasma was determined in treated and untreated rats. On the day of the experiment, rats were anesthetized with 1% to 2% halothane (Ayerst Laboratories Inc) and respired mechanically with a mixture of 95% oxygen in 5% room air. Plastic catheters were implanted under aseptic conditions in a jugular vein (advanced to the tip of the heart) and a carotid artery to administer drugs and collect blood samples, respectively.

After a 30-minute stabilization period, phasic arterial pressure and heart rate were recorded for 1 hour with a PC-based data acquisition system, described elsewhere.³⁰ Synthetic Ang-(1–7) was infused intravenously through the jugular vein at a rate of 278 nmol · kg^-1 · min^-1 for 15 minutes. Samples of arterial blood (500 µL) were collected from the carotid at times 0, 0.5, 1.0, 2.0, 5.0, and 10 minutes after cessation of the infusion of Ang-(1–7) in prechilled tubes containing a mixture of inhibitors described elsewhere³¹ (see below). The dead space (60 µL) in the arterial catheter was cleared of any saline or blood before sample collection. A blood sample was also withdrawn from the arterial catheter before infusion of Ang-(1–7) for measurement of baseline plasma concentrations of Ang-(1–7) by radioimmunoassay.

Data Analysis
The metabolic clearance rate of Ang-(1–7) (MCR_Ang-(1–7)) was calculated with the equation: infusion rate of Ang-(1–7) (nmol · kg^-1 · min^-1) divided by the plasma concentration of Ang-(1–7) (nmol · mL^-1), as described previously.³² At steady state, the rate of removal of Ang-(1–7) is equal to the infusion rate.^{33 34} The large doses of Ang-(1–7) used in these experiments prevented changes in the endogenous production of Ang-(1–7) from interfering in the calculation of the MCR_Ang-(1-7). The half-life (t_1/2) of Ang-(1–7) in the circulation was calculated with the equation t_1/2=0.693/K_e, where K_e is the elimination rate constant.³⁴

Ang-(1–7) Radioimmunoassay
Plasma concentrations of Ang-(1–7) were determined in arterial blood as described in detail by our laboratory elsewhere.^{31 35} Briefly, blood was collected in a cocktail of protease inhibitors [25-mmol/L ethylenediaminetetraacetic acid, 0.44-mmol/L o-phenanthroline, 1 mmol/L 4-chloromercuribenzoic acid, and 0.12 mmol/L pepstatin A], as described by us in detail elsewhere.³¹ The minimum detectable level of the assay was 4 fmol/tube; the intra-assay coefficient of variation averaged 9%.

Ang-(1–7) Metabolism
The metabolism of ¹²⁵I-Ang-(1–7) was determined in pulmonary membranes prepared by homogenization of SHR lung tissue (1:10 wt/vol, previously frozen at -80°C) in 20 mmol/L HEPES, 300 mmol/L mannitol, pH 7.4, and centrifuged at 30 000g for 20 minutes at 4°C. The resultant pellet was homogenized again in the HEPES buffer with a Potter-Elverjhem Teflon pestle and recentrifuged. The assay contained 10 µg protein of pulmonary membrane and 5 nmol/L ¹²⁵I-Ang-(1–7) (2200 Curies/mmol) in a 0.1 mL with or without 10 µmol/L lisinopril. The reaction was terminated with 80% acetonitrile/0.4% phosphoric acid and stored at -80°C until high-performance liquid chromatography (HPLC) analysis (see below). Synthesis and HPLC purification of ¹²⁵I-Ang-(1–7) has been described previously.³⁶ The t_1/2 for Ang-(1–7) was calculated with a plotting and statistical package (GraphPad).

HPLC Analysis
Separation of Ang-(1–7) and Ang-(1–5) was achieved by HPLC with 0.1% phosphoric acid/water (mobile phase A) and 80% acetonitrile/0.1% phosphoric acid (mobile phase B). The analysis was performed on an Applied Biosystems 400 HPLC (ABI) equipped with a narrow-bore Nova-Pak C₁₈ column (Waters, 2.1x150 mm) and an Aquapore C₈ guard column (Applied Biosystems, 3.2x15 mm). The gradient consisted of 15% mobile phase B for 2 minutes, 15% to 30% B linear for 15 minutes, and 30% mobile phase B for 10 minutes at a flow rate of 0.3 mL/min at ambient temperature. HPLC fractions were collected at a 1-minute interval and counted in a gamma counter (Packard Instrument Co).

Statistical Analysis
All data are expressed as mean±SEM. Differences between drug treatments were analyzed by 1- or 2-way analysis of variance (ANOVA) followed by Scheffé's post hoc test or by unpaired Student's t test for the in vitro metabolism. Values of P0.05 were considered statistically significant.

Drugs
Ang-(1–7) was purchased from Bachem Inc. Losartan and lisinopril were a gift from Merck & Co, Inc.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Effects of Treatments on Hemodynamic Actions of Ang-(1–7)
Figure 1 shows the time course of the changes in mean arterial pressure produced by a 15-minute infusion of Ang-(1–7) in SD, SHR, and TG⁺ rats in the 4 treatment groups. Among animals given vehicle (tap water), the biphasic effects of Ang-(1–7) are most marked in the hypertensive SHR and TG⁺ rats. A small depressor response is present in SD rats, whereas in TG⁺ hypertensive animals the pressor component lasted longer than that determined in SHR. In the presence of lisinopril, Ang-(1–7) produced a pressor effect with no evidence of a depressor component for the 15 minutes of Ang-(1–7) infusion (middle panel of Figure 1). In contrast, treatment with losartan abolishes the pressor component of the response while having no effect on the vasodilator phase of the pressor response. Ang-(1–7) had no effect on SD, SHR, and TG⁺ rats medicated with the combination of lisinopril and losartan (rightmost panel of Figure 1).

View larger version (24K): [in this window] [in a new window]   Figure 1. Time course of changes in mean arterial pressure (MAP) produced by infusion of angiotensin-(1–7) (278 nmol/kg per minute) in anesthetized SD, SHR, and [mRen-2]27 transgenic hypertensive rats (TG+) for the various treatment groups (Vehicle, left panel; lisinopril or losartan, middle panel; and combination lisinopril and losartan therapy, right panel). Baseline values for MAP, heart rate, and number of animals per group are: SD-Vehicle, 105±1 mm Hg; 388±12 bpm, n=4; SD-Lisinopril, 72±1 mm Hg, 366±16 bpm, n=5; SD-Losartan, 94±1 mm Hg, 411±10 bpm, n=5; SD-Combination, 65±3 mm Hg, 351±27 bpm, n=5; SHR-Vehicle, 131±2 mm Hg, 317±9 bpm, n=5; SHR-Lisinopril, 78±3 mm Hg, 272±10 bpm; SHR-Losartan, 102±4 mm Hg, 283±18 bpm, n=5; SHR-Combination, 80±7 mm Hg, 314±20 bpm, n=5; TG+-Vehicle, 156±11 mm Hg, 370±16 bpm, n=4; TG+-Lisinopril, 83±3 mm Hg, 393±13 bpm, n=5; TG+-Losartan, 103±3 mm Hg, 399±16 bpm, n=5; TG+-Combination, 81±4 mm Hg, 369±12 bpm, n=5.

Effect of Ang-(1–7) Infusion on Plasma Ang-(1–7) Concentrations
Baseline plasma concentrations of Ang-(1–7) before administration of Ang-(1–7) were significantly higher in SHR and TG⁺ rats compared with vehicle-treated SD animals (Table). Treatment with lisinopril, losartan, or both agents in combination was associated with significant increases in plasma levels of Ang-(1–7) when compared with corresponding vehicle-treated rats. Baseline plasma Ang-(1–7) levels were higher, however, in TG⁺ given lisinopril than in similarly treated SD rats (Table).

View this table: [in this window] [in a new window]   Table 1. Baseline Values of Ang-(1–7) in Various Treatment Groups

Figure 2 illustrates the steady-state plasma concentrations of Ang-(1–7) achieved at the end of the 15-minute infusion period for each strain and treatment regimen. Among strains, the highest values of Ang-(1–7) were observed in TG⁺. Within each strain, however, the highest concentrations of Ang-(1–7) are present in rats treated with lisinopril either alone or in combination with losartan. Compared with vehicle-treated rats, the final plasma concentrations of Ang-(1–7) in lisinopril-treated rats increased by 68% in SD (P<0.02), 76% in SHR (P<0.0001), and 55% (P<0.005) in TG⁺. Plasma concentrations of Ang-(1–7) are not significantly increased in relation to their corresponding vehicle control values for animals treated with losartan.

View larger version (33K): [in this window] [in a new window]   Figure 2. Average concentrations of plasma Ang-(1–7) in SD, SHR, and TG+ after completion of an intravenous 15-minute infusion of 250 µg · kg-1 · min-1 in the various treatment groups. Values are mean±1 SE of mean, and data were analyzed by ANOVA. *P<0.05 compared with corresponding vehicle-treated animals.

Half-life of Ang-(1–7) in SD, SHR, and TG⁺ Rats
Half-life (t_1/2) values of Ang-(1–7) in the circulation are shown in Figure 3. The t_1/2 values for Ang-(1–7) averaged 10±1, 10±1, and 9±1 seconds in vehicle-treated SD, SHR, and TG⁺ rats. Long-term exposure to losartan had no effect on the half-life of Ang-(1–7). In lisinopril-treated rats, irrespective of the strain, the t_1/2 of Ang-(1–7) increased 4- to 6-fold (P<0.0001) compared with their corresponding untreated control group. Combined lisinopril and losartan treatment produced high t_1/2 values of Ang-(1–7), but these values are not different from those measured in the corresponding strain given only lisinopril therapy.

View larger version (30K): [in this window] [in a new window]   Figure 3. Differential effects of long-term treatment with lisinopril and combination of lisinopril and losartan in the half-life of Ang-(1–7) in SD, SHR, and TG+ rats. Values are mean±1 SE of mean, and data were analyzed by ANOVA. *P<0.05 compared with corresponding vehicle-treated animals.

The plasma clearance of Ang-(1–7) averaged 6.5±0.9 L · min^-1 · kg^-1 in vehicle-treated SD rats. The MCR_Ang-(1–7) was reduced by 39% (4.0±0.2 L · min^-1 · kg^-1, P<0.001) in vehicle-treated SHR and by 60% (2.6±0.1 L · min^-1 · kg^-1, P<0.001) in vehicle-treated TG⁺ rats. Long-term treatment with losartan had no effect on the MCR_{Ang- (1–7)}, whereas the clearance of Ang-(1–7) was significantly reduced in SD (3.7±0.2 L · min^-1 · kg^-1, P<0.001), SHR (2.2±0.1 L · min^-1 · kg^-1, P<0.01), and TG⁺ rats (1.7±0.1 L · min^-1 · kg^-1, P<0.001) after treatment with lisinopril. Combination therapy had no further effect on the reduced MCR_Ang-(1–7) found in lisinopril-treated rats.

Figure 4 shows the effects of the various treatment protocols on the MCR_Ang-(1–7) of SD, SHR, and TG⁺ as a function of the final concentration of Ang-(1–7) at the completion of the infusion of Ang-(1–7). For the 3 groups as a whole, MCR_Ang-(1–7) is inversely correlated with the steady-state levels of Ang-(1–7) achieved by the infusion of the peptide [r =-0.91 (F=43.02, P<0.0001)]. Interestingly, the correlations within each strain were similar for the various treatments and did not deviate significantly (P>0.05) from the overall relation as a whole (Figure 4). From inspection of Figure 4 it can be appreciated that the MCR_Ang-(1–7) in lisinopril-treated SD rats is significantly higher than the values achieved in either lisinopril-treated SHR or lisinopril-treated TG⁺ rats. Moreover, vehicle-treated TG⁺ rats show a substantial reduction in MCR_Ang-(1–7) because their clearance values fall within the MCR_Ang-(1–7) determined between SD and SHR rats given the combination of lisinopril and losartan. These findings suggest that plasma concentrations of Ang-(1–7) are markedly influenced by metabolic activity of ACE. In keeping with this interpretation, baseline levels of Ang-(1–7) correlated with both the steady-state concentration of Ang-(1–7) at the end of the infusion period (r=0.60, P<0.04) and the MCR_Ang-(1–7) (r=0.55, P<0.06).

View larger version (12K): [in this window] [in a new window]   Figure 4. Relation of mean clearance rate of angiotensin-(1–7) (MCRAng-(1–7)) as a function of steady-state level of Ang-(1–7) in SD, ( and solid lines), SHR ( with dashed lines), and TG+ ( and continuous line). For each strain, the location of each point from left to right is vehicle-treated, losartan-treated, lisinopril-treated, and lisinopril-losartan–treated rats. Values are mean±SE of 5 rats for each strain.

Ang-(1–7) Metabolism
The observation that lisinopril as a single agent had a profound effect on the t_1/2 values of Ang-(1–7) in the circulation led to the analysis of the metabolism of the peptide in pulmonary membranes of untreated SHR, a tissue with high peptidase activity. As shown in Figure 5, ¹²⁵I-Ang-(1–7) was degraded by peptidase activities within 15 minutes. The t_1/2 of the peptide in the in vitro preparation averaged 3.0±0.4 minutes (n=3); consistent with previous studies,²⁰ the primary metabolite resulting from the hydrolysis of Ang-(1–7) was identified as Ang-(1–5). Addition of 10 µmol/L lisinopril significantly attenuated the metabolism of Ang-(1–7) (+Lis) and abolished the generation of Ang-(1–5). In the presence of the ACE inhibitor, the t_1/2 of ¹²⁵I-Ang-(1–7) increased approximately 15 fold (43±3 minutes, P<0.01 versus control). A similar pattern of ¹²⁵I-Ang-(1–7) metabolism was found in pulmonary membranes of SD rats (data not shown).

View larger version (13K): [in this window] [in a new window]   Figure 5. Time course for disappearance of 125I-Ang-(1–7) with and without the ACE inhibitor lisinopril (+Lis) in SHR pulmonary membranes. 125I-Ang-(1–7) (5 nmol/L final concentration) was incubated with 10 µg protein of membranes at 37°C. Separation of 125I-Ang-(1–7), Ang-(1–5), and other metabolites was achieved by HPLC with a phosphoric acid/acetonitrile gradient as described in Methods. Data are mean of duplicate determinations from 3 animals.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
These studies demonstrate an important role of ACE in the in vivo clearance of Ang-(1–7). They provide a new dimension to the understanding of the mechanisms by which ACE contributes to the regulation of blood pressure because the prevailing concentrations of Ang-(1–7) in plasma in both normotensive and hypertensive rats appear to be predominantly determined by the mechanisms that control the inactivation of the peptide by degradation. These observations in vivo are consistent with kinetic studies that ACE exhibits a high specificity constant (K_cat/Km) for Ang-(1–7) comparable to that reported for bradykinin.^{20 21} Moreover, this interpretation agrees with the concurrent demonstration that ACE was the predominant enzyme involved in the metabolism of Ang-(1–7) by pulmonary membranes from SHR and SD rats. Our studies also demonstrate that the AT₁ receptor does not contribute to the clearance of Ang-(1–7) from the circulation. To our knowledge, these data demonstrate for the first time a novel role of ACE in determining the plasma clearance of Ang-(1–7) in vivo. In addition, the finding that the half-life of Ang-(1–7) in the circulation of normotensive and hypertensive rats is one-fourth that reported for Ang II³³ explains the need to use high doses of the peptide in experiments that initially characterized the biological actions of the heptapeptide.¹

Several precautions were taken to obtain accurate estimates of the plasma clearance of Ang-(1–7) because determination of this variable assumes that a steady-state equilibrium is achieved at the time of estimating the rate of peptide disappearance from the plasma compartment.³⁷ First, our data showed that Ang-(1–7) was removed from the circulation with a t_1/2 ranging between 9 and 10 seconds in untreated rats from all 3 strains. Thus infusion of Ang-(1–7) for a 15-minute period greatly exceeded the t_1/2 of the peptide in vivo. Ang-(1–7) was given at a high dose to ensure that endogenous production would not have artificially altered the concentration of Ang-(1–7) achieved at the end of the infusion period.³⁸ This precaution resulted in steady-state levels of Ang-(1–7) exceeding by >1000-fold the baseline levels of the peptide before infusion. Third, our data showed that hemodynamic effects produced by a high dose of Ang-(1–7) had no significant influence on the determination of the plasma clearance because differences in arterial pressure were not correlated with changes in the plasma clearance of the heptapeptide.

Baseline plasma concentrations of Ang-(1–7) were significantly higher in vehicle-treated hypertensive SHR and TG⁺ rats compared with normotensive vehicle-treated SD rats. Yet, the t_1/2 values for Ang-(1–7) were approximately the same in all 3 strains given vehicle. The baseline values of Ang-(1–7) were not significantly correlated (r=0.45, P>0.10) with the half-life of the peptide, which suggest that plasma levels of Ang-(1–7) reflect both synthesis and degradation in conditions in which disposal mechanisms are not inhibited. The finding that losartan treatment increases baseline concentrations of Ang-(1–7) is consistent with this interpretation as AT₁ receptor blockade disinhibits the negative feedback of Ang II on renin release.³⁹ In contrast, inhibition of ACE unmasked the role of the enzyme in the metabolism of the heptapeptide. Lisinopril therapy caused significant increases in baseline and steady-state levels of Ang-(1–7) as well as the half-life of the peptide in the circulation. As expected, these changes were accompanied by a marked reduction in the MCR_Ang-(1–7) in all 3 strains. The comparative effects of lisinopril on t_1/2 and clearance among the strains argues against a direct relation between the blood pressure disorder and the mechanism for removal of circulating Ang-(1–7). However, the decline in blood pressure after treatment was correlated with a change in MCR_Ang-(1–7) for each strain (SD: r=0.73, P<0.0008; SHR: r=0.63, P<0.003; TG⁺: r=0.75, P<0.0005). Because we observed a greater decline in blood pressure with lisinopril than losartan and no further decline with the combined regimen, the reduction in MCR_Ang-(1–7) (or increased t_1/2) may, in part, contribute to the greater effect of the ACE inhibitor. We have previously shown that Ang-(1–7) blockade partially reverses the decrease in blood pressure in both lisinopril/losartan^{29 40} and lisinopril-treated SHR (unpublished observations). Moreover, kinins do not contribute to the blood pressure–lowering actions of long-term ACE inhibitor or lisinopril/losartan treatment in SHR.^{40 41} Further studies comparing the reduction in blood pressure to changes in MCR_Ang-(1–7) with lower doses of lisinopril are necessary to fully address this relation.

Interestingly, the hemodynamic effects obtained by the infusion of Ang-(1–7) suggest that at high doses, Ang-(1–7) acts at AT₁ receptor sites to produce pressor responses because the AT₁ antagonist losartan blocks this response.³⁰ In contrast to SD rats, the pressor response of Ang-(1–7) in both hypertensive strains may be attributed, in part, to the lower clearance rates of the peptide. As discussed previously,¹ the depressor response observed in the hypertensive animals appears to require an activated renin-angiotensin system. The depressor component was not attenuated by losartan, which supports action at a non-AT₁, non-AT₂ receptor site.^{7 42 43} The combined regimen of lisinopril and losartan may reduce blood pressure to the point where Ang-(1–7) exhibits no further depressor action. In support of the former concept, recent studies demonstrate that removal of circulating Ang-(1–7) by antibody infusion or neprilysin inhibition increased blood pressure in SHR with long-term treatment with lisinopril and losartan.^{29 40}

The findings that both hypertensive strains had a reduced MCR_Ang-(1–7) compared with the SD are noteworthy because it suggests that endogenous inhibition of metabolism may contribute to the vasopressor actions of Ang-(1–7). As shown in Figure 4, the MCR_Ang-(1–7) in untreated SHR was lower than that determined in SD rats given lisinopril alone or in combination with losartan. The more severe hypertension found in vehicle-treated TG⁺ rats was associated with an even lower MCR_Ang-(1–7). These data suggest that hypertension may be associated with activation of endogenous inhibitors of ACE expression or activity. Although various studies have reported the presence of endogenous inhibitors of ACE,^{44 45 46} further work will be required to ascertain the nature and role of this mechanism in hypertension. Interestingly, a recent study suggests that whereas the N-domain of human somatic ACE is involved in the degradation of Ang-(1–7), the peptide may act as an endogenous inhibitor of the C-domain.²² The possibility thus exists that the reduced MCR_Ang-(1–7) in vehicle-treated SHR and TG⁺ rats may reflect an action of Ang-(1–7) or other endogenous inhibitors on the activity of ACE. This interpretation does not negate the possible contribution of other receptor subtypes in the removal of Ang-(1–7) from the circulation. On the other hand, this is consistent with the finding that baseline levels of Ang-(1–7) were higher in hypertensive compared with normotensive vehicle-treated strains.

The determination of the half-life of Ang-(1–7) has provided a first insight into the fate of Ang-(1–7) in the circulation. The demonstration that the t_1/2 of Ang-(1–7) is approximately 4- to 6-fold less than Ang II (t_1/2 of 45 seconds)³³ puts to rest the argument that the actions of Ang-(1–7) are outside the range of physiological responses. Several endogenous vasodilators including bradykinin and nitric oxide also share a short half-life (10 and 6 seconds, respectively).^{47 48 49} The short half-life of vasodilator peptides, in contrast to that of vasoconstrictor peptides, may be one mechanism that the system adapts to maintain vascular tone in a constricted rather than a vasodilator state. In conclusion, these findings bear importance in the understanding of the mechanism regulating the opposing actions of Ang II and Ang-(1–7) in the regulation of arterial pressure. A progressive unfolding of the intrinsic properties and mechanisms of Ang-(1–7) continues to bear fruit in expanding the scope of the understanding of the complex role that the renin-angiotensin system plays in the regulation of tissue perfusion and blood pressure.

	Acknowledgments

 
This research was supported in part by grants 1PO1-HL-51952, HL-50066, and HL-56973 from the National Heart, Lung, and Blood Institutes of the National Insitutes of Health.

Received February 27, 1998; first decision March 19, 1998; accepted May 11, 1998.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Ferrario CM, Chappell MC, Tallant EA, Brosnihan KB, Diz DI. Counterregulatory actions of angiotensin-(1–7). Hypertension. 1997;30:535–541.[Abstract/Free Full Text]

2. DelliPizzi A, Hilchey SD, Bell-Quilley CP. Natriuretic actions of angiotensin-(1–7). Br J Pharmacol. 1994;111:1–3.[Medline] [Order article via Infotrieve]

3. Hilchey SD, Bell-Quilley CP. Association between the natriuretic action of angiotensin-(1–7) and selective stimulation of renal prostaglandin I₂ release. Hypertension. 1995;25:1238–1244.[Abstract/Free Full Text]

4. Handa RK, Ferrario CM, Strandhoy JW. Renal actions of angiotensin-(1–7) in vivo and in vitro studies. Am J Physiol. 1996;270:F141–F147.[Abstract/Free Full Text]

5. Forster C, le Tran Y. Angiotensin-(1–7) and the rat aorta: modulation by the endothelium. J Cardiovasc Pharmacol. 1997;30:676–682.[Medline] [Order article via Infotrieve]

6. Porsti I, Bara AT, Busse R, Hecker M. Release of nitric oxide by angiotensin-(1–7) from porcine coronary endothelium: implications for a novel angiotensin receptor. Br J Pharmacol. 1994;111:652–654.[Medline] [Order article via Infotrieve]

7. Brosnihan KB, Li P, Ferrario CM. Angiotensin-(1–7) dilates canine coronary arteries through kinins and nitric oxide. Hypertension. 1996;27:523–528.[Abstract/Free Full Text]

8. Osei SY, Ahima RS, Minkes RK, Weaver JP, Khosla MC, Kadowitz PJ. Differential responses to angiotensin-(1–7) in the feline mesenteric and hindquarters vascular beds. Eur J Pharmacol. 1993;234:35–42.[Medline] [Order article via Infotrieve]

9. Paula RD, Lima CV, Khosla MC, Santos RAS. Angiotensin-(1–7) potentiates the hypotensive effect of bradykinin in conscious rats. Hypertension. 1995;26:1154–1159.[Abstract/Free Full Text]

10. Benter IF, Diz DI, Ferrario CM. Pressor and reflex sensitivity is altered in spontaneously hypertensive rats treated with angiotensin-(1–7). Hypertension. 1995;26:1138–1144.[Abstract/Free Full Text]

11. Campagnole-Santos MJ, Heringer SB, Batista EN, Khosla MC, Santos RAS. Differential baroreceptor reflex modulation by centrally infused angiotensin peptides. Am J Physiol. 1992;263:R89–R94.[Abstract/Free Full Text]

12. Oliveira DR, Santos RAS, Santos GFP, Khosla MC, Campagnole-Santos MJ. Changes in the baroreflex control of heart rate produced by central infusion of selective angiotensin antagonists in hypertensive rats. Hypertension. 1996;27:1284–1290.[Abstract/Free Full Text]

13. Benter IF, Ferrario CM, Morris M, Diz DI. Antihypertensive actions of angiotensin-(I-7) in spontaneously hypertensive rats. Am J Physiol (Heart Circ Physiol). 1995;269:H313–H319.[Abstract/Free Full Text]

14. Moriguchi A, Tallant EA, Matsumura K, Reilly TM, Walton H, Ganten D, Ferrario CM. Opposing actions of Angiotensin-(1–7) and Angiotensin II in the brain of transgenic hypertensive rats. Hypertension. 1995;25:1260–1265.[Abstract/Free Full Text]

15. Lima CV, Paula RD, Resende FL, Khosla MC, Santos RAS. Potentiation of the hypotensive effect of bradykinin by short-term infusion of angiotensin-(1–7) in normotensive and hypertensive rats. Hypertension. 1997;30:542–548.[Abstract/Free Full Text]

16. Okolicany J, McEnroe GH, Koh GY, Lewiski JA, Maack T. Clearance receptor and neutral endopeptidase-mediated metabolism of atrial natriuretic factor. Am J Physiol. 1992;263:F546–F553.[Abstract/Free Full Text]

17. Almeida FA, Suzuki RM, Scarborough RM, Lewicki JA, Maack T. Clearance function of type C receptors of atrial natriuretic factor in rats. Am J Physiol. 1989;256:R469–R479.[Abstract/Free Full Text]

18. Ashwell G, Harford J. Carbohydrate-specific receptors in the liver. Annu Rev Biochem. 1982;51:531–534.[Medline] [Order article via Infotrieve]

19. Golstein JL, Brown MS. The low-density lipoprotein pathway and its relation to atherosclerosis. Annu Rev Biochem. 1977;81:382–395.

20. Chappell MC, Pirro NT, Sykes A, Ferrario CM. Metabolism of angiotensin-(1–7) by angiotensin converting enzyme. Hypertension. 1998;31:362–367.[Abstract/Free Full Text]

21. Li P, Chappell MC, Ferrario CM, Brosnihan KB. Angiotensin-(1–7) augments bradykinin-induced vasodilation by competing with ACE and releasing nitric oxide. Hypertension. 1997;29:394–400.[Abstract/Free Full Text]

22. Deddish PA, Marcic B, Jackman HL, Wang H-Z, Skidgel RA, Erdös EG. N-domain specific substrate and C-domain inhibitors of angiotensin-converting enzyme: angiotensin-(1–7) and Keto-ACE. Hypertension. 1998;31:912–917.[Abstract/Free Full Text]

23. Lawrence AC, Clark IJ, Campbell DJ. Increased angiotensin-(1–7) in hypophysial-portal plasma of conscious sheep. Neuroendocrinology. 1992;55:105–114.[Medline] [Order article via Infotrieve]

24. Campbell DJ, Lawrence AC, Towrie A, Kladis A, Valentijn AJ. Differential regulation of angiotensin peptide levels in plasma and kidney of the rat. Hypertension. 1991;18:763–773.[Abstract/Free Full Text]

25. Kohara K, Brosnihan KB, Chappell MC, Khosla MC, Ferrario CM. Angiotensin-(1–7): a member of circulating angiotensin peptides. Hypertension. 1991;17:131–138.[Abstract/Free Full Text]

26. Kohara K, Brosnihan KB, Ferrario CM. Angiotensin-(1–7) in the spontaneously hypertensive rat. Peptides. 1993;14:883–891.[Medline] [Order article via Infotrieve]

27. Luque M, Martin P, Martell N, Fernandez C, Brosnihan KB, Ferrario CM. Effects of captopril related to increased levels of prostacyclin and angiotensin-(1–7) in essential hypertension. J Hypertens. 1996;14:799–805.[Medline] [Order article via Infotrieve]

28. Senanayake PD, Moriguchi A, Kumagai H, Ganten D, Ferrario CM, Brosnihan KB. Increased expression of angiotensin peptides in the brain of transgenic hypertensive rats. Peptides. 1994;15:919–926.[Medline] [Order article via Infotrieve]

29. Iyer SN, Chappell MC, Averill DB, Diz DI, Ferrario CM. Vasodepressor actions of angiotensin-(1–7) unmasked during combined treatment with lisinopril and losartan. Hypertension. 1998;31:699–705.[Abstract/Free Full Text]

30. Benter IF, Diz DI, Ferrario CM. Cardiovascular actions of angiotensin-(1–7). Peptides. 1993;14:679–684.[Medline] [Order article via Infotrieve]

31. Kohara K, Tabuchi Y, Senanayake P, Brosnihan KB, Ferrario CM. Reassessment of plasma angiotensins measurement: effects of protease inhibitors and sample handling procedures. Peptides. 1991;12:1135–1141.[Medline] [Order article via Infotrieve]

32. Vernace MA, Mento PF, Maita ME, Wilkes BM. Effects of angiotensin receptor subtype inhibitors on plasma angiotensin clearance. Hypertension. 1994;23:853–856.[Abstract/Free Full Text]

33. Iyer SN, Chappell MC, Brosnihan KB, Ferrario CM. Role of AT₁ and AT₂ receptors in the plasma clearance of angiotensin II. J Cardiovasc Pharmacol. 1998;31:464–469.[Medline] [Order article via Infotrieve]

34. Naden RP, Coultrup S, Arant BS, Rosenfeld CR. Metabolic clearance of angiotensin II in pregnant and non pregnant sheep. Am J Physiol (Endocrinol Metab). 1985;249:E49–E55.[Abstract/Free Full Text]

35. Nakamoto H, Ferrario CM, Fuller SB, Robaczwski DL, Winicov E, Dean RH. Angiotensin-(1–7) and nitric oxide interaction in renovascular hypertension. Hypertension. 1995;25:796–802.[Abstract/Free Full Text]

36. Chappell MC, Brosnihan KB, Welches WR, Ferrario CM. Characterization by high performance liquid chromatography of angiotensin peptides in the plasma and cerebrospinal fluid of the dog. Peptides. 1987;8:939–942.[Medline] [Order article via Infotrieve]

37. van Kats JP, de Lannoy LM, Jan Danser AH, van Meegen JR, Verdouw PD, Schalekamp MADH. Angiotensin II type 1 (AT₁) receptor-mediated accumulation of angiotensin II in tissues and its intracellular half-life in vivo. Hypertension. 1997;30:42–49.[Abstract/Free Full Text]

38. Champlain JD, Genest J, Veyratt R, Boucher R. Factors controlling renin in man. Arch Intern Med. 1966;117:355–363.[Abstract/Free Full Text]

39. Timmermans PBMWM, Wong PC, Chiu AT, Herblin WF, Benfield P, Carini DJ, Lee RJ, Wexler RR, Saye JM, Smith RD. Angiotensin II receptors and angiotensin II receptor antagonists. Pharmacol Rev. 1993;45:205–251.[Medline] [Order article via Infotrieve]

40. Iyer SN, Ferrario CM, Chappell MC. Angiotensin-(1–7) contributes to the antihypertensive effects of blockade of the renin-angiotensin system. Hypertension. 1998;31:356–361.[Abstract/Free Full Text]

41. Cachofeiro V, Maeso R, Rodrigo E, Navarro J, Ruilope LM, Lahera V. Nitric oxide and prostaglandins in the prolonged effects of losartan and ramipril in hypertension. Hypertension. 1995;26:236–243.[Abstract/Free Full Text]

42. Pucci ML, Tong X, Miller KB, Guan H, Nasjletti A. Calcium- and protein kinase C-dependent basal tone in the aorta of hypertensive rats. Hypertension. 1995;25:752–757.[Abstract/Free Full Text]

43. Tallant EA, Lu X, Weiss RB, Chappell MC, Ferrario CM. Bovine aortic endothelial cells contain an angiotensin-(1–7) receptor. Hypertension. 1997;29:388–392.[Abstract/Free Full Text]

44. Erdos EG. Angiotensin I converting enzyme and the changes in our concepts through the years. Hypertension. 1992;16:363–370.[Abstract/Free Full Text]

45. Minshall RD, Tan F, Nakamura F, Rabito SF, Becker RP, Marcic B, Erdos EG. Potentiation of the actions of bradykinin by angiotensin I-converting enzyme inhibitors: the role of expressed human bradykinin B₂ receptors and angiotensin I-converting enzyme in CHO cells. Circ Res. 1997;81:848–856.[Abstract/Free Full Text]

46. Cerpa-Poljak A, Lahnstein J, Mason KE, Smythe GA, Duncan MW. Mass spectrometric identification and quantification of hemorphins extracted from human adrenal and pheochromocytoma tissue. J Neurochem. 1997;68:1712–1719.[Medline] [Order article via Infotrieve]

47. Decarie A, Raymond P, Gervais N, Couture R, Adam A. Serum interspecies differences in metabolic pathways of bradykinin and [des-Arg9]BK: influence of enalaprilat. Am J Physiol. 1997;271:H1340–H1347.

48. Kelm M, Schrader J. Control of coronary vascular tone by nitric oxide. Circ Res. 1990;66:1561–1575.[Abstract/Free Full Text]

49. Ignarro LJ. Endothelium-derived nitric oxide: actions and properties. FASEB J. 1989;3:31–36.[Abstract]

This article has been cited by other articles:

				L. D. Kluskens, S. A. Nelemans, R. Rink, L. de Vries, A. Meter-Arkema, Y. Wang, T. Walther, A. Kuipers, G. N. Moll, and M. Haas Angiotensin-(1-7) with Thioether Bridge: An Angiotensin-Converting Enzyme-Resistant, Potent Angiotensin-(1-7) Analog J. Pharmacol. Exp. Ther., March 1, 2009; 328(3): 849 - 854. [Abstract] [Full Text] [PDF]

				I. Hamming, H. van Goor, A. J. Turner, C. A. Rushworth, A. A. Michaud, P. Corvol, and G. Navis Differential regulation of renal angiotensin-converting enzyme (ACE) and ACE2 during ACE inhibition and dietary sodium restriction in healthy rats Exp Physiol, May 1, 2008; 93(5): 631 - 638. [Abstract] [Full Text] [PDF]

				C. Tikellis, K. Bialkowski, J. Pete, K. Sheehy, Q. Su, C. Johnston, M. E. Cooper, and M. C. Thomas ACE2 Deficiency Modifies Renoprotection Afforded by ACE Inhibition in Experimental Diabetes Diabetes, April 1, 2008; 57(4): 1018 - 1025. [Abstract] [Full Text] [PDF]

				A. Reyes-Engel, L. Morcillo, F. J. Aranda, M. Ruiz, M. J. Gaitan, A. Mayor-Olea, P. Aranda, and C. M. Ferrario Influence of Gender and Genetic Variability on Plasma Angiotensin Peptides Journal of Renin-Angiotensin-Aldosterone System, June 1, 2006; 7(2): 92 - 97. [Abstract] [PDF]

				C. M. Ferrario, A. J. Trask, and J. A. Jessup Advances in biochemical and functional roles of angiotensin-converting enzyme 2 and angiotensin-(1-7) in regulation of cardiovascular function Am J Physiol Heart Circ Physiol, December 1, 2005; 289(6): H2281 - H2290. [Abstract] [Full Text] [PDF]

				A. Sakima, D. B. Averill, P. E. Gallagher, S. O. Kasper, E. N. Tommasi, C. M. Ferrario, and D. I. Diz Impaired Heart Rate Baroreflex in Older Rats: Role of Endogenous Angiotensin-(1-7) at the Nucleus Tractus Solitarii Hypertension, August 1, 2005; 46(2): 333 - 340. [Abstract] [Full Text] [PDF]

				J. G. Modrall, J. Sadjadi, K. B. Brosnihan, P. E. Gallagher, C.-h. Yu, G. L. Kramer, K. E. Bernstein, and M. C. Chappell Depletion of Tissue Angiotensin-Converting Enzyme Differentially Influences the Intrarenal and Urinary Expression of Angiotensin Peptides Hypertension, April 1, 2004; 43(4): 849 - 853. [Abstract] [Full Text] [PDF]

				J. Stegbauer, V. Oberhauser, O. Vonend, and L. C. Rump Angiotensin-(1-7) modulates vascular resistance and sympathetic neurotransmission in kidneys of spontaneously hypertensive rats Cardiovasc Res, February 1, 2004; 61(2): 352 - 359. [Abstract] [Full Text] [PDF]

				C. Wamberg, R. R. Plovsing, N. C. F. Sandgaard, and P. Bie Effects of different angiotensins during acute, double blockade of the renin system in conscious dogs Am J Physiol Regulatory Integrative Comp Physiol, November 1, 2003; 285(5): R971 - R980. [Abstract] [Full Text] [PDF]

				G. Vauquelin, Y. Michotte, I. Smolders, S. Sarre, G. Ebinger, A. Dupont, and P. Vanderheyden Cellular targets for angiotensin II fragments: pharmacological and molecular evidence Journal of Renin-Angiotensin-Aldosterone System, December 1, 2002; 3(4): 195 - 204. [Abstract] [PDF]

				I. Kucharewicz, R. Pawlak, T. Matys, D. Pawlak, and W. Buczko Antithrombotic Effect of Captopril and Losartan Is Mediated by Angiotensin-(1-7) Hypertension, November 1, 2002; 40(5): 774 - 779. [Abstract] [Full Text] [PDF]

				C. N. Shrimpton, A. I. Smith, and R. A. Lew Soluble Metalloendopeptidases and Neuroendocrine Signaling Endocr. Rev., October 1, 2002; 23(5): 647 - 664. [Abstract] [Full Text] [PDF]

				A. Goette, U. Lendeckel, and H. U Klein Signal transduction systems and atrial fibrillation Cardiovasc Res, May 1, 2002; 54(2): 247 - 258. [Abstract] [Full Text] [PDF]

				T. Mustafa, Joo Hyung Lee, Siew Yeen Chai, A. L Albiston, S. G McDowall, and F. A. Mendelsohn Bioactive angiotensin peptides: focus on angiotensin IV Journal of Renin-Angiotensin-Aldosterone System, December 1, 2001; 2(4): 205 - 210. [PDF]

				B. Tom, R. de Vries, P. R. Saxena, and A.H. J. Danser Bradykinin Potentiation by Angiotensin-(1-7) and ACE Inhibitors Correlates With ACE C- and N-Domain Blockade Hypertension, July 1, 2001; 38(1): 95 - 99. [Abstract] [Full Text] [PDF]

				S. N. Iyer, D. B. Averill, M. C. Chappell, K. Yamada, A. J. Allred, and C. M. Ferrario Contribution of Angiotensin-(1-7) to Blood Pressure Regulation in Salt-Depleted Hypertensive Rats Hypertension, September 1, 2000; 36(3): 417 - 422. [Abstract] [Full Text] [PDF]

				E. A. Tallant, D. I. Diz, and C. M. Ferrario Antiproliferative Actions of Angiotensin-(1-7) in Vascular Smooth Muscle Hypertension, October 1, 1999; 34(4): 950 - 957. [Abstract] [Full Text] [PDF]

				W. Linz, P. Wohlfart, B. A Scholkens, T. Malinski, and G. Wiemer Interactions among ACE, kinins and NO Cardiovasc Res, August 15, 1999; 43(3): 549 - 561. [Full Text] [PDF]

				A. J. M. Roks, P. P. van Geel, Y. M. Pinto, H. Buikema, R. H. Henning, D. de Zeeuw, and W. H. van Gilst Angiotensin-(1–7) Is a Modulator of the Human Renin-Angiotensin System Hypertension, August 1, 1999; 34(2): 296 - 301. [Abstract] [Full Text] [PDF]

				T. Traynor, T. Yang, Y. G. Huang, J. H. Krege, J. P. Briggs, O. Smithies, and J. Schnermann Tubuloglomerular feedback in ACE-deficient mice Am J Physiol Renal Physiol, May 1, 1999; 276(5): F751 - F757. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Yamada, K. Articles by Ferrario, C. M. Search for Related Content PubMed PubMed Citation Articles by Yamada, K. Articles by Ferrario, C. M.