Angiotensin-Converting Enzyme Gene Polymorphism Has No Influence on the Circulating Renin-Angiotensin-Aldosterone System or Blood Pressure in Normotensive Subjects
Background Angiotensin-converting enzyme (ACE) is involved in the metabolism of two major vasoactive peptides, converting angiotensin (Ang) I into Ang II and inactivating bradykinin. An insertion/deletion (I/D) polymorphism is present in the 16th intron of the ACE gene and is strongly associated with plasma and cellular ACE levels. Contrasting with the lack of relation between ACE gene polymorphism and blood pressure level, a large case-control study has shown that the deletion marker allele of the ACE gene was associated with an increased risk of myocardial infarction. The pathophysiological link between ACE gene polymorphism and cardiovascular events remains hypothetical. One hypothesis is that this polymorphism influences Ang II and bradykinin concentrations in the peripheral and/or local circulations through its effects on ACE levels in plasma and endothelial cells. The aim of this study was to investigate the effect of the ACE gene I/D polymorphism on blood pressure, plasma active renin, and aldosterone regulation in normal subjects.
Methods and Results Twenty-four normotensive male volunteers homozygous for the ACE I/D polymorphism (12 DD and 12 II) received a renin inhibitor infusion (remikiren 0.1 mg · kg−1 · h−1 for 130 minutes) to suppress endogenous Ang I and Ang II production. Forty minutes after initiating the remikiren infusion, an exogenous Ang I infusion was begun and increased gradually every 15 minutes from 1 to 10 ng · kg−1 · min−1. Median (range) plasma ACE levels (mU/mL) were 39 (32 to 57) and 24 (12 to 30) in the DD and II groups, respectively. Remikiren suppressed plasma Ang I and Ang II, increased plasma active renin (from 23±12 to 154±161 pg/mL), decreased plasma aldosterone (from 106±42 to 82±33 pg/mL), and slightly decreased diastolic blood pressure (from −2.4±2.7 mm Hg). The blood pressure and hormonal responses to Ang I infusion after renin inhibition and the slope of the rise in plasma Ang II with increasing Ang I dose were identical in both groups, as was the plasma Ang I/Ang II ratio before (DD, 2.09±1.04; II, 2.59±0.76) and after (DD, 0.15±0.13; II, 0.09±0.03) combined renin inhibitor and Ang I infusion.
Conclusions Despite its association with a major difference in plasma ACE levels, the ACE I/D polymorphism did not influence the Ang II and plasma aldosterone production, plasma active renin decrease, or diastolic blood pressure increase induced by exogenous Ang I infusion, suggesting that ACE has no limiting influence on systemic Ang II generation and effects under these experimental conditions.
Angiotensin-converting enzyme (ACE; kininase II, dipeptidyl carboxypeptidase I, and EC 3.4.15-1), an endothelial ectoenzyme secreted in plasma, is involved in the metabolism of two major vasoactive peptides, converting angiotensin (Ang) I into the pressor peptide Ang II and inactivating the vasodilatory peptide bradykinin.1 2
Family studies suggest that approximately 50% of the interindividual variability of plasma ACE is attributable to a major gene polymorphism.3 4 Cloning of human ACE cDNA and restriction fragment length polymorphism analysis led to the description of an insertion/deletion (I/D) polymorphism of the ACE gene that consists of the presence or absence of a 287-bp DNA fragment located in intron 16.5 This ACE gene I/D polymorphism is strongly associated with serum ACE levels and accounts for a large part of the total serum ACE variance.4 5 These observations have been extended to the membrane-bound form of ACE by using circulating T-lymphocytes, where ACE levels have also been shown to be genetically determined and associated with the I/D polymorphism.6 The ACE I/D polymorphism is probably only a neutral marker in linkage disequilibrium with an as yet unidentified causal variant that alters ACE gene transcription.3 4 5 6
Despite the well-documented importance of ACE function in blood pressure regulation,7 the plasma ACE level is not a strong determinant of blood pressure level in humans.8 9 10 Zee et al11 initially proposed that the insertion allele could be a marker of hypertension; however, a more recent analysis of their data has shown that this association was, in fact, a result of an age-related loss of the deletion allele.12 The results of several other studies using various approaches have suggested that the ACE gene polymorphism did not play an important role in essential hypertension. These studies included a sib-pair analysis of a microsatellite polymorphism of the growth hormone gene close to the ACE gene locus13 and analysis of the I/D polymorphism distribution in the four-corner approach of Harrap et al14 and in the Dutch Hypertension and Offspring Study.15 Contrasting with the lack of relation between the ACE gene polymorphism and blood pressure level, Cambien et al16 17 have shown in a large case-control study (Etude Cas-Temoins sur l’Infarctus du Myocarde; ECTIM) that the ACE gene I/D polymorphism is associated with an increased risk of myocardial infarction, particularly in the subgroup at low cardiovascular risk. ACE gene polymorphism is associated with parental history of ischemic heart disease,18 19 ischemic or dilated cardiomyopathy,20 risk of sudden death in hypertrophic cardiomyopathy,21 a higher risk of restenosis after coronary angioplasty in patients with myocardial infarction,22 and with left ventricular hypertrophy in patients with essential hypertension.23 In addition, ACE gene polymorphism or plasma ACE levels are associated with microalbuminuria or nephropathy in type I diabetic patients24 and with the occurrence of coronary heart disease in type II diabetes.25 As yet, the pathophysiological mechanisms linking the ACE gene polymorphism and these cardiovascular events remain hypothetical. One plausible hypothesis is that the ACE gene polymorphism influences Ang II and bradykinin concentrations in the peripheral and/or local circulations through its effects on ACE levels in plasma and endothelial cells.16
The aim of this study was to investigate the effect of the ACE gene I/D polymorphism on the systemic conversion of Ang I to Ang II and consequently on blood pressure and plasma renin and aldosterone regulation in young healthy male subjects. Systemic conversion of Ang I to Ang II was explored under basal conditions and during the infusion of pharmacological doses of Ang I after blockade of endogenous Ang I production by the intravenous administration of a renin inhibitor. Blockade of the first enzymatic reaction of the renin-angiotensin system allowed precise control of the Ang I delivery rate to the circulation to investigate the blood pressure and hormonal effects of circulating and vascular membrane-bound ACE in subjects homozygous for the deletion (DD) and insertion (II) alleles.
One hundred and four normotensive healthy male volunteers (age, 18 to 35 years) attending the Broussais Clinical Investigation Center had their ACE (I/D) genotypes determined by enzymatic DNA amplification (see below). Insertion and deletion allele frequencies in the whole group of subjects were 0.40 and 0.60, respectively, which are similar to reported allele frequencies in Caucasians.5 Fifty-six of the 104 subjects were homozygotes and therefore eligible for participation in this investigation. Twenty-seven agreed to participate in the study: 14 DD and 13 II subjects.
The 27 selected subjects were instructed to follow a normal sodium diet; 14 days before the study, each subject underwent a routine outpatient clinical and laboratory evaluation. The protocol was approved by the Comité Consultatif de Protection des Personnes se prêtant à des Recherches Biomédicales (Hôpital Cochin, Paris, France), and all subjects gave written informed consent.
The volunteers were instructed to come to the Clinical Investigation Center at 8 am on the study day, after a light caffeine-free breakfast at 7 am, and were seated in a comfortable armchair. An intravenous catheter was placed in a left antecubital vein for continuous infusions of both the renin inhibitor and Ang I, and an indwelling cannula was inserted in the right brachial vein for blood sampling.
Two DD subjects were excluded from the study before drug administration because of a vasovagal episode during catheter insertion, and one II subject was excluded because of an uncomplicated extravasation during renin inhibitor infusion. All of the 24 remaining subjects (12 DD and 12 II) underwent the entire procedure. The study design is shown in Fig 1⇓.
After 1 hour’s rest, a priming intravenous dose of the renin inhibitor remikiren (0.3 mg/kg solubilized in 50 mL 0.9% NaCl) was infused over the 10-minute period that preceded a 2-hour continuous infusion at the rate of 0.1 mg · kg−1 · h−1 (Becton Dickinson Infusion Systems, Division Vial Medical) so that endogenous Ang I and Ang II production was suppressed during the experiment. Remikiren was graciously provided by Drs F.R. Buhler and R. Jones from Hoffmann-La Roche Laboratories, Basel, Switzerland.
Forty minutes after the beginning of the remikiren infusion (time 0), a continuous exogenous Ang I infusion (H2N-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-OH diluted in 130 mL 0.9% NaCl; Clinalfa AG) was started and gradually increased every 15 minutes from 1 to 10 ng · kg−1 · min−1 over 90 minutes. Both remikiren and Ang I infusions were well tolerated; a mild spontaneously regressive headache was the only adverse event reported.
Blood pressure was monitored every 2 minutes during the procedure with an automatic blood pressure recorder (Press Mate BP 8800, Colin Co).
The baseline plasma level of each hormonal parameter was calculated as the mean of two consecutive determinations performed before remikiren infusion (times −55 and −40 minutes). Blood was also sampled just before Ang I infusion (time 0) and then every 15 minutes for the following 90 minutes (Fig 1⇑); active renin, Ang I, Ang II, and aldosterone levels were measured in each plasma sample. Plasma renin activity (PRA) was measured three times (baseline, time 0, and time 90 minutes). Heparinized tubes were used to collect blood for plasma active renin and aldosterone determinations. For the measurement of plasma angiotensins, blood was rapidly collected (within 10 seconds) in prechilled tubes containing a mixture of 62.5 mmol/L EDTA, 100 μmol/L remikiren, and 100 μmol/L enalaprilat.26 27 Apart from plasma angiotensins, which could be determined in only 10 DD and 8 II subjects, all other hormonal determinations were obtained in all 24 subjects.
Detection of the I/D Polymorphism of the ACE Gene by Enzymatic Amplification
High-molecular-weight DNA was isolated from peripheral blood leukocytes using proteinase K digestion of nuclei, phenol extraction, and ethanol precipitation. DNA concentrations were measured by absorbance at 260 nm.
The ACE gene I/D polymorphism was detected by using the polymerase chain reaction (PCR) technique using oligonucleotide primers flanking the insertion as described,28 with minor modifications. Amplification reactions were carried out in a final volume of 20 μL, containing 100 μg genomic DNA, 2 μL 10× PCR buffer (500 mmol/L KCl, 100 mmol/L Tris-HCl [pH 8.4], 15 mmol/L MgCl2, and 1 mg/L gelatin), 5 pmol/L of each primer, 1 μL dNTP, and 1 U Taq polymerase (Cetus). The DNA was amplified for 30 cycles with denaturation at 94°C for 1 minute and annealed at 60°C for 45 seconds with extension at 72°C for 30 seconds using a PTC-100 thermal cycler (Perkin-Elmer Corporation). PCR products were analyzed by 2% agarose gel electrophoresis and visualized by ethidium bromide staining. The PCR products are a 490-bp fragment in the presence of the insertion allele and a 190-bp fragment in the absence of the insertion allele (ie, the deletion allele). Determination of the ACE I/D genotype was performed twice in all subjects with consistent results.
For plasma angiotensin measurements, blood samples were immediately centrifuged at 3500 rpm at 4°C and stored at −80°C. After extraction on phenylsilylsilica columns (Bondelut PH, Analytichem) according to the procedure of Nussberger et al,29 the dried extracts containing angiotensins were diluted in 220 μL of 0.1 mol/L Tris-HCl buffer, pH 7.5, containing 2 g/L BSA. Recoveries of angiotensins were 98.5±3.5%.26 For the Ang I radioimmunoassay, we used a polyclonal antibody that cross-reacts 100% with des-Asp1 Ang I and less than 0.25% with Ang II, Ang III, and their later analogues.30 For the Ang II radioimmunoassay, we used a monoclonal antibody (a gift from D. Simon and B. Pau, Sanofi, Montpellier, France) that cross-reacts 190% with des-Asp1 Ang II, 154% with Ang (3-8), 51% with Ang (4-8), less than 1% with Ang I, 2% with Ang (1-9), and 1% with Ang (2-10).31 No cross-reactivity was detected with the angiotensin peptides lacking phenylalanine in the carboxy terminus. In both standard curves, 0.5 pg/tube could be detected, and 50% displacement of the tracer was achieved at 5 and 10 pg/tube for Ang I and Ang II, respectively.
In preliminary in vitro experiments, the Ang I concentration of the infusion fluid was measured at the extremity of the infusion tubing at the same sampling times as for the in vivo protocol. Samples of Ang I infusion fluid were also subjected to the same storage and extraction processes as in the protocol. Ang I concentrations remained stable during the whole procedure and within 10% of the calculated theoretical Ang I concentration value, thereby showing that there was no significant loss of the infused Ang I by adsorption to the catheter and tubing.
ACE activity was quantified at baseline and at the end of the experiment (time 90 minutes) by a spectrophotometric method according to Cushman and Cheung32 with slight modifications.
Active renin was measured by a commercially available radioimmunometric assay using two monoclonal antibodies, 3E8 and 4G1 (ERIA, Diagnostics Pasteur). The radioactive 4G1 antibody specifically recognizes active renin, and 3E8 is the immobilized antibody that recognizes total renin.33
PRA was determined on 250-μL plasma samples, pH 7.4, obtained by adding a 3-mol/L Tris-HCl buffer, pH 7.2, containing 200 mmol/L EDTA (1:10) and 2.8 mmol/L PMSF to the plasma samples. The samples were incubated at 37°C, and the concentration of Ang I was measured by radioimmunoassay.
Plasma aldosterone was measured by radioimmunoassay using 125I-aldosterone as a tracer (Coat-a-count Aldosterone, Radioimmunology Behring Diagnostic Products Corp).
Blood pressure and hormonal changes induced by the administration of remikiren and Ang I were analyzed by using graphical and statistical methods. Two separate periods were considered for statistical analysis: period I, corresponding to the remikiren infusion (time −40 minutes to time 0), and period II, corresponding to the combined infusions of remikiren and exogenous Ang I (time 0 to time 90 minutes).
Blood Pressure Analysis
Baseline diastolic blood pressure (DBP) was defined as the average of the 16 DBP values recorded during the last 30 minutes of the rest period before starting the remikiren infusion.
During period I, DBP was defined as the average of the eight DBP values recorded during the last 16 minutes before Ang I infusion (from time −16 minutes to time 0). The first ANOVA was done on DBP values (baseline versus period I) with one repeated factor (time) and one grouping factor (genotype).
The mean DBP value obtained during remikiren infusion (period I) served as a new reference DBP value before Ang I infusion. During period II, DBP was defined as the average of the three measurements recorded during the last 6 minutes of each Ang I dose. The second ANOVA was done on the absolute variations of DBP (DBP during each Ang I dose minus the reference DBP of period I) with one repeated factor (time) and one grouping factor (genotype).
We also calculated for each subject the slope of the regression line that best fit the plot of the variations in DBP values (every 2 minutes) versus time (from time 0 onward), and then averaged these calculated values in each group of subjects. The individual time interval necessary to reverse the blood pressure fall induced by renin blockade was calculated by applying the equation of each subject’s regression line for y(variation in DBP)=0. The percentage of the observed maximal blood pressure increase at the dose of 10 ng · kg−1 · min−1 Ang I was plotted against each Ang I dose, and the Ang I dose necessary to obtain 50% of the maximal blood pressure effect (D50) was then determined graphically for each subject and averaged in each group.
For each hormonal parameter, two separate ANOVAs corresponding to each period were done with one repeated factor (time) and one grouping factor (genotype).
During period II, plasma active renin, Ang I, Ang II, and aldosterone changes were monitored from time 0 to time 90 minutes. In each subject, the slope of the regression line that best fit the plot of each of these log-transformed hormonal parameters versus time (from time 0 onward) was calculated and then averaged in each of the two groups.
For each subject, the percentage of the maximal observed effect on aldosterone and active renin induced by 10 ng · kg−1 · min−1 Ang I was plotted against each Ang I dose to determine the corresponding D50 for each subject; these values were then averaged in each group.
Initial clinical characteristics of the two groups of subjects and average slopes for hormonal parameters and DBP were compared by using an unpaired Student’s t test. The assumptions of ANOVA (homogeneity of variance and normality) were verified for each variable, and natural logarithmic transformation was applied where appropriate. The regression coefficient was estimated by the least-squares method. Calculations were done with statview ii statistical software (Apple Macintosh, Abacus Concepts Inc). Data are expressed as mean±1 SD in the tables and mean±1 SEM in the graphs. A probability value of less than.05 was considered significant.
The main clinical and biological characteristics of the DD and II subjects are summarized in Table 1⇓. There was no significant difference in age, baseline DBP, plasma creatinine and potassium levels, or sodium and potassium dietary intake (as estimated by their respective urinary excretions) between DD and II subjects. According to the ACE genotype, baseline median ACE activity was higher in the DD group (39 mU/mL) than the II group (24 mU/mL) with no overlap (DD, 32 to 57 versus II, 12 to 30 mU/mL). Plasma ACE levels remained stable in each subject during the entire investigation (not shown).
Baseline plasma Ang I and Ang II levels and Ang I/Ang II ratios were not significantly different between the two groups despite the large differences in plasma ACE activity (Table 2⇓). Similarly, no significant differences were observed between genotypes concerning baseline DBP, plasma aldosterone and active renin levels, or PRA (Table 2⇓).
Effects of Remikiren Infusion (Period I)
After 40 minutes of remikiren infusion, PRA, plasma Ang I, and consequently plasma Ang II levels had decreased toward undetectable values in both DD and II groups (Table 2⇑ and Fig 2⇓), confirming complete renin blockade and interruption of endogenous Ang I and Ang II production.
A slight but significant decrease in plasma aldosterone levels followed renin inhibition in both DD and II groups (Table 2⇑ and Fig 3⇓). The relative decrease in plasma aldosterone levels was similar in both groups (DD, −19±19% versus II, −24±19%, NS). The fall in plasma Ang II induced an abrupt and significant increase in plasma active renin levels in both groups (Table 2⇑ and Fig 3⇓), with levels reaching 538±325% and 454±336% of baseline values in DD and II subjects, respectively (NS).
Remikiren induced a slight but significant fall in DBP that reached a plateau within the first 10 minutes of the infusion in both groups (DD, −2.9±2.5 versus II, −1.7±2.9 mm Hg, NS; Table 2⇑ and Fig 4⇓).
Effects of Ang I Infusion (Period II)
Since for all tested parameters there was only a time effect (no genotype effect and no interaction of time with genotype), only F values of the time effect are reported.
Reversal of the Effects of Renin Inhibition
Superimposition of the exogenous Ang I infusion provoked very rapid hemodynamic and hormonal responses that were linked to the instantaneous conversion of the inactive peptide Ang I to the active peptide Ang II.
In both genotypes, plasma Ang II levels increased from zero toward baseline values (pre-remikiren) during the 15-minute infusion of the first Ang I dose (1 ng · kg−1 · min−1; Fig 2⇑). In parallel, DBP increased very rapidly within the first 4 minutes of the Ang I infusion, attaining its pre-remikiren baseline value in both DD and II subjects during the same time interval (Fig 4⇑). In both groups, complete reversal of renin blockade (DBP criterion) was achieved, and the median time interval necessary to reverse the DBP fall due to renin blockade was similar (DD, 10.5 versus II, 7.0 minutes, NS). The first Ang I dose increased DBP by 3.9±2.8 mm Hg in the DD group and by 2.7±2.6 mm Hg in the II group (NS).
Effects of Ang I Infusion on Plasma Ang I and Ang II Levels
Increasing the dose of Ang I induced further significant increases in plasma Ang II levels in a dose-dependent fashion (F6,96=395, P<.001; Fig 2⇑). Mean slopes of log-transformed plasma Ang II concentrations versus time (from time 0 onward) did not significantly differ (DD, 0.033±0.004 versus II, 0.033±0.004, NS). There was no tendency in either group for plasma Ang II levels to plateau. At the end of the Ang I infusion, plasma Ang II levels had increased from their baseline values in each group by 10-fold (DD, 44±16 and II, 46±16 pg/mL; Fig 2⇑). In contrast, plasma Ang I levels increased very slowly (F6,96=41, P<.001; Fig 2⇑) and did not reach their pre-remikiren baseline values even at the 10 ng · kg−1 · min−1 Ang I dose (DD, 6.68±7.37 and II, 4.40±2.65 pg/mL), where they remained significantly lower than their baseline values (F1,16=8.6, P=.01 versus baseline levels; Fig 2⇑). Mean slopes of log-transformed plasma Ang I concentrations versus time did not significantly differ between the two groups (DD, 0.014±0.006 versus II, 0.012±0.004, NS). Consequently, the Ang I/Ang II ratio was much lower at the end of the exogenous Ang I infusion (DD, 0.15±0.13 and II, 0.09±0.03) than under basal conditions (DD, 2.09±1.04 and II, 2.59±0.76) but was similar between both groups.
Effects of Ang I Infusion on Blood Pressure, Plasma Aldosterone, and Plasma Active Renin Levels
During Ang I infusion, the rise in plasma Ang II caused a steep and significant increase in DBP in a dose-dependent fashion (F5,110=93, P<.001; Fig 4⇑). At each Ang I dose, DBP reached a new plateau within 6 minutes. The absolute DBP increase with time did not differ significantly between groups and reached 18±7 and 17±5 mm Hg at the end of the exogenous Ang I infusion in the DD and II subjects, respectively. Mean slopes of DBP increase versus time, reflecting the kinetics of Ang I to Ang II conversion and the Ang II interaction with its vascular receptor, were similar in both groups (DD, 0.22±0.07 versus II, 0.22±0.04 mm Hg · min−1, NS). We calculated a statistical power of 80% for detecting a 5-mm Hg DBP difference between the two groups with an α risk of 5%. When Ang I infusion was stopped, DBP returned to its baseline value within 2 to 4 minutes in both groups (not shown).
In parallel to the rise in plasma Ang II, plasma aldosterone levels increased significantly in a dose-dependent fashion in both groups (F6,132=153, P<.001; Fig 3⇑), reaching 218±98% and 296±160% of their baseline values at the end of the Ang I infusion in the DD and II groups, respectively. Mean slopes of log-transformed plasma aldosterone levels versus time did not significantly differ between the two groups (DD, 0.013±0.004 versus II, 0.015±0.004, NS).
In parallel to the increase in plasma Ang II, a significant dose-dependent fall in plasma active renin levels was observed in both DD and II groups (F6,132=102, P<.001; Fig 3⇑). Final plasma active renin levels at time 90 minutes (DD, 64±60 and II, 42±18 pg/mL) remained significantly higher than their respective baseline levels (F1,22=133, P=.0001). Mean slopes of log-transformed plasma active renin levels versus time did not significantly differ (DD, −0.011±0.003 versus II, −0.009±0.005, NS). PRA remained totally suppressed at time 90 minutes (not shown).
When plotting the percentage of the maximum effect (induced by Ang I 10 ng · kg−1 · min−1) against Ang I dose, hyperbolic dose-response curves were obtained for the three target parameters (DBP, aldosterone, and active renin; Fig 5⇓). Table 3⇓ shows that the average values for the corresponding D50 did not differ significantly between II and DD subjects and that within each group the sensitivity of the three parameters to Ang I infusion was similar.
Following the evidence that plasma ACE levels and the ACE gene I/D polymorphism do not seem to be determinants of blood pressure levels, although they may be cardiovascular risk markers, we investigated the consequences of exogenously infused Ang I on the appearance and biological effects of plasma Ang II in homozygous normotensive volunteers of both ACE genotypes with contrasting plasma ACE levels.
Inhibition of the first step in the renin-angiotensin cascade by remikiren allowed Ang I metabolism to be studied independently of endogenous Ang I production and of the prior renin secretion status and its fluctuations. A single slow infusion of remikiren to healthy subjects, at a similar dosage over 210 minutes, induced a prolonged and almost complete blockade of endogenous Ang I and Ang II production in an earlier experiment.27 In addition to changes in blood pressure and plasma aldosterone levels, the degree of reversal of the massive increase in plasma active renin levels following remikiren constituted a third parameter for assessing the biological effects of the Ang II produced from the exogenous Ang I.
Complete renin inhibition was achieved by the remikiren infusion, as demonstrated by suppression of circulating PRA and plasma Ang I and Ang II levels. Decreases in Ang I and Ang II levels were associated with a mild decrease in plasma aldosterone level, a significant fall in DBP, and an increase in plasma active renin levels due to the interruption of the Ang II feedback loop.27 The multiplicity of blood pressure measurements (every 2 minutes) allowed the detection of the slight decrease in DBP (roughly 3 mm Hg) secondary to the inhibition of the renin-angiotensin system. In the absence of a placebo-treated group a time effect could possibly be considered, but blood pressure stability during the last 30 minutes of the rest period prior to the remikiren infusion and the rapid contemporaneous reversibility of the DBP fall with the exogenous Ang I infusion are two strong indirect arguments against this interpretation. The reversion of DBP to its baseline level occurred within the first 10 minutes of the initial Ang I dose, a time corresponding approximately to the latent period necessary to detect a blood pressure effect after renin blockade at the beginning of remikiren infusion. These small changes in DBP induced by renin blockade and detected by the precision of our methodology favor a contributive role of the renin-angiotensin system in the determination of basal blood pressure levels in sodium-repleted, normotensive volunteers, a physiological issue for which contradictory results have been obtained.34 35
Remikiren-induced suppression of endogenous Ang I avoided the dilution of the exogenously infused Ang I in an endogenous pool, the exogenous peptide being immediately exposed to its metabolism, thus combining conversion and degradation. Each Ang I dose was infused at a constant rate during 15 minutes, a duration greater than the four to five Ang I half-lives36 necessary to reach steady-state plasma Ang I concentrations. After an abrupt increase during the first minutes, DBP reached a new plateau for each Ang I dose. Thus, blood pressure and hormonal measurements were made at plateau concentrations of exogenous Ang I.
Concerning the influence of ACE on Ang II and blood pressure levels, which was the main goal of the study, our results show that despite its association with a major difference in plasma ACE levels, the ACE gene I/D polymorphism did not influence the observed increases in plasma Ang II and aldosterone levels and DBP or the decrease in active renin following exogenous Ang I administration. In basal conditions, plasma Ang II levels and the Ang I/Ang II ratio were similar in both genotypes despite large differences in plasma ACE activity. During exogenous Ang I infusion in the absence of endogenous peptide, each dose of Ang I delivered to the central venous compartment was immediately converted to the active peptide Ang II, and the magnitude and kinetics of this conversion were not influenced by the ACE gene I/D polymorphism. Consequently, there was no significant difference between DD and II subjects concerning hormonal and hemodynamic Ang II–dependent responses to the exogenous Ang I infusion.
Under these experimental conditions, ACE appears to have no limiting influence on the systemic generation of Ang II. In an integrated view of the renin-angiotensin system, if the in vivo conversion of Ang I to Ang II by ACE in the pulmonary and forearm circulation is not a rate-limiting step, contrary to the reaction of renin to angiotensinogen in plasma,37 then, any change in the governing variable of the system, ie, plasma renin concentration, will directly influence the plasma Ang I concentration, and consequently the Ang II plasma level, in the effluent blood.
Our results confirm the findings of Admiraal et al36 38 concerning the intense metabolism of Ang I in the whole body. Despite constant infusion of pharmacologically active doses of human Ang I in conjunction with the renin inhibitor infusion, only very low levels of Ang I could be detected at the contralateral forearm venous site, even at the highest exogenous Ang I dose. At baseline, the plasma Ang I/Ang II ratio was 2.3±0.9 in the whole group (DD and II, n=18); after renin blockade and exogenous Ang I infusion, the ratio decreased greatly to 0.12±0.10 at the end of the infusion. In the absence of endogenously generated Ang I, this low ratio reflects the high conversion and degradation rates of the infused exogenous Ang I during a single passage through, successively, the pulmonary capillary bed and the peripheral vascular bed of the contralateral forearm. During the infusion of exogenous Ang I or 125I–Ang I to essential hypertensive patients in the absence of renin inhibition, Admiraal et al36 found an endogenous Ang I/Ang II ratio of 2.5 in the antecubital vein and a 125I–Ang I/125I–Ang II ratio at the same site of 0.14 after exogenous 125I–Ang I infusion. Interestingly enough, the 125I–Ang I/125I–Ang II ratio in the renal vein was much higher than in the forearm territory, suggesting that the amount of regional ACE may differentially influence Ang I conversion in organs in which endothelial ACE is less abundant, such as the kidneys.39 40 In our experiment, the existence of a permanently low Ang I/Ang II ratio in the DD and II groups during the infusion of increasing doses of Ang I confirms that the enzymatic conversion of Ang I to Ang II in these experimental conditions was not limited by the availability of ACE.
There are several differences between the experimental conditions of the present study and the physiological situation. In our study, ACE activity was investigated in vivo in the absence of endogenous Ang I by documenting the conversion of exogenously infused Ang I to Ang II in the plasma and pulmonary and brachial circulations. A localized, unique, and exogenous source of Ang I differs from the generalized endogenous production of Ang I by the renin-angiotensinogen reaction in blood, interstitial fluid, and perhaps tissues. The difference between the two situations is well illustrated by the observation of an Ang I/Ang II ratio of only 0.1 to 0.3 in forearm blood following exogenous high-dose Ang I infusion after blockade of endogenous Ang I production, but of roughly 2 when Ang I is continuously produced by renin.
Under our experimental conditions, the substrate (Ang I) concentration remained stable during each infusion period, and the substrate-to-enzyme ratio at the entry of the two main sites of conversion, ie, the pulmonary and contralateral forearm circulations, was very low, varying very slightly with time. Under these conditions, Ang I conversion apparently follows first-order kinetics (Fig 2⇑), and since the substrate concentration is very low compared with that of the enzyme, the influence of enzyme concentration is minimal or cannot be detected. In the physiological situation, however, Ang I is continuously produced at each conversion site by the renin-angiotensinogen reaction in plasma and interstitial tissues. Consequently, the substrate-to-enzyme ratio varies greatly, depending on both locally generated Ang I and ACE concentrations at a given vascular site. This ratio is probably higher in the large arterial vessels and in the forearm circulation, where blood sampling is performed, than in the pulmonary circulation. Therefore, physiologically, Ang I may be differently converted depending on local ACE concentration at a given vascular site, although, because of blood recirculation, the overall rate of conversion remains the same in individuals with different vascular and plasma ACE contents.3 4 5 6 Moreover, physiologically, other tissue non-ACE–dependent enzymatic pathways can generate Ang II from Ang I or angiotensinogen.41 42 43 44 However, the extent to which these non-ACE–dependent pathways contribute to Ang II generation or secretion in plasma remains to be established.
Another interesting result emerging from this study is the synchronism of the pharmacodynamic responses (aldosterone and DBP increases and renin decrease) of the target organs to the newly generated Ang II, which was demonstrated by the exact superimposition of the hyperbolic dose-response curves of the three Ang II–dependent parameters and the similarity of the corresponding D50 values, which were around 3 ng · kg−1 · min−1 Ang I. This observation can be integrated within the physiology of the renin-angiotensin system in subjects on a normal sodium diet. Any subtle variation in renin secretion modifies Ang I generation, which is possibly influenced by the genetically determined plasma angiotensinogen levels.45 The newly generated Ang I is immediately converted to Ang II, which produces different effects of similar intensity and kinetics at each of its main sites of action. The absence of differences between the DD and II groups concerning blood pressure reactivity to Ang II, as suggested by the identical relation between these variables in the two genotype groups, mitigates against any potential downregulation of vascular Ang II receptors that theoretically would be observed in DD subjects if this genotype had a major influence on accelerating a local Ang II production.
In conclusion, after renin blockade in normotensive male subjects, there was no detectable influence of the ACE I/D polymorphism on the conversion of intravenously infused Ang I into Ang II or on subsequent Ang II–mediated biological effects. This provides a reasonable explanation for the observed absence of influence of plasma ACE levels and genotype on blood pressure levels. These results do not eliminate potential differences in Ang I conversion rates at specific sites, such as the coronary and renal vessels, where the influence of the ACE gene I/D polymorphism on the amount of ACE still needs to be directly investigated. If the amount of available ACE is locally limited in these territories, a relatively higher amount in the DD genotype than in the II genotype could facilitate the occurrence of vascular lesions secondary to long-term exposure to higher or lower local levels of Ang II and bradykinin, respectively. The results of this clinical investigation illustrate the fact that the physiopathological link between the DD genotype and the risk of myocardial infarction, trying therefore to explain the ECTIM results by a limiting role of ACE to convert Ang I to Ang II or through an increased bradykinin degradation, is not yet certain. Case-control studies are useful to detect new risk factors, and the ECTIM study has convincingly suggested that the DD genotype is a predictive factor for myocardial infarction.16 However, this methodology has intrinsic weaknesses, of which many examples are available.46 Prospective epidemiological studies as well as local pharmacological investigations are necessary before it can be accepted that the DD genotype and the high levels of ACE with which it is associated are risk factors that directly influence the incidence of myocardial infarction.16 17 The ACE I/D polymorphism could be a neutral marker in linkage disequilibrium with an as yet unknown causal variant4 that may be responsible for an excess of cardiovascular and renal events related to the physiological properties of ACE. The causal variant could also be, at least theoretically, a variant of another gene closely linked to the ACE gene and directly responsible for an excess of cardiovascular events, independent of the physiological roles of both plasma and endothelial ACE.
This work was supported by a joint grant from INSERM (Paris, France) and Merck, Sharp & Dohme (USA). The authors wish to thank the nursing staff of the Clinical Investigation Center at the Broussais Hospital, particularly Danièle Ménard, RN, who ran the protocol. The technical contribution of Christiane Dollin, who performed the assays, was also much appreciated. The authors acknowledge Martin Day, MD, for editorial help.
- Received October 17, 1994.
- Revision received December 13, 1994.
- Accepted December 27, 1994.
- Copyright © 1995 by American Heart Association
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