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Metabolism of bradykinin by the rat coronary vascular bed

Marie-Josée Dumoulin, Albert Adam, Charles Blais Jr., Daniel Lamontagne
DOI: http://dx.doi.org/10.1016/S0008-6363(98)00006-6 229-236 First published online: 1 April 1998

Abstract

Objective: To study the metabolism of bradykinin (BK) after a single passage through the coronary bed in isolated Langendorff rat hearts. Methods: BK was infused into the aortic flow line to obtain a final concentration of 10 nM, and the coronary effluent was collected to quantify BK and des-Arg9-BK by competitive enzyme immunoassay. The nature of immunoreactive material was confirmed by immunograms after HPLC separation. The experiments were performed with hearts perfused at either one of the following coronary flow rates: 1, 5 or 10 ml/min. Results: BK recovery without inhibitors was 86.3±2.9, 60.8±6.3, and 29.6±6.8% at 10, 5, and 1 ml/min, respectively. The Vmax/Km ratios at these coronary flow rates were 2.19±0.72, 4.81±0.64, and 2.59±0.33 min−1 g−1, respectively. The angiotensin-converting enzyme (ACE) inhibitor, enalaprilat (130 nM), reduced BK degradation at all flow rates. Inhibition of neutral endopeptidase with retrothiorphan (25 nM) had no effect on BK degradation. However, the combined treatment with enalaprilat and retrothiorphan reduced BK degradation to lower values than enalaprilat alone. The effect of enzyme inhibitors on BK recovery was inversely related to coronary flow: inhibiting BK degradation markedly increased BK recovery at 1 ml/min, but had no effect at 10 ml/min. The kininase I metabolite of BK, des-Arg9-BK, could not be detected under these experimental conditions. Conclusions: ACE is the major enzyme responsible for BK degradation during a single passage through the coronary bed. Neutral endopeptidase contributes to BK degradation only when ACE activity is impaired. The effect of enzyme inhibitors on the coronary concentration of BK is highly dependent on coronary flow rate.

Keywords
  • Bradykinin
  • Metabolism
  • Angiotensin-converting enzyme
  • Neutral endopeptidase
  • Coronary bed

Time for primary review 25 days.

1 Introduction

The nanopeptide bradykinin (BK) has important actions on blood vessels, the heart and kidneys. By far the most important hemodynamic effect of BK in vivo is the vasorelaxation produced by endothelial release of nitric oxide, prostaglandins [1]and endothelium-derived hyperpolarizing factor [2]. Bradykinin is also involved in pain reaction and can increase capillary permeability and produce edema [3]. BK, generated from kininogens by kallikreins is rapidly inactivated in blood and tissues by different amino and carboxypeptidases [4]. At least three of these enzymes are important from a pharmacological point of view [4]: angiotensin-converting enzyme (ACE or kininase II, EC 3.4.15.1) which acts as a dipeptidyl carboxypeptidase to remove the carboxy-terminal dipeptide Phe8-Arg9, neutral endopeptidase (NEP) which cleaves the same bond in BK in vitro and in vivo as ACE to inactivate BK, and kininase I which removes Arg9 residue from BK to produce the active metabolite des-Arg9-BK, an endogenous agonist of B1 kinin receptors [3].

Several studies have shown that the ACE inhibitors protect the heart from tissue damage and arrhythmias caused by ischemia and reperfusion [5, 6]. This protective effect of ACE inhibitors could result from a reduction in local angiotensin II production or increased local bradykinin concentration secondary to inhibition of its catabolism. The latter hypothesis is supported by the fact that exogenous BK exerts a cardioprotective action in isolated hearts against the deleterious effects of ischemia/reperfusion [7].

Neutral endopeptidase (EC 3.4.24.11) is a metallopeptidase involved in the metabolism of several peptides such as enkephalins, atrial natriuretic peptide (ANP) and BK [8]. It degrades also angiotensin I and II [8]and endothelins [9]in vitro and appears to participate in these processes in vivo [10]as well. This enzyme is located in the lungs, brain, intestine and the brush border of the renal proximal tubule [11]. NEP may also be located in rat mesenteric vascular bed [12]. This enzyme appears to be responsible for much of the ANP catabolism in vivo [13]and kinin catabolism in the kidneys [14]. Its contribution in BK catabolism in the coronary vascular bed is currently unknown.

Kinin metabolism in the heart is poorly understood. The aim of our study was to characterize the metabolism of BK after a single passage through the coronary bed in an isolated Langendorff rat heart and to assess the relative contribution of ACE and NEP in this metabolism. To study the enzyme involved in the carboxy-terminal metabolism of BK, an immunological method using highly specific polyclonal antibodies was used. To confirm that the measured immunoreactivity corresponds to the native BK, the aminopeptidase enzymatic activity in the metabolism of BK was assessed using high performance liquid chromatography (HPLC) followed by immunoreactivity profile of the amino-truncated metabolites.

Interestingly, a recent study reported that a chronic treatment of rats with the AT1-receptor antagonist, losartan, reduced ACE activity [15]. Therefore, the effect of losartan was compared with that of enzyme inhibitors.

2 Materials and methods

2.1 Animals and methods

Male Sprague-Dawley rats weighing between 275 and 300 g were used in the present study. Experiments were conducted according to the guidelines from the Canadian Council on Animal Care. Rats were anesthetized with sodium pentobarbital, 65 mg/kg body weight, injected intraperitoneally. The hearts were rapidly excised and immersed into ice-cold buffer containing heparin (20 UI/ml) before being perfused at constant flow rates through the aorta according to the Langendorff model. The perfusion solution was a modified Krebs–Henseleit (KH) buffer continuously gassed with O2/CO2 (95:5) at a temperature of 37°C, pH 7.4, containing (mmol/l) NaCl 118, KCl 4, CaCl2 2.5, KH2PO4 1.2, MgSO4 1, NaHCO324, d-glucose 5, and pyruvate 2. BK and drugs (enalaprilat, retrothiorphan and losartan) were infused with syringe pumps (Sage Instruments, model 355) into the aortic flow line at 1/100th of the coronary flow rate. Coronary perfusion pressure (CPP) was measured with a pressure transducer connected to a sidearm of the aortic flow line. Isovolumetric developed left ventricular pressure (DLVP) was measured with a fluid-filled latex balloon inserted into the left ventricle and connected to a second pressure transducer. Heart rate (HR) was derived from the DLVP. These variables were recorded on a Nihon-Kohden (Japan) polygraph system.

Hearts were allowed to stabilize for 20 min at 10 ml/min. The experiments were performed with hearts perfused at either one of the following coronary flow rates: 1, 5 or 10 ml/min. BK and/or drugs were infused into the aortic flow line for 34 min to obtain a final concentration of 10 nM for BK (10 ng/ml), 0.13 μM for enalaprilat, 25 nM for retrothiorphan and 1 μM for losartan. Five min before the drug infusion, the flow rate was either reduced to 5 ml/min or 1 ml/min or kept at 10 ml/min. In the experiments performed at 1 and 5 ml/min, flow rate was returned to 10 ml/min for the last 7 min of drug infusion. Perfusate fractions of 2 ml were collected into tubes containing ice-cold trifluoroacetic acid (TFA, 100 μl of a 10% solution per ml of perfusate) under constant agitation.

2.2 Purification and quantification of BK and des-Arg9-BK from perfusate

The perfusate was purified by chromatography on C8-silica columns (Waters, Milford MA, USA) as described earlier [16]. The eluate containing kinins was evaporated to dryness in a Speed Vac system (Savant, Farmingdale NJ, USA). The residue was dissolved in 500 μl of a Tris-HCl buffer (50 mmol/l) containing Tween 20 (0.5 ml/l) and NaCl (100 mmol/l). Residual immunoreactive BK and formed immunoreactive des-Arg9-BK were quantified by a competitive enzyme immunoassay as previously described [16, 17]. Both assays used highly specific polyclonal rabbit immunoglobulins raised against the carboxy-terminal end of BK and des-Arg10-kallidin, digoxigenin-labelled peptide as tracer, and alkaline phosphatase-labelled IgGs anti-digoxigenin with chemiluminescent substrate, LUMI-PHOS® (Boehringer Mannheim, Germany) to detect and quantify the immune complexes. On a molar basis, the polyclonal anti-BK purified IgGs exhibited no cross-reactivity with des-Arg9-BK. Likewise, anti-des-Arg10-kallidin IgGs showed no cross-reactivity with BK. Typical calibration curves were characterized by a half-maximal saturation value of 0.78 pmol/ml for BK and 1.53 pmol/ml for des-Arg9-BK.

2.3 High performance liquid chromatography

A HPLC system (Waters Associates, Milford, MA, USA) consisting of a Model 600 Multisolvent Delivery System and a model 484 Turnable Absorbance Detector was used. BK and four products of the amino-truncated peptide (BK(5–9), BK(4–9), BK(3–9), BK(2–9)) were separated on a reverse phase column (Vydac C18 5 μ, 4.6×250 mm; Hesperia, CA, USA) within 45 min, with a linear gradient from 80% solvent A/20% solvent B to 65% solvent A/35% solvent B at a constant flow rate of 0.7 ml/min, and were detected at 214 nm. Solvent A was 0.025% heptafluorobutyric anhydride (HFBA; vol/vol) in distilled water and solvent B was 0.025% HFBA (vol/vol) in 90% acetonitrile −10% distilled water. Fractions of 0.7 ml were collected, evaporated to dryness in Speed Vac Concentrator and then frozen at −80°C until immunoreactivity profile determination. BK and NH2-terminal metabolites were identified by comparison of their retention time with that of reference peptides [18]. This study of immunoreactivity profile was assessed at a coronary flow rate of 1 ml/min, under which condition BK catabolism was maximal.

2.4 Volume of distribution of BK

The volume of distribution of BK was measured at 1, 5, and 10 ml/min with a method adapted from [19]. Hearts were mounted and allowed to stabilize as described above. Tritiated BK ([2,3-prolyl-3,4-3H(N)]-BK, DuPont NEN, Boston MA) was added to cold BK to obtain a final concentration in the heart of 10 nM BK with 10 000 DPM/ml. This solution was infused for 5 to 6 min, which was enough to ensure that the radioactivity overflowing the heart had reached a plateau. The hearts were then removed from the perfusion setup, quickly rinsed in 10 ml KH buffer, and wiped dry. 50-mg samples (in triplicates) from the atria, the right ventricle, and the left ventricle were digested in 0.5 ml hyamine hydroxide (1 M in methanol) at 50°C for 24 h, and bleached with 0.25 ml H2O2 (30.9%) at 50°C for 3 h. A liquid scintillation cocktail was then added (10 ml CytoScint, ICN, Costa Mesa, CA) and the samples counted for 5 min. The volume of distribution of BK (VDBK, in ml/g fresh heart) was calculated with the following equation [19]: Embedded Image where DPMHeart is the radioactive disintegration min−1 g−1 in heart samples, DPMKH is the radioactive disintegration min−1 g−1 of the inflowing KH buffer, and ρKH the density of KH buffer (1.004 g/ml). The VDBK in the total heart was calculated with the weighted sum of the different VDBK measured in the atria and ventricles. There was no difference in the measured VDBK among the different cardiac structures.

2.5 Materials and reagents

Bradykinin and des-Arg9-BK were obtained from Peninsula Laboratories (Belmont, CA, USA). Enalaprilat and losartan were a kind gift from Merck Frosst Canada (Kirkland, Québec, Canada). Retrothiorphan was a generous gift from Dr. Bernard P. Roques (Laboratoire de pharmacochimie moléculaire et structurale, Université René Descartes, Paris, France). Sodium pentobarbital (Somnotol) was purchased from MTC Pharmaceuticals (Mississauga, NJ, USA). Ethanol of HPLC grade was obtained from J.T. Baker (Phillipsburg, NJ, USA). HFBA and TFA were purchased from Pierce (Rockford, IL, USA). Acetonitrile (HPLC grade) and other chemicals of analytical grade were obtained from Fisher Scientific (Montréal, Québec, Canada).

2.6 Statistical analysis

All results were expressed as mean±SEM. BK samples were assayed in triplicates. Vmax/Km was calculated with the following equation [20, 21]: Embedded Image where t is the transit time and %recovery is the percent of BK recovered after a single passage through the coronary bed. The transit time was calculated by the following equation: Embedded Image where VDBK and flow refer to the volume of distribution of BK and coronary flow rate, respectively. Vmax/Km was corrected for the heart mass. The statistical significance was evaluated using an analysis of variance followed by a Tukey HSD post hoc test (Systat for Windows, version 6.1). p<0.05 was considered statistically significant.

3 Results

The concentration of BK in the perfusate before the infusion of exogenous BK was under the detection limit of the quantification method for every coronary flow rate, and did not rise above this limit in time-matched control experiment without infusion of BK either in the absence or presence of enzyme inhibitors (data not shown). Reducing coronary flow rate from 10 to 1 ml/min was accompanied by changes in HR (from 298±10 to 94±28 beats/min, p<0.05), CPP (from 49±3 to 15±4 mmHg, p<0.05), and DLVP (from 84±3 to 19±2 mmHg, p<0.05). These changes were less severe when flow rate was reduced from 10 to 5 ml/min (HR: from 300±10 to 211±18 beats/min, p<0.05; CPP: from 52±2 to 22±2 mmHg, p<0.05; DLVP: from 75±3 to 27±4 mmHg, p<0.05). Recovery of infused BK in the absence of inhibitors was related to the coronary flow rate: recoveries of 86.3±2.9%, 60.8±6.3% and 29.6±6.8% were measured at 10, 5 and 1 ml/min, respectively (Fig. 1). At the end of the experiments, when the flow rate returned to 10 ml/min, BK recovery was similar in all groups (Fig. 1). The VDBK measured at 1, 5, and 10 ml/min was 0.426±0.019, 0.274±0.022, and 0.316±0.026 ml/g, respectively (n=5 per group). The VDBK at 1 ml/min was significantly higher than that at 5 and 10 ml/min, whereas no significant difference was found in the VDBK between 5 and 10 ml/min.

Fig. 1

Recovery (in %) of infused bradykinin (BK) into the aortic flow line of isolated rat hearts perfused at either 1 (closed circles, n=5), 5 (open squares, n=3), or 10 ml/min (open diamonds, n=3) as a function of time. The data points shown at −5 min represent the low endogenous concentrations of BK in the perfusate before the beginning of BK infusion, which started at 0 min. Perfusion rate was restored to 10 ml/min after 27 min in groups originally perfused at 5 or 1 ml/min. * p<0.05 compared with corresponding values in the group perfused at 1 ml/min.

At 1 ml/min, BK recovery in the presence of the ACE inhibitor, enalaprilat, was significantly enhanced (59.9±2.6%, Fig. 2). The AT1 antagonist, losartan, did not modify BK recovery. Inhibition of NEP with retrothiorphan also had no effect on BK recovery. However, the combined treatment with enalaprilat and retrothiorphan improved markedly BK recovery (81.2±5.2%, Fig. 2). Qualitatively similar results but of reduced magnitude were observed at 5 ml/min (Fig. 3). In contrast, BK recovery was unaffected by the enzyme inhibitors at 10 ml/min (Fig. 4).

Fig. 4

Recovery (in %) of infused bradykinin (BK) into the aortic flow line of isolated rat hearts perfused at 10 ml/min in absence (CTL, closed circles, n=3) or in presence of either enalaprilat (ENA, open squares, n=4), retrothiorphan (RTH, open circles, n=3), enalaprilat plus retrothiorphan (closed triangles, n=3), or losartan (LOS, open diamonds, n=5). The time-events described in the legend of Fig. 1 also apply to the present figure.

Fig. 3

Recovery (in %) of infused bradykinin (BK) into the aortic flow line of isolated rat hearts perfused at 5 ml/min in absence (CTL, closed circles, n=3) or in presence of either enalaprilat (ENA, open squares, n=3), retrothiorphan (RTH, open circles, n=3), enalaprilat plus retrothiorphan (closed triangles, n=3), or losartan (LOS, open diamonds, n=5). The time-events described in the legend of Fig. 1 also apply to the present figure. * p<0.05 compared with corresponding values in the control group.

Fig. 2

Recovery (in %) of infused bradykinin (BK) into the aortic flow line of isolated rat hearts perfused at 1 ml/min in absence (CTL, closed circles, n=5) or in presence of either enalaprilat (ENA, open squares, n=4), retrothiorphan (RTH, open circles, n=5), enalaprilat plus retrothiorphan (closed triangles, n=5), or losartan (LOS, open diamonds, n=5). The time-events described in the legend of Fig. 1 also apply to the present figure. * p<0.05 compared with corresponding values in the control group. # p<0.05 compared with corresponding values in the enalaprilat-treated group.

The effect of coronary flow rate and the different treatments on Vmax/Km is illustrated in Fig. 5. Vmax/Km values were found to be significantly higher in hearts perfused at 5 ml/min, with the exception of enalaprilat and combined enalaprilat and retrothiorphan-treated hearts in which Vmax/Km values were comparable among the groups perfused at different flow rates (Fig. 5, panel A). However, this difference was no longer observed when flow rate was restored to 10 ml/min (Fig. 5, panel B). Vmax/Km was reduced in the presence of enalaprilat in all hearts, but the reduction reached the statistical level of significance only in hearts perfused at 5 ml/min (Fig. 5, panel A). The combined treatment with enalaprilat and retrothiorphan significantly reduced Vmax/Km regardless of coronary flow rate (Fig. 5, panels A and B). The effect of flow reduction on HR, CPP, and DLVP in treated hearts was comparable with that in untreated hearts (data not shown).

Fig. 5

Panel A shows the Vmax/Km ratio (min−1 g−1) for bradykinin in control hearts (CTL) and hearts treated with either enalaprilat (ENA), retrothiorphan (RTH), enalaprilat+retrothiorphan, and losartan (LOS), perfused at either 1 (black columns), 5 (hatched columns), or 10 ml/min (open columns). Panel B shows the Vmax/Km ratio in the corresponding groups after perfusion rate was restored to 10 ml/min. ⊗ p<0.05 compared with the corresponding hearts perfused at 1 ml/min; † p<0.05 compared with RTH and LOS; # p<0.05 compared with CTL, RTH, and LOS; § p<0.05 compared with the corresponding hearts perfused at 1 and 10 ml/min; * p<0.05 compared with CTL.

HPLC analysis performed with the perfusate collected from hearts without treatment (panel B), with enalaprilat (panel C), and with enalaprilat and retrothiorphan (panel D), clearly showed that immunoreactivity was mainly detected at the retention time corresponding to native BK (Fig. 6). This native BK constituted 93.1, 93.4 and 91.7% of immunoreactive BK for hearts without treatment, treated with enalaprilat, and treated with enalaprilat and retrothiorphan, respectively, with only a minor peak (<5%) eluting in the position of BK(2–9) detected in all groups.

Fig. 6

Panel A shows retention time for standard peptide: BK(5–9) (1), 28 min; BK(4–9) (2), 32 min; BK(3–9) (3), 35 min; BK(2–9) (4), 36 min and BK (5), 41 min. Panels B, C and D represent the profiles of immunoreactivity after reverse-phase HPLC separation of BK and amino-terminal truncated metabolites generated after a single passage through the coronary bed without treatment (panel B), with enalaprilat (panel C), and with enalaprilat and retrothiorphan (panel D).

Immunoreactive des-Arg9-BK could not be detected in the perfusate collected during these experimental conditions.

4 Discussion

The main actions of ACE are conversion of angiotensin I to angiotensin II and degradation of kinins. In the present study, we used an inhibitor to assess the contribution of ACE in the catabolism of BK following a single passage through the coronary bed. BK degradation was reduced in the presence of enalaprilat, which resulted in enhanced recovery at the lower coronary flow rates. These results suggest that kininase II plays an important role in BK catabolism in the coronary bed. The presence of the NEP inhibitor, retrothiorphan, had no effect on BK degradation and recovery. On the other hand, the combined treatment with enalaprilat and retrothiorphan was more efficacious than enalaprilat alone to inhibit BK degradation. Therefore, NEP contributes to the catabolism of BK only when ACE activity is inhibited. These results could be explained by differences in the Vmax and Km values of BK for ACE and NEP. For example, the Km values of BK for human ACE is much lower than that for NEP: Km of BK for human or rabbit NEP is 120 μM whereas that for ACE was estimated to be 1.0 μM [22].

The relative proportion of ACE and NEP activities in the rat differs in several tissues such as the heart, the skeletal muscle [23], the lung [24]and the kidneys [25]. For example, in the rat heart and skeletal muscles, ACE activity is higher than that of NEP [23]. In addition, it has been shown that ACE is a more important metabolic pathway of pulmonary BK than NEP [26]. Prechel et al. [21]demonstrated that the aminopeptidase P (prolidase) and ACE can fully account for the metabolism of BK in the pulmonary circulation and suggested that NEP is not involved in the pulmonary degradation of BK in vivo. NEP has been found immunocytochemically in lung epithelial cells but not in lung vascular endothelial cells that would be directly in contact with circulating BK [11]. However, NEP is responsible for 75% of renal BK metabolism in the rat. Although animal kidneys are generally very rich in ACE, the rat seems to be the exception [27]. ACE, mainly an endothelial enzyme, is responsible for BK metabolism within the vascular compartment, whereas NEP, mainly an epithelial enzyme, is involved in its catabolism in the kidneys. Yamada et al. have reported a heavy ACE labelling in rat coronary vessels by quantitative autoradiography [28]. This concentration of ACE in rat coronary bed could explain the higher contribution of ACE in BK catabolism, compared with NEP, in our experiments.

Our study has demonstrated a clear effect of flow on BK recovery: higher flow rates were accompanied by a higher recovery. This cannot be due to an effect of flow on the enzymatic machinery since BK degradation rates were comparable in hearts perfused at 1 and 10 ml/min. Rather, the effect of flow on BK recovery is likely due to transit time: at low flow, a longer time is needed for BK to travel through the coronary bed, resulting in a longer exposure of BK to the metabolizing enzymes. These observations suggest that coronary flow rate is an important factor to consider when studying the contribution of kinins in the cardioprotective effects of enzyme inhibitors like ACE inhibitors. In some groups, BK degradation rate in hearts perfused at 5 ml/min was higher than in hearts perfused at either 1 or 10 ml/min. This cannot be explained by change in the tissue distribution of BK at this particular flow rate since the VDBK measured at 5 ml/min, although significantly lower than that at 1 ml/min, was comparable to that at 10 ml/min. Interestingly, the VDBK measured at 5 and 10 ml/min were close to the extracellular space measured in the rabbit heart [19]. On the other hand, the higher VDBK at 1 ml/min may be explained by a slight edema at this low flow rate.

The active metabolite of BK, des-Arg9-BK, was not detected under our experimental conditions, even in the presence of enzyme inhibitors. This is in agreement with the small portion of BK metabolised into des-Arg9-BK (1.4%) in rat serum [29]as well as in normal rat heart membranes (1%, unpublished data). However, these results contrast with those of Lamontagne et al. [30]who observed an enhanced release of des-Arg9-BK with enalaprilat upon reperfusion of the isolated rat heart following a 20-min total ischemia. Thus, BK metabolism in severely ischemic hearts differs from that of non-ischemic hearts. Although highly speculative, release of lysosomal enzymes capable of forming des-Arg9-BK [31]during ischemia may explain this discrepancy.

The AT1 antagonist losartan did not interfere with BK metabolism in our study. These results are in agreement with those observed with normal rat heart membranes (unpublished data). Schieffer et al. [15]have reported that a chronic treatment with losartan diminished the activity of cardiac ACE in hypertrophied rat hearts. Our result with an acute administration clearly shows that this AT1 antagonist does not interfere in the cardiac BK metabolism. This observation implies that the effect of a chronic treatment with losartan on cardiac ACE activity may be indirect and time-dependent.

Ryan et al. [32]identified a prolidase (aminopeptidase P) that cleaved the NH2-terminal Arg1-Pro2 bond of BK in the pulmonary circulation, yielding an amino-terminal truncated peptide devoid of pharmacological activity. Since the antisera used in our analytical methodology recognises specifically the carboxy-terminal end of BK, we verified whether the measured carboxy-terminal immunoreactivity corresponded to native and pharmacologically active peptide. HPLC separation followed by immunoreactivity profile showed clearly that the measured immunoreactivity after a single passage through the coronary bed either with enalaprilat, with enalaprilat and retrothiorphan or without treatment, corresponded to more than 90% of native BK. Thus, the contribution of the aminopeptidase enzymatic activity was weak in our model.

In conclusion, BK recovery during a single passage through the coronary bed is related to coronary flow rate, which is not due to change in degradation rates, but rather to different transit times. An important contribution of ACE to BK catabolism was observed in this model. NEP contributes to the degradation of BK only when the activity of ACE is impaired. The immunoreactivity profile of BK showed that the measured immunoreactivity in the perfusate with or without treatment corresponds to more than 90% of native BK. No significant contribution of kininase I could be observed.

Acknowledgements

This study was supported by the Losartan Medical School Grant, the Pharmaceutical Manufacturers Association of Canada-Medical Research Council of Canada (PMAC-MRCC), the Medical Research Council of Canada (MRCC), and the Heart and Stroke Foundation of Québec. MJD received a studentship from the Fonds FCAR.

References

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