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Faculties of 1 Pharmacy,
3 Arts and Sciences, and
4 Medicine, The respective role of angiotensin-converting
enzyme (ACE) and neutral endopeptidase 24.11 (NEP) in the degradation
of bradykinin (BK) has been studied in the infarcted and hypertrophied
rat heart. Myocardial infarction (MI) was induced in rats by left
descendant coronary artery ligature. Animals were killed, and hearts
were sampled 1, 4, and 35 days post-MI. BK metabolism was assessed by
incubating synthetic BK with heart membranes from sham hearts and
infarcted (scar) and noninfarcted regions of infarcted hearts. The
half-life
(t1/2)
of BK showed significant differences among the three types of tissue at
4 days [sham heart (114 ± 7 s) > noninfarcted region (85 ± 4 s) > infarcted region (28 ± 2 s)] and 35 days
post-MI [sham heart (143 ± 6 s) = noninfarcted region (137 ± 9 s) > infarcted region (55 ± 4 s)]. No difference was observed at 1 day post-MI. The participation of ACE and NEP in the
metabolism of BK was defined by preincubation of the membrane preparations with enalaprilat, an ACE inhibitor, and omapatrilat, a
vasopeptidase inhibitor that acts by combined inhibition of NEP and
ACE. Enalaprilat significantly prevented the rapid degradation of BK in
every tissue type and at every sampling time. Moreover, omapatrilat
significantly increased the
t1/2 of BK
compared with enalaprilat in every tissue type and at every sampling
time. These results demonstrate that experimental MI followed by left ventricular dysfunction significantly modifies the metabolism of
exogenous BK by heart membranes. ACE and NEP participate in the
degradation of BK since both enalaprilat and omapatrilat have potentiating effects on the
t1/2 of BK.
angiotensin-converting enzyme; vasopeptidase inhibitor; left
ventricular hypertrophy
ANGIOTENSIN-CONVERTING ENZYME (ACE) inhibitors have
proven to prolong the survival of postinfarction patients with left
ventricular dysfunction (25). Several mechanisms have been proposed to
explain this effect, one of which is the prevention of adverse
ventricular remodeling postinfarction. Although the inhibition of the
production of angiotensin II plays a role in this effect, there is
mounting evidence that at least part of the beneficial effects of ACE
inhibitors postinfarction are the result of the inhibition of
bradykinin (BK) metabolism, which in turn increases nitric oxide and
prostaglandin levels (18). Paradoxically, the cardiac metabolism of BK
in the acute, subacute, and chronic postinfarction periods has never been measured. Also, the effects of ACE inhibitors on the cardiac metabolism of BK in these settings have never been evaluated.
It has been proposed that a potentiation of the effect of BK by ACE
inhibitors in the postinfarction period could be an important mechanism
by which ACE inhibitors prevent scar expansion postinfarction (19). The
effect of ACE inhibitors on BK metabolism in the area of the necrosis
and scar appears then to be particularly important to evaluate.
Although ACE inhibitors reduce cardiac collagen production in the
postinfarction period (19, 37), which in turn should promote scar
expansion, ACE inhibitors might also reduce scar expansion, thus
contributing to their beneficial effects. The effect of ACE inhibitors
on BK metabolism in the remaining viable myocardium also appears to be
important, because there is mounting evidence that BK plays an
essential role in preventing chronic left ventricular dilatation
postinfarction (19).
BK is the prototype of kinins, a family of powerful bioactive autacoids
released from their precursors called kininogens (4, 27). BK exerts its
pharmacological activities by activating the
B2 receptors, which are widely
distributed throughout mammalian tissues (4, 13). In the past several
years, there has been a renewed interest in BK because of its
cardiovascular effects, consisting mainly of vasodilatory and
anti-proliferative effects (18). BK is a short-lived peptide because of
its rapid metabolism by different peptidases. In vitro, many enzymes
are susceptible to metabolize BK. These enzymes belong mainly to
metallopeptidases but also to serine peptidases and proteases,
astacin-like metallopeptidases, and cathepsins (12). The nature of the
enzymes involved in the metabolism of BK in vivo and their relative
importance depend on the biological medium considered. Recently, we
have shown that ACE (peptidyl dipeptidase A, kininase II; EC 3.4.15.1)
is the main enzyme responsible for the metabolism of BK, not only in rat and human serum (9) but also at the coronary bed level (11) and in
cardiac membrane preparations of normal hearts of different animal
species (5). First, ACE metabolizes BK into BK-(1 A new class of compounds, the vasopeptidase inhibitors, has recently
been developed that not only inhibits the activity of ACE but also
inhibits the activity of NEP (28, 35). In addition to their protective
effect on natriuretic peptides (28, 31), these dual ACE/NEP inhibitors
would be expected to increase BK levels more than ACE inhibitors alone.
These drugs are now being investigated in clinical trials for use in
hypertension and congestive heart failure, and their use in the early
and late postinfarction period is being considered. Given the indirect
evidence for a role of BK in the cardioprotective effects of ACE
inhibitors, it would appear essential to evaluate the effects of these
new ACE/NEP inhibitors on the metabolism of BK in both healthy and pathological cardiac tissue. Omapatrilat (28) is a member of this new
class of therapeutic agents (vasopeptidase inhibitors), and, in this
study, its effects on BK metabolism in healthy and postinfarction
(early and late) cardiac tissue were evaluated and compared with the
effects of an ACE inhibitor. The experimental model used is clinically
relevant, that of the postinfarction rat (26).
Drugs, Peptides, and Reagents
ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
7) and, in a second
step, BK-(17) is degraded into BK-(15) (12). Besides ACE, other
enzymes metabolize BK. Kininase I, a generic name for different plasma
and cell membrane carboxypeptidases, is responsible for the metabolism
of BK into its active metabolite des-Arg9-BK (12, 32). In the serum
as well as in the heart, the kininase I pathway is a minor metabolic
pathway of BK, which becomes evident only when ACE is inhibited (5, 9).
Finally, we have also demonstrated that neutral endopeptidase 24.11 (NEP, neprilysin; EC 3.4.24.11) plays an important role in the
degradation of BK at the endothelial level. Like the kininase I
pathway, the NEP pathway becomes evident only if ACE has been inhibited
previously (11). NEP metabolizes BK into BK- (17), and then
BK-(17) is cleaved into BK-(14) (12).
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Surgery and Animal Death
All of the animal experiments followed the guidelines of the Canadian Council on Animal Care and were approved by the Animal Care Ethics Committee of the Institute of Cardiology of Montreal. Myocardial infarction (MI) was induced in 200- to 250-g male Wistar rats (Charles River, St. Constant, QC) through ligation of the left descendant coronary artery as described earlier (3, 26). The rats were anesthetized with 3% halothane. During surgery, they were artificially ventilated with humidified room air supplemented with oxygen, and the halothane concentration was gradually decreased to 1%. A Harvard Rodent ventilator (Harvard Apparatus, South Natick, MA) set at 2 ml, 70 strokes/min, and a Fluotec 3 halothane vaporizer (Cyprane) were used for this procedure. The heart was quickly exteriorized through a left-sided thoracotomy, and the left descendant coronary artery was ligated ~2 mm from its origin. The heart was then replaced in its normal position in the thorax, and the incision was closed with a Mikron wound clip applicator (Clay Adams) after the crest was gently pressed to expel air from the cavity to avoid a pneumothorax. Once awakened after surgery, the rats were injected with 0.01-0.02 mg/kg buprenorphine to reduce the pain during recovery.On days 1,
4, and
35 after the surgery, the surviving
rats were killed to obtain the heart. The rats were anesthetized using an intramuscular injection of ketamine (50 mg/kg) and xylazine (10 mg/kg). Before death, during the same anesthesia, an electrocardiogram (ECG) was performed, and the intraventricular pressures were measured by inserting a Millar Mikro-Tip Catheter Transducer (Millar
Instruments, Houston, TX) with a pressure sensor at the tip in both the
jugular vein and carotid artery and advancing it in the right and left ventricle, respectively. The ECG and pressures were recorded on a Gould
2,600S recorder (Gould, Cleveland, OH). The rats were classified as
having shammed, small, medium, or large MIs according to the ECG
readings taken before death. The presence of a Q wave in the lead I and
lead aVL derivative signaled an MI. The heights of the R waves in the
V1, V2, and V5 derivatives were summed, and their total value was used
as a criterion for further classification. A value inferior to 0.6 mV
indicated a large infarction, a value >1.0 mV indicated a
sham or small infarction, and a value between 0.6 and 0.85 mV indicated
a medium infarction. Sham hearts and hearts with small infarctions were
pooled and compared with the second group consisting of medium and
large infarctions. Before the heart was removed, 1,000 U/kg of heparin
were injected in the jugular vein. The heart was then excised and
carefully perfused with a 37°C saline solution to remove all blood.
Once this was completed, the infarcted hearts were dissected into two
pieces: the infarcted area of the left ventricular wall (scar) and the remainder of the heart (the noninfarcted area). In this model, the
infarcted area is not clearly visible 24 h postinfarction and always
involves the free wall of the left ventricle, and the septum is never
implicated in the infarct heart. Hearts sampled 24 h postinfarction had
the left ventricular wall dissected free of the septum. The left
ventricular wall was then considered the infarcted area and the septum
as viable myocardium. All portions of the hearts
were frozen at 
80°C until used for biochemical investigations.
Preparation of the Total Heart Membrane Suspensions
To assess the metabolism of BK by enzymes located on cardiac cell membranes, membranes were extracted from the hearts following a procedure previously used by Kinoshita et al. (15) to study the metabolism of angiotensin I and more recently by Blais et al. (5) to assess the metabolism of BK in the normal rat heart. The noninfarcted and infarcted portions of each heart were thawed, weighed, and then cut into 3- to 4-mm pieces. These pieces were placed in a 50 mM Tris · HCl buffer, pH 7.4, at 4°C (10 ml/g of tissue) and were homogenized with a Polytron homogenizer (Brinkmann Instruments, Rexdale, ON) at setting 8 for 15 s. The homogenate was centrifuged at 40,000 g for 20 min at 4°C. After centrifugation, the tissue pellet consisting of membranes was separated from the cytosolic supernatant. The membranes were resuspended in a 50 mM Tris · HCl buffer, pH 7.4, containing 100 mM NaCl, at 4°C. A Wheaton Potter-Elvehjem tissue grinder (Fisher Scientific, Pittsburgh, PA), driven by a T-line motorized stirrer (Talboys Engineering, Emerson, NJ) turning at setting 8 for 60 s, was used for this procedure. During resuspension, the fibrous tissue was discarded from the infarcted pieces. The resulting membrane suspension was assayed for its 5'-nucleotidase activity (1). The protein concentration of the membrane suspensions was determined by the bicinchoninic acid method using bovine serum albumin as the standard.BK Metabolism
Incubation of BK with the total heart membrane suspensions. The membrane suspensions were diluted with a 50 mM Tris · HCl buffer, pH 7.4, containing 100 mM NaCl to obtain a 5 mg/ml protein concentration. The membrane suspensions coming from the infarcted scar pieces were pooled two by two on the basis of an equal protein concentration to obtain a sufficient suspension volume for the incubation procedure. The metabolic profile of BK was measured at 37°C in the same conditions previously used for normal hearts (5). Briefly, 10 µl of saline containing 500 ng of synthetic BK were added to 990 µl of the heart membrane suspension. The final concentration of BK in this suspension was 471 nM. After various incubation periods at 37°C, ranging between 2 and 20 min, the reaction was stopped by precipitating the membrane proteins through the addition of cold (4°C) ethanol at a final concentration of 80% vol/vol. In two sets of parallel experiments, and before the synthetic BK was added, the membrane suspensions were preincubated for 15 min at 37°C either with enalaprilat or with omapatrilat. The inhibitor concentrations were, respectively, 130 nM for enalaprilat and 510 nM for omapatrilat. The precipitated samples were centrifuged 15 min at 4°C and 2,000 g. The clear supernatant containing BK and its metabolites was evaporated to dryness in a Speed Vac Concentrator (Savant, Farmingdale, NY). The residues were stored at80°C until quantification of the residual BK was performed.
Quantification of BK. Immunoreactive BK was quantified in the residues of the evaporated ethanolic extracts using a highly specific enzyme immunoassay developed in our laboratory (8). This assay used highly specific polyclonal rabbit IgG raised against the carboxy-terminal end of BK, digoxigenin-labeled peptide as tracer, and alkaline phosphatase-labeled anti-digoxigenin Fab fragments with the substrate p-nitrophenyl phosphate to detect and quantify the immune complexes. Each sample was measured in triplicate. Typical calibration curves were characterized by half-maximal saturation values of 0.78 pmol/ml. This method was precise and accurate.
Kinetic parameter analysis. BK
hydrolysis rate constant (k) was
evaluated with the first-order equation [BK]=
[BK]0 × e
kt, where [BK] is
the concentration of BK at a given time and
[BK]0 is
[BK] at time (t) = 0. The BK half-life
(t1/2)
was represented as
t1/2 = ln(2)/k (20). The different
t1/2
values were expressed for 1 mg of protein.
ACE relative activity was estimated from
k with
(k+) or without
(k)
enalaprilat using the equation %ACE = 100 × (1 k+/k).
NEP relative activity was estimated by using
k with
(k+) or without
(k)
omapatrilat and by subtracting the ACE relative activity, following the
equation %NEP = [100 × (1 k+/k)] %ACE. This subtraction assumed that both enalaprilat and omapatrilat inhibit ACE to the same extent. In fact, the concentration chosen for each inhibitor was above its inhibitory constant so that
both inhibitors fully inhibited ACE.
Separation and Identification of BK Amino-Truncated Metabolites
To assess if the immunoreactive BK measured at the t1/2 corresponds to the native amino-terminal peptide, immunograms after HPLC were designed for each incubation condition (5). Briefly, in the conditions described above, BK was incubated with the heart membrane preparations for a period corresponding to the calculated t1/2. Incubations were performed either in the presence of enalaprilat, in the presence of omapatrilat, or without inhibitor. After precipitation of proteins with cold ethanol and centrifugation, the ethanolic extracts were separated in two parts. After evaporation, the first part was used for the quantification of immunoreactive BK. The second part was dissolved in 0.025% HFBA (vol/vol) in distilled water before HPLC separation. An HPLC system (Waters Associates, Milford, MA) consisting of a model 600 Multisolvent Delivery System and a model 484 Tunable Absorbance Detector was employed for HPLC analysis. BK and four products of the amino-terminal enzymatic cleavage of BK were separated on a reverse-phase column (Vydac C18 5 µm, 4.6 × 250 mm; Hesperia, CA) at a constant flow rate of 0.7 ml/min using a 45-min linear gradient from 80% solvent A-20% solvent B to 65% solvent A-35% solvent B. Solvent A was 0.025% HFBA (vol/vol) in distilled water, and solvent B was 0.025% HFBA (vol/vol) in 90% acetonitrile-10% distilled water. The column effluent was monitored continuously at 214 nm. Fractions of 0.7 ml were collected, evaporated to dryness in a Speed Vac Concentrator, and then frozen at80°C until immunoreactivity profile determination. BK and
metabolites were identified by comparing their retention times with
those of reference peptides.
Measurement of ACE and NEP Activity
The membrane suspensions used for BK metabolism were solubilized in 8 mM CHAPS (6). ACE activity was measured using the method of Cushman and Cheung (7), and NEP activity was measured using the method of Nortier et al. (22). Each sample was quantified in duplicate for both assays. ACE activity was expressed as picomoles of hippuric acid per minute per milligram of protein, and NEP activity was expressed as picomoles of AMC per minute per milligram of protein.Immunohistochemistry of NEP and ACE Expression
Expression of NEP and ACE was determined immunohistochemically. The hearts of the rats were fixed in a 10% Formalin-phosphate-buffered solution. The hearts were embedded in paraffin, sectioned (6-µm width) with a microtome along the cross section of the specimen at the midpoint between the apex and the base of the heart, and applied on glass slides. The sections were deparaffinized in xylene and ethanol baths, and endogenous peroxidase activity was quenched in a methanol-hydrogen peroxide solution. A nonspecific antibody binding was prevented by preincubating the tissues with a 5% goat serum treatment. Sections were exposed to the primary antibodies of rabbit polyclonal anti-rat NEP IgG (1:500 dilution) raised and purified in our laboratory (P. Crine, unpublished observations) and monoclonal anti-rat ACE IgM (1:500 dilution) kindly provided by Dr. R. Auerbach (University of Wisconsin, Madison, WI) and were used according to Dr. Auerbach's recommendations (2). A purified nonspecific goat IgG (1:500 dilution; Santa Cruz) was used as a primary negative control. The secondary antibodies were biotinylated goat anti-rabbit IgG (1:400 dilution; Vector Laboratories) and goat anti-mouse IgM (1:100 dilution; Vector Laboratories). Revelation of bound antibodies was achieved with an avidin-peroxidase complex (Vector Laboratories), and antibodies were counterstained in Gill's hematoxylin solution. NEP and ACE expression (brown staining) was evaluated for each segment by using a dedicated 3CCD video microscope adapted to a customized software.Statistical Analysis
All data are expressed as means ± SE. Different models of ANOVA were used to analyze the data. Two-way factorial analysis with days and tissues, sham and noninfarcted, or infarcted and noninfarcted were used with Scheffé's contrasts. A two-way analysis with the factor days and a repeated factor, tissues (infarcted and noninfarcted), was also used with paired t-tests using Bonferroni inequality. Finally, in some cases, a one-way analysis with appropriate contrasts was used. In view of the multiple analysis performed on the data, the significance level was fixed at 1%. Because most of the ANOVA showed interaction, only the results of the analysis of contrasts are reported.| |
RESULTS |
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Hemodynamic Characteristics
Hearts with an MI had a significant increase in left ventricular end-diastolic pressure (LVEDP). On day 1, MI hearts had an LVEDP of 9 ± 2 mmHg, on day 4 an LVEDP of 9 ± 1 mmHg, and on day 35 an LVEDP of 7 ± 2 mmHg; all of these pressures were significant (P < 0.01) compared with sham-operated controls (day 1: 1 ± 2 mmHg; day 4: 3 ± 1 mmHg; day 35: 1 ± 2 mmHg, respectively).Effect of MI on the Metabolism of BK
The t1/2 of exogenous BK measured in cardiac membranes from the sham hearts was consistent throughout the 5-wk study period, at between 114 ± 7 to 143 ± 6 s (Fig. 1). The t1/2 in the viable portion of the infarcted hearts was similar to that of sham at 1 day postinfarction (107 ± 8 s, n = 21; and 123 ± 6 s, n = 10, respectively) but was significantly decreased by 4 days (85 ± 4 s, n = 20 in MI vs. 114 ± 7 s, n = 11 in sham; P < 0.01). BK was thus metabolized 1.3 times faster in the viable portion of the MI hearts compared with sham hearts. However, by 35 days postinfarction, the t1/2 had returned to levels similar to those of the sham group (137 ± 9 s, n = 14 and 143 ± 6 s, n = 10, respectively). The infarcted portion of the heart had no difference in BK t1/2 compared with sham or viable myocardium of MI hearts 1 day postinfarction. By 4 days postinfarction, BK t1/2 was decreased markedly to 28 ± 2 s (n = 10), levels that were ~25% of those of sham and 33% of those of the noninfarcted portion of the same hearts (P < 0.01). By 35 days postinfarction, BK t1/2 in the infarcted region had nearly doubled (55 ± 4 s; n = 7) compared with 4 days postinfarction (28 ± 2 s, n = 10; P < 0.01) but remained less than one-half of that of the noninfarcted portion of the same hearts and of sham hearts (P < 0.01). BK was thus metabolized 2.5 times faster in the infarcted region at 35 days compared with sham or the viable myocardium of these hearts.
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Effect of Enalaprilat and Omapatrilat on BK t1/2
The preincubation of the membrane preparations with enalaprilat increased BK t1/2 significantly (P < 0.01) in every tissue type and at every sampling time after surgery (Fig. 2). Omapatrilat increased BK t1/2 significantly even more than did enalaprilat (P < 0.01; Fig. 2). In sham hearts, the effect of both inhibitors remained unchanged over time (Fig. 3). However, in the infarcted and noninfarcted portions of the MI hearts, the effects of both inhibitors increased over time (P < 0.01; Fig. 3). In infarcted pieces, the effect of both inhibitors on BK t1/2 was the greatest at 35 days postinfarction (163 ± 8 s for enalaprilat and 199 ± 14 s for omapatrilat; n = 7) compared with day 1 (36 ± 8 and 68 ± 7 s, respectively; n = 11; P < 0.01) or day 4 (35 ± 4 and 43 ± 4 s, respectively; n = 10; P < 0.01) after infarction. In the noninfarcted pieces, an increase in effect of both inhibitors on BK t1/2 occurred earlier, at 4 days postinfarction (126 ± 7 s for enalaprilat and 185 ± 12 s for omapatrilat; n = 20) vs. 1 day postinfarction (63 ± 10 and 97 ± 14 s, respectively; n = 21; P < 0.01). By 35 days postinfarction, the increase in effect of enalaprilat on BK t1/2 in noninfarcted portions of MI hearts was no longer significant compared with day 1. However, the increase in effect of omapatrilat on BK t1/2 compared with day 1 (97 ± 14 s, n = 21) was maintained up to 35 days (168 ± 14 s, n = 14; P < 0.01).
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Relative Contribution of ACE and NEP on BK Metabolism
The effect of enalaprilat on the BK t1/2 was used as a measure of the involvement of ACE in the metabolism of BK. The difference between the effects of omapatrilat and enalaprilat was used as a reflection of the involvement of NEP in this metabolism (Fig. 4). In sham hearts, the additive effect of NEP on BK metabolism remained unchanged throughout the study period. In the noninfarcted pieces of MI hearts, the involvement of NEP increased gradually over time such that it was significantly higher (P < 0.01) by 35 days postinfarction (69 ± 7 s, n = 14) compared with 1 day postinfarction (34 ± 6 s, n = 21). In infarcted pieces, NEP inhibition significantly prolonged the t1/2 of BK 1 day postinfarction (32 ± 5 s, n = 11); however, by 4 days postinfarction, this was no longer true (8 ± 1 s, n = 10). The effect of NEP inhibition on BK t1/2 was reestablished by 35 days postinfarction (37 ± 9 s, n = 7).
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If one adjusts the additive effects of ACE and NEP inhibition to BK
t1/2
without drugs, the relative contribution of ACE and NEP on BK
metabolism in the various tissues at any given point in time can be
calculated (Table 1). The relative
importance of ACE and NEP on the metabolism of BK varies according to
the nature of the tissue and over time. When compared with NEP, ACE played a greater role in the metabolism of BK in every tissue and at
every time point evaluated. In the sham hearts, the relative participation of ACE remained unchanged over time. In the infarcted pieces, the relative participation of ACE to the metabolism of BK was
similar at day 1 postinfarction
compared with sham; however, it rose steadily over time, more than
tripling between days 1 and
35 (P < 0.01) such that, at day 35, it was
two times that of sham (P < 0.01).
In the noninfarcted pieces, the relative participation of ACE was
similar to that of sham on day 1 postinfarction. It then rose from day
1 to day 4 postinfarction (P < 0.01) and then returned to basal levels by day 35.
The relative importance of NEP in sham or noninfarcted pieces of
myocardium was similar at day 1 postinfarction and did not vary over time. In the infarcted pieces, the
relative participation of NEP was similar at day
1 postinfarction compared with the other two tissues,
but it decreased over time (P < 0.01) such that it was lower than the sham hearts by
days 4 and
35 postinfarction
(P < 0.01).
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Evolution of ACE and NEP Enzymatic Activities Over Time
Both ACE and NEP enzymatic activity exhibited important variations according to the nature of the tissue and its sampling time (Fig. 5). In the sham hearts, ACE activity remained unchanged over time from 164 ± 8 pmol · min1 · mg
protein1
(n = 9) on day
1 to 156 ± 6 pmol · min1 · mg
protein1
(n = 9) on day
35. NEP activity was similar at day
1 and day 4 (24.9 ± 1.9 pmol · min1 · mg
protein1,
n = 10 and 25.4 ± 1.9 pmol · min1 · mg
protein1,
n = 11, respectively), and it
decreased to 17.1 ± 1.8 pmol · min1 · mg
protein1
(n = 9) on day
35, but it did not reach statistical significance (P > 0.01).
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In the infarcted pieces from MI hearts, the ACE activity was similar on
day 1 (120 ± 11 pmol · min1 · mg
protein1,
n = 15) compared with sham. It then
increased progressively over time such that by 4 days postinfarction
the highest values for any tissue were recorded (357 ± 19 pmol · min1 · mg
protein1,
n = 8;
P < 0.01). By 35 days
postinfarction, ACE activity (338 ± 15 pmol · min1 · mg
protein1,
n = 8) remained similar to
day 4. The activity of NEP was higher in the infarcted pieces compared with sham on the three time points (P < 0.01). Moreover, NEP activity
was similar after 1 and 4 days, but it decreased at
day 35 (41.7 ± 3.7 pmol · min1 · mg
protein1,
n = 7) compared with
day 4 (70.7 ± 5.7 pmol · min1 · mg
protein1,
n = 4;
P < 0.01).
In the noninfarcted pieces from MI hearts, ACE activity was higher on
day 1 than in sham
(P < 0.01), but it remained
unchanged over time from 205 ± 17 pmol · min1 · mg
protein1
(n = 20) on day
1 to 143 ± 15 pmol · min1 · mg
protein1
(n = 14) on day
35. NEP activity was lower at day
4 (31.2 ± 1.9 pmol · min1 · mg
protein1,
n = 17) and day
35 (30.8 ± 4.2 pmol · min1 · mg
protein1,
n = 10) compared with
day 1 (45.4 ± 2.7 pmol · min1 · mg
protein1,
n = 17;
P < 0.01).
Amino-Terminal Metabolism of BK in the Heart Membrane Preparations
HPLC analysis performed with residual BK after incubation with membranes coming from 4-day infarcted pieces indicated that the immunoreactive BK measured at the t1/2, in the absence and in the presence of enalaprilat or omapatrilat (Fig. 6), corresponds to the native peptide. In fact, >95% of the detected immunoreactive BK corresponds to the non-amino-truncated B2 agonist. Similar recovery values were measured for sham hearts and the noninfarcted parts of the infarcted hearts.
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Protein Expression of NEP and ACE in Normal and Hypertrophied Hearts
In the absence of cardiac injury in normal hearts, we could not detect the expression of NEP immunohistochemically with an antibody that specifically recognizes the expression of this protein (Fig. 7D). We also analyzed the expression of NEP on the hearts of rats with an MI at 35 days postprocedure. In the noninfarcted region of the heart, we could not detect the expression of NEP (Fig. 7E); however, in the infarcted area of the heart, we observed a clear and localized expression of NEP in some of the cardiomyocytes (Fig. 7F). In each study, a purified nonspecific goat IgG was used as a primary negative control, and in each case we could not detect any positive staining (Fig. 7, A-C). In a similar manner, we also evaluated the expression of ACE. In all hearts (normal and infarcted), we observed a ubiquitous expression of ACE on cardiomyocytes and endothelial cells (Fig. 8, D-F). In each study, a purified nonspecific goat IgG was used as a primary negative control, and in each case we could not detect any positive staining (Fig. 8, A-C).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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In this study, we demonstrated for the first time an alteration in the metabolism of BK by cardiac membranes during the acute, subacute, and chronic postinfarction periods. These alterations varied according to the time postinfarction and the tissue (infarcted or noninfarcted) being studied. Moreover, we showed clearly that an ACE inhibitor, and even more so the new vasopeptidase inhibitor omapatrilat, a dual ACE/NEP inhibitor, prevents the degradation of this vasodilatory and antiproliferative peptide at each time point and in each tissue. Again, these effects varied according to the tissue and the time point postinfarction, and, at each time of our experimental protocol, the effect of omapatrilat was significantly more important than that of enalaprilat on the degradation of BK not only by sham and noninfarcted tissues but also by the scar.
The rat postinfarction model has been used widely to study the pathophysiological processes involved in acute and chronic postinfarction left ventricular remodeling (23, 24). In the early postinfarction, there is an acute inflammatory process in the area of the scar that involves cardiac necrosis and infiltration by numerous inflammatory cell types. This inflammatory process intensifies within the next few days and then gradually disappears as a fibrotic scar develops. The noninfarcted area is under significant hemodynamic stress and intense neurohumoral stimulation during the acute and subacute postinfarction periods (23, 24, 29). Later in postinfarction, hemodynamic stress and neurohumoral activation abate in those tissues in which beneficial compensatory ventricular remodeling occurs. A number of studies suggest that BK may play a critical role in promoting beneficial remodeling and that the cardioprotective effects of ACE inhibitors in this setting are largely due to their prolongation of BK t1/2 (17, 19). In those tissues where adverse ventricular remodeling occurs or MI size is very large, hemodynamic stress and neurohumoral activation as well as low-level inflammation, as reflected by a rise in systemic cytokines, persist. In this study, mild to moderate left ventricular dysfunction accompanied the induction of the MI, suggesting at least partial compensatory ventricular remodeling.
In the heart, endogenous kinins can be produced locally during an MI from two mechanisms. First, because heart possesses an independent kallikrein-kinin system (21), BK can originate from the tissue itself. Second, BK can also originate from plasma. Indeed, in a previous clinical study, we have shown a decrease of prekallikrein and plasma kininogens in the postinfarction period in humans (A. Adam, unpublished observation). In this paper, we applied to the postinfarction period the same experimental in vitro metabolic approach that we had used to define the metabolism of BK in the normal heart of different animal species (5). We clearly showed that MI significantly shortens the t1/2 of BK. In the infarcted zone, this decrease only becomes evident 4 days postinfarction, a time at which the infarcted zone is scarring and the acute inflammatory response is still important. It also persists until at least 35 days postinfarction, a time at which the inflammatory response has largely abated. In the noninfarcted portions of the MI hearts, BK t1/2 also only decreases by 4 days postinfarction and returns to the level of sham by 35 days postinfarction, a period during which hypertrophy of the remaining viable heart is well established.
Because BK was incubated with membrane preparations and t1/2 was expressed per milligram of total membrane proteins, the metabolic changes documented cannot be attributed to an artefactual dilution factor or to soluble intracellular or extracellular enzymes modified during and after MI but rather to enzyme activities modified at the plasmatic cell membrane level. Membrane preparations used in this protocol are representative of normal cardiac muscle because they are composed of at least 75% cardiomyocyte membranes (36). However, we cannot exclude that membranes prepared from infarcted and hypertrophied samples contain membranes of cells other than cardiomyocytes. That is particularly true 4 days post-MI when infiltrating inflammatory cells (neutrophils, macrophages, and fibroblasts) are at their peak in the necrotic zone (33) and could release cytokines that could upregulate the enzyme activity in an autocrine or paracrine way (16).
Preincubation of membranes with an ACE inhibitor significantly increased the t1/2 of BK in the different tissue samples evaluated. The potentiating effect of an ACE inhibitor was similar in sham and noninfarcted tissues. In the infarcted portion of the heart, however, the effect of ACE inhibitor on the BK t1/2 was less important 1 and 4 days post-MI. Four days post-MI, BK t1/2 was markedly decreased in these membranes, and preincubation with enalaprilat did not succeed in normalizing t1/2. In these infarcted pieces, the effect of the ACE inhibitor was mainly evident 35 days postinfarction, a time at which hypertrophy of the remaining viable myocardium has developed. At that time, ACE inhibitor increased BK t1/2 fourfold. These results are consistent with those of Johnston et al. (14), who, using a quantitative autoradiographic method, showed an increased ACE expression in rat heart 4 wk postinfarction. This increase was particularly important in the fibrous scar tissue of the infarcted area. However, when the relative participation of ACE in the metabolism of BK is calculated from the t1/2 values in the presence and in the absence of ACE inhibitor, the values of both kinetic parameters show clearly that, although important in the metabolism of BK, ACE is not the unique enzyme responsible for the inactivation of BK, and the relative participation of these other enzymes varies according to the tissue and timing postinfarction. Differences in the activation of other enzymes (5, 15) would thus explain the only incomplete correlation between ACE activity and the relative contribution of ACE inhibitor on BK metabolism.
Among the other enzymes potentially responsible for the degradation of BK, NEP must be considered as a serious candidate. Recently, in defining the metabolism of BK by the coronary vascular bed of normal rat heart, we have shown that coperfusion of retrothiorphan, a highly specific NEP inhibitor, with BK did not modify its metabolism. However, when perfused in the presence of enalaprilat, retrothiorphan significantly increased the recovery of BK by 36% when compared with enalaprilat alone (11). These in vitro results with normal coronary endothelium show clearly that, when ACE is inhibited, NEP takes over and plays an important role in the metabolism of BK. The behavior of both enzymes can be explained by their respective affinity for BK. These results constitute the experimental basis for the simultaneous inhibition of both enzymes by a vasopeptidase inhibitor that exhibits similar inhibitory potency for both ACE (IC50 = 5 nM) and NEP (IC50 = 9 nM). Our results obtained using cardiac membranes in normal and pathological hearts not only confirms but also extends these previous observations at the normal coronary endothelium level.
When heart membranes were preincubated with omapatrilat, an increase in BK t1/2 occurred that was greater than that measured in the presence of the ACE inhibitor. Although omapatrilat reduced the metabolism of BK in all tissues evaluated, the simultaneous inhibition of NEP and ACE was particularly effective in prolonging BK t1/2 in the infarcted portion of the heart 1 day post-MI. At that time, the relative participation of NEP in the degradation of BK averaged that of ACE. This observation suggests that the use of omapatrilat may be particularly beneficial early in the postinfarction period, when BK appears to be involved in the regulation of the acute inflammatory reaction that stabilizes the scar. Although still significant, the protective effect of omapatrilat on BK was less important in the infarcted zone at 4 and 35 days postinfarction and in the noninfarcted zone, as well as in sham hearts, at all time points measured. The superiority of omapatrilat over a simple ACE inhibitor in reducing the degradation of BK could explain some of the experimental data recently obtained in vivo, which could involve the effects of endogenous BK.
In the rat, inhibition of NEP protected the heart against ischemia-reperfusion injury, as evidenced by a significant reduction of MI size and a tendency toward a reduction of reperfusion arrhythmias (38). These effects of NEP inhibitor were blocked by the B2-receptor-antagonist icatibant, suggesting that accumulation of kinins participates in the cardioprotective effect of NEP inhibitor. However, because NEP is also involved in the metabolism of natriuretic peptides, it has been shown that natriuretic peptides could be responsible, at least in part, for the cardiovascular effects of these new vasopeptidase inhibitors (28, 31, 34). In another study, it was reported that inhibitors of NEP and endopeptidase 24.15 produced cardioprotective effects comparable to those of ramipril in a rabbit model of ischemia-reperfusion (30). It was also demonstrated that a combination of ACE and endopeptidase inhibition produced more protection than when either type of inhibitor was used alone. Trippodo et al. (34) reported that, in hamsters with cardiomyopathy, the combined inhibition of ACE and NEP caused a significant decrease in left ventricular pressure and total peripheral vascular resistance and an increase in cardiac output compared with vehicle or either ACE inhibitor and NEP inhibitor alone. Finally, omapatrilat has been found to prolong the survival of cardiomyopathic hamsters more than did an ACE inhibitor, suggesting superior cardioprotective effects in this model of left ventricular dysfunction (accepted for the American Heart Association presentation).
We also measured the enzyme activity of ACE and NEP; however, the
values measured are difficult to put in relation with the t1/2 of BK
or the relative participation of both of these metallopeptidases.
Indeed, when we inhibit both enzymes, we do not totally block the
degradation of BK (Fig. 9). This was
particularly evident 4 days postinfarction in the scar. In the
infarcted part as well as in the noninfarcted part of the infarcted
heart, we cannot exclude the induction of other membrane enzymes
capable of metabolizing BK by the cytokines released at the local
inflammatory site or activated systemically. Moreover, we
do not know the nature, the properties, and the importance of these
enzymes that account for the rest of BK metabolism in each tissue
sample.
|
Finally, using a polyclonal antibody directed against rat NEP, we could find evidence of the presence of this enzyme in cardiomyocytes in the infarcted border zone 35 days postinfarction in amounts greater than in sham myocardium, suggesting that NEP is particularly active in this area at that time period. Moreover, immunohistochemical analysis using an anti-ACE antibody allowed us to confirm the presence of ACE not only in the endothelium but also in cardiomyocytes (14, 33).
Our results on the effects of an ACE inhibitor and the vasopeptidase inhibitor omapatrilat on the metabolism of BK may have pathophysiological significance and can be used as a rational basis for further studies in the acute and chronic postinfarction setting. Because the heart contains the precursors of BK and their tissue activator (21), our observations for exogenous BK may be transposed to endogenous BK. However, in that case, membrane enzymes and also circulating enzymes released from the membranes and cytosol during ischemia must be considered.
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ACKNOWLEDGEMENTS |
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We thank Dr. R. Auerbach (University of Wisconsin, Madison, WI) for the generous gift of the monoclonal anti-rat angiotensin-converting enzyme antibody.
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FOOTNOTES |
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This work was supported by the Pharmaceutical Manufacturers Association of Canada-Medical Research Council of Canada. C. Blais, Jr., is the recipient of a scholarship from the Fonds de la recherche en santé du Québec. The contributions of the first and third authors are equal.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for correspondence and reprint requests: A. Adam, Faculté de Pharmacie, Université de Montréal, 2900, Boul. Édouard-Montpetit, C.P. 6128, Succursale Centre-ville, Montréal, Québec, Canada H3C 3J7 (E-mail: adama{at}pharm.umontreal.ca).
Received 3 September 1998; accepted in final form 15 January 1999.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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