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(The FASEB Journal. 2002;16:1237-1241.)
© 2002 FASEB

AT1 receptor blockade increases cardiac bradykinin via neutral endopeptidase after induction of myocardial infarction in rats

THOMAS WALTHER1, WOLF-E. SIEMS*, DORIAN HAUKE, FRANK SPILLMANN, ANDREAS DENDORFER{dagger}, WINFRIED KRAUSE*, HEINZ-PETER SCHULTHEISS and CARSTEN TSCHÖPE

Department of Cardiology and Pneumology, Free University of Berlin;
* Institute of Molecular Pharmacology, Berlin, Germany; and
{dagger} Institute of Pharmacology, Medical University of Lübeck, Germany

1Correspondence: University Hospital Benjamin Franklin, Department of Cardiology and Pneumology, Free University of Berlin, Hindenburgdamm 30, D-12200 Berlin Germany. E-mail: thomas.walther{at}ukbf.fu-berlin.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
ACE inhibition protects the heart against ischemic injury by reducing angiotensin II and promoting bradykinin (BK) accumulation. Since neutral endopeptidase (NEP) metabolizes BK, we determined its activity after induction of myocardial infarction (MI) and examined whether it is influenced by treatment with an ACE inhibitor or AT1 receptor blocker. Rats were studied 6 days and 3 wk after coronary occlusion. Starting 48 h after MI induction, additional animals were treated with the ACE inhibitor quinapril (2 mg·kg-1·day-1) or the AT1 blocker irbesartan (50 mg·kg-1·day-1). Animals were hemodynamically characterized. Finally, NEP-specific activity and BK concentrations were detected in homogenates of heart compartments. Quinapril and irbesartan treatment improved left ventricular function 6 days and 3 wk after MI induction, and NEP activity was elevated only in the infarcted area of untreated compared with sham-operated rats. After 6 days, irbesartan reversed this increase by 80% and quinapril by 35%. Quinapril had no effect after 3 wk, whereas irbesartan almost completely blocked the increased NEP activity in the infarcted area and concomitantly induced a further rise in the BK concentrations. These results indicate mechanisms of NEP regulation influenced by the AT1 receptor. Our data suggest that NEP is more decisive than ACE in mediating BK degradation and may indicate BK involvement in the cardioprotective effects of AT1 antagonists.—Walther, T., Siems, W.-E., Hauke, D., Spillmann, F., Dendorfer, A., Krause, W., Schultheiss, H.-P., Tschöpe, C. AT1 receptor blockade increases cardiac bradykinin via neutral endopeptidase after induction of myocardial infarction in rats.


Key Words: bradykinin • enzymes • hemodynamics • inhibitors • neutral endopeptidase • renin-angiotensin system


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
REGULATION OF CARDIAC peptidases has become a major area of investigation in the last 20 years, since angiotensin-converting enzyme (ACE) inhibition improves the clinical outcome in patients with acute and chronic heart failure by a reduction of angiotensin II (ANGII) and an accumulation of the vasodilator bradykinin (BK). Another member of the family of cardiovascularly active peptidases is neutral endopeptidase (NEP) 3.4.24.11, which is expressed in many mammalian tissues such as the brain, kidney and heart. NEP acts as an endopeptidase, cleaving peptide bonds on the amino side of hydrophobic amino acid residues. Typical substrates besides BK are enkephalins, tachykinins, atrial natriuretic peptide (ANP), and endothelins (1) . Several authors found beneficial cardiac effects after NEP inhibition alone or combined with ACE inhibition in animal models with acute and chronic heart failure (2 , 3) .

After treatment with an NEP inhibitor, Trippodo and co-workers (3) found improved left ventricular (LV) function in hamsters with cardiomyopathy, whereas Marie et al. (2) found a reduced extracellular matrix formation and cardiac hypertrophy in a rat model of myocardial infarction (MI). However, less is known about the regulation and localization of NEP under these conditions.

For this reason, we first investigated cardiac NEP activity in different heart areas after coronary occlusion in a rat model of MI and determined whether this peptidase is regulated by ACE inhibitor treatment with quinapril or AT1 receptor blockade with irbesartan.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
Experiments were performed in male Sprague Dawley rats weighing 300–330 g (Charles River, Germany). They had free access to water and standard chow under a 12 h light/dark cycle.

Surgical procedures
NEP distribution and activity were determined in a rat model of MI, 6 days and 3 wk after coronary occlusion. Starting 48 h after MI induction, additional groups of animals were treated by gavage with quinapril (2 mg·kg-1·day-1) or irbesartan (50 mg·kg-1·day-1). All groups were compared with time-matched sham-operated controls (n=10 for each group). MI was induced by permanent ligation of the left descending coronary artery as described by Tschöpe et al. (4) .

LV pressure (LVP), LV end-diastolic pressure (LVEDP), and the dP/dtmax via a Millar tip catheter (2F) system in anesthetized (Ketanest®, 50 mg/kg; Parke Davis, Berlin, Germany; 2% Rompun®, 5 mg/kg; Medistar, Holzwickede, Germany; intraperitoneal), ventilated, open-chest animals were recorded at the end points of the study (4) .

The right ventricle (RV), interventricular septum (S), and infarcted and noninfarcted area of the LV were macroscopically separated after killing the animals. The tissue samples were homogenized with a Braun Potter in 50 mM Tris (pH 7.4), then filtered through Nylon gaze and stored until use at -80°C. Finally, homogenates were used to detect NEP-specific activity.

Measurement of NEP activity
NEP activity was measured using high-performance liquid chromatography (HPLC) to monitor [D-Ala2,Leu5]enkephalin (DALEK, 100 µM) degradation and concomitant Tyr-D-Ala-Gly (TAG) formation in the presence of the APN inhibitor bestatin (10-4 M) and the ACE inhibitor lisinopril (10-6 M) (5) . Fifty microliters of heart homogenate were used for TAG detection after incubation for 180 min at 37°C. Reaction specificity was characterized by NEP suppression with 10-6 M phosphoramidon (Sigma, Munich, Germany). Protein contents were measured using the Bradford method.

Measurement of bradykinin concentrations
Tissue samples were homogenized in 4 mol/L of guanidine thiocyanate, 1% trifluoroacetic acid. Kinins were adsorbed on phenyl-silica (Isolute SPE, International Sorbent Technology, U.K.) and eluted in 50% acetonitrile and 0.1% trifluoroacetic acid. The samples were lyophilized and reconstituted in radioimmunoassay (RIA) buffer. The recovery of [125I-Tyr8]-BK by this procedure was 95%. BK was quantitated by a specific RIA, as described previously (6) . The antiserum displayed a 36% cross-reactivity to T-kinin and had no affinity to smaller kinin fragments such as [1–8]-, [1–7]-, or [1–5]-BK.

Statistics
Results are expressed as mean values ± SE. Statistical comparisons were made by two-way ANOVA and a Tukey HSD test. Statistical significance was considered to be P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Quinapril- and irbesartan-treated rats had significantly better LV function than untreated rats 6 days and 3 wk after MI induction. Although all parameters of LV function are significantly improved by irbesartan after 3 wk, quinapril led to an LVP increase without significantly changing the LVEDP or dP/dt max (Table 1) .


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  		Table 1. Hemodynamic characterization of rats 6 days and 3 wk after induction of myocardial infarction (MI) and sham operationa

NEP activity was measured in homogenates of isolated heart compartments 6 days (Fig. 1 ) and 3 wk (Fig. 2 ) after MI induction. Untreated rats showed an NEP activity increase of ~ 100% specifically in the infarcted LV area compared with sham-operated rats. This rise was significantly reduced by quinapril treatment 6 days, but not 3 wk, after MI induction. In contrast, irbesartan treatment reduced this elevated NEP activity in the infarcted area to levels of sham-operated rats both 6 days and 3 wk after MI induction.



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  		Figure 1. NEP activity was measured in homogenates of isolated heart compartments 6 days after MI induction. Quantification of NEP activity was done by HPLC (each group: n=10). *P < 0.05 sham vs. infarct; §P < 0.05 infarct vs. infarct + quinapril; #P < 0.05 infarct vs. infarct + irbesartan; ##P < 0.01 infarct vs. infarct + irbesartan.



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  		Figure 2. NEP activity was measured in homogenates of isolated heart compartments 3 wk after MI induction. Quantification of NEP activity was done by HPLC (each group: n=10). *P < 0.05 sham vs. infarct; §P < 0.05 infarct vs. infarct + quinapril; #P < 0.05 infarct vs. infarct + irbesartan.

NEP activity in areas remote from the infarction (RV, S, noninfarcted LV) was found to be slightly, but not significantly, higher in untreated than in sham-operated rats at each time point. Whereas NEP activity was reduced by quinapril only during the first week after MI induction, it was lower in irbesartan than in control and sham animals after 6 days and 3 wk. This reduction was also significantly greater than in quinapril-treated animals.

Whereas cardiac BK was largely unaffected in areas remote from the infarction in all groups 3 wk after MI, it was significantly increased in the scar of control and quinapril rats. The elevation was even greater in irbesartan-treated animals (Fig. 3 ).



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  		Figure 3. BK concentrations in the right ventricle (RV), the noninfarcted left ventricle (LV) and the infracted LV 3 wk after myocardial infarction (each group: n=5). *P < 0.05 sham vs. infarct; #P < 0.05 infarct vs. infarct + irbesartan.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The regulation of NEP, an important cardiac peptidase, has not been investigated after MI. Furthermore, neither ACE inhibition nor AT1 receptor blockade was known to influence the regulation of NEP until recently.

Our results demonstrate increased NEP activity for infarcted but not for noninfarcted LV areas in untreated rats 6 days and 3 wk after MI induction. Although the role of cardiac NEP in the repair of infarcted myocardium is not clear, the natriuretic peptide and/or kinin system may be involved through this up-regulation, since NEP is responsible for the breakdown of ANP/BNP and BK. Thus, the increased NEP activity in the infarcted area would reduce not only ANP concentrations (7) but also BK levels. However, we found an increase of myocardial BK levels in infracted areas of untreated rats, indicating that kinin concentrations in the myocardium are partially determined by factors other than kinin-degrading enzymes. The net concentration of the peptide is therefore dependent on the balance of kinin formation and degradation. Since elevated KLK levels (8) and increased BK B1 and B2 receptor expression (4 , 9) have been detected after MI, the enhanced NEP activity described by our data is not sufficient to normalize BK concentrations elevated by stimulation of the kallikrein-kinin system. Our data show that the AT1 blockade can further increase the BK peptide concentration via an ACE inhibition-independent reduction of NEP activity. This additional increase is important, since the kinin axis has been shown to have cardioprotective effects. The role of NEP in this context is demonstrated by the finding that positive effects of NEP inhibition were reduced by cotreatment with the B2 receptor antagonist icatibant (10 , 11) .

At the same time, the cardioprotective effects of AT1 blockers have proved to be partly dependent on B2 receptor stimulation since they could be reduced by additional administration of icatibant (12 , 13) . This cannot be explained by direct ACE-dependent effects of BK catabolism. Tsutsumi et al. (14) proposed a pathway for AT2 receptor-mediated, BK-dependent cGMP synthesis after AT1 receptor antagonism. However, we found an AT1 receptor-dependent decrease in NEP activity correlating with elevated BK concentrations in the LV scar after AT1 blockade, indicating an additional pathway by which the AT1 blockade leads to an ACE-independent BK accumulation (Fig. 3) . This pathway is AT2 independent since both ACE inhibition and AT1 blockade, the former leading to a lack of stimulation and the latter to overstimulation of the AT2 receptor, result in a reduction of NEP activity that could be detected at day 6 after MI induction.

Although our data cannot definitively differentiate between AT2-dependent BK formation and BK accumulation mediated by NEP inhibition, a reduction in MI-induced AT2 expression by AT1 inhibition (15) indicates additive mechanisms of BK increase dominated by the NEP-controlled pathway. The importance of NEP in BK metabolism is substantiated by Blais et al. (16) , who showed that omapatrilat- but not enalapril-treated rats had higher cardiac BK concentrations than untreated infarcted animals. Furthermore, since NEP is the main peptidase controlling the metabolism of cardiac BK in vitro, catabolizing ~80% of it (17) , we suggest that ACE inhibition does not reduce BK degradation only by direct blockade of catabolic ACE activity but may reduce it indirectly by down-regulation of NEP via the AT1 receptor (Fig. 4 ).



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  		Figure 4. The principal scheme demonstrates the interaction between the renin-angiotensin system and NEP indicating an indirect influence of ACE inhibition on BK catabolism by AT1 blockade. The signaling pathway from the AT1 receptor to NEP could be direct or may involve the AT2 receptor.

Thus, our data add a further piece in the puzzling interaction between the RAS and the KKS (18) . Although no data are available to describe the pathway from the AT1 receptor to the NEP gene, our results indicate the existence of an AT1-NEP axis that could influence especially the cardioprotective properties of the kallikrein-kinin system after MI.


   ACKNOWLEDGMENTS
 
This study was supported by the Deutsche Forschungsgemeinschaft (DFG; TS-64/2–1 and WA-1441/1–1) and by grants from Goedecke/Parke Davis, Germany, and Bristol-Myers Squibb.

Received for publication January 7, 2002. Revision received April 18, 2002.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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  8. Hashimoto, K., Hamamoto, H., Honda, Y., Hirose, M., Furukawa, S., Kimura, E. (1978) Changes in components of kinin system and hemodynamic in acute myocardial infarct. Am. Heart J. 95,619-626[CrossRef][Medline]
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  14. Tsutsumi, Y., Matsubara, H., Masaki, H., Kurihara, H., Murasawa, S., Takai, S., Miyazaki, M., Nozawa, Y., Ozono, R., Nakagawa, K., et al (1999) Angiotensin II type 2 receptor overexpression activates the vascular kinin system and causes vasodilation. J. Clin. Invest. 104,925-935[Medline]
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