Introduction

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Inhibitors of angiotensin converting enzyme (EC 3.4.25.1, ACE) are clinically useful for the treatment of hypertension and cardiac failure, being able to attenuate ventricular remodeling and reduce mortality in heart failure patients (Brown and Vaughan, 1998). Inhibitors of neutral endopeptidase 24.11 (EC 3.4.24.11, NEP) induce diuresis and natriuresis in humans (Richards et al., 1990; Schmitt et al., 1994) and have beneficial effects in animal models of heart failure (Seymour et al., 1993; Rademaker et al., 1996) and in patients with congestive cardiac failure (Elsner et al., 1992). Thus, simultaneous inhibition of ACE and NEP offers the possibility of improved therapy for hypertension and cardiac failure (Favrat et al., 1995; Trippodo et al., 1995).

ACE and NEP are zinc-containing metalloendopeptidases involved in the metabolism of a variety of biological peptides (Erdos, 1990; Roques et al., 1993). Both ACE and NEP participate in the metabolism of angiotensin (Ang) and bradykinin (BK) peptides, and NEP also metabolizes atrial natriuretic peptide (Erdos, 1990; Roques et al., 1993). ACE converts the inactive Ang I to Ang II, and NEP metabolizes both Ang II and Ang I (Richards et al., 1992; Yamamoto et al., 1992) (Fig. 1). Both enzymes metabolize BK-(1-9) to BK-(1-7) (Erdos, 1990; Roques et al., 1993) and also cleave BK-(1-7) to release smaller fragments (Fig. 1). ACE inhibitors prevent the pressor response to Ang I, and both ACE and NEP inhibitors potentiate the depressor effects of BK-(1-9) (Ondetti et al., 1977; Yang et al., 1997). Moreover, NEP inhibition enhances the pressor response to Ang II and reduces the clearance of infused Ang II in humans (Richards et al., 1992). NEP inhibition also reduces conversion of Ang I to Ang-(1-7) and increases plasma levels of Ang I and Ang II in rats infused with Ang I (Yamamoto et al., 1992).

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Diagrammatic representation of participation by NEP and ACE in metabolism of BK-(1-9) and Ang I. Alternate pathway of conversion of Ang I to Ang II by serine protease activity is also shown.

Both ACE and NEP have a widespread tissue distribution, including the heart, vascular endothelium, and the brush border of proximal tubule cells of the kidney (Bruneval et al., 1986; Erdos, 1990; Roques et al., 1993; Graf et al., 1995). Given the colocalization of ACE and NEP in many tissues, one would predict interactions between the effects of ACE and NEP inhibitors on Ang and BK peptide levels during simultaneous ACE and NEP inhibition. Simultaneous administration of ACE and NEP inhibitors may have a synergistic effect on BK-(1-9) levels. Moreover, increased Ang I and Ang II levels in response to NEP inhibition may counteract the effects of simultaneous ACE inhibition on Ang II levels.

The purpose of this study was to characterize the interactions between the effects of ACE and NEP inhibition on Ang and BK peptides in rats with myocardial infarction. The NEP inhibitor ecadotril, N-(S)-[2-[(acetylthio)methyl]-1-oxo-3-phenylpropyl]-glycine benzylester, formerly called sinorphan, is the orally active prodrug of (S)-thiorphan. We investigated the dose-related effects of ecadotril on circulating levels of Ang-(1-7), Ang II, and Ang I; tissue levels of Ang II, Ang I, BK-(1-7), and BK-(1-9); and urine kinin levels in rats administered ecadotril alone and in rats simultaneously administered the ACE inhibitor perindopril. In addition to measurement of peptide levels, we calculated the Ang-(1-7)/Ang I ratio, which provides an index of the rate of conversion of Ang I to Ang-(1-7), the Ang II/Ang I ratio, which provides an index of the rate of conversion of Ang I to Ang II, and the BK-(1-7)/BK-(1-9) ratio, which provides an index of the rate of BK-(1-9) metabolism to BK-(1-7). The dose of perindopril used in this study (0.2 mg/kg/day) was submaximal with respect to its effects on Ang and BK peptides, blood pressure, and cardiac hypertrophy (Campbell et al., 1994; Duncan et al., 1996). We chose a submaximal dose of perindopril for these studies to enable the detection of possible additive effects of NEP and ACE inhibition.

	Materials and Methods

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Animals. Male Sprague-Dawley rats (104-230 g) were allowed free access to tap water and standard rat chow containing 0.25% sodium and 0.76% potassium (GR2; Clarke-King & Co., Gladesville, NSW, Australia). This study was performed in accordance with the guidelines of the Animal Experimentation Ethics Committee of St. Vincent's Hospital.

Measurement of Renin, Angiotensinogen, and ACE in Plasma. Trunk blood for measurement of renin, angiotensinogen, ACE, and NEP was collected into heparinized tubes on ice and then centrifuged, and the plasma was rapidly frozen on dry ice and stored at 80°C. The plasma concentrations of active renin, angiotensinogen, and ACE were measured as described previously (Campbell et al., 1991).

Extraction and RIA of Ang and BK Peptides. Plasma levels of Ang-(1-7), Ang II, and Ang I were measured as described previously (Campbell et al., 1995). Briefly, trunk blood (2-3 ml) was rapidly collected into tubes containing 0.5 ml of inhibitor solution (1 mM renin inhibitor acetyl-His-Pro-Phe-Val-Sta-Leu-Phe-NH₂, 146 µM pepstatin, 50 mM 1,10-phenanthroline, 125 mM ethylenediaminetetra-acetate, 2 g/liter neomycin sulfate, 2% dimethyl sulfoxide, and 2% ethanol in water) at 4°C. The blood was centrifuged, and the plasma (1-2 ml) was immediately extracted with Sep-Pak C₁₈ cartridges (Waters Chromatography Division, Milford, MA). Tissues homogenized in GTC/TFA were processed as described previously and extracted with Sep-Pak C₁₈ cartridges (Campbell et al., 1995). For the measurement of BK peptides in urine, 1 ml of freshly thawed urine was added to 10 ml GTC/TFA and extracted with Sep-Pak C₁₈ cartridges (Anastasopoulos et al., 1998). Peptides were acetylated and treated with piperidine before HPLC and assay of HPLC fractions by N-terminal directed radioimmunoassay (RIA) (Campbell et al., 1995). Data were corrected for recovery as reported elsewhere (Campbell et al., 1995; Anastasopoulos et al., 1998).

Measurement of Sodium, Potassium, and cGMP in Urine. Urine sodium and potassium were measured by autoanalyzer by the Department of Chemical Pathology, St. Vincent's Hospital. cGMP was measured by RIA using reagents from Amersham International (Buckinghamshire, UK).

In Vitro Autoradiography. Cryostat sections of kidney (20 µm) were cut and mounted on gelatin-coated slides. In vitro autoradiography of binding to NEP was performed using ¹²⁵I-RB104 (2-[(3-iodo-4-hydroxy)phenylmethyl]-4-N-[3-(hydroxyamino-3-oxo-1-phenylmethyl)propyl]amino-4-oxobutanoic acid) (Fournié-Zaluski et al., 1992) as described previously (Campbell et al., 1998). RB104 was a generous gift from Dr. B. Roques, Université René Descartes (Paris, France).

Statistical Analysis. All data are expressed as mean ± S.E.M. Both experiments 1 and 2 included vehicle-treated infarct rats. Comparisons of vehicle-treated sham-operated and vehicle-treated infarct rats from experiment 2 were by t test, except for blood pressure, where analysis was by repeated measures ANOVA. Comparisons of infarct rats administered drugs with vehicle-treated infarct rats were by one-way ANOVA for experiments 1 and 2 separately, using Dunnett's test for multiple comparisons with the vehicle-treated control. In addition, the effects of ecadotril in perindopril-treated rats in experiment 2 were analyzed similarly using Dunnett's test for multiple comparisons with the perindopril-treated control. When more than half of the samples comprising a mean had values below the minimum detectable, the sample mean is shown as less than the minimum detectable. Where values were below the minimum detectable, they were set at half the minimum detectable for statistical calculations. Logarithmic transformation of the data was performed when required to obtain similar variances between groups. All tests were two-tailed. Differences were considered significant at P < .05. Statistical analyses were performed using SuperANOVA (Abacus Concepts, Inc., Berkely, CA).

	Results

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As described in Materials and Methods, these data were obtained from two separate experiments. Absolute values are presented for vehicle-treated sham-operated and vehicle-treated infarct rats from experiment 2 (Tables 1 and 2). The effects of drug administration in infarct rats are presented as absolute values for heart weight/body weight ratio, plasma Ang-(1-7) levels, plasma Ang-(1-7)/Ang I ratio, aortic Ang I levels, and aortic Ang II/Ang I ratio; otherwise, data are presented as percentages of the mean of the relevant vehicle-treated infarct rats to correct for differences between mean values for vehicle-treated infarct rats in experiments 1 and 2 (Figs. 2-8; Tables 3 and 4).

View larger version (27K): [in this window] [in a new window]   Fig. 2.   Inhibition of NEP in vivo in rats with myocardial infarction. Bar graphs show effects of ecadotril administration alone (open columns) and together with 0.2 mg/kg/day perindopril (closed columns) on 125I-RB104 binding to kidney sections in vitro. Each value represents mean ± S.E.M. **P < .01 compared with vehicle-treated control, P < .01 comparison of rats administered both ecadotril and perindopril with perindopril-treated control, n = 6 to 10 per group.

Comparison of Sham-Operated and Vehicle-Treated Infarct Rats. Vehicle-treated infarct rats had lower blood pressures than sham-operated rats from day 2 to day 27, as assessed by repeated measures ANOVA (Table 1). Vehicle-treated infarct rats also had lower body weight and higher heart weight/body weight ratio than sham-operated rats (Table 1). Vehicle-treated infarct rats had higher urine cGMP excretion, consistent with increased natriuretic peptide levels due to cardiac failure, although infarct and sham-operated rats had similar lung weight/body weight ratios. Urine excretion of BK-(1-7) and BK-(1-8) were higher in vehicle-treated infarct than sham-operated rats. Infarct rats also had lower plasma Ang I levels, associated with a higher plasma Ang II/Ang I ratio, although plasma Ang II, renin, angiotensinogen, and ACE levels were no different from those of sham-operated rats (Table 1). Infarct rats had increased Ang II levels and Ang II/Ang I ratio in heart and reduced BK-(1-7) levels and BK-(1-7)/BK-(1-9) ratio in kidney and lung (Table 2).

Inhibition of NEP ¹²⁵I-RB104 binding to kidney sections was similar for sham-operated and vehicle-treated infarct rats (data not shown). Ecadotril produced dose-related occupancy of renal NEP, as determined by binding of ¹²⁵I-RB104 to kidney sections from rats treated with ecadotril alone and from perindopril-treated rats (Fig. 2). Although perindopril alone reduced ¹²⁵I-RB104 binding by ~45%, this decrease did not achieve statistical significance, and the failure of perindopril alone to modify urine cGMP excretion and the BK-(1-7)/BK-(1-9) ratio, presented below, indicates that perindopril did not produce NEP inhibition. Another indicator of NEP inhibition was the plasma Ang-(1-7)/Ang I ratio (Table 4). Ang-(1-7) levels were below the limit of detection (<3 fmol/ml) for vehicle-treated rats and rats treated with ecadotril alone and were ~ 40 fmol/ml in perindopril-treated rats (Table 4). Ecadotril decreased plasma Ang-(1-7) levels in perindopril-treated rats, and the Ang-(1-7)/Ang I ratio was decreased by 66 and 76% by 10 and 100 mg/kg/day ecadotril, respectively (Table 4), consistent with inhibition of NEP-mediated conversion of circulating Ang I to Ang-(1-7).

Blood Pressure, Body Weight, and Heart Weight/Body Weight Ratio. Neither drug nor their combination affected blood pressure or body weight (data not shown). Perindopril alone reduced heart weight/body weight ratio by 5%, and 100 mg/kg/day ecadotril alone reduced heart weight/body weight ratio by 8%, neither effect being statistically significant. However, the combination of perindopril with 10 and 100 mg/kg/day ecadotril reduced heart weight/body weight ratio by 10% (Fig. 3), although the reduction in heart weight/body weight ratio by combined NEP/ACE inhibition was not statistically significantly different from that seen with perindopril alone.

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Bar graphs show effects of ecadotril administration alone (open columns) and together with 0.2 mg/kg/day perindopril (closed columns) on heart weight/body weight ratio in rats with myocardial infarction. Each value represents mean ± S.E.M. *P < .05 compared with vehicle-treated control, n = 10 per group.

Urine Volume, Electrolytes, cGMP, and BK Peptides. Ecadotril increased urine cGMP and BK-(1-9) excretion and reduced BK-(1-7) excretion in rats administered ecadotril alone and in perindopril-treated rats, although the effect on BK-(1-7) excretion was statistically significant only in perindopril-treated rats (Table 3). Perindopril potentiated the effect of ecadotril on both urine cGMP and BK-(1-9) excretion; whereas 100 mg/kg/day ecadotril was required to increase cGMP and BK-(1-9) excretion in rats administered ecadotril alone, 10 mg/kg/day ecadotril increased cGMP excretion, and 1 mg/kg/day increased BK-(1-9) excretion in perindopril-treated rats (Table 3). Ecadotril produced a similar reduction of the urine BK-(1-7)/BK-(1-9) ratio in rats administered ecadotril alone and in perindopril-treated rats (Table 3). Ecadotril did not influence urine volume or sodium or potassium excretion, apart from a small decrease in urine volume in perindopril-treated rats administered 0.1 mg/kg/day ecadotril (Table 3). Perindopril alone did not affect urine volume, sodium, potassium, BK-(1-7), BK-(1-9), or cGMP excretion. However, perindopril alone reduced urine BK-(1-8) excretion, and BK-(1-8) excretion was normalized in perindopril-treated rats administered ecadotril (Table 3). Ecadotril alone did not modify urine BK-(1-8) excretion.

Plasma Renin, Angiotensinogen, ACE, Ang II, and Ang I. Ecadotril alone was without effect on plasma levels of renin, angiotensinogen, or ACE, except for a 4-fold increase in plasma renin at 100 mg/kg/day ecadotril (data not shown). Perindopril increased plasma renin by 60-100-fold, reduced plasma angiotensinogen by 50%, and reduced plasma ACE activity by 70% (data not shown). Ecadotril did not modify plasma renin, angiotensinogen, or ACE levels in perindopril-treated rats (data not shown).

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Bar graphs show effects of ecadotril administration alone (open columns) and together with 0.2 mg/kg/day perindopril (closed columns) on plasma levels of Ang II and Ang I, and plasma Ang II/Ang I ratio in rats with myocardial infarction. Each value represents mean ± S.E.M. *P < .05 and **P < .01 compared with vehicle-treated control, P < .01 comparison of rats administered both ecadotril and perindopril with perindopril-treated control, n = 9 to 10 per group.

Tissue Angiotensin Peptides. Perindopril alone reduced Ang II levels in kidney and increased Ang I levels in kidney, heart, aorta, and lung, associated with a reduction in Ang II/Ang I ratio in these tissues (Figs. 5 and 6; Table 4). Ecadotril alone did not modify Ang II or Ang I levels in any tissue (Figs. 5 and 6; Table 4). However, ecadotril increased Ang II levels in kidney, aorta, and lung of perindopril-treated rats (Figs. 5 and 6).

View larger version (18K): [in this window] [in a new window]   Fig. 5.   Bar graphs show effects of ecadotril administration alone (open columns) and together with 0.2 mg/kg/day perindopril (closed columns) on kidney levels of Ang II and Ang I, and kidney Ang II/Ang I ratio in rats with myocardial infarction. Each value represents mean ± S.E.M. **P < .01 compared with vehicle-treated control, P < .01 comparison of rats administered both ecadotril and perindopril with perindopril-treated control, n = 9 to 10 per group.

View larger version (16K): [in this window] [in a new window]   Fig. 6.   Bar graphs show effects of ecadotril administration alone (open columns) and together with 0.2 mg/kg/day perindopril (closed columns) on Ang II levels in heart, aorta, and lung in rats with myocardial infarction. Each value represents mean ± S.E.M. *P < .05 and **P < .01 compared with vehicle-treated control, P < .01 comparison of rats administered both ecadotril and perindopril with perindopril-treated control, n = 10 per group.

Tissue BK Peptides. Perindopril alone increased renal BK-(1-7) and BK-(1-9) levels, without effect on the renal BK-(1-7)/BK-(1-9) ratio (Fig. 7). Ecadotril alone had no effect on renal BK-(1-7) and BK-(1-9) levels, and ecadotril did not modify BK peptide levels in kidney of perindopril-treated rats (Fig. 7). Whereas neither ecadotril nor perindopril alone modified BK peptides in heart, the combination of ecadotril and perindopril increased BK-(1-9) levels and reduced the BK-(1-7)/BK-(1-9) ratio in heart (Fig. 8). Neither perindopril nor ecadotril alone, nor their combination, modified BK-(1-7) or BK-(1-9) levels or the BK-(1-7)/BK-(1-9) ratio in lung or aorta, except for an increase in lung BK-(1-7) levels (248% of control) at 100 mg/kg/day ecadotril and an increase in aorta BK-(1-7)/BK-(1-9) ratio (258% of control) at 10 mg/kg/day ecadotril (data not shown).

View larger version (19K): [in this window] [in a new window]   Fig. 7.   Bar graphs show effects of ecadotril administration alone (open columns) and together with 0.2 mg/kg/day perindopril (closed columns) on kidney levels of BK-(1-7) and BK-(1-9), and kidney BK-(1-7)/BK-(1-9) ratio in rats with myocardial infarction. Each value represents mean ± S.E.M. *P < .05 compared with vehicle-treated control, n = 9 to 10 per group.

View larger version (19K): [in this window] [in a new window]   Fig. 8.   Bar graphs show effects of ecadotril administration alone (open columns) and together with 0.2 mg/kg/day perindopril (closed columns) on heart levels of BK-(1-7) and BK-(1-9), and heart BK-(1-7)/BK-(1-9) ratio in rats with myocardial infarction. Each value represents mean ± S.E.M. *P < .05 and **P < .01 compared with vehicle-treated control, P < .05, comparison of rats administered both ecadotril and perindopril with perindopril-treated control, n = 9 to 10 per group.

	Discussion

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This study demonstrated important interactions between NEP and ACE inhibition in an established model of cardiac failure, where ECG criteria were used to select rats with infarcts of moderate size. The increased heart weight/body weight ratio and urine cGMP excretion, together with similar lung weight/body weight ratio in vehicle-treated infarct rats, compared with sham-operated rats, were consistent with a state of compensated heart failure in vehicle-treated infarct rats. The effects of 0.2 mg/kg/day perindopril alone were similar to those we reported previously for this dose of perindopril in rats with myocardial infarction (Duncan et al., 1996). Kidney was the only tissue to show a reduction in Ang II levels in response to perindopril; however, perindopril increased Ang I levels and reduced Ang II/Ang I ratio in plasma and tissues. We showed previously that renal Ang II levels are most sensitive to ACE inhibition (Campbell et al., 1994). Perindopril also increased BK-(1-9) levels in kidney and reduced urine BK-(1-8) excretion. The main effects of ecadotril were to increase urine cGMP and BK-(1-9) excretion and to reduce the BK-(1-7)/BK-(1-9) ratio in urine. In addition to summation of the effects of separate NEP and ACE inhibition, combined NEP/ACE inhibition achieved many effects not seen with separate NEP and ACE inhibition. These included reduction of heart weight/body weight ratio, increase in cardiac BK-(1-9) levels, and reduction of cardiac BK-(1-7)/BK-(1-9) ratio. Perindopril potentiated the effects of ecadotril on urine cGMP and BK-(1-9) excretion. Moreover, ecadotril reduced plasma Ang-(1-7) levels and increased Ang II levels in plasma, kidney, aorta, and lung of perindopril-treated rats.

In a previous study of rats with myocardial infarction, we showed that 2 mg/kg/day perindopril reduced Ang II levels in kidney, heart, and lung and also increased BK-(1-9) levels in blood, although not in tissues (Duncan et al., 1996). Whereas 0.2 mg/kg/day perindopril failed to normalize heart weight/body weight ratio in this and our previous study (Duncan et al., 1996), we showed that 2 mg/kg/day perindopril normalized heart weight/body weight ratio in rats with myocardial infarction (Duncan et al., 1996). Thus, the effects of combined NEP/ACE inhibition on heart weight/body weight ratio in the present study were similar to those of a 10-fold higher dose of ACE inhibitor alone. It is likely that different interactions between ecadotril and perindopril would have occurred for alternative doses of perindopril. We chose a submaximal dose of perindopril for these studies to enable the detection of possible additive effects of NEP and ACE inhibition.

Ecadotril is the orally active prodrug of (S)-thiorphan. (S)-Thiorphan inhibits both NEP (K_i, 4 nM) and ACE (K_i, 140 nM) (Roques et al., 1993). We found that the highest dose of ecadotril (100 mg/kg/day) inhibited ACE, as indicated by the reduction of plasma Ang II/Ang I ratio, associated with a rise in plasma levels of renin and Ang I. The normal plasma ACE activity in rats administered 100 mg/kg/day ecadotril was probably due to loss of inhibitory activity due to oxidation of the sulfhydryl-containing (S)-thiorphan during storage of plasma before assay, as has been described for the sulfhydryl-containing ACE inhibitor captopril (Boomsma et al., 1981). The failure of tissue Ang peptide levels to show evidence of ACE inhibition by ecadotril alone may have been due to lower drug levels in tissue than in plasma.

Many studies show that NEP inhibition potentiates both the plasma levels of administered natriuretic peptide and the effects of natriuretic peptide administration (Smits et al., 1990; Rademaker et al., 1996). Whereas the effects of NEP inhibition on endogenous plasma atrial natriuretic peptide levels depend on the experimental model (Smits et al., 1990; Stasch et al., 1996), NEP inhibition produces consistent increases in urine cGMP excretion (Dussaule et al., 1991; Favrat et al., 1995; Stasch et al., 1996), as shown in the present study. The potentiation by perindopril of the urine cGMP response to ecadotril suggests that perindopril may potentiate the enhancement by ecadotril of the renal effects of natriuretic peptides. It is also possible that the potentiation by perindopril of the urine cGMP response to ecadotril was due to the potentiation by perindopril of urine BK-(1-9) excretion, given that kinins also increase cGMP levels (Bhoola et al., 1992). Contrary to previous reports (Richards et al., 1990; Dussaule et al., 1991; Pham et al., 1993; Schmitt et al., 1994; Stasch et al., 1996), we failed to observe a diuretic or natriuretic response to ecadotril. This may have been due to our experimental design, whereby we collected urine for 6 h after a water load. Moreover, urine volume and sodium excretion may become tied to water and sodium intake after 4 weeks of NEP inhibition. Our finding that ecadotril markedly suppressed urine BK-(1-7)/BK-(1-9) ratio supports previous studies demonstrating a major role for NEP in metabolism of kinins in urine (Ura et al., 1987; Pham et al., 1993). We are unable to explain the reduction of urine BK-(1-8) excretion by perindopril alone or the increase in urine BK-(1-8) excretion by perindopril-treated rats administered ecadotril.

Despite marked effects on the levels of BK peptides in urine, ecadotril had little effect on BK peptide levels in kidney. By contrast, perindopril alone increased renal levels of BK-(1-7) and BK-(1-9). These data indicate that the relative contribution of ACE and NEP to kinin metabolism in renal tissue was different from that in urine. Whereas NEP was the major pathway of BK-(1-9) metabolism in urine, ACE was the major pathway of BK-(1-9) metabolism in renal tissue. Moreover, the failure of combined NEP/ACE inhibition to completely suppress the BK-(1-7)/BK-(1-9) ratio in kidney and other tissues indicates that enzymes other than NEP and ACE play an important role in BK-(1-9) metabolism in tissue.

Apart from urine, the most consistent effects of combined NEP/ACE inhibition on kinin peptide levels were in heart. Although 0.2 mg/kg/day perindopril alone failed to influence cardiac BK-(1-9) levels or the cardiac BK-(1-7)/BK-(1-9) ratio in the present study, we showed previously that higher doses of perindopril increased cardiac BK-(1-9) levels in normal rats and reduced cardiac BK-(1-7)/BK-(1-9) ratio in both normal and infarct rats (Campbell et al., 1994; Duncan et al., 1996). Our previous studies and the present data emphasize that both NEP and ACE have important roles in BK-(1-9) metabolism in heart. NEP immunostaining and activity have been found on the surface of cultured neonatal rat myocytes and endothelial cells of the human coronary vasculature (Piedimonte et al., 1994; Graf et al., 1995). BK-(1-9) has cardioprotective actions, and many studies demonstrate a role for kinins in mediating the cardiac effects of ACE inhibitors (Linz et al., 1995; Liu et al., 1997), including the prevention of cardiac hypertrophy by ACE inhibitors (Linz and Schölkens, 1992; McDonald et al., 1995). Kinins may also mediate the cardiac effects of NEP inhibition. BK-(1-9) receptor antagonism prevented the protective effects of NEP inhibition on ischemia reperfusion injury in the rat heart (Schriefer et al., 1996; Yang et al., 1997) and on isoproterenol-induced myocardial hypoperfusion (Piedimonte et al., 1994). Thus, the increased cardiac BK-(1-9) levels in rats administered combined NEP/ACE inhibition in the present study may have contributed to the reduction in heart weight/body weight ratio in these rats.

Several of the effects of NEP inhibition observed in this study may compromise the therapeutic effects of simultaneous ACE inhibition. ACE inhibition increased plasma Ang-(1-7) levels, whereas simultaneous NEP inhibition reduced these levels. These data confirm the role of NEP in conversion of Ang I to Ang-(1-7) (Yamamoto et al., 1992). Recent studies suggest that increased Ang-(1-7) levels may participate in the therapeutic effects of ACE inhibition. Ang-(1-7) potentiates the vasodepressor effects of BK-(1-9) (Paula et al., 1995; Li et al., 1997), and Ang-(1-7) antagonism causes partial reversal of the hypotensive effects of combined ACE inhibition/type 1 Ang II receptor antagonism in spontaneously hypertensive rats (Iyer et al., 1998). Thus, the reduction in ACE inhibitor-induced increase in Ang-(1-7) levels by simultaneous NEP inhibition may compromise part of the hemodynamic effects of ACE inhibition.

Additional evidence that NEP inhibition may compromise the therapeutic effects of simultaneous ACE inhibition was the ecadotril-induced increase in Ang II levels in plasma and tissues of perindopril-treated infarct rats. Perindopril reduced Ang II levels in kidney, but simultaneous ecadotril administration increased renal Ang II levels to those of vehicle-treated infarct rats. Moreover, ecadotril administration to perindopril-treated rats increased plasma, aorta, and lung Ang II levels above those of vehicle-treated infarct rats. We similarly showed that combined NEP/ACE inhibition increased Ang II levels in plasma, aorta, and lung of normal rats (Campbell et al., 1998). NEP inhibitor-induced increase in Ang II levels in plasma and tissues may compromise the beneficial therapeutic effects of ACE inhibition. The cause of the increased Ang II levels in plasma and tissues of perindopril-treated rats administered 100 mg/kg/day ecadotril is uncertain, given that ecadotril alone did not increase Ang II levels at this dose. NEP may play an important role in metabolism of circulating Ang II and Ang I (Richards et al., 1992; Yamamoto et al., 1992). During ACE inhibition, simultaneous NEP inhibition may lead to diversion of the elevated Ang I levels to conversion by incompletely inhibited ACE or to alternative serine protease-mediated pathways of conversion to Ang II (Fig. 1) (Campbell, 1993; Campbell et al., 1994) and, together with the inhibition of Ang II degradation by NEP, thus account for the increased Ang II levels in plasma and tissues observed during combined NEP/ACE inhibition in the present study.

Our study demonstrates that combined NEP/ACE inhibition produces important effects beyond the summation of the effects of separate NEP and ACE inhibition. Perindopril potentiated the ecadotril-induced increase in urine cGMP and BK-(1-9) excretion. Moreover, increased BK-(1-9) levels in heart may have contributed to the reduction of cardiac remodeling by combined NEP/ACE inhibition. However, reduction in plasma Ang-(1-7) levels and increase in Ang II levels in plasma and tissues may compromise the therapeutic effects of combined NEP/ACE inhibition.