The renin-angiotensin-aldosterone system (RAAS) and natriuretic peptide system are both intimately involved in the pathophysiology of congestive heart failure (1). The RAAS promotes vasoconstriction and salt and water retention (thereby increasing cardiac filling pressures and cardiac workload), whereas the natriuretic peptide system exerts potentially beneficial natriuretic, diuretic, and vasodilating effects (1). Although both systems are activated in heart failure, and plasma levels of the natriuretic peptides increase in parallel with the severity of cardiac dysfunction (2), vasoconstriction and salt retention predominate in overt heart failure. This is thought to be attributable, at least in part, to the antagonizing effects of the activated RAAS overwhelming the response to the natriuretic peptides. The natriuretic peptide system, however, still protects the failing heart to some degree, because the administration of atrial natriuretic peptide (ANP) antibodies increased right atrial pressure, left ventricular end-diastolic pressure, and systemic vascular resistance in rats with myocardial infarction (3). Furthermore, despite already elevated levels, administration of ANP (4) and brain natriuretic peptide (BNP; 5) to patients with heart failure resulted in beneficial cardiovascular and renal effects.
The activity of these two systems is largely controlled by two related metallopeptidases, angiotensin-converting enzyme (ACE) and neutral endopeptidase 24.11 (NEP 24.11). Specific inhibitors of ACE, the principal enzyme responsible for angiotensin II (Ang II) formation, are now established therapy for heart failure (6). ACE inhibition can, however, result in a decline in renal function (7). The enzyme NEP 24.11 cleaves and inactivates the natriuretic peptides (8), and inhibition of NEP has been shown to increase endogenous ANP and BNP levels in association with beneficial hemodynamic and renal effects in heart failure (9-11). The opposing natures of the RAAS and natriuretic peptide systems suggest that coinhibition of these two enzymes may be a valuable therapeutic approach in human heart failure. Indeed, a number of recent studies examining combined ACE and NEP inhibition in models of heart failure have demonstrated additional hemodynamic and renal benefits over those achieved with either inhibitor alone (12-15).
As well as degrading the natriuretic peptides, NEP has also been shown to cleave both Ang I and Ang II in vitro (16), and therefore NEP inhibition might be expected to increase Ang II levels. Indeed, NEP inhibition has been shown to enhance infused Ang II levels in essential hypertension (17). It is therefore imperative that both the degree and duration of Ang II repression be closely examined during combined ACE and NEP inhibition. In addition, there is some evidence that the metabolism of ANP and BNP differs. Unlike ANP, the molecular composition of BNP differs considerably among species and is cleaved by NEP at several different sites and different rates in a species-specific manner (18). Furthermore, there is some controversy regarding the involvement of ACE in the metabolism of the natriuretic peptides. Whereas ANP is not considered to be a substrate for ACE (18,19), studies examining the effect of ACE on BNP degradation have shown both negative (18) and positive (19) results in vitro. In rats, Vanneste et al. (19) observed that the plasma half-life of labeled porcine BNP increased from 1.2 to 7 min after captopril administration and to 15.3 min in rats receiving both captopril and the NEP inhibitor, phosphoramidon.
Surprisingly, no studies have previously examined the response of either plasma Ang II or both natriuretic peptides during coinhibition with ACE and NEP. In view of the shared substrates and possible differences in the metabolism of ANP and BNP by both ACE and NEP, we have set out in this study to detail the combined hemodynamic and renal dynamics of dual NEP and ACE inhibition in sheep with pacing-induced heart failure, with full assessment of the two major relevant hormone systems-the RAAS and natriuretic peptide system.
Eight Coopworth ewes (body weight, 40-49 kg) were instrumented as previously described (20) via a left lateral thoracotomy. Under general anesthesia [induced by thiopentone (17 mg/kg) and maintained with halothane and nitrous oxide], two polyvinyl chloride catheters were inserted in the left atrium for blood sampling and pressure (LAP) determination; a Konigsberg (P 6.0) high-fidelity pressure-tip transducer was inserted in the aorta for measurement of mean arterial pressure (MAP); an electromagnetic flow probe was placed around the ascending aorta to measure cardiac output (CO); a 7F Swan-Ganz catheter was inserted in the pulmonary artery for infusions; and a 7F His-bundle electrode was stitched subepicardially to the wall of the left ventricle for subsequent left ventricular pacing by using an external pacemaker made in our department. All leads were externalized through individual incisions in the neck. An indwelling bladder catheter was inserted per urethra for subsequent urine collections. The animals were allowed to recover for ≥14 days before commencing the study protocol. During the experiments, the animals were held in metabolic cages, had free access to water, and ate a diet of chaff and sheep pellets (containing ∼40 mmol/day sodium; 200 mmol/day potassium) supplemented with a further 40 mmol of sodium administered orally each morning as NaCl tablets by using an applicator.
Heart failure was induced by 7 days of rapid left ventricular pacing at 225 beats/min (20). On days 8, 10, 12, and 14 of pacing, the sheep received, in random order, a vehicle control (0.9% saline), an NEP inhibitor (SCH32615, 5-mg/kg bolus + 1 mg/kg/h infusion for 3 h), an ACE inhibitor (captopril, 25-mg bolus + 2 mg/h infusion for 3 h), and the combination of both agents. All infusions were administered in a total volume of 60 ml via the pulmonary artery catheter, commencing at 1000 h.
Hemodynamic recordings (MAP, LAP, and CO) were performed at 15-min intervals from the hour before infusion (baseline) to 1 h after infusion, after which half-hourly measurements were made for a further hour. All measurements were made with the sheep standing quietly in the metabolic cage. The left atrial catheter was connected to a Statham P50 strain-gauge transducer (Mennen Medical Inc., Clarence, NY, U.S.A.) positioned at the level of the atria and linked to a hemodynamic monitor (M17294; Mennen-Greatbatch Ltd, Rehevot, Israel) for pressure determination relative to atmospheric pressure. The Konigsberg pressure transducer was connected to a preamplifier before display by the monitor. Hemodynamic measurements were determined by on-line computer-assisted analysis by using methods previously described (21).
Blood samples were drawn from the left atrium at 30 min and immediately before infusion (baseline), at 30, 60, 120, and 180 min during the 3-h infusion period, and at 30, 60, and 120 min after infusion. The blood was taken into tubes on ice, centrifuged at 4°C, and stored at −80°C before assay of ANP (22), BNP (23), cyclic 3′,5′-guanosine monophosphate (cyclic GMP; 22), plasma renin activity (PRA), aldosterone, and cortisol (20). Samples taken for the measurement of Ang II were placed into chilled tubes with a mixture of EDTA, o-phenanthroline, NaCl, neomycin sulfate, ethanol, and the renin inhibitor enalkarin (to prevent in vitro generation and degradation of Ang II). After centrifugation at 4°C, tubes were snap frozen and stored at −80°C. Plasma Ang II levels were determined by radioimmunoassay by using an antiserum raised locally in New Zealand White rabbits to Ang II conjugated to bovine serum albumin (BSA). Cross-reactivity of the antisera was as follows: Ang II, 100%; Ang III, 141%; Ang I, 0.11%; Ang II pentapeptide and Ang II hexapeptide, 77%; des Asp Ang I, 0.08%. Ang II standards (Peninsula Laboratories, Belmont, CA, U.S.A.) were dispensed to give 0-400 pg/tube. Plasma samples and standards were pipetted into double their volume of ethanol in duplicate followed by centrifugation and transfer of supernatants to another tube. Supernatants were dried under air for 2 h at 45°C and reconstituted with 400 μl buffer ready for assay. Fifty microliters of the antiserum (at a dilution of 1:7500) was added to all sample and standard tubes. [125I]-Ang II (50 μl), prepared by the chloramine T method and purified by HPLC, was added to give 5,000 cpm/tube. Tubes were incubated for 24 h at 4°C. Separation of bound from free label was performed by using dextran-coated charcoal (1 ml) at 4°C. Tubes were then centrifuged, the supernatant aspirated, and the charcoal pellet (free tracer) counted in a gamma counter. The detection limit of the assay was <8.5 pM. The intraassay CV was 8.2% measured at 33 pM and interassay CV 11.1% at 30 pM.
All samples from each animal were measured in the same assay to avoid interassay variability. Hematocrit concentrations were measured with every blood sample taken.
Blood samples for analysis of plasma sodium, potassium, and creatinine concentrations were taken immediately before infusion (baseline), at the end of infusion, and 2 h after infusion. Urine volume and samples for the measurement of sodium, potassium, and creatinine excretion were collected hourly from the hour before infusion (baseline) to 2 h after infusion.
The study protocol was approved by the Animal Ethics Committee of the Christchurch School of Medicine.
Results are expressed as mean ± standard error of the mean (SEM). Baseline hemodynamic and hormone values represent the mean of the four and two measurements, respectively, made within the hour immediately before infusion. Statistical analysis was performed by repeated-measures analysis of variance (ANOVA) by using the BMDP P2V package. Baseline data from the vehicle, SCH32615, captopril, and combined treatment days were compared. Treatment and time differences between all four study days were determined by using a two-way ANOVA. Significance was assumed when p < 0.05. Where significant differences were identified by ANOVA, Fisher's protected least-significant difference tests were used to identify time-points significantly different from control.
There were no significant intergroup differences in pretreatment baseline data for any variable. After 7 days of rapid left ventricular pacing, all sheep exhibited the hemodynamic and hormonal hallmarks of established heart failure (20). MAP and CO were reduced, whereas LAP and plasma ANP, BNP, cyclic guanosine monophosphate (cGMP), PRA, Ang II, and aldosterone levels were increased.
Plasma ANP levels were significantly increased during SCH32615 (p < 0.001; 86% increase at 3 h of treatment), reduced during captopril (p < 0.01; 24% decrease), and showed a modest increase in response to combined inhibition (p < 0.001; 25% increase). ANP levels dropped toward baseline concentrations after cessation of SCH32615, and to levels significantly below baseline after cessation of the combined treatment (Fig. 1). Plasma BNP responded similarly to ANP, increasing during SCH32615 (p < 0.001; by 76%), decreased during captopril (p < 0.01; by 23%), and increased slightly during combined inhibition (p < 0.001; by 26%). BNP levels also decreased after cessation of SCH32615 and the combined treatment (p < 0.001; Fig. 1). The changes observed in natriuretic peptide concentrations during the different treatments were associated with similar changes in the plasma levels of their putative second messenger, cGMP. Compared with control levels, plasma cGMP levels were increased significantly during SCH32615 (p < 0.001) to a lesser extent during combined inhibition (p < 0.001) and tended to decreased during captopril (0.1 > p > 0.05; Fig. 1). As with the natriuretic peptides, plasma cGMP decreased after termination of SCH32615 and the combined treatment.
PRA increased rapidly after initiation of captopril and combined inhibition (both p < 0.001) and was unaltered compared with control during SCH32615 (Fig. 2). Plasma ANG II levels were reduced similarly during captopril and combined treatment (both p < 0.05) and were still equally and significantly lower than control concentrations at 2 h after infusion. Ang II levels were not significantly different from control data during SCH32615 (Fig. 2). Plasma aldosterone (Fig. 2) and cortisol (Table 1) levels were not significantly affected by any treatment compared with vehicle control.
Compared with control data, MAP was significantly reduced by SCH32615 (p < 0.001) and further still by captopril and the combined treatment (both p < 0.001; Fig. 3). Left atrial pressure was reduced to a similar extent by both SCH32615 and captopril (both p < 0.001) and decreased further during combined inhibition (p < 0.001; Fig. 3). MAP and LAP were still significantly lower than control levels at 2 h after infusion of all active treatments. Cardiac output was significantly increased by all active treatments (all p < 0.001; Fig. 3; particularly in the first 2 h of treatment), with the response to captopril and combined inhibition more rapid than to SCH32615 alone. Cardiac output had returned to control levels at 2 h after infusion of all active treatments. Calculated total peripheral resistance (CTPR) was significantly reduced by SCH32615 (p < 0.001) and further by captopril and combined treatment (both p < 0.001; Fig. 3). Hematocrit was increased relative to control by SCH32615 and captopril (both p < 0.001) and significantly more so by the combined treatment (p < 0.001; Table 1).
Compared with control data, SCH32615 and combined inhibition significantly increased urine volume (sixfold; p < 0.01; and 5.6-fold; p < 0.05, respectively) and urine sodium (both >35-fold; both p < 0.05) and potassium excretion (p < 0.01 and p < 0.05, respectively), whereas captopril administration did not significantly alter these renal indices (Fig. 4). Urine creatinine excretion increased significantly during SCH32615 (p < 0.01), decreased during captopril (p < 0.05), and was unaltered during combined inhibition (Fig. 4). Plasma creatinine levels were increased during captopril (p < 0.05) and were not significantly affected by either SCH32615 or combined treatment (Table 1). Creatinine clearance increased significantly during SCH32615 (p < 0.5), tended to decrease during captopril (0.1 > p > 0.5), and was unaltered during combined inhibition (Table 1). Plasma sodium and potassium levels were not altered by any treatment (data not shown).
Our vehicle-controlled study directly compares for the first time the effect of combined ACE and NEP inhibition on plasma levels of Ang II and both natriuretic peptides in heart failure. Plasma ANP and BNP levels were similarly increased during SCH32615 and to a lesser extent during combined inhibition but decreased during captopril. Captopril and combined inhibition induced identical increases in plasma renin activity and reductions in angiotensin II, whereas levels were unchanged during SCH32615. Combined inhibition produced hemodynamic effects (a reduction in MAP and CTPR and increase in cardiac output) similar to those of ACE inhibition but with an additional reduction in preload (LAP) greater than that observed with either treatment alone. Coinhibition was associated with a marked diuresis and natriuresis similar to that observed during NEP inhibition with preservation of the glomerular filtration rate, despite a significantly greater decrease in blood pressure. These results are consistent with concurrent inhibition of the RAAS and enhancement of the natriuretic peptide system and indicate a more favorable profile of hemodynamic and renal responses than those achieved with either inhibitor alone.
In agreement with observations in humans (2), we find circulating plasma concentrations of ANP to be higher than concurrent BNP levels in sheep, and both natriuretic peptides to be significantly increased in HF. Further increases in plasma ANP levels in response to NEP inhibition have been widely reported in both experimental (9-11) and human heart failure (24) as a result of reduced degradation of the endogenous peptide. In addition, a limited number of studies have also observed increases in plasma BNP levels in response to NEP inhibition (9,25). In our study, although the absolute increase in plasma ANP in response to NEP inhibition was greater than that of BNP, the proportional increases of ANP and BNP were similar (1.9- and 1.8-fold, respectively). These findings are consistent with in vitro data (8), showing that the enzyme has a similar affinity for both ANP and porcine [identical to ovine (26)] BNP and supports the concept that NEP contributes significantly to the clearance of both peptides in sheep. In direct contrast to NEP inhibition, ACE inhibition reduced both plasma ANP and BNP concentrations in our study (by 24 and 23%, respectively). Similar observations have been reported during ACE inhibition in heart failure in humans (27) and are presumed to be a consequence of improved hemodynamic function, namely reduced intracardiac pressures (as evidenced by the decreases in LAP and MAP in our study), thus leading to reduced secretion (28). Although our results do not support a major role for ACE in the metabolism of ANP or BNP, they do indicate that, at least in sheep, both peptides respond similarly to hemodynamic unloading associated with ACE inhibition. The responses of plasma ANP and BNP to coinhibition of NEP and ACE were intermediate to those of each inhibitor separately and are consistent with the effects of both protection of the endogenous natriuretic peptide from enzymatic degradation and reduced secretion (as intracardiac pressure decreases). A similar pattern of plasma ANP responses was observed by Seymour et al. (14) in response to separate and combined inhibition of NEP and ACE in dogs with pacing-induced heart failure. We found no difference in the pattern of response of plasma ANP to that of BNP during either NEP, ACE, or combined inhibition in our study. However, these findings may not apply to humans and other species in which the affinity of the enzymes for the peptides may differ in a species-specific manner. Indeed, Lang et al. (27) observed a smaller reduction in plasma BNP levels (10% decrease) compared with that of ANP (32% decrease) during captopril administration in patients with heart failure. This difference, however, may be related to the comparatively longer plasma half-life of BNP relative to ANP in humans [because of the lower affinity of NEP and the natriuretic peptide clearance-receptor for human BNP (8)] after reduced secretion of both peptides. We found the changes in the natriuretic peptides levels to be associated with similar changes in plasma concentrations of their intracellular second messenger, cyclic GMP.
A significant difference between NEP and ACE inhibition concerns the response of PRA. Whereas captopril resulted in a marked increase in PRA, a well-established consequence of ACE inhibition, NEP inhibition tended to reduce PRA despite the decrease in arterial pressure. This may reflect increased flow of sodium through the macula densa or a direct effect of raised natriuretic peptide levels suppressing renin secretion or both (29). Coinhibition in our study produced a increase in PRA identical to that of ACE inhibition alone. Plasma angiotensin II levels were similarly reduced (in both degree and duration) by captopril and combined inhibition and unchanged by NEP inhibition alone. Our failure to see any increase in Ang II levels during NEP inhibition alone, or any difference in the response to captopril or combined inhibition, does not support a principal role for NEP in the metabolism of Ang II in the paced sheep. However, these findings may not apply to humans and other species where affinity of species-specific forms of the enzyme for Ang II may differ. Indeed, the response of Ang II must be closely examined when dual ACE and NEP inhibition is trialed in humans.
It has been suggested that ACE inhibition exerts much of its effects through reducing tissue-angiotensin levels. Pinto et al. (30) have reported the activation of the tissue renin-angiotensin system in the hearts of rats after myocardial infarction, and it is thought that cardiac angiotensin II may not only impair diastolic relaxation (31) but also may be a mediator of ventricular hypertrophy through its growth-promoting effects. On the other hand, both ANP and BNP have been reported to inhibit angiotensin II-stimulated proliferation of cardiac fibroblasts (32). It has been reported that long-term NEP inhibition in the spontaneously hypertensive rat reduced cardiac mass in the absence of hypotensive activity (33), whereas in a study in rats with myocardial infarction, the mixed NEP and ACE inhibitor alatriopril, reduced cardiac hypertrophy with greater efficacy than captopril (34). Furthermore, it has been shown that NEP inhibition influences coronary hemodynamics (flow and resistance) in a manner similar to ACE inhibition (35) and thus could also have profound effects on blood flow in the ischemic heart. These results suggest that not only may mixed NEP and ACE inhibitors have immediate beneficial effects on systemic hemodynamics and renal function but may also have additive long-term effects inhibiting hypertrophy and improving cardiac function.
The hemodynamic effects of separate NEP and ACE inhibition in our study were both characterized by reductions in preload (to a similar extent) and afterload (to a greater extent by captopril) and increase in cardiac output (the effect more rapid with captopril). Similar hemodynamic responses during ACE inhibition have been well documented in both experimental (36) and human heart failure (37), whereas previous studies reporting the effects of NEP inhibition in heart failure have consistently observed decreases in atrial pressure and, less consistently, decreases in blood pressure and increases in cardiac index (9,10). Combined inhibition in our study produced hemodynamic effects similar to that observed during ACE inhibition alone but with an additional and significantly greater decrease in preload. This enhanced reduction in LAP during combined treatment may be mediated by a greater decrease in circulating volume and venous return (as judged by the larger increase in hematocrit than was achieved with either treatment separately). Previous studies in experimental heart failure have also demonstrated the beneficial hemodynamic effects of coinhibition of NEP and ACE. In dogs with pacing-induced heart failure, combined NEP and ACE inhibition resulted in greater increases in cardiac output and stroke volume and reductions in blood pressure and systemic vascular resistance than was achieved with either treatment alone (14), whereas in cardiomyopathic hamsters, coinhibition produced significant reductions in preload and afterload that were not observed when the ACE or NEP inhibitors were administered separately (12). The rapidly growing interest in coinhibition of NEP and ACE has led to the development of agents that simultaneously inhibit both enzymes. In another study in cardiomyopathic hamsters with the dual inhibitor, BMS-182657, Trippodo et al. (13) observed significantly greater reductions in left ventricular end-diastolic pressure, arterial pressure, and peripheral resistance and increase in cardiac index than was attained by separate inhibitors. These results, in a variety of species and models, all demonstrate additional hemodynamic effects of combined NEP and ACE inhibition in heart failure.
NEP inhibition was associated with a marked natriuresis and diuresis in our study. Enhanced urine sodium excretion in heart failure after NEP inhibition has been reported previously (9,11) and is thought to be the result of both the increase in endogenous natriuretic peptide levels and, perhaps more important, of the protection of these peptides from degradation within the kidney [particularly within the tubular brush-border membranes where the enzyme is most concentrated (38)]. This view is supported by results from studies that observed increases in urinary ANP levels in association with a significant natriuresis after NEP inhibition (11), as well as combined NEP and ACE inhibition (39) in experimental heart failure. The increase in urine sodium excretion in this investigation may be mediated by glomerular and tubular mechanisms, as it was associated with an increase in glomerular filtration rate (indicated by the increase in endogenous creatinine clearance). In contrast, captopril administration was associated with a decline in creatinine clearance. These results are in agreement with clinical studies that have demonstrated that ACE inhibition in some patients results in a decline in renal function resulting in hypotension and reduced glomerular filtration at low perfusion pressures (7). The renal response to coinhibition of NEP and ACE was characterized by significant increases in urine volume and sodium excretion similar to that observed during NEP inhibition alone, and the maintenance of creatinine clearance (GFR), despite the marked decreases in MAP. Indeed it is remarkable that these decreases in blood pressure (and presumably renal perfusion pressure) were associated with such an impressive natriuresis during combined inhibition. Presumably this dramatic shift in the pressure-natriuresis curve relates to reduced intrarenal degradation of the natriuretic peptides in association with sustained inhibition of renal Ang II. It is interesting to note that the natriuresis during combined inhibition was equal to that of NEP inhibition alone, although plasma ANP and BNP levels were lower, suggesting the importance of the intrarenal natriuretic peptide/Ang II ratio. The mitigation of the decrease in GFR during combined inhibition compared with ACE inhibition alone in our study, is an important (potential) major advantage in the clinical setting. A possible explanation for this is that GFR depends on the maintenance of glomerular efferent tone, which is largely Ang II dependent in low-pressure states. The decrease in Ang II levels during ACE inhibition results in the reduction of efferent tone, reducing intraglomerular pressure and ultimately GFR. On the other hand, ANP (and therefore NEP inhibition) preserves efferent tone and GFR (40), offsetting the reduction in Ang II levels. Our results are in agreement with others who have previously reported that coinhibition of NEP and ACE maintains sodium excretion at lower renal-perfusion pressures (14,39), as well as increases the glomerular filtration rate (15) in experimental heart failure.
In summary, NEP and ACE inhibition, both separately and in combination, affect plasma concentrations of ANP and BNP in a proportionately similar manner in sheep with heart failure. Plasma Ang II levels are similarly reduced during ACE and combined ACE/NEP inhibition. Compared with ACE inhibition alone, cotreatment with an NEP inhibitor further improved filling pressures, induced a diuresis and natriuresis, and maintained GFR (despite a marked decrease in arterial pressure), presumably because of the protection of endogenous ANP and BNP from enzymatic degradation. Coinhibition of NEP 24.11 and ACE has therapeutic value in heart failure beyond ACE inhibition alone.
Acknowledgment: This study was supported by grants from the the National Heart Foundation of New Zealand and The Health Research Council of New Zealand. We are grateful to the staff of the Christchurch School of Medicine Animal Laboratory for care of the animals and the staff of the Endocrine, Steroid and Biochemistry Laboratories for hormone and biochemical assays.
1. Svanegaard J, Johansen JB, Thayssen P, Haghfelt T. Neurohormonal systems during progression of heart failure: a review. Cardiology
2. Mukoyama M, Nakao K, Hosoda K, et al. Brain natriuretic peptide as a novel cardiac hormone in humans: evidence for an exquisite dual natriuretic peptide system, atrial natriuretic peptide and brain natriuretic peptide. J Clin Invest
3. Drexler H, Hirth C, Stasch HP, Lu W, Neuser D, Just H. Vasodilatory action of atrial natriuretic factor in a rat model of chronic heart failure as determined by monoclonal ANF antibody. Circ Res
4. Herrmann HC, Palacios IF, Dec GW, Scheer JM, Fifer MA. Effects of atrial natriuretic factor on coronary hemodynamic and myocardial energetics in patients with heart failure. Am Heart J
5. Yoshimura M, Yasue H, Morita E, et al. Hemodynamic, renal, and hormonal responses to brain natriuretic peptide in patients with congestive heart failure. Circulation
6. CONSENSUS Trial Study Group. Effects of enalapril on mortality in severe congestive heart failure. N Engl J Med
7. Pierpont GL, Francis GS, Cohn JN. Effect of captopril on renal function in patients with congestive heart failure. N Engl J Med
8. Kenny AJ, Bourne A, Ingram J. Hydrolysis of human and pig brain natriuretic peptides, urodilatin, C-type natriuretic peptide and some C-receptor ligands by endopeptidase-24.11. Biochem J
9. Rademaker MT, Charles CJ, Espiner EA, Nicholls MG, Richards AM, Kosoglou T. Neutral endopeptidase inhibition: augmented atrial and brain natriuretic peptide, haemodynamic and natriuretic responses in ovine heart failure. Clin Sci
10. Fitzpatrick MA, Rademaker MT, Charles CJ, et al. Acute hemodynamic, hormonal and renal effects of neutral endopeptidase inhibition in ovine heart failure. J Cardiovasc Pharmacol
11. Seymour AA, Asaad MM, Lanoce VM, Fennell SA, Cheung HS, Rogers WL. Inhibition of neutral endopeptidase 3.24.11 in conscious dogs with pacing induced heart failure. Cardiovasc Res
12. Trippodo NC, Fox M, Natarajan V, Panchal BC, Dorso CR, Assad MM. Combined inhibition of neutral endopeptidase and angiotensin-converting enzyme in cardiomyopathic hamsters with compensated heart failure. J Pharmacol Exp Ther
13. Trippodo NC, Robl JA, Assad MM, et al. Cardiovascular effects of the novel dual inhibitor of neutral endopeptidase and angiotensin-converting enzyme BMS-182657 in experimental hypertension and heart failure. J Pharmacol Exp Ther
14. Seymour AA, Asaad MM, Lanoce VM, Langebacher KM, Fennell SA, Rogers WL. Systemic hemodynamics, renal function and hormonal levels during inhibition of neutral endopeptidase 188.8.131.52 and angiotensin-converting enzyme in conscious dogs with pacing-induced heart failure. J Pharmacol Exp Ther
15. Margulies KB, Perrella MA, McKinley LJ, Burnett JC. Angiotensin inhibition potentiates the renal response to neutral endopeptidase inhibition in dogs with congestive heart failure. J Clin Invest
16. Shimamori Y, Watanabe Y, Fujimoto Y. Specificity of a membrane bound neutral endopeptidase from rat kidney. Chem Pharmacol Bull
17. Richards AM, Wittert GA, Crozier IG, et al. Chronic inhibition of endopeptidase 24.11 in essential hypertension-evidence for enhanced atrial natriuretic peptide and angiotensin II. J Hypertens
18. Norman JA, Little D, Bolger M, di Donato G. Degradation of brain natriuretic peptide by neutral endopeptidase: species specific sites of proteolysis determined by mass spectrometry. Biochem Biophys Res Commun
19. Vanneste Y, Pauwels S, Lambotte L, Deschodt-Lanckman M. In vivo metabolism of brain natriuretic peptide in the rat involves endopeptidase 24.11 and angiotensin converting enzyme. Biochem Biophys Res Commun
20. Fitzpatrick MA, Nicholls MG, Espiner EA, et al. Neurohumoral changes during the onset and offset of ovine heart failure: role of ANP. Am J Physiol
21. Fitzpatrick MA, Rademaker MT, CFrampton CM, et al. Hemodynamic and hormonal effects of renin inhibition in ovine heart failure. Am J Physiol
22. Charles CJ, Espiner EA, Cameron VA, Richards AM. Hemodynamic, renal, and endocrine actions of ANF in sheep: effect of 24-h, low-dose infusions. Am J Physiol
23. Yandle TG, Aitken GD, Fisher SF, Rademaker MT, Espiner EA, Nicholls MG. Ovine brain natriuretic peptide: identity measurement and regulation [Abstract]. J Hypertens
24. Northridge DB, Jardine AG, Findlay IN, Archibald M, Dilly SG, Dargie HJ. Inhibition of the metabolism of atrial natriuretic factor causes diuresis and natriuresis
in chronic heart failure. Am J Hypertens
25. Lang CC, Motwani JG, Coutie WJR, Struthers AD. Clearance of brain natriuretic peptide in patients with chronic heart failure: indirect evidence for a neutral endopeptidase mechanism but against an atrial natriuretic peptide clearance receptor mechanism. Clin Sci
26. Aitken GD, George P, Espiner EA. Characterisation of ovine natriuretic peptide genomic DNA sequences [Abstract]. J Hypertens
27. Lang CC, Motwani JG, Rahman AR, Coutie WJ, Struthers AD. Effect of angiotensin-converting enzyme on plasma natriuretic peptide levels in patients with heart failure. Clin Sci
28. Lang CC, Choy AMJ, Henderson IS, Coutie WJR, Struthers AD. Effect of haemodialysis on plasma levels of brain natriuretic peptide in patients with chronic renal failure. Clin Sci
29. Obana K, Naruse M, Naruse K, et al. Synthetic rat atrial natriuretic factor inhibits in vitro and in vivo renin secretion in rats. Endocrinology
30. Pinto YM, De Smet BGJL, Van Gilst WH, et al. Selective and time related activation of the cardiac renin-angiotensin system after experimental heart failure: relation to ventricular function and morphology. Cardiovasc Res
31. Schunkert H, Dzau VJ, Tang SS, Hirsch AT, Apstein CS, Lorell BH. Increased rat cardiac angiotensin converting enzyme activity and mRNA expression in pressure overload left ventricular hypertrophy. J Clin Invest
32. Fujisaki H, Ito H, Hirata Y, et al. Natriuretic peptides inhibit angiotensin II-induced proliferation of rat cardiac fibroblasts by blocking endothelin-1 expression. J Clin Invest
33. Monopoli A, Ongini E, Cigola E, Olivetti G. The neutral endopeptidase inhibitor, SCH 34826, reduces left ventricular hypertrophy in spontaneously hypertensive rats. J Cardiovasc Pharmacol
34. Bralet J, Marie C, Mossiat C, Lecomte J, Gros C, Schwartz J. Effects of alatriopril, a mixed inhibitor of atriopeptidase and angiotensin I-converting enzyme, on cardiac hypertrophy and hormonal responses in rats with myocardial infarction: comparison with captopril. J Pharmacol Exp Ther
35. Maxwell AJ, Husseini WK, Piedimonte G, Hoffman JIE. Effects of inhibiting endopeptidase and kininase II on coronary and systemic hemodynamics in rats. Am J Physiol
36. Fitzpatrick MA, Rademaker MT, Charles CJ, et al. Comparison of the effect of renin inhibition and angiotensin-converting enzyme inhibition in ovine heart failure. J Cardiovasc Pharmacol
37. Manthey J, Osterziel J, Rohrig N, et al. Ramipril and captopril in patients with heart failure: effects on hemodynamics and vasoconstrictor systems. Am J Cardiol
38. Seymour AA, Abboa-Offei BE, Smith PL, Mathers PD, Asaad MM, Rogers WL. Potentiation of natriuretic peptides by neutral endopeptidase inhibitors. Clin Exp Pharmacol Physiol
39. Gonzalez W, Beslot F, Laboulandine I, Fournie-Zaluski M-C, Roques B-P, Michel J-P. Inhibition of both angiotensin-converting enzyme and neutral endopeptidase by S21402 (RB105) in rats with experimental myocardial infarction. J Pharmacol Exp Ther
40. Marin-Grez M, Fleming JT, Steinhausen M. Atrial natriuretic peptide causes pre-glomerular vasodilation and post-glomerular vasoconstriction in rat kidney. Nature