Hemodynamic and hormonal effects of neutral endopeptidase inhibitor SCH 39370 in sheep.

Whole body clearance of atrial natriuretic factor is due to both receptor uptake and enzymatic degradation initiated by neutral endopeptidase 24.11. The effects of neutral endopeptidase inhibition have been studied in seven sodium-replete sheep using SCH 39370, a specific and potent inhibitor of neutral endopeptidase, in the presence or absence of exogenous hormone [rat ANF-(101-126), 2.4 pmol/kg/min for 2 hours]. SCH 39370 alone (2.5 mg/kg bolus) increased plasma atrial natriuretic factor and plasma cyclic GMP levels, lowered arterial pressure for periods beyond changes in plasma atrial natriuretic factor or cyclic GMP, and suppressed both plasma aldosterone and cortisol levels when compared with vehicle injections. The effects of SCH 39370 were similar to or exceeded those of atrial natriuretic factor infusions, which induced significantly greater increases in plasma atrial natriuretic factor (p = 0.01). Neither agent alone was natriuretic. When SCH 39370 and atrial natriuretic factor were given together, plasma cyclic GMP but not atrial natriuretic factor levels were increased (p = 0.013) compared with atrial natriuretic factor infusion alone, and the half-life was prolonged (p = 0.002) in the presence of SCH 39370. The hypotensive response was greater than that induced by atrial natriuretic factor alone (p = 0.03) but not different from SCH 39370 alone. Inhibitory effects of SCH 39370 on aldosterone levels were similar in the presence of absence of exogenous atrial natriuretic factor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of neutral endopeptidase amplifies the effects of endogenous atrial natriuretic peptide on blood pressure and fluid partition.

Neutral endopeptidase (EC 3.4.24.11) is a wide-spread enzyme that degrades atrial natriuretic peptide (ANP). We studied the effects of a potent neutral endopeptidase inhibitor, SQ 28,603, given intravenously (30 mg/kg over 45 min) to anesthetized, bilaterally nephrectomized Sprague-Dawley rats. Infusion of vehicle alone was accompanied by a modest increase, 3.2 +/- 2.2% (mean +/- SE), in mean arterial blood pressure (MAP) and a slight rise in hematocrit (Hct) of 0.9 +/- 0.7%. After administration of SQ 28,603, MAP fell 3.2 +/- 0.5%, and Hct rose 4.9 +/- 0.5%, both significantly different from the changes with vehicle alone; the lesser increase in plasma protein concentration (2.5 +/- 0.4%) suggested an increase in vascular permeability to both plasma protein and fluid similar to that caused by ANP. When SQ 28,603 was given to rats pretreated with rabbit antirat ANP antiserum, blood pressure rose by 3.8 +/- 0.5%, and Hct increased by 1.0 +/- 0.4%, values very similar to those observed with vehicle alone. Inhibition of neutral endopeptidase therefore amplifies the actions of endogenous ANP on blood pressure and fluid partition.     

Delayed metabolism of human brain natriuretic peptide reflects resistance to neutral endopeptidase

Metabolism of natriuretic peptides is regulated by two degradative pathways: uptake by the clearance receptor (natriuretic peptide receptor C--NPR-C) and hydrolysis by neutral endopeptidase (NEP). Affinity studies favour a dominant role of NPR-C in hormone degradation in several species but do not account for the efficacy of NEP inhibitors in vivo, nor the uniquely prolonged half life (t((1/2))) of human brain natriuretic peptide (hBNP). Postulating that (1) delayed metabolism of hBNP reflects resistance to NEP and (2) interactions between NPR-C and NEP increase enzyme activity, we have used purified ovine and human NEP, plus ovine lung plasma membranes to study the relative importance of receptor and enzyme pathways. We have also related the findings to hormone metabolism in vivo. Binding affinities of atrial natriuretic peptide (ANP), hBNP and ovine BNP (oBNP) to oNPR-C were similar (K(d)=8-16 pM). In contrast, unlike ANP and oBNP, hBNP was not significantly degraded by purified oNEP or plasma membranes. Despite similar (and high) affinity of oNPR-C for oBNP and hBNP, the t((1/2)) of hBNP (12.7 min) was more than fourfold that of oBNP (2.6 min). Although we found no evidence for receptor-enzyme interaction, our results show that the delayed metabolism of hBNP reflects resistance to NEP. These findings have important implications for future treatment strategies in human disease.     

Natriuretic peptides in the pathophysiology of congestive heart failure

A hallmark of congestive heart failure (CHF) is the activation of the cardiac endocrine system, in particular atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP). The natriuretic peptides are a group of structurally similar but genetically distinct peptides that have diverse actions in cardiovascular, renal, and endocrine homeostasis. ANP and BNP are of myocardial cell origin and C-type natriuretic peptide (CNP) is of endothelial origin. ANP and BNP bind to the natriuretic peptide-A receptor (NPR-A), which, via 3',5'-cyclic guanosine monophosphate (cGMP), mediates natriuresis, vasodilatation, renin inhibition, antimitogenesis, and lusitropic properties. CNP lacks natriuretic actions but possesses vasodilating and growth inhibiting actions via the guanylyl cyclase-linked natriuretic peptide-B receptor. All three peptides are cleared by the natriuretic peptide-C receptor and degraded by the ectoenzyme neutral endopeptidase 24.11, both of which are widely expressed in kidney, lung, and vascular wall. Recently, a fourth member of the natriuretic peptide, Dendroaspis natriuretic peptide (DNP) has been reported to be present in human plasma and atrial myocardium and is elevated in plasma of human CHF.     

Basal and volume expansion-stimulated plasma atrial natriuretic peptide concentrations and hemodynamics in conscious rats: effects of SCH 39.370, an endopeptidase inhibitor, and C-ANF-(4-23), a clearance receptor ligand.

The effects of a neutral endopeptidase (NEP) inhibitor, SCH 39.370, and a clearance receptor ligand, C-atrial natriuretic factor-(4-23) [C-ANF-(4-23)] on the plasma concentration of atrial natriuretic peptide (ANP) and hemodynamics under basal conditions and during increased circulating ANP levels produced by acute volume loading in conscious rats were studied. Measurements of plasma immunoreactive N-terminal fragment of pro-ANP (IR-NT-ANP) concentrations were used to characterize the endogenous secretion of the biologically active peptide in response to drug infusions and volume expansion. Infusion of SCH 39.370 increased plasma IR-ANP levels dose-dependently in conscious normotensive Wistar rats; maximal increases of 17% (P less than 0.02) after the dose of 3 mg/kg, iv, and 67% (P less than 0.002) after the dose of 10 mg/kg, iv, SCH 39.370 were noted. Similarly, infusion of C-ANF-(4-23) alone (30 micrograms/kg, iv bolus, followed by infusion of 3 micrograms/kg.min for 30 min) increased plasma IR-ANP levels by 37% (P less than 0.001). Given in combination, SCH 39.370 and C-ANF-(4-23) produced a greater increase in plasma IR-ANP concentration (83%; P less than 0.001) than when either substance was infused alone. Neither SCH 39.370 nor C-ANF-(4-23), alone or in combination, had any effect on basal plasma IR-NT-ANP concentrations. The combination reduced mean arterial pressure (8 +/- 2 mm Hg; P less than 0.01) and right atrial pressure (0.67 +/- 0.20 mm Hg; P less than 0.01), while administration of SCH 39.370 or C-ANF-(4-23) alone had no effect on mean arterial pressure, heart rate, or right atrial pressure in conscious rats. Acute volume expansion with 0.9% saline (1.1 ml/100 g BW) resulted in an increase in right atrial pressure (2.7 +/- 0.2 mm Hg; P less than 0.001) as well as in plasma IR-ANP (55%; P less than 0.001) and IR-NT-ANP concentrations (24%; P less than 0.03). Volume expansion in rats pretreated with SCH 39.370 resulted in a greater increase in plasma IR-ANP concentrations than in control animals; the relative ANP increases corresponding to the 2.5-mm Hg increase in right atrial pressure were 1.48-, 1.69-, and 2.28-fold in control, 3 mg/kg SCH 39.370-treated, and 10 mg/kg SCH 39.370-treated groups, respectively. When the relation between changes from control in plasma IR-ANP and right atrial pressure in response to acute volume expansion was analyzed in the presence of C-ANF-(4-23), no difference was noted between control and treated rats.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cardiovascular effects of natriuretic peptides and their interrelation with endothelin-1

The natriuretic peptides are a group of structurally related but genetically distinct peptides. Four types of natriuretic peptides have been found thus far: atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP) and Dendroaspis natriuretic peptide (DNP). ANP and BNP are secreted mainly from the heart and function as hormones with vasodilatory and natriuretic effects. CNP originates mainly from endothelial cells with a paracrine effect to induce vasodilation. Other effects of natriuretic peptides including negative inotropy, antimitogenic and anticoagulation have been described. Three types of natriuretic peptide receptors mediate their functions, and among them two are cGMP-coupled. Clearance of natriuretic peptides is via its clearance receptor through the action of neutral endopeptidases. Natriuretic peptides interact with other vasoactive peptides including endothelin. The putative role of natriuretic peptides in the pathophysiology of various cardiovascular diseases including congestive heart failure, hypertension, ischemic heart disease, and cardiomyopathy are discussed. Natriuretic peptide plasma levels are used for the diagnosis and therapeutic follow-up of congestive heart failure patients. Increasing the levels of natriuretic peptides by natriuretic peptide mimetics and neutral endopeptidase inhibitors may provide a new therapeutic strategy for the treatment of cardiovascular diseases such as congestive heart failure and hypertension.     

Potentiation of renal effects of atrial natriuretic factor-(99-126) by SQ 29,072

Depressor and renal activities of atrial natriuretic factor-(99-126) were determined in conscious, unrestrained spontaneously hypertensive rats treated with a neutral endopeptidase inhibitor, SQ 29,072 (7-[[2-(mercaptomethyl)-1-oxo-3-phenylpropyl]amino]heptanoic acid). SQ 29,072 (100 mumol/kg i.v.) prolonged the transient depressor effects of the peptide for as long as 2 hours. During the first hour after 3, 10, and 30 nmol/kg atrial natriuretic factor, urinary excretion of cyclic 3'5' guanosine monophosphate was significantly increased by 9.2 +/- 3.4, 13.0 +/- 2.2, and 12.7 +/- 4.2 nmol/kg/hr, respectively, in vehicle-treated rats and by 26.9 +/- 7.9, 52.1 +/- 11.1, and 46.4 +/- 12.2 nmol/kg/hr, respectively, in rats given 100 mumol/kg SQ 29,072. During the first hour after 3 and 10 nmol/kg atrial natriuretic factor-(99-126), the sodium loss was 161 +/- 56 and 139 +/- 42 mueq/kg/hr in vehicle-treated rats and was significantly greater (694 +/- 316 and 1,038 +/- 135 mueq/kg/hr) in rats given 100 mumol/kg SQ 29,072. After administration of 3, 10, and 30 mumol/kg SQ 29,072, the area over the curves of the depressor responses to 3 nmol/kg of the peptide increased from 297 +/- 70 to 306 +/- 108, 440 +/- 143, and 669 +/- 186 mm Hg.min, respectively, while the concurrent natriuretic responses rose from 161 +/- 56 to 250 +/- 88, 332 +/- 142, 464 +/- 164, and 694 +/- 316 mueq/kg/hr. In summary, the neutral endopeptidase inhibitor SQ 29,072 increased the magnitudes and especially the durations of the depressor, natriuretic, and cyclic guanosine monophosphate responses to exogenous atrial natriuretic factor-(99-126) in conscious spontaneously hypertensive rats, presumably by inhibition of degradation of atrial natriuretic factor in vivo. In conclusion, neutral endopeptidase inhibition offers an important new technique for enhancement and prolongation of the biological lifetime of atrial natriuretic factor.     

Neutral endopeptidase inhibition augments vascular actions of bradykinin in patients treated with angiotensin-converting enzyme inhibition

Angiotensin-converting enzyme and neutral endopeptidase (EC 3.4.24.11; neprilysin) are metallopeptidases present on the endothelium that metabolize bradykinin. Inhibitors of angiotensin-converting enzyme potentiate bradykinin-mediated vasodilatation and endothelial tissue plasminogen activator release. Combined angiotensin-converting enzyme and neutral endopeptidase inhibition may have additional beneficial cardiovascular effects mediated through bradykinin potentiation. We investigated the effects of local neutral endopeptidase inhibition on the vascular actions of bradykinin in heart failure patients maintained on chronic angiotensin-converting enzyme inhibition. Ten patients received intrabrachial infusion of thiorphan (30 nmol/min), a neutral endopeptidase inhibitor, in a randomized double-blind placebo-controlled crossover trial. Thiorphan was coinfused with Lys-des-Arg9-bradykinin (1 to 10 nmol/min), bradykinin (30 to 300 pmol/min), atrial natriuretic peptide (10 to 100 pmol/min), and sodium nitroprusside (2 to 8 mug/min). Bradykinin, atrial natriuretic peptide, and sodium nitroprusside caused dose-dependent vasodilatation (peak blood flow 14.4+/-2.2, 3.6+/-0.6, and 8.6+/-1.3 mL per 100 mL/min, respectively; P<0.0001). Bradykinin caused dose-dependent increases in tissue plasminogen activator antigen and activity (peak concentration 31.8+/-3.4 ng/mL and 21.9+/-7.6 IU/mL, respectively; P<0.001) and estimated antigen and activity release (peak release 152+/-46 ng per 100 mL/min and 154+/-22 IU/100 mL/min, respectively; P<0.005). Compared with placebo, thiorphan augmented bradykinin-mediated vasodilatation (1.4-fold; P<0.0001) and net tissue plasminogen activator release (1.5-fold; P<0.005). Neutral endopeptidase contributes to bradykinin metabolism in heart failure patients maintained on angiotensin-converting enzyme inhibitor therapy. Our findings may explain some of the clinical effects of combined angiotensin-converting enzyme and neutral endopeptidase inhibition, including the greater vasodepressor effect observed with combined therapy when compared with angiotensin-converting enzyme inhibition alone.     

Neutral endopeptidase (EC 3.4.24.11) in cirrhotic liver: a new target to treat portal hypertension?

BACKGROUND/AIMS: In liver cirrhosis atrial natriuretic peptide (ANP) decreases portal vascular resistance and tributary flow. The enzyme neutral endopeptidase (NEP) degrades ANP and bradykinin and generates endothelin-1 from big-endothelin. We determined the effects of NEP inhibition by candoxatrilat on hormonal status, liver function and arterial and portal pressures in rats with CCl4-induced cirrhosis. METHODS: Two groups of seven control rats received 1 ml 5% glucose solution alone or containing 10 mg/kg candoxatrilat; three groups of 10 ascitic cirrhotic rats received placebo, 5 or 10 mg/kg candoxatrilat. NEP protein concentration and immunostaining were analyzed in normal and cirrhotic livers. RESULTS: In cirrhotic rats 10 mg/kg candoxatrilat significantly increased steady-state indocyanine green clearance (a parameter reflecting liver plasma flow) (P<0.01), decreased portal pressure (P<0.01), had no effect on arterial pressure and plasma renin activity but increased ANP plasma levels (P<0.05) and urinary excretions (P<0.01) of ANP and cGMP. In the cytosol fraction of rat cirrhotic livers a 280% increase in NEP content was found (P<0.01), chiefly localized in desmin-positive myofibroblast-like cells of fibrous septa. CONCLUSIONS: Candoxatrilat has few effects on systemic hemodynamics and hormonal status; its portal hypotensive action depends on effects exerted on intrahepatic vascular resistance.     

Dual inhibition of angiotensin-converting enzyme and neutral endopeptidase by the orally active inhibitor mixanpril: a potential therapeutic approach in hypertension.

In the treatment of cardiovascular disease, it could be of therapeutic interest to associate the hypotensive effects due to the inhibition of angiotensin II formation with the diuretic and natriuretic responses induced by the protection of the endogenous atrial natriuretic peptide (ANP). Investigation of this hypothesis requires an orally active compound able to simultaneously inhibit angiotensin-converting enzyme (ACE) and neutral endopeptidase (NEP), which is involved in renal ANP metabolism. Such compounds have been rationally designed by taking into account the structural characteristics of the active site of both peptidases. Among them, RB 105, N-[(2S,3R)-2-mercaptomethyl-1-oxo-3-phenylbutyl]-(S)-alanine, inhibited NEP and ACE with Ki values of 1.7 +/- 0.3 nM and 4.2 +/- 0.5 nM, respectively. Intravenous infusion of RB 105 in conscious spontaneously hypertensive rats prevented the pressor response to exogenous angiotensin I and potentiated the natriuretic response to ANP. Infusion of RB 105, at 2.5, 5, 10, 25, and 50 mg/kg per hr decreased blood pressure dose-dependently in conscious catheterized spontaneously hypertensive rats and increased diuresis and natriuresis. Infusion of RB 105 as a bolus of 25 mg/kg followed by 25 mg/kg per hr similarly decreased blood pressure and increased natriuresis in three different models of hypertension (renovascular, deoxycorticosterone acetate-salt, and spontaneously hypertensive rats). Mixanpril, a lipophilic prodrug of RB 105 (ED50 values when given orally to mice, 0.7 mg/kg for NEP; 7 mg/kg for ACE), elicited dose-dependent hypotensive effects of long duration in spontaneously hypertensive rats after oral administration [-37 mmHg for 50 mg/kg twice a day (1 mmHg = 133 Pa) and is therefore the first dual NEP/ACE inhibitor potentially useful for clinical investigations.     

Enkephalinase inhibition increases plasma atrial natriuretic peptide levels, glomerular filtration rate, and urinary sodium excretion in rats with reduced renal mass

To investigate the in vivo effects of inhibition of endopeptidase 24.11, an enkephalinase enzyme shown to be involved in atrial natriuretic peptide (ANP) breakdown in vitro, we infused phosphoramidon, a specific inhibitor of endopeptidase 24.11, into rats with reduced renal mass (and chronic extracellular volume expansion) and into normal rats. Relative to baseline values in rats with remnant kidneys, phosphoramidon led to elevations of plasma ANP levels and concomitant increases in urinary sodium excretion, fractional excretion of sodium, glomerular filtration rate, filtration fraction, and urinary cyclic GMP excretion. Similar changes in renal function and urinary cyclic GMP excretion were obtained with thiorphan, another endopeptidase 24.11 inhibitor. These enhanced ANP levels and renal actions were not observed with phosphoramidon in normal rats. These results show that plasma ANP levels can be modulated in rats with reduced renal mass by inhibition of endopeptidase 24.11.     

Endogenous natriuretic peptides participate in renal and humoral actions of acute vasopeptidase inhibition in experimental mild heart failure

Mild heart failure is characterized by increases in atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) in the absence of activation of the renin-angiotensin-aldosterone system (RAAS). Vasopeptidase (VP) inhibitors are novel molecules that coinhibit neutral endopeptidase 24.11, which degrades the natriuretic peptides (NPs) and ACE. In a well-characterized canine model of mild heart failure produced by ventricular pacing at 180 bpm for 10 days, we defined the renal and humoral actions of acute VP inhibition with omapatrilat (OMA, n=6) and acute ACE inhibition (n=5) alone with fosinoprilat. We also sought to determine whether the NPs participate in the renal actions of acute VP inhibition by the administration of OMA together with an intrarenal administration of the NP receptor antagonist HS-142-1 (n=5). OMA resulted in a greater natriuretic response than did ACE inhibition in association with increases in plasma cGMP, ANP, BNP, urinary cGMP, urinary ANP excretion, and glomerular filtration rate (P<0.05 for OMA versus ACE inhibition). Plasma renin activity was increased only in the group subjected to ACE inhibition. Administration of intrarenal HS-142-1 attenuated the renal properties of OMA in association with a decrease in urinary cGMP excretion despite similar increases in plasma ANP and BNP. This study provides new insight into a unique new pharmacological agent that has beneficial renal actions in experimental mild heart failure beyond the actions that are observed with ACE inhibition alone and that are linked to the NP system.     

Effectiveness of endopeptidase inhibition (candoxatril) in congestive heart failure.

Candoxatril is a novel, orally active inhibitor of neutral endopeptidase EC 3.4.24.11, the enzyme that degrades atrial natriuretic peptide (ANP). The acute and chronic (10 days treatment) hemodynamic and hormonal effects of candoxatril (150 mg twice daily) in 12 patients with moderately severe congestive heart failure were investigated in a randomized, placebo-controlled, double-blind study. On study day 1, candoxatril acutely increased plasma ANP levels, suppressed aldosterone and decreased right atrial and pulmonary capillary wedge pressures. After 10 days of treatment, basal ANP was increased and basal aldosterone was decreased. Body weight was reduced, most likely reflecting chronic natriuretic or diuretic effects, or both, and there was a trend toward increased cardiac index and reduced preload values. On study day 10, the acute effects of candoxatril were similar to those on day 1 (i.e., ANP was further increased, aldosterone was suppressed, and right and left ventricular filling pressures were decreased). Thus, candoxatril may offer a new and effective therapeutic approach in the treatment of heart failure.     

Restorative effect of atrial natriuretic peptide or chronic neutral endopeptidase inhibition on blunted cardiopulmonary vagal reflexes in aged rats

Arterial baroreflex function diminishes with age, but whether cardiopulmonary vagal reflexes are similarly altered with physiological aging has not been fully elucidated. In this study, predominantly cardiac high pressure mechanoreceptor-activated (ramp baroreflex) and cardiopulmonary chemoreceptor-activated (von Bezold-Jarisch reflex) vagal reflexes in conscious, instrumented rats were impaired by 30% to 40% (P<0.05) in 24-month-old (n=12) compared with 6-month-old rats (n=12). To determine whether this is a restorable deficit, the influence of atrial natriuretic peptide (ANP), either by infusion or blockade of its breakdown, was studied. ANP infusion was previously shown to enhance Bezold-Jarisch reflex and ramp baroreflex bradycardia in young adult rats. The present study confirmed that vagal reflex augmentation by ANP (50 pmol/kg per minute) also occurs in old rats (increased by 60+/-18% (Bezold-Jarisch reflex) and 91+/-15% (ramp baroreflex; P<0.05). Direct vagal stimulation in anesthetized animals showed that the target for ANP was not the cardiac vagus itself in old rats (n=7), although in young rats only, we confirmed the published finding that ANP enhances vagal bradycardia (by 58+/-14%, n=7). Neutral endopeptidase 24.11 degrades ANP and several other peptides. The neutral endopeptidase inhibitor candoxatrilat (5 mg/kg per day IV for 7 to 9 days) restored vagal reflex bradycardia in old rats (n=6) to levels similar to those in young neutral endopeptidase inhibitor-treated rats (n=6). Impaired cardiopulmonary vagal reflex control of heart rate is thus a feature of normal aging, and this deficit may be ameliorated by either ANP infusion or chronic neutral endopeptidase inhibition.     

Effects of a dual inhibitor of angiotensin converting enzyme and neutral endopeptidase, MDL 100,240, on endocrine and renal functions in healthy volunteers

OBJECTIVE: To investigate the endocrine and renal effects of the dual inhibitor of angiotensin converting enzyme and neutral endopeptidase, MDL 100,240. DESIGN: A randomized, placebo-controlled, crossover study was performed in 12 healthy volunteers. METHODS: MDL 100,240 was administered intravenously over 20 min at single doses of 6.25 and 25 mg in subjects with a sodium intake of 280 (n = 6) or 80 (n = 6) mmol/day. Measurements were taken of supine and standing blood pressure, plasma angiotensin converting enzyme activity, angiotensin II, atrial natriuretic peptide, urinary atrial natriuretic peptide and cyclic GMP excretion, effective renal plasma flow and the glomerular filtration rate as p-aminohippurate and inulin clearances, electrolytes and segmental tubular function by endogenous lithium clearance. RESULTS: Supine systolic blood pressure was consistently decreased by MDL 100,240, particularly after the high dose and during the low-salt intake. Diastolic blood pressure and heart rate did not change. Plasma angiotensin converting enzyme activity decreased rapidly and dose-dependently. In both the high- and the low-salt treatment groups, plasma angiotensin II levels fell and renin activity rose accordingly, while plasma atrial natriuretic peptide levels remained unchanged. In contrast, urinary atrial natriuretic peptide excretion increased dose-dependently under both diets, as did urinary cyclic GMP excretion. Effective renal plasma flow and the glomerular filtration rate did not change. The urinary flow rate increased markedly during the first 2 h following administration of either dose of MDL 100,240 (P < 0.001) and, similarly, sodium excretion tended to increase from 0 to 4 h after the dose (P = 0.07). Potassium excretion remained stable. Proximal and distal fractional sodium reabsorption were not significantly altered by the treatment. Uric acid excretion was increased. The safety and clinical tolerance of MDL 100,240 were good. CONCLUSIONS: The increased fall in blood pressure in normal volunteers together with the preservation of renal hemodynamics and the increased urinary volume, atrial natriuretic peptide and cyclic GMP excretion distinguish MDL 100,240 as a double-enzyme inhibitor from inhibitors of the angiotensin converting enzyme alone. The differences appear to be due, at least in part, to increased renal exposure to atrial natriuretic peptide following neutral endopeptidase blockade.     

Guanylyl cyclase receptors mediate cardiopulmonary vagal reflex actions of ANP

Atrial natriuretic peptide (ANP) potentiates vagal cardiopulmonary reflexes due to chemosensory (Bezold-Jarisch [B-J] reflex) or mechanosensory (ramp baroreflex) activation. The ANP receptor mediating these actions is unknown. We examined the role of particulate guanylyl-cyclase (pGC) receptors in ANP-induced enhancement of cardiopulmonary vagal reflexes. Cardiopulmonary baroreceptor reflex function was assessed by bradycardic responses to ramp blood pressure rises after rapid intravenous methoxamine (100 micro g/kg bolus dose). The B-J reflex was evoked by 3 intravenous doses of serotonin (1 to 10 micro g/kg). In conscious, chronically instrumented rats (n=9), these tests were performed on each animal during randomized infusions of rat ANP (150 ng/kg per minute IV), saline (270 micro L/h IV), the pGC receptor antagonist HS-142-1 (3 mg/kg IV), or combined HS-142-1+ANP treatment. HS-142-1 alone attenuated normal B-J reflex (by 33+/-8%, P<0.05) but not ramp baroreflex responses. As we showed previously, ANP enhanced baroreflex and B-J reflex bradycardia (by approximately 140% and approximately 30%, respectively, P<0.05), compared with saline infusion. These ANP effects were completely blocked by HS-142-1, demonstrating that the cardiopulmonary vagal reflex actions of ANP occurred through pGC natriuretic peptide receptors. Additionally, we have provided evidence for the first time that pGC natriuretic peptide receptors are essential for the full expression of the B-J reflex but not for that of cardiopulmonary vagal baroreflexes. This tonic interaction between pGC natriuretic peptide receptors and cardiopulmonary chemosensitive receptors may be important during pathophysiological activation of B-J reflex, such as with myocardial infarction.     

Essential biochemistry and physiology of (NT-pro)BNP

Brain natriuretic peptide (BNP) is a 32 amino acid cardiac natriuretic peptide hormone originally isolated from porcine brain tissue. The human BNP gene is located on chromosome 1 and encodes the prohormone proBNP. The biologically active BNP and the remaining part of the prohormone, NT-proBNP (76 amino acids) can be measured by immunoassay in human blood. Cardiac myocytes constitute the major source of BNP related peptides. The main stimulus for peptide synthesis and secretion is myocyte stretch. Recently, cardiac fibroblasts have also been shown to produce BNP. Other neurohormones may stimulate cardiac BNP production in different cardiac cell types. In contrast to atrial natriuretic peptides (ANP/NT-proANP), which originate mainly from atrial tissue, BNP related peptides are produced mainly from ventricular myocytes. Ventricular (NT-pro)BNP production is strongly upregulated in cardiac failure and locally in the area surrounding a myocardial infarction. In peripheral organs BNP binds to the natriuretic peptide receptor type A causing increased intracellular cGMP production. The biological effects include diuresis, vasodilatation, inhibition of renin and aldosterone production and of cardiac and vascular myocyte growth. In mice BNP gene knockout leads to cardiac fibrosis, gene over-expression to hypotension and bone malformations. BNP is cleared from plasma through binding to the natriuretic peptide clearance receptor type C, but it seems relatively resistant to proteolysis by neutral endopeptidase NEP 24.11. Clearance mechanisms for NT-proBNP await further study. While the plasma concentration of NT-proBNP and BNP is approximately equal in normal controls, NT-proBNP plasma concentration is 2-10 times higher than BNP in patients with heart failure. This relative change in peptide levels may be explained by shifts in cardiac secretion and/or clearance mechanisms.     

Modulating effects of atrial natriuretic peptide on vascular permeability and blood pressure by inhibition of cyclic phosphodiesterase GMP in the rat]

Atrial natriuretic peptide lowers arterial pressure and increases hematocrit through reduction in plasma volume caused by a transcapillary shift of plasma fluid and protein toward the interstitium. Cyclic GMP, the second messenger of atrial natriuretic peptide is catabolized by cGMP-phosphodiesterase; therefore we examined the consequences of inhibition of the phosphodiesterase on these responses using the specific cGMP inhibitor M&B 22.948. In anesthetized, bilaterally nephrectomized rats, a 45-min infusion of atrial natriuretic peptide (1 microgram/kg/min) reduced arterial pressure by 7.6 +/- 1.5% and increased hematocrit by 9 +/- 0.6% (both p < 0.01), leading to a calculated decrease in plasma volume of 14.4 +/- 0.9%. Infusion of M&B 22.948 (0.68 mg/kg/min) did not affect hematocrit and lowered arterial pressure by 8.1 +/- 0.5% (p < 0.01), an effect similar to that observed following administration of sodium nitroprusside (10 micrograms/kg/min). Simultaneous infusion of atrial natriuretic peptide and M&B 22.948 had additive arterial pressure lowering effects (-15.9 +/- 1.1%; p < 0.01 vs atrial natriuretic peptide or M&B 22.948 alone), while the increase in hematocrit of 9.4 +/- 0.7% was identical to that seen with atrial natriuretic peptide alone. Thus, M&B 22.948 amplified atrial natriuretic peptide effects on arterial pressure, but not on vascular permeability. These findings indicate differential regulation of atrial natriuretic peptide effects by inhibition of the cGMP-phosphodiesterase.     

Neutral endopeptidase 24.11 inhibition reduces pulmonary vascular remodeling in rats exposed to chronic hypoxia.

Inhibition of the metabolism of endogenous atrial natriuretic peptide (ANP), by continuous infusion of a specific inhibitor of neutral endopeptidase (membrane metalloendopeptidase E.C. 3.4.24.11), UK 73,967 (candoxatrilat), was undertaken in rats, in which chronic hypoxia was used as a stimulus to induce pulmonary hypertension and right ventricular hypertrophy. Inhibition of neutral endopeptidase 24.11 with low-dose and high-dose UK 73,967 (NEI) increased endogenous plasma ANP by greater than 155% during the development of pulmonary hypertension. NEI treatment reduced mean pulmonary arterial pressure in hypoxia as follows: vehicle 26.6 +/- 4.0 mm Hg; low-dose NEI 22.7 +/- 1.9 mm Hg, and high-dose NEI 22.6 +/- 2.5 mm Hg (both p less than 0.01 compared with hypoxic vehicle); however, it was without effect on pulmonary arterial pressure in normoxia (17.6 +/- 2.2 mm Hg) or on systemic blood pressure. The development of right ventricular hypertrophy was also reduced in both groups treated with NEI (right ventricular weight/left ventricular weight: 0.43 +/- 0.03 vehicle; 0.40 +/- 0.02 low-dose NEI and 0.40 +/- 0.02 high-dose NEI, both p less than 0.05 compared with vehicle). Remodelling of the pulmonary vasculature, characterized by extension of the muscle within the small pulmonary arteries toward the periphery of the lung, was reduced by NEI treatment (percentage of thick-walled peripheral vessels; 19.2 +/- 3.1% vehicle; 10.4 +/- 2.3% low-dose NEI and 8.1 +/- 1.8% high-dose NEI, both p less than 0.001 compared with vehicle). In the isolated blood perfused rat lung pulsed doses of NEI had no effect on pulmonary vascular tone in the absence of ANP. Specific inhibition of the enzyme neutral endopeptidase reduces vascular remodelling, the development of pulmonary hypertension, and right ventricular hypertrophy. Endogenous ANP modulates vascular remodelling in vivo. Retarding the metabolism of endogenous ANP through inhibition of neutral endopeptidase 24.11 represents a potential approach toward therapy. g     

Renal and antihypertensive effects of neutral endopeptidase inhibition in transgenic rats with an extra renin gene

The cardiovascular consequences of neutral endopeptidase (NEP) inhibition with the NEP inhibition ecadotril were evaluated by determining acute and long-term effects of the compound on hemodynamic, hormonal, renal, and structural parameters in hypertensive transgenic rats harboring a mouse renin gene (TGR (m(Ren2)27)) and in normotensive controls (Sprague-Dawley rats, SDR). Acute administration of ecadotril (10 and 30 mg/kg, orally) produced a dose-dependent decrease in systolic blood pressure with a maximal effect of −23 mm Hg between 2 and 4 h after oral administration. The NEP activity in plasma was significantly inhibited and the plasma levels of atrial (ANP) and brain (BNP) natriuretic peptides and their second messenger, cyclic GMP, were distinctly raised after oral administration. In addition, ecadotril (10 and 30 mg/kg, orally) produced a dose-dependent increase in the urinary excretion of sodium and cyclic GMF. These effects were more pronounced in TGR (mRen2)27 than in the normotensive SDR without an activated natriuretic peptide system. In the long-term study, the systolic pressure in control TG (m(Ren2)27) rats increasdd from 213 ± 5 to 255 ± 7 mm Hg, whereas, in animals treated with ecadotril (30 mg/kg, orally twice daily), the blood pressure increased only from 213 ± 5 to 227 ± 6 mm Hg during the observation period of 13 weeks. The increases in heart weight and in kidney weight were also delayed. At the end of the study, cyclic GMP was elevated and ANP tended to be higher, whereas plasma renin activity had decreased. These data indicate a beneficial pharmacological profile of neutral endopeptidase inhibition that could prove useful in the treatment of cardiovascular diseases like hypertension.