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While regional plasma concentrations of the endocrine hormones atrial and brain natriuretic peptide (ANP and BNP) have been studied, there are few reports of regional changes in the largely paracrine C-type natriuretic peptide (CNP) and its amino terminal fragment NT-CNP. Accordingly, we have performed trans-organ arteriovenous sampling for measurement of plasma ANP, BNP, CNP and NT-CNP in anesthetized sheep before and after induction of experimental heart failure. ANP and BNP plasma concentrations are sourced from a single organ (the heart) and are subject to substantial extraction across most tissue beds. In contrast, our data demonstrate that multiple tissues including liver, heart, hind limb and kidney contribute to circulating CNP. Given that arteriovenous gradients for NT-CNP were similar, this is likely to represent de novo secretion. Circulating levels of CNP and NT-CNP were raised in heart failure but to a much lesser degree than ANP and BNP. There was no evidence of net extraction of CNP or NT-CNP across any tissue bed.

Clinical heart failure, often the result of myocardial infarction, may be preceded by a period of compensated left ventricular impairment. There is substantial need for an experimental model that reflects this human condition. In sheep, coronary artery ligation produced consistent left ventricular anteroapical myocardial infarctions resulting in chronic (5 wk), stable hemodynamic changes compared with sham controls, including reductions in ejection fraction (51 +/- 2 vs. 30 +/- 5%, P < 0.001), cardiac output (6.3 +/- 0.2 vs. 5.1 +/- 0.2 l/min, P < 0.01), and arterial pressure (93 +/- 2 vs. 79 +/- 3 mmHg, P < 0.001), and increases in cardiac preload (left atrial pressure, 3.3 +/- 0.1 vs. 8.3 +/- 1.3 mmHg, P < 0.001). These changes were associated with acute and sustained increases in plasma concentrations of atrial natriuretic peptide (ANP; 5 wk, 11 +/- 2 vs. 27 +/- 5 pmol/l, P < 0.001), brain natriuretic peptide (BNP; 3 +/- 0.2 vs. 11 +/- 2 pmol/l, P < 0.001), and amino-terminal pro-brain natriuretic peptide (NT-BNP; 17 +/- 3 vs. 42 +/- 12 pmol/l, P < 0.001). Significant correlations were observed between plasma levels of the natriuretic peptides (ANP, day 7 to week 5 samples; BNP and NT-BNP, day 1 to week 5 samples) and changes in left ventricular volumes and ejection fraction. In contrast, renin activity, aldosterone, catecholamines, and endothelin were not chronically elevated postinfarction and were not related to indexes of ventricular function. Coronary artery ligation in sheep produces the pathological, hemodynamic, and neurohormonal characteristics of compensated left ventricular impairment secondary to myocardial infarction. Plasma concentrations of the cardiac natriuretic peptides are sensitive markers of left ventricular dysfunction. This is a reproducible model that reflects the clinical condition and should prove suitable for investigating the pathophysiology of, and experimental therapies in, early left ventricular dysfunction.

Few studies have addressed the possibility that brain natriuretic peptide (BNP) possesses a profile of bioactivity that is distinct from that of atrial natriuretic peptide (ANP). Accordingly, we assessed the biologic actions of BNP in the setting of maximal or near-maximal ANP-induced biologic activity. Background ANP infusions (7.5 pmol/kg/min) administered on all study days, increased plasma ANP (approximately 120 pM) and cyclic guanosine monophosphate (GMP) levels (approximately 40 nM), and induced significant decreases in arterial pressure and cardiac output associated with increased heart rate, hematocrit, diuresis, and natriuresis. Increasing the dose twofold after 1 h (experiment 1, n = 5) showed no enhancement of these actions despite a further twofold increase in plasma ANP and cyclic GMP (both p values <0.001). Addition of low-dose BNP (2 pmol/kg/min) after 1 h background infusion (experiment 2, n = 8), increased plasma BNP levels (30 pM, p < 0.001) but caused no significant effects on the hemodynamic, renal, or hormonal indices measured. In conclusion, in the setting of maximal hemodynamic, renal, and endocrine responses to high-dose background infusions of ANP, coinfusion of BNP exhibits no enhancement of, or additional, biologic activity. This study provides no evidence for unique short-term biologic actions of ANP and BNP.

We examined for the first time the specific roles of angiotensin II and the natriuretic peptides during inhibition of angiotensin-converting enzyme (captopril, 25 mg bolus + 6 mg/3 h infusion) and endopeptidase 24.11 (SCH32615, 5 mg/kg bolus + 3 mg/kg/3 h infusion), both separately and in combination, in eight sheep with pacing-induced heart failure. Plasma atrial and brain natriuretic peptide levels were similarly increased by SCH32615 and to a lesser extent during combined inhibition but decreased with captopril. Captopril and combined inhibition induced identical increases in plasma renin activity and reductions in angiotensin II, whereas neither was changed by SCH32615 alone. Mean arterial pressure and peripheral resistance decreased during SCH32615 and further still during captopril and combined treatment. Left atrial pressure was reduced to a similar extent by SCH32615 and captopril alone and reduced further by combined inhibition. Cardiac output increased during all treatments. Urine volume and sodium excretion were significantly increased during SCH32615 and combined inhibition. Creatinine clearance increased during SCH32615, decreased during captopril, and was maintained during combined treatment. In conclusion, compared with captopril alone, cotreatment with an endopeptidase 24.11 inhibitor further improved filling pressures and induced a diuresis and natriuresis with preservation of renal glomerular filtration.

The effects of separate and combined endopeptidase inhibition (by SCH-32615) and natriuretic peptide receptor C blockade [by C-ANP-(4-23)] on the clearance and bioactivity of atrial (ANP) and brain (BNP) natriuretic peptides was investigated in eight sheep with heart failure. SCH-32615 and C-ANP-(4-23) administered separately induced significant and proportionate dose-dependent rises in plasma ANP, BNP, and guanosine 3',5'-cyclic monophosphate (cGMP) levels. Associated with these changes were reductions in arterial pressure, left atrial pressure, and peripheral resistance and increases in cardiac output, urine volume, sodium excretion, and creatinine clearance. SCH-32615 induced greater diuresis and natriuresis than C-ANP-(4-23). Combined administration of SCH-32615 and C-ANP-(4-23) induced greater than additive rises in plasma ANP, BNP, and cGMP concentrations, with enhanced hemodynamic effects, diuresis, and natriuresis and reduced plasma aldosterone levels. In conclusion, we find that the enzymatic and receptor clearance pathways contribute equally to the metabolism of endogenous ANP and BNP in sheep with heart failure. Combined inhibition of both degradative pathways was associated with enhanced hormonal, hemodynamic, and renal effects and may have greater potential therapeutic value than either agent separately.

1. Whereas many studies have detailed the effects of exogenous atrial natriuretic peptide (ANP) infusions in heart failure, and a limited number have examined the effects of brain natriuretic peptide (BNP), none have directly compared the bioactivity of similar doses of ANP and BNP under standard conditions of impaired cardiac function. We compared the hormonal, haemodynamic and renal effects of 3 h infusions of ANP, BNP and a vehicle control in eight sheep with pacing-induced heart failure (225 beats/min for 8-12 days). 2. Infusion of ANP and BNP increased plasma ANP (P < 0.001) (276 +/- 27 versus control 142 +/- 26 pmol/l) and BNP (P < 0.001) (257 +/- 34 versus control 45 +/- 5 pmol/l) respectively, in association with increased cyclic 3',5'-guanosine monophosphate [control, 40 +/- 6; ANP, 53 +/- 6 (P < 0.05); BNP, 57 +/- 7 nmol/l (P < 0.001)]. Metabolic clearance rate and half-life were similar for both peptides. Infusion of ANP and BNP similarly reduced mean arterial pressure [control, 73.0 +/- 1.6; ANP, 67.6 +/- 1.2 (P < 0.01); BNP, 65.7 +/- 1.7 mmHg (P < 0.001)], left atrial pressure (both P < 0.05) (control, 22.0 +/- 0.7; ANP, 19.9 +/- 1.0; BNP, 19.8 +/- 0.9 mmHg) and peripheral resistance [control, 50.3 +/- 4.1 mmHg l-1 min-1; ANP, 46.0 +/- 2.8 (P < 0.05); BNP, 43.8 +/- 4.5 (P < 0.01)], and increased urine volume (2-3-fold, both P < 0.05), sodium excretion (> 10-fold, both P < 0.01) and haematocrit levels relative to control (both P < 0.05). Infused BNP tended to raise plasma ANP levels (by 28 pmol/l), while ANP increased plasma BNP (by 18 pmol/l). Plasma aldosterone was reduced by approximately 40% by both peptides (both P < 0.05). 3. In conclusion, ANP and BNP are both powerfully, natriuretic, similarly suppress aldosterone and appear equipotent in reducing preload and afterload in this model of pacing-induced heart failure.

To examine tissue and plasma atrial (ANP) and brain natriuretic peptide (BNP) responses to left ventricular hypertrophy (LVH) 7 sheep underwent suprarenal aortic banding (20 mmHg initial pressure differential). Median survival time was 15 days. Proximal mean aortic pressure (MAP) increased from 65.1 +/- 5.0 mmHg (baseline) to 111.6 +/- 7.5 mmHg (day 7, p < 0.0001). Distal systolic aortic pressure fell from 85.5 +/- 8.7 mmHg (baseline) to 55.6 +/- 6.4 mmHg (day 7, p = 0.0002). Maximal plasma ANP (26.9 +/- 3.6 vs 10.1 +/- 1.2 pmol/L, p = 0.005) and BNP (15.3 +/- 3.6 vs. 3.5 +/- 1.0 pmol/L, p = 0.006) were recorded at 15 +/- 4.0 days. Coarctation induced rapid increases in PRA and plasma aldosterone and a fall in urinary sodium. Post-mortem examination of hearts confirmed LVH. Compared with controls, tissue ANP concentration was reduced in left atrium (p = 0.04) and LV (p = 0.04). BNP concentration was reduced in left atrium (p = 0.02) but tended to be higher in LV. In conclusion, suprarenal aortic coarctation leads to progressive hypertension resulting in LVH, progressive increases in plasma ANP and BNP and, in most cases, death from heart failure.

1. Atrial and brain natriuretic peptide are both circulating hormones subject to degradation by neutral endopeptidase 24.11. Whereas endogenous levels of atrial natriuretic peptide are increased by neutral endopeptidase inhibition in most pathophysiological states, the effect on brain natriuretic peptide and the influence of cardiac status is less clear. To further evaluate the role of neutral endopeptidase 24.11, we directly compared the responses of atrial and brain natriuretic peptide, together with the effects on other vasoactive hormones, haemodynamics and renal indices, to a neutral endopeptidase inhibitor, SCH32615, and a vehicle control in eight conscious sheep before and during pacing-induced heart failure. 2. In normal animals, SCH32615 significantly increased concentrations of plasma atrial natriuretic peptide (22 +/- 5 pmol/l compared with 14 +/- 2 pmol/l in control, 1.6-fold increase) and brain natriuretic peptide (6.5 +/- 1.2 pmol/l compared with 4.1 +/- 0.7 pmol/l in control, 1.6-fold increase), whereas in heart failure, plasma levels of atrial natriuretic peptide (306 +/- 38 pmol/l compared with 187 +/- 25 pmol/l in control, 1.6-fold increase) and brain natriuretic peptide (93 +/- 11 pmol/l compared with 55 +/- 9 pmol/l in control, 1.7-fold increase) were elevated to a significantly greater absolute, but proportionately similar, extent. In both normal and heart-failed animals, SCH32615 induced reductions in mean arterial pressure and left atrial pressure and increases in haematocrit, plasma cGMP and endogenous creatinine clearance. However, only in heart failure did neutral endopeptidase inhibition induce a significant and marked natriuresis (> 10-fold increase) and diuresis (4-fold increase), together with suppression of renin activity and haemodynamic effects including decreased peripheral resistance and raised cardiac output. 3. In conclusion, neutral endopeptidase inhibition increases plasma concentrations of atrial and brain natriuretic peptide to a proportionately similar extent in both normal and heart-failed sheep. The striking natriuresis and diuresis and additional haemodynamic effects demonstrated in sheep with heart failure, where natriuretic peptide levels are elevated compared with normal sheep, supports the concept that neutral endopeptidase inhibition augments endogenous atrial and brain natriuretic peptide.

Although atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) appear to share functional homology, there is doubt concerning a possible endocrine function for C-type natriuretic peptide (CNP) and the relative potency of species-specific forms of these hormones. Accordingly, we have examined the biological effects, interactions, and pharmacokinetics of equimolar doses (0.5 followed by 2.5 pmol.kg-1.min-1, each for 2 h) of species-specific forms of ANP, BNP-26, and CNP-22 in vehicle-controlled studies in normal conscious sheep. Although pharmacokinetics (metabolic clearance rates of 5.7 +/- 1.17, 7.5 +/- 1.36, and 4.7 +/- 0.71 l/min and half-lives of 3.9 +/- 0.42, 2.5 +/- 0.21, and 2.0 +/- 0.18 min for ANP, BNP, and CNP, respectively) are similar, the biological effects and actions on endogenous natriuretic peptide levels differ. Plasma BNP was significantly increased by CNP infusion (P < 0.0001), as was CNP by BNP infusions (P = 0.0009). Compared with ANP and BNP, which were equipotent in stimulating plasma guanosine 3',5'-cyclic monophosphate (cGMP; P < 0.0001 for both) and lowering arterial pressure (P < 0.05 for both) and cardiac output, CNP infusions induced only a small increment in cGMP and had no significant hemodynamic actions. In contrast, all three peptides suppressed plasma aldosterone levels (P < 0.05 for each), yet none induced significant natriuresis. Actions of CNP to increase BNP (and ANP) may account for the observed bioactivity of CNP. The findings show that potentially important interactions occur among all three hormones that need to be considered when interpreting the effects of individual peptides, particularly CNP.

The responses of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) were investigated in six conscious sheep paced at 120, 155, 190, and 225 beats/min for 1.5 h at each rate and at 180, 225, and 180 beats/min for 4 days at each rate. Increased pacing reduced arterial pressure, cardiac output, and urine and Na excretion and increased left atrial pressure and plasma ANP, BNP, and C-type natriuretic peptide, with delayed activation of the renin-angiotensin system (RAS). Acute pacing increased plasma ANP and BNP levels 8.6- and 3.6-fold, respectively (both P < 0.001), whereas chronic pacing increased ANP and BNP 7.8- and 9-fold, respectively (both P < 0.001). Thus the ANP-to-BNP ratio increased during acute pacing (P < 0.001) and decreased proportionately during chronic pacing (P < 0.05). Reduction in pacing improved hemodynamic status, reduced natriuretic peptides (BNP less than ANP), normalized the RAS, and induced diuresis and natriuresis. In conclusion, BNP is less responsive than ANP to acute changes in intracardiac pressure but is proportionately more responsive to chronic hemodynamic changes such as occur in congestive heart failure.

Whereas the short-term effects of atrial natriuretic factor (ANF) are well documented, less is known concerning the possible hypotensive action of sustained low-dose ANF infusions. Accordingly, we have examined the sequential renal, hormonal, and hemodynamic responses to 48-h low-dose ANF infusions in eight normotensive conscious sheep and evaluated the effects of these infusions on autonomic (baroreceptor), adrenocortical, and pressor responsiveness to exogenous stimulation. Plasma ANF levels tended to rise in response to ANF infusions, but the difference between ANF and control day levels was not significantly different. Systolic arterial pressure (SAP) was significantly lower after ANF infusion (P = 0.01) and was associated with a reduction in calculated total peripheral resistance (CTPR, P = 0.007). Mean arterial pressure also tended to be lower (P = 0.08) in response to ANF. No change was seen in urinary volume or sodium excretion or in plasma angiotensin II (ANG II), aldosterone, or cortisol levels. ANF significantly attenuated the pressor response to exogenous ANG II (P = 0.01) but did not affect the adrenocortical responsiveness or autonomic (baroreceptor) responsiveness to exogenous stimulation. This study demonstrates that chronic low-dose infusions of ANF that barely elevate plasma ANF levels induce significant cardiovascular effects, including lowering of SAP associated with a fall in CTPR and attenuation of pressor responsiveness to physiological increments in ANG II, thus providing further support for the importance of ANF in blood pressure homeostasis in normal sheep.

C-type natriuretic peptide (CNP) and its specific receptor (ANPRB) are richly distributed throughout the brain, especially the anterior pituitary and hypothalamus but not in tissue of nonneural origin. These findings suggest that the actions of CNP, unlike atrial natriuretic factor (ANF), are largely confined to the brain where CNP may participate in the central regulation of hemodynamics and salt and water balance. Therefore, we have studied the hemodynamic, renal, and hormonal effects of continuous intracerebroventricular infusions of CNP (5 micrograms/h for 4 h) in a vehicle-controlled study and compared the responses to those of ANF in normal conscious sheep. Hemodynamic and hormonal responses to ANF were not different from control infusions. There was a trend for urinary sodium and potassium excretion to increase throughout the control infusion but not on the ANF day. Water intake during control infusion (358 +/- 160 ml/4 h) was almost 3-fold that ingested during ANF (127 +/- 89 ml/4 h, NS). In contrast, CNP induced a prompt fall in mean arterial pressure (mean decrement 5 mm Hg), arterial pressure remaining below time control values for the remainder of the infusion (P = 0.006). Rises in both heart rate and PRA observed on the control day tended to be attenuated by CNP. Urine electrolyte response to CNP was similar to that observed with ANF. Compared with control infusions, the responses of both plasma aldosterone (P = 0.006) and cortisol (P = 0.043) were significantly different. Following CNP-induced hypotension, plasma cortisol and aldosterone increased abruptly at 30 min after which values fell to control or lower levels until the infusion was terminated. These studies show that intracerebroventricular CNP lowers arterial pressure without increasing heart rate and also suppresses the adrenocortical response, whereas ANF given under the same conditions has no significant effects. These data support the hypothesis that CNP plays an important role in the central regulation of blood pressure and hormones.

The effects of neutral endopeptidase inhibition (NEP-I) were studied in 6 conscious sheep with heart failure (HF) induced by rapid ventricular pacing for 7 days. Measurements were performed 1 h before and for 6 h after intravenous (i.v.) bolus administration of vehicle and SCH 39370 (1.25 and 5 mg/kg) on separate days. After the higher dose, an index of serum NEP activity decreased from 0.83 +/- 0.05 to 0.13 +/- 0.07 nmol/ml/min (p less than 0.001) at 1 h and then returned to control levels at 6 h. Plasma atrial natriuretic peptide (ANP) and cyclic GMP rose from 328 +/- 28 and 20.2 +/- 4.3 to a peak of 570 +/- 65 pmol/L (p less than 0.001) and 28.7 +/- 6.3 nmol/L (p less than 0.05) respectively. Natriuresis and diuresis were significant and left atrial pressure (LAP) decreased from 21.9 +/- 1.1 to 20.1 +/- 0.8 mm Hg (p less than 0.05). Despite high endogenous ANP levels in HF, NEP-I further increases both ANP and its "second messenger." Its natriuretic and hemodynamic effects are consistent with enhanced ANP activity in renal and vascular tissues, suggesting that NEP-I may be useful for treating HF.

Plasma volume expansion stimulates cardiac secretion of atrial natriuretic factor (ANF) and also increases the ANF concentration in cerebrospinal fluid. In order to determine whether brain ANF is involved in the compensatory response to hypervolemia or the regulation of cardiac secretion of ANF, we have studied the integrated hemodynamic, renal, and hormonal response to acute volume expansion (15 ml/kg Dextran over 30 min) in five sheep given nonimmune serum (control) and ANF antiserum by intracerebroventricular (icv) injections on separate days. Dextran loading caused similar decreases in hematocrit and increases in central venous and mean arterial pressures on both study days. Heart rate was higher after antiserum injections (P less than 0.05). Dextran loading increased plasma ANF on the control (20 pmol/liter maximal mean increment above baseline) but not on the antiserum day (P less than 0.01). The diuresis (P less than 0.01) and natriuresis (P less than 0.05) observed on the control day was inhibited by icv antiserum. Plasma aldosterone and cortisol levels showed similar falls in response to the dextran load on both days. These experiments show that icv ANF antiserum inhibits both the increase in cardiac secretion of ANF and the renal response to plasma volume expansion without affecting hemodynamic status. These data support the hypothesis that the brain ANF system is important in the systemic responses to volume loading.

Atrial natriuretic factor (ANF) cleaved between Cys105 and Phe106 is the primary metabolite of ANF and circulates in human plasma. Because the role of this metabolite in vivo and its possible interaction with intact ANF are unclear, we studied the biologic effects of a 2-h infusion of rat cleaved ANF101-105/106-126 (15 pmol/kg/min) or vehicle alone in six normal sheep. Infusions of cleaved ANF increased venous plasma levels of cleaved ANF from less than 5 to 260 pmol/L and induced a progressive and significant increase in plasma cyclic GMP (p = 0.025) without significantly affecting plasma ANF levels. These changes were associated with a small (nonsignificant) decrease in arterial pressure and a significant increase in heart rate (HR) and sympathetic nervous activity and were followed by activation of the renin-angiotensin-aldosterone (RAA) axis after infusions were terminated. Unlike ANF itself, cleaved ANF was not natriuretic and did not reduce plasma volume or right atrial pressure. Calculated metabolic clearance rate (MCR) (1.47 +/- 0.4 L/min) and disappearance rate of cleaved ANF from plasma (4.8 +/- 0.37 min) were similar to values reported previously for intact ANF in sheep. These studies show that cleaved ANF stimulates guanylate cyclase and alters hemodynamics and the RAA system in vivo.

The integrative and sustained effects of small increments in atrial natriuretic factor (ANF) on hemodynamic, renal, and hormonal function are still unclear. Accordingly, we have studied the sequential effects of 24-h, low-dose infusion (2.4 pmol.kg-1.min-1) of rat Ile-ANF or vehicle alone (control) in six normal sheep receiving a constant sodium intake. Compared with control observations, ANF infusions induced a threefold rise in venous plasma ANF and a sustained twofold rise in plasma guanosine 3',5'-cyclic monophosphate. Systolic arterial and right atrial pressures fell promptly and remained significantly reduced for the duration of the ANF infusions. These changes were associated with a sustained fall in plasma volume but no activation of renin-angiotensin-aldosterone system. In contrast to the other actions, ANF-induced natriuresis, which was significant in the first 8 h of infusion, was not sustained. These studies show that significant biological effects occur in response to small ANF increments (20-25 pmol/l) and indicate that hemodynamic factors, especially fall in plasma volume, are important in determining the renal response even at near-normal plasma ANF concentrations.