(Hypertension. 1995;26:89-94.)
© 1995 American Heart Association, Inc.
Articles |
Endopeptidase Inhibition in Angiotensin-Induced Hypertension
Effect of SCH 39370 in Sheep
From the Department of Endocrinology, Christchurch (New Zealand) Hospital, and Schering-Plough Research, Bloomfield, NJ (E.J.S.).
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Abstract |
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Abstract To assess the efficacy of neutral endopeptidase 24.11 inhibition in the setting of elevated plasma levels of angiotensin II (Ang II), we studied the hemodynamic, renal, and hormonal effects of bolus injections of the potent and specific neutral endopeptidase inhibitor SCH 39370 or vehicle (control) in 10 sheep with Ang II–induced hypertension. Ang II infusion (5 ng/kg per minute for 6 days) sufficient to increase plasma Ang II levels 50% to 100% induced a consistent rise in mean arterial pressure (mean increment, 15 mm Hg; P<.0001) and increased plasma atrial natriuretic peptide (P=.017) and its second messenger cGMP (P=.049). Compared with time-matched control observations after vehicle alone, SCH 39370 (2.5 mg/kg) further increased plasma atrial natriuretic peptide (P=.0006), cGMP (P=.006), and plasma Ang II (P=.054). Systolic and mean arterial pressures tended to fall after SCH 39370, but these changes were not significant compared with control. No significant changes were observed in urinary volume and sodium excretion. Viewed in relation to previous studies in normotensive sheep, the current findings indicate that the vasodepressor response to neutral endopeptidase inhibition is blunted in hyperangiotensinemic sheep, in which neutral endopeptidase inhibition further augments plasma Ang II levels.
Key Words: renin-angiotensin system • natriuretic peptides • blood pressure • hypertension, experimental
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Introduction |
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Atrial natriuretic peptide (ANP) has potent effects on renal, cardiovascular, and hormonal systems. Attention has focused on the therapeutic potential of ANP in the treatment of hypertension and heart failure. However, the use of exogenous ANP is constrained because of its polypeptide nature (necessitating parenteral administration) and short plasma half-life.1 ANP is cleared from plasma by at least two mechanisms: specific receptor uptake2 and enzymatic degradation.3 4 The relative importance of these two pathways is unclear.5 Evidence suggests6 7 that the neutral endopeptidase EC 3.4.24.11 (NEP) initiates the cleavage of ANP to an inactive metabolite known as cleaved ANP.8 9 A variety of NEP inhibitors have been shown to impair clearance of ANP and enhance the biological effects of ANP in both normotensive animals10 and humans.11 12
A variety of animal models have been developed to mimic the different characteristics of human hypertension. Many of these hypertensive animal models have been used in the evaluation of NEP inhibitors. Several studies have shown that NEP inhibitors lower blood pressure in low-renin models of hypertension, such as the deoxycorticosterone acetate–salt rat, but have significantly less hypotensive action in the spontaneously hypertensive rat.13 14 15 However, to our knowledge the efficacy of NEP inhibition has not been studied in an animal model of hypertension with elevated plasma angiotensin II (Ang II) levels, as seen for example in human renovascular hypertension and high-renin essential hypertension. Because NEP inhibitors may further increase plasma Ang II in the setting of hyperangiotensinemia,16 this question requires more formal study.
Accordingly, using sodium-replete sheep we developed a model in which hypertension was induced by long-term low-dose Ang II infusion. We then used this model to assess the hemodynamic, renal, and hormonal effects of the specific and potent NEP inhibitor SCH 39370.
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Methods |
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Ten Coopworth ewes (body weight, 45 to 64 kg) were housed in an air-conditioned, light-controlled room. With sheep under general anesthesia (induced by 20 mg/kg IV thiopental and maintained by halothane, nitrous oxide, and oxygen), a carotid artery was cannulated (Angiocath, 16-gauge, Becton Dickinson) for direct measurement of arterial pressure and heart rate. Three polyethylene catheters were placed in the jugular veins for infusion, blood sampling, and measurement of right atrial pressure (RAP). A Foley catheter (14-gauge) was placed through the urethra into the urinary bladder. The animals were allowed to recover for at least 7 days before experiments were begun.
During the study and beginning at least 5 days before experiments, the animals received a standard diet of sheep nuts and chaff supplemented with 90 mmol sodium administered orally each day as sodium chloride tablets. This provided a daily intake of 100 mmol sodium and 180 mmol potassium. Animals were held in metabolic crates with free access to water.
Each sheep underwent 2 days (days -1 and 0) of baseline recordings immediately before Ang II infusions were begun. Daily recordings were continued from day -1 to the end of the Ang II infusion (day 6). Immediately after recordings on day 0, Ang II (Hypertensin [CIBA-Geigy] in Haemaccel [Behring]) was administered (5 ng/kg per minute IV) in a volume of 2 mL/h for 6 days.
Arterial pressure and RAP were measured daily from 10 to 10:30 AM via the carotid and right atrial cannulas, respectively, with the use of Statham pressure transducers (Spectramed Medical Products) and a chart recorder (Astromed Inc). Heart rate was measured from the arterial pressure trace at preset intervals, and the pressure recordings were manually integrated over 5-minute time periods. Plasma volume was measured by the Evans blue method17 and body weight recorded on days 0 and 6 of infusion.
Daily (24-hour) urine collections were made for the duration of the experiment. Volume was recorded before samples were analyzed for sodium, potassium, and creatinine excretion.
Venous blood samples were drawn at 10 AM on days -1, 0, 1, 3, and 5. Samples were centrifuged at 4°C, and plasma was stored at -20°C (-80°C for ANP) before assay for ANP,18 cGMP,18 Ang II,19 aldosterone,20 and cortisol.21 Hematocrit was determined by the microhematocrit technique at the time of sampling.
On days 5 and 6 of Ang II infusion after a 2-hour baseline hemodynamic recording period, an intravenous bolus injection (10 mL) of either SCH 39370 (2.5 mg/kg, active day) or vehicle (1 mol/L Tris-HCl, pH 7.4, control day) was given in random order at 11 AM. SCH 39370 (N-{N-[1-(S)-carboxy-3-phenylpropyl]-(S)-phenylalanyl}-(S)-isoserine) is a specific and potent inhibitor of NEP. The SCH 39370 concentration required for 50% inhibition of the hydrolysis of [3H]Leu-enkephalin (IC50) is 11.2 nmol/L. SCH 39370 completely prevents the degradation of 10 µmol ANP-(99-126) by NEP at 100 nmol/L with an IC50 value of 5 nmol/L. In contrast, SCH 39370 shows no inhibitory activity against several other proteases, including angiotensin-converting enzyme and carboxypeptidase A at 1 and 10 µmol/L, respectively.13 Arterial pressure, RAP, and heart rate were measured continuously for 4 hours after the bolus as outlined above. Additional urine samples were collected hourly at 1, 2, 3, and 4 hours after bolus administration. Venous blood was drawn at 60, 30, and 0 minutes before bolus and at 15, 30, 45, 60, 90, 120, 150, 180, and 240 minutes after bolus and assayed as above. Additional blood was drawn into heparin immediately before and 30 and 120 minutes after bolus for assessment of NEP inhibitor levels in plasma.22 The total blood volume withdrawn did not exceed 200 mL/d on days 5 and 6 and did not exceed 20 mL on other study days.
The study protocol was approved by the Animal Ethics Committee of the Christchurch School of Medicine.
All results are expressed as mean±SEM. One-way ANOVA with repeated measures (BMDP program 2V)23 was used for assessment of changes with time in response to Ang II infusions (day -1 to day 6). Two-way ANOVA with repeated measures was used for comparison of time and treatment effects with and without SCH 39370 (days 5 and 6). Statistical significance was assumed at a value of P<.05.
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Results |
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All studies were performed without incident, and data collection was complete.
Development of Hypertension
As shown in Fig 1, Ang II infusion significantly
increased plasma Ang II levels (P=.026) and mean arterial
pressure (MAP, P<.0001). MAP continued to rise for 3 to 4
days of infusion, after which it plateaued at a level approximately 15
mm Hg above preinfusion levels. Heart rate and RAP were not
significantly affected (Table 1). Plasma volume tended
to rise after 6 days of infusion (3458±303 mL on day 0 and 3723±329
mL on day 6, P=NS), and body weight tended to fall
(51.6±1.77 kg on day 0 and 50.8±1.59 kg on day 6,
P=NS).
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Plasma ANP (P=.017) and plasma cGMP (P=.049) both rose in response to Ang II–induced hypertension (Fig 1). Plasma aldosterone, cortisol, sodium, and potassium levels were variable and showed no significant change (Table 1). Likewise, urinary volume excretion rates were not significantly affected by Ang II infusion (Table 1). However, urinary sodium (P=.075) and potassium (P=.07) excretion rates tended to be reduced in response to Ang II infusion (Table 1).
Effect of NEP Inhibition
Plasma taken 30 minutes after SCH 39370 showed inhibition of 80%
of the activity of exogenous NEP and 40% of the activity of exogenous
NEP at 120 minutes after bolus compared with time-matched control day
observations (P<.0001). Whereas baseline (prebolus) plasma
ANP levels tended to be lower on the active day compared with the
control day (Fig 2, P=NS), SCH 39370 induced
a prompt and progressive increase in both plasma ANP
(P=.0006) and cGMP (P=.006), peaking within 60
minutes after bolus (Fig 2). Plasma Ang II tended to rise in response
to SCH 39370, whereas there was no consistent change on the control
day. However, the difference between the 2 days did not achieve
statistical significance (P=.054). Plasma aldosterone and
cortisol (Table 2) were not significantly altered by SCH
39370.
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) or control (vehicle, 
) in 10
sheep receiving Ang II infusions (mean±SEM). Plasma ANP
(P=.0006) and cyclic GMP (P=.006) were
significantly elevated by SCH 39370.
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Systolic arterial pressure and MAP tended to fall on the active day although these changes were not significantly different from control (Fig 3). Heart rate (Fig 3), RAP, and hematocrit (Table 2) were not significantly altered by SCH 39370. Likewise, SCH 39370 did not affect urinary volume, sodium, potassium, or creatinine excretion rates (Table 2).
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Discussion |
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These studies show that long-term (6-day) infusions of Ang II given to sodium-replete normal sheep at a dose sufficient to raise basal plasma Ang II levels 50% to 100% induce a consistent rise in arterial pressure of approximately 15 mm Hg. These changes were associated with increases in plasma ANP and cGMP. A single dose of the NEP inhibitor SCH 39370 given to the Ang II–induced hypertensive sheep increased plasma ANP, cGMP, and Ang II levels and was associated with a small but nonsignificant fall in arterial pressure.
Previous studies have shown that in addition to its short-term (vasoconstrictor) pressor action, Ang II also raises blood pressure gradually when given by constant infusion at doses below the threshold for short-term pressor action.24 The mechanism of the slow pressor effect is not clear. Sodium retention and fluid volume expansion, stimulation of the sympathetic nervous system, central nervous system actions, and resetting of baroreceptor reflexes have been invoked. Several studies have demonstrated that Ang II–induced hypertension is salt dependent.25 26 Krieger et al26 showed that long-term low-dose Ang II infusion (3 ng/kg per minute for 7 days) given to dogs receiving a low salt intake (approximately 0.5 mmol sodium/kg per day) had no effect on blood pressure, whereas a switch to high salt intake (approximately 7 mmol sodium/kg per day) induced a gradual rise in blood pressure. They demonstrated sodium retention on day 1 of high salt intake, after which the dogs achieved sodium balance by the second day. The same authors27 subsequently reported that when body weight (and hence fluid volume) is maintained constant, hypertension does not develop, suggesting that the rise in arterial pressure depended on fluid volume expansion. The combined results of these two studies indicate that there is a close relationship between sodium and water retention, blood volume expansion, and cardiac output elevation in the initiation of Ang II hypertension. A similar trend was observed in the present studies on sheep ingesting fluids freely and 2 mmol sodium/kg per day. Trends for plasma volume to increase and urinary sodium excretion to fall below preinfusion levels were associated with increases in plasma ANP and cGMP. However, unlike the previous studies in which plasma aldosterone fell, we observed no significant changes in plasma aldosterone or plasma sodium or potassium, presumably because sodium retention (which diminishes the adrenal response to Ang II) and hypokalemia were avoided.
Previous studies on the effects of NEP inhibition in hypertensive animals have reported discrepant findings. Generally, NEP inhibitors have been shown to be more efficacious in low-renin models of hypertension, such as the deoxycorticosterone acetate–salt rat, compared with spontaneously hypertensive rats.13 14 15 However, the effects of NEP inhibition in animal models of hypertension characterized by enhanced renin-angiotensin-aldosterone activity and hence high circulating Ang II levels have not been reported. Our current findings in hypertensive sheep can be compared with our previous studies in normotensive sheep using an identical experimental protocol. As seen in Fig 4 the effects of NEP inhibition on plasma ANP and cGMP observed in the present study are similar to those previously reported in normotensive sheep.10 However, the fall in arterial pressure (maximal mean decrement of 4% for systolic arterial pressure) observed in the present study in Ang II–induced hypertensive sheep was less than that observed previously (maximal mean decrement of 10%) in normotensive sheep. Thus increased baseline plasma ANP and cGMP levels and raised arterial pressure in the Ang II–infused sheep do not appear to potentiate the effects of the NEP inhibition.
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ANP is not the sole vasoactive substrate for NEP. Others include Ang I and Ang II, bradykinin, and substance P.28 29 Hence the net hemodynamic effect of NEP inhibition may well depend on the proportional effect on vasopressor as opposed to vasodilator substrates. It has been previously shown that inhibition of Ang II degradation appears to be an important action of SCH 39370 administered to rats.30 Richards et al11 have shown that dosing healthy men with the NEP inhibitor candoxatril reduces the metabolic clearance rate of Ang II during stepwise infusions of Ang II that raise plasma Ang II levels threefold to fivefold. The authors raised the possibility that the therapeutic efficacy of NEP inhibition in hypertension and heart failure may be modulated by baseline activity of the renin-angiotensin-aldosterone system. Therefore, patients with high-renin hypertensive states may exhibit significant enhancement of plasma Ang II with administration of an NEP inhibitor, thereby maintaining or exacerbating the hypertensive state. The results of the present study appear to support this hypothesis. SCH 39370 bolus administration, in addition to increasing plasma ANP, also significantly increased plasma Ang II. This may explain why the reduction in arterial pressure in the present study was less than that previously observed in normotensive sheep, in which no changes in plasma Ang II were observed (Fig 4).
Previous studies have demonstrated prominent renal effects of NEP inhibitors, particularly natriuresis.11 12 15 No renal effects were observed in the present study, despite a trend for mild sodium retention induced by Ang II infusion. However, major fluctuations in basal sodium excretion in sheep make interpretation difficult, at least in the setting of elevations in plasma ANP levels as small as seen in the present study. Several studies have demonstrated a blunted renal response to exogenous ANP and NEP inhibition in congestive heart failure, effects attributed to enhanced renin-angiotensin-aldosterone activity.31 32 Margulies et al32 demonstrated that long-term inhibition of Ang II generation with an angiotensin-converting enzyme inhibitor potentiates the renal responses to NEP inhibition in congestive heart failure, whereas low-dose intrarenal Ang II abolished the potentiated responses to NEP inhibition. Thus, enhanced circulating levels of Ang II may have contributed to the lack of renal response to SCH 39370 in the present study.
In conclusion, we have developed a sheep model of hypertension induced by long-term low-dose Ang II infusion sufficient to elevate plasma Ang II levels twofold. MAP is maintained approximately 15 mm Hg above baseline associated with increased plasma ANP and cGMP. Bolus doses of SCH 39370 induced significant rises in plasma ANP and cGMP but only a small (nonsignificant) vasodepressor response. Viewed in relation to previous studies in normotensive sheep, these data suggest that a blunted hypotensive response in hyperangiotensinemic sheep may be caused by augmentation of plasma Ang II levels by NEP inhibition.
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Acknowledgments |
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We are grateful to the Health Research Council and National Heart Foundation of New Zealand for grants supporting this study, to Andrew Campbell for technical assistance with animal experiments, and to the staff of the Christchurch Endocrine Laboratories for hormone assays.
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Footnotes |
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Reprint requests to C.J. Charles, Department of Endocrinology, Christchurch School of Medicine, PO Box 4345, Christchurch, New Zealand.
Received July 26, 1994; first decision November 15, 1994; accepted February 20, 1995.
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