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Hypertension. 1997;30:184-190

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(Hypertension. 1997;30:184-190.)
© 1997 American Heart Association, Inc.

Articles

Neutral Endopeptidase Regulates C-Type Natriuretic Peptide Metabolism But Does Not Potentiate Its Bioactivity In Vivo

Roland R. Brandt; Michael T. Mattingly; Alfredo L. Clavell; Paul L. Barclay; ; John C. Burnett, Jr

From the Cardiorenal Research Laboratory, Division of Cardiovascular Diseases, Mayo Clinic and Foundation, Rochester, Minn; and Pfizer Central Research, Sandwich, Kent, UK.

Correspondence to John C. Burnett, Jr, MD, Cardiorenal Research Laboratory, Mayo Clinic and Foundation, 200 First St SW, Rochester, MN 55905. E-mail burnett.john{at}mayo.edu


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Abstract C-type natriuretic peptide (CNP) is a newly described 22–amino acid peptide of endothelial and renal cell origin with selective cardiovascular actions. Recent in vitro studies have reported that CNP is the most susceptible of all natriuretic peptides to enzymatic degradation by neutral endopeptidase 24.11 (NEP). The present study was undertaken to define the role of NEP in total and regional CNP metabolism and the modulatory actions of NEP inhibition on the biological actions of CNP. CNP (10 ng · kg-1 · min-1) followed by candoxatrilat (240 µg · kg-1 bolus and 8 µg · kg-1 · min-1), a potent and selective NEP inhibitor, was administered intravenously to a group of anesthetized mongrel dogs (group 1) to permit calculation of total metabolic clearance rate (MCR); results were compared with those in a group receiving vehicle infusion followed by candoxatrilat (group 2; both groups, n=7). NEP inhibition increased circulating CNP achieved by exogenous infusion and reduced total MCR in group 1. The regional CNP MCRs increased after CNP administration. While the pulmonary MCR did not change during concomitant candoxatrilat infusion, renal MCR was suppressed. Hemodynamic changes were not different between groups. A mild natriuretic and diuretic effect in association with an increase in circulating and urinary ANP levels was not different between groups. Urinary CNP excretion did not change with CNP infusion but markedly increased after NEP inhibition. We conclude that (1) circulating CNP achieved by exogenous CNP infusion is regulated by NEP in vivo, (2) regional MCRs are heterogeneous with NEP inhibition, (3) NEP inhibition does not potentiate acute cardiovascular actions of CNP, and (4) a mild natriuretic and diuretic effect observed with CNP and NEP inhibition may be ANP dependent.


Key Words: natriuretic peptides • metabolism • kidney • lung • natriuresis • cyclic GMP


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
C-type natriuretic peptide is a 22–amino acid peptide with structural homology to ANP and BNP.1 CNP was initially isolated from porcine brain2 but has also been found in the kidney and intestine in rats and humans.3 4 Recently, CNP presence, gene expression, and secretion have been demonstrated from bovine and human endothelial cells5 6 and renal tubular cells,4 and CNP is detectable in human and canine plasma.5 7 Studies with cells transfected for NPRs have reported that CNP is a specific ligand for the NPR-B receptor as opposed to the receptor for ANP, termed NPR-A.8 9 This receptor is expressed in vascular smooth muscle cells, has been demonstrated in the human kidney,9 10 11 and is linked to particulate guanylate cyclase.8 CNP has potent in vivo and in vitro biological actions that are distinct from ANP and BNP, including potent venous and selective arterial vasodilation, antiproliferative actions, and minimal natriuretic effects.2 7 12 13 14 15 We have previously shown that the natriuretic peptide clearance receptor in vivo participates in CNP clearance metabolism and that clearance receptor antagonism potentiates the cardiovascular actions of CNP.16

NEP 24.11 (EC 3.4.24.11) is a zinc-dependent peptidase that is present in numerous tissues, including lung, kidney, endothelial cells, and plasma, and is most prominent in brush border vesicles of proximal renal tubular cells.17 18 19 20 NEP degrades the cardiac hormone ANP,19 21 and NEP inhibition potentiates the renal actions of ANP.22 A recent study has shown that CNP is highly susceptible to degradation by NEP in vitro.23 However, one should be cautious in extrapolating such data to the in vivo metabolism of CNP, where peptide concentrations and the accessibility of the enzyme may be crucial factors. A preliminary study has reported that the total MCR of CNP in normal humans appears to be similar to that of ANP.24 However, the importance of NEP in the in vivo metabolism of CNP and in its bioactivity remains poorly defined.

The present study was therefore designed with two objectives: (1) to determine the functional significance of NEP for total, pulmonary, renal, and peripheral MCRs, recognizing the wide distribution of NEP in lung, kidney, and blood vessels, and (2) to determine for the first time whether NEP inhibition potentiates the biological actions of CNP in vivo.


*    Methods
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 up arrowAbstract
 up arrowIntroduction
 *Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Surgical Preparation
Experiments were performed in two groups (both n=7) of male mongrel dogs weighing between 18 and 22.5 kg. Animals were maintained on a standard laboratory diet (Lab Canine Diet 5006, Purina Mills) and were fasted overnight on the evening before the experiment with free access to drinking water. All animals were treated and cared for in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Acute studies were performed in dogs anesthetized with pentobarbital sodium (30 mg · kg-1 IV). Supplemental nonhypotensive doses of pentobarbital were given as needed. After intubation with a cuffed endotracheal tube, dogs were mechanically ventilated (Harvard respirator, Harvard Apparatus) with 5 L · min-1 of supplemental oxygen. The right external jugular vein was cannulated, and a flow-directed balloon-tipped thermodilution catheter (model 93A-131-7F, American Edwards Laboratory) was advanced into the pulmonary artery for hemodynamic measurements. The left kidney was exposed through a retroperitoneal flank incision, a calibrated noncannulating electromagnetic flow probe was placed carefully around the renal artery and connected to a flowmeter (model FM 5010, Carolina Medical Electronics) for continuous monitoring of renal blood flow, and a curved 23-gauge needle was inserted into the renal vein for direct sampling of renal venous blood. The ureter was cannulated, and urine was collected on ice for measurement of urine volume, flow rate, and inulin, sodium, and natriuretic peptide concentrations. After a left thoracotomy incision was made at the fourth intercostal space and retraction of the left lung, a small pulmonary vein branch was cannulated for blood sampling. The right femoral vessels were exposed, a calibrated noncannulating electromagnetic flow probe was placed around the femoral artery and connected to a flowmeter for continuous monitoring of femoral blood flow, and a curved 23-gauge needle was inserted into the femoral vein for direct sampling of femoral venous blood. Finally, two peripheral veins were cannulated for drug infusion. Arterial blood pressure measurement and arterial blood sampling were performed through a peripheral artery catheter advanced into the aorta. At the conclusion of the surgical preparation, a priming solution of inulin (ICN Biomedicals) dissolved in isotonic saline was injected, followed by a continuous infusion to achieve a steady state plasma concentration of approximately 30 mg · dL-1. Dogs were suspended in the prone position, and a recovery period of 60 minutes was allowed after surgery. Body temperature was maintained by external warming (infrared heating lamp, Sylvania; heating pad model K-1-3, Gorman-Rupp Industries Division).

In Vivo Experimental Protocol
At the end of the equilibration period, one 30-minute baseline clearance (C1) was obtained. Each clearance period lasting 30 minutes consisted of hemodynamic measurements and withdrawal of 25 mL arterial blood for hormonal analysis at the midpoint of each clearance period. Additional blood samples were collected simultaneously through the femoral vein, renal vein, pulmonary vein, and pulmonary artery catheters for determination of regional clearances and extraction of CNP. For calculation of the MCR of CNP, as well as to determine whether NEP inhibition would potentiate the biological actions of CNP, an intravenous infusion of synthetic CNP (Peninsula Laboratories Inc) was started at a constant rate of 10 ng · kg-1 · min-1 (group 1) and continued throughout the experiment (C2), while group 2 received vehicle only. Subsequently, candoxatrilat (Pfizer Central Research) was given intravenously as a loading dose (240 µg · kg-1) followed by a continuous infusion (8 µg · kg-1 · min-1) to obtain the third and final clearance period (C3). Candoxatrilat (cis-4-{[2-carboxy-3-(2-methoxyethoxy)propyl]-1-cyclopentanecarbonylamino}-1-cyclohexane carboxylic acid, UK-73,967) is a potent, competitive, selective NEP inhibitor (Ki=14 nmol/L).25 This concentration of candoxatrilat has previously been shown to have no effect on other proteases representative of the zinc-, aspartyl-, and serine-dependent classes of enzymes. The dosing regimen was chosen to achieve plasma drug levels of 1200 ng · mL-1, which is 16 times the concentration required to achieve 95% inhibition of NEP (IC95) in vitro.26 Clearance periods C2 and C3 were each preceded by a 15-minute lead-in period to allow for equilibration. The plasma half-life of CNP in humans was found to be 2.6 minutes.24 On the basis of this information, a 15-minute lead-in period equal to five half-lives of CNP was chosen to precede clearance periods C2 and C3 to allow for equilibration. Arterial blood was also sampled at the end of each clearance period and assayed for CNP. CNP concentrations at the end of each clearance period were not different from midclearance levels, verifying that calculations for MCRs were performed at steady state.

Hemodynamic measurements included heart rate, mean arterial pressure (MAP), right atrial pressure (RAP), pulmonary capillary wedge pressure, femoral blood flow, renal blood flow, and cardiac output (CO), which was determined by the thermodilution method using a CO computer (model COM-1, American Edwards Laboratory) in triplicate and then averaged. Systemic vascular resistance (SVR) was calculated as SVR=([MAP-RAP]-1 · CO).

Analytical Methods
Plasma for electrolyte and inulin measurement was obtained from blood collected in heparinized tubes. Blood for hormonal analysis was collected into prechilled EDTA tubes, immediately placed on ice, and centrifuged for 10 minutes at 2500 rpm and 4°C; the plasma was decanted and stored in polystyrene tubes at -20°C until analysis. Urine undergoing measurement for cGMP was heated to 90°C to inhibit degradative enzymatic activity before being stored at -20°C. Plasma and urine sodium were measured using ion-selective electrodes (Beckman Synchron Elise), and plasma and urine lithium levels were determined by flame-emission spectrophotometry (model 357, Instrumentation Laboratory). Plasma and urine inulin concentrations were measured by the anthrone method,27 and the glomerular filtration rate (GFR) was estimated as the clearance of inulin. Proximal Fractional Sodium Reabsorption=1-(Lithium Clearance/GFR) and Distal Fractional Sodium Reabsorption=(Lithium Clearance-Sodium Clearance)/Lithium Clearance were approximated using the lithium clearance technique.28

Plasma samples for cGMP were extracted with ethanol. The radioimmunoassay techniques for hormonal measurements were performed as reported previously by our laboratory and included CNP,7 ANP,29 cGMP,30 plasma renin activity,31 and aldosterone.28 For the CNP radioimmunoassay, plasma and urine samples were extracted by the Vycor glass technique by mixing 1 mL plasma with 0.5 mL Vycor glass suspension for 1 hour at 4°C. The Vycor was washed with water, and the CNP was eluted from the Vycor glass with 60% acetone in 50 mmol/L HCl. Eluates were lyophilized on a Speed-Vac instrument (Savant), and pellets were resuspended in radioimmunoassay buffer (0.1 mol/L sodium phosphate, 0.05 mol/L sodium chloride, 0.1% sodium azide, 0.1% bovine serum albumin). A specific antibody to human CNP recognizing the COOH-terminal CNP1-22 (Peninsula Laboratories Inc) was used in the assay. Intra-assay and interassay variability were both determined to be 5.2%, and the lower limit of detection was 2 pg per tube (6 pg · mL-1 in sample). The cross-reactivity of the ANP and CNP assays to the respective other natriuretic peptide was evaluated by addition of synthetic ANP or CNP to the CNP and ANP assay in concentrations ranging from 0.5 to 500 pg · mL-1. There was no detectable immunoreactivity demonstrating a cross-reactivity of <1%. All samples from an individual animal were subjected to a single thawing only and underwent extraction and radioimmunoassay together. In vitro stability tests were performed to examine the effects of storage on plasma CNP levels. Canine blood was collected into prechilled EDTA tubes, immediately placed on ice, and centrifuged for 10 minutes at 2500 rpm and 4°C, and the plasma was decanted and divided into multiple aliquots; for some experiments 125 pg · mL-1 of synthetic CNP was added to the plasma aliquots. The reference samples (n=5) were extracted and assayed immediately, while the remaining aliquots (n=5) were stored at -20°C over a period of 1 month before assay. The CNP concentration in samples centrifuged, extracted, and assayed immediately after collection was 8.8±0.5 pg · mL-1 and 115.0±2.0 pg · mL-1 after addition of 125 pg · mL-1 of synthetic CNP to plasma samples. From these data, the recovery of CNP was determined to be 85.0±1.6%. The CNP concentration in samples stored over a period of 1 month was not different from that in the reference samples (121.4±3.3 versus 115.0±2.0 pg · mL-1, P=NS). Additional peptidase inhibitors such as aprotinin did not enhance stability when compared with EDTA alone. These results indicate that CNP immunoreactivity remained stable for at least 1 month under these storage conditions with EDTA. The mean CNP concentration of the infusate solution was 104±7% of that originally calculated on a weight basis. Total MCR of CNP was calculated according to the theoretical CNP concentrations in the infusate.

Calculations were as follows: Total MCR of CNP=Infusion Rate of CNP/([CNP]infused-[CNP]baseline), Pulmonary MCR=Cardiac Outputx(Pulmonary Artery Concentration-Pulmonary Vein Concentration)/Pulmonary Artery Concentration, Renal MCR=Renal Blood Flowx(Renal Artery Concentration-Renal Vein Concentration)/Renal Artery Concentration, Femoral MCR=Femoral Blood Flowx(Femoral Artery Concentration-Femoral Vein Concentration)/Femoral Artery Concentration, Urinary CNP Excretion=Urine Flow RatexUrinary CNP Concentration, Urinary ANP Excretion=Urine Flow RatexUrinary ANP Concentration, and Urinary cGMP Excretion=Urine Flow RatexUrinary cGMP Concentration. A negative value for the calculated regional clearance denotes a net secretion across the respective vascular territory.

Statistical Analysis
Results of quantitative studies are expressed as mean±SEM. Statistical comparisons within each group were carried out using ANOVA for repeated measures followed by Fisher's least-significant difference test when appropriate. Data between groups were analyzed by Student's unpaired t test. Statistical significance was accepted at a value of P<.05.


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 *Results
down arrowDiscussion
 down arrowReferences
 
Hormonal Parameters and Metabolic CNP Clearances
The hormonal parameters and total and regional metabolic CNP clearance rates are illustrated in Table 1Down and Figs 1Down and 2Down. There was no difference between groups at baseline. At baseline, CNP was present in the circulation at low concentrations. Low-dose CNP infusion in group 1 resulted in a significant increase in CNP levels that was further potentiated by concomitant NEP inhibition. NEP inhibition with candoxatrilat decreased total MCR in group 1 from 1258±300 to 651±162 mL · min-1 (P<.05, Fig 1Down). Calculated pulmonary, renal, and femoral MCRs all increased during CNP infusion (Fig 2Down). Administration of candoxatrilat decreased only renal MCR, while pulmonary and femoral MCRs did not change. In contrast, in group 2 there were no significant changes of CNP levels and respective MCRs (data not shown) during the experiment. ANP and plasma cGMP levels were not affected by low-dose CNP infusion but increased significantly in both groups after administration of candoxatrilat. Plasma renin activity did not change in either group throughout the experiment, and plasma aldosterone was not different between groups but increased in group 2 during the course of the experiment.


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  		Table 1. Hormonal Responses to CNP and NEP Inhibition



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  		Figure 1. Plasma CNP and total MCR (TMCR) of CNP at baseline, during CNP infusion, and combined CNP and NEP inhibitor (NEPI) infusion (group 1). Data are expressed as mean±SEM. *P<.05 vs baseline, {dagger}P<.05 vs CNP infusion. See "Methods" for calculations.



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  		Figure 2. Pulmonary (solid bars), renal (hatched bars), and femoral (open bars) MCR of plasma CNP at baseline, during CNP infusion, and combined CNP and NEP inhibitor (NEPI) infusion (group 1). Data are expressed as mean±SEM. *P<.05 vs baseline, {dagger}P<.05 vs CNP infusion. See "Methods" for calculations.

Renal Hemodynamic and Excretory Responses
Renal hemodynamic and excretory responses are presented in Table 2Down. There was no difference between groups at baseline. In group 1, urinary sodium excretion increased with low-dose CNP infusion, which was further potentiated by candoxatrilat administration. NEP inhibition alone (group 2) was also natriuretic, and this was not significantly different from that observed in the presence of CNP. In both groups, the natriuretic effect was associated with a decrease in distal fractional sodium reabsorption without any significant changes in proximal sodium reabsorption. Urine flow rate increased modestly in both groups with candoxatrilat administration. These natriuretic and diuretic effects were observed in the absence of any significant changes of renal blood flow or glomerular filtration rate. Urinary CNP excretion increased more than 100-fold in group 1 with NEP inhibition but did not change in group 2. Urinary ANP excretion increased in both groups with administration of candoxatrilat. No significant changes were observed for urinary cGMP excretion in either group.


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  		Table 2. Renal Responses to CNP and NEP Inhibition

Hemodynamic Parameters
The systemic hemodynamic effects observed in both study groups are listed in Table 3Down. There was no difference between groups at baseline. Heart rate did not change throughout the course of the experiment in either group. The decrease in cardiac output occurred to a similar degree in both CNP- and vehicle-treated dogs. Mean arterial pressure increased in the vehicle group but remained unchanged in the CNP group. Right atrial and pulmonary capillary wedge pressures were not different between groups, and CNP infusion decreased filling pressures, an effect that was not further potentiated by addition of candoxatrilat. In contrast, right atrial pressure did not change in the vehicle group. Femoral blood flow decreased significantly in the final clearance period in group 1 and nonsignificantly in group 2.


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  		Table 3. Hemodynamic Responses to CNP and NEP Inhibition


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
To our knowledge, this is the first study to comprehensively investigate the role of NEP under steady state conditions in the in vivo regulation of CNP MCRs and the biological actions of CNP; the findings provide evidence that exogenous CNP is inactivated by NEP in vivo, suggesting that there may be an important role for NEP in endogenous CNP metabolism. This is in accordance with a recent in vitro study showing that CNP is more rapidly hydrolyzed by NEP than any other natriuretic peptide.23 However, susceptibility of a peptide to hydrolysis does not guarantee inactivation in vivo; therefore, it was important to establish the effects of NEP inhibition on the biological response to CNP in the whole animal. The present data demonstrate that the NEP inhibitor candoxatrilat decreases total MCR of CNP by increasing plasma CNP levels. The calculated total MCR in the present study appeared to be similar to that previously reported for CNP in normal humans, taking into account the different body weights.24 NEP inhibition did not completely suppress total MCR, suggesting alternative clearance mechanisms for CNP metabolism such as glomerular filtration and receptor-mediated clearance.8 9 16 In the systemic circulation, the combined decrease in renal and femoral MCRs approximates what would be expected to account for the decrease in total MCR with regard to the portion of the cardiac output supplying both vascular territories. Other vascular territories may also contribute to total MCR according to their regional blood flow.

CNP secretion has been demonstrated from vascular endothelial cells,5 6 and NEP activity is present on vascular endothelium.8 9 This makes the pulmonary circulation, with its large capillary endothelial surface area, a potentially important organ in CNP metabolism. The pulmonary MCR of CNP at baseline was close to zero, indicating a net balance between CNP secretion and clearance. During CNP infusion, pulmonary MCR became significantly positive. While total MCR was significantly decreased by NEP inhibition, candoxatrilat did not suppress pulmonary MCR. This finding would either indicate a relatively low NEP activity in the pulmonary vascular bed as opposed to lung tissue17 or reflect alternative clearance mechanisms such as the receptor-mediated pathway.8 9 This seems to be consistent with a previous study from our laboratory demonstrating that under similar experimental conditions, clearance receptor blockade significantly decreased pulmonary MCR.16 The major role of the clearance receptor compared with NEP in the removal of circulating CNP may be explained with biochemical properties of both systems. CNP binds to clearance receptors with a dissociation constant in the 0.1 nmol/L range, compared with a Michaelis constant of NEP for CNP in the micromolar range.8 9 23 Consequently, CNP will bind preferentially to clearance receptors, even if it has equal access to NEP; this gives support to the hypothesis that in the pulmonary circulation, clearance receptor blockade has a higher impact compared with NEP inhibition. The renal and hindlimb MCRs tended to be negative at baseline, suggesting a possible net secretion of CNP across these circulations. CNP infusion increased the MCR in both vascular territories. In contrast to pulmonary MCR, renal MCR was suppressed by NEP inhibition. The heterogeneous response to NEP between different vascular territories may have been related to different anatomic and hemodynamic parameters, such as capillary surface area and flow rates, or may be related to a differential pattern of NEP activity in vascular territories. Indeed, renal tissue contains a higher NEP activity when compared with pulmonary tissue, which would make the renal circulation more susceptible to NEP inhibition.17 While renal MCR was more responsive to NEP inhibition compared with clearance receptor blockade, the hindlimb circulation did not respond well to either blockade.16 Taken together, the present studies underscore the differential pattern of enzymatic and receptor-mediated CNP clearance in different vascular territories.

Plasma ANP levels increased during combined CNP and candoxatrilat infusion despite significant decreases in right and left atrial pressures, suggesting that decreases in ANP degradation, rather than hemodynamically mediated increases in ANP secretion, were responsible. This is in agreement with a previous study in humans showing that acute increases in plasma CNP were associated with small but significant increments in plasma ANP.24 The small cross-reactivity of CNP in the ANP assay (<1%) could not account for this observation. The mechanism by which plasma ANP is increased by CNP may involve competition between both peptides for clearance receptors or NEP binding sites. Because the concomitant increase in plasma cGMP during coinfusion of CNP and candoxatrilat corresponds with that expected for the observed increment in ANP, most of the increase in plasma cGMP is likely to be independent of CNP increments. CNP is a specific ligand for a transmembranous guanylate cyclase, termed NPR-B receptor, predominantly located on vascular smooth muscle cells and sparse in the vascular endothelium.8 9 10 Failure to observe significant plasma cGMP increments with CNP infusion alone may be attributable to lack of access of circulating CNP to vascular smooth muscle cells and poor cGMP egress into the circulation through a barrier of endothelial cells.

Plasma renin activity did not change throughout the experiment in either group, suggesting that CNP may have no effect on renin release in vivo. The in vitro effect of CNP on cultured juxtaglomerular cells remains to be investigated. Plasma aldosterone levels were not different between groups but increased in the vehicle group during the experiment. Previous reports of the effects of CNP on aldosterone are inconsistent. Stingo et al12 reported increased aldosterone secretion after CNP infusions in anesthetized dogs, and Hunt et al24 have shown a significant decrease in plasma aldosterone in normal humans, whereas Charles et al32 saw little change in conscious sheep. In vitro, a weak inhibitory effect of CNP on aldosterone secretion was observed in bovine adrenal glomerulosa cell cultures.33 The perceived changes in plasma aldosterone may be the result of direct and indirect effects of CNP that are influenced by the duration of the CNP infusion, anesthesia, and species examined.

ANP is cleared from the circulation by the kidney through glomerular filtration.34 35 Although the kidney is known to contain a high concentration of NEP in proximal nephron segments,17 19 renal NEP does not appear to play a major role in ANP clearance from plasma.36 A similar conclusion for the renal CNP clearance may be drawn from the present study. Glomerular filtered ANP is normally degraded rapidly by renal tubular NEP, and little intact ANP appears in the urine.34 35 The detection of low concentrations of CNP in canine urine confirms an earlier report about the presence of CNP immunoreactivity in human urine.4 Urinary CNP excretion did not change during CNP infusion despite a much higher glomerular filtered load of CNP. Therefore, the fractional excretion of CNP, a marker of tubular handling of CNP, actually decreased during CNP infusion, consistent with the nonsaturable nature of the renal tubular NEP system.17 19 After candoxatrilat administration, increases in urinary ANP and CNP excretions were observed, which is in agreement with previous reports.22 34 In the presence of NEP inhibition, glomerular filtered ANP and CNP would be protected from tubular degradation, permitted to reach the more distal nephron segments, and excreted in the urine. Candoxatrilat alone did not increase urinary CNP excretion. Conceivably, additional intrarenal clearance mechanisms compensating for renal NEP inhibition could account for this finding. Alternatively, most of the urinary CNP may be derived from renal CNP production beyond the proximal tubules,37 thus evading tubular NEP and making NEP inhibition ineffective.

CNP infusion alone caused a mild natriuretic effect that was not different between groups. A natriuretic response to CNP was not observed in previous experiments from our group.7 Our finding may be due to anesthesia or a time effect that previously has been shown to cause some mild natriuresis.38 The increase in sodium and water excretion with candoxatrilat was not different between groups and occurred in the absence of any increases in glomerular filtration rate or renal blood flow, indicating that a decrease in tubular sodium reabsorption was probably responsible for the observed natriuresis. The lithium clearance technique localized this effect to the distal tubules. The renal effects in both groups were associated with an increase in urinary ANP excretion and occurred in the presence of circulating ANP levels that have been previously associated with natriuresis and diuresis.28 The degree of the increase in urinary ANP exceeded the increase in plasma ANP concentration, indicating that urinary ANP evades degradation by NEP in the proximal nephron and may play some role in natriuresis at the distal nephron site.22 26 34 While CNP has not been associated with natriuresis as observed with ANP,7 24 data from the present study suggest that although NEP inhibition decreases renal tubular and systemic CNP degradation, the natriuretic and diuretic actions of candoxatrilat in the presence or absence of CNP are not different and may only be dependent on inhibition of systemic or localized renal tubular ANP degradation. While the physiological role of CNP continues to evolve, the presence of both the peptide and its receptor in the kidney suggests a role for CNP at the renal level.4 11 CNP has been shown to possess antiproliferative properties in cultured vascular smooth muscle cells,15 and this certainly raises the question of whether CNP could be a growth regulator of tubular epithelial cells.

The study failed to show a vasodepressor effect of a low-dose CNP infusion alone or in combination with candoxatrilat. Reports of a CNP effect on mean arterial blood pressure have been inconsistent. While a decrease in mean arterial blood pressure was noted during continuous but not bolus infusion of CNP in anesthetized dogs,12 no hypotensive action was noted in conscious sheep and normal humans.24 32 39 A preliminary report showed that NEP inhibition potentiated the hypotensive action of high-dose CNP bolus infusions in conscious monkeys,40 although the relevance to endogenous low CNP levels in cardiovascular physiology is uncertain. Apart from anesthesia, these discrepancies may be explained by species differences, duration of CNP infusion, volume status of the study objects, and volume replacements during the experiment. Nevertheless, our study indicates a relative action of CNP in lowering blood pressure because mean arterial pressure rose in the vehicle group while no change occurred in the CNP group. One might have anticipated a more pronounced hemodynamic effect after administration of candoxatrilat raised circulating CNP to levels that have been previously associated with a decrease in mean arterial blood pressure in a similar experimental model.7 However, NEP hydrolyzes a number of vasoactive peptides,21 and the contribution of these other mediators to the observed cardiovascular effects must be considered. Specifically, NEP has a well-documented role in the inactivation of the vasoconstrictor hormone endothelin-1,41 42 and this might have confounded the CNP action. While right atrial pressure did not change in the vehicle group, there was a significant decrease in the CNP group. This hemodynamic response to CNP may be the result of venous pooling, thereby decreasing venous return in accordance with the specific venodilator actions of CNP in isolated canine veins.7 13 However, such a specific venodilator action has not yet been demonstrated in vivo. A peripheral vascular action is consistent with the report that CNP increases cGMP production in vascular strips and isolated vascular smooth muscle cells.15 The mechanism whereby cardiac output is decreased by CNP remains unclear but may involve a decrease in venous return. This is supported by the observation that cardiac filling pressures tended to decrease during CNP infusion. The possibility of a time-dependent phenomenon must be considered, since cardiac output also decreased in the vehicle group and also has been shown to decrease in the absence of pharmacological agents in the same type of experimental preparation during comparable time periods.38

In summary, the present study reports for the first time on the role of NEP in the action and degradation of circulating CNP achieved by exogenous infusion. The major findings are that NEP inhibition increased circulating CNP and decreased total MCR but did not potentiate the hemodynamic actions of a low-dose CNP infusion. The regional circulations showed a heterogeneous response to NEP inhibition. The observed renal effects during coadministration of CNP and candoxatrilat were most likely related to inactivation of systemic and localized renal ANP degradation. In conclusion, this study suggests that there may be an important role for NEP in endogenous CNP metabolism.


*    Selected Abbreviations and Acronyms
 
ANP = atrial natriuretic peptide
BNP = brain natriuretic peptide
cGMP = cyclic GMP
CNP = C-type natriuretic peptide
MCR = metabolic clearance rate
NEP = neutral endopeptidase 24.11
NPR = natriuretic peptide receptor


*    Acknowledgments
 
This work was supported by grants from the National Heart, Lung, and Blood Institute (HL-36634), the Minnesota Affiliate of the American Heart Association (MHA 112), the National Kidney Foundation of the Upper Midwest, and the Mayo Foundation. The authors gratefully acknowledge the expert technical assistance of Lawrence L. Aarhus and Denise M. Heublein.


*    Footnotes
 
Presented in part at the 9th Scientific Meeting, The American Society of Hypertension, New York, NY, May 11-14, 1994; published in abstract form in Am J Hypertens. 1994;7:139A.

Received August 2, 1996; first decision August 21, 1996; accepted August 21, 1996.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 up arrowDiscussion
 *References
 

  1. Arimura JJ, Minamino N, Kangawa K, Matsuo H. Isolation and identification of C-type natriuretic peptide in chicken brain. Biochem Biophys Res Commun. 1991;174:142-148.[Medline] [Order article via Infotrieve]
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