Metabolism Clinical and Experimental JULY 1992
VOL 41, NO 7
PRELIMINARY REPORT
Neutral Endopeptidase Inhibition Increases the Urinary Excretion and Plasma Levels of Endothelin Zaid Abassi, Eliyahu Golomb,
and Harry R. Keiser
pathway for removal of the circulating potent vasoconstrictor peptide, endothelin (ET), is unclear. To determine the contribution of neutral endopeptidase (NEP) to ET metabolism, urinary excretion (UsrV) and plasma levels of ET (Pm) were measured after infusion of the NEP inhibitor (NEP-I), SQ 29,072 (30 mg/kg [n = lo] and 60 mg/kg [n = S]), in anesthetized Munich Wistar rats. Both doses significantly increased U&I at 30, 60, and 90 minutes; the maximal effect was obtained 30 minutes after infusion, and the response was longer in rats pretreated with the higher dose of the inhibitor. PR. increased 36% and 55% (P 5 .05) at 30 and 120 minutes after injection of the larger dose of SQ 29,072. We also studied the effect of NEP-I on the excretion of exogenous ET after the infusion of 1251-ET(1 pCi) in rats pretreated with either NEP-I or vehicle. In rats treated with the lower dose of the inhibitor, urinary radioactivity increased 2.1- and 1.5-fold (P 5 .05 Y control) after 30 and 60 minutes, respectively. After the higher dose of NEP-I, urinary radioactivity increased 2.7- and 1.7-fold (P < .05). The distribution of the urinary radioactivity as defined by high-performance liquid chromatography (HPLC) showed that intact labeled ET accounts for only 6% to 9% of the total counts recovered in urine from control rats, while the remainder was either free iodine or other products of hydrolysis. Intact ET increased significantly in urine from rats pretreated with NEP-I, to 50% to 56% of total radioactivity in urine. These results demonstrate that the inhibition of NEP increases the urinary excretion and plasma levels of ET, and that NEP plays an important role in the metabolism of ET. This is a US government work. There are no restrictions on its use.
The
E
NDOTHELIN (ET) is a recently discovered, 21-amino acid peptide secreted from endothelial cells.* ET is a very potent vasoconstrictor that may be involved in the regulation of vascular tone.’ It decreases renal blood flow and glomerular filtration rate.2 Although much has been learned about the structure, synthesis, release, and mechanism of action of ET, little is known regarding its metabolism. Recently, Vijayaraghavan et al3 reported that ET is a good substrate for neutral endopeptidase (NEP) 24.11, an enzyme implicated in the degradation of many peptides. The enzyme is located on the basolateral surface of many cells, including the brush border of the renal proximal tubule. Thus, NEP is ideally located to play an important role in the metabolism of ET. To determine the significance of NEP in ET metabolism in vivo, we examined the effect of inhibition of NEP on the inactivation of ET from either endogenous or exogenous sources in rats. MATERIALS
AND
METHODS
lZ51-ET-1 was purchased from Peninsula Labs (Belmont, CA), and the NEP inhibitor (NEP-I), SQ 29,072, was supplied by Squibb (Princeton, NJ). ET levels were determined by a highly sensitive radioimmunoassay (RIA), using a commercial kit (Amersham, Arlington Heights. IL). Me?abo/ism, Vol41, No 7 (July), 1992: pp 683-685
Effect on NEP-I on the Metabolism of Endogenous ET Munich Wistar rats were anesthetized with inactin (100 mgikg, intraperitoneally [IP]) (BYK Guldens, Konstanz, Germany) and prepared for studies of renal clearance. This species was selected because NEP in rodents is found predominantly in the brush border of the kidney, and its inhibition is therefore expected to exert a maximal effect on urinary excretion of the enzyme’s substrate. After a tracheostomy, polyethylene tubes were inserted into the carotid artery for monitoring blood pressure and for periodic blood sampling, into the jugular vein for infusion, and into the urinary bladder for urine collection. Following 60 minutes of equilibration, a 60-minute baseline urine collection was obtained for the measurement of basal urinary flow and ET excretion (UnrV). Then, a single dose of either 30 mg/kg or 60 mg/kg of NEP-I SQ 29.072 dissolved in 1% NaHCO, was infused intrave-
From the Hypertension-Endocrine Branch of the Nation& Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD. Address reprint requests to Zaid Abassi, PhD, Bldg IO, Room 8C103, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892. This is a US government work. There are no restrictions on its use. 00260495/9214107-0001$0.0010 683
684
ABASSI, GOLOMB, AND KEISER
nously and urine was collected into preweighed vials kept on ice for four successive 30-minute periods. Similar doses of NEP-I were used in other studies.“,5 An identical volume of vehicle (0.5 mL) was infused into control rats. To avoid volume depletion, urinary losses and blood samples were replaced at the end of each period by infusion of 0.9% saline and blood cells, respectively. Blood samples (1.5 mL) for the assay of plasma ET (PET) were collected in tubes containing EDTA (3 mg/mL) and aprotonin (500 kU/mL) 30 and 120 minutes after infusion of the inhibitor.
Table 1. Effect of NEP-I SO 29,072 on Urine Radioactivity of Rats Injected With ‘*%ET, % Administered
Radioactivity
Recovered in Urine
Control (n = 6)
Intact ET as % of Radioactivity 30 Minutes
in Urine
30 Minutes
60 Minutes
60 Minutes
0.20 r 0.04
0.38 + 0.04
0.42 2 0.05*
0.56 -e 0.03*
50 2 4”
35 2 4*
0.54 ? O.lO*
0.63 f 0.05*
56 + 4*
51 + 5*t
921
6+1
NEP-I (30 mg/kg, ” = 7) NEP-I (60 mg/kg,
Effect of NEP-I on the Metabolism of Exogenous ET To evaluate the effect of NEP-I on the metabolism of exogenous ET, 1 &i of lzI-ET was infused into another group of rats pretreated with either 30 mgikg or 60 mgikg of the NEP-I or with an equal volume ofvehicle. Urine was collected for two consecutive 30-minute periods, and the distribution of the excreted radioactivity was determined via fractionation on a C-2 cartridge minicolumn (Waters, Milford, MA). After separation, fractions were further analyzed on a reverse-phase high-performance liquid chromatography (HPLC) column (p Bondapak Cl& 3.9 x 300 mm, Waters, Milford, MA) using a linear gradient of 10% to 60% acetonitrile in 0.1% trifluoroacetic acid (TFA). ANOVAwas used for statistical evaluation of repeated measurements. When there was a significant change by ANOVA, a comparison within the group was made with an unpaired t test. P values of .OSor less were considered significant. RESULTS
NOTE. Rats were administered either SQ 29,072 or vehicle 5 minutes before injection of ‘25l-ET, 1 &i (IV). and radioactivity was recovered in urine 30 and 60 minutes later. Intact ET was separated from free ‘*? by C-2 cartridge minicolumn, and was further analyzed by HPLC. *P 5 .05,
compared with control.
tP 2 .05, compared with rats pretreated with 30 mg/kg SQ 29,072.
SQ 29,072 (60 mg/kg) increased PET by 36% (from 9.7 & 0.7 to 13.2 2 0.5, P I .05) and 5.5% (to 15.0 ? 1.7, P I .05) at 30 and 120 minutes after injection of the larger dose of inhibitor, while the smaller dose of NEP-I increased PET by 13% (to 11.0 +- 1.0) and 35% (13.1 t 1.2 pg/mL, P I .06). Infusion of the vehicle had no effect on PET. Glomerular filtration rate and blood pressure remained unchanged throughout the experiment. Effect of NEP-I on Exogenous ET Metabolism
Effect of NEP-I SQ 29,072 on Endogenous ETMetabolism Both doses of NEP-I significantly
n = 7)
(P I .Ol) increased
UETV in rats at 30, 60, and 90 minutes, but not at 120 minutes, when compared with control rats (Fig 1). The maximal effect of the inhibitor was obtained 30 minutes after infusion. The UETV response was longer in rats treated with the higher dose of the inhibitor.
In control rats, only a small fraction of infused radioactivity was recovered in the urine after either 30 minutes (0.2% ? 0.04%) or 60 minutes (0.4% 2 0.04%). In rats treated with the lower dose of NEP-I, total urinary radioactivity increased to 0.42% c 0.05% and 0.56% * 0.03% of the total radioactivity infused after 30 and 60 minutes, respectively (P I .05 v control). After the larger dose of NEP-I, urinary radioactivity increased to 0.54% * 0.1% and 0.63% i 0.05% (P I .05 v control) (Table 1). Intact labeled ET accounted for only 6% to 9% of the total counts recovered in urine from control rats, while the remainder was either free iodine or other products of ET hydrolysis (Fig 2). Intact ET increased significantly (P I ,001) in urine from rats pretreated with low-dose NEP-I, to 50% t 4% and 35% ? 4% of total radioactivity in the urine, while in rats pretreated with high-dose NEP-I, it increased to 56% ? 4% and 51% 2 5% at 30 and 120 minutes, respectively (P I .OOl) (Table 1). DISCUSSION
0
30
60
90
120
TIME (min) Fig 1. UETV in rats before and after administration of either 30 mg/ kg or 60 mg/ kg of the NEP-I, SO 29,072, or an equal amount of vehicle. Data represent the mean * SEM. *P s .05 compared with vehicle. +P 5 .05 compared with lower dose of NEP-I.
In vitro studies have shown that purified NEP from rat kidney membranes rapidly cleaves ET-l, -2, and -3. Vijayaraghavan et al3 showed that the affinities of NEP for ET in vitro are among the highest reported for this enzyme. In this study, we show that a similar degradation pathway occurs in vivo by measuring both the UETV and PET following the administration of a specific NEP-I, SQ 29,072. It was shown previously that bilateral nephrectomy significantly delays the disappearance of ET from plasma and prolongs the half-life of ET in plasma. These changes were associated with prolonged blood pressure elevation after ET administration in rats.6 The authors concluded that the
METABOLISM
685
OF ENDOTHELIN
3000 p
I-
SyntheticET
1
2000
1000
0 0
5
10
Time
after
15
injection
20
25
3.0
(mins)
Fig 2. Reverse-phase HPLC of radioactivity in urine after infusion of ‘WET. Intact ET was separated from its hydrolysis products by a C-2 cartridge minicolumn. followed by HPLC and elution with a linear gradient of 10% to 60% acetonitrile in 0.1% TFA.
kidneys play a role in the removal of ET from the circulation, without further identifying the clearance mechanism. Our findings, that inhibition of NEP significantly increases UurV and PET of endogenous ET and enhances UETV of intact ET. support the conclusion that the kidneys play an
important role in the inactivation of ET and suggest that NEP is a significant part of this inactivation process. Other studies have reported that renal NEP is involved in the metabolism of other peptides, such as atria1 natriuretic factor (ANF). The higher V max and lower Km of NEP for ET than for ANF suggest that the inactivation of ET is more rapid than that of ANF. The high efficiency of NEP in degrading ET may be physiologically important because of the fact that ET is the most potent vasoconstrictor. Many previous studies have shown that the administration of NEP-I to normal rats and patients with heart failure improves renal function and renal responsiveness to infused ANF.4.5 Most of those studies related the improvement to inhibition of ANF degradation. That explanation is contrary to other studies showing that the renal response to both endogenous and exogenous ANF is blunted in heart failure.’ Our finding that NEP inhibition increases and prolongs both Par and UErV of intact ET may bear strongly on such improvement, especially in light of increasing information about its involvement in the regulation of water and sodium balance,8.9 since ET has been found to have diuretic and natriuretic effects at physiological levels.*” Furthermore, increased levels of PETwere found in patients suffering from renal failure. Such increases may contribute to the hypertension found in this condition, and could be attributed to impaired renal NEP activity.rO In conclusion, the increased UmV and Pm after inhibition of NEP indicate that this enzyme plays a major role in the metabolism of ET.
REFERENCES
1 Yanagisawa M, Kurihara H, Kimura S, et al: A novel potent vasoactive peptide produced by vascular endothelial cells. Nature 322-411-415, 1988 2 Badr KF. Murray JJ, Breyer MD, et al: Mesangial cell, glomerular and vascular responses to endothelin in the rat kidney. J Clin Invest 83:336-342. 1989 3 Vijayaraghavan J, Scicli AG, Carretero OA. et al: The hydrolysis of endothelins by neutral endopeptidase 24. I1 (Enkephalinase). J Biol Chem 265:14150-14155. 1990 4 Seymour AA. Fennell SA, Swerdel JN: Potentiation of renal effects of atrial natriuretic factor (99-126) by SQ29,072. Hypertension 14:87-97. 1989 5 Northridge DB, Findley IN, Jardine A, et al: Acute effects of atriopeptidase inhibition on plasma atrial natriuretic factor in chronic heart failure. J Am Coll Cardiol 13:76A, 1989 (abstr)
6. Kohno M, Muirakawa K, Yasunari K, et al: Prolonged blood pressure elevation after endothelin administration in bilaterally nephrectomized rats. Metabolism 38:712-713, 1989 7. Cody RJ, Atlas SA, Laragh JH, et al: Atrial natriuretic factor in normal subjects and in heart failure patients. J Clin Invest 78:1362-1374, 1986 8. Miller WL, Redfield MM, Burnett JC: Integrated cardiac. renal and endocrine actions of endothelin. J Clin Invest X3:317-320. 1989 9. King AJ, Brenner BM, Anderson S: Endothelin: A potent renal and systemic vasoconstrictor peptide. Am J Physiol256:FlO5 lF10.58,1989 10. Predel HG, Meyer-Lehnert H, Backer A. et al: Plasma concentrations of endothelin in patients with ahnormal vascular reactivity. Life Sci 47: 1837-1843, lY90
VOL 41, NO 7
PRELIMINARY REPORT
Neutral Endopeptidase Inhibition Increases the Urinary Excretion and Plasma Levels of Endothelin Zaid Abassi, Eliyahu Golomb,
and Harry R. Keiser
pathway for removal of the circulating potent vasoconstrictor peptide, endothelin (ET), is unclear. To determine the contribution of neutral endopeptidase (NEP) to ET metabolism, urinary excretion (UsrV) and plasma levels of ET (Pm) were measured after infusion of the NEP inhibitor (NEP-I), SQ 29,072 (30 mg/kg [n = lo] and 60 mg/kg [n = S]), in anesthetized Munich Wistar rats. Both doses significantly increased U&I at 30, 60, and 90 minutes; the maximal effect was obtained 30 minutes after infusion, and the response was longer in rats pretreated with the higher dose of the inhibitor. PR. increased 36% and 55% (P 5 .05) at 30 and 120 minutes after injection of the larger dose of SQ 29,072. We also studied the effect of NEP-I on the excretion of exogenous ET after the infusion of 1251-ET(1 pCi) in rats pretreated with either NEP-I or vehicle. In rats treated with the lower dose of the inhibitor, urinary radioactivity increased 2.1- and 1.5-fold (P 5 .05 Y control) after 30 and 60 minutes, respectively. After the higher dose of NEP-I, urinary radioactivity increased 2.7- and 1.7-fold (P < .05). The distribution of the urinary radioactivity as defined by high-performance liquid chromatography (HPLC) showed that intact labeled ET accounts for only 6% to 9% of the total counts recovered in urine from control rats, while the remainder was either free iodine or other products of hydrolysis. Intact ET increased significantly in urine from rats pretreated with NEP-I, to 50% to 56% of total radioactivity in urine. These results demonstrate that the inhibition of NEP increases the urinary excretion and plasma levels of ET, and that NEP plays an important role in the metabolism of ET. This is a US government work. There are no restrictions on its use.
The
E
NDOTHELIN (ET) is a recently discovered, 21-amino acid peptide secreted from endothelial cells.* ET is a very potent vasoconstrictor that may be involved in the regulation of vascular tone.’ It decreases renal blood flow and glomerular filtration rate.2 Although much has been learned about the structure, synthesis, release, and mechanism of action of ET, little is known regarding its metabolism. Recently, Vijayaraghavan et al3 reported that ET is a good substrate for neutral endopeptidase (NEP) 24.11, an enzyme implicated in the degradation of many peptides. The enzyme is located on the basolateral surface of many cells, including the brush border of the renal proximal tubule. Thus, NEP is ideally located to play an important role in the metabolism of ET. To determine the significance of NEP in ET metabolism in vivo, we examined the effect of inhibition of NEP on the inactivation of ET from either endogenous or exogenous sources in rats. MATERIALS
AND
METHODS
lZ51-ET-1 was purchased from Peninsula Labs (Belmont, CA), and the NEP inhibitor (NEP-I), SQ 29,072, was supplied by Squibb (Princeton, NJ). ET levels were determined by a highly sensitive radioimmunoassay (RIA), using a commercial kit (Amersham, Arlington Heights. IL). Me?abo/ism, Vol41, No 7 (July), 1992: pp 683-685
Effect on NEP-I on the Metabolism of Endogenous ET Munich Wistar rats were anesthetized with inactin (100 mgikg, intraperitoneally [IP]) (BYK Guldens, Konstanz, Germany) and prepared for studies of renal clearance. This species was selected because NEP in rodents is found predominantly in the brush border of the kidney, and its inhibition is therefore expected to exert a maximal effect on urinary excretion of the enzyme’s substrate. After a tracheostomy, polyethylene tubes were inserted into the carotid artery for monitoring blood pressure and for periodic blood sampling, into the jugular vein for infusion, and into the urinary bladder for urine collection. Following 60 minutes of equilibration, a 60-minute baseline urine collection was obtained for the measurement of basal urinary flow and ET excretion (UnrV). Then, a single dose of either 30 mg/kg or 60 mg/kg of NEP-I SQ 29.072 dissolved in 1% NaHCO, was infused intrave-
From the Hypertension-Endocrine Branch of the Nation& Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD. Address reprint requests to Zaid Abassi, PhD, Bldg IO, Room 8C103, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892. This is a US government work. There are no restrictions on its use. 00260495/9214107-0001$0.0010 683
684
ABASSI, GOLOMB, AND KEISER
nously and urine was collected into preweighed vials kept on ice for four successive 30-minute periods. Similar doses of NEP-I were used in other studies.“,5 An identical volume of vehicle (0.5 mL) was infused into control rats. To avoid volume depletion, urinary losses and blood samples were replaced at the end of each period by infusion of 0.9% saline and blood cells, respectively. Blood samples (1.5 mL) for the assay of plasma ET (PET) were collected in tubes containing EDTA (3 mg/mL) and aprotonin (500 kU/mL) 30 and 120 minutes after infusion of the inhibitor.
Table 1. Effect of NEP-I SO 29,072 on Urine Radioactivity of Rats Injected With ‘*%ET, % Administered
Radioactivity
Recovered in Urine
Control (n = 6)
Intact ET as % of Radioactivity 30 Minutes
in Urine
30 Minutes
60 Minutes
60 Minutes
0.20 r 0.04
0.38 + 0.04
0.42 2 0.05*
0.56 -e 0.03*
50 2 4”
35 2 4*
0.54 ? O.lO*
0.63 f 0.05*
56 + 4*
51 + 5*t
921
6+1
NEP-I (30 mg/kg, ” = 7) NEP-I (60 mg/kg,
Effect of NEP-I on the Metabolism of Exogenous ET To evaluate the effect of NEP-I on the metabolism of exogenous ET, 1 &i of lzI-ET was infused into another group of rats pretreated with either 30 mgikg or 60 mgikg of the NEP-I or with an equal volume ofvehicle. Urine was collected for two consecutive 30-minute periods, and the distribution of the excreted radioactivity was determined via fractionation on a C-2 cartridge minicolumn (Waters, Milford, MA). After separation, fractions were further analyzed on a reverse-phase high-performance liquid chromatography (HPLC) column (p Bondapak Cl& 3.9 x 300 mm, Waters, Milford, MA) using a linear gradient of 10% to 60% acetonitrile in 0.1% trifluoroacetic acid (TFA). ANOVAwas used for statistical evaluation of repeated measurements. When there was a significant change by ANOVA, a comparison within the group was made with an unpaired t test. P values of .OSor less were considered significant. RESULTS
NOTE. Rats were administered either SQ 29,072 or vehicle 5 minutes before injection of ‘25l-ET, 1 &i (IV). and radioactivity was recovered in urine 30 and 60 minutes later. Intact ET was separated from free ‘*? by C-2 cartridge minicolumn, and was further analyzed by HPLC. *P 5 .05,
compared with control.
tP 2 .05, compared with rats pretreated with 30 mg/kg SQ 29,072.
SQ 29,072 (60 mg/kg) increased PET by 36% (from 9.7 & 0.7 to 13.2 2 0.5, P I .05) and 5.5% (to 15.0 ? 1.7, P I .05) at 30 and 120 minutes after injection of the larger dose of inhibitor, while the smaller dose of NEP-I increased PET by 13% (to 11.0 +- 1.0) and 35% (13.1 t 1.2 pg/mL, P I .06). Infusion of the vehicle had no effect on PET. Glomerular filtration rate and blood pressure remained unchanged throughout the experiment. Effect of NEP-I on Exogenous ET Metabolism
Effect of NEP-I SQ 29,072 on Endogenous ETMetabolism Both doses of NEP-I significantly
n = 7)
(P I .Ol) increased
UETV in rats at 30, 60, and 90 minutes, but not at 120 minutes, when compared with control rats (Fig 1). The maximal effect of the inhibitor was obtained 30 minutes after infusion. The UETV response was longer in rats treated with the higher dose of the inhibitor.
In control rats, only a small fraction of infused radioactivity was recovered in the urine after either 30 minutes (0.2% ? 0.04%) or 60 minutes (0.4% 2 0.04%). In rats treated with the lower dose of NEP-I, total urinary radioactivity increased to 0.42% c 0.05% and 0.56% * 0.03% of the total radioactivity infused after 30 and 60 minutes, respectively (P I .05 v control). After the larger dose of NEP-I, urinary radioactivity increased to 0.54% * 0.1% and 0.63% i 0.05% (P I .05 v control) (Table 1). Intact labeled ET accounted for only 6% to 9% of the total counts recovered in urine from control rats, while the remainder was either free iodine or other products of ET hydrolysis (Fig 2). Intact ET increased significantly (P I ,001) in urine from rats pretreated with low-dose NEP-I, to 50% t 4% and 35% ? 4% of total radioactivity in the urine, while in rats pretreated with high-dose NEP-I, it increased to 56% ? 4% and 51% 2 5% at 30 and 120 minutes, respectively (P I .OOl) (Table 1). DISCUSSION
0
30
60
90
120
TIME (min) Fig 1. UETV in rats before and after administration of either 30 mg/ kg or 60 mg/ kg of the NEP-I, SO 29,072, or an equal amount of vehicle. Data represent the mean * SEM. *P s .05 compared with vehicle. +P 5 .05 compared with lower dose of NEP-I.
In vitro studies have shown that purified NEP from rat kidney membranes rapidly cleaves ET-l, -2, and -3. Vijayaraghavan et al3 showed that the affinities of NEP for ET in vitro are among the highest reported for this enzyme. In this study, we show that a similar degradation pathway occurs in vivo by measuring both the UETV and PET following the administration of a specific NEP-I, SQ 29,072. It was shown previously that bilateral nephrectomy significantly delays the disappearance of ET from plasma and prolongs the half-life of ET in plasma. These changes were associated with prolonged blood pressure elevation after ET administration in rats.6 The authors concluded that the
METABOLISM
685
OF ENDOTHELIN
3000 p
I-
SyntheticET
1
2000
1000
0 0
5
10
Time
after
15
injection
20
25
3.0
(mins)
Fig 2. Reverse-phase HPLC of radioactivity in urine after infusion of ‘WET. Intact ET was separated from its hydrolysis products by a C-2 cartridge minicolumn. followed by HPLC and elution with a linear gradient of 10% to 60% acetonitrile in 0.1% TFA.
kidneys play a role in the removal of ET from the circulation, without further identifying the clearance mechanism. Our findings, that inhibition of NEP significantly increases UurV and PET of endogenous ET and enhances UETV of intact ET. support the conclusion that the kidneys play an
important role in the inactivation of ET and suggest that NEP is a significant part of this inactivation process. Other studies have reported that renal NEP is involved in the metabolism of other peptides, such as atria1 natriuretic factor (ANF). The higher V max and lower Km of NEP for ET than for ANF suggest that the inactivation of ET is more rapid than that of ANF. The high efficiency of NEP in degrading ET may be physiologically important because of the fact that ET is the most potent vasoconstrictor. Many previous studies have shown that the administration of NEP-I to normal rats and patients with heart failure improves renal function and renal responsiveness to infused ANF.4.5 Most of those studies related the improvement to inhibition of ANF degradation. That explanation is contrary to other studies showing that the renal response to both endogenous and exogenous ANF is blunted in heart failure.’ Our finding that NEP inhibition increases and prolongs both Par and UErV of intact ET may bear strongly on such improvement, especially in light of increasing information about its involvement in the regulation of water and sodium balance,8.9 since ET has been found to have diuretic and natriuretic effects at physiological levels.*” Furthermore, increased levels of PETwere found in patients suffering from renal failure. Such increases may contribute to the hypertension found in this condition, and could be attributed to impaired renal NEP activity.rO In conclusion, the increased UmV and Pm after inhibition of NEP indicate that this enzyme plays a major role in the metabolism of ET.
REFERENCES
1 Yanagisawa M, Kurihara H, Kimura S, et al: A novel potent vasoactive peptide produced by vascular endothelial cells. Nature 322-411-415, 1988 2 Badr KF. Murray JJ, Breyer MD, et al: Mesangial cell, glomerular and vascular responses to endothelin in the rat kidney. J Clin Invest 83:336-342. 1989 3 Vijayaraghavan J, Scicli AG, Carretero OA. et al: The hydrolysis of endothelins by neutral endopeptidase 24. I1 (Enkephalinase). J Biol Chem 265:14150-14155. 1990 4 Seymour AA. Fennell SA, Swerdel JN: Potentiation of renal effects of atrial natriuretic factor (99-126) by SQ29,072. Hypertension 14:87-97. 1989 5 Northridge DB, Findley IN, Jardine A, et al: Acute effects of atriopeptidase inhibition on plasma atrial natriuretic factor in chronic heart failure. J Am Coll Cardiol 13:76A, 1989 (abstr)
6. Kohno M, Muirakawa K, Yasunari K, et al: Prolonged blood pressure elevation after endothelin administration in bilaterally nephrectomized rats. Metabolism 38:712-713, 1989 7. Cody RJ, Atlas SA, Laragh JH, et al: Atrial natriuretic factor in normal subjects and in heart failure patients. J Clin Invest 78:1362-1374, 1986 8. Miller WL, Redfield MM, Burnett JC: Integrated cardiac. renal and endocrine actions of endothelin. J Clin Invest X3:317-320. 1989 9. King AJ, Brenner BM, Anderson S: Endothelin: A potent renal and systemic vasoconstrictor peptide. Am J Physiol256:FlO5 lF10.58,1989 10. Predel HG, Meyer-Lehnert H, Backer A. et al: Plasma concentrations of endothelin in patients with ahnormal vascular reactivity. Life Sci 47: 1837-1843, lY90
