Effects of Natriuretic Peptides and Neutral Endopeptidase 24.11 Inhibition in Isolated Perfused Rat Lung 1- 3
LAN ZHAO, J. M. B. HUGHES, and ROBERT J. D. WINTER Introduction SUMMARY Wehave studied the acute effect of brain natriuretic peptide (BNP)and atrial natriuretic Atrial natriuretic peptide (ANP) has a peptide (ANP) on pulmonary vascular tone in normoxla and acute hypoxia In the absence and presrole in regulating pulmonary vascular ence of a specific Inhibitor of neutral endopeptidase 24.11 (NEI, UK 73,967, candoxatrllat; Pfizer) tone, and it may be involvedin the pathoIn the Isolated and blood-perfused rat lung preparation. Baseline pulmonary artery pressure (Ppa) physiology of hypoxia-induced pulmowas 16.4 ± 0.3 mm Hg In lungs from normoxlc control rats and 22.5 ± 0.3 mm Hg In lungs from nary hypertension (1-4). There is inrats kept In hypoxia (FIOz = 10%) for 7 days. Acute hypoxic pulmonary vasoconstriction (HPV .:\Ppa) was similar In normoxlc control rats (9.5 ± 0.6 mm Hg) and chronically hypoxic rats (9.8 ± 0.9 mm creased synthesis and releaseof ANP and Hg). NEI at 0.07 and 0.2 mg had no effect on baseline Ppa or HPV .:\Ppa. Synthetic BNP at 10 nM increased plasma ANP in both exhad no effect on baseline Ppa but produced a 2.8 ± 0.2 mm Hg reduction In HPV .:\Ppa alone and perimental and clinical pulmonary 2.7 ± 0.2 mm Hg reduction In the presence of 0.07 mg NEI In normoxlc control rats. In contrast, hypertension (5, 6). We and others have ANP at 10 nM produced a significantly greater decrease In HPV .:\Ppa In the presence of 0.07 mg found that physiologic doses of synthetNEI (4.8 ± 0.3 mm Hg, p < 0.05) compared with ANP alone (2.9 ± 0.4 mm Hg), and similar results ic ANP attenuated both developing and were also observed In chronically hypoxic rats. Thus, BNP has a vasodilator effect similar to that fully established pulmonary hypertenof ANP In the pulmonary circulation. Inhibition of neutral endopeptidase 24.11 augments the effects sion and pulmonary vascular remodelof ANP on HPV but does not Influence the pulmonary vascular responses to BNP. ing in rats (7-10). The therapeutic appliAM REV RESPIR DIS 1992; 146:1198-1201 cation of these findings is limited by the short plasma half-life of less than 5 min in all species studied (11, 12). The circulating form of ANP is a 28- serine-dependentclasses (21). In vivo, UK Analysis of human and rat peptide seresidue peptide containing a 17-residue 73,967 is a potent inhibitor of renal and quences for BNP has shown remarkable disulfide-linked ring. The transient bio- extrarenal pathways of ANP metabolism homology with the structure of ANP, logic responses and rapid plasma disap- and is capable of producing a two- to particularly within the ring structure pearance of ANP is brought about by two three-fold increment in plasma ANP (22, formed in both peptides by disulfide principal pathways: degradation by the 23). We have shown that chronic infu- bridging (25). The functional effects of clearance-receptor mechanism (13, 14) sion of UK 73,967increases plasma ANP BNP have not yet been fully defined: in and proteolytic cleavage of the peptide and attenuates pulmonary remodeling, vivo natriuresis and vasorelaxant effects by neutral endopeptidase 24.11 (NE 24.11) pulmonary hypertension, and right ven- have been observed, and studies in cul(15). Neutral endopeptidase 24.11 (mem- tricular hypertrophy in chronic hypoxic tured cells have suggested that BNP acts brane metalloendopeptidase, enkepha- rats (24). via guanylate cyclase-coupled ANP relinase; EC 3.4.24.11), a zinc-dependent Brain natriuretic peptide (BNP), a nov- ceptors (26, 29). Limited information expeptidase purified from renal brush bor- el natriuretic peptide, isolated from por- ists about the effects of BNP or its meder, cleaves the Cys'-Phe" amide bond cine brain, has either 26 or 32 amino acid tabolism in the pulmonary circulation. of ANP, generating the inactive metabo- residues (25).BNP is also synthesizedand The aim of this study was to investigate lite in vitro (15-18). This endopeptidase secreted from the heart in the pig and rat the effects of UK 73,967, an inhibitor of is widely distributed in peripheral tissues (26, 27). In humans a major source of neutral endopeptidase 24.11, and BNP on and at high concentration in lung and circulating BNP is also the heart: in the pulmonary vascular reactivity and to exin kidney (19). Inhibitors of NE 24.11, normal heart the tissue BNP level in the amine the neutral endopeptidase inhibisuch as phosphoramidon and thiophan, ventricle is about 13 times higher than tion interaction with ANP, BNP, and have been reported to prolong the half- in the atrial tissue (28). Furthermore, the life of ANP in vivo and to potentiate the BNP/ANP ratio in the ventricle was conrenal and hemodynamic effects of exog- siderably higher than in the atrium, rais- (Received in original form January 21, 1992 and enous ANP (18, 20). However, these in- ing the possibility that the ventricle in revised form June 15, 1992) hibitors actively inhibit other systems, produces and secretes BNP, especially in From the Department of Medicine, Respiratosuch as the angiotensin converting en- pathophysiologic conditions, such as ry Division, Royal Postgraduate Medical School, zyme (ACE). UK 73,967 (candoxatrilat; heart failure, when the plasma concen- Hammersmith Hospital, London, United Kingdom. Pfizer), the active enantiomer of UK tration is known to increase. Previous z Supported by a grant from the British Heart 69,578, is a potent specific inhibitor of studies have shown that BNP is secreted Foundation. 3 Correspondence and requests for reprints neutral endopeptidase, and in vitro no into the perfusate of the isolated perfused should be addressed to Dr. Robert Winter, Departsignificant inhibitory activity has been heart in a manner similar to ANP (26), ment of Medicine (Respiratory Division), Royal demonstrated toward mammalian pro- further suggesting that BNP may play an Postgraduate Medical School, Hammersmith Hosteases representative of the zinc- and important role as a circulating factor. pital, London W12 ONN, UK. 1
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sodium nitroprusside on pulmonary vascular tone in normoxia and acute hypoxia. Methods Animals and Environmental Chamber Specified pathogen-free albino male Wistar rats (weight range 200 to 250 g) were used throughout. Chronically hypoxic rats were placed in a normobaric hypoxic environmental chamber for 7 days, where the fractional inspired oxygen concentration (F102) was maintained at 100,10, with excesshumidity and carbon dioxide removed by means of scrub filters (30). Gas was sampled periodically and analyzed by mass spectrometer, and the fraction of inspired carbon dioxide (FIe02) was shown to be less than 0.04% at all times. Relative humidity was always less than ambient, and temperature was constantly maintained within 10 C of air temperature. Normoxic control rats were placed in the same room and exposed to the same light-dark cycle. Food, water, and standard laboratory chow weregiven to all animals ad libitum.
Isolated and Blood-Perfused Rat Lung The isolated rat lung preparation of Emery and coworkers was used with minor modification (31). Rats were anesthetized with pentobarbitone (6 mg/IOO g body weight, intraperitoneally). Blood was taken from a normoxie donor rat so that the hematocrit was identical in all groups. The lungs were left in situ after the trachea was cannulated. A metal cannula was inserted into the pulmonary artery through the right ventricle and tied with a ligature. The left atrium was cannulated directly with a preformed cannula, the pressure difference across all cannula being less than 1 mm Hg at flow rates up to 20 ml/min. Blood from control rats was heparinized and perfused into the pulmonary artery by means of a Watson Marlow roller pump (503U; Falmouth, Devon, UK) and a plastic reservoir suspended in the water bath; blood returned to the reservoir from the left atrial cannula. Reservoir temperature was maintained at 380 C. The pulmonary artery pressure was measured using a Gould pressure transducer (P23XL) and a three-channel thermal array recorder (Gould RE 550; Gould, Essex, UK). Nonpulsatile blood flow was kept constant at 18 ml/min, which gave pulmonary artery pressures within the normal range (16.4 ± mm Hg, n = 18 for control rats). Drugs and Reagents Synthetic ANP (rat 3-28, Bachem, CA), sodium nitroprusside (SNP; Sigma Chemical Company, St. Louis, MO) and BNP (rat 32, Peninsula, CA) weredissolved in saline (0.9% NaCI). The neutral endopeptidase inhibitor (NEI) UK 73,967 (candoxatrilat, Pfizer) was also diluted with saline. Ventilation and Reactivity to Hypoxia The lungs were ventilated with 5% CO 2 in air, and blood pH was adjusted by the addition of small quantities of sodium bicarbonate
(1 mmol/L) to maintain reservoir pH at 7.40. Hypoxic vasoconstriction (HPV) was assessed by changing the ventilating gas to 2% O2, 5% CO 2, and 93% N2 and allowing the pulmonary artery pressureto reach a plateau (~Ppa). The interval between successive tests was 8 min. The lungs wereventilated using a pump for small animals at a constant rate (32 breaths/min) to a maximum end-expiratory pressure of 4 mm Hg, giving a tidal volume between 5 and 7 ml. Intratracheal pressure was recorded continuously.
Effects of NElon Pulmonary Vasoreactivity Successivehypoxic challenges (usually three) were given until the increment in pulmonary artery pressure was constant. Pulsed doses of either UK 73,967 (0.07 and 0.2 mg) or vehicle (0.9% NaCl) in a volume less than 20 JlIwere administered into the reservoir during normoxie ventilation to assess their effects on baseline Ppa or during the stable phase of vasoconstriction during hypoxic ventilation to assess their effects on HPV ~Ppa. Effects of AN~ BN~ and SNP on Pulmonary Vasoreactivity Synthetic ANP (300 ng, 10 nM final reservoir concentration) and BNP (360 ng, 10 nM final reservoir concentration) were injected in a volume of 20 IIIinto the pulmonary inflow tubing; SNP (10-11 M final reservoir concentration) was administered into the reservoir during normoxic ventilation or during the stable phase of HPV in the absence and presence of 0.07 mg NEI in separate groups of rats. Data Manipulation and Statistical Analysis Resting Ppa and HPV dPpa were expressed as mean ± standard error of the mean. The effect of drugs on the HPV ~Ppa was expressed as primary data (mm Hg). Comparisons between groups were made using analysis of variance and Student's t test as appropriate. Results
Baseline Pulmonary Artery Pressure and Hypoxic Pulmonary Vasoconstriction In normoxic control rats baseline Ppa was 16.4 ± 0.3 mm Hg (n = 18); HPV L\.Ppa was 9.5 ± 0.6 mm Hg (n = 14). Baseline Ppa was significantly higher in 7-day chronically hypoxic rats (n = 8, 22.5 ± 0.3 mm Hg) compared with normoxie control rats (p < 0.001), but HPV L\.Ppa was similar to that in normoxic control rats (n = 5, 9.8 ± 0.9 mm Hg). Effects of NElon Pulmonary Vasoreactivity Pulsed doses of UK 73,967 (0.07 and 0.2 mg) had no effect on baseline Ppa or HPV L\.Ppa.
Effects of ANP on Pulmonary Reactivity In normoxic control rats, 10 nM ANP had no effect on baseline Ppa. In the stable phase of HPV, 300 ng ANP produced a decrease in HPV L\.Ppa of 2.9 ± 0.4 mm Hg (n = 6). In the presence of 0.07 mg NEI, ANP produced a greater decrease in HPV L\.Ppa of 4.8 ± 0.3 mm Hg (p < 0.05)compared with 300ng ANP alone (figure la and table 1). Similar results were also observed in the 7-day chronically hypoxic rats: HPV L\.Ppa decreased by 2.8 ± 0.3 mm Hg with 300 ng ANP and 5.0 ± 1.6mm Hg (p < 0.05) with 300 ng ANP in the presence of 0.07 mg NEI (all n = 4; figure 2). Effects of BNP on Pulmonary Vasoreactivity BNP at 10 nM had no effect on baseline Ppa. BNP produced 2.8 ± 0.2 mm Hg decreases in HPV L\.Ppa alone and 2.7 ± 0.2 mm Hg decreases in HPV L\.Ppa in the presence of 0.07 mg NEI in normoxie control rats (figure 1b and table 1). Note the longer duration of action of BNP during acute hypoxic vasoconstriction (figure lb) compared withANP (figure la). Effects of SNP on Pulmonary Vasoreactivity SNP at 100 pM had no effects on baseline Ppa, and SNP produced similar falls in HPV L\.Ppa either in the absence of 0.07 mg NEI (5.4 ± 0.4 mm Hg) or in the presence of 0.07 mg NEI (5.4 ± 0.4 mm Hg, n = 4) (table 1). Discussion
Acute hypoxia is a potent stimulus for ANP release from isolated hearts and in vivo (32, 33). The increased plasma ANP observed in rats with hypoxic pulmonary hypertension has been attributed to stimulation of atrial ANP releaseby increased right atrial pressure and distension, coupled with increased ventricular synthesis and secretion in association with hypertrophy (1, 5). In this experiment the isolated and blood-perfused lung preparation excluded the atrium and ventricle, so little or no endogenous ANP was released into the circulation in response to acute hypoxia. This permitted investigation of the effect of inhibition of neutral endopeptidase 24.11 in the pulmonary circulation alone or in the presence of exogenous synthetic ANP. Little information is available about the influence of hypoxia on the synthesis and secretion of BNP; however, the exclusion of the ventricle and atrium, major sites of
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and hypoxic pulmonary vasoconstriction after 7 days of chronic hypoxia exposure suggest that the enzyme activity of neutral endopeptidase 24.11 itself is not induced by chronic hypoxia. Inhibition of neutral endopeptidase 24.11 had no effects on the pulmonary circulation at any dose tested but potentiated the vasodilator effects of ANP during acute hypoxic vasoconstriction, as shown in figure 1a. The data imply that neutral endopeptidase 24.11 is involved in the metabolism of ANP in rat lung and that its inhibition by UK 73,967 may retard this enzymatic degradation. To investigate the specificity of UK 73,967, we studied the effects of sodium nitroprusside, a pulmonary vasodilator that acts via intracellular guanosine 3',5'-cyclic monophosphate (cGMP) (34) but is not a substrate for NE 24.11. The vasodilating effects of SNP during acute HPV response in the presence and absence of UK 73,967 were similar, showing that the effect of UK 73,967 in the pulmonary circulation was specific to ANP. We found that BNP has vasodilator effects in the pulmonary circulation when pulmonary vascular tone was increased during hypoxic ventilation (figure 1b). Natriuretic peptide receptors have been classified into two categories, C (13) and B receptors, which are coupled with particulate guanylate cyclase (35). The vasodilator effects of ANP are mediated by activation of particulate guanylate cyclase with consequent enhancement in production of intracellular guanosine 5'-cyclic monophosphate (cGMP) (36). Recent studies have indicated that human BNP (hBNP) is relatively specific for the B receptor, and the binding ability of hBNP to the C receptor in human lung is 14 times lower than that of a-hANP (28). Nakao and colleagues examined cGMP production of hBNP in cultured human mesangial bovine endothelial cells and found that the potency for cGMP production of hBNP is equal to
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Time (min) Fig. 1. Representative hypoxic challenges in a single isolated perfused lung preparation, plotting pulmonary artery pressure (Ppa) against time in minutes. (a) Left panel shows no effect of ANP on resting tone (before hypoxic challenge); center panel shows a vasodilator effect when ANP is given during hypoxic vasoconstriction; right panel shows an additional vasodilator effect when NEI is given together with AN? (b) Similar to a except that BNP was substituted for AN? No potentiation of the BNP vasodilatation by NEI is seen.
synthesis, also allowed study of the effect of BNP on the pulmonary vasoreactivity, In keeping with its known vasodilator action in the pulmonary circulation, 300 ng synthetic ANP caused vasodilatation during acute hypoxic pulmonary vasoconstriction in both normoxic control and chronically hypoxic rats. The vasodilator effect of ANP disappeared quickly (about 3 min) after the injection, as seen in figure la, consistent with the rapid clearance of exogenous ANP during its passage across the lung, by the clearance receptor mechanism or by neutral endopeptidase 24.11. ANP at 300 ng did not alter baseline pulmonary artery pressure, but it attenuated acute hypox-
ic pulmonary vasoconstriction. Thus, as previously suggested, ANP may act as a modulating factor in the pulmonary vascular bed when pulmonary vascular tone is increased by stimulation of hypoxia. This agrees with previous studies by ourselves and others in which chronic infusion of ANP (300 ng/h/rat) attenuated the hypoxic pulmonary hypertension in rats exposed to chronic hypoxic for 7 days but was without effect on pulmonary artery pressure in normoxic control rats (7, 8). The similar effects of both ANP and NElon resting pulmonary tone
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TABLE 1 ACUTE EFFECTS OF SYNTHETIC ANP, BNP, AND SNP ON PULMONARY VASCULAR TONE IN NORMOXIA AND ACUTE HYPOXIA IN THE PRESENCE AND ABSENCE OF NEI HPV (.6.Ppa) HPV (.6.Ppa) (mm Hg) (mm Hg, 0.07 mg NEf) ANP, 10 nM BNP, 10 nM SNP, 100 pM • p
-2.9 ± 0.4 -2.8 ± 0.2 -5.4 ± 0.4
< 0.05 compared
with ANP alone.
-4.8 ± 0.3* -2.7 ± 0.2 -5.4 ± 0.4
Fig. 2. ANP at 300 ng (open bars, n = 6) produced a similar reduction in HPV APpa in normoxic control (2.9 ± 0.4 mm Hg) and chronically hypoxic rats (2.8 ± 0.3 mm Hg). With 0.07mg NEI, ANP produced 4.8 ± 0.3 mm Hg decreases in HPV APpa in normoxic control rats and 5.0 ± 1.6mm Hg in chronically hypoxic rats(shadedbars,n = 6). *p< 0.05compared with ANP group.
E
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C.
LAN ZHAO, J. M. B. HUGHES, and ROBERT J. D. WINTER Introduction SUMMARY Wehave studied the acute effect of brain natriuretic peptide (BNP)and atrial natriuretic Atrial natriuretic peptide (ANP) has a peptide (ANP) on pulmonary vascular tone in normoxla and acute hypoxia In the absence and presrole in regulating pulmonary vascular ence of a specific Inhibitor of neutral endopeptidase 24.11 (NEI, UK 73,967, candoxatrllat; Pfizer) tone, and it may be involvedin the pathoIn the Isolated and blood-perfused rat lung preparation. Baseline pulmonary artery pressure (Ppa) physiology of hypoxia-induced pulmowas 16.4 ± 0.3 mm Hg In lungs from normoxlc control rats and 22.5 ± 0.3 mm Hg In lungs from nary hypertension (1-4). There is inrats kept In hypoxia (FIOz = 10%) for 7 days. Acute hypoxic pulmonary vasoconstriction (HPV .:\Ppa) was similar In normoxlc control rats (9.5 ± 0.6 mm Hg) and chronically hypoxic rats (9.8 ± 0.9 mm creased synthesis and releaseof ANP and Hg). NEI at 0.07 and 0.2 mg had no effect on baseline Ppa or HPV .:\Ppa. Synthetic BNP at 10 nM increased plasma ANP in both exhad no effect on baseline Ppa but produced a 2.8 ± 0.2 mm Hg reduction In HPV .:\Ppa alone and perimental and clinical pulmonary 2.7 ± 0.2 mm Hg reduction In the presence of 0.07 mg NEI In normoxlc control rats. In contrast, hypertension (5, 6). We and others have ANP at 10 nM produced a significantly greater decrease In HPV .:\Ppa In the presence of 0.07 mg found that physiologic doses of synthetNEI (4.8 ± 0.3 mm Hg, p < 0.05) compared with ANP alone (2.9 ± 0.4 mm Hg), and similar results ic ANP attenuated both developing and were also observed In chronically hypoxic rats. Thus, BNP has a vasodilator effect similar to that fully established pulmonary hypertenof ANP In the pulmonary circulation. Inhibition of neutral endopeptidase 24.11 augments the effects sion and pulmonary vascular remodelof ANP on HPV but does not Influence the pulmonary vascular responses to BNP. ing in rats (7-10). The therapeutic appliAM REV RESPIR DIS 1992; 146:1198-1201 cation of these findings is limited by the short plasma half-life of less than 5 min in all species studied (11, 12). The circulating form of ANP is a 28- serine-dependentclasses (21). In vivo, UK Analysis of human and rat peptide seresidue peptide containing a 17-residue 73,967 is a potent inhibitor of renal and quences for BNP has shown remarkable disulfide-linked ring. The transient bio- extrarenal pathways of ANP metabolism homology with the structure of ANP, logic responses and rapid plasma disap- and is capable of producing a two- to particularly within the ring structure pearance of ANP is brought about by two three-fold increment in plasma ANP (22, formed in both peptides by disulfide principal pathways: degradation by the 23). We have shown that chronic infu- bridging (25). The functional effects of clearance-receptor mechanism (13, 14) sion of UK 73,967increases plasma ANP BNP have not yet been fully defined: in and proteolytic cleavage of the peptide and attenuates pulmonary remodeling, vivo natriuresis and vasorelaxant effects by neutral endopeptidase 24.11 (NE 24.11) pulmonary hypertension, and right ven- have been observed, and studies in cul(15). Neutral endopeptidase 24.11 (mem- tricular hypertrophy in chronic hypoxic tured cells have suggested that BNP acts brane metalloendopeptidase, enkepha- rats (24). via guanylate cyclase-coupled ANP relinase; EC 3.4.24.11), a zinc-dependent Brain natriuretic peptide (BNP), a nov- ceptors (26, 29). Limited information expeptidase purified from renal brush bor- el natriuretic peptide, isolated from por- ists about the effects of BNP or its meder, cleaves the Cys'-Phe" amide bond cine brain, has either 26 or 32 amino acid tabolism in the pulmonary circulation. of ANP, generating the inactive metabo- residues (25).BNP is also synthesizedand The aim of this study was to investigate lite in vitro (15-18). This endopeptidase secreted from the heart in the pig and rat the effects of UK 73,967, an inhibitor of is widely distributed in peripheral tissues (26, 27). In humans a major source of neutral endopeptidase 24.11, and BNP on and at high concentration in lung and circulating BNP is also the heart: in the pulmonary vascular reactivity and to exin kidney (19). Inhibitors of NE 24.11, normal heart the tissue BNP level in the amine the neutral endopeptidase inhibisuch as phosphoramidon and thiophan, ventricle is about 13 times higher than tion interaction with ANP, BNP, and have been reported to prolong the half- in the atrial tissue (28). Furthermore, the life of ANP in vivo and to potentiate the BNP/ANP ratio in the ventricle was conrenal and hemodynamic effects of exog- siderably higher than in the atrium, rais- (Received in original form January 21, 1992 and enous ANP (18, 20). However, these in- ing the possibility that the ventricle in revised form June 15, 1992) hibitors actively inhibit other systems, produces and secretes BNP, especially in From the Department of Medicine, Respiratosuch as the angiotensin converting en- pathophysiologic conditions, such as ry Division, Royal Postgraduate Medical School, zyme (ACE). UK 73,967 (candoxatrilat; heart failure, when the plasma concen- Hammersmith Hospital, London, United Kingdom. Pfizer), the active enantiomer of UK tration is known to increase. Previous z Supported by a grant from the British Heart 69,578, is a potent specific inhibitor of studies have shown that BNP is secreted Foundation. 3 Correspondence and requests for reprints neutral endopeptidase, and in vitro no into the perfusate of the isolated perfused should be addressed to Dr. Robert Winter, Departsignificant inhibitory activity has been heart in a manner similar to ANP (26), ment of Medicine (Respiratory Division), Royal demonstrated toward mammalian pro- further suggesting that BNP may play an Postgraduate Medical School, Hammersmith Hosteases representative of the zinc- and important role as a circulating factor. pital, London W12 ONN, UK. 1
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sodium nitroprusside on pulmonary vascular tone in normoxia and acute hypoxia. Methods Animals and Environmental Chamber Specified pathogen-free albino male Wistar rats (weight range 200 to 250 g) were used throughout. Chronically hypoxic rats were placed in a normobaric hypoxic environmental chamber for 7 days, where the fractional inspired oxygen concentration (F102) was maintained at 100,10, with excesshumidity and carbon dioxide removed by means of scrub filters (30). Gas was sampled periodically and analyzed by mass spectrometer, and the fraction of inspired carbon dioxide (FIe02) was shown to be less than 0.04% at all times. Relative humidity was always less than ambient, and temperature was constantly maintained within 10 C of air temperature. Normoxic control rats were placed in the same room and exposed to the same light-dark cycle. Food, water, and standard laboratory chow weregiven to all animals ad libitum.
Isolated and Blood-Perfused Rat Lung The isolated rat lung preparation of Emery and coworkers was used with minor modification (31). Rats were anesthetized with pentobarbitone (6 mg/IOO g body weight, intraperitoneally). Blood was taken from a normoxie donor rat so that the hematocrit was identical in all groups. The lungs were left in situ after the trachea was cannulated. A metal cannula was inserted into the pulmonary artery through the right ventricle and tied with a ligature. The left atrium was cannulated directly with a preformed cannula, the pressure difference across all cannula being less than 1 mm Hg at flow rates up to 20 ml/min. Blood from control rats was heparinized and perfused into the pulmonary artery by means of a Watson Marlow roller pump (503U; Falmouth, Devon, UK) and a plastic reservoir suspended in the water bath; blood returned to the reservoir from the left atrial cannula. Reservoir temperature was maintained at 380 C. The pulmonary artery pressure was measured using a Gould pressure transducer (P23XL) and a three-channel thermal array recorder (Gould RE 550; Gould, Essex, UK). Nonpulsatile blood flow was kept constant at 18 ml/min, which gave pulmonary artery pressures within the normal range (16.4 ± mm Hg, n = 18 for control rats). Drugs and Reagents Synthetic ANP (rat 3-28, Bachem, CA), sodium nitroprusside (SNP; Sigma Chemical Company, St. Louis, MO) and BNP (rat 32, Peninsula, CA) weredissolved in saline (0.9% NaCI). The neutral endopeptidase inhibitor (NEI) UK 73,967 (candoxatrilat, Pfizer) was also diluted with saline. Ventilation and Reactivity to Hypoxia The lungs were ventilated with 5% CO 2 in air, and blood pH was adjusted by the addition of small quantities of sodium bicarbonate
(1 mmol/L) to maintain reservoir pH at 7.40. Hypoxic vasoconstriction (HPV) was assessed by changing the ventilating gas to 2% O2, 5% CO 2, and 93% N2 and allowing the pulmonary artery pressureto reach a plateau (~Ppa). The interval between successive tests was 8 min. The lungs wereventilated using a pump for small animals at a constant rate (32 breaths/min) to a maximum end-expiratory pressure of 4 mm Hg, giving a tidal volume between 5 and 7 ml. Intratracheal pressure was recorded continuously.
Effects of NElon Pulmonary Vasoreactivity Successivehypoxic challenges (usually three) were given until the increment in pulmonary artery pressure was constant. Pulsed doses of either UK 73,967 (0.07 and 0.2 mg) or vehicle (0.9% NaCl) in a volume less than 20 JlIwere administered into the reservoir during normoxie ventilation to assess their effects on baseline Ppa or during the stable phase of vasoconstriction during hypoxic ventilation to assess their effects on HPV ~Ppa. Effects of AN~ BN~ and SNP on Pulmonary Vasoreactivity Synthetic ANP (300 ng, 10 nM final reservoir concentration) and BNP (360 ng, 10 nM final reservoir concentration) were injected in a volume of 20 IIIinto the pulmonary inflow tubing; SNP (10-11 M final reservoir concentration) was administered into the reservoir during normoxic ventilation or during the stable phase of HPV in the absence and presence of 0.07 mg NEI in separate groups of rats. Data Manipulation and Statistical Analysis Resting Ppa and HPV dPpa were expressed as mean ± standard error of the mean. The effect of drugs on the HPV ~Ppa was expressed as primary data (mm Hg). Comparisons between groups were made using analysis of variance and Student's t test as appropriate. Results
Baseline Pulmonary Artery Pressure and Hypoxic Pulmonary Vasoconstriction In normoxic control rats baseline Ppa was 16.4 ± 0.3 mm Hg (n = 18); HPV L\.Ppa was 9.5 ± 0.6 mm Hg (n = 14). Baseline Ppa was significantly higher in 7-day chronically hypoxic rats (n = 8, 22.5 ± 0.3 mm Hg) compared with normoxie control rats (p < 0.001), but HPV L\.Ppa was similar to that in normoxic control rats (n = 5, 9.8 ± 0.9 mm Hg). Effects of NElon Pulmonary Vasoreactivity Pulsed doses of UK 73,967 (0.07 and 0.2 mg) had no effect on baseline Ppa or HPV L\.Ppa.
Effects of ANP on Pulmonary Reactivity In normoxic control rats, 10 nM ANP had no effect on baseline Ppa. In the stable phase of HPV, 300 ng ANP produced a decrease in HPV L\.Ppa of 2.9 ± 0.4 mm Hg (n = 6). In the presence of 0.07 mg NEI, ANP produced a greater decrease in HPV L\.Ppa of 4.8 ± 0.3 mm Hg (p < 0.05)compared with 300ng ANP alone (figure la and table 1). Similar results were also observed in the 7-day chronically hypoxic rats: HPV L\.Ppa decreased by 2.8 ± 0.3 mm Hg with 300 ng ANP and 5.0 ± 1.6mm Hg (p < 0.05) with 300 ng ANP in the presence of 0.07 mg NEI (all n = 4; figure 2). Effects of BNP on Pulmonary Vasoreactivity BNP at 10 nM had no effect on baseline Ppa. BNP produced 2.8 ± 0.2 mm Hg decreases in HPV L\.Ppa alone and 2.7 ± 0.2 mm Hg decreases in HPV L\.Ppa in the presence of 0.07 mg NEI in normoxie control rats (figure 1b and table 1). Note the longer duration of action of BNP during acute hypoxic vasoconstriction (figure lb) compared withANP (figure la). Effects of SNP on Pulmonary Vasoreactivity SNP at 100 pM had no effects on baseline Ppa, and SNP produced similar falls in HPV L\.Ppa either in the absence of 0.07 mg NEI (5.4 ± 0.4 mm Hg) or in the presence of 0.07 mg NEI (5.4 ± 0.4 mm Hg, n = 4) (table 1). Discussion
Acute hypoxia is a potent stimulus for ANP release from isolated hearts and in vivo (32, 33). The increased plasma ANP observed in rats with hypoxic pulmonary hypertension has been attributed to stimulation of atrial ANP releaseby increased right atrial pressure and distension, coupled with increased ventricular synthesis and secretion in association with hypertrophy (1, 5). In this experiment the isolated and blood-perfused lung preparation excluded the atrium and ventricle, so little or no endogenous ANP was released into the circulation in response to acute hypoxia. This permitted investigation of the effect of inhibition of neutral endopeptidase 24.11 in the pulmonary circulation alone or in the presence of exogenous synthetic ANP. Little information is available about the influence of hypoxia on the synthesis and secretion of BNP; however, the exclusion of the ventricle and atrium, major sites of
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a :I: E
g
and hypoxic pulmonary vasoconstriction after 7 days of chronic hypoxia exposure suggest that the enzyme activity of neutral endopeptidase 24.11 itself is not induced by chronic hypoxia. Inhibition of neutral endopeptidase 24.11 had no effects on the pulmonary circulation at any dose tested but potentiated the vasodilator effects of ANP during acute hypoxic vasoconstriction, as shown in figure 1a. The data imply that neutral endopeptidase 24.11 is involved in the metabolism of ANP in rat lung and that its inhibition by UK 73,967 may retard this enzymatic degradation. To investigate the specificity of UK 73,967, we studied the effects of sodium nitroprusside, a pulmonary vasodilator that acts via intracellular guanosine 3',5'-cyclic monophosphate (cGMP) (34) but is not a substrate for NE 24.11. The vasodilating effects of SNP during acute HPV response in the presence and absence of UK 73,967 were similar, showing that the effect of UK 73,967 in the pulmonary circulation was specific to ANP. We found that BNP has vasodilator effects in the pulmonary circulation when pulmonary vascular tone was increased during hypoxic ventilation (figure 1b). Natriuretic peptide receptors have been classified into two categories, C (13) and B receptors, which are coupled with particulate guanylate cyclase (35). The vasodilator effects of ANP are mediated by activation of particulate guanylate cyclase with consequent enhancement in production of intracellular guanosine 5'-cyclic monophosphate (cGMP) (36). Recent studies have indicated that human BNP (hBNP) is relatively specific for the B receptor, and the binding ability of hBNP to the C receptor in human lung is 14 times lower than that of a-hANP (28). Nakao and colleagues examined cGMP production of hBNP in cultured human mesangial bovine endothelial cells and found that the potency for cGMP production of hBNP is equal to
(8)
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0.07mg NEI 300ng ANP
300ng ANP
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360ng BNP
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Time (min) Fig. 1. Representative hypoxic challenges in a single isolated perfused lung preparation, plotting pulmonary artery pressure (Ppa) against time in minutes. (a) Left panel shows no effect of ANP on resting tone (before hypoxic challenge); center panel shows a vasodilator effect when ANP is given during hypoxic vasoconstriction; right panel shows an additional vasodilator effect when NEI is given together with AN? (b) Similar to a except that BNP was substituted for AN? No potentiation of the BNP vasodilatation by NEI is seen.
synthesis, also allowed study of the effect of BNP on the pulmonary vasoreactivity, In keeping with its known vasodilator action in the pulmonary circulation, 300 ng synthetic ANP caused vasodilatation during acute hypoxic pulmonary vasoconstriction in both normoxic control and chronically hypoxic rats. The vasodilator effect of ANP disappeared quickly (about 3 min) after the injection, as seen in figure la, consistent with the rapid clearance of exogenous ANP during its passage across the lung, by the clearance receptor mechanism or by neutral endopeptidase 24.11. ANP at 300 ng did not alter baseline pulmonary artery pressure, but it attenuated acute hypox-
ic pulmonary vasoconstriction. Thus, as previously suggested, ANP may act as a modulating factor in the pulmonary vascular bed when pulmonary vascular tone is increased by stimulation of hypoxia. This agrees with previous studies by ourselves and others in which chronic infusion of ANP (300 ng/h/rat) attenuated the hypoxic pulmonary hypertension in rats exposed to chronic hypoxic for 7 days but was without effect on pulmonary artery pressure in normoxic control rats (7, 8). The similar effects of both ANP and NElon resting pulmonary tone
-
normoxic
hypoxic
C)
:::I:
TABLE 1 ACUTE EFFECTS OF SYNTHETIC ANP, BNP, AND SNP ON PULMONARY VASCULAR TONE IN NORMOXIA AND ACUTE HYPOXIA IN THE PRESENCE AND ABSENCE OF NEI HPV (.6.Ppa) HPV (.6.Ppa) (mm Hg) (mm Hg, 0.07 mg NEf) ANP, 10 nM BNP, 10 nM SNP, 100 pM • p
-2.9 ± 0.4 -2.8 ± 0.2 -5.4 ± 0.4
< 0.05 compared
with ANP alone.
-4.8 ± 0.3* -2.7 ± 0.2 -5.4 ± 0.4
Fig. 2. ANP at 300 ng (open bars, n = 6) produced a similar reduction in HPV APpa in normoxic control (2.9 ± 0.4 mm Hg) and chronically hypoxic rats (2.8 ± 0.3 mm Hg). With 0.07mg NEI, ANP produced 4.8 ± 0.3 mm Hg decreases in HPV APpa in normoxic control rats and 5.0 ± 1.6mm Hg in chronically hypoxic rats(shadedbars,n = 6). *p< 0.05compared with ANP group.
E
E as c.
C.
