Jean-Pierre Valentin, Changbin Michael Κ Humphreys
Qiu, Eckehart
Wiedemann,
Neutral endopeptidase (EC 3.4.24.11) is a wide spread enzyme that degrades atrial natriuretic pep tide (ANP). We studied the effects of a potent neu tral endopeptidase inhibitor, SQ 28,603, given intravenously (30 m g / k g over 45 min) to anesthe tized, bilaterally nephrectomized Sprague-Dawley rats. Infusion of vehicle alone was accompanied by a modest increase, 3.2 ± 2.2% (mean ± SE), in mean arterial blood pressure (MAP) and a slight rise in hematocrit (Hct) of 0.9 ± 0.7%. After administration of SQ 28,603, MAP fell 3.2 ± 0.5%, and Hct rose 4.9 ± 0.5%, both significantly different from the changes with vehicle alone; the lesser increase in plasma protein concentration (2.5 ± 0.4%) suggested
A
and
an increase in vascular permeability to both plasma protein and fluid similar to that caused by ANP. When SQ 28,603 was given to rats pretreated with rabbit antirat ANP antiserum, blood pressure rose by 3.8 ± 0.5%, and Hct increased by 1.0 ± 0.4%, values very similar to those observed with vehicle alone. Inhibition of neutral endopeptidase there fore amplifies the actions of endogenous ANP on blood pressure and fluid partition. Am J Hypertens 1992;5:88-91 KEY WORDS: Hormone, catabolism, enzyme inhibi tion, nephrectomized, vascular permeability, blood pressure.
trial natriuretic peptide (ANP) is a potent di uretic, natriuretic, and vasodilator sub stance. Furthermore, infusion of ANP is as sociated with an increase in hematocrit in both intact and bilaterally nephrectomized animals. " This rise in hematocrit results from plasma volume con traction caused by a transcapillary shift of plasma fluid and protein to the interstitium. Neutral metalloendo1
1
David Gardner,
3
4-6
peptidase (NEP, EC 3.4.24.11) is a widely distributed enzyme that participates in ANP catabolism. Potent inhibitors of NEP have been reported; these compounds induce a series of responses such as diuresis, natriuresis and increase in cGMP excretion that mimic the effect of administered ANP. However, these responses are not systematically associated with an increase in plasma ANP concentration. ' In the present study conducted using anephric rats, we evaluated the effect of SQ 28,603, a highly potent and specific NEP inhibitor, on blood pressure and on transcapillary shift of plasma fluid to the interstitium. In addition, we assessed the role of ANP in these responses using antiserum directed against rat ANP. 7,8
7 8
9
From the Divisions of Nephrology (J-PV, CQ, MHH) and Endocri nology (EW), San Francisco General Hospital, and Metabolic Research Unit (DG), University of California, San Francisco, California 9 4 1 4 3 . SQ 28,603 was generously provided by Dr. James R. Powell, Squibb Institute for Medical Research, Princeton, NJ. This work was sup ported by grant DK 31623 from the National Institutes of Health, Bethesda, MD, and grant-in-aid 891134 from the American Heart Association. Dr. Valentin was the recipient of a fellowship award from ICI-Pharma, Paris, France. Address correspondence and reprint requests to Michael H. Humphreys, Division of Nephrology, Room 350, Bldg. 100, San Fran cisco General Hospital, San Francisco, CA 94110.
10
METHODS We carried out studies in male Sprague-Dawley rats (Bantin-Kingman, Fremont, CA) weighing 250 to 360 g, and allowed free access to food and water until the day
Downloaded from https://academic.oup.com/ajh/article-abstract/5/2/88/139134 by BIUS Jussieu user on 28 January 2019
Inhibition of Neutral Endopeptidase Amplifies the Effects of Endogenous Atrial Natriuretic Peptide on Blood Pressure and Fluid Partition
441
11
f
f
f
12
RESULTS As shown in Figure 1, the intravenous infusion of the NEPI elicited a reduction in mean arterial pressure (MAP) which was significantly different from the small increase observed in the vehicle group (—3.2 ± 0.5% ν + 3.2 ± 2.2% in the vehicle group; Ρ < .01). A reversible increase in hematocrit was observed after infusion of SQ 28,603: hematocrit rose + 4 . 9 ± 0.5% from a basal value of 44.1 ± 1.7% (P < .001). This effect was signifi cantly (P < .01) greater than that measured in the vehi cle group ( + 0 . 9 ± 0.7%). The increase in plasma pro tein concentration (PPC) induced by the 45-min SQ 28,603 infusion ( + 2 . 5 ± 0.4% from a basal value of 4.78 ± 0.06 g/dL; Ρ < . 0 1 ) was of smaller magnitude than the corresponding change in hematocrit. The decrease in plasma volume calculated from the change in hematocrit amounted to 8 ± 0.9%, whereas it was only 1.6 ± 1.2% for the vehicle. Such a decrease in plasma volume should have increased PPC by approxi mately 9 % , much greater than the observed increase of only 2.5%, suggesting that some loss of plasma protein may have occurred in response to SQ 28,603 infusion. These effects closely match those observed following infusion of A N P . " No significant change in MAP (105 ± 3 ν 103 ± 5 mm Hg), hematocrit (39.1 ± 0.6 ν 38.9 ± 0.4%) and PPC (4.38 ± 0.09 ν 4.35 ± 0.08 g/dL) was observed following the administration of the rabbit anti-rat ANP antiserum at the midpoint of the control period. As de picted in Figure 1, when the neutral endopeptidase in hibitor was infused into rats pretreated with the anti serum, MAP increased by 3.8 ± 0.5%, an effect not different from that observed following administration of the vehicle. As also illustrated in Figure 1, no signifi cant increase in Hct or PPC occurred ( + 1 ± 0.4 and - 0 . 2 ± 0 . 5 % ; P = NS). 4
6
DISCUSSION The results of the present studies demonstrate that infu sion of SQ 28,603, a highly potent neutral endopepti dase inhibitor, is associated with a reversible increase in hematocrit. Because our experiments were carried out in bilaterally nephrectomized animals, negative fluid balance resulting from diuresis and/or natriuresis caused in some way by inhibition of the neutral endopeptidase " cannot account for our results. Con sequently, the observed increase in hematocrit probably resulted from a shift of plasma fluid from the intravascu lar to the extravascular compartment. Furthermore, our results suggest that infusion of SQ 28,603 may increase the vasacular permeability to proteins. This initially was suggested by the observation that the increase in hemat ocrit was much greater on a percentage basis than the increase in PPC. The increase in hematocrit was caused by an estimated decrease in plasma volume of 8%. If this 9,10
7
10
Downloaded from https://academic.oup.com/ajh/article-abstract/5/2/88/139134 by BIUS Jussieu user on 28 January 2019
of the experiment. Animals were anesthetized with an intraperitoneal injection of 100 m g / k g of Inactin (BykGulden, Konstanz, Germany) and placed on a heated table to maintain rectal temperature at 37 ± 0.5 °C. Ani mals underwent tracheostomy and breathed spontane ously; they were prepared for acute experimentation as previously described. Briefly, catheters were inserted into a femoral artery and vein and the right carotid ar tery for sampling blood, infusion of fluids and drugs, and continuous measurement of arterial pressure via a Statham P23 ID pressure transducer (Gould Instru ments, Oxnard, CA) connected to a polygraph (model 7D, Grass Instrument Co., Quincy, MA). Both kidneys were then removed through bilateral retroperitoneal flank incisions. During the surgical preparation, rats re ceived a constant intravenous infusion of plasma sub stitute (Hespan, 6% hetastarch in 0.9% sodium chloride, DuPont Pharmaceuticals, Wilmington, DE) at a rate of 40 //L/min via a syringe pump (Harvard Apparatus, South Natick, MA) until a total volume of 0.5% body weight was administered to replace estimated fluid losses. Thereafter, the infusion rate was reduced to 10 //L/min for the duration of the studies. Experiments were started 45 to 60 min after completion of surgical procedures. Following a 45-min control period, rats received either a neutral endopeptidase inhibitor (SQ 28,603) (NEPI) (3 m g / k g priming dose followed by 0.6 m g / k g / m i n , η = 6) or the vehicle (n = 8). To test the importance of ANP on the response to administered SQ 28,603, the drug was infused in rats given rabbit ANP antiserum (n = 6). The preparation and efficacy of this antiserum have been previously described. A vol ume of 70 //L of the antiserum was injected in the mid dle of the control period, at the beginning of the experi mental period and again at the beginning of the recovery period. The infusion of SQ 28,603 or vehicle was at a rate of 10 //L/min for 45 min. After the experi mental period, a 45-minute recovery period was ob tained. Three 50 //L samples of blood were taken at 15, 30, and 45 min during the basal, experimental, and re covery periods, respectively, for determination of he matocrit and plasma protein concentration. Hematocrit was measured in duplicate on each blood sample by spinning blood at 12,000 rpm in a microcentrifuge (Clay Adams, Parsippany, NJ) for 3 min. Plasma protein con centration was estimated in duplicate by refractometry (National Instrument Co., Baltimore, MD). Estimated changes in plasma volume were calculated according to the following formula: dV = ( 1 0 0 / 1 0 0 - H i ) X [100 X ( H j — H ) / H ] , where dV is the percent change in plasma volume, and Hj and H the initial and final he matocrit, respectively. Data are expressed as means ± SE. Two way analysis of variance and Student's t test were used to assess significance among and be tween groups. Ρ < .05 was considered statistically signif icant.
Φ c (0
Ο
PLASMA PROTEIN CONCENTRATION
vehicle
decrease in plasma volume was the result of loss of plasma water alone, then PPC should have increased by 9 % . Instead, we observed an increase of only 2.5%, suggesting that plasma proteins as well as water leaked out of the vascular tree in response to SQ 28,603 infu sion. However, additional studies are required to assess this question directly. The neutral endopeptidase inhibitor caused a slight but significant decrease in blood pressure compared to the small increase observed in animals receiving vehicle infusion. This is likely a real finding de spite the small changes involved, since treatment with ANP antiserum completely blocked this response, sug gesting that it could be referred to an effect of SQ 28,603 to amplify this effect of endogenous, circulating ANP. Neutral endopeptidase cleaves a variety of peptides including ANP, enkephalins, bradykinin and endothe lin. The effects observed after neutral endopeptidase inhibition may reflect inhibition of the degradation of one or several of these peptides. The observation of hemoconcentration and possibly plasma protein extrav asation identical to that observed as a result of ANP i n f u s i o n led us to question whether ANP could ac count for our results. To test this directly, we carried out experiments on the vasodilator and hemoconcentrating effect of SQ 28,603 in rats pretreated with rabbit anti serum raised against rat ANP. Infusion of SQ 28,603 in animals given this antiserum had major effects. No change in hematocrit, plasma protein concentration and blood pressure occurred over the time course of these experiments; indeed, the effect on each of these vari 13,14
4-6
SQ28,603
SQ + ANP-Ab
ables was nearly superimposable on the trivial change observed in rats receiving vehicle alone (Figure 1). This observation provides evidence that the effects of SQ 28,603 to decrease blood pressure and to increase vascular permeability resulting in hemoconcentration are due to its ability to inhibit the degradation of ANP. Neutral endopeptidase is a ubiquitous enzyme present in brain, kidney, thyroid, parts of the intestine, lung, male genital tract, neutrophils, and fibroblasts, as well as in plasma. In our studies the effects attributed to ANP cannot be explained by an inhibition of intrarenal ANP degradation since our experiments were pre formed in bilaterally nephrectomized animals, but rather to inhibition of extrarenal ANP catabolism. Fur thermore, nephrectomy reduces plasma ANP clearance only slightly and actually enhances the protective action of neutral endopeptidase inhibitors. In summary, infusion of SQ 28,603 elicits an ANPmediated decrease in blood pressure and increase in vascular permeability resulting in a decrease in plasma volume. Thus, neutral endopeptidase inhibitors may be useful agents to explore the various actions of ANP in normal animals and in pathophysiologic conditions such as hypertension. 7,8
8
REFERENCES Brenner BM, Ballermann BJ, Gunning ME, Zeidel ML: Diverse biological actions of atrial natriuretic peptide. Physiol Reviews 1990;70:665-699.
Downloaded from https://academic.oup.com/ajh/article-abstract/5/2/88/139134 by BIUS Jussieu user on 28 January 2019
FIGURE 1. The maximal percent change in blood pressure, hematocrit, and plasma protein concentration after vehicle infusion, infusion of SQ 28,603, or infusion of SQ 28,603 in the presence of anti-ANP anti serum. * = values during SQ 28,603 to be significantly different from re sults with vehicle orSQ + anti-ANP Ab(P
Qiu, Eckehart
Wiedemann,
Neutral endopeptidase (EC 3.4.24.11) is a wide spread enzyme that degrades atrial natriuretic pep tide (ANP). We studied the effects of a potent neu tral endopeptidase inhibitor, SQ 28,603, given intravenously (30 m g / k g over 45 min) to anesthe tized, bilaterally nephrectomized Sprague-Dawley rats. Infusion of vehicle alone was accompanied by a modest increase, 3.2 ± 2.2% (mean ± SE), in mean arterial blood pressure (MAP) and a slight rise in hematocrit (Hct) of 0.9 ± 0.7%. After administration of SQ 28,603, MAP fell 3.2 ± 0.5%, and Hct rose 4.9 ± 0.5%, both significantly different from the changes with vehicle alone; the lesser increase in plasma protein concentration (2.5 ± 0.4%) suggested
A
and
an increase in vascular permeability to both plasma protein and fluid similar to that caused by ANP. When SQ 28,603 was given to rats pretreated with rabbit antirat ANP antiserum, blood pressure rose by 3.8 ± 0.5%, and Hct increased by 1.0 ± 0.4%, values very similar to those observed with vehicle alone. Inhibition of neutral endopeptidase there fore amplifies the actions of endogenous ANP on blood pressure and fluid partition. Am J Hypertens 1992;5:88-91 KEY WORDS: Hormone, catabolism, enzyme inhibi tion, nephrectomized, vascular permeability, blood pressure.
trial natriuretic peptide (ANP) is a potent di uretic, natriuretic, and vasodilator sub stance. Furthermore, infusion of ANP is as sociated with an increase in hematocrit in both intact and bilaterally nephrectomized animals. " This rise in hematocrit results from plasma volume con traction caused by a transcapillary shift of plasma fluid and protein to the interstitium. Neutral metalloendo1
1
David Gardner,
3
4-6
peptidase (NEP, EC 3.4.24.11) is a widely distributed enzyme that participates in ANP catabolism. Potent inhibitors of NEP have been reported; these compounds induce a series of responses such as diuresis, natriuresis and increase in cGMP excretion that mimic the effect of administered ANP. However, these responses are not systematically associated with an increase in plasma ANP concentration. ' In the present study conducted using anephric rats, we evaluated the effect of SQ 28,603, a highly potent and specific NEP inhibitor, on blood pressure and on transcapillary shift of plasma fluid to the interstitium. In addition, we assessed the role of ANP in these responses using antiserum directed against rat ANP. 7,8
7 8
9
From the Divisions of Nephrology (J-PV, CQ, MHH) and Endocri nology (EW), San Francisco General Hospital, and Metabolic Research Unit (DG), University of California, San Francisco, California 9 4 1 4 3 . SQ 28,603 was generously provided by Dr. James R. Powell, Squibb Institute for Medical Research, Princeton, NJ. This work was sup ported by grant DK 31623 from the National Institutes of Health, Bethesda, MD, and grant-in-aid 891134 from the American Heart Association. Dr. Valentin was the recipient of a fellowship award from ICI-Pharma, Paris, France. Address correspondence and reprint requests to Michael H. Humphreys, Division of Nephrology, Room 350, Bldg. 100, San Fran cisco General Hospital, San Francisco, CA 94110.
10
METHODS We carried out studies in male Sprague-Dawley rats (Bantin-Kingman, Fremont, CA) weighing 250 to 360 g, and allowed free access to food and water until the day
Downloaded from https://academic.oup.com/ajh/article-abstract/5/2/88/139134 by BIUS Jussieu user on 28 January 2019
Inhibition of Neutral Endopeptidase Amplifies the Effects of Endogenous Atrial Natriuretic Peptide on Blood Pressure and Fluid Partition
441
11
f
f
f
12
RESULTS As shown in Figure 1, the intravenous infusion of the NEPI elicited a reduction in mean arterial pressure (MAP) which was significantly different from the small increase observed in the vehicle group (—3.2 ± 0.5% ν + 3.2 ± 2.2% in the vehicle group; Ρ < .01). A reversible increase in hematocrit was observed after infusion of SQ 28,603: hematocrit rose + 4 . 9 ± 0.5% from a basal value of 44.1 ± 1.7% (P < .001). This effect was signifi cantly (P < .01) greater than that measured in the vehi cle group ( + 0 . 9 ± 0.7%). The increase in plasma pro tein concentration (PPC) induced by the 45-min SQ 28,603 infusion ( + 2 . 5 ± 0.4% from a basal value of 4.78 ± 0.06 g/dL; Ρ < . 0 1 ) was of smaller magnitude than the corresponding change in hematocrit. The decrease in plasma volume calculated from the change in hematocrit amounted to 8 ± 0.9%, whereas it was only 1.6 ± 1.2% for the vehicle. Such a decrease in plasma volume should have increased PPC by approxi mately 9 % , much greater than the observed increase of only 2.5%, suggesting that some loss of plasma protein may have occurred in response to SQ 28,603 infusion. These effects closely match those observed following infusion of A N P . " No significant change in MAP (105 ± 3 ν 103 ± 5 mm Hg), hematocrit (39.1 ± 0.6 ν 38.9 ± 0.4%) and PPC (4.38 ± 0.09 ν 4.35 ± 0.08 g/dL) was observed following the administration of the rabbit anti-rat ANP antiserum at the midpoint of the control period. As de picted in Figure 1, when the neutral endopeptidase in hibitor was infused into rats pretreated with the anti serum, MAP increased by 3.8 ± 0.5%, an effect not different from that observed following administration of the vehicle. As also illustrated in Figure 1, no signifi cant increase in Hct or PPC occurred ( + 1 ± 0.4 and - 0 . 2 ± 0 . 5 % ; P = NS). 4
6
DISCUSSION The results of the present studies demonstrate that infu sion of SQ 28,603, a highly potent neutral endopepti dase inhibitor, is associated with a reversible increase in hematocrit. Because our experiments were carried out in bilaterally nephrectomized animals, negative fluid balance resulting from diuresis and/or natriuresis caused in some way by inhibition of the neutral endopeptidase " cannot account for our results. Con sequently, the observed increase in hematocrit probably resulted from a shift of plasma fluid from the intravascu lar to the extravascular compartment. Furthermore, our results suggest that infusion of SQ 28,603 may increase the vasacular permeability to proteins. This initially was suggested by the observation that the increase in hemat ocrit was much greater on a percentage basis than the increase in PPC. The increase in hematocrit was caused by an estimated decrease in plasma volume of 8%. If this 9,10
7
10
Downloaded from https://academic.oup.com/ajh/article-abstract/5/2/88/139134 by BIUS Jussieu user on 28 January 2019
of the experiment. Animals were anesthetized with an intraperitoneal injection of 100 m g / k g of Inactin (BykGulden, Konstanz, Germany) and placed on a heated table to maintain rectal temperature at 37 ± 0.5 °C. Ani mals underwent tracheostomy and breathed spontane ously; they were prepared for acute experimentation as previously described. Briefly, catheters were inserted into a femoral artery and vein and the right carotid ar tery for sampling blood, infusion of fluids and drugs, and continuous measurement of arterial pressure via a Statham P23 ID pressure transducer (Gould Instru ments, Oxnard, CA) connected to a polygraph (model 7D, Grass Instrument Co., Quincy, MA). Both kidneys were then removed through bilateral retroperitoneal flank incisions. During the surgical preparation, rats re ceived a constant intravenous infusion of plasma sub stitute (Hespan, 6% hetastarch in 0.9% sodium chloride, DuPont Pharmaceuticals, Wilmington, DE) at a rate of 40 //L/min via a syringe pump (Harvard Apparatus, South Natick, MA) until a total volume of 0.5% body weight was administered to replace estimated fluid losses. Thereafter, the infusion rate was reduced to 10 //L/min for the duration of the studies. Experiments were started 45 to 60 min after completion of surgical procedures. Following a 45-min control period, rats received either a neutral endopeptidase inhibitor (SQ 28,603) (NEPI) (3 m g / k g priming dose followed by 0.6 m g / k g / m i n , η = 6) or the vehicle (n = 8). To test the importance of ANP on the response to administered SQ 28,603, the drug was infused in rats given rabbit ANP antiserum (n = 6). The preparation and efficacy of this antiserum have been previously described. A vol ume of 70 //L of the antiserum was injected in the mid dle of the control period, at the beginning of the experi mental period and again at the beginning of the recovery period. The infusion of SQ 28,603 or vehicle was at a rate of 10 //L/min for 45 min. After the experi mental period, a 45-minute recovery period was ob tained. Three 50 //L samples of blood were taken at 15, 30, and 45 min during the basal, experimental, and re covery periods, respectively, for determination of he matocrit and plasma protein concentration. Hematocrit was measured in duplicate on each blood sample by spinning blood at 12,000 rpm in a microcentrifuge (Clay Adams, Parsippany, NJ) for 3 min. Plasma protein con centration was estimated in duplicate by refractometry (National Instrument Co., Baltimore, MD). Estimated changes in plasma volume were calculated according to the following formula: dV = ( 1 0 0 / 1 0 0 - H i ) X [100 X ( H j — H ) / H ] , where dV is the percent change in plasma volume, and Hj and H the initial and final he matocrit, respectively. Data are expressed as means ± SE. Two way analysis of variance and Student's t test were used to assess significance among and be tween groups. Ρ < .05 was considered statistically signif icant.
Φ c (0
Ο
PLASMA PROTEIN CONCENTRATION
vehicle
decrease in plasma volume was the result of loss of plasma water alone, then PPC should have increased by 9 % . Instead, we observed an increase of only 2.5%, suggesting that plasma proteins as well as water leaked out of the vascular tree in response to SQ 28,603 infu sion. However, additional studies are required to assess this question directly. The neutral endopeptidase inhibitor caused a slight but significant decrease in blood pressure compared to the small increase observed in animals receiving vehicle infusion. This is likely a real finding de spite the small changes involved, since treatment with ANP antiserum completely blocked this response, sug gesting that it could be referred to an effect of SQ 28,603 to amplify this effect of endogenous, circulating ANP. Neutral endopeptidase cleaves a variety of peptides including ANP, enkephalins, bradykinin and endothe lin. The effects observed after neutral endopeptidase inhibition may reflect inhibition of the degradation of one or several of these peptides. The observation of hemoconcentration and possibly plasma protein extrav asation identical to that observed as a result of ANP i n f u s i o n led us to question whether ANP could ac count for our results. To test this directly, we carried out experiments on the vasodilator and hemoconcentrating effect of SQ 28,603 in rats pretreated with rabbit anti serum raised against rat ANP. Infusion of SQ 28,603 in animals given this antiserum had major effects. No change in hematocrit, plasma protein concentration and blood pressure occurred over the time course of these experiments; indeed, the effect on each of these vari 13,14
4-6
SQ28,603
SQ + ANP-Ab
ables was nearly superimposable on the trivial change observed in rats receiving vehicle alone (Figure 1). This observation provides evidence that the effects of SQ 28,603 to decrease blood pressure and to increase vascular permeability resulting in hemoconcentration are due to its ability to inhibit the degradation of ANP. Neutral endopeptidase is a ubiquitous enzyme present in brain, kidney, thyroid, parts of the intestine, lung, male genital tract, neutrophils, and fibroblasts, as well as in plasma. In our studies the effects attributed to ANP cannot be explained by an inhibition of intrarenal ANP degradation since our experiments were pre formed in bilaterally nephrectomized animals, but rather to inhibition of extrarenal ANP catabolism. Fur thermore, nephrectomy reduces plasma ANP clearance only slightly and actually enhances the protective action of neutral endopeptidase inhibitors. In summary, infusion of SQ 28,603 elicits an ANPmediated decrease in blood pressure and increase in vascular permeability resulting in a decrease in plasma volume. Thus, neutral endopeptidase inhibitors may be useful agents to explore the various actions of ANP in normal animals and in pathophysiologic conditions such as hypertension. 7,8
8
REFERENCES Brenner BM, Ballermann BJ, Gunning ME, Zeidel ML: Diverse biological actions of atrial natriuretic peptide. Physiol Reviews 1990;70:665-699.
Downloaded from https://academic.oup.com/ajh/article-abstract/5/2/88/139134 by BIUS Jussieu user on 28 January 2019
FIGURE 1. The maximal percent change in blood pressure, hematocrit, and plasma protein concentration after vehicle infusion, infusion of SQ 28,603, or infusion of SQ 28,603 in the presence of anti-ANP anti serum. * = values during SQ 28,603 to be significantly different from re sults with vehicle orSQ + anti-ANP Ab(P
