Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
461 High-Dose Atrial Natriuretic Factor Enhances Albumin Escape From the Systemic but Not the Pulmonary Circulation Robert S. Zimmerman, Nick C. Trippodo, Allan A. MacPhee, Alberto J. Martinez, and R. Wayne Barbee Downloaded from http://circres.ahajournals.org/ by guest on June 12, 2017 Atrial natriuretic factor (ANF) causes plasma fluid to shift out of the circulation and enhances the escape of radiolabeled albumin. Examination of the mechanisms by which ANF alters microcirculatory fluid and protein transfer will likely require studies in localized vascular regions. This study was aimed at determining the specific organs in which ANF increases the escape of albumin. Anesthetized, splenectomized rats that had both kidneys removed were infused with vehicle alone or rat ANF-(99-126) at 0.025, 0.05, 0.1, or 0.5 p,g * min` * kg` for 2 hours (n=8 per group). Total red cell and plasma volumes were measured with chromium51-labeled erythrocytes and iodine-125-labeled albumin, respectively. At the end of 2 hours, the rats were frozen in liquid nitrogen, and organ blood volumes and tissue 1251-albumin were determined. ANF decreased plasma volume at infusion rates of 0.1 and 0.5 ,ugg min-l kg`. ANF increased the rate at which 121-albumin escaped from the overall circulation at infusion rates of 0.1 and 0.5 p,g* min-l* kg`. At an ANF infusion rate of 0.1 ,ug* min-l kg-', the albumin escape rate increased in the gastrointestinal tract, skeletal muscle, heart, and lungs. At an infusion rate of 0.5 jgg min-1 *kg-', the albumin escape rate increased in the gastrointestinal tract, muscle, and skin, but not the lungs. These findings suggest that at pathophysiological levels, ANF shifts protein out of the circulation in peripheral vascular beds and the lungs and may contribute to pulmonary edema in states such as congestive heart failure. At pharmacological levels, ANF may be protective of the lungs by preventing increased pulmonary albumin escape. (Circulation Research 1990;67:461-468) A trial natriuretic factor (ANF) is elevated in certain disease states such as congestive heart failure, which is associated with both pulmonary and peripheral edema.' Furthermore, some investigators have used ANF in treatment of patients with conditions such as congestive heart failure.2 Because short-term administration of pharmacological doses of ANF results in both enhanced net capillary filtration3-7 and attenuated net capillary absorption,8 it is important to determine whether ANF has a generalized effect on fluid exchange or if its action is confined to certain vascular regions. It is also important to determine whether the effects of ANF on fluid filtration in specific vascular compartFrom the Division of Research, Alton Ochsner Medical Foundation, New Orleans, La. Supported in part by grants from the National Heart, Lung, and Blood Institute (HL-29952), the Barton Hypertension Fund, and the American Heart Association, Louisiana Affiliate. Address for correspondence: Dr. Robert S. Zimmerman, Ochsner Clinic, Department of Endocrinology, 1514 Jefferson Highway, New Orleans, LA 70121. Received September 15, 1988; accepted March 30, 1990. ments differ at pathophysiological and pharmacological infusion rates of ANF. Parkes et a19 recently reported that the decreased plasma volume observed during the first day of long-term infusion of ANF in sheep was associated with an increased rate of escape of `1P-albumin from the circulation. In this present study, we reasoned that an enhanced escape of `1P-albumin from the circulation would be accompanied by increased fluid filtration and that if the escape rate of `251-albumin could be quantitated in specific organs, information with regard to the regional distribution of ANF's action on capillary fluid shift could be obtained. We hypothesized that ANF may contribute to edema formation at pathophysiological levels by enhancing albumin escape from the vasculature in both the pulmonary and peripheral vascular beds but may be protective of the lung at pharmacological levels by preventing increased pulmonary albumin escape by decreasing venous return.7 Therefore, the present study was performed in splenectomized, nephrectomized, anesthetized rats to determine the total-body and organ-specific albumin escape rates in rats infused 462 Circulation Research Vol 67, No 2, August 1990 with ANF at physiological, pathophysiological, and pharmacological doses. Downloaded from http://circres.ahajournals.org/ by guest on June 12, 2017 Materials and Methods Protocol Male Sprague-Dawley rats (Harlan SpragueDawley, Indianapolis) weighing 280-391 g were anesthetized with Inactin (100 mg/kg i.p.) (BYK Gulden, Konstanz, FRG). A 240-gauge polyethylene tube was placed in the trachea to facilitate breathing. To eliminate renal fluid and albumin loss and to ensure that changes in hematocrit were not the result of splenic contraction, both kidneys and the spleen were surgically removed through a midline incision. To facilitate later separation of frozen organs, a thin plastic sheet was positioned between the liver and other viscera before suturing the abdominal incision. By using a femoral approach, catheters were placed in the abdominal aorta for measuring mean arterial pressure and collecting blood samples, in the thoracic inferior vena cava for measuring central venous pressure, and in the abdominal inferior vena cava for injections and infusions. At 15 minutes after surgery, 51Cr-tagged erythrocytes (-1 ,Ci) were injected intravenously. Five minutes later, 1'5I-human serum albumin (-1.5 ,Ci) was injected. In four groups of eight rats, an intravenous infusion of rat ANF (99-126; Peninsula, Belmont, Calif.) at 0.025 ,ugg* min-l* kg-', 0.05 ,gg min-l kg-', 0.1 ,gg min-1 * kg-', and 0.5 g * min-l * kg`1 in 0.9% NaCl (vehicle) was commenced at a flow rate of 5 ,ul/min. The vehicle alone was infused in a separate group of eight rats. Blood samples for measuring hematocrit and "Cr and '`'I radioactivities were collected at 10, 60, and 120 minutes after the start of ANF or vehicle infusion. Mean arterial and central venous pressures were monitored continuously with Gould-Statham transducers (Gould, Cleveland) and a Grass recorder (Quincy, Mass.). Immediately after the last blood sample was taken, the rat was quickly frozen by immersing it in liquid nitrogen for 90 seconds.'0 The thoracic and abdominal viscera and samples of skin and skeletal muscle were dissected and separated from one another while they remained frozen. They were weighed and, together with washings from thawed blood for each organ, were placed in separate vials for determination of 51Cr and 1251 radioactivities. Red Cell, Plasma, and Blood Volumes Erythrocytes were labeled with sodium 51chromate in saline (DuPont Medical Products, Wilmington, Del.) as previously described'1 and mixed in 0.9% NaCl solution to a hematocrit of 40-55%; approximately 1.5 ,Ci of 1251-human serum albumin (Mallinckrodt, St. Louis) was injected intravenously in a volume of 0.15 ml. At 15 minutes after injection of 5"Cr-erythrocytes (10 minutes after injection of 125I-albumin and 10 minutes after the start of ANF or vehicle infusion), triplicate blood samples were taken from the catheter in the abdominal aorta into glass capillary tubes calibrated to 24 1l. Duplicate blood samples were taken at 60 and 120 minutes. "Largevessel" hematocrit (LVHct) was determined with a manual hematocrit reader (Damon/IEC Division, Needham Heights, Mass.). By using a Minaxi AutoGamma 5000 series gamma counter (Packard Instrument Co., Downers Grove, Ill.) the 51Cr and 1251 radioactivities were determined for the blood samples, as well as for triplicate glass capillary tubes containing 24-,l samples of the mixture of 51Crerythrocytes and 10-gul samples of the solution of 1251-albumin used for injections. The counting rates were corrected for background activity and spillover across channels with the COMPUSPHERE software package (Packard). For the first blood sample, red cell volume (RCV), plasma volume (PV), and blood volume (BV) were determined according to the following formulas: RCV=51Cr activity injected (counts per minute) xLVHct/blood 51Cr activity concentration (counts per minute per milliliter); PV= 125j activity injected x (1 -LVHct)/blood "`I activity concentration; and BV= RCV+PV. The whole-body F cell ratio, which represents the ratio of whole-body hematocrit to LVHct, was determined by (RCV/BV)/LVHct. For subsequent blood samples, the following formulas were used: BV=[(51Cr activity injected-cumulative sampling loss of 51Cr activity)/blood 51Cr activity concentration]/F cells; RCV=RCV measured during the first sample-RCV lost through sampling. RCV lost through sampling= MCr activity lost through sampling/! Cr activity per milliliter RCV; 51Cr activity per milliliter RCV=`Cr activity injected/RCV measured during first sample; PV=BV-RCV. Organ and Tissue Blood Volume Corrected 51Cr and '`'I radioactivities for organs and tissues were determined as described above. Organ blood volume (OBV) was calculated according to OBV=(organ 51Cr activity/blood 51Cr activity concentration)/organ F cells, where blood 51Cr activity represents the blood sample taken immediately before the rat was frozen and organ F cells is the ratio of organ hematocrit to LVHct. The organ F cell ratios were determined in separate groups of rats as follows. Male Sprague-Dawley rats weighing 312-370 g were anesthetized and surgically prepared as described above. However, in this series, the infusion of vehicle (n=3) or ANF (n=2; 0.5 ,gg min-1 * kg-') (at the same rates and duration described above) was commenced before labeled erythrocytes and albumin were injected. At 10 and 5 minutes before blood samples were drawn and the rats were frozen, `Cr-erythrocytes and "'I-albumin were injected, respectively, as described above. Therefore, unlike the experiments described above, whereby 1251albumin was allowed to escape from the circulation for 2 hours before the rats were frozen, the rats were frozen in liquid nitrogen at 5 minutes after injection of I-albumin. Based on the whole-body escape rate of 5I-albumin from the circulation as measured in the Zimmerman et al ANF Enhances Albumin Escape experiments described above, it was estimated that approximately 1% of the injected `251-albumin would have crossed the capillaries into the tissues by 5 minutes after injection (see "Results"). Therefore, except for the liver, whose sinusoids are highly permeable to albumin,12 the level of 125I radioactivity measured in each organ at 5 minutes after injection of `251-albumin provided an estimate of the organ plasma volume, while the organ 51Cr activity was a measure of organ erythrocyte volume. Each organ hematocrit was determined according to standard relations (see "Appendix"), which reduced algebraically to the following formula: Organ hematocrit= Downloaded from http://circres.ahajournals.org/ by guest on June 12, 2017 1+(1-LVHct) LVHct x ratio where ratio is the ratio between 51Cr activity/1251 activity in each organ and 51Cr activity/"25I activity in blood, that is, (Cr/I) organ/(Cr/I) blood. This formula, applied to an example of a set of data given by Jodal and Lundgren,13 yielded the same value for tissue hematocrit as reported by these authors, although they used a different method of calculation. Each organ F cell ratio was then determined, and the results were 1.02, 0.96, 0.83, 0.82, and 0.83 for heart, lung, gastrointestinal tract, skin, and skeletal muscle, respectively. The respective coefficients of variation were 1.8%, 1.6%, 6.8%, 3.1%, and 2.2%. This technique does not adequately reflect the F cell ratio for the liver because of very rapid albumin escape into the sinusoids. Hence, we assumed the liver hematocrit was equal to LVHct, which gives an F cell ratio of 1. "'I-Albumin Escape Rate and Organ Mass The rate at which 1251-human serum albumin escaped from the total circulation (`251-AERt) was determined according to `25P-AER,=[(net 125I activity injected-total plasma 125I activity at 120 minutes)/net `1I activity injected]/2 hours x 100, where net 1251 activity injected is the total 125I activity injected minus the cumulative `25I activity removed from the circulation by blood sampling. Total plasma `I activity is the product of plasma 1251 activity concentration and plasma volume at 120 minutes after the start of ANF or vehicle infusion and 120 minutes after injection of 125`-albumin. The rate at which 125`-albumin escaped from the circulation in each organ (`25IjAER ) was determined according to the formula ` I-AER.=tissue `251-albumin activity/net `25I activity injected/2 hours x 100/corrected organ weight. Tissue 125`-albumin activity was taken as the difference in total organ 125I activity and organ plasma `I activity, where the latter was the product of organ plasma volume and plasma `25I activity concentration at 120 minutes. Organ plasma volume was determined as organ blood volumex[1-(organ F cellsxLVHct)]. Organ weight was corrected by subtracting estimated organ blood weight, taken as organ blood volume xblood specific gravity (1.06), from organ wet weight. The `251-albumin activity that accumulated 463 TABLE 1. Weight, F Cell Ratio, and Atrial Natriuretic Factor (ANF) Concentration in Rats Infused With Saline and With ANF in Saline F cell ANF Weight Rat group ratio (g) (pg/mi) 1 317+6 (8) 0.85+±0.01 (8) 57±5 (15) 2 322±5 (8) 0.86±0.01 (8) 146±62 (10) 3 317±6 (8) 0.83±0.02 (8) 336±78 (9)* 4 321±5 (8) 0.84±0.01 (8) 1,232±199 (6)* 5 325±6 (8) 0.81±0.01 (8) 7,734±675 (8)* Values are mean±SEM. Group 1 rats were infused with saline, group 2 was infused with ANF in saline at 0.025 ig. min kg-1, group 3 with ANF in saline at 0.05 ,ug . min-l . kg-', group 4 with ANF in saline at 0.1 yg* min 1 kg-1, and group 5 with ANF in saline at 0.5 gg * min- 1* kg`. ANF concentrations were determined in a separate group of rats. Number of rats in each group are shown in parentheses after the value. *p<0.05 vs. vehicle infusion. in the tissues in whole organs was expressed as tissue `I activity/total 125I activity detected in all organs studiedx 100. Total `I activity detected in all organs represented the activity in blood and tissue. Total skeletal muscle and skin masses were determined as 47% and 20% body weight, respectively. These latter values were determined by completely dissecting one rat weighing 350 g and boiling the muscle from the bones to determine skeletal weight; they compare reasonably well to values determined from previously reported data.14 ANF levels were determined in a separate group of nephrectomized, splenectomized, anesthetized rats after saline infusion or ANF infusion at the same rates and concentrations used in the albumin escape studies. Approximately 2.5 ml blood was obtained after 2 hours of infusion. ANF concentrations were determined by radioimmunoassay after extraction on a C18 Sep-Pak cartridge (Millipore Corp., Milford, Mass.) and elution with 1 ml of 60% acetonitrile in 0.2% ammonium acetate.15 The interassay and intra-assay coefficients of variation were 15% and 6%, respectively. Recovery was 75%. Calculations were accomplished with a spreadsheet software package. Data were analyzed with a one-way analysis of variance. Subsequent comparisons between vehicle- and ANF-infused groups were analyzed by Dunnett's test for one-sided comparisons between treatment and control means. Values of p<0.05 were considered significant. Data are expressed as mean±SEM. Results ANF in control and ANFlevels Circulating infused animals are indicated in Table 1. Body weight and whole-body F cell ratios were similar in the groups and are listed in Table 1. After 2 hours of ANF infusion, mean arterial pressure was significantly lower than control levels of 107±4 mm Hg only at an ANF infusion dose of 0.5 ,gg kg.* min-1, which decreased mean arterial pressure to 95 ± 2 mm Hg (p<0.05) as illustrated in Figure 1. ANF infusion for 2 464 Circulation Research Vol 67, No 2, August 1990 Hemodynamic and Blood Volume Effects of ANF Infusion 120 100 MAP (mm H19) 80 60 40 20 0--0.1 ACVP (mm Hg) -02 -0.3 -0.4: J6 T TL1J pared to control pared to control Downloaded from http://circres.ahajournals.org/ by guest on June 12, 2017 -0.5' 20 18 16 14 RCV 12, 10m (mL/kg) 8. PV (mL/kg) H mm ri FIGURE 1. Changes in mean arteralpressure (MAP), central venous pressure (CVP), red cell volume (RCV), and plasma volume (PV) in anesthetized, nephrectomized, splenectomized rats receiving intravenous infusion (S 1/min) of 0. 9% NaCl (vehicle) or rat atrial natriuretic factor (ANF)-(99-126). n=8 per group. 28. 24 20 16 12' 8' 4' 0.1 .025 0.05 ANF Dose, pggkglmln hours increased LVHct from 50.7+0.3% (control) to 54.6+1%. at 0.1 ,g min- *kg-1 ANF infusion (p<O.O5) and to 56.3 ±0.5% at an ANF infusion rate of 0.5 ,gg min` * kg` (p<0.05); however, lower infusion rates did not significantly affect the LVHct. The increase in hematocrit resulted from a shift in plasma volume from the intravascular space with no change in red cell volume as illustrated in Figure 1. Plasma volume decreased from control levels of 24.9+1.0 to 22.4+1.0 ml kg` after 2 hours of ANF infusion at 0.1 jig * min-l kg` (p<0.05). Plasma volume decreased from 27.0+0.7 to 23.0+0.6 ml-1* kg1 at an ANF infusion rate of 0.5 ,jg. min-1 kg`1 (p<O.OS). No change in plasma volume was observed at lower infusion doses of ANF. Despite the change in plasma volume observed at an ANF infusion rate of 0.1 g min-l kg-l, a significant change in central '. observed only at an ANF infusion rate of 0.5 ,ug. min` kg`. The total-body albumin escape rate did not change at the lowest levels of ANF infusion but increased by 56% at an ANF infusion rate of 0.1 jig * min`-* kg` and by 127% at an ANF infusion rate of 0.5 ,g * min-1 kg1 (Figure 2). ANF selectively enhanced the organ albumin escape rate only at the two highest infusion rates of ANF as shown in Figure 3. At an ANF infusion rate of 0.1 gg min-1 * kg- 1, the albumin escape rate was increased in the heart, lung, gastrointestinal tract, and muscle. At an infusion rate of 0.5 ,ug * min` * kg-', the albumin escape rate increased in the gastrointestinal tract, muscle, and skin, but the lungs were protected. Although the albumin escape rate per gram of tissue suggests that muscle accounts for only a small fraction of the total-body albumin escape, when total organ venous pressure was Zimmerman et al ANF Enhances Albumin Escape 465 present study was aimed at determining the specific organs in which ANF enhances protein transfer. ANF was infused in anesthetized, anephric, and splenectomized rats for 2 hours at physiological, pathophysiological, and pharmacological doses. Plasma volume decreased, while the rate of escape of 1251-albumin from the circulation increased at ANF infusion rates of 0.1 and 0.5 jig. min-l* kg`. At a pathophysiological ANF infusion rate (0.1 ,jg. min-l* kg-'), labeled albumin escaped mainly into the muscle and gastrointestinal tract but also significantly increased in the lung. At pharmacological levels of ANF infusion (0.5 ,gg min`' kg-'), the greatest increase in escaped albumin accumulated in the tissues of the skeletal muscle, skin, and gastrointestinal tract, but not in the lungs. A similar increase in the 1251-albumin content of skeletal muscle after ANF infusion was previously reported by Winquist et a116 in conscious rats. These findings suggest that the action of ANF to shift protein out of the circulation is not selective for various peripheral vascular beds. At high ANF infusion rates, it is likely that the lungs are protected by decreases in pulmonary pressures, as suggested by the decrease in central venous pressure. Irrespective of the mechanism by which ANF increased the transmicrocirculatory movement of albumin, it is reasonable to assume that the increased tissue content of `2I-albumin was attended by some increase in tissue fluid as well. For instance, if ANF facilitated the transmicrocirculatory movement of 12I-albumin by diffusion or vesicular exchange, an enhancement of fluid filtration would likely occur because of the changes in microcirculatory oncotic forces. Another possibility is that ANF altered the microcirculatory hydrostatic forces in favor of filtration, and the increased transport of albumin reflected transmicrocirculatory albumin flux that was directly TOTAL BODY ALBUMIN ESCAPE RATE c 'g 5z 0- Downloaded from http://circres.ahajournals.org/ by guest on June 12, 2017 ANF Infusion Rate, pg/kg/mln FIGURE 2. Rate at which l2SI-albumin escaped from the total circulation over a 2-hour period in the groups of rats featured in Figure 1. ANF, atrial natriuretic factor. accumulation is measured, as shown in Figure 4, muscle albumin escape accounts for the highest percentage of albumin escape. Organ plasma volume is illustrated in Figure 5. Plasma volume significantly decreased in heart and muscle at an ANF infusion rate of 0.1 gg* min`f . kg` and decreased in heart and lung at an ANF infusion rate of 0.5 ,gg min`f . kg-'. Organ blood volume as a percentage of total blood volume is shown in Table 2. No change in blood volume distribution was observed at any dose of ANF infusion. Discussion To obtain direct evidence regarding the mechanisms by which ANF shifts fluid and protein out of the vascular system will likely require studies of regional or local microcirculations. Therefore, this ORGAN ALBUMIN ESCAPE RATES 1.0] 0.8 Heart 0.4 0.3 0.6 GI.. 0.2 9- 0.1 . CD 0.4 0.12~ 1.2 0.10 FIGURE 3. Rate at which escaped l2SI-albumin (2SI-ALB) accumulated in selected tissues over a 2-hour period in the groups of rats featured in Figure 1. GI, gastrointestinal tract; ANF, atrial natriuretic factor. 1.0 Lung m -J Muscle 0.6: 'I18 u'I 0.06 0.04 0.2 p0O.05 0.4 9- p0.0 1 0 compared compared to to 0.16 0.14 control control 0.12 0.10 z) Skin z Liver 0.08 0.04 0.0:4um 0.2 0 .1 0.02 0 0.025 0.05 05 ANF Dose (pg/kg/min) 0 0.025 0.05 0.1 0.5 466 Circulation Research Vol 67, No 2, August 1990 TOTAL ORGAN ALBUMIN ACCUMULATION 15.0 12.5 1.0 0.8 * Heart 0.6- Gl 10.0 7.5. 0.4.. c 5.0 E 2.5- z FIGURE 4. 125I-Albumin activity that accumulated in tissue over a 2-hour period in selected whole organs in the rats featured in Figure 1. GI, gastrointestinal tract; ANF, atrial natriuretic factor. 2.0- .0 1.6 ,fl 1 1.2- Lung Muscle DI 0.8 7.5 5.0- I0 3.0 'p0O.05 compared to control 10 2.5 2.0 Liver **pO.Ol compared to control 8 6- nfi Skin 1.5 Downloaded from http://circres.ahajournals.org/ by guest on June 12, 2017 1.0 0 0.025 0.05 0 0.5 0.1 0.025 0.05 0.1 0.5 ANF Dose (pig/kg/min) coupled to the outward volume flow.'2 Finally, it is possible that ANF altered capillary hydraulic conductivity, as was observed by Huxley et al17 in vessels isolated from the frog mesentery, but this was not addressed in this study. These observations do not exclude the possibility that there were markedly unequal regional differences in coupling of albumin and fluid transport; however, this is unlikely because in most tissues, convection appears to be the dominant mechanism for the transmicrocirculatory transport of macromolecules with dimensions similar to albumin.12 To distinguish the `PI-albumin activity in the tissues from that in the plasma, we determined the plasma volume in each organ. The product of organ plasma volume and plasma concentration of 12I-albumin activity yielded the amount of '1I-albumin activity in the plasma; this subtracted from the total `PI-albumin activity in each organ gave the amount that accumu- lated in the tissues. A critical aspect of this approach is the measurement of organ plasma volume, which in turn depends on the determination of organ hematocrit. Partly because the hematocrit in the microcirculation is less than that in large vessels18 and partly because of albumin leakage from the vascular space, the whole-body hematocrit (measured RCV/[measured RCV+measured PV]) is less than LVHct.1920 If the ratio of whole-body hematocrit to LVHct (F cell ratio) is known for a certain set of experimental conditions, this parameter can be used to convert LVHct to the whole-body value. This same approach can be used to convert LVHct to organ hematocrit, if the organ F cell ratio is known for each organ. To this end, the organ F cell ratios were determined in preliminary experiments, and these values were used to determine organ hematocrit in the rats included in the main study. The organ F cell ratios determined in the ORGAN PLASMA VOLUME ANF Infusion Rate Vehicle 0.1 E0 pg/kg/min E _2 0.5 * E 0~. Heart Lung Liver S Organ pg/kg/min px0.05 compared to Vehicle FIGURE 5. Organ plasma volume during vehicle and atrial natriuretic factor (ANF) infusion at 0.1 and 0.5 pg/kg/min. Organ plasma volumes at 0.025 and 0.05 ,uglkglmin were not significantly different from vehicle infusion. Zimmerman et al ANF Enhances Albumin Escape 467 TABLE 2. Organ Blood Volume as a Percent of Total Blood Volume ANF infusion rate (jig. min- . kg-') Heart Lung Liver GI Muscle Skin (%) (%) (%) (%) (%) (%) 8.2±0.4 21.9+0.6 20.6+1.0 6.5+0.6 10.1+0.6 11.2+0.6 Vehicle 22.1±1.3 10.3±0.6 6.1±0.2 22.6±1.1 8.3±0.4 0.025 11.0±0.5 23.0±1.0 8.4±0.4 22.2±0.7 7.3±0.8 10.4±0.6 11.1±0.3 0.05 22.0±1.0 22.0±1.3 7.3±0.5 8.5±0.3 0.1 10.9±0.7 11.2±0.5 20.7±0.8 8.2±0.3 19.6±0.7 6.6±0.4 0.5 8.9±0.6 10.1±0.6 Values are mean±SEM. Organ blood volume reported as a percent of total blood volume in rats infused with saline and atrial natriuretic factor (ANF) at the indicated infusion rates. No significant change was noted in any group by one-way analysis of variance. GI, gastrointestinal tract. Downloaded from http://circres.ahajournals.org/ by guest on June 12, 2017 preliminary studies for the three rats receiving vehicle were similar to those in the two rats receiving a pharmacological dose of ANF. In the main study, the average whole-body F cell ratio was similar in the vehicle and ANF groups (Table 1). These considerations support the assumption that ANF did not change the F cell ratio and that the data obtained in the preliminary experiments could be applied to all groups in the main study. Circulating ANF levels in rats infused with 0.1 ,ggmin-lkg-1 were 1,232+199 pg/ml. These levels are within the range of ANF levels previously reported from this laboratory for rats with experimental heart failure.15 It is interesting that at ANF levels seen in heart failure, albumin escape is enhanced in the lung, whereas at pharmacological levels of ANF, the lung is protected from albumin escape. Although there may be differential effects of ANF on the peripheral and pulmonary vasculature, this is unlikely because no differences are seen at the lower infusion rates. It is most likely that the differential effect of ANF on the pulmonary vasculature and the peripheral circulation noted at pharmacological infusion rates of ANF are due to the overall hemodynamic effects of ANF. At the highest ANF infusion rate, central venous pressure significantly decreased, most likely reflecting a decrease in venous return. This decrease in venous return most likely resulted in a decrease in pulmonary artery pressure, as previously shown during similar infusion rates of ANF in the dog.2' Studies in isolated perfused guinea pig lung have suggested that ANF protects against chemically induced pulmonary edema.22 Whether this protective effect was partially mediated by a direct action of ANF to prevent capillary damage, as was suggested by Imamura et al,22 or by an action of ANF on the ratio of arterial to venous resistance, as was reported in a preliminary study in perfused dog lung lobe,23 remains to be investigated. From the results of our study, it might be speculated that this hormone could be a potential etiologic factor in the pulmonary edema associated with certain disease states, such as congestive heart failure. At pharmacological levels, however, ANF may be beneficial in heart failure by helping to distribute retained body fluid away from the lungs and into the innocuous areas of the interstitial spaces of skin and skeletal muscle. This last finding is in agreement with a recent study by Williamson et al,24 showing that ANF does not promote albumin escape in the lungs at pharmacological infusion doses. Unlike the previous study, however, we have also evaluated organ-specific albumin escape rates at pathophysiological ANF infusion doses and found that ANF does enhance albumin escape at these levels. Finally, although not a primary aim in this study, the data confirmed earlier findings in rats with intact kidneys and spleens that ANF did not cause a major redistribution of blood volume to the splanchnic region,10 as shown in Table 2. The splanchnic circulation contains the greatest vascular capacitance of the circulation,25 and dilation of the splanchnic capacitance vessels would be expected to cause a redistribution of blood volume into these organs, as was observed in ganglionic blocked rats.'0 The lack of such an effect by ANF supports our contention that ANF is not a potent dilator of capacitance vessels7 and does not lower cardiac output via a redistribution of blood volume. Appendix Calculations used for deriving the equation in "Materials and Methods" for organ hematocrit are defined as follows. Abbreviations ORCV, organ red cell volume; OPV, organ plasma volume; OBV, organ blood volume; OHct, organ hematocrit. Note: Volume is in milliliters. 1251 and 51Cr activities are in counts per minute; units of activity concentration are in counts per minute per milliliter. Calculations 51Cr activity per milliliter erythrocytes= 51Cr activity in 0.024 ml blood sample/ (A-i) (Hct blood sample x 0.024 ml) 2I activity per milliliter plasma= 'I activity in 0.024 ml blood sample/ [(1-Hct blood sample)xO.024 ml] (A-2) ORCV=organ 5`Cr activity/51Cr activity per milliliter erythrocytes (A-3) 468 Circulation Research Vol 67, No 2, August 1990 OPV=organ 1251 activity/125I activity per milliliter plasma (A-4) OBV=ORCV+OPV (A-5) OHct= ORCV/OBV (A- 6) Acknowledgments The authors wish to thank Dr. Edward D. Frohlich for his continued support and advice and Ms. Jean Wood for assistance in preparing the manuscript. References Downloaded from http://circres.ahajournals.org/ by guest on June 12, 2017 1. Burnett JC Jr, Kao PC, Hu DC, Heser DW, Heublein D, Granger JP, Opgenorth TI, Reeder GS: Atrial natriuretic peptide elevation in congestive heart failure in the human. Science 1986;231:1145-1147 2. Cody RJ, Atlas SA, Laragh JH, Kubo SH, Covit AB, Ryman KS, Shaknovich A, Pondolfino K, Clark M, Camargo M, Scarborough RM, Kewicki JA: Atrial natriuretic factor in normal subjects and heart failure patients: Plasma levels and renal, hormonal, and hemodynamic responses to peptide infusion. J Clin Invest 1986;78:1362-1374 3. Maack T, Marion DN, Camargo MJF, Kleinert HD, Laragh JH, Vaughan ED Jr, Atlas SA: Effects of auriculin (atrial natriuretic factor) on blood j,ressure, renal function, and the reninaldosterone system in dogs. Am JMed 1984;77:1069-1075 4. Weidmann P, Hasler L, Gnadinger MP, Lang RE, Uehlinger DE, Shaw S, Rascher W, Reubi FC: Blood levels and renal effects of atrial natriuretic peptide in normal man. J Clin Invest 1986;77:734-742 5. Fluckiger JP, Waeber B, Matsueda G, Delaloye B, Nussberger J, Brunner HR: Effect of atriopeptin III on hematocrit and volemia of nephrectomized rats. Am J Physiol 1986;251(Heart Circ Physiol 20):H880-H883 6. Almeida FA, Suzuki M, Maack T: Atrial natriuretic factor increases hematocrit and decreases plasma volume in nephrectomized rats. Life Sci 1986;39:1193-1199 7. Trippodo NC, Cole FE, Frohlich ED, MacPhee AA: Atrial natriuretic peptide decreases circulatory capacitance in areflexic rats. Circ Res 1986;59:291-296 8. Trippodo NC, Barbee RW: Atrial natriuretic factor decreases whole-body capillary absorption in rats. Am J Physiol 1987; 252(Regulatory Integrative Comp Physiol 21):R915-R920 9. Parkes DG, Coghlan JP, McDougall JG, Scoggins BA: Longterm hemodynamic actions of atrial natriuretic factor (99-126) in conscious sheep. Am J Physiol 1988;254(Heart Circ Physiol 23):H811-H815 10. Trippodo NC, Kardon MB, Pegram BL, Cole FE, MacPhee AA: Acute haemodynamic effects of the atrial natriuretic hormone in rats. J Hypertens 1986;4(suppl 2):S35-S40 11. Trippodo NC: Total circulatory capacity in the rat: Effects of epinephrine and vasopressin on compliance and unstressed volume. Circ Res 1981;49:923-931 12. Taylor AE, Granger DN: Exchange of macromolecules across the microcirculation, in Renken EM, Michel CC (eds): Hand- book of Physiology, Section 2: The Cardiovascular System, Volume 1V Part I. Bethesda, Md., American Physiological Society, 1984, chap 11, pp 467-520 13. Jodal M, Lundgren 0: Plasma skimming in the intestinal tract. Acta Physiol Scand 1970;80:50-60 14. Pitts GC, Bull LS: Exercise, dietary obesity, and growth in the rat. Am J Physiol 1977;232(Regulatory Integrative Comp Physiol 1):R38-R44 15. Chien YW, Barbee RW, MacPhee AA, Frohlich ED, Trippodo NC: Increased ANF secretion after volume expansion is preserved in rats with heart failure. Am J Physiol 1988; 254:R185-R191 16. Winquist RJ, Baskin EP, Faison EP, Clair J, Gould R: The effects of atrial natriuretic peptide on hematocrit and vascular permeability in conscious and anesthetized rats, in Halpern W, Pegram B, Brayden J, Mackey K, McLaughlin M, Osol G (eds): Second International Symposium on Resistance Arteries. New York, Perinatology Press, 1988, pp 435-441 17. Huxley VH, Tucker VL, Verburg KM, Freeman RH: Increased capillary hydraulic conductivity induced by atrial natriuretic peptide. Circ Res 1987;60:304-307 18. Klitzman B, Duling BR: Microvascular hematocrit and red cell flow in resting and contracting striated muscle. Am J Physiol 1979;237(Heart Circ Physiol 6):H481-H490 19. Gregersen MI, Rawson RA: Blood volume. Physiol Rev 1959; 39:307-342 20. Wang L: Plasma volume, cell volume, total blood volume and Fcells factor in the normal and splenectomized Sherman rat. Am J Physiol 1959;196:188-192 21. Zimmerman RS, Schirger JA, Edwards BS, Schwab TR, Heublein DM, Burnett JC Jr: Cardio-renal-endocrine dynamics during stepwise infusion of physiologic and pharmacologic concentrations of atrial natriuretic factor in the dog. Circ Res 1987;61:63-69 22. Imamura T, Ohnuma N, Iwasa F, Furuya M, Hayashi Y, Inomata N, Ishihara T, Noguchi T: Protective effect of alpha-human atrial natriuretic polypeptide (alpha-hANP) on chemical-induced pulmonary edema. Life Sci 1988;42:403-414 23. Faisal MM, Hall JE: Pulmonary vascular effects of atrial natriuretic factor II (ANF) in the isolated canine lung lobe. Am J Hypertens 1988;1:97A 24. Williamson JR, Holmberg SW, Chang K, Marvel J, Sutera SP, Needleman P: Mechanisms underlying atriopeptin-induced increases in hematocrit and vascular permeation in rats. Circ Res 1989;64:890-899 25. Rothe CF: Reflex control of veins and vascular capacitance. Physiol Rev 1983;63:1281-1342 KEY WORDS * hematocrit * serum albumin * plasma volume * congestive heart failure * rat High-dose atrial natriuretic factor enhances albumin escape from the systemic but not the pulmonary circulation. R S Zimmerman, N C Trippodo, A A MacPhee, A J Martinez and R W Barbee Downloaded from http://circres.ahajournals.org/ by guest on June 12, 2017 Circ Res. 1990;67:461-468 doi: 10.1161/01.RES.67.2.461 Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 1990 American Heart Association, Inc. All rights reserved. Print ISSN: 0009-7330. Online ISSN: 1524-4571 The online version of this article, along with updated information and services, is located on the World Wide Web at: http://circres.ahajournals.org/content/67/2/461 Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Circulation Research can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. Further information about this process is available in the Permissions and Rights Question and Answer document. Reprints: Information about reprints can be found online at: http://www.lww.com/reprints Subscriptions: Information about subscribing to Circulation Research is online at: http://circres.ahajournals.org//subscriptions/