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1027 Effect of Endothelin on Plasma Volume and Albumin Escape Robert S. Zimmerman, Alberto J. Martinez, Michael Maymind, and R. Wayne Barbee Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017 Previous studies have demonstrated that endothelin-1 (ET-1) is a potent vasoconstrictor that decreases cardiac output and increases hematocrit. The present study was designed to determine if the rise in hematocrit and decrease in cardiac output are in part due to shifts of plasma from the vascular space to the interstitial space. Red blood cell volume and plasma volume were determined by using chromium51-labeled erythrocytes and iodine-125-labeled albumin, respectively, in anesthetized, nephrectomized, splenectomized rats. The present study demonstrates that ET-1 increases mean arterial pressure and hematocrit. This effect is associated with an increase in total-body albumin escape, which is reflected by a marked reduction in whole-body plasma volume. ET-1 enhanced albumin escape primarily in the liver, lung, and heart at low doses. At high doses, albumin escape increased primarily in the liver, heart, and gastrointestinal tract but not the lung. The present study demonstrates that ET-1 increases hematocrit independent of splenic contraction or renal losses by enhancing loss of plasma volume to the interstitial space without affecting red blood cell volume. Because of the profound pressor effects of ET-1, it is likely that the plasma loss results from increased capillary hydrostatic pressure. (Circulation Research 1992;70:1027-1034) KEY WoRDs * hematocrit * radioiodinated T he endothelin family of peptides (ET-1, ET-2, and ET-3) has recently been described.12 ET-134 and ET-35 have been shown to cause marked vasoconstriction, resulting in increases in mean arterial pressure when injected into dogs or rats. The increased vascular resistance is associated with a decrease in cardiac output. Several possible explanations for the decreased cardiac output have been suggested, including coronary artery vasoconstriction decreasing myocardial contractility,6 arterial baroreceptor stimulation,4 and marked increases in cardiac afterload.3-5 It is possible, however, that other factors are important in the decrease in cardiac output observed during endothelin infusion. One hypothesis not previously addressed is that endothelin may decrease preload by reducing plasma volume. This could occur via renal fluid losses or by enhancing transudation of fluid from the intravascular to interstitial spaces. Although very low doses of ET-3 have been shown to cause a very modest natriuresis,5 higher doses of endothelin decrease both sodium excretion and urine flow by the kidney.3-5 Despite the antidiuresis observed at higher endothelin infusion rates, a dramatic rise in hematocrit has been observed by several investigators during endothelin infusion.3-5 This increase in hematocrit could be caused either by transudation of fluid out of the circulation or by an increase in red blood cell volume that is due to splenic From the Division of Research, Alton Ochsner Medical Foundation, New Orleans, La. Supported in part by grants from the Ochsner Foundation, the American Heart Association of Louisiana, and the National Heart, Lung, and Blood Institute (HL-29952). Address for correspondence: Dr. Robert S. Zimmerman, Ochsner Clinic, Department of Endocrinology, 1514 Jefferson Highway, New Orleans, LA 70121. Received April 19, 1991; accepted January 13, 1992. serum albumin * rats contracture. The previously observed increase in hematocrit from 48% to 54%5 would represent a 21.4% decrease in plasma volume7 if splenic contracture did not occur. This decrease in plasma volume could dramatically decrease cardiac output. To address this issue, Goetz et a14 performed a splenectomy in one dog and found that the increase in hematocrit caused by ET-1 was not observed. Based on this observation, he concluded that the ET-1induced increase in hematocrit resulted from splenic contracture enhancing red blood cell release rather than from decreases in plasma volume. Because of the decrease in cardiac output and the potential marked increase in capillary pressures that could enhance transudation of fluid out of the circulation, we hypothesized that ET-1 increases hematocrit by increasing transudation of fluid out of the circulation. To evaluate this hypothesis, we evaluated the effect of endothelin on blood and plasma volume and albumin escape in nephrectomized, splenectomized rats. Materials and Methods Protocol Male Sprague-Dawley rats (Harlan Sprague Dawley, Inc., Indianapolis, Ind.) weighing 312-370 g were anesthetized with 100 mg/kg i.p. Inactin (BYK Gulden, Konstanz, FRG). A 240-gauge polyethylene tube was placed in the trachea to facilitate breathing. To eliminate labeled albumin loss via the kidney and to prevent dilution of chromium-51-labeled erythrocytes as a 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 1028 Circulation Research Vol 70, No 5 May 1992 Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017 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 (CVP), and in the abdominal inferior vena cava for injections and infusions. Fifteen minutes after surgery, 51Cr-tagged erythrocytes (-1 ,Ci) were injected into the inferior vena cava. Ten minutes later, 1251-human serum albumin (- 1.5 M£Ci) was injected in a volume of 0.15 ml (Mallinckrodt, St. Louis, Mo.). In two separate groups of eight rats, an intravenous infusion of ET-1 (Peptides International, Inc., Louisville, Ky.) in 0.9% sodium chloride (vehicle) at 75 ng * min-l' kg-1 and 100 ng* minm kg-', respectively, was begun at a delivery rate of 0.01 ml/min. The vehicle alone was infused in a separate group of eight rats. Blood samples for measuring hematocrit and 5tCr and `zI radioactivities were collected at 0 and 60 minutes after the start of ET-1 or vehicle infusion. Mean arterial and central venous pressures were monitored continuously with GouldStatham transducers (Gould, Cleveland, Ohio) and a recorder (Grass Instrument Co., Quincy, Mass.). Immediately after the last blood sample was taken, the rats were quickly frozen by immersion in liquid nitrogen for 90 seconds.8 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 125J radioactivities. Red Blood Cell, Plasma, and Blood Volumes Erythrocytes were labeled with sodium 51-chromate in saline (DuPont Medical Products, Wilmington, Del.) as previously described9 and mixed in 0.9% sodium chloride solution to a hematocrit of 40-55. At 15 minutes after injection of 5`Cr-erythrocytes (5 minutes after injection of "2I-albumin), triplicate blood samples were taken from the catheter in the abdominal aorta into glass capillary tubes calibrated to 24 pl, and ET-1 or saline infusion was initiated. Duplicate blood samples were taken at 60 minutes. "Large-vessel" hematocrit (LVHct) was determined with a manual hematocrit reader (Damon/IEC Division, Needham Heights, Mass.). By using a Minaxi Auto-Gamma 5000 series gamma counter (Packard Instrument Co., Downers Grove, Ill.), the 5MCr and 125J radioactivities were determined for the blood samples as well as standards (triplicate glass capillary tubes containing 24-pli samples of the mixture of 51Cr-erythrocytes and 15-gl samples of the solution of `1I-albumin used for injections). The counting rates were corrected for background activity and spillover across channels using the COMPUSPHERE software package (Packard). For the first blood sample, red blood cell volume (RCV), plasma volume (PV), and blood volume (BV) were determined according to the following formulas: RCV=51Cr activity injected (counts per minute) x LVHct-.+blood 51Cr activity concentration (counts per minute per milliliter); PV=1251 activity injected x (1-LVHct) *. blood 125J activity concentration; and BV=RCV+PV. The whole-body Fce,is 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=[(5`Cr activity injected-cumulative sampling loss of `1Cr activity) blood `1Cr activity concentration]+ F,,,; + RCV=RCV measured during the first sample-RCV lost through sampling; RCV lost through sampling=51Cr activity lost through sampling ±. `Cr activity per milliliter RCV; 51Cr activity per ml RCV=51Cr activity injected-RCV measured during first sample; PV= BV-RCV. Organ and Tissue Blood Volume Corrected 51Cr and 125J radioactivities for organs and tissues were determined as described above. Organ blood volume (OBV) was calculated according to OBV=(organ `1Cr activity+ blood 5'Cr activity concentration) .organ Fce,,,, where blood 51Cr activity represented the blood sample taken immediately before the rat was frozen and organ Fcells was the ratio of organ hematocrit to LVHct. The organ Fceiis ratios were determined in separate groups of rats as previously described10 by injecting 51Cr-erythrocytes and `251-albumin 15 and 5 minutes, respectively, before drawing blood samples and freezing the rats. In previous experiments, the Fce,,s ratios 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%.10 Based on the whole-body escape rate of 125I-albumin from the circulation as measured in the experiments described previously, it was estimated that -1% of the injected `PI-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,10 the level of `2I radioactivity measured in each organ at 5 minutes after injection of `PI-albumin provided an estimate of the organ plasma volume, whereas the organ 51Cr activity was a measure of organ erythrocyte volume. Each organ hematocrit was determined according to standard relations,10 which reduced algebraically to the following formula: Organ hematocrit 1 1 +(1-LVHct) LVHct x ratio where ratio is the ratio between 51Cr activity/`2I activity in each organ and `1Cr activity/12I activity in blood; i.e., (Cr/I) organ. (Cr/I) blood. This technique does not adequately reflect the Fcells ratio for the liver because of very rapid albumin escape into the sinusoids. Hence, we assumed that the liver hematocrit was equal to LVHct, which gives an F,ell, ratio of one. To determine if the organ Fc,, ratios changed during ET-1 infusion at the highest dose used in the study, 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 100 ng * min- 1 kg` endothelin-1 (n=3) (at the same rates and duration described above) was begun 1 hour before injecting labeled erythrocytes and albumin. No significant differences in organ F,11s ratios between ET-1 and vehicleinfused groups were obtained. = Zimmerman et al Endothelin Decreases Plasma Volume "5I-Albumin Escape Rate and Organ Mass Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017 The rate at which `251-human serum albumin escaped from the total circulation (`PI-AERt) was determined according to `PI-AERt= [(net `2I activity injected-total plasma `2I activity at 60 minutes) net 1251 activity injected] ÷1 hourx 100, where net '5I activity injected is the total 125I activity injected less the cumulative `2I activity removed from the circulation by blood sampling. Total plasma `2I activity is the product of plasma '25I activity concentration and plasma volume at 60 minutes after the start of ET-1 or vehicle infusion and 60 minutes after injection of `PI-albumin. The rate at which '251-albumin escaped from the circulation in each organ (`PI-AER0) was determined according to the formula 1251-AERo= (tissue 1251-albumin activity.-net 125I activity injected) +1 hourxlO00 corrected organ weight. Tissue `PI-albumin activity was taken as the difference in total organ 1251 activity and organ plasma 125I activity, where the latter was the product of organ plasma volume and plasma 125I activity concentration at 60 minutes. Organ plasma volume was determined as organ blood volume x [1- (organ Fce,is X LVHct)]. 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 `PI-albumin activity that accumulated in the tissues in whole organs was expressed as tissue 1251 activity * total 1251 activity detected in all organs studiedxlO0. Total 1251 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 previously determined by completely dissecting one rat weighing 350 g and boiling the muscle from the bones to determine skeletal weight.10 In two separate groups of rats after anesthesia, nephrectomy, and splenectomy as described above, a thermocouple microprobe (Sensortek, Clifton, N.J.) was placed in the aortic arch and connected to a cardiotherm 500 (Columbus Instruments, Columbus, Ohio) for measurement of cardiac output. ET-1 was infused at 75 ng/kg/min (n=5) and 100 ng/kg/min (n=5) for 60 minutes for determination of cardiac output, colloid osmotic pressure, albumin concentration, and atrial natriuretic factor (ANF) concentration. A separate group of rats was necessary to avoid the effects of blood withdrawal and dextrose injection on plasma volume and cardiac output. In two additional groups of rats, the same protocol was performed as outlined above, and 3 ml blood was obtained before and after ET-1 infusion at 75 ng/kg/min (n=5) and 100 ng/kg/min (n=5) for 60 minutes for measurement of ET-1. Colloid osmotic pressure was determined utilizing a colloid osmometer (model 4400, Wescor Inc., Logan, Utah). Albumin concentrations were determined by an automatic analyzer (model 747, Hitachi, Tokyo). Cardiac output was determined in duplicate, and numbers were averaged. Immunoreactive endothelin was determined by radioimmunoassay using a radioimmunoassay kit specific for endothelin-1-21 (Amersham Laboratories, UK). In this kit, cross-reactivity is 0.4% between ET-1 and big ET-1, 144% between ET-1 and ET-2, and 52% between ET-1 TABLE 1. Body Weight and Whole-Body F,,S Ratio fused With Saline or Endothelin-1 in Saline Body weight (g) ET-1 infusion 326+6 0 ng/kg/min 334±7 75 ng/kg/min 333±5 100 ng/kg/min Values are mean±SEM. ET-1, endothelin-1. 1029 in Rats In- FCel ratio 0.79±0.02 0.78±0.02 0.81±0.01 and ET-3. Blood was collected in chilled EDTA tubes, centrifuged at 4°C for 20 minutes, and frozen at -70°C until assay. One milliliter of plasma was preacidified with 1 ml of 0.1% trifluoroacetic acid (TFA) before extraction on a Sep-Pak C18 cartridge (Millipore Corp., Milford, Mass.). The cartridge was activated with 4 ml of 60% acetonitrile in 0.1% TFA followed by 0.1% TFA alone. Two milliliters of the plasma sample was applied to the column. The columns were washed with 0.1% TFA. The sample was eluted with 3 ml of 60% acetonitrile in 0.1% TFA. The eluate was dried on a Speed Vac (Savant, Hicksville, N.Y.) overnight. Samples were resuspended in 250 ,ul assay buffer. Antiserum (100 ,ul) for ET-1-21 was incubated with the sample and standards for 4 hours. One hundred microliters of 1`I-ET-3 tracer was subsequently added and incubated for 18 hours. Two hundred fifty microliters of donkey anti-rabbit serum coated on polymer particles was incubated for 10 minutes. Tubes were centrifuged for 10 minutes at 2,500g. The supernatant was aspirated, and the pellet was counted for radioactivity on a gamma counter (Auto-Gamma 5000 series, Packard), and log-logit analysis was performed using a software package (SECURIA II, Packard). The intra-assay coefficient of variation was 4.8%. The interassay coefficient of variation was 13.8%. Circulating levels of rat-ANF (r-ANF) were determined by radioimmunoassay after blood collection in 3-mI plastic syringes (containing potassium EDTA) and centrifugation at 4°C. The r-ANF was assayed after extraction on a C18 Sep-Pak cartridge (Millipore) as previously described.1 Recorded values were not corrected for recovery (-75%). The interassay and intra-assay coefficients of variation were 15% and 6%, respectively. Calculations were accomplished using a software spreadsheet package. Data were analyzed with a oneway analysis of variance. Subsequent comparisons between vehicle and ET-1-infused groups were analyzed by Dunnett's test for comparisons between treatment and control means. Values of p<0.05 were considered significant. Data are expressed as mean+± SEM. Results The body weights and Fc,i1s ratios were similar in the three groups analyzed, as shown in Table 1. The hemodynamic and blood volume effects of ET-1 are illustrated in Figure 1. At 60 minutes, mean arterial pressure increased from 103 + 2 mm Hg during vehicle infusion to 125 ±2 mm Hg during ET- 1 infusion at 75 ng* min` . kg-1 (p<0.05) and to 132±+3 mm Hg during ET-1 infusion at 100 ng- min-l . kg-1 (p<o.oS). Although there was a tendency for a decrease in heart rate (from 337+±9 to 312±12 beats per minute after 60 minutes of ET-1 infusion at 75 ng * min-11 kg-'), this effect was not significant. At the highest ET-1 infusion Circulation Research Vol 70, No 5 May 1992 1030 -70 ~~~~~~35 20 16.4 RCV 30 4ii~~~~~~~iPVA ~ 12 (mLl1kg) (mL/1kg) 8.A 0 5 140 50 -HCT 201 10 100 75 0 100 * FIGURE 1. Bar graphs showing effect of endothelin-1 (ET-1) infusions of 0 nglkg/min (vehicle, open bar), 75 ng/kg/min (hatched bar), and 100 nglkg/ min (dotted bar) for 60 minutes on red blood cell volume (RCV), plasma volume (PV), hematocrit (HCT), mean arterial pressure (MAP), and central venous pressure (CVP) in rats. 40 15.()3 75 0 * 25 20 * 60 - * 120 ... CVP (mm Hg) -1 I * p<0.05 compared to 20 -2 0 75 100 L~~~~~. 75 0 100 ET-1 Dose (ng / kg / min) Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017 rate, heart rate was 355 + 15 beats per minute, which was not significantly different from vehicle infusion at 60 minutes. A marked reduction in CVP was observed during ET-1 infusion. After 60 minutes of infusion, CVP decreased from -0.16+0.23 mm Hg during vehicle infusion to - 1.20±0.15 mm Hg during ET-1 infusion at 75 ng - min` *kg` and to -1.63±0.16 mm Hg during ET-1 infusion at 100 ng * min` kg-l ET-1 infusion for 60 minutes markedly increased hematocrit from 47% (vehicle) to 57% (p<0.05) during ET-1 infusion at 75 ng * min-1 * kg`1 and to 60% (p<0.05) during ET-1 infusion at 100 ng- min-m1 kg-1. The increase in hematocrit resulted from a marked decrease in plasma volume from 30.8±0.7 ml/kg (vehicle) to 25.2±0.7 ml/kg (p<0.05) and 20.9±1.0 ml/kg (p<O.O5) after 60 minutes of ET-1 infusion at 75 and 100 ng . min`1 kg-', respectively, with no change in red blood cell volume. The decrease in plasma volume also was associated with a decrease in total-body blood volume from 49.5±1 ml/kg (vehicle) to 45.0±0.8 ml/kg (p<0.05) and 39.6 ml/kg (p<0.05) after 60 minutes of ET-1 infusion at 75 and 100 ng . min-1 kg-1. The decrease in blood volume was not associated with a change in red blood cell volume. The total-body albumin escape rate increased from 15.4±0.5% * hr-1 during vehicle infusion to 17.6± 0.5% * hr-1 (p<0.05) and 23.1±1.0% * hr-1 (p<0.05) after ET-1 infusion at 75 and 100 ng * min-m1 kg-', respectively (Figure 2). The organ albumin escape rates are illustrated in Figure 3. ET-1 enhanced the albumin escape rate in the heart, lung, and liver, at an infusion rate of 75 ng * min1 kg-'. At an infusion rate of 100 ng * min 1 * kg-1, ET-1 enhanced the albumin escape rate in the heart, liver, and gastrointestinal tract, but not in the lung. The organ plasma volume decreased significantly in the heart, lung, liver, and muscle at an ET-1 infusion rate of 75 ng * min . kg-'. At the higher ET-1 infusion dose, plasma volume decreased in all the organs assessed (Figure 4). - 0.6 IA-I 0.25- (1 .0IJfliquii~~~~~~0 - 0.20 - 0.15 - 0.10 -... 0.4 0.2 -.... ~~~~~~~~~0.0 Muscle 0 0) Lung a3.5 3.0 0.06 0.05 - o 0) 2.5 - ..o... 0.0 2.0 01.5 ICLII0 -1 1.75 ifs0.50.6~~~~~~~~~00 1 C 24 ,l 22 (D 0 a CL 20 -) C: -. E: 0.37EA0fI - 12 0.2 0.1 .0 10< 864Z 2.- -2 ~~~~~~ ~~~~~0.06 0.4 0~~~~~~~~~~~~00 18' 16 14 ..0 Zn 0.0 100 ET-1 Infusion Rate (ng I kg l min) FIGURE 2. Bargraph showing effect of endothelin-l (ET-1) infusions of 0 ng/kglmin (vehicle, open bar), 75 nglkglmin (hatched bar), and 100 nglkg/min (dotted bar) for 60 minutes on the total-body albumin escape rate in rats. 0.02 Livmnesaerae intehat ugie,gskrints 0 5 10 7 0 tract* 0 co),mpacle,ad 0.7a . soVeic 0.08e st ET-1 Dose (ng / kg / min) FIGURE 3. Bar graphs showing effect of endothelin-] (ET-1) infusions of 0 ng/kg/min (vehicle, open bar), 75 nglkglmin (hatched bar), and 100 ng/kg/min (dotted bar) for 60 minutes on albumin escape rates in the heart, lung, liver, gastrointestinal tract (GI), muscle, and skin of the rat. *~ 50- Zimmerman et al Endothelin Decreases Plasma Volume 2.5 - ET-1 Infusion Rate 2.0 * C 1.0 L- U~~~1i:I:.. 0 1.8 0 1.4 >2 1.0 2J X__X_____m_____________ _E Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017 o 0.6 m 0.2 E 5 4 02 3 2 1 -00 0 Heart Lung I ~~~~~~~~~~~~~~~~~~~~~~~~~~~~1 Vehicle 75 nglkg/miiin 100nglkg/nmin * p<O.05 comipared to Vehicle g [lu75n/kIm *~~~~ 0.5 C)' M 5 >2 1.5 CL E a 1031 Liver Splanchnic * FIGURE 4. Bar graphs showing effect of endothelin-1 (ET-i) infusion for 60 minutes on organ plasma volume (PV), red blood cell volume, and blood volume in rats. _____T__ Skin Muscle Organ The organ blood volume decreased only in the liver after ET-1 infusion at 75 ng min` *kg-l. At 100 ng * min`. kg-1, ET-1 decreased blood volume in the lung, liver, and muscle (Figure 4). Organ red blood cell volume did not significantly change in any of the organs studied during ET-1 infusion. Organ blood volume as a percentage of total blood volume is shown in Figure 5. At the lowest ET-1 infusion rate, a decrease in liver blood volume compared with total blood volume was observed, suggesting shunting of blood away from the liver or increased loss of blood volume in the liver compared with other organs. At the ET-1 infusion rate of 100 ng min- kg-1, an increase in heart blood volume was observed, which suggests that pooling of blood in the heart occurs at high ET-1 infusion doses. ET-1 and ANF levels after ET-1 infusion are illustrated in Figure 6. ET-1 increased from 33±6 to 42+5 pg/ml (p<0.05) at an ET-1 infusion dose of 75 mg/kg/ min and from 44±6 to 55+6 pg/ml (p<0.05) at an ET-1 infusion dose of 100 ng/kg/min. Circulating levels of ANF increased from 51±3 to 183±26 pg/ml (n=3, p<O.OS) during ET-1 infusion at 75 ng/kg/min and from 72±22 to 457±49 pg/ml (p<0.05) during ET-1 infusion at 100 ng/kg/min. 60 50 ET-1 Infusion Rate 40 ET-1 Vehicle 75 ng I kg l min 10 T _ o 0100 ng/kg/min * p<O.05 compared to Vehicle 2520- 400 m I- 15- AE300- 10- m 0 5- 1000 1 -w--- Heart -_ Lung A Liver Splanchnic Muscle Skin FIGURE 5. Bar graphs showing effect of endothelin-1 (ET-I) infusion for 60 minutes on organ blood volume (OBV) represented as a percentage of total blood volume (TBV) in rats. 500 - 400 - 300 - 200- 200 01.1 n_ 1 30 20- 100 _ 0 75 0 0 ET-1 Dose (nglkg/min) FIGURE 6. Bargraphs showing effect of endothelin-i (ET-I) infusion for 60 minutes on circulating levels of ET-I and atrial natriuretic factor (ANF) in rats. *p<0.05 compared with vehicle (0 nglkglmin). 1032 Circulation Research Vol 70, No 5 May 1992 TABLE 2. Cardiac Output, Colloid Osmotic Pressure, and Albumin Concentration During Endothelin-1 Infusion in Rats Albumin COP CO ET-1 infusion (mm Hg) (ml/min) (mg/dl) Lower infusion rate 13.9+0.3 Baseline 107±5 3.1+0.1 18.0+0.6* 75 ng/kg/min 70±6* 3.8+0.1* Higher infusion rate 13.7±0.3 Baseline 125±3 3.1+0.1 100 ng/kg/min 57±4* 19.5±0.7* 4.0±0.1* Values are mean±SEM. ET-1, endothelin-1; CO, cardiac output; COP, colloid osmotic pressure. *p<0.05 compared with corresponding baseline value. Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017 Cardiac output, serum albumin, and colloid osmotic pressure are shown in Table 2. Cardiac output decreased from 107+5 to 70±6 ml/min (p<0.05) at an ET-1 infusion dose of 75 ng/kg/min and from 125±3 to 57±4 ml/min (p<0.05) during ET-1 infusion at 100 ng/kg/min. The serum albumin concentration increased from 3.1±0.1 to 3.8±0.1 mg/dl (p<0.05) at an ET-1 infusion rate of 75 ng/kg/min and from 3.1±0.1 to 4.0±0.1 mg/dl at an ET-1 infusion rate of 100 ng/kg/ min. The colloid osmotic pressure increased from 13.9 ±0.3 to 18.0± 0.6 mm Hg (p<0.05) at an ET-1 infusion rate of 75 ng/kg/min and from 13.7±0.3 to 19.5±0.7 mm Hg (p<O.OS) at an ET-1 infusion rate of 100 ng/kg/min. Discussion The aim of the present study was to determine the effect of ET-1 infusion on systemic hemodynamics and body fluid shifts. The study demonstrates that ET-1 has potent pressor activity associated with reductions in both CVP and cardiac output. The reduction in CVP and increase in hematocrit were associated with a loss of plasma from the intravascular space, because no change in total-body red blood cell volume was observed. The loss of plasma volume was associated with a dosedependent increase in the albumin escape rate from the intravascular space. At lower ET-1 infusion rates, albumin escaped primarily in the heart, lung, and liver, whereas the higher ET-1 infusion rates caused increased albumin escape in the heart, liver, and gastrointestinal tract but not the lung. A marked reduction in cardiac output was observed in the present study. This finding is in agreement with previous studies in our laboratory512 and others.34 The decrease in cardiac output has generally been thought to be secondary to the marked increase in systemic vascular resistance. The present study importantly shows that cardiac output is also affected by a marked loss of blood volume that is due to transudation of fluid out of the vascular space. The pressor effect of endothelin has been well described in previous studies in this laboratory5 and others.3.4 The decrease in CVP is most consistent with a decrease in total-body blood volume, which was dramatically demonstrated in the present study. At the highest ET-1 infusion rate, blood volume decreased 20%. The decrease in blood volume resulted almost exclusively from a 33% decrease in plasma volume. No change in total-body red blood cell volume was observed. The marked reduction in plasma volume resulted in the dramatic increase in whole-body hematocrit from 47% to 60%. These observations are particularly surprising because previous studies by Goetz et a14 in the dog have suggested that the ET-1-induced rise in hematocrit resulted from splenic contracture rather than shifts of plasma out of the circulation. Their conclusion was based on observations in one splenectomized dog that did not develop an increase in hematocrit during a 60-minute ET-1 infusion. The present study is in marked contrast to this previous study and accurately measures plasma volume and blood volume effects during ET-1 infusion. Because the rats were splenectomized, the increase in hematocrit observed in the present study could not have occurred because of splenic contracture. The loss of intravascular plasma volume accounts entirely for the observed increase in hematocrit. It is unlikely that the spleen significantly contributes to hematocrit elevation in rats, because we have found similar increases in hematocrit (not greater) during ET-3 infusion in nonsplenectomized rats.5 It is possible that ET-1 affects dog vascular fluids in a different way than rat vascular fluids and that the spleen in dogs, unlike rats, may contribute significantly to the increased hematocrit during ET-1 infusion. The rate of albumin escape from the circulation likely reflects fluid transfer, because in most tissues convection appears to be the dominant mechanism for transmicrocirculatory transport of molecules with dimensions similar to albumin. 13 The present study demonstrates a marked increase in whole-body albumin escape. We have previously found that ANF also increases albumin escape in the rat.'0 ET-1 and ANF most likely increase whole-body albumin escape by different mechanisms. ANF probably increases albumin escape by increases in capillary permeability'4 rather than by increases in capillary hydrostatic pressure via venular construction.15 ET-1, on the other hand, may increase albumin escape via transmission of increased systemic arterial pressure to the capillaries. It is possible that ET-1 increases capillary permeability, but this is unlikely, since studies in the kidney have found that ET-1 decreases the ultrafiltration coefficient.'6.'7 At an ET-1 infusion rate of 75 ng kg` * min-1, organ blood volume decreased only in the liver. A decrease in organ blood volume can be caused by shunting of blood away from the organ; this shunting is due to enhanced vasoconstriction in arteries supplying the organ and/or loss of plasma volume into the interstitial space in the capillaries. The observation that liver plasma volume significantly decreased without a significant change in red blood cell volume suggests that the decrease in organ blood volume in the liver resulted primarily from loss of plasma in the liver and not from shunting of blood away from the liver. At an ET-1 infusion rate of 100 ng kg-l min-', organ blood volume decreased in the lung, liver, and muscle. Organ plasma volume decreased in all organs, and organ red blood cell volume did not change. The albumin escape rate increased in the heart, liver, and gastrointestinal tract. These observations suggest that the decrease in blood volume in the affected organs resulted primarily from a decrease in plasma volume rather than preferential shunting of - Zimmerman et al Endothelin Decreases Plasma Volume Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017 blood away from the affected organs. If preferential shunting had occurred, a significant decrease in organ red blood cell volume would be expected and was not observed. The increase in albumin escape in the heart, liver, and gastrointestinal tract suggests that these organs are primarily responsible for the loss of plasma volume at the higher ET-1 infusion rates. It is likely that a vasoconstrictor such as vasopressin and epinephrine would have similar effects on plasma volume as shown in the present study. Indeed, studies by Trippodo9 have shown that epinephrine infusion and epinephrine plus vasopressin infusion in the rat cause a marked reduction in plasma volume that is due to transcapillary shifts of fluid out of the intravascular space. The effects of vasoconstrictors on the albumin escape rate are controversial. Haraldsson18 has shown that norepinephrine decreases the albumin escape rate in perfused rat hind limbs, presumably because of decreased capillary surface area. In contrast, Parving et al19 found that angiotensin II infusion in humans increases the transcapillary albumin escape rate. Presumably, there are tissue-specific effects of these vasoconstrictors on large arterioles affecting systemic vascular resistance and small arterioles controlling capillary surface area. The relative participation of these two segments of the microcirculation will determine the average capillary hydrostatic pressure and ultimately affect albumin escape rate. Although these studies seem contradictory, both are consistent with findings observed in the present study. No change in muscle albumin escape rate was observed during ET-1 infusion; however, because of increases in the albumin escape rate in the gastrointestinal tract, lungs, and liver, a marked increase in albumin escape was observed in the body as a whole. Taken together, these studies suggest that other vasoconstrictors would have effects similar to the effects of endothelin. It is of interest that at lower ET-1 infusion rates albumin escape increased in the lung but that at higher ET-1 infusion rates no increase in pulmonary albumin escape was observed. This observation at first seems perplexing because pulmonary plasma volume decreases more at higher ET-1 infusion rates than at the lower ET-1 infusion rate. Taken together, however, these observations suggest that at the higher ET-1 infusion rate significant plasma loss occurs in nonpulmonary vascular beds (i.e., the liver and gastrointestinal tract), which causes a generalized loss of plasma from the total circulation, resulting in a decrease in pulmonary plasma volume. This interpretation of the data is supported by studies in our laboratory5 and others3,4 that have shown a decrease in CVP or right atrial pressure at high endothelin infusion rates. Indeed, a dramatic loss of plasma volume was observed in all of the organs evaluated. Several possible mechanisms could cause this decrease in pulmonary albumin escape at the highest ET-1 infusion rate. First, it is possible that the higher loss of plasma volume in the nonpulmonary beds at the highest ET-1 infusion rate increased capillary plasma oncotic pressure sufficiently in the lung to decrease albumin escape in that bed. A second possibility is that high ET-1 infusion rates decreased the filtration coefficient across the pulmonary vasculature, causing a decrease in the transudation of fluid into the lungs at higher doses. 1033 Although studies by King et al'6 and Munger et al17 have demonstrated that ET-1 can decrease the ultrafiltration coefficient in the kidney, Barnard et a120 found no change in the ultrafiltration coefficient caused by ET-1 in the rat lung. The most likely explanation for this finding is that at higher ET-1 infusion rates the pulmonary arterial circulation has increased vasoconstriction, causing decreased transudation of fluid and albumin into the lung that is due to reduced capillary hydrostatic pressure. This finding is supported by preliminary studies in the rat lung by Barnard et a120 who found increased pulmonary vascular resistance caused by constriction of only the small arteries after infusion of 10`8 M ET-1. ET-1 affected pulmonary albumin escape in a manner similar to ANF, despite opposite effects on systemic arterial pressure. We have previously shown that at an ANF infusion rate of 0.1 gg/kg/min, albumin escape increases in the lungs but that at higher ANF infusion rates (0.5 gg/kg/min) the lung is protected from increased albumin escape.'0 It is likely that the protective effect of higher doses of ANF on the pulmonary circulation may also be due to loss of significant amounts of plasma volume before the blood enters the pulmonary circulation. The 11 pg/ml change in circulating levels of endothelin observed in the present study is not very different from the change of 7 pg/ml of circulating ET-1 that we have previously observed during hemorrhage (R.S. Zimmerman, M. Maymind, and R.W. Barbee, manuscript submitted), suggesting that the dose of ET-1 infused was a pathophysiological dose of endothelin. Indeed, preliminary studies by Vemulapalli et a121 have found changes of 30 pg/ml of circulating ET-1 during hemorrhage in the rat, supporting the concept that pathophysiological ET-1 doses were used in the present study. The observed increase in ANF during endothelin infusion has previously been observed by two studies from our laboratory5'12 and from other laboratories.3'4 ANF most likely increased through hemodynamic effects or by direct stimulation of ANF release. Although CVP decreased in the present study, ET-1 has been shown to increase pulmonary capillary wedge pressure, a marker for left atrial pressure, in the dog,3 without affecting right atrial pressure. Previous studies have demonstrated that increases in atrial pressure have been shown to increase ANF release.22 It is also possible that endothelin may augment circulating ANF by directly stimulating its release. In vitro studies have demonstrated that ET-1 stimulates ANF release from atrial myocytes in culture.23 Furthermore, preliminary studies by Dananberg et a124 have shown that cardiac endothelial cell destruction blocks atrial tension-induced ANF release but that addition of ET-1 restores tension-induced ANF release, suggesting that ET-1 may be a chemical link between atrial stretch and ANF release. The observation that colloid osmotic pressure and serum albumin increases in the present study suggests that the albumin escape observed in the present study is associated with greater movement of free water than albumin, resulting in increased osmotic pressure. The increased osmotic pressure may then offset the increased vascular pressure, which results in the marked third spacing of both plasma and albumin observed during ET-1 infusion. 1034 Circulation Research Vol 70, No 5 May 1992 The present study demonstrates marked fluid shifts caused by ET-1 infusion in rats. The present study for the first time observes marked loss of intravascular plasma volume, which causes the marked increase in hematocrit noted during ET-1 infusion and contributes to the fall in cardiac output as previously described.3-5 Identification of the vascular beds where this fluid shift occurs during ET-1 infusion is an important first step in identifying the mechanisms by which ET-1 enhances intravascular fluid loss. Acknowledgments The authors would like to thank Jean Wood for her help in the preparation of this manuscript, Dr. N.C. Trippodo for assistance in protocol design, and Dr. G. Navar at the Tulane Medical School for use of his colloid osmometer. References Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017 1. Yanagisawa M, Kurihara H, Kimura S, Tomobe Y, Kobayashi M, Mitsui Y, Yazaki Y, Goto K, Masaki T: A novel and potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 1988;332:411-415 2. Inoue A, Yanagisawa M, Kimura S, Kasuya Y, Miyauchi T, Goto K, Masaki T: The human endothelin family: Three structurally and pharmacologically distinct isopeptides predicted by three separate genes. Proc Natl Acad Sci U S A 1989;86:2863-2867 3. Miller WL, Redfield MM, Burnett JC Jr: Integrated cardiac, renal and endocrine actions of endothelin. J Clin Invest 1989;83:317-320 4. Goetz KL, Wang BC, Madwed JB, Zhu JL, Lendley RJ Jr: Cardiovascular, renal and endocrine responses to intravenous endothelin in conscious dogs. Am JPhysiol 1988;255:R1064-R1068 5. Zimmerman RS, Martinez AJ, MacPhee AA, Barbee RW: Cardiorenal effects of endothelin-3 in the rat. Life Sci 1990;47:2323-2332 6. Kurihara H, Yoshizumi M, Sugiyama T, Yamaoki K, Nagai R, Takaku T, Satoh H, Inui J, Yanagasawa M, Masaki T, Yazaki Y: The possible role of endothelin-1 in the pathogenesis of coronary vasospasm. J Cardiovasc Pharmacol 1989;13(suppl 5):S132-S137 7. Davies R, Slater DH, Forsling ML, Payne N: The response of arginine vasopressin and plasma renin to postural change in normal man, with observations on syncope. Clin Sci 1976;51: 267-274 8. 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 9. Trippodo NC: Total circulatory capacity in the rat: Effects of epinephrine and vasopressin on compliance and unstressed volume. Circ Res 1981;49:923-931 10. Zimmerman RS, Trippodo NC, MacPhee AA, Martinez AJ, Barbee RW: High-dose atrial natriuretic factor enhances albumin escape from the systemic but not the pulmonary circulation. Circ Res 1990;67:461-468 11. Barbee RW, Trippodo NC: The contribution of atrial natriuretic factor to acute volume natriuresis in rats. Am J Physiol 1987;253: F1129-1135 12. Zimmerman RS, Martinez AJ, MacPhee AA, Barbee RW: Atrial natriuretic factor blocks the pressor action of endothelin. J Cardiovasc Pharmacol 1990;16:865-870 13. Taylor AE, Granger DN: Exchange of macromolecules across the microcirculation, in Renken EM, Michel CC (eds): Handbook of Physiology, Section 2: The Cardiovascular System, Volume IV, Part 1. Bethesda, Md, American Physiological Society, 1984, pp 467-520 14. Huxley VH, Tucker VL, Verburg KM, Freeman RH: Increased capillary hydraulic conductivity induced by atrial natriuretic peptide. Circ Res 1987;60:304-307 15. Harrison-Bernard LM, Carmines PK, Barbee RW: Effects of intravenous atrial natriuretic factor (ANF) infusion on rat cremaster microcirculation. (abstract) FASEB J 1991;5:A672 16. King AJ, Brenner BM, Anderson S: Endothelin: A potent renal and systemic vasoconstrictor peptide. Am J Physiol 1989;256: F1051-F1058 17. Munger KA, Sugiura M, Inagami T, Takahashi K, Badr KF: Mechanisms of endothelin-induced natriuresis in the rat. (abstract) Clin Res 1989;37:497A 18. Haraldsson B: Effects of noradrenaline on the transcapillary passage of albumin, fluid and CrEDTA in the perfused rat hindlimb. Acta Physiol Scand 1985;125:561-571 19. Parving H1-H, Nielsen SL, Lassen NA: Increased transcapillary escape rate of albumin, IgG, and IgM during angiotensin-IIinduced hypertension in man. Scand J Clin Lab Invest 1974;34: 111-118 20. Barnard JW, Barman SA, Adkins WK, Longenecker GL, Taylor AE: Characterization of endothelin-1 effects on the rat pulmonary circulation. (abstract) FASEB J 1990;4:A272 21. Vemulapalli S, Chiu PJS, Rivelli M, Cedeno K, Coleman W, Sybertz EJ: Tumor necrosis factor and hemorrhage produce elevation of plasma endothelin in rats (abstract). FASEB J 1990;4:A456 22. Zimmerman RS, Edwards BS, Schwab TR, Heublein DM, Burnett JC Jr: Cardiorenal-endocrine dynamics during and following volume expansion. Am J Physiol 1987;252:R336-R340 23. Fukuda Y, Hirata Y, Yoshimi H, Takatsugu K, Kobayashi Y, Yanagisawa M, Masaki T: Endothelin is a potent secretagogue for atrial natriuretic peptide in cultured rat atrial myocytes. Biochem Biophys Res Commun 1988;155:167-172 24. Dananberg J, Sider RS, Webb RC, Grekin RJ: Atrial tension is an important mediator of ANF secretion induced by adrenergic stimulation. (abstract) Progr Abstr Endocr Soc 1989;71:183 Effect of endothelin on plasma volume and albumin escape. R S Zimmerman, A J Martinez, M Maymind and R W Barbee Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017 Circ Res. 1992;70:1027-1034 doi: 10.1161/01.RES.70.5.1027 Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 1992 American Heart Association, Inc. All rights reserved. Print ISSN: 0009-7330. 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