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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
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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
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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
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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)
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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
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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.
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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
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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.
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R S Zimmerman, A J Martinez, M Maymind and R W Barbee
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Circ Res. 1992;70:1027-1034
doi: 10.1161/01.RES.70.5.1027
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