Download Print - Circulation Research

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/