Kinetics of endothelin-1 binding in the dog liver microcirculation
in vivo
Jocelyn
Dupuis1,
Andreas
J.
Schwab2,
André
Simard2,
Peter
Cernacek1,
Duncan J.
Stewart3, and
Carl A.
Goresky
,2,4,5
1 Department of Medicine,
Montreal Heart Institute, Montreal H1T 1C8,
2 McGill University Medical
Clinic in the Montreal General Hospital, Departments of
4 Medicine and
5 Physiology, McGill University,
Montreal, Quebec H3G 1A4, and
3 Department of Medicine, St.
Michael's Hospital, Toronto, Ontario, Canada M5B 1W8
 |
ABSTRACT |
Endothelin-1
(ET-1) is a 21-amino acid peptide produced by vascular endothelial
cells that acts as a potent constrictor of hepatic sinusoids. Hepatic
binding of tracer 125I-labeled
ET-1 was investigated in anesthetized dogs with the multiple-indicator
dilution technique with simultaneous measurements of unlabeled
immunoreactive ET-1 plasma levels. Despite 80% binding to albumin,
tracer 125I-ET-1 was avidly
extracted by the liver, with only 15 ± 6% of the peptide surviving
passage through the organ. Exchange of ET-1 between plasma and binding
sites, probably located on the surface of liver cells, was
quantitatively described by a barrier-limited, space-distributed
variable transit time model. Reversible and irreversible parallel
binding sites were found. Reversible and irreversible plasma clearances
of unbound 125I-ET-1 were 0.084 ± 0.033 ml · s
1 · g
liver
1 and 0.17 ± 0.09 ml · s
1 · g
liver
1, respectively, and
the dissociation rate constant for reversible binding was 0.24 ± 0.12 s
1. The specific
ETA receptor antagonist BMS-182874
did not modify binding to either site. The nonspecific
ETA/ETB
antagonist LU-224332 dose-dependently reduced irreversible binding
only. ET-1 levels in the hepatic vein were significantly lower than in
the portal vein but were not different from those in the hepatic
artery. The ratio between hepatic vein and portal vein levels (0.64 ± 0.31) was considerably higher than survival fractions, suggesting a substantial simultaneous release of newly synthesized or stored ET-1
by the liver. These results demonstrate both substantial clearance and
production of ET-1 by the intact liver. Hepatic ET-1 clearance is
mediated by the ETB receptor, with
the presence of reversible, nonspecific ET-1 binding at the liver
surface
endothelin clearance; endothelin receptor antagonist; protein
binding; multiple indicator-dilution technique; vasoactive peptide; receptor-binding kinetics
 |
INTRODUCTION |
ENDOTHELIN-1 (ET-1) is a 21-amino acid peptide
produced by vascular endothelial cells and secreted preferentially in a
paracrine fashion, with a smaller luminal secretion resulting in
measurable plasma levels of this peptide. Its actions are mediated by
two well-characterized receptor subtypes,
ETA and
ETB (7, 28). ET-1 is a potent
constrictor of the hepatic sinusoids (15), promotes glycogenolysis
(31), and stimulates the release of humoral mediators by Kupffer cells
(14). Hepatic stellate cells (also called Ito cells, fat-storing cells,
or lipocytes), which are located in the Disse space where they act in a
fashion similar to pericytes, are believed to be involved in hepatic
vasoconstriction mediated by ET-1 (38). Hepatic stellate cells in
culture contract in the presence of ET-1 in a dose-dependent way (24).
Hepatocytes also possess high-affinity ET-1 binding sites that activate
the phosphoinositide signal-transduction pathway (31). In contrast to
cells from other organs, liver-derived cells respond to quite low
levels of this peptide (14, 31).
Chronic liver disease is associated with increased serum ET-1 levels
(26) and, in animal models, with an increase in hepatic ET-1 production
(13). This may contribute to and/or reflect the disease process. The
mixed ETA and
ETB receptor antagonist bosentan has been shown to be effective in reducing carbon
tetrachloride liver injury in rats, suggesting a contributing role of
ET-1 in the pathological process (23). These findings connote the
importance of endothelin for liver function and support a necessary
entry of circulating ET-1 into the Disse space to interact with target cells.
In the rat, intravenously injected radioactive ET-1 has a very short
plasma half-life of 7 min (1, 9, 32) and appears rapidly in tissues,
predominantly in the lungs, followed in importance by the liver and the
kidneys (1, 32). In the liver, it is deposited predominantly in hepatic
stellate cells, followed by endothelial cells and to a lesser extent
hepatocytes (10, 16). The autoradiographic lobular distribution of the
immobilized ET-1 follows a gradient, with ET-1 levels diminishing from
the portal to the centrolobular zone (16), a behavior typical for
substances metabolized by the liver (19). In isolated rat liver cells, the binding of labeled ET-1 also occurs mainly in hepatic stellate cells, followed by the endothelial cells and hepatocytes (24). ET-1 is
internalized and metabolized rapidly by hepatocytes in culture
(11).
These observations suggest that the liver is an important site for the
metabolism of circulating ET-1; on the other hand, the liver may be
particularly sensitive to variations in the circulating levels of this
peptide. To understand the dynamics of the hepatic action of ET-1, it
is necessary to investigate its kinetic behavior, including the binding
kinetics in a single pass through an organ. However, the
above-mentioned studies were conducted in time frames of several
minutes to hours. Similarly, receptor-binding studies were ordinarily
performed with incubation times of 10-60 min, with subsequent
washing with buffers (10-12, 31, 35). These times are far longer
than the transit time of plasma through an organ, which is on the order
of seconds. To obtain an accurate picture of the dynamics of endothelin
turnover in the mammalian body, the underlying events must be well
understood, including their temporal behavior. In this study, we used
the multiple-indicator dilution technique to assess reversible and
irreversible binding of circulating ET-1 in livers of anesthetized dogs.
 |
MATERIALS AND METHODS |
The experiments were performed in anesthetized dogs with the multiple
indicator-dilution technique. Three groups of animals were studied: in
the first group (n = 9), no endothelin
receptor antagonists were used; in the second group
(n = 11), a nonspecific ETA/ETB
receptor antagonist was administered; and in the third group
(n = 5), a specific
ETA receptor antagonist was administered.
Animal preparation.
Mongrel dogs were anesthetized with pentobarbital sodium (50 mg/kg iv),
intubated, and allowed to spontaneously breathe room air. A median
laparotomy was performed, and catheters were positioned in the portal
vein (injection site) and in the hepatic vein (collection site) as
previously described (17). To ensure adequate ventilation and
hemodynamic stability, arterial blood pressure was continuously monitored through a right femoral arterial line, and arterial blood
gases were obtained for analysis from the same line. A continuous electrocardiographic tracing was also recorded. For the nine
experiments done in the absence of endothelin-receptor antagonists,
3-ml blood samples were taken from the portal vein, the hepatic vein,
and the femoral artery for measurement of plasma immunoreactive ET-1 levels as previously described (5), and after completion of the
experiment, the liver was surgically removed for determination of its
wet weight.
Multiple-indicator dilution experiments.
An injection mixture was prepared as previously described (4) that
contained the various tracers to be studied. It was composed of the
following three noneliminated tracers:
51Cr-labeled red blood cells (25 µCi), a marker of the vascular space; Evans blue-labeled albumin (5 mg Evans blue in 1 ml water), a marker of the plasma proteins of blood
with limited diffusion into the Disse space; and
[14C]sucrose (50 µCi), a tracer that diffuses into the Disse space. The fourth and
only eliminated tracer in the preparation was
125I-labeled ET-1 (5 µCi,
specific activity 2,200 Ci/mM). Red blood cells were added
to match the hematocrit of the dog. A minimal volume of 4-ml injection
mixture was prepared; 2 ml were kept to serve as the portal vein
injection bolus; the remainder was used to prepare three dose standards
by adding 0.1 ml injection mixture to 0.9 ml of venous blood. Isotope
crossover standards were prepared by adding small amounts of each
indicator separately to venous blood.
This mixture (2 ml) was injected as a bolus through the portal vein
catheter into the portal vein, and timed samples were simultaneously
collected from a hepatic vein through the hepatic vein catheter, with a
peristaltic pump and a rapid sample collector at 1-s intervals for a
total of 40 s (17). To prevent clotting of the samples, each collection
tube was preloaded with 5 ml heparin (10,000 IU/ml; Heparin Leo, Leo
Laboratories, Ajax, ON). All tubes, including the dose and crossover
standards, were then processed in the same fashion to determine the
activity of each tracer. The tubes were swirled for ~1 s
(Vortex-Genie, Fisher Scientific, Ottawa, ON), an aliquot of 0.1 ml of
blood was transferred to a new tube containing 1.5 ml of physiological
saline, and 0.2 ml 1.5 M TCA was added to precipitate the proteins. The
tubes were then inserted into a gamma counter (Cobra 5002, Canberra-Packard, Meriden, CT) for the determination of
51Cr and
125I activity. After settling of
the precipitate by gravity, 0.3 ml of supernatant was added to 5 ml
aqueous liquid scintillation cocktail (Ready Safe, Beckman Instruments,
Fullerton, CA) for determination of
14C activity in a liquid
scintillation spectrometer (LS5801, Beckman). The remaining blood
sample was centrifuged 10 min at 870 g, and 0.2 ml of the resulting plasma
was added to 1 ml of physiological saline in a standard disposable
spectrophotometer cuvette (1-cm path length; Fisher Scientific,
Montreal, PQ) for determination of Evans blue absorbance at
620-740 nm in a spectrophotometer (HP 8452A, Hewlett Packard, Palo
Alto, CA).
Effect of endothelin receptor antagonists on hepatic ET-1 kinetics.
To gain more insight into the mechanisms responsible for hepatic ET-1
clearance, additional experiments were performed with intraportal
injections of either the highly specific
ETA receptor antagonist BMS-182874
[constant of inhibition
(Ki) for
ETA was 55 nM and for
ETB was 50 mM; Bristol-Myers
Squibb, Princeton, NJ], or the mixed
ETA/ETB
receptor antagonist LU-224332
(Ki for ETA was 3.5 nM and for
ETB was 7.2 nM; kindly provided by
Michael Kirchengast, Knoll, Ludwigshafen, Germany). After completion of a first indicator-dilution experiment, the antagonists were directly injected into the portal vein followed by a second indicator-dilution experiment 5 min later. For each animal studied, only one injection of
either BMS-182874 (50-800 mg) or LU-224332 (1-50 mg) was used.
Detection of 125I-labeled metabolites in
hepatic effluent.
To determine whether 125I-labeled
ET-1 metabolites were present in the collected samples, we used the TCA
precipitation method. With the use of this technique with HPLC control,
Gandhi et al. (12) have shown that intact
125I-ET-1 precipitates with
albumin in the resulting pellet, whereas metabolites remain in the
supernatant. Accordingly, to detect any metabolites of
125I-ET-1, radioactivity was
measured in 200-µl aliquots of the supernatant resulting from TCA
precipitation in all samples from five experiments.
Binding of ET-1 to plasma proteins.
We added 0.2 µCi of 125I-ET-1
(0.008 fM) to 300 ml of 4% PBS or dog plasma with increasing
quantities of unlabeled ET-1, from 0.4 fM to 4.0 µM (final
concentrations of ET-1 from 0.04 pol/l to 12 µmol/l); this resulted
in nine serial samples for each experimental condition. Ultrafiltration
was performed as follows (30): the solutions were placed in the
reservoirs of ultrafiltration units (Millipore Ultrafree-MC filter
units, 10,000 NMWL) that had been pretreated with
Sigmacoat (Sigma, St. Louis, MO) to prevent nonspecific ET-1 binding.
The units were then centrifuged at 2,000 g for 20 min, resulting in an
ultrafiltrate volume representing about 40% of the total. Aliquots of
the unfiltered solutions and of the ultrafiltrates as well as the empty
filter units were counted to determine the bound fraction of ET-1 as
well as binding to the filter units. For all experiments combined,
nonspecific binding to the filters was <1%.
Moment analysis of indicator dilution curves.
The radioactivity determined in each outflow sample was divided by that
determined in the samples from the injection mixture (corrected for the
1:10 sample dilution) and by the injection volume (22). Outflow
profiles were thus presented as outflow tracer blood concentrations,
normalized by dividing by the amount of tracer in the injection
mixture, which corresponds to fractional recoveries per milliliter of
blood. Single-pass dilution curves were obtained by truncating the late
portion of outflow profiles in which recirculation is apparent and
applying exponential extrapolation (17). The areas under the curves
were calculated by numerical integration (22). For noneliminated
substances (red blood cells, albumin, and sucrose), this integral is
equal to the inverse of blood flow (17). Recovery of
125I-ET-1 was determined as the
ratio between the area under the curve of its outflow profile and that
of noneliminated substances (expected to be equal to each other). The
mean transit time of each tracer through the system is then calculated
as the time integral of the product of fractional recovery and time,
divided by the time integrals of the fractional recoveries (22). The liver mean transit times were found by subtracting the transit time of
the inflow and outflow catheters from those through the whole system.
Modeling of reference indicator kinetics.
Outflow profiles for noneliminated indicators, such as red blood cells,
sucrose, or albumin, can be described with the delayed-wave flow-limited model. In this model, the axial diffusional gradient is
neglected, whereas exchange between the plasma, interstitial, and/or
hepatocyte space is quasi-instantaneous (17). With the use of the red
blood cells curve (CRBC) as a
reference, any diffusible tracer profile
(Cdiff) can be represented in
this model with the following equation
|
(1)
|
where
diff is the ratio between the
extravascular and intravascular spaces accessed by the tracer and
t0 is a common
large-vessel transit time (17). The equation implies that, after the
large-vessel transit time, each point on the tracer outflow curve will
be delayed in time relative to the corresponding point on the red blood
cells curve by the factor (1 +
diff), and its magnitude will
be decreased by the factor 1/(1 +
diff). To take into account
the distortion imposed by the input and collection catheters (21), the
above relation (Eq. 1) was modified
by deconvolution/convolution as previously developed (22). In the
present experiments, the outflow catheter and the attached pump were
the same as those characterized in this previous study.
Optimized parameters were obtained by a nonlinear least-squares
procedure from Visual Numerics (Houston, TX) (22). The fit being
performed simultaneously for all diffusible tracer profiles, the
optimized parameters were
t0 and a space
ratio, as defined above, for each tracer, for albumin
(
alb) and for sucrose
(
suc).
Modeling of ET-1 kinetics.
Evaluation of the microcirculatory exchanges of ET-1 between liver cell
and plasma was based on the barrier-limited space-distributed variable
transit time model developed by Goresky et al. (19). The model
describes the relationship between the outflow profile for the
substance under study,
CET-1(t), and that of a
reference substance,
Cref(t), which is not
taken up by the cells but behaves similarly in all other respects.
Transfer of plasma ET-1 between the vascular and the Disse space was
assumed to be extremely rapid (flow limited).
The original model includes three parameters describing influx and
efflux across the liver cell membranes and intracellular sequestration
or enzymic transformation. It has been proposed that this model is also
applicable to the interaction between polypeptide hormones and cell
surface receptors, as it occurs in receptor-mediated endocytosis (29).
In this case, reversible association with and dissociation from cell
surface receptors replaces influx and efflux, and sequestration from
the receptor to the cell interior replaces intracellular sequestration.
This model will be referred to as the one receptor (or one binding site) model. However, for reasons discussed in RESULTS, we
favor a model where removal of tracer ET-1 from plasma occurs by means of irreversible binding to specific receptors, and reversible binding
to different binding sites without sequestration occurs in parallel
(Fig. 1). This will be the parallel binding
site model. It has been shown previously that sequestration from the
plasma space and sequestration from the intracellular space yield
identical outflow profiles with appropriate parameter transformation
(18).

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Fig. 1.
Schematic representation of model of endothelin-1 (ET-1) receptor
binding at level of a single sinusoid;
kpr, transfer
coefficient for association to reversible binding site;
krp, transfer
coefficient for dissociation from reversible binding site;
kpi, transfer
coefficient for association to irreversable binding
site.
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|
Previous experiments have suggested considerable reversible binding of
endothelin to plasma albumin (34). Because of the limited access of
plasma protein to the Disse space of the liver, such a binding could
influence the outflow profile of
125I-ET-1, and it needed to be
addressed in this study. We therefore used a composite hypothetical
reference indicator for which the apparent volume of distribution is
related to those of sucrose (representing free plasma
125I-ET-1) and albumin
(representing 125I-ET-1 bound to
plasma proteins). Because of the very high single-pass 125I-ET-1 extraction (86%),
binding of 125I-ET-1 to proteins
was assumed to occur with very high association and dissociation rate
constants (at least one order of magnitude faster than interaction with
surface receptors) and thus no rate limiting effect on
125I-ET-1 exchange between plasma
and parenchymal cells. The appropriate reference curve for
125I-ET-1 can then be calculated
as a transformation similar to Eq. 1
applied to any diffusible tracer profile (here albumin was chosen) (37)
|
(2)
|
where
ref is the apparent space ratio
between 125I-ET-1 Disse space and
sinusoidal plasma space, calculated as
|
(3)
|
and
fu is
125I-ET-1 unbound fraction.
By analogy to the original model by Goresky et al. (19) with
appropriate parameter transformation (18), the outflow profiles for
125I-ET-1 in the parallel binding
site model can be described by the following relation
|
(4)
|
Because
the fenestrated endothelial cells of the liver do not constitute a
barrier to ET-1, the modeling of this process will be analogous to that
of an enzymic process at the surface of the endothelial cells as
considered by Goresky et al. (20) but with the use of an appropriate
reference. The parameters describing the behavior of
125I-ET-1 tracer in this model are
coefficients of tracer transfer between a mobile (plasma) compartment
(p), represented by the combination of the vascular and Disse plasma
spaces, and a stationary compartment (r), representing reversible
binding sites on the surface of parenchymal cells. The transfer
coefficient for rapid receptor binding or association is
kpr,
that for dissociation from receptors is
krp, that for
irreversible sequestration is kpi, all with
units of a reciprocal time
(s
1). Again,
Eq. 4 was modified by
deconvolution/convolution (22). The parameters were optimized with a
least-square algorithm.
The unidirectional plasma clearances per gram liver for
125I-ET-1 for reversible binding
(CLpr) and irreversible
sequestration (CLpi) were
computed as the products of influx transfer coefficient and the sum of
sinusoidal plasma and Disse spaces as shown below
|
(5)
|
|
(6)
|
where
Hct is hematocrit, F is liver blood flow, and
suc
is sucrose mean transit time.
 |
RESULTS |
Binding of ET-1 to plasma proteins.
The ultrafiltration experiments revealed considerable and similar
binding of 125I-ET-1 to BSA and
dog plasma proteins. The unbound fraction was similar for all
concentrations of ET-1 (from 0.04 pmol/l to 12 µmol/l), with a mean
of fu = 0.20 ± 0.04 for BSA and
fu = 0.22 ± 0.05 for dog plasma.
Outflow profile of noneliminated indicators.
Representative sets of dilution curves obtained after injection of the
tracers into the portal vein and collection from a hepatic vein are
shown in Fig. 2. The typical behavior of
the noneliminated tracers, red blood cells, albumin, and sucrose has been extensively described previously (17). According to the delayed
wave flow limited model, each tracer is delayed and diluted compared
with the intravascular reference, red blood cells, by virtue of its own
larger volume of distribution in the liver. The results of the analysis
of the noneliminated reference indicators are assembled in Table
1. The recoveries for the noneliminated tracers were essentially complete within experimental error (their areas under the curves being equal). The parameters obtained from the
simultaneous fit of red blood cells, albumin, and sucrose experimental
profiles are compiled in Table 2.

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Fig. 2.
Representative sets of outflow dilution curves from control conditions
(A and
B) and after either nonspecific
ET-receptor antagonist (C) or
specific ETA receptor antagonist
(D). Data are truncated before
recirculation occurs, and extrapolation (dashed lines) was according to
terminal slopes on this logarithmic representation.
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Table 2.
Parameters obtained from the simultaneous fit of red blood cells, albumin and
sucrose experimental profiles
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|
Effect of the endothelin receptor antagonists on the recovery and
outflow profile of 125I-ET-1.
In the absence of endothelin receptor antagonists, apart from its much
smaller fractional recovery, tracer
125I-ET-1 exhibits an outflow
profile closely related to that of tracer albumin. This is consistent
with the finding of 80% binding of ET-1 to serum albumin and supports
the use of albumin as a reference indicator for the description of
tracer 125I-ET-1 kinetics in the
liver. The mean single-pass hepatic fractional recovery of
125I-ET-1 in the absence of
antagonists was quite small (Table 3), with
an overall mean of 0.15 ± 0.06 for the 25 experiments performed in
the absence of antagonists. The specific
ETA receptor antagonist BMS-182874
did not modify tracer 125I-ET-1
outflow profiles and recoveries, even at the very high dose of 400 mg
(Fig. 2 and Table 3). On the other hand, the mixed ETA/ETB
receptor antagonist LU-224332 caused an upward shift in the
125I-ET-1 outflow profile and an
increase in 125I-ET-1 recovery,
which depended on the dose and attained a range between 0.76 and 0.80 at the highest dose of 50 mg. The analysis of
125I-ET-1 outflow profile after
administration of the mixed antagonist then becomes critical in the
choice of a model that will best describe hepatic kinetics of this
peptide. The upslope portion of the
125I-ET-1 curve, from appearance
to peak (Fig. 2, top right), is below that of albumin, with mild progressive separation of the curves
attributed to the mild hepatic removal of
125I-ET-1. The downslope portion
of the 125I-ET-1 curve, however,
progressively approaches that of albumin and, later in time, crosses
over. This pattern is typical of substances that exhibit reversible
hepatic binding. The late returning component represents a
progressively decreasing instantaneous extraction compared with the
vascular reference. We found no detectable
125I-ET-1 metabolites in the
supernatants of TCA precipitation of the hepatic effluent, suggesting
that this small returning component represents intact
125I-ET-1.
Endothelin binding in the liver.
ET-1 is known to bind to its receptors with high affinity and a slow
dissociation rate. Accordingly, one would not expect any returning
component, and if a reversible binding site did exist, the returning
component should be proportionally reduced after receptor blockade.
This is not supported by the present data; despite substantial
reduction in hepatic 125I-ET-1
removal after injection of the mixed antagonist, the returning component of 125I-ET-1 persisted
and even became more prominent on the outflow profile as
125I-ET-1 recovery increased. The
one reversible binding site model was therefore rejected and a two
binding site model was adopted (Fig. 1). This model describes a
irreversible binding site in parallel with a reversible binding site
without sequestration.
The optimal parameters obtained from the fits of this model are
compiled in Table 3. The variation of the clearances with liver blood
flow for the nine experiments performed without endothelin-receptor antagonists is shown in Fig. 3. The Pearson
correlation coefficients obtained by performing error-weighted
regression between the clearances and liver blood flow are 0.90 and
0.99 for CLpr and
CLpi, respectively. This behavior,
previously described for rubidium uptake (22), is tentatively
attributed to flow recruitment of sinusoids.

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Fig. 3.
Variation of influx reversible binding
(CLpr) and irreversible binding
(CLpi) clearances per gram liver
with flow. Dashed line is linear regression of data. Error bars
represent standard deviations as measures of reliability from fit.
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The effects of the antagonists on hepatic
125I-ET-1 extraction and the
model-derived transfer coefficients are more easily appreciated when
the ratios of these parameters, measured before and after injection of
the antagonists, are plotted as a function of the increasing antagonist
doses (Fig. 4). Hepatic
125I-ET-1 extraction was
dose-dependently decreased by the mixed ETA/ETB
antagonist but remained unaffected by comparatively large doses of the
specific ETA antagonist. The
transfer coefficient for binding of
125I-ET-1 to the irreversible site
(kpi) behaved
similarly, exhibiting a marked dose-dependent reduction only with the
mixed ET-1 receptor antagonist. The fitted coefficients for association
to (kpr) and dissociation from
(krp) the
reversible binding site show considerable scatter around the line of
unity, with large standard errors (Fig. 4). No dose-dependent effect is
evident for either antagonist. For the specific
ETA antagonist, the ratios for
kpr tend to be higher than the line of unity, and for the experiments performed with
the mixed antagonist, the ratios for both
kpr and
krp tend to be
lower that the line of unity.

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Fig. 4.
Variations in ET-1-binding kinetic parameters in dog liver as a
function of doses of nonspecific endothelin receptor and specific
ETA receptor antagonists.
Parameters are expressed as ratio of value obtained before and after
injection of antagonist.
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With the use of the model-derived parameters, the experimental hepatic
outflow profile for 125I-ET-1 can
be divided into its throughput and returning components (Fig.
5). The former represents
125I-ET-1 that has transited
through the liver without any interaction with binding sites, whereas
the latter is composed of
125I-ET-1 returning to circulation
after reversible binding. After administration of the nonspecific
antagonist, both throughput and returning components were increased.

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Fig. 5.
Theoretical outflow profiles of ET-1, illustrating throughput and
returning components in control conditions (A) and after
administration of the nonspecific endothelin-receptor antagonist
(B).
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|
Immunoreactive ET-1 levels.
The mean values for ET-1 levels were 0.46 ± 0.35 pmol/l in the
portal vein, 0.36 ± 0.36 pmol/l in the hepatic vein, and 0.35 ± 0.37 pmol/l in the hepatic artery (Fig. 6).
The average ratio to portal vein ET-1 levels was 0.64 ± 0.33 for
hepatic vein levels and 0.59 ± 0.31 for hepatic artery levels. ET-1
levels in the hepatic vein or the hepatic artery were significantly
lower than the ones in the portal vein (paired
t-test; probability that levels are
the same is P < 0.01 in both
cases), whereas no significant difference was found between hepatic
artery and hepatic vein. One subject had markedly elevated levels of
over 1 pmol/l with no evident hemodynamic or macroscopic hepatic
abnormality during the experiment. Exclusion of this animal from the
statistical analysis did not modify the above interpretation, ET-1
levels being 0.39 ± 0.21 pmol/l in the portal vein, 0.24 ± 0.22 pmol/l in hepatic vein, and 0.26 ± 0.22 pmol/l in
hepatic artery.

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Fig. 6.
Individual immunoreactive ET-1 levels in portal vein (PV), hepatic vein
(HV), and hepatic artery (HA) from each of 9 experiments without
endothelin antagonists
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 |
DISCUSSION |
ET-1 removal by the liver.
We found that ~85% of circulating tracer
125I-ET-1 is retained by the liver
in a single passage. This represents the highest proportion of removal
of ET-1 by an organ reported to date; with the use of a similar
technique we previously found that the dog lung removes 33% of
circulating 125I-ET-1 (4) while
the human lung removes 46% (5). The dog myocardium shows a much
smaller extraction at 15% (3). If the organ clearance of
125I-ET-1 is computed (clearance = organ flow × fractional extraction), the lung remains the major
site for 125I-ET-1 clearance
because it accommodates the whole cardiac output, whereas the hepatic
blood flow represents ~25-30% of the total (33).
The pulmonary removal of ET-1 is mediated through the endothelial
ETB receptors in dogs (2) and rats
(9), with complete inhibition of ET-1 removal after administration of
the specific ETB antagonist
BQ-788. The liver and liver-derived cells are rich in both receptor
subtypes (23, 24). Hepatic stellate cells show both kinds of receptors,
whereas endothelial cells and Kupffer cells only express the
ETB subtype (24). Rat hepatocytes
demonstrate high-affinity ET-1 binding sites (11, 31) of both the
ETA and
ETB receptor subtypes (25).
Hepatic uptake of ET-1 in rats was found unaffected by the
ETB antagonist BQ-788 (9). The mechanism responsible for ET-1 removal in the liver may consequently be
different from that in other organs, with the potential involvement of
multiple receptor subtypes.
The present experiments demonstrate that circulating
125I-ET-1 is rapidly extracted by
the liver. A small portion of
125I-ET-1 retained by the liver
returns rapidly into the circulation, representing unchanged ET-1
because no metabolites were detected in the hepatic effluent. This is
in agreement with observations in rats, in which no appreciable amount
of degraded forms of 125I-ET-1
have been found in the blood for up to 60 min after intravenous injection (32). The experimental data were quite similar to those
obtained with substrates such as galactose or norepinephrine (18, 19).
However, the observed binding reversibility stands in sharp contrast to
previous observations of virtually irreversible binding of ET-1 to
ETA and
ETB receptors (31, 35).
The hypothesis was therefore put forward that two modes of ET-1 binding
exist, one rapid and reversible (occurring on the order of seconds) and
one tight and virtually irreversible. In contrast to the case of the
liver, reversible retention of
125I-ET-1 was not observed in
similar multiple-indicator dilution experiments in the canine
myocardium (3) and lung (2). According to this, extraction of
125I-ET-1 as observed in the liver
(this study), in the myocardium (3), and in the lung (2) may represent
quasi-irreversible binding to surface receptors. With the use of
endothelin-receptor antagonists, it was demonstrated that the
irreversible binding site responsible for hepatic ET-1 clearance is the
ETB receptor. This result is in
accordance with observations in dog lungs, in which ET-1 extraction is
exclusively mediated by the endothelial ETB receptor with no detectable
reversible binding by indicator-dilution experiments (2). It is of note
that previous hepatic endothelin-receptor binding studies were
performed with long incubation times (between 10 min and several
hours), with subsequent washing with buffers. Under such conditions,
reversible binding with dissociation rate constants as high as those
reported here would have been missed. Our results contradict those in
rats where hepatic retention of ET-1 was not affected by the
ETB antagonist BQ-788 (9).
The reversible binding site was not dose dependently affected by the
nonspecific antagonist, suggesting that this second site is neither an
ETA nor an
ETB receptor. Because no other
type of endothelin receptors have been described in mammals, this most likely represents nonspecific binding at the liver surface. The reason
why the fitted transfer coefficients for the reversible hepatic binding
site tended to be lower, but not dose dependent, with the nonspecific
antagonist is unclear. The greater variability and standard errors in
the fitted parameters may suggest that this represents experimental
variability. Such nonspecific reversible liver binding has previously
been postulated for the polypeptide hormone somatostatin, with the use
of similar multiple-indicator dilution experiments in the perfused rat
liver (29). This is in agreement with the reported absence of
somatostatin receptors from the sinusoidal surface of hepatocytes
(8).
Multiple indicator dilution experiments with the peptide hormones
glucagon and epidermal growth factor have been found to conform to the
same barrier-limited model (29). However, an excess of nonlabeled
peptide completely suppressed reversible binding as indicated by the
abolishment of the returning component in the curves. These peptides
are therefore believed to undergo reversible binding to cell-surface
receptors, and the one binding site variant of the model was adopted.
This is in agreement with reversible binding to cell-surface receptors
demonstrated with biophysical measurements with the use of receptors
from cancer cell membranes (36) or expressed in single cells (27).
From experiments with human ETB
receptors expressed in single cells (35), a bimolecular rate constant
for irreversible ET-1 binding of
~10
7 M/s
can be derived. With this value and the value for irreversible hepatic
ET-1 clearance of 0.17 ml · s
1 · g
1
from the present work, the receptor density is ~17 pM/g liver.
The majority of labeled 125I-ET-1
is retained in the liver. This is consistent with previous
electron-microscopic autoradiographic observations obtained from liver
cell cultures showing internalization of labeled ET-1, particularly
into hepatic stellate cells (10, 16). A time-dependent reduction of the
grain density observed in endothelial and Kupffer cells may be
interpreted as a return of unchanged ET-1 after interaction with
surface receptors (10). This process, however, is much slower than the
return of tracer into the Disse space as assessed with the
multiple-indicator dilution technique. The retained ET-1 is most likely
metabolized because enzymes capable of ET-1 degradation have been
identified both on the cell surface and in the intracellular
compartment of liver-derived cells (12).
ET-1 binding to albumin.
The hepatic outflow profile of
125I-ET-1 was closely related to
that of albumin, suggesting a substantial binding of ET-1 to plasma
proteins. This was confirmed by ultrafiltration experiments that
demonstrated that 80% of ET-1 was bound to albumin. Others have
demonstrated qualitatively important and reversible binding of ET-1 to
plasma albumin with the use of gel electrophoresis (34). This binding
must, however, show a fast dissociation rate constant because ~85%
of circulating ET-1 is extracted within a single transit time through
the liver. These observations were therefore incorporated into the
modeling analysis by assuming that dissociation of the ET-1-albumin
complex had no rate-limiting effect on ET-1 exchanges in the liver and
by adjusting the proportion of the Disse space accessible to ET-1 to
reflect the partial exclusion of protein-bound ET-1 from this space.
This resulted in a good quality of fit.
ET-1 production by the liver.
The liver cells have the ability to produce ET-1 that can be used
locally in a paracrine fashion but that may also be released into the
systemic circulation, where it could act as a circulating hormone. We
found a significant statistical difference in the measured ET-1 levels
between the portal and the hepatic veins, indicative of a falling ET-1
gradient along the acinar flow path. This difference is, however, of
much smaller magnitude than that expected from the important tracer
extraction of 85%. This suggests a substantial simultaneous release of
ET-1 by the liver into the circulation in excess of what is predicted
from returning tracer. ET-1 may either be newly synthesized in the
liver or stored for time periods largely exceeding the time of sample
collection. We cannot, however, quantify this release because we have
not measured hepatic artery and portal blood flow separately in our preparation. Our results nevertheless firmly establish for the first
time, in vivo, that the liver not only has the ability to remove but
also to release ET-1 into circulation. This view is confirmed by the
recent finding of ET-1 mRNA in the rat liver, which was most abundant
in endothelial cells, and increased ninefold 3 h after a bolus infusion
of endotoxin into a mesenteric vein (6).
Our results demonstrate both substantial clearance and production of
ET-1 by the intact liver. Hepatic ET-1 clearance is mediated by the
ETB receptor, with the presence of
reversible, nonspecific ET-1 binding at the liver surface. Whether
alterations of hepatic ET-1 metabolism in chronic liver disease may
contribute to the observed increase in circulating ET-1 levels through
a reduction in clearance, an increase in production, or a combination
of both will require further studies.
 |
ACKNOWLEDGEMENTS |
We thank Eva Ibrahim, Bruce Ritchie, and Kay Lumsden for expert
technical assistance as well as Diane Campeau for help in typing this manuscript.
 |
FOOTNOTES |
Deceased 21 March 1996.
This work was supported by the Medical Research Council of Canada, the
Fonds de la recherche en santé du Québec, the Quebec Heart
and Stroke Foundation, the Fonds de recherche de l'Institut de
Cardiologie de Montréal, and the Fast Foundation.
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: J. Dupuis,
Research Center, Montreal Heart Institute, Montreal, PQ, Canada H1T
1C8.
Received 11 February 1998; accepted in final form 10 June 1999.
 |
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