Am J Physiol Heart Circ Physiol
281: H1075–H1084, 2001.
Hemorrhagic shock primes the hepatic portal circulation
for the vasoconstrictive effects of endothelin-1
BENEDIKT H. J. PANNEN,1 STEPHAN SCHROLL,1 TORSTEN LOOP,1
MICHAEL BAUER,2 ALEXANDER HOETZEL,1 AND KLAUS K. GEIGER1
1
Department of Anesthesiology and Critical Care Medicine, University of Freiburg,
D-79106 Freiburg; and 2Department of Anesthesiology and Critical Care
Medicine, University of the Saarland, D-66421 Homburg, Germany
Received 30 November 2000; accepted in final form 24 April 2001
associated with a poor
prognosis, even if adequate volume resuscitation results
in a restoration of systemic hemodynamics. For example,
a recently published prospective study (17) in patients
with hemorrhagic shock revealed an overall mortality of
⬎50%. This high mortality rate seems primarily to be due
to the subsequent development of multiple organ failure
(39). Among those patients that develop multiple organ
failure, the incidence of liver failure has been reported to
exceed 60% (39). Moreover, based on the central role the
liver plays in the organism’s metabolic and immunological response to injury (37), the occurrence of liver failure
is frequently associated with a further deterioration of
the prognosis (19).
This raises the question of which pathophysiological
mechanisms may be responsible for the development of
liver injury after hemorrhagic shock and resuscitation
(HSR). Although various factors have been implicated,
accumulating evidence suggests that the occurrence of
hepatic microcirculatory failure may be one major determinant. For example, Wang et al. (47) described a
progressive impairment of hepatic microvascular blood
flow after hemorrhagic shock despite adequate fluid
resuscitation. In addition, physical prevention of microvascular shutdown using a flow-controlled reperfusion mode largely prevents parenchymal cell necrosis
after ischemia-reperfusion of the liver, i.e., the degree
of microcirculatory failure determines the extent of
lethal hepatocyte injury (10). Moreover, the occurrence
of sinusoidal perfusion failure after HSR is associated
with deteriorations of the hepatic mitochondrial redox
state and bile flow, enzyme release from the liver, and
hepatocyte necrosis (2, 36).
It is of particular interest to note that recent evidence from our and other laboratories suggests that
endothelins (ETs), a family of isopeptides that can
exert potent and long-acting vasoconstriction (49), may
play a crucial role in the dysregulation of hepatic
microvascular perfusion (12). Exogenous ETs can
cause a sustained increase in total portal resistance
and a decrease in portal flow in the normal liver (14,
51). A video microscopic study (8) of the hepatic microcirculation has revealed that these vasoconstrictive
effects of ETs involve both extrasinusoidal and sinusoidal sites. The pattern of microcirculatory disturbances caused by exogenously administered ETs
closely resemble the pattern of changes observed in the
liver under pathological conditions such as endotoxemia (30, 35) or ischemia followed by reperfusion (21).
Moreover, under these conditions, ET levels are increased, and administration of ET antiserum or pharmacological blockade of ET receptors reduces microvascular injury in the liver (16, 32, 33, 48).
Address for reprint requests and other correspondence: B. H. J.
Pannen, Anaesthesiologische Universitaetsklinik, Hugstetterstrasse 55, D-79106 Freiburg, Germany (E-mail: [email protected].
uni-freiburg.de).
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. Section 1734
solely to indicate this fact.
endothelin receptors; microcirculation; portal vein; pressureflow relationship; sarafotoxin 6c
HEMORRHAGIC SHOCK IS FREQUENTLY
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0363-6135/01 $5.00 Copyright © 2001 the American Physiological Society
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Pannen, Benedikt H. J., Stephan Schroll, Torsten
Loop, Michael Bauer, Alexander Hoetzel, and Klaus K.
Geiger. Hemorrhagic shock primes the hepatic portal circulation for the vasoconstrictive effects of endothelin-1. Am J
Physiol Heart Circ Physiol 281: H1075–H1084, 2001.—To
test whether hemorrhagic shock and resuscitation (HSR)
alters the vascular responsiveness of the portohepatic circulation to endothelins (ETs), we studied the macro- and microcirculatory effects of the preferential ETA receptor agonist
ET-1 and of the selective ETB receptor agonist sarafotoxin 6c
(S6c) after 1 h of hemorrhagic hypotension and 5 h of volume
resuscitation in the isolated perfused rat liver ex vivo using
portal pressure-flow relationships and epifluorescence microscopy. Although HSR did not cause major disturbances of
hepatic perfusion per se, the response to ET-1 (0.5 ⫻ 10⫺9 M)
was enhanced, leading to greater increases in portal driving
pressure, total portal resistance, and zero-flow pressures and
more pronounced decreases in portal flow, sinusoidal diameters, and hepatic oxygen delivery compared with timematched sham shock controls. In sharp contrast, the constrictive response to S6c (0.25 ⫻ 10⫺9 M) remained unchanged.
Thus HSR primes the portohepatic circulation for the vasoconstrictive effects of ET-1 but does not alter the effects of the
ETB receptor agonist S6c. The enhanced sinusoidal response
may contribute to the subsequent development of hepatic
microcirculatory failure after secondary insults that are associated with increased generation of ET-1.
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PORTAL CONTRACTILE RESPONSE AFTER HEMORRHAGIC SHOCK
MATERIALS AND METHODS
Animals. Male Sprague-Dawley rats (Charles River; Sulzfeld, Germany), weighing between 300 and 350 g, were used
for all experiments. The experimental protocol was approved
by the local animal care and use committee, and all animals
received humane care according to the criteria outlined in the
National Institutes of Health Guide for the Care and Use of
Laboratory Animals (NIH Publication 86-23, Revised 1985).
Rats were fasted for 6 h before the induction of anesthesia
but allowed free access to water. After anesthesia was induced with an intraperitoneal injection of pentobarbital sodium (50 mg/kg body wt), animals were placed supine on a
heating pad (38–40°C) to maintain body temperature. After a
tail vein was cannulated, an infusion of Ringer solution (10
ml 䡠 kg⫺1 䡠 h⫺1) was started to compensate for evaporative
losses during the surgical preparation. Anesthesia was maintained with supplemental intravenous bolus injections of
pentobarbital sodium (5 mg/kg body wt) when indicated by
any evidence of spontaneous muscle activity or lessening of
the anesthetic plane. A tracheotomy was performed, and
animals were allowed to breathe spontaneously. A fluid-filled
polyethylene-50 catheter was inserted into the left carotid
artery and connected to a pressure transducer (MX 860,
Medex Medical; Lancashire, UK), and arterial blood pressure
was continuously measured.
Hemorrhagic shock protocol. A nonlethal nonheparinized
pressure-controlled model of compensated HSR was used.
After surgical instrumentation and a stabilization period of
10 min, animals in shock groups were bled to a mean arterial
pressure of 40 mmHg in ⬍5 min. The rate of blood withdrawal was 2 ml/min. Shed blood was collected in syringes
containing citrate-phosphate-dextrose solution (0.14 ml/ml of
blood) (Sigma; St. Louis, MO). Mean arterial pressure was
AJP-Heart Circ Physiol • VOL
maintained at 40 ⫾ 4 mmHg by intermittent withdrawal of
0.3- to 0.4-ml aliquots of blood or infusion of respective
aliquots of Ringer solution. After 60 min of hemorrhagic
hypotension, resuscitation of the animals was performed
with 60% of the shed blood withdrawn (reinfused during the
first 20 min of resuscitation), and twice the maximal bleedout
volume was given as Ringer solution during the first hour of
resuscitation, i.e., 200% of the shed blood volume. During the
second hour of resuscitation, the infusion rate of the Ringer
solution was lowered to a volume equaling the maximal
bleedout volume, i.e., 100% of the shed blood volume. Thus
the total volume of Ringer solution administered during the
first 2 h of resuscitation equals 300% of the shed blood
withdrawn and 500% of the amount of shed blood reinfused.
For the remaining experimental period, the infusion rate of
Ringer solution was kept constant at the basal rate of 10
ml 䡠 kg⫺1 䡠 h⫺1. This resuscitation regimen ensures complete
recovery and maintenance of mean arterial pressure until
the end of the experiment. Time-matched sham shock animals were anesthetized, completely instrumented as described above, and received a constant infusion of Ringer
solution of 10 ml 䡠 kg⫺1 䡠 h⫺1 during the whole observation
period but did not undergo hemorrhage (sham shock groups).
Isolated perfused liver system. A pressure-limited recirculating isolated liver perfusion system was employed as previously described (10, 34). Briefly, the perfusate [5% rat red
blood cells (RBC) in Krebs-Henseleit bicarbonate buffer
(KHB); pH 7.4] was pumped from an outflow reservoir
through a Silastic tubing oxygenator (gassed with a mixture
of 95% O2-5% CO2) into an overflow chamber that served as
the inflow reservoir. A heat exchanger was used to warm the
perfusate to 37°C. An ultrasonic in-line flow probe (Transonic; Ithaca, NY) was placed ahead of the inlet cannula and
connected to a flowmeter (T-206, Transonic) to measure the
total flow rate (Qt). Portal inlet pressure (Pinlet) was measured via a pressure transducer (Transpac Transducer, Abbott; Wiesbaden, Germany) connected to a T-fitting in the
inlet cannula. The outflow cannula drained into the outflow
reservoir. Another pressure transducer was connected to a
second T-fitting in the vena caval outlet cannula to measure
outlet pressure (Poutlet). Both pressure transducers were calibrated simultaneously and zeroed against a column of fluid
open to the atmosphere at the level of the cannula tips. The
two cannulas were positioned at the level of the abdominal
posterior vena cava. Signals were recorded using the Windaq
200 personal computer-based data-acquisition system (Dataq
Instruments; Akron, OH). Total portal resistance (Rt) was
calculated from (Pinlet ⫺ Poutlet)/Qt.
Multiple-point P-Q relationships were generated essentially as we have previously described (35). The flow rate was
adjusted to 0, 2.5, 5, 10, 15, 20, and 25 ml/min, and Pinlet was
measured under steady-state conditions including direct
measurement of the inlet pressure at zero flow (PQ⫽0). During the generation of each P-Q relationship, Poutlet was continuously monitored and kept at a value of ⫺1 cmH2O by
adjusting the height of the outflow reservoir. The slope of the
P-Q relationship (slopePQR) reflects the flow-dependent incremental resistance of the portohepatic system.
Allogeneic rat blood was obtained for each experiment
from a donor animal after cannulation of the left carotid
artery under pentobarbital anesthesia. RBC were separated
by centrifugation at 3,000 g for 3 min. The supernatant
(plasma and buffy coat) was discarded. The RBC pellet was
washed in 0.9% saline solution, resuspended in an equal
volume of KHB, filtered through a Pall PL50 leucocyte removal filter (Pall Newquay; Cornwall, UK), and stored at 4°C
for no longer than 30 min before use.
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However, it remains unclear how an initial insult
such as compensated and reversible HSR may lead to
the subsequent development of progressive hepatic microcirculatory and organ failure. Evidence suggests
that subtle changes occur in cells or even organs after
exposure to a primary minor stimulus that produces
hardly detectable injury in itself, e.g., short periods of
ischemia, but that may aggravate the response to a
secondary insult (13, 22). Consequently, in addition to
increased generation, an enhanced response to ETs
could contribute to this phenomenon, as previously
shown in other models of hepatic stress such as chronic
ethanol consumption or endotoxemia (6, 35). Therefore,
we hypothesized that HSR, even if it is not primarily
associated with profound impairments of sinusoidal
perfusion, may alter the vascular responsiveness of the
portohepatic circulation and prime the liver for the
deleterious vasoconstrictive effects of ETs.
To test this hypothesis, we used a rat model of in vivo
HSR and studied the effect of hemorrhagic shock on
the intrinsic portal contractile response to ETs in the
isolated perfused liver ex vivo. This methodological
approach allows the combined analysis of 1) total portal resistance, 2) flow-dependent and -independent
components of portal resistance using multiple-point
pressure-flow (P-Q) relationships, and 3) hepatic microcirculation via direct in situ video microscopy without any confounding systemic hemodynamic effects of
ETs.
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PORTAL CONTRACTILE RESPONSE AFTER HEMORRHAGIC SHOCK
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previously described (34) using digitized frame-by-frame
analysis with the Lobulus image analysis system (Medvis;
Homburg, Germany) at a specimen-to-monitor ratio of
⫻2,400 (32). Pre- and posttreatment measurements were
performed within the same segments of the same sinusoids.
Hepatic oxygen delivery and consumption. To obtain estimates of total hepatic oxygen delivery (DO2) and consumption
(VO2), 200 ␮l of perfusate were simultaneously withdrawn
from the inflow and outflow tubing of the isolated perfused
liver system into gas-tight syringes. Subsequently, hemoglobin concentration, oxygen saturation, and oxygen partial pressure were determined within each sample using
an automated blood gas analysis system (ABL 625, Radiometer Medical A/S; Copenhagen, Denmark), which had
been calibrated for rat blood using an OSM 3 hemoximeter
(Radiometer Medical A/S). Estimates of hepatic DO2 and
VO2 were derived using standard and previously described
equations (31).
Data analysis. Data are presented as means ⫾ SE. Raw
data at baseline are provided in Table 1. Statistical differences from baseline were determined using a paired t-test.
Differences between shock and sham shock groups were
tested using an unpaired t-test within each experimental
series. When criteria for parametric tests were not met
(Kolomogorov-Smirnow test for normality and/or LeveneMediane test for equal variance failed), the respective
nonparametric tests (i.e., Wilcoxon signed-rank test or
Mann-Whitney rank sum test) were used. All individual
P-Q relationships were analyzed by linear regression to
obtain the regression slope. A P value ⬍0.05 was considered to indicate a significant difference. All statistical tests
were performed using the SigmaStat software package
(Jandel Scientific; San Rafael, CA).
RESULTS
Hepatic macrohemodynamics, microhemodynamics,
and oxygenation after HSR. The model of compensated
hemorrhagic shock used in the present study did not
cause major disturbances of portal venous hemodynamics in the isolated perfused liver after 5 h of resusTable 1. Hepatic macrohemodynamics,
microhemodynamics, and oxygenation in sham shock
and shock groups at baseline
Pinlet ⫺ Poutlet, cmH2O
Qt, ml/min
Rt, cmH2O 䡠 min 䡠 ml⫺1
PQ⫽0, cmH2O
SlopePQR, cmH2O 䡠 min 䡠 ml⫺1
DS, ␮m
VRBC, ␮m/s
DO2, ␮l/min
VO2, ␮l/min
Sham Shock
Shock
8.8 ⫾ 0.34
20.1 ⫾ 0.04
0.44 ⫾ 0.017
1.9 ⫾ 0.15
0.27 ⫾ 0.019
12.3 ⫾ 0.35
406 ⫾ 30
710 ⫾ 26.7
361 ⫾ 12.7
9.2 ⫾ 0.50
20.1 ⫾ 0.03
0.46 ⫾ 0.025
1.6 ⫾ 0.21
0.27 ⫾ 0.017
12.5 ⫾ 0.34
407 ⫾ 41
783 ⫾ 24.4
374 ⫾ 19.9
Values are means ⫾ SE. Livers were obtained from rats subjected
to hemorrhagic shock (1 h; mean arterial pressure: 40 ⫾ 4 mmHg)
and volume resuscitation (for 5 h; shock group, n ⫽ 19 experiments)
or from time-matched sham shock animals (sham shock group,
n ⫽ 20 experiments), and isolated and perfused via the portal vein.
After a stabilization period of 15 min, all baseline measurements
were obtained. Pinlet ⫺ Poutlet, inlet pressure minus outlet pressure;
Qt, total portal flow rate; Rt, total portal resistance; PQ⫽0, zero-flow
inlet pressure; slopePQR, regression slope of multiple-point pressureflow relationships; Ds, sinusoid diameter; VRBC, red blood cell velocity; DO2, hepatic oxygen delivery; VO2, hepatic oxygen consumption.
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Experimental protocol. On the basis of the time course of
shock-induced hepatic expression of vasoactive mediators (3,
5), baseline measurements and all subsequent pharmacological interventions were performed at 6 h after shock induction (i.e., 1-h shock, 5-h resuscitation; shock groups) or in
time-matched sham shock experiments (1-h sham shock, 5-h
sham resuscitation; sham shock groups). At 30 min before
baseline, a laparotomy was performed, and livers were isolated and perfused in situ via the portal vein essentially as
previously described (10, 34). The initial flow rate was set to
30 ml/min, and the perfusate hematocrit was adjusted to
⬃5% by addition of rat RBC prepared as described above.
Fluorescein isothiocyanate-labeled bovine serum albumin
was added to the perfusate for assessment of sinusoidal
boundaries and visualization of RBC in the sinusoids via
negative contrast (26). Subsequently, the flow rate was reduced to 20 ml/min, and, after a 15-min stabilization period,
all baseline measurements were obtained.
Three different series of experiments were conducted. In
the first series, immediately after baseline, a single dose of
the preferential ETA receptor agonist ET-1 (Sigma) was
added to the perfusate of livers isolated from either sham
shock or shock animals (n ⫽ 9 per group) to achieve a final
ET-1 concentration of 0.5 ⫻ 10⫺9 M. In a second experimental series, the selective ETB receptor agonist sarafotoxin 6c
(S6c; Sigma) was added (sham shock, n ⫽ 6 experiments/
group; shock, n ⫽ 5 experiments/group). A S6c perfusate
concentration of 0.25 ⫻ 10⫺9 M was chosen, because pilot
experiments with sham control livers had shown that the
peak increase in Rt induced by this S6c dose was similar in
magnitude to the peak response to an ET-1 perfusate concentration of 0.5 ⫻ 10⫺9 M. In a third series, 1 ml of vehicle
(H2O) was added to the perfusate of livers from either sham
shock or shock animals as a control (n ⫽ 5 per group).
Posttreatment measurements were performed after 10 min,
because pilot experiments have shown that the macro- and
microhemodynamic effects of ET-1 and S6c reached a stable
plateau after 5 min and that this lasted for ⬃5–10 min.
Epifluorescence liver microscopy. Videomicroscopy of the
liver was performed essentially as reported in detail previously (10, 34). Briefly, the liver preparation was positioned on
the stage of a Zeiss Axiotech fluorescence microscope (Axiotech Vario 100 HD; Carl Zeiss; Jena, Germany) and viewed
with a ⫻40 water-immersion objective (Zeiss Achroplan, Carl
Zeiss). The surface of the left liver lobe was epi-illuminated
with a fiber-optic illuminator (KL 1500, Schott Glaswerke;
Wiesbaden, Germany) similar to the oblique transillumination procedure described by MacPhee et al. (24), and a randomly chosen acinus was brought into focus so that sinusoids
of zone 3 could be observed. This region of the acinus was
chosen because the parallel arrangement of sinusoids permits unambiguous quantitative assessment of the sinusoidal
microcirculation (11). With the same area in focus, the liver
was epi-illuminated with a 100-W mercury lamp with 450–
490 nm of excitation and 520-nm emission band-pass filters,
which allows visualization of fluorescein isothiocyanate-conjugated albumin within hepatic sinusoids. All microscopic
images were projected onto a charge-coupled device video
camera (FK 6990 IQ-S, Pieper; Schwerte, Germany). On-line
digital contrast enhancement was performed on all images
for improved clarity using an Argus-20 image processor
(Hamamatsu Photonics; Hamamatsu, Japan). Processed images were recorded for off-line analysis using a S-VHS video
recorder (Panasonic AG 7350E, Matsushita Electrical Industrial; Osaka, Japan).
Assessment of sinusoid diameter (Ds) and RBC velocity
(VRBC) was done off-line during video playback as we have
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PORTAL CONTRACTILE RESPONSE AFTER HEMORRHAGIC SHOCK
Fig. 1. Effect of hemorrhagic shock on changes in portal driving
pressure (inlet-outlet pressure) in isolated perfused rat livers in
response to either vehicle (1 ml H2O), endothelin-1 (ET-1; 0.5 ⫻ 10⫺9
M), a preferential ETA receptor agonist, or sarafotoxin 6c (S6c;
0.25 ⫻ 10⫺9 M), a selective ETB receptor agonist. Posttreatment
measurements were performed 10 min after pharmacological intervention. Livers were obtained from rats subjected to hemorrhagic
shock (1 h; mean arterial pressure: 40 ⫾ 4 mmHg) and volume
resuscitation (5 h; shock groups) or from time-matched sham shock
animals. Data represent means ⫾ SE; n ⫽ 5–9 experiments/group.
*P ⬍ 0.05 vs. respective baseline value (within each group); #P ⬍
0.05 vs. respective sham shock group (within each series).
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Fig. 2. Effect of hemorrhagic shock on changes in total portal flow
rate in isolated perfused rat livers in response to either vehicle (1 ml
H2O), ET-1 (0.5 ⫻ 10⫺9 M), or S6c (0.25 ⫻ 10⫺9 M). Posttreatment
measurements were performed 10 min after pharmacological intervention. Livers were obtained from rats subjected to hemorrhagic
shock and volume resuscitation (shock groups) or from time-matched
sham shock animals. Data represent means ⫾ SE; n ⫽ 5–9 experiments/group. *P ⬍ 0.05 vs. respective baseline value (within each
group); #P ⬍ 0.05 vs. respective sham shock group (within each
series).
both vasoactive agents caused increases in the flowindependent (Fig. 4) and flow-dependent components
(Fig. 5) of Rt in sham control and shock groups. HSR
did not alter the effect of ET-1 or S6c on the slopePQR
(Fig. 5). However, the changes in PQ⫽0 were differentially affected after shock (Fig. 4). Whereas HSR did
not influence the magnitude of the increase in PQ⫽0
Fig. 3. Effect of hemorrhagic shock on changes in total portal vascular resistance in isolated perfused rat livers in response to either
vehicle (1 ml H2O), ET-1 (0.5 ⫻ 10⫺9 M), or S6c (0.25 ⫻ 10⫺9 M).
Posttreatment measurements were performed 10 min after pharmacological intervention. Livers were obtained from rats subjected to
hemorrhagic shock and volume resuscitation (shock groups) or from
time-matched sham shock animals. Data represent means ⫾ SE; n ⫽
5–9 experiments/group. *P ⬍ 0.05 vs. respective baseline value
(within each group); #P ⬍ 0.05 vs. respective sham shock group
(within each series).
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citation. This is illustrated by the experimental data
summarized in Table 1. At this time point, all macroand microhemodynamic parameters measured, as well
as hepatic DO2 and VO2, did not significantly differ
between the shock and sham shock groups.
Effect of HSR on the change in total portal vein
resistance in response to ET-1 and S6c. Whereas vehicle did not cause any major changes in the macrohemodynamic parameters measured, administration of
ET-1 increased Pinlet ⫺ Poutlet (Fig. 1), caused a decrease in Qt (Fig. 2), and caused a respective increase
in Rt (Fig. 3) in all experimental groups. However, the
resistive response to ET-1 was more pronounced in
livers from animals that underwent HSR compared
with livers from sham shock animals (Figs. 1–3). Addition of the preferential ETB receptor agonist S6c to
the perfusate also resulted in increases in Pinlet ⫺
Poutlet and Rt (Figs. 1 and 3) and decreases in Qt
compared with baseline (Fig. 2). Despite this similarity, livers from shock animals showed different behaviors in response to the two vasoconstrictive agents. In
contrast with the enhancing effect of HSR on the portal
contractile response to ET-1, the macrohemodynamic
changes induced by S6c were either unaffected (Qt and
Rt; Figs. 2 and 3) or even attenuated (Pinlet ⫺ Poutlet;
Fig. 1) after HSR compared with the respective sham
control group.
Effect of HSR on ET-1- and S6c-induced changes in
portal vein P-Q relationships. The administration of
vehicle to the perfusate did not have a significant effect
on PQ⫽0 (Fig. 4) or the slopePQR in livers from sham
shock controls or shock animals (Fig. 5). In contrast,
PORTAL CONTRACTILE RESPONSE AFTER HEMORRHAGIC SHOCK
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groups (Fig. 8, A and B). In contrast, both ET-1 and S6c
caused substantial reductions in DO2 (Fig. 8A).
Whereas the negative effect of S6c on DO2 tended to be
attenuated after HSR, the impairment of the hepatic
oxygen supply in response to ET-1 was more pronounced under these conditions compared with respective sham shock controls (Fig. 8A). Although the
changes in VO2 did not reach statistical significance,
there was a tendency toward a lower VO2 in particular
after ET-1 administration in the shock group, whereas
VO2 tended to increase on S6c administration after
shock (Fig. 8B).
DISCUSSION
after S6c, the increase in PQ⫽0 in response to ET-1 was
much more pronounced after shock compared with
sham shock.
Effect of HSR on the hepatic microcirculatory response to ET-1 and S6c. Direct microscopic observation
of the hepatic microcirculation within the intact organ
revealed a significant narrowing of sinusoids in response to ET-1 that could not be observed in the vehicle-treated groups (Table 2 and Fig. 6). The magnitude
of sinusoidal narrowing after ET-1 was much more
pronounced in livers from animals that underwent
HSR compared with those that had been obtained from
sham shock animals (Fig. 6). Representative highpower video micrographs illustrating the enhanced
ET-1-induced decrease in sinusoidal dimensions that
could be observed after HSR are depicted in Fig. 7.
Whereas S6c did also cause a reduction in Ds, its
microcirculatory effects were not altered by HSR, i.e.,
the decrease in Ds was similar in the sham and the
shock group (Table 2 and Fig. 6). Whereas VRBC remained unchanged in the two vehicle groups, administration of ET-1 caused a decrease in VRBC in the sham
control group that could not be observed in the shock
group (⫺106 ⫾ 37 vs. ⫺6 ⫾ 40 ␮m/s, P ⬍ 0.05). In
contrast, addition of S6c to the perfusate resulted in an
increase in VRBC in livers from sham shock animals
(169 ⫾ 48 ␮m/s, P ⬍ 0.05) but did not cause any
significant changes in VRBC in livers from shock animals.
Influence of HSR on the changes in hepatic oxygenation after ET-1 and S6c. Estimates of total hepatic
DO2 and VO2 remained unchanged in the two vehicle
AJP-Heart Circ Physiol • VOL
Fig. 5. Effect of hemorrhagic shock on changes in the calculated
regression slope of multiple-point pressure-flow relationships
(slopePQR) in isolated perfused rat livers in response to either vehicle
(1 ml H2O), ET-1 (0.5 ⫻ 10⫺9 M), or S6c (0.25 ⫻ 10⫺9 M). Posttreatment measurements were performed 10 min after pharmacological
intervention. The inlet pressure was measured under steady-state
conditions at multiple levels of portal flow (0, 2.5, 5, 10, 15, 20, and
25 ml/min) while the outlet pressure was kept constant at a value of
⫺1 cmH2O. Slopes of individual pressure-flow relationships of each
organ at each pre- and posttreatment time point were calculated over
the entire range of flow. Livers were obtained from rats subjected to
hemorrhagic shock and volume resuscitation (shock groups) or from
time-matched sham shock animals. Data represent means ⫾ SE; n ⫽
5–9 experiments/group. *P ⬍ 0.05 vs. respective baseline value
(within each group).
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Fig. 4. Effect of hemorrhagic shock on changes in zero-flow portal
inlet pressure (PQ⫽0) in isolated perfused rat livers in response to
either vehicle (1 ml H2O), ET-1 (0.5 ⫻ 10⫺9 M), or S6c (0.25 ⫻ 10⫺9
M). Posttreatment measurements were performed 10 min after pharmacological intervention. The inlet pressure was measured under
steady-state conditions while the outlet pressure was kept constant
at a value of ⫺1 cmH2O. Livers were obtained from rats subjected to
hemorrhagic shock and volume resuscitation (shock groups) or from
time-matched sham shock animals. Data represent means ⫾ SE; n ⫽
5–9 experiments/group. *P ⬍ 0.05 vs. respective baseline value
(within each group); #P ⬍ 0.05 vs. respective sham shock group
(within each series).
Accumulating evidence suggests that even a subtle
initial insult that does not cause overt disturbances in
organ perfusion, function, or integrity may nevertheless significantly alter the responsiveness to a secondary noxious stimulus, thus leading to the subsequent
development of organ dysfunction or failure (13, 23).
However, the pathophysiological mechanisms responsible for this “two-hit” phenomenon remain to be identified. Maintenance of the integrity of the microcirculation is crucial to prevent liver injury (10). Therefore,
in addition to changes in hepatic metabolic activity,
energy status, or immune function, alterations in the
control of nutritive microvascular blood flow could provide a mechanistical basis for the negative priming
effect exerted by hemorrhagic shock (28). In this re-
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PORTAL CONTRACTILE RESPONSE AFTER HEMORRHAGIC SHOCK
Table 2. Mean Ds in individual isolated perfused rat
livers from sham shock or shock animals before and
after vehicle, ET-1, or S6c
Sham Shock
Baseline
Shock
10 min
10 min
12.1 ⫾ 2.76
10.1 ⫾ 0.65
10.2 ⫾ 0.69
15.2 ⫾ 1.25
11.8 ⫾ 1.06
12.9 ⫾ 2.78*
9.7 ⫾ 0.83
10.4 ⫾ 0.64
15.8 ⫾ 1.04
12.4 ⫾ 1.64
12.2 ⫾ 1.73
12.1 ⫾ 1.01
14.4 ⫾ 0.19
15.8 ⫾ 0.82
13.0 ⫾ 2.30
14.7 ⫾ 1.03
10.0 ⫾ 0.75
15.3 ⫾ 1.57
12.9 ⫾ 1.12
9.4 ⫾ 1.86*
12.0 ⫾ 0.71
7.4 ⫾ 0.66*
14.5 ⫾ 1.47
9.1 ⫾ 0.99
10.1 ⫾ 1.08*
6.7 ⫾ 1.04*
9.2 ⫾ 1.54*
9.4 ⫾ 1.14*
12.9 ⫾ 1.11
13.2 ⫾ 0.18
12.7 ⫾ 0.36
8.5 ⫾ 0.80
9.7 ⫾ 0.45
8.2 ⫾ 0.62*
8.5 ⫾ 0.61*
9.0 ⫾ 0.40*
5.7 ⫾ 0.70*
6.7 ⫾ 0.42*
Vehicle
6.6 ⫾ 0.38
13.4 ⫾ 0.29
13.5 ⫾ 0.60
11.5 ⫾ 0.83
15.8 ⫾ 2.12
7.4 ⫾ 0.44
12.7 ⫾ 0.34*
13.9 ⫾ 0.72
11.3 ⫾ 0.66
14.6 ⫾ 1.87
11.0 ⫾ 0.45
10.5 ⫾ 1.68
14.5 ⫾ 0.39
9.0 ⫾ 0.85
11.0 ⫾ 0.89
8.5 ⫾ 0.62
12.1 ⫾ 1.18
13.8 ⫾ 1.34
11.7 ⫾ 1.21
9.6 ⫾ 0.60
8.8 ⫾ 1.63*
10.6 ⫾ 0.60*
6.3 ⫾ 0.64*
11.5 ⫾ 1.01
6.4 ⫾ 0.76*
12.9 ⫾ 0.82
11.4 ⫾ 0.65
8.8 ⫾ 1.08*
ET-1
S6c
16.3 ⫾ 1.51
15.3 ⫾ 0.54
14.7 ⫾ 0.82
12.6 ⫾ 1.51
12.6 ⫾ 0.83
11.9 ⫾ 1.52
11.0 ⫾ 0.83*
11.5 ⫾ 0.40*
10.7 ⫾ 1.10*
7.1 ⫾ 0.94*
9.1 ⫾ 0.85*
8.0 ⫾ 1.04*
Values are means ⫾ SE of all sinusoids analyzed in each individual
liver. Livers were obtained from rats subjected to hemorrhagic shock
(1 h; mean arterial pressure: 40 ⫾ 4 mmHg) and volume resuscitation (for 5 h; Shock group, n ⫽ 19 experiments) or from timematched sham shock animals (sham shock group, n ⫽ 20 experiments), and isolated and perfused via the portal vein. After a
stabilization period of 15 min, baseline measurements were performed. Posttreatment measurements were obtained at 10 min after
the administration of either vehicle (1 ml H2O), endothelin-1 (ET-1;
0.5 ⫻ 10⫺9 M), or sarafotoxin 6c (S6c; 0.25 ⫻ 10⫺9 M). * P ⬍ 0.05 vs.
respective baseline value (within each experiment).
gard, it is of particular interest to note, as a recently
published study by Garrison et al. (15) showed, that
HSR can substantially enhance the microvascular responsiveness of the small intestinal circulation to bacterial endotoxin. Thus it was the aim of the present
study to test the hypothesis that hemorrhagic shock,
even if it is not primarily associated with major impairments of sinusoidal perfusion, may prime the portohepatic circulation for the vasoconstrictive effects of ETs.
The combined analysis of portal macro- and microhemodynamics revealed that HSR enhances the portal
vasoconstrictive response to ET-1 and that this increase in responsiveness occurs predominantly within
the sinusoidal compartment. These conclusions are derived from the following observations.
Administration of ET-1 to livers from sham shock
animals increased Rt and caused a respective decrease
in portal flow. This is in agreement with previous
results from our and other laboratories (8, 45) and
confirms that ET-1 acts as a potent vasoconstrictor
within the portal venous circulation of the liver. Although HSR in itself did not cause major disturbances
AJP-Heart Circ Physiol • VOL
Fig. 6. Effect of hemorrhagic shock on changes in sinusoid diameter
in isolated perfused rat livers in response to either vehicle (1 ml
H2O), ET-1 (0.5 ⫻ 10⫺9 M), or S6c (0.25 ⫻ 10⫺9 M). Posttreatment
measurements were performed 10 min after pharmacological intervention. Sinusoid diameters were determined using in situ epifluorescence videomicroscopy. Pre- and posttreatment measurements
were performed within the same segments of the same sinusoids.
Livers were obtained from rats subjected to hemorrhagic shock and
volume resuscitation (shock groups) or from time-matched sham
shock animals. Data represent means ⫾ SE; n ⫽ 5–9 experiments/
group. *P ⬍ 0.05 vs. respective baseline value (within each group);
#P ⬍ 0.05 vs. respective sham shock group (within each series).
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Baseline
of hepatic perfusion under these experimental conditions, the responsiveness to ET-1 was substantially
enhanced, i.e., the increase in Pinlet ⫺ Poutlet and Rt as
well as the decrease in Qt after ET-1 was much more
pronounced in livers from animals that underwent
HSR compared with livers from sham shock animals. A
similar increase in the portohepatic contractile response to ETs has also been described after chronic
ethanol consumption (6) or endotoxemia (35). It is
important to note that these are very different pathological entities. For example, whereas endotoxemia
causes a generalized inflammatory response, inflammation is a delayed and much less pronounced event
after HSR. Therefore, the similar increase in the contractile response to ET-1 observed under these different conditions may be considered to reflect a rather
uniform general remodeling of the hepatic hemodynamic responsiveness that occurs after diverse pathological stimuli.
Identification of the sites of altered resistance regulation could contribute to the characterization of the
mechanisms responsible for the HSR-mediated increase in the portohepatic contractile response to ET-1.
Thus we combined the analysis of portal venous P-Q
relationships with the direct observation of the sinusoidal microcirculation using epifluorescence video microscopy. Previous studies (1, 27) have provided substantial indirect evidence that changes in the flowindependent component of portal venous resistance,
i.e., PQ⫽0, primarily reflect changes in resistance that
occur in a downstream sinusoidal or postsinusoidal
compartment. On the other hand, changes in the flow-
PORTAL CONTRACTILE RESPONSE AFTER HEMORRHAGIC SHOCK
H1081
dependent component of portal resistance, i.e., of the
slopePQR, reflect changes in resistance that occur upstream from the site of the PQ⫽0 in a vessel where a
positive PQ⫽0 higher than the actual Poutlet exists (9,
27). Therefore, the increase in PQ⫽0 and slopePQR,
associated with a respective decrease in Ds and VRBC
that could be observed in livers from sham shock animals in response to ET-1, strongly supports the results
of previous studies (8, 50) in the normal liver: that
ET-1 acts at both sinusoidal and extrasinusoidal sites.
It is of great interest to note that the changes in the
components of Rt after ET-1 were differentially affected by HSR. Whereas the ET-1-induced increase in
PQ⫽0 was much greater after HSR compared with
sham, the increase in slopePQR was comparable in both
groups. On the basis of the assumptions summarized
above, these data would suggest that the enhancement
of the contractile response to ET-1 after HSR occurred
primarily within the sinusoidal compartment of the
liver. This is further supported by the fact that in situ
microscopic measurements of Ds did indeed reveal that
the sinusoidal narrowing caused by ET-1 was much
more pronounced after HSR compared with sham
while, at the same time, the reduction in VRBC was
attenuated. This raises the question of which factors
could be responsible for the fact that manifestation of
the enhanced responsiveness to ET-1 after HSR occurred primarily within the sinusoidal compartment.
On one hand, this could be the result of a HSRinduced increase in the contractility of hepatic stellate
cells (HSC) (46). These nonparenchymal liver cells are
localized within the space of Dissé and have been
suggested to act as liver-specific pericytes with vasoAJP-Heart Circ Physiol • VOL
regulatory properties at the level of the sinusoids (18,
38). For example, HSC can actively contract in response to ET-1 in vitro as well as in the intact liver in
situ (20, 50). Moreover, activation of HSC has been
shown to be associated with a specific increase in the
sensitivity to ET-1 (40, 43). Therefore, the results of
the present study would be internally consistent with
the notion that the enhancement of the ET-1-mediated
sinusoidal narrowing after HSR may result from a
hypercontractile state of HSC. On the other hand,
McCuskey (25) recently suggested that diameters of
sinusoids might also decrease due to passive recoil
when inflow is reduced via an increase in presinusoidal
resistance and intrasinusoidal distending pressure
falls. Thus the enhanced sinusoidal narrowing in response to ET-1 after HSR could also reflect a hypercontractility of smooth muscle cells located within the
terminal portal venules. However, portal venular constriction, e.g., in response to the ␣1-adrenoreceptor
agonist phenylephrine, has been shown to be associated with an isolated decrease in slopePQR in the absence of any significant changes in PQ⫽0 (35). Because
this was not the case under the experimental conditions of the present study, the sum of these data favor
a predominant role of the sinusoidal compartment in
the HSR-induced enhancement of the vasoconstrictive
response to ET-1.
The biological effects of ETs are mediated through
different receptors that are heterogeneously expressed
among liver cells. This raises the question of which
receptors could be involved in the sensitizing process
after HSR. Whereas HSC and hepatocytes express ETA
and ETB receptors, only the ETB receptor can be found
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Fig. 7. Representative high-power in
situ video micrographs of sinusoids as
viewed by the oblique transillumination technique at baseline (A and C)
and 10 min after the administration of
ET-1 (0.5 ⫻ 10⫺9 M; B and D). The
liver depicted in A and B was obtained
from a sham shock animal, and the
liver shown in C and D was from an
animal that underwent hemorrhagic
shock. Note that ET-1 causes only a
moderate decrease in sinusoid diameters after sham shock (B), whereas the
sinusoidal narrowing observed in response to ET-1 is much more pronounced after HSR (D).
H1082
PORTAL CONTRACTILE RESPONSE AFTER HEMORRHAGIC SHOCK
on Kupffer and endothelial cells (18). Both ETA and
ETB receptor agonists may cause contraction of the
respective hepatic target cells (42, 50). However, the
relative contribution of the two receptors may largely
vary depending on the actual experimental or pathological conditions. Thus we also studied the effect of
HSR on the contractile response to the selective ETB
receptor agonist S6c. In sharp contrast with the enhancing effect of HSR on the response to ET-1, the
portohepatic hemodynamic effects of S6c were unchanged or even slightly attenuated after shock. In this
regard, it is of particular interest to note that Reinher
et al. (40) recently proposed that activation of HSC
may induce the appearance of a high-affinity ETA receptor subtype, whereas in quiescent HSC a low-affinity ETA receptor subtype prevails (40). However, these
putative receptor subtypes have not been dissected at
the molecular level, and neither ETA nor ETB receptor
AJP-Heart Circ Physiol • VOL
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Fig. 8. Effect of hemorrhagic shock on changes in estimates of total
hepatic oxygen delivery (A) and consumption (B) in isolated perfused
rat livers in response to either vehicle (1 ml H2O), ET-1 (0.5 ⫻ 10⫺9
M), or S6c (0.25 ⫻ 10⫺9 M). Hemoglobin concentration, oxygen
saturation, and oxygen partial pressure were determined within
perfusate samples withdrawn from the inflow and outflow tubing to
calculate estimates of oxygen delivery and consumption. Posttreatment measurements were performed 10 min after pharmacological
intervention. Livers were obtained from rats subjected to hemorrhagic shock and volume resuscitation (shock groups) or from timematched sham shock animals. Data represent means ⫾ SE; n ⫽ 5–9
experiments/group. *P ⬍ 0.05 vs. respective baseline value (within
each group); #P ⬍ 0.05 vs. respective sham shock group (within each
series).
antagonists were used in the present study. Therefore,
the sensitizing effect of HSR on the contractile response to ETs cannot be ascribed to a specific ET
receptor. In addition, it is important to note that only a
single dose of ET-1 and S6c was tested. On the basis of
the results of previous dose-response determinations of
ET-1 in the portal circulation (7), an ET-1 dose was
chosen that caused a half-maximal increase in portal
resistance to allow the detection of both increases as
well as decreases in the contractile response. Consequently, a dose of S6c was chosen for comparison,
which caused an increase in Rt in the normal liver of a
similar magnitude as that of ET-1 did. However, based
on this experimental design, we cannot exclude the
possibility that the differential effects of HSR on the
portohepatic contractile response may vary depending
on the actual concentration of ET-1 or S6c.
The results of the present study were obtained using
an isolated perfused liver system. This experimental
approach was chosen to allow the analysis of the intrinsic hepatic hemodynamic response to ETs in the
absence of confounding factors such as varying levels of
anesthesia, changes in sympathetic outflow, differences in hematocrit, or systemic hemodynamics. Despite these advantages, the use of such a system may
be associated with significant alterations of hepatic
perfusion. For example, in agreement with previous
results, mean Ds were ⬃0.5–2 ␮m wider, and Ds
showed a larger variability in the isolated perfused
liver in vitro compared with the normally perfused
liver in vivo (4, 8, 51). Therefore, factors that could
contribute to these phenomena, such as an inhomogeneous washout of vasoactive mediators, denervation,
or altered intra- and extravascular pressure gradients,
may in turn have affected the response to the agonists.
Consequently, further in vitro and in vivo studies will
be needed to more precisely define the mechanisms of
the sensitization of the liver to ET-1 after HSR.
Various pathological conditions that frequently follow upon hemorrhagic shock, such as endotoxemia,
hypoxemia, or recurrent periods of hemorrhagic hypotension, have been shown to result in increases in
systemic or hepatic levels of ET-1 (29, 41, 44, 48, 52).
On the basis of the results of the present study, exposure of the liver to increased amounts of ET-1 after
HSR-mediated priming of the portohepatic circulation
may cause critical perturbations of nutritive hepatic
blood flow. The potential functional significance of
these findings is further supported by the fact that the
increased responsiveness to ET-1 was associated with
respective impairments of hepatic oxygenation.
In conclusion, our results demonstrate that HSR
primes the portohepatic circulation for the vasoconstrictive effects of ET-1 via a mechanism that predominantly involves the sinusoidal compartment. These
findings could, at least in part, explain why compensated and reversible HSR frequently leads to the subsequent development of progressive hepatic microcirculatory and organ failure in response to a secondary
insult.
PORTAL CONTRACTILE RESPONSE AFTER HEMORRHAGIC SHOCK
The authors thank Karl-Heinz Kopp for helpful discussions.
This work was supported by The Deutsche Forschungsgemeinschaft PA 533/2-2 and PA 533/3-1 (to B. H. J. Pannen) and BA
1601/1-2 (to M. Bauer).
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