Center for Surgical Research and Department of Surgery, University of Alabama at Birmingham, Birmingham, Alabama 35294
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ABSTRACT |
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Gut-derived norepinephrine (NE) has been shown to play a
critical role in producing hepatocellular dysfunction in early sepsis, but it is not known whether 2-adrenoceptor activation
mediates this dysfunction. We infused normal male adult rats with NE,
NE plus the specific
2-adrenergic antagonist rauwolscine
(RW), or vehicle (normal saline) for 2 h. Hepatocellular function
was determined by in vivo indocyanine green (ICG) clearance. An
isolated perfused liver preparation was also used to assess
hepatocellular function by in vitro ICG clearance; NE alone or with RW
was added to the perfusate. Rats were subjected to sepsis by cecal
ligation and puncture (CLP). At 1 h after CLP, RW was infused for
15 min. At 5 h after CLP, we measured hepatocellular function and
serum tumor necrosis factor-
(TNF-
) levels. Intraportal NE
infusion in normal rats produced hepatocellular dysfunction, which was
prevented by RW and NE infusion. This is confirmed by findings with the isolated perfused liver preparation. RW administration in early sepsis
maintained hepatocellular function and downregulated TNF-
production
at 5 h after CLP. These results suggest that NE-induced hepatocellular dysfunction in early sepsis is mediated by
2-adrenoceptor activation, which appears to upregulate
TNF-
production. Modulation of hepatic responsiveness to NE by
2-adrenergic antagonists should provide a novel approach
for maintaining cell and organ functions during sepsis.
indocyanine green clearance; rauwolscine; cecal ligation and
puncture; isolated perfused rat liver; tumor necrosis factor-
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INTRODUCTION |
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BECAUSE OF ITS
IMPORTANT ROLE in metabolism and host defense mechanisms, the
liver has been extensively studied during sepsis and septic shock and
is thought to be a major organ in the development of multiple organ
failure under such conditions (41). Although hepatic
failure is generally thought to be a late complication following
pulmonary and renal failures (2), previous studies (34, 36) have shown that hepatocellular dysfunction occurs very early after the onset of sepsis and that the depression of hepatocellular function does not appear to be due to a reduction in
hepatic perfusion in early sepsis. It is therefore important to
determine the factor(s) (released early after the onset of sepsis) that
might be responsible for producing hepatocellular dysfunction. In this
regard, proinflammatory cytokines such as tumor necrosis factor-
(TNF-
) have been implicated (31, 32) as important
mediators responsible for producing cellular dysfunction and metabolic
alterations during sepsis. Kupffer cells are known to be a major source
of proinflammatory cytokine release during sepsis as well as under
other adverse circulatory conditions (5, 16, 26). It has
been suggested (25) that the gut may be the "motor"
for initiating multiple organ dysfunction after injury. In this regard,
we (49) have recently demonstrated that the gut becomes a
significant source of norepinephrine (NE) release during sepsis and
gut-derived NE plays a crucial role in depressing hepatocellular
function during the early stage of sepsis. Moreover, Spengler et al.
(28, 29) reported that stimulation of peritoneal macrophages with NE augments TNF-
production, which appears to be
mediated by an
-adrenergic mechanism. Based on these observations, we have postulated that gut-derived NE produces hepatocellular dysfunction during sepsis via its upregulatory effect on TNF-
production by Kupffer cells, which is mediated by the activation of
2-adrenoceptors in the liver (17). Although
perfusion of the isolated liver with NE, at a concentration similar to
that observed during sepsis (10), produces hepatocellular
dysfunction, it remains unknown whether or not hepatocellular
dysfunction observed under such conditions is mediated at least in part
by the activation of
2-adrenoceptors. The objectives of
this study therefore were to determine 1) whether
coadministration of the specific
2-adrenergic antagonist
rauwolscine (RW) prevents NE-induced hepatocellular dysfunction and
2) whether administration of RW early after the onset of
sepsis maintains hepatocellular function and downregulates the
proinflammatory cytokine TNF-
.
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MATERIALS AND METHODS |
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Experimental animals. Adult male Sprague-Dawley rats (275-325 g), purchased from Charles River Laboratories (Wilmington, MA), were used in the present study. All surgery was performed using aseptic procedures with the exception of the induction of sepsis by cecal ligation and puncture (CLP). The experiments described below were performed in adherence to the Guide for the Care and Use of Laboratory Animals [DHHS Publication No. (NIH) 85-23, Revised 1985, Office of Science and Health Reports, Bethesda, MD 20892]. This project was approved by the Institutional Animal Care and Use Committee of the University of Alabama at Birmingham.
Intraportal infusion of NE or NE plus RW in normal animals.
Rats were fasted overnight but allowed water ad libitum before the
experiment. The animals were anesthetized with isoflurane inhalation
during the cannulation of a femoral vein, and the anesthesia was
maintained by intravenous pentobarbital sodium (30 mg/kg body wt)
during the entire experimental period. A 4-cm midline incision was
performed, and a branch of the superior mesenteric vein was isolated
and cannuated with PE-10 tubing (Becton Dickinson, Parsippany, NJ). The
tip of the catheter was advanced toward the liver, reaching the portal
vein. After the catheter was secured, the abdominal wall was closed in
layers. The left femoral artery was also cannuated with PE-50 tubing
and connected to a blood pressure analyzer (Micro-Med, Louisville, KY)
to monitor the mean arterial pressure (MAP) and heart rate during the
infusion of various agents. NE (20 µM, Sigma, St. Louis, MO) or NE
(20 µM) and the specific 2-adrenergic antagonist RW (1 mM, Tocris, Ballwin, MO) were infused via the portal catheter by a
Harvard pump. The infusion rate was set at 13 µl/min, and the total
infusion time was 2 h. Control animals were infused only with
normal saline via the portal vein at a rate of 13 µl/min for 2 h. Portal blood flow was reported to be ~13
ml · min
1 · liver
1 in the
rat (35). Thus infusion of 20 µM NE solution at
a rate of 13 µl/min would increase portal blood levels of NE to ~20
nM, which is similar to that observed during sepsis (10,
49). This concentration of NE (~20 nM) was used because our
(49) recent study indicated that 20 nM NE reduces
hepatocellular function using an isolated perfused liver preparation.
The dose of RW used in this study was 50-fold higher than NE to
completely block the
2-adrenergic effects of this catecholamine.
Determination of hepatocellular function and cardiac output. Hepatocellular function was determined using an in vivo indocyanine green (ICG) clearance technique at the completion of the 2-h NE infusion as we (34) previously described in detail. In brief, rats were anesthetized with isoflurane inhalation, and a 2.4-French fiberoptic catheter was inserted to the level of the aortic arch via the right carotid artery. Three doses of ICG (0.167, 0.333, and 0.833 mg/kg body wt) were administered via the jugular vein catheter, and the ICG concentration in the circulation was recorded each second for 5 min using an in vivo hemoreflectometer and computer-assisted data acquisition. An e raised to a second-order polynomial ([ICG] =ea + bt + ct2) was used to determine the initial velocity of ICG clearance. The maximal velocity of the ICG clearance (Vmax) and the Michaelis constant (Km) were determined from the Lineweaver-Burk plot (41, 43). It should be noted that Vmax represents the number of functional ICG carriers or transporters of the active hepatocellular ICG transport system and Km represents the efficiency of the active transport process (41). Cardiac output was determined by using a dye dilution technique immediately at the end of the 2-h infusion as previously described (34).
Isolated perfused liver preparation and in vitro ICG clearance. An isolated perfused rat liver preparation was used as described in detail previously (48, 49). In brief, the anesthetized rats underwent a longitudinal midline and transverse subcostal incision. The common bile duct was cannulated with a PE-10 catheter for collecting the bile. The distal vena cava and distal portal vein were ligated with sutures. The portal vein was then immediately cannulated with a 16-gauge silicon catheter, and perfusion was initiated within 1 min. The catheter was connected to a three-limb tube that was attached to a perfusion pump and to a syringe filled with 3 ml normal saline containing heparin (20 U/ml). A blood pressure analyzer was connected to the portal catheter using PE-50 tubing to monitor the portal venous perfusion pressure. While the portal catheter was secured, 3 ml heparin-saline were injected into the portal vein. This was immediately followed by perfusion with Krebs-Henseleit buffer with 0.1% glucose and 0.5% BSA (fraction V) gassed with 95% O2-5% CO2 at a consistent rate of 35 ml/min for 60 min (49). NE (20 nM) alone or plus RW (1 µM) was added to the perfusate and present throughout the entire period of perfusion. Before the determination of in vitro ICG clearance (a measure of hepatocellular function), the isolated liver was perfused with Krebs-Henseleit buffer for 30 min without recirculation and then perfused with an additional 400 ml of perfusate containing 8 mg ICG with recirculation. Samples of effluent (1 ml each) were collected every 5 min for 30 min after ICG administration. The ICG concentration in the effluent was determined using a spectrophotometer at a wavelength of 800 nm (33). The difference in ICG content between different time points was the amount of ICG taken up by the liver. At the end of the experiment, the livers were harvested for determination of dry weight, and hepatic ICG clearance was expressed as micrograms per gram of dry liver.
Animal model of sepsis.
Polymicrobial sepsis was induced by CLP as previously described
(6, 42). In brief, male adult Sprague-Dawley rats were fasted overnight but allowed water ad libitum before the experiment. The animals were then anesthetized with isoflurane inhalation, and a
2-cm midline incision was performed. The cecum was exposed, ligated
just distal to the ileocecal valve to avoid intestinal obstruction, and
then punctured twice with an 18-gauge needle. The puncture was squeezed
to expel a small amount of fecal material, and the abdominal incision
was closed in two layers. Sham-operated rats underwent the same
surgical procedure except that the cecum was neither ligated nor
punctured. All animals received normal saline (3 ml/100 g body wt)
subcutaneously immediately after the operation to provide fluid
resuscitation. At 1 h after CLP, RW at 1 mg/kg body wt
(9) in 1 ml normal saline or 1 ml normal saline alone was
infused via the femoral venous catheter over 15 min. In addition, a
femoral artery was also cannulated with PE-50 tubing, which was
connected with a blood pressure analyzer to monitor MAP and heart rate
during RW infusion. RW administration did not significantly alter MAP
or heart rate (data not shown). At 5 h after CLP, hepatocellular
function and serum levels of TNF- were determined.
Assay of serum TNF-.
After the determination of hepatocellular function, blood samples (~1
ml) were collected by cardiac puncture. The samples were put on ice for
10 min and then centrifuged at 1,200 g for 10 min, and the
serum was stored at
70°C until assayed. The serum level of TNF-
was measured using an ELISA kit (BioSource International, Camarillo,
CA) according to the manufacturer's suggested protocol.
Statistical analysis.
Data are presented as means ± SE. One-way ANOVA and Tukey's test
were employed for comparison among different groups of animals. Differences were considered significant at P 0.05.
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RESULTS |
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Effects of intraportal administration of NE or NE plus RW on
hepatocellular function in normal animals.
As indicated in Fig. 1,
Vmax and Km decreased by
44% (P < 0.05) and 48% (P < 0.05),
respectively, at the end of the 2-h intraportal infusion of 20 µM NE
compared with vehicle (normal saline)-infused animals. However,
infusion of NE (20 µM) in combination with RW (1 mM) maintained
hepatocellular function. Vmax and
Km increased by 61% (P < 0.05)
and 65% (P < 0.05), respectively, at the completion of the 2-h infusion of NE plus RW compared with NE-infused animals (Fig. 1). The effects of NE or NE plus RW on hepatocellular function were not due to changes in systemic hemodynamic parameters, because cardiac output was not altered under such conditions (37.4 ± 0.6, 37 ± 0.4, and 37.2 ± 0.5 ml · min1 · 100 g body wt
1
in normal saline-, NE-, or NE plus RW-infused animals, respectively; n = 6/group). Similarly, both MAP and heart rate did
not change significantly after intraportal administration of NE or NE
plus RW (data not shown).
|
Effects of NE or NE plus RW on hepatocellular function using an
isolated perfused liver preparation from normal animals.
As shown in Fig. 2, the perfused livers
isolated from normal animals demonstrated effective ICG clearance. The
addition of 20 nM NE to the perfusate, however, significantly reduced
ICG clearance at 40-60 min after its addition (by 44-48%,
P < 0.05). The decreased ICG clearance induced by NE
was attenuated by coadministration of 1 µM RW. There was no
significant difference in ICG clearance between vehicle and NE plus RW
groups (Fig. 2). Additionally, there was no significant difference in
bile production among livers perfused with vehicle, NE, or NE plus RW
(Fig. 3), indicating that the viability
of the liver was not altered by administration of NE or RW.
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Effects of RW on hepatocellular function and circulating levels of
TNF- during early sepsis.
At 5 h after the onset of sepsis (i.e., the early stage of
polymicrobial sepsis; 41), Vmax decreased by
57% compared with sham-operated animals (P < 0.05, Fig. 4A). Intravenous
administration of RW at 1 h after CLP, however, increased
Vmax by 107% (P < 0.05) compared with septic animals treated with vehicle (normal saline) and
was not different from sham-operated animals (Fig. 4A).
Similarly, Km decreased by 57% at 5 h
after CLP compared with sham-operated animals, and administration of RW
attenuated the decrease in Km (Fig.
4B). As indicated in Fig. 5,
circulating levels of TNF-
increased approximately fourfold at
5 h after the onset of sepsis (P < 0.05).
Administration of RW at 1 h after CLP, however, reduced the serum
levels of TNF-
by 68% (P < 0.05), and the levels
were similar to the sham group (Fig. 5).
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DISCUSSION |
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Studies (34, 36, 41) have shown that hepatocellular
function, as assessed by in vivo ICG clearance, is depressed early after the onset of sepsis and is further reduced with the progression of sepsis. The depression of hepatocellular function in early sepsis
does not appear to be due to any reduction in regional perfusion,
because total hepatic blood flow increases significantly under those
conditions (35, 36). Thus it appears that hepatocellular dysfunction observed during early sepsis may be due to factors other
than a disturbance in hepatic perfusion. In this regard, the
proinflammatory cytokine TNF- has been suggested (37,
41) to play an important role in producing hepatocellular
dysfunction. Although it has been shown that the gut is capable of
producing proinflammatory cytokines after hemorrhagic shock or systemic inflammation (7, 15, 24), our (19) recent
studies have indicated that the gut does not appear to contribute to
the elevated levels of proinflammatory cytokines, e.g., TNF-
,
interleukin-1
(IL-1
), and IL-6, observed during sepsis. Several
lines of evidence (15, 17, 27, 32) suggest that the
activation of Kupffer cells is responsible for proinflammatory cytokine
release and subsequently hepatocellular dysfunction during sepsis. Our
(16) recent studies suggest that Kupffer cells are the
major source of proinflammatory cytokines such as TNF-
, IL-1
and
IL-6 in early sepsis. The number of Kupffer cells in male adult
rats was reduced in vivo by intravenous injection of gadolinium
chloride (GdCl3; 10 mg/kg) 48 h before CLP (i.e., an
animal model of polymicrobial sepsis). The results indicate that the
circulating levels of TNF-
increased from nondetectible in
sham-operated animals to 148.9 ± 30.3 pg/ml at 5 h after
CLP. Prior administration of GdCl3, however, significantly
attenuated the increase in circulating TNF-
levels to 35.1 ± 20.2 pg/ml (P < 0.05, n = 5/group).
Moreover, both IL-1
and IL-6 levels increased significantly at
5 h after CLP in Kupffer cell-intact animals, and Kupffer cell
reduction by GdCl3 significantly lowered circulating levels
of IL-1
and IL-6 (16). These results indicate that
Kupffer cells are indeed responsible for producing proinflammatory
cytokines in early sepsis. Under septic conditions, there is an
increase in peripheral sympathetic nerve activity and peripheral
adrenergic stimulation, resulting in elevated plasma levels of
catecholamines. In support of this, studies (10, 21) have
indicated that plasma levels of NE increased significantly as early as
30 min after the onset of sepsis. Additionally, mesenteric organs
(primarily the gut) have been demonstrated (1, 8, 49) to
contribute substantially to total body NE production under normal
conditions as well as during sepsis. Because of the intimate connection
of the gut and liver via portal circulation, it is important to examine
whether gut-derived NE affects hepatocellular function during sepsis.
To this end, our (49) recent studies have indicated that
gut-derived NE plays an important role in depressing hepatocellular
function in the early stage of sepsis. Because stimulation of
macrophage
-adrenoceptors appears to contribute to the production of
TNF-
in vitro (28, 29), we hypothesized that
hepatocellular dysfunction observed during the early stage of sepsis is
mediated by the activation of
2-adrenoceptors in the
liver (presumably on Kupffer cells). The present study was therefore
conducted to test this hypothesis and to determine whether modulation
of hepatic responsiveness to NE stimulation by the specific
2-adrenergic antagonist RW attenuates hepatocellular dysfunction in early sepsis.
Our results indicate that intraportal infusion of NE in normal animals
for 2 h produced hepatocellular dysfunction. In addition, perfusion of NE in the isolated livers resulted in a significant decrease in ICG clearance. Administration of NE in combination with the
specific 2-adrenergic antagonist RW, however, prevented NE-induced decrease in ICG clearance during in vivo and in vitro conditions. The studies by Fessler et al. (9) have also
shown that pretreatment with RW significantly reduced intestinal and hepatic injury as well as TNF-
levels after endotoxic shock. In
addition, Kotanidou et al. (20) reported that
administration of another
2-adrenergic antagonist,
urethane, significantly improved the survival rate and reduced TNF-
release after lethal endotoxemia. Our present data also demonstrated
that intravenous administration of RW early after the onset of sepsis
attenuated sepsis-induced hepatocellular dysfunction, which was
associated with downregulation of the proinflammatory cytokine TNF-
.
These results, taken together, would suggest that NE-induced
hepatocellular dysfunction during sepsis appears to be mediated by the
activation of
2-adrenoceptors in the liver (presumably
on Kupffer cells).
It should be noted that we did not directly determine the specific role
of Kupffer cell 2-adrenoceptors in upregulating TNF-
in the present study. However, our recent results have indicated that
NE increases TNF-
release in cultured Kupffer cells as well as in
isolated perfused liver preparation, which can be blocked by a specific
2-adrenergic antagonist (50). In addition,
incubation of Kupffer cells with the
2-adrenergic
agonist clonidine upregulates TNF-
in the cell culture system. Thus
NE-induced release of TNF-
from Kupffer cells is mediated by the
activation of
2-adrenoceptors (50).
Furthermore, the gut appears to be the major source of NE release
during sepsis (49). With regard to the source of TNF-
in sepsis, our studies have indicated that Kupffer cells are
responsible for the release of TNF-
(50) as well as
other proinflammatory cytokines, such as IL-1
and IL-6
(16), during the early stage of sepsis. Moreover, studies
from our laboratory (41) have demonstrated that TNF-
is
indeed responsible for producing hepatocellular dysfunction observed
during the early stage of sepsis. These results along with the findings
in the present study suggest that gut-derived NE produces
hepatocellular dysfunction during sepsis via its upregulatory effect on
TNF-
production by Kupffer cells, which is mediated by the
activation of
2-adrenoceptors. It could be argued that
NE-induced upregulation of TNF-
may be due to an increase in
endotoxin. This does not appear to be the case, because our
(50) recent studies have indicated that intraportal
administration of NE did not significantly alter plasma levels of
endotoxin. This would suggest that NE-induced upregulation of TNF-
is endotoxin independent.
In the present study, ICG clearance was used as a measure of
hepatocellular function because it is an extremely sensitive and early
indicator of alterations in hepatocellular function in sepsis
(41, 42). This technique is distinct from assessment of
plasma liver enzymes, as it measures hepatocellular function, rather
than injury, and thus reflects hepatocellular dysfunction as opposed to
hepatocellular damage. We (36, 39) have reported that
hepatic clearance of this dye decreased at 1.5 h, whereas circulating levels of liver transaminases (alanine aminotransferase and
aspartate aminotransferase) did not increase until 10 h after the
onset of sepsis. In addition, a cytosolic liver enzyme, -glutathione S-transferase, a sensitive indicator of alterations of
hepatocyte integrity, increased only as early as 5 h after CLP
(18). Although sepsis-induced hepatocellular dysfunction
appears to be due to multiple mediators, our (41) findings
have suggested that upregulation of TNF-
plays an important role in
depressing hepatocellular function under such conditions. In this
regard, administration of a low dose of TNF-
, which does not alter
cardiac output and hepatic perfusion, produced hepatocellular
dysfunction (32). In addition, administration of
pentoxifylline, which has been shown to downregulate TNF-
(30), maintained hepatocellular function at both 2 and
5 h after the onset of sepsis (38). Moreover, TNF-
is upregulated in Kupffer cells before the occurrence of hepatocellular
dysfunction after the onset of sepsis (37). These results,
taken together, suggest that TNF-
does play a major role in
producing hepatocellular dysfunction after the onset of sepsis.
Although we only determined serum levels of TNF-
at 5 h after
CLP in the present study, we (19, 41) have previously reported that this proinflammatory cytokine increased at various time
points after CLP. However, it remains unknown whether administration of
RW at 1 h after CLP reduces TNF-
levels at 20 h after the onset of sepsis. In a separate study (50), we have
reported that administration of NE via the portal vein in normal
animals and in the isolated perfused liver significantly increased
TNF-
production, which was prevented by coadministation of the
2-adrenergic antagonist yohimbine.
Studies (28) have demonstrated that
2-adrenergic agonists NE and UK-14304 enhanced
endotoxin-stimulated TNF-
production by peritoneal macrophages. At
the transcriptional level,
2-adrenergic agonists
increase TNF-
mRNA accumulation, which can be blocked by the
2-adrenergic antagonist yohimbine (28). In
support of these findings, our studies indicate that intraportal
administration of NE upregulated TNF-
gene expression in Kupffer
cells as well as cellular and plasma levels of this cytokine, while
coadministration of NE and the
2-adrenergic antagonist
yohimbine prevented the increase in cellular and plasma TNF-
levels
(50). Studies (14, 28) have also shown that
TNF-
mRNA induced by
2-adrenergic agonists in
peritoneal macrophages is endotoxin dependent. However, we have shown
that stimulation of Kupffer cell
2-adrenoceptors by NE
or another
2-adrenergic agonist, clonidine, without the presence of endotoxin also upregulates TNF-
production in a Kupffer cell culture system (50). The fact that
2-adrenergic antagonists, such as idazoxan
(3), CH-39083 (12), or RW (9)
inhibit TNF-
production after endotoxemia suggests that NE enhances
TNF-
production via the stimulation of
2-adrenoceptors (presumably on Kupffer cells). Although
it remains unknown whether infusion of NE alters
2-adrenoceptors, we have conducted a preliminary study
to determine whether Kupffer cell
2-adrenoceptor maximum binding capacity (Bmax; i.e., the maximal receptor number)
and dissociation constant (Kd) are altered
during early sepsis. To determine this, the receptor binding assay was
used as we (11, 44) previously described. Briefly, freshly
isolated Kupffer cells (106) from sham and septic animals
at 2 h after CLP were incubated with [3H]yohimbine
(a radioactively labeled
2-adrenoceptor antagonist; sp
act 79.2 Ci/mmol; DuPont NEN; final concentration, 2-64 nM in a
volume of 200 µl) with or without 10 µM of unlabeled yohimbine for
30 min at 37°C in an assay buffer (40 mM Tris · HCl and 10 mM
MgCl2, pH 7.5) (28). Depending on the
concentration of [3H]yohimbine, nonspecific binding was
found to be 20-65% of total binding capacity. The values of
Bmax and Kd were determined by Scatchard analysis (47). The results (average of 2 rats in
each group) indicate that Bmax was 18.8 fmol/106 cells in sham animals and increased to 24.3 fmol/106 cells at 2 h after CLP (increased by 29%).
In contrast, Kd decreased from 46.9 nM in sham
animals to 17.3 nM at 2 h after CLP (decreased by 63%). Because
Kd represents 1/affinity, the decreased
Kd in septic animals suggests an increase in
receptor affinity. Thus Kupffer cell
2-adrenoceptor
binding capacity and affinity increase during early sepsis. In
contrast, Kupffer cell cAMP levels decreased from 7.31 ± 0.14 pmol/5 × 106 cells in sham-operated animals
(n = 5) to 3.82 ± 1.70 pmol/5 × 106 cells at 2 h after CLP (n = 4;
P = 0.05; decreased by 48%). Since stimulation of
2-adrenoceptors reduces adenylate cyclase activity via
the inhibitory GTP-binding protein (23), the decreased
Kupffer cell cAMP level during early sepsis appears to be due to the
increased
2-adrenoceptor binding capacity and affinity.
In view of the previous reports (10, 21) demonstrating
that circulating levels of NE increase significantly during sepsis, we
propose that the increased binding capacity of
2-adrenoceptors on Kupffer cells during sepsis despite
the elevated levels of NE plays a critical role in upregulating TNF-
production. Thus blockade of Kupffer cell
2-adrenoceptors should reduce TNF-
release and
prevent hepatocellular dysfunction. The present study indicates that in vivo and in vitro administration of RW protects hepatocellular function
and attenuates TNF-
production during early sepsis as well as after
NE administration. Thus administration of
2-adrenergic antagonists appears to be a novel approach for maintaining cellular functions during sepsis. It should be noted that portal infusion of the
2-adrenergic antagonist RW at the doses used in this
study did not alter the cardiac output, MAP, or heart rate. However, although administration of RW in septic animals prevented the occurrence of hepatocellular dysfunction and upregulation of TNF-
, future studies are needed to determine whether the blockade of
2-adrenoceptors by RW in normal rats alters ICG
clearance and TNF-
production.
Intraportal administration of NE was performed, because recent study (49) has clearly indicated that the gut is the major source of NE release during sepsis. However, it could be argued that infusion of NE via the portal vein may cause maldistribution of hepatic perfusion. Although some redistribution of hepatic blood flow might have occurred under such conditions, we (45) have recently conducted studies to produce systemic NE levels similar to those observed in sepsis by implantation of a peritoneal miniosmotic pump (consistently releasing NE). The results (45) indicate that sustained elevation of systemic levels of NE produces significant depression in hepatocellular function as evidenced by reduced Vmax and Km of ICG clearance. The findings that perfusion of the isolated livers with NE reduced ICG clearance but did not alter alanine aminotransferase (49) or bile production (Fig. 3) suggest that NE at the concentration used in the present study does not appear to cause significant maldistribution of hepatic flow. Moreover, in the early stage of sepsis when plasma levels of NE increased to ~20 nM (10), hepatocellular dysfunction occurred despite the fact that hepatocellular perfusion increased as demonstrated by various techniques such as radioactive microspheres, laser Doppler flowmetry, galactose clearance, and colloid carbon infusion (41). Thus it is unlikely that NE-induced hepatocellular dysfunction is mainly due to significant maldistribution of hepatic blood flow by this catecholamine.
Because NE and RW may affect hepatic perfusion, it could be argued that the change of hepatic blood flow may have altered in vivo ICG clearance in the present study. However, this does not appear to be the case. Paumgartner et al. (27) suggested that the capacity of liver to remove ICG has a maximal limit. Their studies (27) also indicate that the classic Michaelis-Menten kinetics (with Lineweaver-Burk plot) could be applied to the initial ICG uptake in the rat and human livers. Paumgartner et al. (27) also postulated that when all hepatocyte receptor/carrier sites for ICG are occupied, removal capacity is at its maximum. Because saturation can theoretically be obtained despite fluctuations in hepatic blood flow and other variables and it is not possible to determine ICG clearance at an extremely high dose of this agent, maximal velocity of ICG clearance can be determined from three or more submaximal doses of ICG. This appears to be an ideal method for evaluating hepatocellular function independent of changes in hepatic perfusion (43). It should be pointed out that hepatocellular dysfunction observed during early stages of sepsis (i.e., 2-10 h after CLP) does not appear to be due to any reduction in hepatic blood flow. This conclusion is based on the findings that hepatic perfusion increases at 2-10 h after CLP and Vmax and Km of ICG clearance decrease at the same time points after the onset of sepsis (41). Thus hepatocellular dysfunction is not a flow-related event during early sepsis. In addition, since hyperdynamic circulation occurs at 2 h and hepatocellular function is depressed at 1.5 h after CLP, it appears that hepatocellular dysfunction, observed in the very early stage of sepsis, is not due to hyperdynamic circulation or hypermetabolism-related events (36).
Although the precise signal transduction mechanism responsible for the
activation of 2-adrenoceptors on macrophages or Kupffer cells remains unknown (28),
2-adrenoceptors
are associated with the inhibition of adenylate cyclase via the
inhibitory GTP-binding protein subunit and subsequent suppression of
intracellular cAMP (4). It is thought that stimulation of
2-adrenoceptors enhances TNF-
production through the
reduction of intracellular cAMP levels (13). In this
regard, our preliminary data have indicated that intracellular cAMP
level in Kupffer cells decreases by 48% at 2 h after the onset of
sepsis. A second mechanism by which TNF-
may be upregulated by NE is
2-adrenoceptor-mediated calcium flux into Kupffer cells,
resulting in increased intracellular calcium levels (4).
Studies (22, 46) have shown that an increase in macrophage
calcium plays an important role in upregulating TNF-
production.
Therefore, maintenance of intracellular cAMP levels and/or blockade of
calcium influx appears to be an effective approach for attenuating the
increased release of this inflammatory cytokine from Kupffer cells due
to NE stimulation. However, it remains to be determined whether the
beneficial effects of RW on TNF-
and hepatocellular function
observed in the current study are indeed due to changes in Kupffer cell
cAMP levels and/or calcium flux during early sepsis.
In summary, our results indicate that administration of the
2-adrenergic agonist NE in normal animals depressed
hepatocellular function. Coadministration of NE with the specific
2-adrenergic antagonist RW, however, prevented the
occurrence of NE-induced hepatocellular dysfunction. Moreover,
intravenous administration of RW early after the onset of sepsis
prevented sepsis-induced hepatocellular dysfunction and downregulated
production of TNF-
. These results, taken together, suggest that
gut-derived NE-induced hepatocellular dysfunction in early sepsis is
mediated by the activation of
2-adrenoceptors, which
appears to be responsible for stimulating Kupffer cells to enhance the
release of TNF-
. Thus modulation of hepatic responsiveness to NE by
2-adrenergic antagonists should provide a novel approach
for maintaining cell and organ functions during sepsis.
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ACKNOWLEDGEMENTS |
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This study was supported by National Institute of General Medical Sciences Grant GM-53008 (P. Wang). P. Wang is the recipient of National Institutes of Health Independent Scientist Award K02-AI-01461.
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FOOTNOTES |
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Address for reprint requests and other correspondence: P. Wang, Center for Surgical Research, Univ. of Alabama at Birmingham, 1670 Univ. Blvd., Volker Hall, Rm. G094P, Birmingham, AL 35294-0019 (E-mail: Ping.Wang{at}ccc.uab.edu).
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.
Received 8 February 2001; accepted in final form 22 May 2001.
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