Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78284-7760
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The onset of liver injury is a pivotal event
during endotoxemia. Lipopolysaccharide (LPS) activates the Kupffer
cells (KC), the resident macrophages of the liver, to generate an
abundance of inflammatory substances, including nitric oxide (NO).
Elevated levels of NO are thought to contribute to the propagation of
liver injury during sepsis. Calcium, a major second messenger in
several cellular signaling events, is required by the KC for the
generation of inducible nitric oxide synthase (iNOS). The purpose of
this study was to determine whether calcium channel antagonists limit hepatic injury and iNOS expression in vivo following LPS exposure and
to evaluate their effects on the regulation of iNOS expression in
cultured KC. In rats subjected to LPS for 6 h, the serum alanine aminotransferase (ALT) level was elevated significantly; this response
was accompanied by an increase in iNOS mRNA formation in the intact
liver. Pretreatment of rats with calcium channel antagonists (i.e.,
diltiazem, nifedipine, or verapamil) before LPS exposure attenuated the
serum ALT level and iNOS mRNA expression in the liver. Pretreatment of
cultured KC with calcium channel antagonists for 1 h followed by the
addition of LPS markedly repressed iNOS protein and mRNA expression.
Time-course studies revealed that calcium channel antagonists were most
effective at inhibiting LPS-induced iNOS mRNA formation by KC when
added before LPS. Treatment of KC with calcium channel antagonists
prior to the addition of LPS decreased nuclear levels of the p65
subunit of nuclear factor-B and prevented the LPS-dependent
degradation of the inhibitory protein I
B
. Thus our
findings indicate that under endotoxemic conditions calcium channel
antagonists limit hepatocellular injury that is accompanied by an
inhibition of LPS-mediated iNOS expression in rat liver KC.
endotoxemia; hepatocellular injury; inducible nitric oxide synthase mRNA; lipopolysaccharide
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
THE ONSET OF LIVER FAILURE consequent to endotoxic shock occurs frequently in the clinical setting. The liver is uniquely suited to protect the systemic circulation from gut-derived lipopolysaccharide (LPS) because of its location proximal to the gastrointestinal tract in the circulatory system (12, 17). Kupffer cells, the resident macrophages of the liver, are positioned in the hepatic sinusoids, where they are exposed continuously to the systemic circulation (51). These cells are potent scavenger cells that, under normal circumstances, avidly clear nonpathogenic amounts of circulating LPS from the blood. However, during pathophysiological episodes such as gut hypoperfusion or chronic inflammation, vast amounts of LPS pass into the liver through the portal vein, resulting in marked morphological changes and activation of sinusoidal and parenchymal cells (17, 19). Early hepatic responses observed during LPS exposure are displayed by Kupffer cells. Initially, Kupffer cells appear swollen and contain increased numbers of lysosomal granules and phagocytic vacuoles; these alterations in Kupffer cell morphology are associated with the onset of endothelial cell damage (17). The morphological changes observed in sinusoidal cells are detectable within 1 h after exposure to LPS, become exaggerated by 4 h, and often persist for up to 48 h. Concomitant with changes in the morphology of sinusoidal cells, hepatocytes also undergo functional and metabolic changes, including alterations in DNA and protein synthesis, that appear to be crucial to the onset of hepatocellular injury and eventual necrosis (17, 22).
During endotoxemic episodes, Kupffer cells, in addition to their
phagocytic functions, also secrete a wide array of inflammatory mediators, including cytokines [e.g., tumor necrosis factor
(TNF)- and interleukins], lipid mediators [e.g.,
platelet-activating factor (PAF)], reactive oxygen, and nitrogen
intermediates [e.g., nitric oxide (NO)] (8, 11, and S. B. Mustafa, B. D. Flickinger, and M. S. Olson, unpublished observations).
Several pathophysiological outcomes observed in the liver during
endotoxemic episodes are thought to be mediated in part by the action
of the endogenous factors (e.g., PAF, TNF-
, NO) synthesized mainly
by Kupffer cells in response to LPS rather than a direct consequence of
LPS association with hepatic cells. Therefore, activation of Kupffer
cells by LPS during the early stages of endotoxemia is a pivotal event that contributes to the propagation of hepatocellular injury (3, 24).
One of the many responses of cells to extracellular stimuli is a tightly regulated transient elevation in the cytosolic free calcium concentration. However, during pathological situations, the uncontrolled influx of calcium into cells may result in irreversible cellular injury. An early study by McLean et al. (34) reported an increase in the number of calcium deposits in areas of liver cell necrosis induced by chemical injury compared with normal liver cells. Furthermore, Sayeed and Maitra (43) observed an increase of cellular exchangeable calcium in the liver during endotoxemia. Moreover, our laboratory has demonstrated previously that perfusion of the rat liver with PAF results in an enhanced mobilization of intracellular calcium pools, most likely from sinusoidal cells (25). These findings are particularly relevant during endotoxemic episodes, as our laboratory has also observed that infusion of LPS into the rat liver in situ results in a rapid increase in tissue PAF levels (R. Duffy-Krywicki and M.S. Olson, unpublished data). In Kupffer cells, calcium is a prominent second messenger molecule involved in several signaling pathways. During endotoxemic episodes the irrepressible influx of calcium into Kupffer cells via L-type voltage-dependent calcium channels results in their activation and subsequent synthesis of several inflammatory mediators (e.g., PAF, NO, and eicosanoids) (7, 11, 18, and Mustafa et al., unpublished observations). In addition, Ikeda et al. (21) recently reported that the calcium ionophore A-23187 significantly enhanced the induction of inducible nitric oxide synthase (iNOS) mRNA formation in LPS-treated Kupffer cells. Since calcium entry into Kupffer cells during endotoxemic episodes is an important prerequisite for their activation, calcium channel antagonists would be expected to be anti-inflammatory in the liver, limiting hepatic tissue damage. Calcium channel antagonists have received considerable attention for their utility as a therapeutic option in the treatment of endotoxemia. In fact, several calcium channel antagonists have been found to protect against cardiovascular failure and to prolong survival time in various animal models of endotoxic shock (29). Moreover, previous studies have demonstrated that several structurally unrelated calcium channel antagonists attenuated chemically induced hepatocellular damage (47). In addition, the dihydropyridine-type calcium channel antagonists appear to reduce hypoxic liver injury and to increase graft survival after liver transplantation by attenuating Kupffer cell activation (46). Accordingly, the present study was designed in two parts. First we evaluated whether three structurally unrelated calcium channel antagonists (i.e., diltiazem, nifedipine, and verapamil) attenuate liver injury and studied their subsequent effect on hepatic iNOS mRNA formation in LPS-treated rats. Second, because of the importance of Kupffer cell activation during endotoxemic episodes, we focused exclusively on an investigation of the pathways by which calcium channel antagonists attenuate LPS-induced NO formation in this particular cell type.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials.
Anti-macrophage iNOS antiserum was obtained from
Transduction Laboratories (Lexington, KY). Rabbit polyclonal antibodies
raised against p65 and IB
were purchased from Santa Cruz Biotech
(Santa Cruz, CA). Goat anti-mouse and goat anti-rabbit IgG horseradish peroxidase conjugate and prestained SDS-PAGE standards were obtained from Bio-Rad (Hercules, CA). Bacterial LPS (from
Escherichia coli serotypes 011:B4 and
055:B5), diltiazem, nifedipine, and verapamil were purchased from Sigma
(St. Louis, MO).
Endotoxin exposure model.
The use of animals during the course of this study conformed to
National Institutes of Health guidelines. Male Sprague-Dawley rats were
anesthetized by an intramuscular injection of 0.35 ml of a
xylazine-ketamine mixture. A 1-cm lower-midline abdominal incision was
made, and a single loop of intestine was brought out of the abdomen. A
1 ml/kg body wt (0.20-0.25 ml) dose of 0.9% saline and 0.1% BSA
with or without 3 mg/ml of LPS (from E. coli serotype 055:B5) was injected into a mesenteric
vein within 3-5 mm of the intestine with a 27-gauge needle over 1 min. In control rats, a solution of 0.9% saline and 0.1% BSA was
infused into the mesenteric vein. In separate experiments, calcium
channel antagonists were dissolved in physiological saline and injected intravenously (1 mg · ml1 · kg
1)
into a tail vein 40 min prior to LPS administration. The rats were
allowed to awaken; food and water were offered ad libitum. After 6 h
rats were reanesthetized, this time with pentobarbital intraperitoneally. Whole blood was removed by inferior vena cava cannulation and allowed to clot and then centrifuged to obtain serum.
Serum levels of alanine aminotransferase (ALT) were assessed using a
commercially available assay kit (Sigma). The abdomen was opened
completely, and the liver was removed and immediately freeze-clamped in
liquid nitrogen and stored at
80°C. Total RNA from whole
liver samples was obtained from 0.5-mg samples of liver, which were
homogenized using the TRIzol method (39).
Isolation and primary culture of rat Kupffer cells. Following enzymatic digestion of the rat liver, Kupffer cells were isolated by centrifugal elutriation as described previously (39). The viability of the Kupffer cell preparation was greater than 95% as determined by trypan blue exclusion. Freshly isolated Kupffer cells were maintained at 37°C in RPMI 1640 culture medium (GIBCO, Grand Island, NY) supplemented with 25 mM HEPES, L-glutamine, and 10% fetal bovine serum (HyClone Laboratories, Logan, UT), 112 U/ml of penicillin, and 112 U/ml of streptomycin in 60-mm tissue culture dishes. All cells were incubated in an atmosphere of 90% air-10% CO2. On the second day of culture, the RPMI medium was changed. For experimental purposes, Kupffer cells were used within 3 days of their establishment in culture.
Preparation of whole cell, nuclear, and cytoplasmic extracts.
Kupffer cells were plated at a density of 1 × 107 cells per 60-mm dish. After 3 days in culture, the cells were rinsed with fresh medium and stimulated
with LPS alone or together with the calcium channel antagonists. The
morphology of cultured Kupffer cells was not altered in the presence of
any of the calcium channel antagonists, as assessed by phase-contrast
microscopy. After treatment, the cells were rinsed with PBS three
times, 500 µl of lysis buffer (50 mM Tris · HCl, pH
7.4) containing 5 mM EDTA, 5 mM EGTA, 1µM leupeptin, 1 µM pepstatin
A, 1 µM aprotonin, and 1 µM phenylmethylsulphonyl fluoride (PMSF)
were added to each dish; and the cells were quickly scraped. The
resulting cell suspensions were subjected to three rapid
freeze-thaw-vortex cycles to disrupt the Kupffer cells completely. A
100-µl sample of the lysed cell preparation was used for protein quantitation, and the remainder of the sample was stored at
80°C until analyzed.
Western blot analysis for iNOS. Before SDS-PAGE, the disrupted cell suspensions were dried using a Savant vacuum centrifuge. Sample pellets were solubilized in buffer containing DTT (0.1 M), 50% glycerol, Tris (0.5 M, pH 6.8), pyronine Y (2.5 mM), and 20% SDS and subjected to SDS-PAGE (7.5% gel). The separated proteins were transferred electrophoretically to polyvinylidine difluoride (PVDF) membranes with the use of a semidry transfer blot system, and the membranes were soaked in Tris-buffered saline (TBS, pH 7.4) containing 5% nonfat dried milk powder for 1 h and incubated with anti-iNOS antibody in 50 mM Tris · HCl, pH 7.4, 150 mM NaCl, 0.01% Tween 20, and 1% BSA for 24 h. The blots were incubated with horseradish peroxidase-labeled goat anti-mouse IgG in the same buffer for 2 h. Finally, the blots were rinsed in 50 mM Tris · HCl, pH 7.4, 150 mM NaCl, and 0.01% Tween 20. Peroxidase labeled-proteins were visualized by incubation with the peroxidase color development reagent 3,3'-diaminobenzidine and hydrogen peroxide.
Western blot analysis for p65 and
IB
.
Cytoplasmic and nuclear samples were resolved using SDS-PAGE (11%
gel). The separated proteins were electrotransferred to PVDF membranes.
The membranes were then incubated in blocking buffer (TBS, pH 7.4)
containing 10% nonfat dried milk powder for 1 h and then exposed to
diluted primary antibodies against the p65 subunit of nuclear factor
(NF)-
B or the inhibitory protein I
B
overnight at
4°C. The membranes were incubated for 1 h at room temperature with
horseradish peroxidase-conjugated anti-rabbit IgG antibody diluted
5,000-fold. Protein bands were visualized with an enhanced
chemiluminescence assay kit.
Northern blot analysis.
Kupffer cells were plated at a density of 1 × 107 cells per 60-mm culture dish.
After three days in culture and the appropriate treatment, total RNA
from cultured rat Kupffer cells was isolated with the use of TRIzol
reagent (GIBCO BRL, Gaithersburg, MD). RNA (3-4 µg) was
separated by electrophoresis on a 0.8% agarose-2.2 M formaldehyde gel
and transferred with the use of a Possiblot (Stratagene, La Jolla, CA)
onto a Magna nylon membrane (Microns Separations, Westborough, MA). A
full-length murine iNOS cDNA probe kindly provided by Dr. S. H. Snyder
(The Johns Hopkins University School of Medicine, Baltimore, MD) was
labeled with a multiprime DNA labeling system using
[-32P]dCTP (sp act,
3,000 Ci/mmol). Northern blot hybridizations were performed in 50%
formamide, 1 M NaCl, 10% dextran sulfate, 50 mM
Tris · HCl, pH 7.5, 0.1% sodium pyrophosphate, and
0.2% Denhardt's solution at 42°C for 16 h. The membranes were
washed twice in 2× standard saline citrate (SSC)-1% SDS at
65°C for 20 min, twice in 0.1× SSC-0.1% SDS at 55°C for
15 min, and finally at room temperature in 0.1× SSC for 15 min.
Radioactivity was visualized using a PhosphorImager (Molecular
Dynamics, Sunnyvale, CA). Control hybridizations were performed with
the use of a 32P end-labeled
oligonucleotide complementary to rat 18S rRNA.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Suppression of LPS-induced liver dysfunction by calcium channel
antagonists.
Liver injury and dysfunction are characteristic of both clinical sepsis
and experimental models of endotoxemia. Exposure of rats to LPS induces
marked alterations in liver structure that include extensive hepatocyte
hypertrophy, nuclear enlargement, granulation, and vacuolization of the
hepatocyte cytoplasm (27). Liver dysfunction in our rat model of
endotoxemia was assessed by the serum level of the enzyme ALT, a
specific marker for hepatic parenchymal cell damage. Treatment of rats
with LPS evoked a significant increase in the circulating serum level
of ALT compared with that detected in saline-treated rats (Fig.
1). Previous findings have shown that
several structurally different calcium channel antagonists protect
against cardiovascular failure and tissue damage and prolong survival
time in various animal models of endotoxemia (5, 29). In light of the
above reports, we utilized our liver-focused model of endotoxemia to
determine whether pretreatment of rats with three structurally
unrelated calcium channel antagonists (i.e., diltiazem, nifedipine, and
verapamil) ameliorated LPS-induced hepatocellular damage. The results
depicted in Fig. 1 indicate that pretreatment of rats with calcium
channel antagonists for 40 min before LPS exposure significantly
reduced the circulating serum level of ALT compared with rats treated
with LPS.
|
Effects of calcium channel antagonists on iNOS mRNA expression in
the intact liver after LPS treatment.
We (Mustafa et al., unpublished observations) and others (27) have
shown that, in the intact rat, exposure to LPS for 6 h causes induction
of iNOS mRNA in the liver. The results represented in Fig.
2 indicate that in rats infused with saline
iNOS mRNA levels in the intact liver were barely detectable at 6 h, in
contrast to the strong iNOS signal observed in livers of rats infused
with LPS for the same interval. Exposure of rats to calcium channel antagonists via tail-vein injection did not induce the expression of
iNOS mRNA observed at 6 h (data not shown). Moreover, pretreatment of
rats with the same calcium channel antagonists via tail-vein injections
for 40 min before LPS administration into a mesenteric vein greatly
minimized iNOS mRNA levels in the intact liver detected at 6 h. All
hepatic cells in the intact liver are capable of expressing iNOS mRNA
over a period of several hours in the presence of LPS and/or cytokines
(Mustafa et al., unpublished observations). We have demonstrated
previously that Kupffer cells isolated from rats exposed to LPS for 6 h
are the first of the hepatic cell types to express iNOS mRNA in
abundance, in contrast to the low level of expression of iNOS mRNA
detected in endothelial cells and hepatocytes at the same time (Mustafa
et al., unpublished observations). Clearly these observations indicate
that calcium channel antagonists not only ameliorate liver injury after
exposure to LPS for 6 h but also attenuate the induction of iNOS gene
expression in the Kupffer cell compartment of the intact liver.
|
Effects of calcium channel antagonists on LPS-induced iNOS protein
and mRNA levels in cultured Kupffer cells.
The principal target of LPS in the liver is the Kupffer cell (26). When
activated, Kupffer cells synthesize and release several potent
inflammatory substances, including cytokines, reactive oxygen and
nitrogen species, PAF, and eicosanoids. These mediators are capable of
influencing neighboring cells, in particular hepatocytes and
endothelial cells. Essentially all hepatic cells, namely endothelial, stellate (Ito), and Kupffer cells, and hepatocytes in culture are
capable of producing NO after stimulation by LPS and/or cytokines (13, 41, and Mustafa et al., unpublished observations). Furthermore, it has
been reported that cytokines (derived from LPS-stimulated Kupffer
cells) are required in addition to LPS for induction of iNOS mRNA in
hepatocytes (3, 13). Therefore, modulation of early Kupffer cell
responses during endotoxemia may ameliorate the deleterious effects of
LPS in the liver. In view of the above observations we sought to
characterize how the signaling pathways involved in the regulation of
LPS-induced iNOS formation in Kupffer cells are modified by exposure to
calcium channel antagonists. As shown in Fig.
3, stimulation of Kupffer cells with LPS
generated an intense signal at 24 h, indicating an abundance of iNOS
protein as detected by Western blot analysis. However, pretreatment of Kupffer cells with calcium channel antagonists for 1 h attenuated LPS-induced iNOS protein levels. We next examined the levels of iNOS
mRNA in Kupffer cells that had been treated with calcium channel
antagonists for 1 h before LPS stimulation for 6 h. In agreement with
our previous studies, treatment of Kupffer cells with LPS resulted in
an abundance of iNOS mRNA formation at 6 h (Fig.
4) (19). In contrast, the levels of iNOS
mRNA expressed in Kupffer cells that had been pretreated with calcium
channel antagonists 1 h before the addition of LPS were greatly
minimized.
|
|
Kinetics of the decrease in iNOS mRNA synthesis by calcium channel
antagonists.
In our earlier studies, we showed that in Kupffer cells LPS induces
iNOS mRNA formation in a transient manner (39). We observed that
maximal levels of iNOS mRNA occurred between 6 and 8 h after the
addition of LPS. Therefore, for an inhibitor to effectively block iNOS
mRNA formation it should be in contact with cells within the 6-h time
frame after exposure to LPS. To confirm this inference, each of the
calcium channel antagonists employed in this study was added to
cultured Kupffer cells before and at different times after the addition
of LPS. The results outlined in Fig. 5
indicate that maximal suppression of LPS-induced iNOS mRNA formation by diltiazem and nifedipine occurred when these antagonists were added to
the cells 1 h before and 1 h after the addition of LPS. When diltiazem
and nifedipine were added later, i.e., several hours after the addition
of LPS, the extent of inhibition of iNOS mRNA progressively decreased.
Interestingly, verapamil exhibited the same extent of inhibition of
LPS-induced iNOS mRNA formation even when added to cells at 2 h after
exposure to LPS. The inhibitory effect of verapamil was greatly
decreased when added to cells that were exposed previously to LPS for 4 h.
|
Calcium channel antagonists attenuate the translocation of p65 into
the nucleus.
Activation of NF-B by LPS is an essential requirement for the onset
of iNOS gene expression in Kupffer cells (39, 52). In its latent state,
NF-
B resides in the cytoplasm bound to its inhibitory proteins
designated I
B. After the interaction of LPS with its cell surface
receptors, NF-
B disassociates from I
B subunits, resulting in the
translocation of NF-
B into the nucleus, where it binds to promoter
regions of its target genes (16). To discern whether the decrease in
LPS-induced iNOS mRNA levels in Kupffer cells in the presence of
calcium channel antagonists was associated with a reduction in p65
nuclear translocation, cytosolic and nuclear p65 levels were assayed by
Western blotting. These studies revealed that cytosolic p65 exhibited
basal expression in vehicle-treated Kupffer cells; furthermore,
cytosolic fractions isolated from LPS-treated Kupffer cells in the
absence and presence of each calcium channel antagonist displayed p65
protein levels. In contrast, nuclear p65 levels were amplified greatly
in LPS-treated Kupffer cells compared with vehicle-treated cells but
were diminished when calcium channel antagonists were added to Kupffer
cells before exposure to LPS (Fig. 6).
These findings imply that incubation of Kupffer cells with structurally
different calcium channel antagonists before LPS exposure abates
LPS-mediated signaling pathways within the Kupffer cell by minimizing
the translocation of NF-
B protein into the nucleus, thereby
attenuating iNOS gene transcription.
|
Cytoplasmic IB
protein is conserved
by calcium channel antagonists.
Activation of NF-
B by inflammatory stimuli with subsequent
translocation from the cytoplasm into the nucleus is preceded by the
phosphorylation and proteolytic degradation of I
B subunits, in
particular I
B
(16). Cytosolic fractions isolated from
unstimulated Kupffer cells exhibited a basal steady-state amount of
I
B
, a major component of I
B, as detected by Western blotting
(Fig. 7). In contrast, the cytoplasmic
levels of I
B
isolated from Kupffer cells treated with LPS were
barely detectable. As depicted in Fig. 7, pretreatment of Kupffer cells
with calcium channel antagonists 1 h before LPS exposure resulted in
the preservation of cytoplasmic I
B
, the levels of which are
comparable to those detected in unstimulated Kupffer cells. This result
suggests that calcium channel antagonists modulate LPS-dependent
signaling pathways in these cells, thereby preventing the degradation
of I
B subunits and subsequent translocation of NF-
B into the
nucleus.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
There is a growing body of evidence indicating that activation of
Kupffer cells in the liver is a pivotal response during the
pathogenesis of endotoxin-associated hepatic tissue dysfunction (3, 24,
40). In particular, the inflammatory and immunomodulating mediators
(e.g., TNF-, interleukins, NO, and oxygen radicals) synthesized and
released by Kupffer cells during endotoxemic episodes mediate
LPS-induced alterations such as fluctuations in metabolic pathways and
necrosis observed in the pathological liver (11, 27). In this study we
have focused on NO, a specific signaling molecule synthesized by
numerous cell types (36). Endothelial cells and neurons possess two
separate isoforms of NOS that are expressed constitutively and in
general release low levels of NO into the microenvironment in transient
bursts (6, 36). In contrast, a wide variety of cell types are capable
of expressing iNOS after exposure to LPS and/or inflammatory substances
(e.g., cytokines, PAF, and arachidonic acid metabolites) (26, 35, 41).
In the liver, under normal physiological conditions, small amounts of NO are produced by a constitutively expressed NOS (eNOS) in sinusoidal endothelial cells that is involved in sustaining sinusoidal tone and blood flow throughout the hepatic microvasculature. Furthermore, iNOS is now known to be induced in all liver cells, including hepatocytes, Kupffer, endothelial, and Ito cells and has been implicated in the pathogenesis of a number of processes, such as acute hepatocellular injury during endotoxemia, ischemia-reperfusion injury, and toxin-mediated liver damage (1, 23, 32, 49). The recognition of NO as a key contributor to the pathogenesis of liver tissue injury during endotoxemia led to the proposal that the pharmacological attenuation of NO generated by parenchymal and nonparenchymal cells may represent a useful adjunct in the treatment of this particular disorder. Curiously, recent reports indicate that inhibition of NO with L-arginine analogs [e.g., NG-nitro-L-arginine methyl ester (L-NAME) and NG-monomethyl-L-arginine (L-NMMA)] actually accelerates hepatic injury (15, 48). The inhibitors used in these studies were nonselective for all NOS isoforms. In fact, both L-NAME and L-NMMA are therapeutically better inhibitors of eNOS than iNOS (48). Moreover, it was suggested that the deleterious effects of NO observed in the liver during these studies were probably due to the loss of the regulatory functions of eNOS. Consistent with these observations, other studies have shown that inhibitors selective for iNOS attenuate hepatocellular injury in rodent models of endotoxemia (9, 48). It is of interest to note that survival studies using iNOS-knockout mice exposed to high doses of endotoxin have revealed conflicting results: Laubach and co-workers (28) reported no significant survival differences between iNOS-knockout and wild-type mice. MacMicking et al. (33) showed that iNOS-knockout and wild-type mice experienced a similar degree of liver damage after exposure to LPS. In contrast, Wei and co-workers (50) found that iNOS-knockout mice were protected from endotoxin exposure. The discrepancies among these early observations have yet to be adequately explained. Although the data available at present are conflicting, the consensus seems to be that in the event of endotoxemia the challenge is to inhibit excess formation of NO derived from iNOS, meanwhile conserving the basal level of NO derived from eNOS, which appears to be critical for maintaining the integrity of the hepatic microvasculature.
Calcium channel antagonists have found widespread use during the last
25 years in the treatment of cardiovascular abnormalities. More
recently, calcium channel antagonists have been used experimentally in
a wider range of disorders outside the cardiovascular system; for
example, it has been reported that diltiazem, nifedipine, and verapamil
attenuate acute hepatocellular damage in the perfused rat liver induced
by D-galactosamine and
CCl4 (10, 47). Also, several
laboratories have reported that calcium channel antagonists prolong the
survival time in various animal models of endotoxic shock (5, 22).
Furthermore, Szabó et al. (45) demonstrated previously that in
anesthetized rats subjected to LPS for 3 h, pretreatment with
nifedipine alleviates the decrease in mean arterial blood pressure and
the vascular hyperreactivity to norepinephrine. We have
observed in the present study that in rats treated with calcium channel
antagonists before LPS exposure, the circulating serum level of a
specific marker for hepatic tissue damage, ALT, was reduced
significantly compared with the level in rats treated with LPS alone.
In parallel with these observations, we noted that exposure of rats to
LPS for 6 h induces the expression of iNOS mRNA in the intact liver.
Treatment of animals with structurally unrelated calcium channel
antagonists before LPS administration minimized the levels of iNOS mRNA
in the intact liver. In a similar context, an earlier report by
Szabó et al. (45) demonstrated that the level of iNOS enzyme
activity in lung homogenates of rats that had been treated with
nifedipine before LPS exposure for 3 h was greatly reduced. Further
studies by these researchers have indicated that nifedipine did not
affect NOS activity in lung homogenates of LPS-treated rats.
Interestingly, recent studies by Mügge and co-workers (37)
revealed that calcium channel antagonists employed clinically (e.g.,
diltiazem, nifedipine, and verapamil) do not inhibit the release of NO
derived from eNOS in bovine aortic endothelial cells. Liver injury
during endotoxemia is not attributable to one particular
proinflammatory substance but occurs as a consequence of the excess
production of several proinflammatory mediators by all hepatic cell
types. Recent studies by Lichtman et al. (30, 31) showed that different
calcium channel antagonists, including nifedipine and verapamil,
inhibited LPS-induced TNF- expression and release by cultured
Kupffer cells. TNF-
is a major proinflammatory cytokine that induces
several features of endotoxic shock. TNF-
, in conjunction with LPS,
induces iNOS mRNA expression in hepatocytes (13). Thus in our
experimental model of endotoxemia it is quite likely that treatment of
rats with calcium channel antagonists before LPS exposure not only decreases iNOS mRNA expression in the intact liver but also very likely
limits the synthesis of TNF-
by Kupffer cells. In sum, the
protective effect of calcium channel antagonists on LPS-mediated tissue
injury in the intact liver (as indicated by a decrease in serum ALT
level) is due most likely to their ability to attenuate not only the
induction of iNOS without compromising the salutary function of eNOS
but also the production of cytokines such as TNF-
.
It has been well documented that the initial activation of Kupffer cells by gut-derived LPS is a pivotal event in the propagation of liver tissue dysfunction observed during endotoxemic episodes (11, 17, 24, 51). We have shown previously that Kupffer cells isolated from livers of rats 6 h after exposure to LPS through a mesenteric vein express the majority of iNOS mRNA, in contrast to low levels of mRNA detected in endothelial cells and hepatocytes at the same time point (Mustafa et al., unpublished observations). Also, hepatocytes produce NO in abundance but require cytokines released from Kupffer cells in addition to LPS for the induction of iNOS mRNA (3, 13). This would explain the low signal of iNOS mRNA in hepatocytes isolated 6 h after LPS exposure. It is our contention that iNOS mRNA is expressed in a specific temporal manner by each of the different hepatic cells in the liver; we have demonstrated that in our liver-focused model of endotoxemia Kupffer cells are the first to express iNOS mRNA (Mustafa et al., unpublished observations). Thus manipulation of Kupffer cell function by pharmacological agents during the early stages of endotoxemia may limit the progression of hepatocellular tissue damage.
Previously, we observed that, in the presence of nifedipine,
LPS-induced nitrite formation by cultured Kupffer cells was attenuated in a dose-dependent manner (unpublished observations). In this study we
have demonstrated that treatment of cultured Kupffer cells with either
diltiazem, nifedipine, or verapamil 1 h before the addition of LPS
markedly decreased iNOS protein and mRNA formation. In addition, we
noted that the extent of the inhibitory action of diltiazem and
nifedipine was greatest when applied before or 1 h after LPS. The
maximal inhibitory effect of verapamil remained apparent even when
added to Kupffer cells 2 h after the addition of LPS. Furthermore, we
observed that in Kupffer cells treated with the aforementioned calcium
channel antagonists before LPS stimulation, the nuclear levels of
NF-B were minimized, thereby limiting LPS-induced iNOS gene
expression. It is interesting to note that verapamil appeared to be
more effective than either diltiazem or nifedipine at inhibiting
LPS-induced iNOS mRNA and protein formation and NF-
B translocation
into the nucleus in cultured Kupffer cells. A similar inhibitory action
of verapamil compared with both diltiazem and nifedipine
was observed during our whole animal studies. In quite different
experimental scenarios, others have reported previously that verapamil
was more effective than either diltiazem or nifedipine at inhibiting
the action of platelet-derived growth factor (PDGF) function in
vascular smooth muscle cells (4). Clearly, verapamil displays a greater
effectiveness in blocking calcium mobilization in different biological
systems than either diltiazem or nifedipine at the same concentration.
We have reported that the addition of LPS to cultured Kupffer cells
evoked a slow transient increase in intracellular calcium that reached
a maximal level after 20 min, returning gradually to baseline values
within a subsequent 20-min interval (Mustafa et al., unpublished
observations). Treatment of cultured Kupffer cells with calcium channel
antagonists has been shown to attenuate the LPS-dependent elevation in
intracellular calcium (20). It has been well documented that IB
degradation is dependent on elevation of intracellular calcium (14, 44,
52). We observed that treatment of Kupffer cells with calcium channel
antagonists before stimulation with LPS resulted in a preservation of
cytoplasmic I
B
protein, a finding supportive of reports in the
literature describing the essential requirement of calcium for I
B
degradation from the NF-
B-I
B complex.
The control of overproduction of inflammatory mediators such as
TNF-, interleukins, PAF, and NO from cells, including Kupffer cells,
should greatly ameliorate tissue injury and facilitate the treatment of
endotoxic shock. In macrophages such as the Kupffer cell, several of
these mediators are regulated primarily at the level of mRNA expression
via the involvement of various transcription factors, e.g., NF-
B
(14, 52). It has been widely reported that LPS-dependent expression of
the iNOS gene in macrophages is wholly dependent on the activation of
NF-
B (52). Translocation of NF-
B from the cytoplasm to the
nucleus can be attenuated by preventing the degradation of the I
B
subunits. In the present study, we have demonstrated that pretreatment
of Kupffer cells with different calcium channel antagonists preserves
the cytoplasmic level of I
B
, thereby limiting the translocation
of NF-
B into the nucleus. On the basis of our previous findings, we
concluded in this study that pretreatment of Kupffer cells with calcium channel antagonists blocked the LPS-dependent increase in intracellular calcium, thereby minimizing the degradation of NF-
B-I
B complex (44).
A previous study by Roth et al. (42) reported that the calcium channel
antagonist manidipine inhibited PDGF-dependent gene transcription of
several cytokines in mesangial cells (42). Furthermore, these
investigators reported that the inhibitory action of manidipine in
mesangial cells was not influenced by the presence or absence of
calcium but due to a modulation of the activation of protein kinase C
(PKC). Also, these authors reported that calcium channel
antagonists attenuate the activity of PDGF-dependent activation of PKC
(42). Stimulation of Kupffer cells by LPS results in TNF- formation
that is accompanied by an increase in the activation of PKC (4, 20). In
a similar context we found previously that pretreatment of Kupffer
cells with the PKC inhibitor staurosporine markedly decreased
LPS-induced iNOS protein formation (unpublished observations).
Additionally, two independent studies have indicated that I
B
phosphorylation is dependent upon PKC activation (2, 44). Thus it is
possible that in addition to blocking the entry of calcium into Kupffer cells, calcium channel antagonists exert a limiting effect on LPS-induced PKC activity in these cells, thereby influencing the intermediary signals (e.g., I
B
phosphorylation and its subsequent degradation) with resultant changes at the level of gene expression (e.g., inhibition of iNOS expression). The unique ability of calcium channel antagonists to modulate gene expression of inflammatory mediators during pathological situations warrants further study to
resolve the mode(s) of action of these pharmacological agents with
regard to their organ-protective mechanisms at the level of gene transcription.
In summary, the data presented in this study delineate a potential protective effect of structurally different calcium channel antagonists on LPS-induced liver tissue injury in the intact animal. Furthermore, we have documented the ability of calcium channel antagonists to manipulate LPS-dependent gene expression in cultured Kupffer cells. Therefore, modulation of hepatic Kupffer cell function with calcium channel antagonists during pathological disorders in clinical situations may prove therapeutically useful.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Lynnette Walters, David Arguello, and Michael DeBuysere for excellent technical assistance. We are grateful to Dr. S. A. K. Harvey for invaluable assistance in preparing this manuscript.
![]() |
FOOTNOTES |
---|
This work was funded by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-33538.
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: M. S. Olson, Dept. of Biochemistry, Univ. of Texas Health Science Center, 7703 Floyd Curl Dr., San Antonio, TX 78284-7760 (E-mail: olson{at}biochem.uthscsa.edu).
Received 21 December 1998; accepted in final form 26 April 1999.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Alexander, B.
The role of nitric oxide in hepatic metabolism.
Nutrition
14:
376-380,
1998[Medline].
2.
Bankey, P.,
A. Carlson,
M. Ortiz,
R. Singh,
and
F. Cerra.
Tumor necrosis factor production by Kupffer cells requires protein kinase C activation.
J. Surg. Res.
49:
256-261,
1990[Medline].
3.
Billiar, T. R.,
R. D. Curran,
D. J. Stuehr,
M. A. West,
B. G. Bentz,
and
R. L. Simmons.
An L-arginine dependent mechanism mediates Kupffer cell influences on hepatocyte protein synthesis in vitro.
J. Exp. Med.
169:
1467-1472,
1989
4.
Block, L. H.,
L. R. Emmons,
E. Vogt,
A. Sachinidis,
W. Vetter,
and
J. Hoppe.
Ca2+-channel blockers inhibit the action of recombinant platelet-derived growth factor vascular smooth muscle cells.
Proc. Natl. Acad. Sci. USA
86:
2388-2392,
1989[Abstract].
5.
Bosson, S.,
M. Kuenzig,
and
S. I. Schwartz.
Verapamil improves cardiac function and increases survival in canine E. coli endotoxic shock.
Circ. Shock
16:
307-316,
1985[Medline].
6.
Bredt, D. S.,
P. M. Hwang,
C. E. Glatt,
C. Lowenstein,
R. R. Reed,
and
S. H. Snyder.
Cloned and expressed nitric oxide synthase structurally resembles cytochrome P-450 reductase.
Nature
351:
714-718,
1991[Medline].
7.
Chao, W.,
A. Siafaka-Kapadai,
M. S. Olson,
and
D. J. Hanahan.
Biosynthesis of platelet-activating factor by cultured rat Kupffer cells stimulated with calcium ionophore A23184.
Biochem. J.
257:
823-829,
1989[Medline].
8.
Clemens, M. G.,
M. Bauer,
C. Gingalewski,
E. Miescher,
and
J. Zhang.
Hepatic intercellular communication in shock and inflammation.
Shock
2:
1-5,
1994.
9.
Corso, C. O.,
Y. Gundersen,
M. Dorger,
P. Lilleaasen,
A. O. Aasen,
and
K. Messmer.
Effects of nitric oxide synthase inhibitors NG-nitro-L-arginine methyl ester and aminoethyl-isothiourea on the liver microcirculation in rat endotoxemia.
J. Hepatol.
28:
61-69,
1998[Medline].
10.
Deakin, C. D.,
E. A. Fagan,
and
R. Williams.
Cytoprotective effects of calcium channel blocker. Mechanisms and potential applications in hepatocellular injury.
J. Hepatol.
12:
251-225,
1991[Medline].
11.
Decker, K.
Biological active products of stimulated liver macrophages (Kupffer cells).
Eur. J. Biochem.
192:
245-261,
1990[Medline].
12.
Frey, D. E.,
L. Pearlstein,
R. L. Fulton,
and
H. C. Polk.
Multiple system organ failure.
Arch. Surg.
115:
136-140,
1980[Abstract].
13.
Geller, D. A.,
A. K. Nussler,
M. Di Silvio,
C. J. Lowenstein,
R. A. Shapiro,
S. C. Wang,
R. L. Simmons,
and
T. R. Billiar.
Cytokines, endotoxin, and glucocorticosteroids regulate the expression of inducible nitric oxide synthase in hepatocytes.
Proc. Natl. Acad. Sci. USA
90:
522-526,
1993[Abstract].
14.
Ghosh, S.,
and
D. Baltimore.
Activation in vitro of NF-B by phosphorylation of its inhibitor I
B.
Nature
344:
678-682,
1990[Medline].
15.
Harbrecht, B. G.,
T. R. Billiar,
J. Stadler,
A. J. Demetris,
J. Ochoa,
R. D. Curran,
and
R. L. Simmons.
Inhibition of nitric oxide synthesis during endotoxemia promotes intrahepatic thrombosis and an oxygen radical-mediated hepatic injury.
J. Leukoc. Biol.
52:
390-394,
1992[Abstract].
16.
Henkel, T.,
T. Machleidt,
I. Alkalay,
M. Kronke,
Y. Ben-Neriah,
and
P. A. Baeuerle.
Rapid proteolysis of IB-
is necessary for activation of transcription factor NF-
B.
Nature
365:
182-185,
1993[Medline].
17.
Hewett, A. J.,
and
R. A. Roth.
Hepatic and extrahepatic pathobiology of bacterial lipopolysaccharides.
Pharmacol. Rev.
45:
381-411,
1993.
18.
Hijoika, T.,
R. L. Rosenberg,
J. L. Lemasters,
and
R. G. Thurman.
Kupffer cells contain voltage-dependent calcium channels.
Mol. Pharmacol.
41:
435-440,
1992[Abstract].
19.
Hirata, K.,
A. Kaneko,
K. Ogawan,
H. Hayasaka,
and
T. Onoe.
Effect of endotoxin on rat liver. Analysis of acid phosphatase isoenzymes in the liver of normal and endotoxin-treated rats.
Lab. Invest.
43:
165-171,
1980[Medline].
20.
Iimuro, Y.,
K. Ikejima,
M. L. Rose,
B. U. Bradford,
and
R. G. Thurman.
Nimodipine, a dihydropyridine-type calcium channel blocker, prevents alcoholic hepatitis caused by chronic intragastric ethanol exposure in the rat.
Hepatology
24:
391-397,
1996[Medline].
21.
Ikeda, K.,
S. Kubo,
K. Hirohashi,
H. Kinoshita,
K. Kaneda,
N. Kawada,
E. F. Sato,
and
M. Inoue.
Mechanisms that regulate nitric oxide production by lipopolysaccharide-stimulated rat Kupffer cells.
Physiol. Chem. Phys. Med. NMR
28:
239-253,
1996[Medline].
22.
Jean, P. A.,
and
R. A. Roth.
Bacterial endotoxin-induced cytotoxicity to rat hepatocytes (HC) cocultured with Kupffer cells (KCs) and neutrophils (PMNs).
Toxicology
13:
427,
1993.
23.
Johnson, M. L.,
and
T. R. Billiar.
Roles of nitric oxide in surgical infection and sepsis.
World J. Surg.
22:
187-196,
1998[Medline].
24.
Kurose, I.,
S. Miura,
H. Higuchi,
N. Watanabe,
Y. Kamegaya,
M. Takaishi,
K. Tomita,
D. Fukumura,
S. Kato,
and
H. Ishii.
Increased nitric oxide synthase activity as a cause of mitochondrial dysfunction in rat hepatocytes: roles for tumor necrosis factor-.
Hepatology
24:
1185-1192,
1996[Medline].
25.
Lapointe, D. S.,
D. J. Hanahan,
and
M. S. Olson.
Mobilization of hepatic calcium pools by platelet-activating factor.
Biochemistry
26:
1568-1574,
1987[Medline].
26.
Laskin, D. L.
Nonparenchymal cells and hepatotoxicity.
Semin. Liver Dis.
10:
293-304,
1990[Medline].
27.
Laskin, D. L.,
M. Rodriguez Del Valle,
D. E. Heck,
S. Hwang,
S. T. Ohnishi,
S. K. Durham,
N. L. Goller,
and
J. D. Laskin.
Hepatic nitric oxide production following acute endotoxemia in rats is mediated by increased inducible nitric oxide synthase gene expression.
Hepatology
22:
223-234,
1995[Medline].
28.
Laubach, V. E.,
E. G. Shesley,
O. Smithies,
and
P. A. Sherman.
Mice lacking inducible nitric oxide synthase are not resistant to lipopolysaccharide-induced death.
Proc. Natl. Acad. Sci. USA
92:
10688-10692,
1995[Abstract].
29.
Lee, H. C.,
and
B. K. B. Lum.
Protective action of calcium entry blockers in endotoxin shock.
Circ. Shock
18:
193-203,
1986[Medline].
30.
Lichtman, S. N.,
J. Wang,
R. T. Currin,
and
J. J. Lemasters.
Induction of TNF- release by bacterial cell wall polymers: comparison of peptidoglycan-polysaccharide with endotoxin.
In: Cells of the Hepatic Sinusoid, edited by E. Wisse,
D. L. Knook,
and K. Wake. Rijswijk, The Netherlands: Kupffer Cell Foundation, 1995, vol. 5, p. 7-9.
31.
Lichtman, S. N.,
J. Wang,
C. Zhang,
and
J. J. Lemasters.
Endocytosis and Ca2+ are required for endotoxin-stimulated TNF- release by rat Kupffer cells.
Am. J. Physiol.
271 (Gastrointest. Liver Physiol. 34):
G920-G928,
1996
32.
Ma, T. T.,
H. Ischiropoulos,
and
C. A. Brass.
Endotoxin-stimulated nitric oxide production increases injury and reduces rat liver chemiluminescence during reperfusion.
Gastroenterology
108:
463-469,
1995[Medline].
33.
MacMicking, J. D.,
C. Nathan,
G. Hom,
N. Chartrain,
D. S. Fletcher,
M. Trumbauer,
K. Stevens,
O. W. Xie,
K. Sokol,
and
N. Hutchinson.
Altered responses to bacterial infection and endotoxic shock in mice lacking inducible nitric oxide synthase.
Cell
81:
641-650,
1995[Medline].
34.
McLean, A. E. M.,
M. McLean,
and
J. D. Judah.
Cellular necrosis in the liver induced and modified by drugs.
Int. Rev. Exp. Pathol.
4:
127-157,
1985.
35.
Moncada, S.,
and
A. Higgs.
The L-arginine-nitric oxide pathway.
N. Engl. J. Med.
329:
2002-2012,
1993
36.
Moncada, S.,
R. J. Palmer,
and
E. A. Higgs.
Biosynthesis of nitric oxide from L-arginine. A pathway for the regulation of cell function and communication.
Biochem. Pharmacol.
38:
1709-1716,
1989[Medline].
37.
Mügge, A.,
T. Peterson,
and
D. G. Harrison.
Release of nitrogen oxides from cultured bovine endothelial cells is not impaired by calcium channel antagonists.
Circulation
83:
1404-1409,
1991[Abstract].
39.
Mustafa, S. B.,
and
M. S. Olson.
Expression of nitric oxide in rat Kupffer cells is regulated by cAMP.
J. Biol. Chem.
273:
5073-5080,
1998
40.
Nolan, J. P.
Endotoxin reticuloendothelial function and liver injury.
Hepatology
1:
458-465,
1981[Medline].
41.
Rockey, D. C.,
and
J. J. Chung.
Regulation of inducible nitric oxide synthase in hepatic sinusoidal endothelial cells.
Am. J. Physiol.
271 (Gastrointest. Liver Physiol. 34):
G260-G267,
1996
42.
Roth, M.,
R. Keul,
L. R. Emmons,
W. H. Horl,
and
L. H. Block.
Manidipine regulates the transcription of cytokine gene.
Proc. Natl. Acad. Sci. USA
89:
4071-4075,
1992[Abstract].
43.
Sayeed, N. M.,
and
S. R. Maitra.
Effect of diltiazem on altered cellular calcium regulation during endotoxic shock.
Am. J. Physiol.
253 (Regulatory Integrative Comp. Physiol. 22):
R549-R554,
1987
44.
Steffan, N. M.,
G. D. Bren,
B. Frantz,
M. J. Tocci,
E. A. O'Neill,
and
C. V. Paya.
Regulation of IB
phosphorylation by PKC- and Ca2+-dependent signal tranduction pathway.
J. Immunol.
155:
4685-4691,
1995[Abstract].
45.
Szabó, C.,
J. A. Mitchell,
S. S. Gross,
C. Thiemermann,
and
J. R. Vane.
Nifedipine inhibits the induction of nitric oxide synthase by bacterial lipopolysaccharide.
J. Pharmacol. Exp. Ther.
265:
674-680,
1993[Abstract].
46.
Takei, Y.,
I. Marzi,
F. C. Kauffman,
R. T. Currin,
J. J. Lemasters,
and
R. G. Thurman.
Prevention of early graft failure by the calcium channel blocker nisoldipine: involvement of Kupffer cells.
Transplantation
50:
14-20,
1990[Medline].
47.
Thurman, R. G.,
E. Apel,
M. Badr,
and
J. J. Lemasters.
Protection of liver by calcium entry blockers.
Ann. NY Acad. Sci.
522:
757-770,
1988[Medline].
48.
Vos, T. A.,
A. S. H. Gouw,
P. A. Klock,
R. Havinga,
H. VanGoor,
S. Huitema,
H. Roelofsen,
F. Kuipers,
P. L. M. Jansen,
and
H. Moshage.
Differential effects of nitric oxide synthase inhibitors on endotoxin-induced liver damage in rats.
Gastroenterology
13:
1323-1333,
1997.
49.
Wang, J. H.,
H. P. Redmond,
Q. D. Wu,
and
D. Bouchier-Hayes.
Nitric oxide mediates hepatocyte injury.
Am. J. Physiol.
275 (Gastrointest. Liver Physiol. 38):
G1117-G1126,
1998
50.
Wei, X. Q.,
I. G. Charles,
A. Smith,
J. Ure,
G. J. Feng,
F. P. Huang,
D. Xu,
W. Muller,
S. Moncada,
and
F. Y. Liew.
Altered immune responses in mice lacking inducible nitric oxide synthase.
Nature
375:
408-411,
1995[Medline].
51.
Winwood, P. J.,
and
M. J. P. Arthur.
Kupffer cells: their activation and role in animal models of liver injury and human liver disease.
Semin. Liver Dis.
13:
50-59,
1993[Medline].
52.
Xie, Q.-W.,
Y. Kashiwabara,
and
C. Nathan.
Role of transcription factor NFB/Rel in the induction of nitric oxide synthase.
J. Biol. Chem.
269:
4705-4708,
1996