THEMES
Nitric Oxide
IV. Determinants of nitric oxide protection and
toxicity in
liver*
Jianrong
Li and
Timothy R.
Billiar
Department of Surgery, University of Pittsburgh School of
Medicine, Pittsburgh, Pennsylvania 15261
 |
ABSTRACT |
Whereas nitric oxide (NO)
produced by constitutive endothelial NO synthase is protective to the
liver, NO produced by the inducible NO synthase (iNOS) can be either
toxic or protective depending on the conditions. The availability of
selective iNOS inhibitors and mice lacking various NOS isoforms made it
possible to begin to elucidate the precise roles of NO in the liver.
Under conditions of redox stress, induced NO contributes to hepatic damage. However, in acute inflammatory conditions associated with cytokine exposure, NO acts as a potent inhibitor of apoptosis in the
liver. Our current understanding of the mechanisms by which NO exerts
both hepatoprotective and hepatotoxic actions is discussed in this
themes article.
inducible nitric oxide synthase; apoptosis; caspases; peroxynitrite
 |
INTRODUCTION |
NITRIC OXIDE (NO), a short-lived free radical,
influences physiological processes in essentially every organ and
tissue. It exhibits a remarkably broad spectrum of functions, including
roles in neurotransmission and memory formation, prevention of blood clotting, regulation of blood pressure, and mediation of
the bactericidal and tumoricidal activity of macrophages. NO is
enzymatically synthesized from
L-arginine by three known NO
synthase isoforms: constitutively expressed endothelial NO synthase
(eNOS or type 3 NOS) and neuronal NO synthase (type 1 NOS) and the
inducible NO synthase (iNOS or type 2 NOS). It is known that NO
mediates many of its physiological functions, but not all, through the
direct heme-dependent activation of soluble guanylate cyclase and the
consequent increase in intracellular cGMP levels. NO and its reaction
products can also modify protein function through
S-nitrosylation of thiol groups or
nitration of tyrosine residues of proteins. Production of NO by
constitutive eNOS is primarily regulated by fluctuation of
intracellular calcium levels. In contrast, iNOS is calcium independent
and is mainly regulated at the transcriptional level. In most cell
systems, iNOS expression requires stimulation by cytokines or microbial products. Under normal conditions, only the constitutive eNOS is
present in the liver and the low level of NO produced by eNOS regulates
hepatic perfusion. iNOS, however, is readily upregulated in the liver
under a number of conditions, including endotoxemia, hemorrhagic shock,
ischemia-reperfusion, sepsis, infection, hepatitis, ozone
exposure, and liver regeneration. Once iNOS is expressed, large amounts
of NO are generated in the liver in a sustained fashion, serving as an
important regulator and effector during inflammation and infection.
Because the liver plays pivotal roles in a large number of metabolic
and immune processes, the physiological and pathophysiological
functions of NO generated in the liver have attracted numerous
investigations from many laboratories. Both cytoprotective and
cytotoxic effects of NO have been demonstrated in the liver. This
themes article focuses on our current understanding of the mechanisms
of these dual roles.
 |
PRODUCTION OF NO IN THE LIVER |
In response to endotoxin, lipopolysaccharide (LPS), or proinflammatory
cytokines such as tumor necrosis factor-
(TNF-
), interleukin
(IL)-1, and interferon-
as well as their combinations, iNOS is rapidly upregulated within hours in hepatocytes and in resident
hepatic macrophages (Kupffer cells) (reviewed in Ref. 21). These
stimuli often act synergistically to induce iNOS expression; however,
IL-1
alone is an effective stimulator of iNOS in hepatocytes. The
cytokine-mediated upregulation of iNOS gene transcription requires the
transcriptional factors nuclear factor-
B (NF-
B) in
human and rat hepatocytes. In addition, hepatic endothelial cells and
Ito cells can also produce NO through iNOS expression. Therefore, in
inflamed liver, hepatocytes are situated in an environment where NO is
generated from surrounding cells as well as from hepatocytes
themselves. The degree, duration, and location of NO production are
probably determined by the stimulus and the cell types stimulated to
express iNOS. For example, iNOS is rapidly upregulated in Kupffer cells
by low doses of LPS with very low expression in hepatocytes. High doses
of LPS upregulate iNOS in multiple cell types, including hepatocytes,
where the upregulation is transient (24-48 h). In contrast,
injection of killed Corynebacterium
parvum in rodents results in a sustained (7-14
days) upregulation of iNOS in almost all hepatocytes. A summary of some
factors that have been shown to regulate iNOS expression and NO
production is listed in Table 1.
 |
CYTOPROTECTIVE FUNCTION OF NO IN THE LIVER |
Cytokines such as TNF-
are produced in the liver under many
inflammatory conditions and are believed to mediate the extensive hepatocellular injury in endotoxemia and in fulminant hepatic failure.
Administration of nonselective NOS inhibitors, such as NG-monomethyl-L-arginine
(L-NMMA) and
NG-nitro-L-arginine methyl ester
(L-NAME), markedly increases
hepatic damage caused by LPS or TNF-
(1), whereas infusion of
pharmacological doses of NO donor confers protection against the injury
(2, 18). These data argue for a protective role of NO in the liver in
endotoxemia without determining the source of NO. Coadministration of
transcriptional inhibitors with endotoxin LPS or TNF-
markedly increases the liver damage and results in rapid and relatively selective lethal liver damage and failure (13). A liver-selective NO
donor has been used to almost completely inhibit liver damage in the
LPS + N-galactosamine model (20).
Because N-galactosamine inhibits iNOS
expression, these data indicate that induction of iNOS may be one of
the protection pathways inhibited by this transcriptional inhibitor.
Nonetheless, the role of iNOS in liver damage during endotoxemia
remains controversial. Thiemermann et al. (22) previously showed that
administration of a relatively iNOS-selective inhibitor, aminoethylisothiourea, attenuated liver damage and dysfunction in
LPS-treated rats. The emergence of several iNOS-specific inhibitors and
the availability of iNOS-deficient mice made it feasible to examine the
specific role of NO derived from eNOS and iNOS under various
conditions. When highly selective and potent iNOS inhibitors, W-1400
and
NG-iminoethyl-L-lysine
(L-NIL), were used, they did not
attenuate the liver damage caused by endotoxin in anesthetized rats,
despite the prevention of circulatory failure (25). This is in line with the observation that there was no difference in liver necrosis between iNOS-deficient animals and wild-type animals receiving endotoxin (15). Interestingly, iNOS-deficient mice did demonstrate increases in polymorphonuclear neutrophil (PMN) influx when challenged with LPS (4), indicating that iNOS expression is associated with a
suppression of PMN accumulation in mice.
We examined the relative contribution of eNOS vs. iNOS to liver
protection in endotoxic rats using direct infusion of nonselective or
iNOS-selective inhibitors into the liver (18). Continuous intraportal
infusion of nonselective NOS inhibitors
(L-NMMA,
L-NAME) using Alzet osmotic
pumps resulted in increased hepatocellular necrosis and neutrophil
infiltration in LPS-challenged rats (18). In contrast, infusion of
iNOS-selective inhibitors
(L-NIL, aminoguanidine) had no
effect on the LPS-induced hepatic necrosis or PMN influx. However,
apoptotic cell death was markedly increased in the presence of either
nonselective inhibitors or iNOS-selective inhibitors, whereas a NO
donor prevented hepatic apoptosis in endotoxemic rats (18).
Interestingly, inhibition of PMN influx by PMN depletion or
anti-intercellular adhesion molecule-1 antibodies did not abrogate the
increased damage seen with nonselective NOS inhibition. Thus the
increased PMN accumulation does not contribute to the damage. Nonetheless, eNOS clearly has a protective role by maintaining perfusion and limiting thrombosis and probably PMN influx. The consequences of iNOS expression are less consistent and can range from
protection against apoptosis and inhibition of PMN influx to toxicity.
Some of the divergent results obtained in various studies may be the
result of model differences.
The anti-apoptotic properties of NO in liver cells have been examined
in some detail. Cultured hepatocytes undergo apoptotic cell death when
exposed to Fas ligand, TNF-
plus the transcriptional inhibitor
actinomycin D, transforming growth factor-
(TGF-
), or prolonged
cell culturing. It has been shown that NO suppresses not only Fas- and
TNF-
-mediated apoptosis but also spontaneous apoptosis of cultured
hepatocytes (10). Both the exogenously produced NO via NO donors and
the NO derived from induction of iNOS with cytokines prevented hepatic
apoptosis. Furthermore, adenovirus-mediated gene transfer of human iNOS
into hepatocytes effectively inhibits TNF-
-mediated apoptosis (24).
These in vitro studies are supported by impressive in vivo results. A
liver-selective NO donor dramatically reduced the massive apoptosis
induced by TNF-
and N-galactosamine
in rats (20). iNOS knockout animals exhibit a significant increase in
hepatocyte apoptosis 24 h after partial hepatectomy, indicating that
iNOS is probably involved in preventing apoptosis during liver
regeneration (19). Although NO induces apoptosis in many cell types, we
found no evidence for apoptosis in hepatocytes exposed to even
millimolar concentrations of NO donors and found that the high
concentration of NO required to kill hepatocytes actually resulted in
necrosis, not apoptosis (unpublished observations). Thus NO is a potent
anti-apoptotic molecule in hepatocytes.
We have begun to examine how NO interacts with the apoptotic signaling
cascade in hepatocytes. In most cell types, the death signals appear to
converge at the level of a unique family of cysteine proteases named
caspases (reviewed in Ref. 23). A total of 14 mammalian caspases have
been identified. These enzymes exist in cells as zymogens and require
proteolytic cleavage into the enzymatically active form. Initiator
caspases, for instance caspase-8, can propagate the apoptotic signal by
activating downstream effector caspases, such as caspase-3, which serve
as executioners in the terminal phase of apoptosis by cleaving key
death substrates [such as poly(ADP-ribose) polymerase,
DNA-dependent kinase, and the inhibitor of the caspase-activated
deoxyribonuclease]. Activated upstream caspases can also cleave
members of the Bcl-2 family, leading to the release of cytochrome
c from mitochondria. The translocated
cytochrome c forms a complex with
apoptotic protease-activating factor 1, dATP, and procaspase-9,
resulting in the activation of caspase-9 that in turn activates
caspase-3. Given the growing evidence that caspase-3-like proteases
play a pivotal role in hepatic apoptosis induced by TNF-
, Fas, and
TGF-
, the relationship between NO and caspases was examined in our
laboratory. It was found that NO suppressed increases in caspase-3-like
protease activity of hepatocytes in response to TNF-
, Fas, or
spontaneous cell death in vitro and following TNF-
plus
N-galactosamine treatment in vivo
(10). The capacity of dithiothreitol to partially reactivate the
caspases is consistent with a NO-mediated redox modification of the
activated caspases (10, 14). Like other cysteine proteases, caspases
require a reduced thiol at the active site, and it is likely that this
cysteine is S-nitrosylated by NO and
thereby inactivated by NO. We have since gone on to show that NO blocks not only the activity of caspases but also their proteolytic activation (specifically caspases-8 and -3) and that these effects can be mimicked
by a cGMP analog (unpublished results). Furthermore, the cGMP effects
appear to be mediated by the activation of protein kinase G (10).
Consistent with the inhibition of caspase activation by NO is the
inhibition of Bcl-2 degradation, prevention of mitochondrial depolarization, and an inhibition of cytochrome
c release in hepatocytes (unpublished
data). Taken together, these studies demonstrate that NO, its reaction
products, or its downstream signaling molecules can interact either
directly or indirectly with the apoptosis-signaling cascade at multiple
levels and thereby suppress hepatic apoptosis. NO-stimulated cGMP
formation inhibits caspase activation by mechanisms that are not yet
clear, whereas NO can directly inhibit caspase activity via
S-nitrosylation. It is likely that the
capacity for hepatocytes to effectively carry out
S-nitrosylation contributes to the
potent anti-apoptotic actions of NO in these cells. For NO to
S-nitrosylate the cysteine residue, it
must first give up an electron; the likely acceptors in hepatocytes
include molecular oxygen and iron-bound proteins.
Besides hepatocytes, other primary cell types that have been shown to
be protected by NO against apoptosis include B lymphocytes, eosinophils, splenocytes, and endothelial cells (reviewed in Ref. 8).
In addition to the inhibition of caspase activation, other mechanisms
by which NO prevents apoptosis include upregulation of Bcl-2,
disruption of the c-Jun
NH2-terminal kinase- signaling pathway, and upregulation of the protective proteins 70-kDa heat shock
protein (HSP70) and heme oxygenase-1. Preexposure of
hepatocytes to high amounts of NO donor results in upregulation of
HSP70 and desensitizes the cells to TNF-
-mediated apoptosis (9).
Although the mechanism by which HSP70 blocks apoptosis has yet to be
resolved, HSP70 has been shown to act downstream of caspase activation
in tumor cell lines following exposure to TNF-
, staurosporine, or the anti-cancer drug doxorubicin (6).
Other studies have shown that NO can protect hepatic cells against
oxidative damage and lipid peroxidation. This includes damage caused by
carbon tetrachloride (16), acetaminophen (11), alcohol (17), and
H2O2-mediated
oxidative stress (7) (Table 2).
Acetaminophen-induced hepatocyte injury was inhibited by exogenous NO
or cytokine-induced NO production (11). Inhibition of NOS resulted in a
severalfold decrease in intracellular reduced glutathione levels and
potentiated acetaminophen-induced hepatotoxicity. In carbon
tetrachloride-induced liver damage, endogenous NO protected the liver
from lipid peroxidation, fibrosis, and damage (16). One mechanism for
the protection against oxidative damage is through the upregulation of
heme oxygenase-1 (7). This results in the production of the potent
antioxidant biliverdin. Direct scavenging of oxygen radicals by NO is
another possibility; however, the ratio between NO and superoxide is
likely to be one important factor.
 |
CYTOTOXICITY OF NO IN THE LIVER |
Whereas iNOS expression can protect the liver in acute hepatic
inflammation, it appears to be detrimental and may account for hepatic
necrosis under conditions of severe redox stress such as
ischemia-reperfusion and hemorrhagic shock (5). As in other insults,
nonspecific NOS inhibition using
L-NAME increases hepatic damage
in resuscitated hemorrhagic shock (3). However, administration of
iNOS-selective inhibitor L-NIL
to rats at the time of resuscitation or the performance of hemorrhagic
shock in iNOS knockout animals was associated with a significant
decrease in hepatic injury compared with control groups (5).
Interestingly, the reduction of injury was associated with a marked
inhibition in NF-
B activation, IL-6 and granulocyte
colony-stimulating factor mRNA levels, PMN influx, and
signal transducer and activator of transcription-3
activation. These data suggest that iNOS-generated NO not only directly
contributes to tissue damage but that it also upregulates the
inflammatory response through specific signaling mechanisms. Because
oxygen radicals are produced during resuscitation, it is likely that NO
reacts with superoxide to form peroxynitrite that causes oxidative injury. This is also supported by observations in a murine model of
warm liver ischemia-reperfusion in which iNOS knockout mice are
significantly protected (V. Lee, M. Johnson, T. Billiar, unpublished results). The direct toxicity by NO through peroxynitrite formation during oxidative stress seems straightforward. Less clear is the mechanism for the activation of NF-
B by NO in this setting. One possibility is the activation of Ras by NO, which
ultimately leads to NF-
B nuclear translocation, as that relationship
has been established in the human Jurkat T cell line and potentiated by redox stress (12).
 |
CONCLUSION |
Whereas NO produced from eNOS is clearly beneficial to the liver for
its role in maintaining perfusion, and preventing platelet adhesion,
thrombosis, and PMN accumulation, the inducible NO production can have
opposite effects, depending on the insult. The determinants for the
effects of iNOS expression appear to include the redox status of liver,
the coproduction of reactive oxygen species, and the type of insult.
The availability of selective iNOS inhibitors and the availability of
iNOS knockout mice have clearly established a role for induced NO in
the prevention of apoptosis in acute inflammation and a damaging effect
in the ischemia-reperfusion type of insults. Further research
should provide a solid basis for therapeutic approaches to either
supplement NO to the liver for its protective effect or suppress iNOS
to prevent liver damage.
 |
ACKNOWLEDGEMENTS |
This work was supported by National Institute of General Medical
Sciences Grants GM-44100 and GM-37753.
 |
FOOTNOTES |
*
Fourth in a series of invited articles on Nitric
Oxide.
Address for reprint requests and other correspondence: T. R. Billiar,
A1010 Presbyterian Univ. Hospital, Pittsburgh, PA 15213 (E-mail:
billiartr{at}mxs.upmc.edu).
 |
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