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
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INTRODUCTION
PRODUCTION OF NO IN...
CYTOPROTECTIVE FUNCTION OF NO...
CYTOTOXICITY OF NO IN...
CONCLUSION
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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
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ABSTRACT
INTRODUCTION
PRODUCTION OF NO IN...
CYTOPROTECTIVE FUNCTION OF NO...
CYTOTOXICITY OF NO IN...
CONCLUSION
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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
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ABSTRACT
INTRODUCTION
PRODUCTION OF NO IN...
CYTOPROTECTIVE FUNCTION OF NO...
CYTOTOXICITY OF NO IN...
CONCLUSION
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In response to endotoxin, lipopolysaccharide (LPS), or proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha ), interleukin (IL)-1, and interferon-gamma 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-1beta alone is an effective stimulator of iNOS in hepatocytes. The cytokine-mediated upregulation of iNOS gene transcription requires the transcriptional factors nuclear factor-kappa B (NF-kappa 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.

                              
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Table 1.   Factors that positively or negatively regulate hepatic iNOS expression


    CYTOPROTECTIVE FUNCTION OF NO IN THE LIVER
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ABSTRACT
INTRODUCTION
PRODUCTION OF NO IN...
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Cytokines such as TNF-alpha 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-alpha (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-alpha 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-alpha plus the transcriptional inhibitor actinomycin D, transforming growth factor-beta (TGF-beta ), or prolonged cell culturing. It has been shown that NO suppresses not only Fas- and TNF-alpha -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-alpha -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-alpha 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-alpha , Fas, and TGF-beta , 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-alpha , Fas, or spontaneous cell death in vitro and following TNF-alpha 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-alpha -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-alpha , 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.

                              
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Table 2.   Role of inducible NO production in liver damage


    CYTOTOXICITY OF NO IN THE LIVER
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ABSTRACT
INTRODUCTION
PRODUCTION OF NO IN...
CYTOPROTECTIVE FUNCTION OF NO...
CYTOTOXICITY OF NO IN...
CONCLUSION
REFERENCES

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-kappa 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-kappa B by NO in this setting. One possibility is the activation of Ras by NO, which ultimately leads to NF-kappa B nuclear translocation, as that relationship has been established in the human Jurkat T cell line and potentiated by redox stress (12).


    CONCLUSION
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ABSTRACT
INTRODUCTION
PRODUCTION OF NO IN...
CYTOPROTECTIVE FUNCTION OF NO...
CYTOTOXICITY OF NO IN...
CONCLUSION
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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).


    REFERENCES
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ABSTRACT
INTRODUCTION
PRODUCTION OF NO IN...
CYTOPROTECTIVE FUNCTION OF NO...
CYTOTOXICITY OF NO IN...
CONCLUSION
REFERENCES

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Am J Physiol Gastroint Liver Physiol 276(5):G1069-G1073
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