Nitric Oxide
Jose M. Prince, Timothy R. Billiar
25
25.1
Introduction
Nitric oxide (NO), first identified as endothelium-de- rived relaxing factor in 1980, is a potent biologic me- diator whose physiologic and pathophysiologic im- portance has exploded over the past almost 25 years [35]. NO, a diffusible, free radical gas, is produced by the enzyme nitric oxide synthase (NOS) (Fig. 25.1) The
L-arginine → NO biosynthetic pathway respon- sible for its production was first fully elucidated in 1987 [52, 59, 102]. The magnitude of this discovery was recognized by Science magazine in 1992 when NO was named molecule of the year [69]. In 1998, Furchgott, Ignarro, and Murad received the Nobel Prize in Medicine for their discoveries on the role of the NO → cGMP system in the cardiovascular sys- tem. Perhaps the best indicator of the importance of NO in biology is the sheer volume of publications on the topic of NO over the past 15 years (over 45,000).
Although originally characterized as a vasodilator, NO has subsequently been implicated in a variety of diverse biological activities. In particular, the influ- ence of NO in hepatic physiology continues to be ac- tively pursued by scientists and physicians around the world. Every new discovery adds to the already complex, and at times conflicting, understanding of the role of NO in the liver.
Many liver diseases and the surgical procedures used as therapy for these diseases result in acute stress and inflammation that can lead to end-organ dysfunction or damage. Common insults experi- enced by these patients include exposure to infec- tious and septic stimuli and reduced perfusion with or without ischemia/reperfusion (I/R) injury. The stress and adaptive responses that characterize each of these insults have unique features; however, most injuries result in the activation of inflammatory cascades, including the upregulation of the induc- ible, or inflammatory isoform of NOS (iNOS) [130].
Studies on the liver have demonstrated a clear di- chotomy for iNOS/NO. On the one hand, NO limits programmed cell death in some acute settings (en-
dotoxemia, regeneration, exposure to death ligands) [64, 101, 109], while on the other hand, promoting hepatic damage in other contexts (I/R, hemorrhagic shock, (HS) [55, 71]. In this chapter, we will summa- rize what is known about NO synthesis in the liver, as well as the role of NO in various liver diseases.
We will begin with an overview of the chemistry of NO in cells.
25.2
Nitric Oxide Synthesis and Signaling
Nitric oxide is produced enzymatically by one of three NO synthases (NOS) from l-arginine [40, 93, 115] (Table 25.1). These enzymes are products of dis- tinct genes that share approximately 50% sequence homology. Their nomenclature is defined by the cel- lular expression patterns that led to their discovery and the order in which the cDNAs were first cloned:
neuronal nNOS (NOS-1), inducible or inflammatory iNOS (NOS-2), and endothelial eNOS (NOS-3) [46].
Important components needed for NOS enzymatic
Fig. 25.1. Nitric oxide (NO) may be protective or toxic to hepa- tocytes in different liver diseases depending upon the specific conditions present
activity include nicotinamide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), heme, and tetrahydrobiopterin (BH
4). Neuronal NOS and eNOS are typically referred to as constitutive because these isoforms are present in tissues under resting conditions. Regulation of these enzymes is predom- inantly post-translational. A dominant mechanism for control involves the Ca
2+-dependent binding of calmodulin to eNOS or nNOS that then transient- ly activates the enzyme to produce small bursts of NO. Numerous other regulatory mechanisms have been identified, and both constitutive isoforms are now known to have a wide range of tissue expres- sion [11, 34]. iNOS is not typically expressed in the cellular resting state (exceptions include intestinal and bronchial epithelial cells, and renal tubular epi- thelial cells) [40, 115]. Instead, gene transcription is activated under acute inflammatory or stress condi- tions, and because calmodulin binds to iNOS in the absence of calcium elevations, the enzyme produces NO in large quantities in a sustained manner [19].
Pro-inflammatory cytokines, microbial products, and oxidant stress are all particularly effective at stimulating iNOS transcription [40, 93, 106, 115].
Not surprisingly, transcription factors activated by these stimuli (e.g., NF- κB, STATs, AP-1, HNF4-α) are all known to be involved in iNOS gene activation [40, 93, 115]. Although iNOS expression was first identified in murine macrophages, it is now known that iNOS can be expressed in virtually every cell type from all mammalian species if appropriately stimulated.
Studies on the liver identified the first non- macrophage cell type with iNOS-like activity, the hepatocyte. The presence of iNOS expression in rat hepatocytes demonstrated the first evidence that parenchymal cells could express iNOS. In addition to hepatocytes, iNOS is expressed in Kupffer cells (resident hepatic macrophages), vascular endothe- lial cells, and stellate cells [8, 23]. The discovery of iNOS expression in the liver led to the first identifi-
cation of iNOS expression in humans [95] and the subsequent cloning of the human iNOS cDNA [42]
and gene [16]. Inducible NOS can be expressed in all cell types in the liver, but hepatocyte expression still appears to be the most prominent and consistent across species. In vitro, IL-1, TNF, IFN- γ, and bacte- rial lipopolysaccharide (LPS) act synergistically to induce iNOS expression maximally in hepatocytes [43]. Interleukin-1 is the most potent single inducer of iNOS in hepatocytes [41], while redox stress fur- ther activates iNOS gene transcription [106].
As we will discuss in greater detail below, hepa- tocyte iNOS expression has been identified in vivo in response to endotoxemia [101], HS [26, 55], hepatic and intestinal I/R [71], and during hepatic regenera- tion [107] or hepatitis [26, 110]. Whereas acute in- sults result in a rapid but transient increase in iNOS expression, more massive and sustained hepatocyte iNOS expression has also been seen following the injection of killed Corynebacterium parvum [6, 7].
In combination with LPS, C. parvum is a known in- ducer of various cytokines including IL-1, TNF- α, and IFN- γ. The exuberant hepatic iNOS expression produced by this model lasts for days with little evi- dence of hepatic injury [7].
25.2.1
The Chemical Actions of NO in Biological Systems
The chemical fate of NO in cells remains the topic of considerable debate due, in large part, to the com- plexities associated with measuring the abundance of short-lived radicals. It is reasonably well accepted that NO, with its one unpaired electron, will react avidly with oxygen, superoxide anion radical (O
2–), and transition metals. These reactions can lead to the modification of proteins, resulting in the activa- tion or inactivation of enzymes, or lead to cellular toxicity through various other means. The chemis- try resulting from these interactions can be separat-
Table 25.1. Characteristics of the NOS family. Three distinct NOS isoforms produce NO by their enzymatic action on
L-arginine
Nitric oxide synthases Characteristic expression pattern Calcium Protein size (kDa)
NOS-1 (nNOS) Constitutive Dependent 161
NOS-2 (iNOS) Inducible Independent 131
NOS-3 (eNOS) Constitutive Dependent 133
ed into nitrosation or oxidation [82, 121]. The chal- lenge in the field has been in defining the pathways to nitrosation or oxidation in intact tissue. One line of thought argues that the chemical fate of NO (i.e., nitrosation vs. oxidation) is dependent on the redox status of the cell. This process will depend not only on cell type, but also on environmental factors such as ischemia. In a resting cell, the interaction of NO with oxygen or ferrous iron can lead to the forma- tion of S-nitrosoglutathione (GSNO) in the presence of adequate GSH levels. Both N
2O
3and GSNO ef- ficiently S-nitrosate thiols on proteins. One conse- quence of reduced GSH levels, as seen during redox stress, would be enhanced peroxynitrite formation resulting from the interaction of NO and O
2–. Glu- tathione may also be involved in the detoxification of peroxynitrite, and thus low GSH levels may favor the formation and toxicity of peroxynitrite. Agents that inhibit GSH synthesis or GSSG → GSH recycling allowed for NO-mediated hepatocyte death [49].
25.2.2
NO Signals Through Cyclic Nucleotides and Alters Gene Expression
Cyclic nucleotides are classical second messengers that transmit signals by activating downstream ki- nases or through the activation of cyclic nucleotide- gated channels [2, 32, 78]. The cyclase that leads to cGMP formation is abundant in hepatocytes [2, 78]. In fact, much of the initial characterization of guanylyl cyclase was in the liver [68]. Nitric oxide directly activates the soluble isoform of guanylyl cyclase in a heme-dependent manner [78]. Ignarro and coworkers first reported in 1987 that NO-releas- ing compounds could elevate cGMP levels in whole liver slices [124]. Subsequently, endogenous NO was found to induce a several-fold increase in cGMP production in hepatocytes [5]. The prototypic target for cGMP is protein kinase G (PKG). This enzyme is comprised of three isoforms: I α, IB, II [78]. Most of the PKG-I targets identified to date modulate the functions of platelets and smooth muscle cells (e.g., phospholambdin and the type I 1,4,5-triphosphate (InsP
3) receptor). In hepatocytes, cGMP-PKG sign- aling is only known to regulate Ca
2+concentrations through the phosphorylation of the InsP
3receptor [30, 108].
One of the means by which NO provides protec- tive actions in hepatocytes is via cGMP signaling [67, 74]. In other cell types, the protective actions of NO are almost entirely cGMP-dependent [28].
Cell-permeable cGMP analogs are highly protective against apoptosis in hepatocytes [74]. In addition to cGMP signaling, NO regulation of gene expression
has begun to be evaluated in the liver by using dif- ferential display, DNA microarray, and proteomic analyses [128]. For example, by using these tech- niques a confirmation of iNOS-induced expression of hemoxygenase-1 (HO-1) has been demonstrated at the mRNA and protein levels both in vitro and in vivo [129]. These observations confirm the up- regulation of this protective gene in hepatocytes by iNOS-derived NO and suggest another mechanism for NO hepatoprotection [131].
25.3
Inflammation and Sepsis
The role of NO in mediating hepatic injury or protec- tion in conditions producing inflammatory signal- ing cascades has at times yielded conflicting results.
In a normal rat liver, NO synthesis inhibition yields increased perfusion pressure, supporting a role for NO in the maintenance of hepatic perfusion during normal physiologic conditions [89]. Although it is clear that iNOS expression is part of the pathophysi- ology of sepsis and shock, unraveling the actions of iNOS in vivo has been very challenging. Much has been learned using non-selective pharmacological NOS inhibitors, selective iNOS inhibitors, and iNOS and eNOS null mice to assess hepatic NOS function.
An initial report on the role of NO in hepatic dam- age in 1990 [6], followed by numerous subsequent reports (reviewed in references [14, 23, 24, 60, 61]) have developed a line of investigation demonstrat- ing that inhibition of eNOS dramatically enhances hepatic injury in sepsis models, shock and I/R. En- dothelial NOS appears to be critical for preserving tissue perfusion and endothelial cell viability [71].
The role of iNOS in HS and I/R contrasts to what is witnessed in endotoxemia [75, 101], lipopolysac- charide/d-galactosamine (LPS/
D-gal) administra- tion [109], hepatic regeneration [107] and cold I/R following transplantation [125]. In each of these latter settings, either the absence or the inhibition of iNOS resulted in enhanced hepatocyte apoptosis.
The anti-apoptotic capacity of NO in the liver was most evident in the TNF/
D-gal model of massive hepatic apoptosis. The use of a liver-selective NO donor showed that NO almost completely inhibited the widespread cell death seen in this model [109].
This suggests that inhibition of apoptosis is one of
the mechanisms of hepatoprotection mediated by
iNOS. However, low levels of iNOS may be required
for the induction of Fas-induced apoptosis as dem-
onstrated by the failure of Fas-activating antibody
to induce liver damage in iNOS-deficient animals
[15]. Additional iNOS-mediated protective effects
include the inhibition of PMN influx [85] and im- proved liver perfusion [103]. Evidence for upregula- tion of iNOS in humans under conditions similar to the animal models is provided by a study of septic patients, demonstrating a fourfold increase in circu- lating levels of reactive nitrogen species [98].
25.4
Ischemia/Reperfusion and Hemorrhagic Shock
In contrast to the iNOS-mediated hepatoprotection seen in sepsis, iNOS may have the opposing action in ischemic conditions depending on the specific re- dox milieu. In studies employing both iNOS-selec- tive inhibitors and iNOS knockout mice, increased iNOS expression increased hepatocellular injury in HS [55] and I/R [71].
This damage is evident within 3–6 h of either resuscitation or reperfusion and hence occurs prior to significant PMN infiltration. In HS, iNOS expres- sion is also associated with enhanced IL-6 produc- tion and NF- κB activation, while iNOS expression in I/R appears to enhance the efficiency of reperfusion.
However, in both models the temporal sequence of events supports direct hepatocellular injury in the presence of iNOS. Expression of iNOS has also been shown to contribute to toxicity in models of alcohol- induced cirrhosis [26] and hepatitis induced by con- canavalin A or acetaminophen [110]. We will first consider the role of iNOS in HS [38, 39].
In mouse HS models, the absence of iNOS (iNOS knockouts), or iNOS inhibition, was associated with a 40%–60% reduction in the resuscitation-induced NF- κB activation, STAT3 activation, and the upreg- ulation of IL-6 and G-CSF in the liver and lungs [55].
Similar observations were made when a compound that scavenges NO was given at the time of resusci- tation [53, 56]. In one study characterizing the up- regulation of iNOS in the liver during HS, quantita- tive RT-PCR found that both the trauma of the vessel cannulation (sham animals) and HS lead to increas- es in iNOS mRNA between 30 and 60 min. A further increase in iNOS expression was seen in animals subjected to shock alone between 60 and 90 min.
iNOS protein appeared by 90 min, localized almost exclusively in hepatocytes, and was most prevalent in cells near the central vein. Electron microscopy revealed that iNOS was distributed in both the cys- tosol and peroxisomes [21]. iNOS inhibition using low doses of the relatively selective iNOS inhibitor N-6-(1-iminoethyl) lysine (L-NIL, 50 µg/kg) reduced liver damage while increasing the expression of heat shock protein 70 [86]. The greater expression of this
protective protein was associated with a reduction in staining for 3-nitrotyrosine in the liver. These re- sults are consistent with studies demonstrating that iNOS contributes to organ damage in resuscitated HS [55]. A pro-inflammatory consequence of iNOS expression is the activation of NF- κB [54]. In addi- tion, iNOS expression in human trauma patients has been demonstrated in one study performing RT- PCR on liver needle biopsies obtained at the time of emergency laparotomy for trauma. Of 18 trauma patients studied, all were positive for iNOS mRNA while biopsies from non-trauma patients were all negative for iNOS expression.
25.4.1
Role of iNOS in Hepatic I/R
One explanation for the conversion of protective ac- tions of iNOS to toxic effects in the setting of hy- poxia/reoxygenation is the concomitant production of reactive oxygen species (ROS). A series of experi- ments in a model of warm hepatic I/R evaluated the consequences of iNOS expression in the setting of I/R isolated to the liver [71]. The model of partial hepatic I/R in mice consists of temporary occlu- sion of the left branches of the hepatic artery and portal vein with a microvascular clip. This permits flow to the right lobes and avoids intestinal conges- tion. One hour of ischemia results in a reproducible level of liver damage upon reperfusion. Both iNOS knockout mice and wild-type mice treated with an iNOS inhibitor (L-NIL) exhibited lower levels of liver damage as assessed by circulating transami- nase levels and histology. Interestingly, the absence of iNOS was associated with a marked delay in mi- crovascular flow upon reperfusion. These results demonstrate a clear difference between HS and I/R in that iNOS was not expressed in I/R until 3 hours after reperfusion. Thus, models of isolated I/R may be useful to study the specific effects of ischemia, but they may not replicate the systemic injury in re- sponse to shock.
25.4.2
eNOS Is Protective in HS and I/R
A consistent finding in studies evaluating the con-
tribution of endothelial NOS is that constitutive NO
production is protective and essential to maintain-
ing perfusion in low flow states. This may explain
the increase in injury seen when non-specific NOS
inhibitors have been used in shock models [50]. The
contribution of eNOS function in either HS [111]
or I/R [71] was evaluated in eNOS knockout mice.
eNOS knockout mice exhibited a dramatic increase in mortality compared to wild-type or iNOS knock- out mice when subjected to HS. Furthermore, liver damage was significantly greater in eNOS knockout mice in the hepatic I/R model when compared to wild-type controls. Thus, in clear contrast to the ac- tions of iNOS, eNOS is protective in shock and I/R.
25.5 Cirrhosis
The role of NO in the liver is not limited to acute set- tings: NO biology is important in chronic conditions such as cirrhosis. Cirrhosis develops in patients sus- taining persistent hepatic injury, which results in permanent scarring that manifests itself with a va- riety of hemodynamic disturbances. Patients with progressive cirrhosis develop portal hypertension and present with a hyperdynamic circulatory state consisting of a low systemic vascular resistance and high cardiac output. The systemic vasodilation re- sults in the body sensing a decreased effective circu- lating volume and responding by secreting antidiu- retic hormone (ADH) and aldosterone along with sympathetic nervous system stimulation. These compensatory actions result in the fluid and salt retention that leads to ascites formation. In 1991, Vallance and Moncada proposed a role for NO in the pathophysiology of liver cirrhosis [117]. In their hypothesis, NO overproduction incited by increased endotoxemia results in a hyperdynamic circulation.
Patients with cirrhosis display high levels of endo- toxin without necessarily demonstrating clinical evidence of infection [79]. Porto-systemic shunting has been suggested to be the mechanism permitting bacterial endotoxins to attain systemic circulatory access [47]. Supporting this line of thought, endo- toxin infusion in humans has been shown to lead to the development of peripheral vasodilation [31, 113]. As we discussed earlier, NO biology has been implicated in sepsis. Patients in septic shock treat- ed with NOS inhibitors demonstrated increases in blood pressure [104]. This finding suggests that the production of NO is at least in part responsible for the hypotension seen in these patients.
In the setting of cirrhosis, increased NO produc- tion has been supported by the detection of elevated nitrate levels in plasma, urine, and ascites samples from humans [10, 22, 48, 57]. Furthermore, the se- verity of liver damage appears to correlate with high nitrate levels. Patients with cirrhosis exhale higher levels of NO with respiration [3]. In one human ex- perimental model, cirrhotic patients infused with a
NOS inhibitor (
L-NMMA) demonstrated improved vasoconstrictor responsiveness in their forearm ar- teries [12]. In animal models of cirrhosis, CCl4-in- duced cirrhotic rats demonstrate increased NO-de- pendent vasodilation in aortic ring specimens with elevated cGMP levels [20]. Additionally, treatment with a NOS inhibitor (
L-NAME) increases systemic vascular resistance in cirrhotic rats. In a rat model of chronic portal hypertension, which involves liga- tion of the portal vein, NO synthesis inhibition re- sults in systemic and splanchnic vascular constric- tion [105]. In vitro, neutrophils and monocytes iso- lated from cirrhotic patients demonstrate increased iNOS activity [70]. Furthermore, this elevated NOS activity appears to be increased in relation to the se- verity of liver dysfunction [37]. Despite these find- ings in vitro, the exact cause of increased NO levels in cirrhosis remains undetermined, with some ob- servations contradicting the role of iNOS as the pri- mary source of increased NO production [29]. Alter- native explanations for increased NO levels include increased eNOS expression or decreased urinary nitrate clearance [36]. Overall, increased NO levels appear to be detrimental in cirrhosis and reduction of NO beneficial, though some contradictory evi- dence does exist. The initial cause of NO production remains contested but may involve endotoxemia- induced cytokine signaling or shear stress-induced signaling.
25.6
Liver Tumors and Apoptosis
Nitric oxide has both mutagenic and tumoricidal effects. Various infectious and inflammatory con- ditions may favor a carcinogenic progression in the liver. For example, liver fluke infestation has been implicated in the occurrence of cholangiocarcinoma [100]. Increased NO production has been implicated in the development of liver cancer in a woodchuck model of chronic viral hepatitis [76]. Similarly, NO levels are elevated in patients with chronic hepatitis who have an associated increased risk of hepato- cellular carcinoma in the setting of viral hepatitis (HBV or HCV) infection [99].
The role of NO biology in cancer remains con-
troversial. NO has both mutagenic and antitumor
effects [123]. The mutagenic effects may be mediat-
ed by nitrosative deamination or oxidation of DNA
bases resulting in DNA mutations. NO may also fa-
cilitate angiogenesis and limit leukocyte infiltration
promoting tumor growth [90, 94, 122]. For example,
vascular endothelial growth factor (VEGF), which
increases the vascular permeability and endothe-
lial growth important in angiogenesis, requires NO/
cGMP signaling within the endothelial compart- ment to promote neovascular growth. On the other hand, there have been numerous studies on the role of NO in the induction or prevention of apoptosis/
necrosis. Hibbs et al. [52] first proposed a cytotoxic role for macrophage-derived NO in 1987, and the Al- bina laboratory first showed in 1993 that NO could increase apoptosis [1]. Stamler and coworkers [80]
first revealed in 1994 the capacity of NO to prevent apoptosis. Many review articles and book chapters have reviewed this topic in detail; we will therefore limit our discussion in this chapter [62, 119].
The effect of NO on cell viability differs based on the degree of redox stress; additionally, different cell types differ considerably in their response to NO. In macrophages, pancreatic islet cells, neurons, ente- rocytes, thymocytes, cardiac myocytes, endothelial cells, and fibroblasts, even low-level NO leads to ap- optosis. In contrast, B lymphocytes, natural killer cells, eosinophils, embryonic motor neurons, pheo- chromocytomas, ovarian follicles, and hepatocytes can be protected by NO against apoptosis induced in various ways [62]. Hepatocytes are unique in that not only are these cells protected by NO, but also necrotic death is not seen until the cells are exposed to supraphysiologic concentrations of NO donors in the millimolar range [63]. This response changes dramatically when the cells are subjected to redox stress. Under these circumstances, NO induces hepatocyte necrosis even at low to moderate con- centrations. In NO-sensitive cell types, the pro-ap- optotic actions of NO involve DNA damage and the activation of p53 [87], the upregulation of Fas and Fas ligand (FasL) [33, 51, 77, 120], and direct effects on mitochondria. The mitochondria may be a par- ticularly important target in susceptible cells and hepatocytes exposed to redox stress. Targets for NO in the mitochondria include cytochrome c oxidase, mitochondrial complexes I, II, III, IV, and V, aconi- tase, creatine kinase, the mitochondrial membrane, mitochondrial DNA, and superoxide dismutase [4].
In some cells, the pro-death signaling is initiated by cGMP [44, 112], while in others (including hepato- cytes) cGMP inhibits apoptosis [67, 74, 114]. Other anti-apoptotic actions of NO include the upregula- tion of anti-apoptotic Bcl-2 family proteins [44, 114], the downregulation of pro-death proteins (BNIP3) [127], and the direct modification of caspases by S- nitrosation [27, 72, 81].
Ligand-dependent apoptosis in hepatocytes takes place via activation of the type II or mitochon- drial-dependent pathway [126]. We will first briefly summarize this pathway to illustrate how NO can block cell death efficiently in hepatocytes. The bind- ing of pro-death ligands (e.g., FasL, TNF, or TRAIL)
to specific receptors can induce oligomerization of the receptors followed by the recruitment of adapter proteins to the cytoplasmic portions of the receptor [92]. Common to all death receptors is the binding of FADD (Fas-associated death domain protein) [9, 17]. The TNF signal transduction pathway is more complex, in that signaling through the p55 recep- tor activates inflammatory pathways distinct from apoptotic pathways [18]. The p55 TNF receptor also recruits TRADD (TNF receptor-associated death domain) as well as RAIDD, and RIP [18]. Recent evidence indicates that the separation of the pro- inflammatory and pro-apoptotic pathways occurs through the formation of distinct post-TNFRI sig- naling complexes. The formation of a pro-death complex requires the formation of a complex incor- porating FADD and caspase-8 within the cytoplasm [88]. The cytoplasmic complex may then lead to the auto-cleavage and activation of caspase-8 [83, 91].
In hepatocytes, caspase-8 then cleaves BID, a pro- death Bcl-2 family member [126]. The BID cleavage product interacts with the mitochondria, leading to loss of membrane potential and release of pro-death signaling proteins, including cytochrome c. Cyto- chrome c supports the formation of the apoptosome, which is comprised of Apaf-1, caspase-9, and ATP, in addition to cytochrome c. This complex activates downstream effector caspases, such as caspase-3 and -7 [45]. Enzymatic cleavage of downstream tar- gets of these effector caspases results in the mor- phological and structural changes characteristic of apoptosis. Hepatocytes are representative of type II cells in that ligand-dependent apoptosis depends on BID cleavage [126]. In contrast, cells utilizing the type I pathway bypass the mitochondria and the proximal caspases directly cleave and activate the downstream effector caspases [116].
Nitric oxide can interfere with this apoptotic signaling pathway in hepatocytes through several mechanisms. At higher levels, NO can upregulate heat shock protein 70, which in turn can interfere with the proximal signaling steps in apoptosis [64].
Caspases are cysteine proteases, and all possess ac-
tive-site cysteine residues essential for their activ-
ity. This cysteine is susceptible to S-nitrosation that
blocks enzyme activity. At least five of the 14 known
caspases are activated in hepatocytes stimulated to
undergo apoptosis [73]. For S-nitrosation to take
place, NO must react with an electron acceptor (e.g.,
oxygen, transition metals). This process is very ef-
ficient in hepatocytes in the absence of redox stress,
and the direct inhibition of caspases seems to be the
dominant mechanism for the inhibition of apopto-
sis in hepatocytes. However, not all of the protection
afforded to hepatocytes by NO is due to S-nitrosa-
tion; cGMP synthesis also accounts for some of the
protective actions [67]. The activation of caspase-3 is inhibited in hepatocytes by NO [67]. Additionally, NO inhibits the proximal steps in apoptosis induced by treatment with TNF and actinomycin D (ActD) by suppressing the loss of mitochondrial membrane potential and the release of cytochrome c [65, 74].
NO blocks the activities of the initiator caspase-8 in a mechanism partially reversed by the reducing agent dithiothreitol (DTT). Furthermore, cleavage of the caspase-8 substrate, BID, and Bcl-2 is also in- hibited by NO [65, 66]. The cleavage product of BID induces pro-apoptotic mitochondrial changes in hepatocytes [126].
25.7
Regeneration
Since the ancient Greeks, man has been fascinated with the capacity of the human liver to regenerate.
In ancient Greek mythology, Prometheus is pun- ished by the gods for giving men the knowledge of fire by having him chained to a stone. Daily, a great bird eats his liver, only to have the liver regrow over- night to be ready for another meal. In actuality, the regenerative process does not happen overnight, but instead involves a complex process of self-renewal.
Many investigators have been interested in identify- ing and defining a potential role for NO in hepatic regeneration. Immediately following partial hepa- tectomy in rats, iNOS expression is induced and NO released [13, 58, 84, 97]. Hepatocytes appear to be the initial source of NO production, followed by Kupffer and endothelial cells [25, 96]. In this set- ting, transcriptional control of iNOS appears to be regulated by NF- κB. One possible explanation for the etiology of increased NO production focuses on shear stress-induced signaling that occurs after al- terations in blood flow after hepatic resection [118].
The increased NO levels may then be hepatoprotec- tive by reducing apoptosis in hepatocytes. Support- ing this argument, iNOS knockout mice subjected to partial hepatectomy demonstrate increased apopto- sis 24 h after hepatic resection [107].
25.8 Conclusion
Nitric oxide is a simple molecule with a complicated role to play in liver pathophysiology. The final effect of NO varies in different liver diseases and depends upon a variety of factors. NO production can be ex- cessive or inadequate resulting in hepatoprotection
or hepatotoxicity, depending upon the overall hepat- ic environment. Given the complexity of NO biology in the liver, successful pharmacologic manipulation of NO synthesis or action to improve patient out- comes for individuals with hepatic disease will have to balance carefully the risks and benefits of alter- ing the complex function of NO. As further research dissects the contradictory evidence obtained in var- ious experimental models, opportunities to harness the potential benefits of NO biology manipulation with NOS isotype-specific and cell-selective agents will continue to provide new avenues of investiga- tion for researchers and hope for patients.
Selected Reading
Furchgott RF, Zawadzki JV. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetyl- choline. Nature 1980;288:373–376. (Classic paper initiating the search for the endothelium-derived relaxing factor re- sponsible for NO discovery.)
Ignarro LJ, Buga GM, Wood KS et al. Endothelium-derived relax- ing factor produced and released from artery and vein is ni- tric oxide. Proc Natl Acad Sci USA 1987;84:9265–9269. (This paper presents some of the initial findings critical in identi- fying NO as endothelium-derived relaxing factor.)
Griffith OW, Stuehr DJ. Nitric oxide synthases: properties and catalytic mechanism. Annu Rev Physiol 1995;57:707–736.
(Very thorough review of the nitric oxide synthases includ- ing protein structure and enzymatic mechanism.)
Kim PK, Zamora R, Petrosko P, Billiar TR. The regulatory role of ni- tric oxide in apoptosis. Int Immunopharmacol 2001;1:1421–
1441. (This review paper focuses on the physiologic impact of NO biology in apoptosis.)
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