Nitric oxide biology and the liver: Report of an AASLD research workshop
Authors: Don C. Rockey, Vijay Shah
Journal: Hepatology (2004)

Nitric oxide is a gaseous molecule essential to numerous biologic processes, including blood vessel relaxation, neurotransmission, host defense, and others. Some of the many unique aspects of this molecule include the mechanism by which this free radical signals via chemical reaction with protein targets as well as the complex biochemistry by which this molecule is generated and regulated. Many advances over the past 2 decades, particularly a heightened focus on NO in the 1990s, has led to a greater understanding of the role of NO in health and disease. Indeed, NO has recently been recognized as critical in the biology of multiple liver processes and diseases. This review summarizes basic aspects of NO biology and diverse areas of liver-related NO biology in the context of the American Association for the Study of Liver Diseases Research Workshop held at the 52nd annual meeting in Boston, Massachusetts, on November 2, 2002. cGMP, guanosine 3′,5′-cyclic monophosphate; NOS, nitric oxide synthase; MPO, myeloperoxidase; NADPH, nicotinamide adenine dinucleotide phosphate; iNOS, inducible nitric oxide synthase; eNOS, endothelial nitric oxide synthase; I/R, ischemia-reperfusion; SOS, sinusoidal obstruction syndrome; SEC, sinusoidal endothelial cell. The biologic actions of NO are mediated through distinct signaling pathways, including activation of soluble guanylate cyclase, interactions with superoxide anion, and cysteine thiol S-nitrosylation, with the relative role of each of these signaling pathways influenced by a number of factors including the quantity of NO, cell type, and surrounding redox state. Of these, the most well characterized is the NO/guanosine 3′,5′-cyclic monophosphate (cGMP) signaling pathway in which NO diffuses from the cell of origin to adjacent cells and binds soluble guanylate cyclase at the heme site. NO binding activates guanylate cyclase–mediated conversion of guanosine triphosphate to cGMP, with cGMP accumulation antagonized by cellular phosphodiesterases that convert cGMP to guanosine 3′,5′-monophosphate.1 cGMP accumulation initiates a number of phosphorylation steps of downstream substrates, mediated through protein kinase G, which include Hsp20 and myosin light chain kinase.2 The sum effect of protein kinase G–mediated phosphorylation in smooth muscle cells is to reduce intracellular calcium levels, thereby modulating a number of cell physiologic functions including cellular relaxation. Other prominent targets of cGMP include the cyclic nucleotide-gated channels and cyclic nucleotide-dependent phosphodiesterases; thus, some of the downstream actions of NO may be mediated through these intermediates as well, depending on the cell type and the specific cell physiologic end point. The NO/cGMP signaling pathway, although in some ways well characterized, remains enigmatic in many respects. These include the precise mechanism by which NO activates the enzyme, the spatial and temporal localization of the guanylate cyclase enzyme with the NO-generating NO synthase (NOS) isoform, and the relative importance and redundancy of the various downstream phosphorylation steps. Indeed, some evidence exists that NO may directly activate channels in smooth muscle rather than signal to channels indirectly via protein kinase G; furthermore, overlapping substrate specificity exists between protein kinase G–dependent signaling and other smooth muscle cell signaling pathways, including the adenosine 3′,5′-cyclic monophosphate–dependent protein kinase and prostaglandin signaling.2 In addition to guanylate cyclase, a number of new NO chemistry targets have also been identified. An elegant example of such a novel aspect of downstream NO signaling (in the vasculature) was provided by Jason P. Eiserich (University of California at Davis, Davis, CA; "Oxidant-Dependent Mechanisms for Modulating Nitric Oxide Signaling"). Eiserich et al. have shown that myeloperoxidase (MPO), a highly expressed hemoprotein in neutrophils, may be important in endothelium-derived NO catabolism and signaling. During neutrophil activation, MPO is released into the extracellular space, where it uses hydrogen peroxide derived from the respiratory burst of NADPH oxidase from neutrophils to catalyze formation of the microbicidal agent hypochlorous acid (HOCl) and other oxidizing species generation.3, 4 Recent studies using rodent models of acute endotoxemia in MPO-deficient mice have shown that MPO, independent of its ability to produce HOCl, can modulate the vascular signaling and vasodilatory functions of NO during acute inflammation by serving as an NO oxidase.5 In these studies, MPO-deficient mice were resistant to endotoxin-induced vascular dysfunction. In wild-type mice, this altered vascular responsiveness was found to be due to catalytic consumption of NO by substrate radicals such as tyrosyl radicals generated by MPO.5 Further, MPO can transform nitrite (NO), a metabolite of NO, into more potent nitrating agents, and this is believed to play a major role in tyrosine nitration in vivo.5, 6 NO itself also reacts with superoxide anion at a near diffusion-limited rate to produce the highly oxidizing and nitrating species peroxynitrite, which may also contribute to tyrosine nitration in vivo.3 Another redox-based chemical reaction relates to NO-mediated nitrosylation of protein thiols residing on specific cysteine residues with flanking polar residues. More than 100 proteins have been identified that may be targets of NO-dependent nitrosylation.7 Protein channels seem to be a particularly prominent S-nitrosylation substrates. Other specific nitrosylation targets include albumin, hemoglobin, and a number of signaling proteins.7 Although the relative biological importance of this more recently described redox-based posttranslational modification on all of these protein targets remains to be fully validated, it is believed that nitrosylation generally inhibits the function or activity of the target protein. Spatial and temporal NO signaling is greatly influenced by the rate and kinetics of NO metabolism. For example, the mechanism by which NO reaches the guanylate cyclase and other targets that resides in neighboring cells, despite the presence of hemoglobin and myoglobin, remains unclear. NO kinetics are highly influenced by blood because oxyhemoglobin scavenges much of the generated NO into nitrate. The ability of NO to signal to platelets, smooth muscle, and leukocytes in vivo, despite the presence of blood, is probably due to several factors, including the ability to generate NO at spatially appropriate sites adequately distant from erythrocytes and the ability to compartmentalize hemoglobin within the erythrocyte. These concepts are supported by observations showing that cell-free hemoglobin scavenges NO in a more efficient manner than hemoglobin contained within erythrocytes.8 Another concept that may allow NO to function in the presence of hemoglobin relates to hemoglobin-mediated delivery of NO. In this scenario, NO is believed to travel to targets downstream from its generation by binding to hemoglobin in the form of S-nitroso-hemoglobin.9 NO is subsequently released from hemoglobin at downstream sites, owing to hypoxia-mediated conformational shifts in hemoglobin. The relative influence of alternative mechanisms of NO bioactivity in the presence of erythrocytes, serum, and hemoglobin remains an area of investigation. NO is generated by NOS family members (Fig. 1), which are bidomain enzymes that use a series of electron transfer steps to generate NO and citrulline via the reaction intermediate, N-hydroxy L-arginine. The mechanism of NO synthesis is not completely understood but appears to involve the transfer of electrons between enzyme cofactors, including flavin adenine dinucleotide, flavin mononucleotide, nicotinamide adenine dinucleotide phosphate (NADPH), tetrahydrobiopterin, and heme. The NOS enzymes are P450-like heme proteins consisting of a reductase domain in the carboxy terminus, where NADPH binds and donates electrons that are transferred through the bound flavins FMN and FAD, to the heme group in the amino terminal oxygenase domain.10 These steps are potentiated by the calcium regulatory protein calmodulin. The oxygenase domain subsequently catalyzes a reaction that transfers the nitrogen from arginine to activated oxygen, thereby generating NO in a reaction that is modulated by tetrahydrobiopterin.10 Subdomains of NOS proteins. The NOS enzymes consist of a carboxy terminal reductase domain (white boxes), which contains consensus binding motifs for NADPH, FAD, FMN, and calmodulin (CAM), and an amino terminal oxygenase domain (gray boxes), which contains the enzymatic active site that has motifs for binding of L-arginine, tetrahydrobiopterin, and heme site as well as zinc (not shown). NADPH-derived electrons are carried from the reductase domain modules to the oxygenase domain, with interdomain and intradomain electron transfer potentiated by the binding of CAM at its consensus site. At the oxygenase domain, oxygen is activated and reduced in a tetrahydrobiopterin (BH4)-dependent manner, allowing for conversion of L-arginine to NO and L-citrulline. Tetrahydrobiopterin stabilizes this process through several potential mechanisms. The 3 isoforms are relatively well conserved except for 2 sites: (1) on the amino terminus of the oxygenase domain (white star), at which eNOS contains acylation sequences and neuronal NOS contains a protein interaction domain, and (2) carboxy terminal to the CAM binding domain (black star), at which eNOS and neuronal NOS contain a loop sequence that appears to negatively regulate CAM binding and may account for the lower affinity of CAM binding to these 2 isoforms. Interestingly, as discussed by Dr. Dennis Stuehr (Lerner Research Institute, Cleveland, OH; "Biochemistry of Nitric Oxide Synthesis"), the NOS heme moiety can bind NO itself in a manner that is regulated by cofactors such as tetrahydrobiopterin and arginine.11 Binding of NO to the heme iron in NOS is relevant because this serves to reversibly inhibit NOS catalysis. This concept has now been characterized in all three NOS isoforms, in which the heme/NO complex formation is associated with an inhibition of electron flux and catalytic activity, as well as inhibition of dimerization.11-13 A kinetic simulation model has been developed by Dr. Stuehr and colleagues that allows for prediction of catalysis and regulation of the different NOS isoforms.12, 13 This model has also been used to predict how the rate of heme reduction modulates neuronal NOS catalysis and has been useful in dissecting the previously mentioned influence of the NO/heme complex on neuronal NOS catalysis.12 An extension of this modeling to other NOS isoforms has allowed for an analysis of the divergent catalytic rates of inducible NOS (iNOS) and endothelial NOS (eNOS).13 Three parameters were identified to account for these marked differences in catalytic behavior of the isoforms: rate of ferric heme reduction, ferric heme dissociation from NO, and ferrous heme/NO oxidation.13 It is anticipated that such kinetic modeling should allow for elucidation of mechanisms and kinetic control parameters of the NOS isoforms from both an enzymologic and physiologic perspective. Molecular regulation of the NOS isoforms is complex.14, 15 Transcriptional regulation occurs for each; additionally, the "constitutive" NOSs are regulated through posttranscriptional and posttranslational mechanisms.14, 15 Regulation of the eNOS isoform appears to be the most complex; eNOS-derived NO is synthesized and released from the vascular endothelium in response to diverse humoral, neural, and mechanical stimuli16 as a result of a complex series of covalent modifications and protein-protein associations16 (Fig. 2). Indeed, this protein has multiple protein interactions, many of which have functional effects on the enzyme. Calmodulin is perhaps the best characterized and promotes electron transfer within the enzyme.17 Caveolin and Hsp90 are additional well-characterized eNOS-associated proteins. The former acts as an inhibitory clamp on eNOS activation; in contradistinction, the latter promotes NOS activation.18-20 Recently, additional putative NOS regulatory proteins have been identified, including the BK2 receptor, NOSIP, and NOSTRIN.15 Recent work, discussed by Dr. Vijay Shah (Mayo Clinic, Rochester, MN; "Regulation of NOS: Overview and Influence of Protein Interactions"), has highlighted a further eNOS-interacting protein, the large guanosine triphosphatase dynamin 2.21, 22 These studies have dissected distinct actions of dynamin on eNOS function via direct protein interactions as well as through modulation of eNOS targeting within cells and, in sum, indicate that dynamin acts as a positive regulator of eNOS function.21, 22 NOS processing and NO signaling. Although NOS isoforms each maintain distinct regulatory mechanisms, globally the enzyme is regulated by extracellular stimuli that signal through receptor-mediated mechanisms. Regulation occurs at the transcriptional level (green dashed arrow), and this is particularly prominent for the iNOS isoform. Regulation also occurs at the posttranslational level (blue dashed arrow), and this mechanism is particularly prominent for the constitutive isoforms (eNOS and nNOS). A third mechanism of regulation occurs at the trafficking and targeting level (red dashed arrows); in this regard, NOS isoforms maintain distinct subcellular locations depending on the specific isoform and cell type of expression (an endothelial cell is shown), including within the Golgi, cytosol, mitochondria, and plasma membrane caveolae. The eNOS isoform, depicted in the figure, is most prominent within golgi and in plasmalemmal caveolae vesicles. Each of the NOS isoenzymes generates NO and L-citrulline from L-arginine, and NO rapidly diffuses abluminally to effector cells (a contractile cell is depicted in pink). Within effector cells, the most prominent signaling pathway involves NO binding and activation of soluble guanylate cyclase (SGC) (red arrow and black lettering). SGC converts guanosine triphosphate (GTP) to cGMP, which promotes diverse physiologic responses through a number of pathways, including protein kinase G (PKG), cGMP-dependent phosphodiesterase (PDE), and cyclic nucleotide-gated channels (CNG). Alternative cGMP-independent NO chemical reactions may occur as well, including direct nitrosylation of cysteine thiol residues of target proteins (green arrows and lettering), or interaction of NO with superoxide anion with generation of peroxynitrite (blue arrows and lettering). Peroxynitrite is capable of nitrating protein tyrosine residues via intermediate nitrogenous radicals. Nitrosylation and nitration reactions influence target protein functions, frequently in an inhibitory manner. eNOS phosphorylation and subcellular targeting are also key determinants in NO generation by this isoform, as highlighted by Thomas Michel and colleagues (Harvard Medical School, Boston, MA; "Endothelial Cell Derived Nitric Oxide"). In cardiac myocytes and in endothelial cells, eNOS targets to specialized lipid-rich signal-transducing domains in the plasma membrane termed caveolae. Michel et al. emphasized that the dynamic targeting of eNOS to plasmalemmal caveolae provides a novel mechanism for enzyme regulation in endothelial cells because the eNOS translocates within the cell in response to receptor activation, including the actions of specific platelet-derived lipid mediators. He provided extensive evidence that one such lipid mediator is sphingosine 1-phosphate.23, 24 Sphingosine 1-phosphate, which binds to the G protein–coupled EDG-1 receptor in endothelial cells, stimulates eNOS activation to a magnitude nearly as great as that elicited by classic eNOS agonists such as bradykinin or vascular endothelial growth factor. Additionally, exposure of cells to vascular endothelial growth factor leads to rapid up-regulation of membrane EDG-1 receptor. Importantly, diverse eNOS agonists appear to signal to eNOS activation via distinct kinase signaling cascades. For example, sphingosine 1-phosphate activates the β isoform of phosphoinositide 3-kinase whereas vascular endothelial growth factor activates both the α and β phosphoinositide 3-kinase, with both agonists culminating in protein kinase B (AKT)-dependent eNOS phosphorylation. Conversely, bradykinin fails to activate either phosphoinositide 3-kinase isoform, indicating a distinct signaling pathway from the aforementioned agonists. Thus, different eNOS agonists converge on eNOS only after activating distinct upstream signaling pathways. Subsequent eNOS phosphorylation occurs at multiple residues, including serine 1179, serine 635, serine 617, serine 116, and threonine 497, with divergent consequences for control of enzyme activity and subcellular targeting.25, 26 For example, phosphorylation of eNOS at serine residue 116 inhibits eNOS activity (and vascular endothelial growth factor causes eNOS activation and a corresponding decrease in phosphorylation at this residue). The immunosuppressant, cyclosporin A, which inhibits the activity of the phosphatase calcineurin, blocks vascular endothelial growth factor–induced eNOS dephosphorylation at serine 116 and thereby reduces eNOS activity.25, 26 Michel et al. postulated that cyclosporin-induced hypertension may be at least in part due to differential serine 116 phosphorylation. Thus, the data emphasize that serine phosphorylation is not associated with eNOS activation under all circumstances and that phosphorylation may actually inhibit eNOS function rather than activate eNOS. In contrast to the "constitutive" NOS isoforms, eNOS and neuronal NOS, iNOS is governed largely at the transcriptional level.27 iNOS is generated by all liver cell types given the appropriate inflammatory stimuli; in contradistinction to eNOS, iNOS binds calmodulin avidly, making changes in intracellular calcium flux less relevant to its regulation and allowing for high levels of "basal" NO production via this isoform.14 A substantial body of literature has examined the role of NO in hepatotoxicity (Table 1). Of the 3 NOS isoforms, it is well known that stress and inflammation, including in the liver, induce iNOS.27 The consequences of high levels of NO production (i.e., that generated by iNOS) on hepatocytes depend on a number of factors; particularly important are the quantity and duration of NO exposure. It has often been viewed that low levels of NO are cytoprotective, whereas higher levels lead to cytotoxicity. Cytotoxicity of NO may also be related to the genesis of oxidative intermediates such as peroxynitrite, a product of NO and superoxide. The mechanisms by which NO can lead to cellular toxicity are multiple and include the following: (1) direct interaction of NO with mitochondrial targets (NO can inactivate cytochrome c oxidase, leading to mitochondrial dysfunction), (2) NO can result in DNA damage, resulting in cell cycle arrest and possibly apoptosis, and (3) NO may activate c-Jun N-terminal kinase, resulting in cell death. Mechanisms by which NO inhibits apoptosis in hepatocytes are multiple (it should be emphasized that NO may stimulate apoptosis in some systems).28-31 This topic was discussed by Timothy Billiar (University of Pittsburgh, Pittsburgh, PA; "Nitric Oxide, the Liver and Apoptosis"), who emphasized that NO protects the liver from injury. He showed that NO inhibits caspase activation directly through apparent redox-based modifications of cysteine residues in caspase active sites, a process known as S-nitrosylation that is particularly efficient in hepatocytes and endothelial cells.32 NO can also activate guanylyl cyclase, which leads to the synthesis of cGMP, which in turn has antiapoptotic activity in hepatocytes. Billiar et al. have shown that NO also protects hepatocytes against apoptosis by way of induction of stress proteins such as Hsp70.29 They have shown that NO also up-regulates heme oxygenase-1 in hepatocytes33 and moreover that carbon monoxide, the breakdown product of bilirubin produced by heme oxygenase-1, is antiapoptotic in hepatocytes, perhaps though positive feedback regulation of iNOS by carbon monoxide. Thus, multiple complementary mechanisms account for the cytoprotective effects of NO in hepatocytes. The liver has remarkable regenerative capacity, regenerating normally after even 70% partial hepatectomy. The pathobiology of regeneration is complex, involving interaction of proinflammatory and anti-inflammatory cytokines.34 Indeed, injury signals themselves appear to trigger hepatocyte regeneration; for example, both tumor necrosis factor α and tumor necrosis factor–induced cytokines, such as interleukin 6, are required to stimulate hepatocyte DNA synthesis. Because proinflammatory cytokines are known to induce iNOS, Dr. Anna Mae Diehl (The Johns Hopkins University School of Medicine, Baltimore, MD; "Nitric Oxide and Liver Regeneration") hypothesized that induction of iNOS might be cytoprotective during liver regeneration. To evaluate this hypothesis, Diehl et al. have examined iNOS-deficient mice subjected to partial hepatectomy.35 Although iNOS-deficient mice do not have an obvious hepatic phenotype at baseline, their mortality was significantly greater than controls after partial hepatectomy. Following partial hepatectomy, iNOS-deficient mice were jaundiced, had elevated alanine aminotransferase levels, and exhibited histologic evidence of liver injury and exaggerated hepatocyte apoptosis, all significantly greater in surviving iNOS-deficient mice than control mice. Additionally, hepatocyte DNA synthesis and mitoses were significantly reduced in conjunction with an increase in the activity of caspase 3 (a terminal apoptosis effector enzyme) in iNOS-deficient mice after partial hepatectomy compared with controls. These data suggest a protective role for NO after partial hepatectomy, perhaps through an NO-mediated inhibition of hepatocyte apoptosis, and are consistent with Dr. Billiar's work showing that NO has antiapoptotic properties in hepatocytes. NO appears to play a multitude of roles in ethanol-mediated liver disease. For example, when infused into the liver, ethanol leads to increased portal pressure via effects on the intrahepatic circulation, which are mediated at least in part by NO.36, 37 Further, inhibition of NO synthesis exacerbated, and arginine supplementation inhibited, ethanol-induced liver injury in animal models,38 although it should be emphasized that controversy exists surrounding the role of NO in the genesis of alcoholic liver disease.39 Dr. Gavin Arteel (University of Louisville, Louisville, KY; "Ethanol-Induced Injury and Nitric Oxide") proposed that NO generated by iNOS could play an injurious role in the initiation and progression of ethanol-induced liver disease, specifically via genesis of reactive nitrogen species such as nitrotyrosine or peroxynitrite (the latter is produced when NO and O [superoxide] combine via a diffusion-limited reaction to form peroxynitrite, which then may be protonated to yield highly reactive ROS/RNS). Nitrotyrosine has been shown to be formed after ethanol exposure in experimental models40; Arteel et al. examined the role of iNOS and NO in iNOS-deficient mice. Enteral ethanol, fed continuously for 4 weeks, resulted in increased liver/body weight ratios and serum alanine aminotransferase levels in wild-type mice; however, these effects were significantly abrogated in iNOS-deficient mice. Enteral ethanol also resulted in accumulation of fat in the liver, mild inflammation, and necrosis in wild-type mice; similar liver lesions were not present in iNOS knockout mice. Additionally, free radical products, including 4-hydroxynonenal and 3-nitrotyrosine, were not found in iNOS knockout mice. Mice lacking NADPH oxidase (p47phox deficient mice) were also protected from ethanol-induced liver injury41 and raised the possibility that free radical oxidant production in ethanol-induced liver disease depends on the production of both O and NO, further raising a potential role in alcoholic liver injury. Importantly, the data contrast with those in other models of liver injury (such as previously described) in which NO appears to be protective and therefore emphasize that the type of injurious stimulus as well as the quantity and duration of NO exposure also play a critical role in subsequent liver injury. A key function of NO in the liver relates to the regulation of sinusoidal blood flow and vascular tone. Dr. Mark Clemens (University of North Carolina at Charlotte, Charlotte, NC; "Nitric Oxide Mediated Regulation of Sinusoidal Blood Flow") and colleagues have reported on a series of elegant in vivo microscopic aspects of the hepatic microcirculation, emphasizing the perisinusoidal anatomic position of hepatic stellate cells and showing their perisinusoidal constrictive functional properties, which are believed to be important in regulation of sinusoidal blood flow.42-44 Dr. Clemens' studies emphasize the perisinusoidal anatomic position of hepatic stellate cells and show their perisinusoidal constrictive functional properties to be important in NO-mediated regulation of sinusoidal blood flow as well as alterations in this process in response to endotoxemia.45 Sinusoidal blood flow becomes altered when the balance of vasoconstrictors and NO becomes disrupted with inadequate NO production, resulting in unopposed contraction of hepatic stellate cells and subsequent sinusoidal constriction. This phenomenon may contribute to a number of hepatic vascular diseases and syndromes, including ischemia-reperfusion (I/R) injury, portal hypertension, and sinusoidal obstruction syndrome (SOS), also known as veno-occlusive disease. Dr. Laurie DeLeve (University of Southern California, Los Angeles, CA; "The Role of Nitric Oxide in Hepatic Veno-occlusive Disease") and colleagues have focused on the role of NO in the latter of these syndromes, SOS.46 SOS typically occurs after exposure to pyrrolizidine alkaloids or after high-dose chemotherapy, particularly after stem cell transplantation. Sinusoidal endothelial cells (SECs) become injured early in SOS47 and subsequently slough and obstruct the sinusoids, ultimately leading to parenchymal necrosis. In the monocrotaline rat model, progressive injury to SECs occurs within the first 72 hours following administration of toxin. In this model, hepatic NO generation was reduced within 24 hours of monocrotaline administration and remained low through early and late SOS. This finding is consistent with injury to SECs and reduced eNOS function. Inhibition of NO synthesis with NG-nitro-L-arginine methyl ester exacerbated SOS, particularly at lower doses of monocrotaline. Additionally, the NO donor V-PYRRO/NO reversed SEC injury and sinusoidal perfusion and prevented the histologic pattern typical of SOS.48 The protective mechanism of V-PYRRO/NO in SOS appears to be by the prevention of monocrotaline-induced up-regulation of matrix metalloproteinase 9, an enzyme regulated by NO in rat mesangial cells,49 which in turn may prevent SEC disruption. These data are consistent with the postulate that NO deficiency contributes to experimental SOS and further that NO supplementation may be a rational therapeutic option. Another vascular syndrome pertinent to NO biology in the liver is that of I/R injury. I/R is a requisite consequence of most types of hepatic resection as well as liver transplantation. Further, there is an emerging body of clinical and experimental data suggesting that sex may influence the outcome following such types of procedures.50 Dr. Matthew Grisham (LSU Health Sciences Center, Shreveport, LA; "Ischemia-Reperfusion Injury and Nitric Oxide") and colleagues have shown that there may be a link between eNOS-derived NO and sex in I/R injury.51-53 It has been previously reported that estradiol stimulates eNOS transcription and expression.54 Therefore, Grisham et al. postulated that estradiol-induced eNOS may have a protective role in experimental models of reduced-size liver transplantation and I/R injury. In studies performed in male and female wild-type and eNOS-deficient (eNOS−/−) mice, all female mice survived indefinitely after surgery whereas all male mice died within 5 days following reduced-size liver transplantation plus I/R. Additionally, liver injury was more severe in male mice than in female mice following surgery. Administration of estradiol to male mice resulted in significant reductions in serum alanine aminotransferase levels at 20 hours following reduced-size liver transplantation plus I/R and in remarkable improvements in survival, whereas treatment of female mice with a selective estradiol receptor antagonist (ICI 182,780) resulted in enhanced liver injury and reduced survival. These observations suggest a protective role of estrogen. Additionally, eNOS messenger RNA levels were markedly increased in female mice and estradiol-treated males 1 hour after reduced-size liver transplantation plus I/R; the elevation was abrogated in female mice treated with the estradiol receptor antagonist. To further support a role for NO, Grisham et al. treated female mice with NG-nitro-L-arginine methyl ester; this resulted in sensitization to the damaging effects of reduced-size liver transplantation plus I/R compared with control female mice not receiving NG-nitro-L-arginine methyl ester. Similar results were found in eNOS−/− female mice. Thus, Grisham et al. concluded that the protective effect of estrogen in reduced-size liver transplantation plus I/R may be mediated through eNOS-generated NO. A growing body of literature has implicated a role for NO in the development of portal hypertension. In most forms of liver injury, portal hypertension is a result of increased intrahepatic resistance.55 It is commonly believed that a close relationship occurs between the progression of liver fibrosis and development of portal hypertension. The connection remains unclear but may be related to capillarization of liver sinusoids, alteration of native intrahepatic vasculature by scar tissue, and enhanced contractility of hepatic stellate cells (also known as Ito cells or lipocytes) in the injured liver.56, 57 After liver injury, stellate cells become activated and not only synthesize abundant extracellular matrix but also acquire smooth muscle isoforms of actin and myosin and enhanced contractile properties.56, 57 The activation process is also associated with enhanced production of endothelin 1 and endothelin receptors.58 Stellate cell contraction is counterbalanced by vasorelaxing agents such as NO.59 In the injured liver, endothelin-1 production is increased whereas NO release from SECs is reduced, ostensibly owing to impaired SEC eNOS activity.60 Because NO antagonizes the effect of endothelin 1, reduced production of NO favors sinusoidal (and vascular wall) constriction by perisinusoidal stellate cells and hepatic vascular smooth muscle cells, respectively. The mechanism of deficient eNOS-derived NO generation remains an area of investigation and may relate to enhanced interaction of eNOS with caveolin in conjunction with diminished interaction of eNOS with calmodulin.61-65 More recent studies have indicated a defect in the signaling pathways responsible for eNOS phosphorylation activation in cirrhotic endothelial cells as well. Finally, it should be emphasized that while eNOS function is down-regulated within the liver, eNOS is up-regulated in the splanchnic circulation in patients with chronic liver disease (see Wiest and Groszmann37 and Rockey60 for more detailed review). Given the apparent defect of eNOS function in SECs of the cirrhotic liver, Dr. Stephano Fiorucci (University of Perugia, Italy; "Nitric Oxide, Hepatic Fibrosis and Portal Hypertension") and colleagues postulated that a pharmacologic approach to deliver NO directly to the liver should reduce intrahepatic resistance.66 Nitrovasodilators have been the focus of current therapeutic approaches aimed at intrahepatic vasodilation but in patients are hampered by systemic complications. A new compound that selectively releases NO in the liver without affecting systemic arterial pressure,67 (acetyloxy) benzoic acid 3-(nitrooxymethyl) phenyl ester (NCX-1000), was designed by adding an NO-releasing moiety to ursodeoxycholic acid, which is selectively metabolized by hepatocytes. NCX-1000 significantly reduced liver fibrosis and ameliorated portal hypertension in a rat model of fibrogenesis and portal hypertension.67 NCX-1000 also inhibited stellate cell contraction and had a relaxing effect similar to the NO donor S-nitroso-N-acetylpenicillamine. These data emphasize that NCX-1000 (which appears to release NO into the liver microcirculation) may serve as a novel therapy for patients with portal hypertension. Recognition of the importance of NO led to the awarding of the Nobel Prize in Medicine in 1998 to the codiscoverers of NO. Not only has the importance of NO been broadly recognized, but the explosion of work over the past decade focusing on the liver emphasizes the critical role that NO plays in liver biology. NO is particularly critical in the areas of injury and hepatotoxicity as well as in regulation of blood flow. The authors thank the speakers and attendees who participated in this workshop.