Nitric oxide in gastrointestinal health and disease

GASTROENTEROLOGY 2004;126:903–913

SPECIAL REPORTS AND REVIEWS Nitric Oxide in Gastrointestinal Health and Disease VIJAY SHAH,*,‡ GREG LYFORD,§ GREG GORES,‡ and GIANRICO FARRUGIA§ *GI Research Unit, §Enteric Neuroscience Program, ‡Advanced Liver Disease Study Group, Mayo Clinic and Foundation, Rochester, Minnesota

Nitric oxide is an intracellular and intercellular messenger with important functions in a number of physiologic and pathobiologic processes within gastroenterology and hepatology, including gastrointestinal tract motility, mucosal function, inflammatory responses, gastrointestinal malignancy, and blood flow regulation. Since the broad review of this topic in GASTROENTEROLOGY more than 10 years ago, a number of advances have been made in the area of NO biology and its relevance to the gastrointestinal system.1 The aim of this review is to focus on our expanded understanding of the role NO plays in human gastrointestinal and hepatic physiology and disease processes by drawing on data from relevant in vitro and animal models as well as observational human studies.

itric oxide (NO) is a free radical signaling molecule generated by a family of P450-like enzymes, termed “NO synthases” (NOS), which generate this molecule through a series of regulated electron transfer steps. Three independent genes encoding neuronal,2 endothelial,3 and inducible NOS4 (nNOS, eNOS, and iNOS, respectively) have been cloned, each with the capacity to generate NO through both complimentary and distinct regulatory mechanisms. Though probably short-lived in biological systems, the lipophilic nature of NO allows it to diffuse quickly, thereby initiating intercellular and intracellular signals.5 The most characterized downstream NO signaling pathway relates to the soluble guanylate cyclase pathway, through which NO was shown to be identical to the previously postulated endothelial relaxing factor.5 Via this pathway, NO binds to the guanylate cyclase heme moiety thereby stimulating the enzyme to generate cGMP, which in turn activates protein kinase G (PKG), with downstream phosphorylation cascades leading to effector functions (Figure 1).6 However, in pathophysiologic conditions and probably during certain normal physiologic conditions, NOmediated signaling may also be independent of guanylate cyclase activation. For example, NO directly regulates

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the function of ion channels, enzymes, and a number of other proteins.7 This regulation appears to occur in part through the nitrosylation of cysteine thiols in target proteins.8 NO may also contribute to cell damage in inflammatory conditions via nitration reactions.9 It has an unmatched electron and as a free radical can scavenge the more reactive superoxide, thereby generating peroxynitrite, which in turn may be responsible for nitration of protein tyrosine residues, mitochondrial energy depletion, and induction of DNA strand breaks.9 It is important to note that these cyclic guanosine monophosphate (cGMP)-dependent and independent actions, though attributed to NO, may occur in varying degrees through alternative nitrogen species and chemistry pathways and are therefore still an area of active investigation.9 These NO signaling pathways contribute to a number of physiologic functions in the gastrointestinal (GI) system including motility and vascular regulation, as well as the disease processes inherent to these functions. While the influence of NO on GI motility is largely controlled by nNOS, NO effects on vascular function in the GI system occurs largely under the purview of eNOS. Additionally, iNOS derived NO production contributes to cellular inflammation and the carcinogenic process. The stimulatory and inhibitory influence of NO on these broad processes of motility, vascular function, and inflammation are outlined below.

Gastrointestinal Motility Biology of nNOS The most prominent role of nNOS-derived NO in GI motility is as an inhibitory nonadrenergic noncholinergic (NANC)10,11 smooth muscle cell relaxant via Abbreviations used in this paper: COX, cyclooxygenase; GMP, guanosine monophosphate; IRI, ischemia–reperfusion injury; NANC, nonadrenergic noncholinergic; NO, nitric oxide; NOS, nitric oxide synthase; PGK, protein kinase G. © 2004 by the American Gastroenterological Association 0016-5085/04/$30.00 doi:10.1053/j.gastro.2003.11.046

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VIP as upstream as well as downstream of nNOS derived-NO generation, thus the interrelationship of NO and VIP remains under investigation.20,21 The diatomic gas and alternative guanylate cyclase binding partner, carbon monoxide, is proposed as a cotransmitter of nNOS-derived NO,17 and crosstalk between the NO and carbon monoxide signaling pathways occurs at multiple levels including transcriptional and posttranslational.22,23 The role of nNOS-derived NO in esophageal, gastric, and intestinal motility and motor disorders is described below. Esophageal Motility and Motor Disorders Figure 1. Nitric oxide signaling and targets for potential therapeutic intervention. The free radical gas, NO, is generated by the NOS isoforms, which convert L-arginine to L-citrulline and NO through a series of enzymatic steps that involve electron transfer and hydroxylation. The primary target of NO is soluble guanylate cyclase. NO binds and activates guanylate cyclase, thereby enhancing the conversion of GTP to cGMP. cGMP acts on its kinase, protein kinase G, thereby enabling a number of downstream physiologic actions including vasodilation, NANC, and inhibition of platelet aggregation. NO interactions with superoxide anion (O2⫺), result in the generation of the oxidative molecule, peroxynitrite (ONOO⫺). NO interactions with protein thiols also appear to regulate a number of target enzymes and proteins (not shown). Sites of potential therapeutic interventions by which to activate or inhibit NO signaling pathways are shown. Potentiation of NO signaling (depicted in squares) may be achieved by providing excess arginine substrate, enhancing signaling pathways that activate NOS function, NO donors, antioxidants, or phosphodiesterase (PDE) inhibitors. Inhibition of NO signaling (depicted in circles) may be achieved by competitive and allosteric inhibitors of NOS activity and inhibitors of guanylate cyclase (GC) function.

nNOS-derived activation of smooth muscle cell guanylate cyclase. Nerves throughout the length of the luminal GI tract express nNOS with highest levels in the pyloric sphincter,12 particularly the soma and processes of myenteric nerves.13 Although initial reports of nNOS were specific to neurons,13 evidence is emerging that nNOS is widely distributed. Six splice variants of nNOS have been described in the luminal GI tract14; the functional significance and differential distribution of these splice variants within the GI tract remains to be determined. Other transmitter molecules such as vasoactive intestinal peptide (VIP),15 adenosine triphosphate,16 and carbon monoxide17 also function in conjunction with NO as NANC inhibitory signals. For example, VIP colocalizes with NOS and appears to have similar inhibitory functions on smooth muscle in the gut. However, VIP possesses relaxing effects in the gastric fundus that is retained in nNOS⫺/⫺, eNOS⫺/⫺, and iNOS⫺/⫺ mice as well as cGMP kinase 1⫺/⫺ mice, suggesting parallel and redundant pathways.18,19 Other studies have targeted

Data from human studies indicate that NO inhibits esophageal smooth muscle function. In normal human subjects, systemic administration of a recombinant human hemoglobin that scavenges NO promotes increased velocity of peristaltic contractions, simultaneous contractions, spontaneous contractions associated with chest pain, and diminished lower esophageal sphincter (LES) relaxation.24 Pharmacologic blockade of NO synthesis reduces the latency between swallows, contraction in the distal esophagus, increases basal LES pressure, increases peristaltic wave pressure, and decreases the number of transient LES relaxations.25 These and other studies suggest that deficient NO may cause esophageal dysmotility. A role for NO in achalasia is suggested by the absence of NOS immunoreactivity and enzymatic activity in LES neurons of patients with achalasia.26 A causative role for deficient NO in achalasia is supported by the phenotype of nNOS⫺/⫺ mice, which exhibit elevated baseline LES pressures and reduced swallow-induced relaxation of the LES27 in contradistinction to eNOS⫺/⫺ mice, which display relaxation responses similar to control animals.28 Therapy for achalasia using NO donors in humans, however, provides only minimal success,29 perhaps because of the inability to provide selective and adequate NO delivery to the target site. Potentiation of the NO– cGMP pathway in human esophageal motor disease has also been attempted using sildenafil, the phosphodiesterase type-5 inhibitor, which decreases swallow-induced contractions in the distal esophagus.30 Although patients with achalasia reported no benefit, chronic administration was associated with symptomatic improvement in some individuals with nutcracker esophagus, esophageal spasm, and hypertensive LES.30 Thus, although NO is critical for NANC-inhibitory signaling and sphincter relaxation in esophagus, more studies will be required to establish the efficacy of NO– cGMP pathway activation as a therapeutic target for achalasia.

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Gastric Motility and Motor Disorders NO promotes gastric accommodation and emptying. In humans, pharmacologic inhibitors of NOS increase frequency of gastric contractions, decrease gastric emptying time, and decrease fundic volume both basally and after meals,31 while NO donors slow gastric emptying and improve accommodation of the proximal stomach.32 NO has also been shown to promote fundic accommodation in animal studies.33 These studies have resulted in preliminary studies on nitrate therapy for functional dyspepsia,34,35 a condition that evidences defective fundic accommodation in a subset of patients. Data to date suggest that NO donors are able to effectively relax the stomach, but the efficacy in functional dyspepsia remains uncertain because symptoms in this condition may not be solely linked to increased tone or inadequate relaxation. A therapeutic role for NO supplementation has also been suggested in diabetic gastroenteropathy. In murine models of diabetes, NANC relaxation and NOS activity are decreased in gastric muscle preparations.36 Interestingly, insulin administration restores NOS expression and improves gastric emptying, while the phosphodiesterase inhibitor sildenafil corrects gastric motor abnormalities in these models, the latter perhaps by decreasing pyloric spasm.36 In diabetic human studies, nitrates improve postprandial gastric accommodation although postprandial symptoms are not improved.37 Hypertrophic pyloric stenosis is also associated with a decrease in nNOS expression within the hypertrophied circular smooth muscle sphincter, while NOS protein remains in the adjacent longitudinal muscle layer, suggesting a specific defect in the sphincter. The neuronal defect in circular smooth muscle may not be limited to nNOS, but may also involve widespread disruption of neuronal elements38 and interstitial cells of Cajal (ICC) networks.39 As a corollary, nNOS⫺/⫺ and PKG⫺/⫺ mice develop marked gastric dilation and a thickened pylorus at a young age,40,41 although these animals do not entirely recapitulate the phenotype of infantile hypertrophic pyloric stenosis. These studies argue for the importance of the NO– cGMP pathway in pyloric sphincter relaxation and suggest that NO donors and phosphodiesterase inhibitors may be an attractive area for future studies for a variety of gastroparetic syndromes in humans. Intestinal Motility and Motor Disorders Studies from animal models have shown a role for NO as an inhibitor of small bowel motility. In humans, NOS blockade results in an increase in fasting motor

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activity.42 As a corollary, postoperative ileus may be a result of increased intestinal NO production because urinary nitrate levels are raised postoperatively,43 perhaps in response to surgical manipulation of the intestine. Indeed, iNOS⫺/⫺ mice exhibit less phagocyte infiltration than controls, and are protected from the reduction in contractility that occurs in response to surgical manipulation.44 Furthermore, NOS inhibitors, when used in combination with adrenergic agents, normalize intestinal transit time in animal models of the postoperative gut.45 However, the link between NO generation and postoperative ileus in humans is not as well established at this juncture.43 Deficient NO generation has also been linked to other small and large intestinal transit disorders including diabetic enteropathy and slow-transit constipation. For example, loss of NOS expression has been described in a patient with long-standing diabetes, together with loss of immunoreactivity for VIP and a decrease in the volume of ICC.46 Based on these and other studies, the role of NO– cGMP supplementation in the treatment of diabetic GI symptoms is currently under investigation. Although the role of NO in colonic motility in humans is not well studied, some studies show reduced NOS expression in slow-transit constipation. However, the selective contribution of NO deficiency to the pathogenic basis of these disorders remains unestablished as other cellular defects are present as well.47– 49 Immunohistochemical12 and electrophysiologic50 evidence, together with the use of topical NO donors show a clear role for NO in relaxation of the anal sphincter. Topical application of glyceryl trinitrate produces a decrement in the resting pressure of the anal sphincter.51 Topical NO donors are also effective in treating chronic anal fissure because they provide relief from fissurerelated pain and promote healing of the fissure itself.52,53 This effect may come through 2 potential mechanisms, decreased anal sphincter tone and increased regional blood flow through vasodilation. Based on these studies, NO donors may also be useful in the treatment of anismus, which is characterized by elevated sphincter tone and disordered defecation.

Vascular Function in the Gastroesophageal System Biology of eNOS eNOS is the key NOS isoform responsible for NO-regulated vasodilation in the GI system. Thus it follows that in the GI system, eNOS expression is most prominent in endothelial cells lining vascular channels,

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throughout the gut, liver, and pancreas, including arterial, venous, and microcirculatory vessels.54 eNOS may also be expressed in gut smooth muscle cells and ICC.55,56 eNOS-derived NO is produced under basal conditions and in response to a variety of stimuli. For example, a number of hormonal, paracrine, and mechanical factors stimulate increases in eNOS-derived NO production from existing eNOS protein, such as shear stress, insulin, bradykinin, and others.57 Integral to this process is the calcium-dependent activation of calmodulin which, in conjunction with additional molecular steps including phosphorylation, regulatory protein interactions, and aminoterminal fatty acid modifications, act in concert to finely tune NO production.58 Furthermore, although “constitutive,” a number of transcriptional and posttranscriptional mechanisms are also important to the sum generation of NO via eNOS.58 eNOS regulates a number of cellular and physiologic functions within the GI and hepatic systems including vasodilation, inhibition of leukocyte, platelet and mast cell adhesion, and protection of mucosal barrier function. Based on these physiologic functions, regional changes in eNOS-derived NO generation may contribute to the pathogenesis of a number of digestive diseases including portal hypertension, ischemia-reperfusion, and gastric mucosal injury, as described below. Gastrointestinal Blood Flow Regulation and Portal Hypertension NO plays a key role in vascular perfusion and regulation by promoting vasodilation via signaling to smooth muscle cell cGMP and PKG.6 The majority of blood flow to the GI system enters from the mesenteric vasculature, and flow regulation at the level of the mesenteric arterioles is a key determinant of total and regional intestinal blood flow.59,60 Additionally, much of the mesenteric inflow is directed to the GI mucosa to facilitate absorption and secretion. The role of NO in these processes is evidenced by the demonstration that inhibitors of NOS decrease splanchnic blood flow by inhibiting NOS at the mesenteric arteriolar level.60 In addition to increasing splanchnic inflow, eNOSderived NO generated from liver endothelial cells increases flow within the sinusoidal channels of the liver itself.57,61,62 This concept is supported by studies showing the expression and regulation of eNOS within hepatic vascular channels57,63 increase in hepatic pressure in response to NOS inhibitors61 and inhibitory influence of NOS isoform overexpression on hepatic vasoconstrictive responses.64,65 NO has also been implicated prominently in hepatic microcirculatory and splanchnic vascular dysfunction,

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Figure 2. The role of NO and NOS isoforms in pathogenesis and complications of portal hypertension. (Left) The vascular contributions to the pathogenesis of portal hypertension include an increase in intrahepatic resistance and an increase in splanchnic inflow. While the former is mediated through a deficiency in eNOS-derived NO generation in the liver, the latter is mediated by an increase in NO generation in the systemic circulation, particularly at the mesenteric arteriole. Most studies implicate eNOS as the relevant NOS isoform in this process, although iNOS has been implicated as well. (Right) Complications of portal hypertension in which NO has been implicated include hepatopulmonary syndrome, hepatic encephalopathy, hepatorenal syndrome, and cirrhotic cardiomyopathy. Each of these complications has been associated with excess NO generation. However, the relevant isoform responsible for excess NO generation in these complications varies, and the therapeutic benefit of NOS inhibition is not yet established.

particularly portal hypertension (Figure 2).62,63,66,67 Although mechanical factors contribute to much of the increased resistance within the liver in portal hypertension, there is clearly a vasculogenic component to the development and perpetuation of this syndrome as well. The vascular component of portal hypertension includes an increase in splanchnic blood flow, as well as an increase in intrahepatic vascular resistance.68 Dysregulation of the NO system appears to play a key role in both these processes. For example, increased NO generation mediates the increased splanchnic blood flow and vasodilation most likely through increased activation of the eNOS isoform,69 –72 although both iNOS and nNOS isoforms have been implicated in this process as well.73,74 The mechanism of activation of eNOS in this process seems to involve both mechanical and humoral factors that signal NOS activation by pathways involving increased intracellular calcium, AKT, molecular chaperones, and others.71,75–77 However, eNOS and NO may not be indispensable in this process as some, though not all, studies in eNOS⫺/⫺ mice have shown that the hyperdynamic circulation and portal hypertension develop even in the absence of eNOS.78,79 In contradistinction to the activation of eNOS in the mesenteric endothelial cells, eNOS function is impaired in the hepatic endothelial cells, thereby contributing to increased intrahepatic resistance and portal hypertension (Figure 2).63,67,80 Additionally, contractile cells in the liver demonstrate impaired ability to dilate in response to NO.81 The signaling events and primary stimuli that

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cause impaired NOS activation in the hepatic sinusoids may involve the inhibitory NOS protein, caveolin,67,80 and/or impaired AKT activation of eNOS.82 Therapeutic approaches aimed at manipulating the NO pathway in portal hypertension and its components have been an area of investigation in both experimental systems and in humans and have met with varying success. Inhibition of NOS signaling pathways in the mesenteric vasculature has been attempted, with the goal of attenuating the hyperdynamic circulatory state. However, initial studies aimed at systemic NOS inhibition to limit portal hypertension and its other complications in humans have not been promising,83 except perhaps in the hepatopulmonary syndrome, in which case an inhibitor of guanylate cyclase, methylene blue, has been shown to reduce the excess pulmonary vasodilation that characterizes this condition.84 Attempts to pharmacologically augment the NOS system in liver to correct hepatic vasoconstriction have been largely impaired by the inability to selectively deliver NO donor agents to the desired site of action.85 However, some potential directions for therapy of portal hypertension and its complications through potentiation of NOS function, both pharmacologically or molecularly, have been investigated in experimental models or in preliminary form in humans.64,65,82,86 – 88

Mesenteric Ischemia–Reperfusion Injury NO inhibits the adhesion and activation of specific hematopoietic cells, thereby acting as a mitigating force against vascular stasis and ensuing ischemic tissue injury. Cells most prominently involved in this process and inhibited by NO include leukocytes and platelets. In the case of leukocyte function, NO inhibits leukocyte adhesion to the vascular endothelium, in part through inhibiting the ability of leukocytes to roll along the vascular channels, a prerequisite step for adhesion.89 The inhibitory influence of NO on leukocyte– endothelium interactions appears to be mediated through adhesion molecule interactions, particularly involving P-selectin and beta integrins (CD11/CD18).90 Additionally, studies using pharmacologic inhibitors implicate signaling pathways involving phospholipase A2, PAF, and leukotriene B4 in this process.91 The inhibitory actions of NO on platelet activation, adhesion, and aggregation are also well established.92 NO-mediated inhibition of platelet aggregation is mediated in large part by cGMP-PI3 kinase-dependent mechanisms, although other pathways independent of cGMP and involving peroxy-

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nitrite-dependent protein nitration have also been proposed.93,94 The inhibitory influence of NO on leukocyte and platelet function has significant import on mesenteric ischemia–reperfusion injury (IRI) because platelet aggregation and interactions of leukocytes with the vascular endothelium contribute to this syndrome.95,96 Experimental studies show that NO production is reduced in mesenteric IRI, inhibition of NOS function mimics the effects of IRI on the gut, and NO donor compounds protect against IRI, perhaps in part by inhibiting the upregulation of P-selectin.95 Owing to the beneficial effects of NO supplementation on experimental IRI, this condition is another potential target of NO donor compounds. Mucosal Permeability and Injury Although the influence of NO on mucosal permeability and protection of barrier function is controversial, experiments using pharmacologic inhibitors of NOS indicate that inhibition of basal NO production enhances epithelial mucosal permeability, thus implicating constitutive NO production in the protection of GI mucosal barrier function.97 The mechanism of this NO protective effect may be through enhanced mucosal blood flow and a stabilizing influence on mast cell function, although direct effects on epithelial cell function may also be important.97,98 However, excess NO production associated with inflammatory states is also characterized by increased epithelial permeability and a loss of mucosal barrier function. Thus, the level of NO generation, relevant isoform-generating NO, redox status of the epithelial cells, and other specifics relating to the cellular milieu may determine the sum effect of NO on mucosal permeability and protection. The potentially protective influence of NO on mucosal barrier function has been extrapolated to a possible beneficial role of NO donor agents in specific disease processes and toxicities associated with impaired mucosal barrier function, such as ulcer disease, nonsteroidal antiinflammatory drug (NSAID) toxicity, and alcohol. For example, clinical studies have shown that coadministration of NO donor agents with NSAIDs may protect from NSAID-induced ulcers, and combination agents in which the NSAID or aspirin is linked to a NO-releasing moiety may result in less mucosal injury, as compared with the use of traditional cyclooxygenase (COX) inhibitor agents, without impairing their beneficial effects, and may even enhance mucosal tissue repair.99 –101 Thus, a compelling target of NO donor compounds relates to protection of gastric mucosal integrity.

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Inflammation and Carcinogenesis Biology of iNOS iNOS is the NOS isoform most broadly implicated in the processes of inflammation and carcinogenesis in the GI system. iNOS expression can be detected within almost any GI cell type, given the appropriate stimulatory milieu, though most notably in inflammatory cells. Some reports also suggest constitutive expression in the epithelia of the normal human colon102 and myenteric neurons.55 iNOS is regulated predominantly at the transcriptional level through mechanisms dependent on NF-␬B.103 However, recent studies are highlighting the importance of additional transcription factors and response elements in the regulation of iNOS gene transcription as well.104 The enzymatic activity of iNOS is not altered by increased calcium transients, owing to its high affinity for calmodulin, which facilitates binding even in the absence of stimulus dependent increases in intracellular calcium. However, putative posttranslational mechanisms of iNOS regulation have also been described.105 iNOS-derived NO generation occurs at orders of magnitude greater than that of other isoforms. However, it has been postulated that other reactive oxygen species may be contemporaneously generated by iNOS, which may contribute to differential molecular targets and cellular influences of iNOS-mediated NO generation. In the GI system, iNOS-derived NO generation has been importantly implicated in epithelial cell injury/apoptosis, host immune defense, and perpetuation of inflammatory responses. The specific functional roles of iNOS-mediated NO on these processes is described later. Colitis and Colon Cancer Although the role of NO in Crohn’s disease is unclear, a role for NO has been proposed in the pathogenesis of ulcerative colitis.106 In human ulcerative colitis, mucosal iNOS expression and activity, serum nitrate levels, and luminal NO generation are increased.106 –109 Levels of nitrotyrosine (a marker of nitrosative stress) are also increased, supporting a pathogenic effect of NO in this condition.107 In animal models of colitis, colonic NO generation is also increased in association with development of colitis, although blockade of NO production in these models has not consistently ameliorated measures of colitis.110,111 Furthermore, induction of colitis in the iNOS⫺/⫺ suggests that iNOS may actually be important in recovery from experimental colitis.112 Other colitides such as collagenous colitis are also associated with increased NO generation,107 although in collagenous colitis, nitrotyrosine staining is not elevated,

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perhaps corresponding to the generally lower degree of inflammation seen in this condition. In addition to its potential role in the pathogenesis of colitides, increased NO generation may also contribute to the reduced colonic motility associated with intestinal inflammation, such as toxic megacolon,113,114 as well as the secretory diarrhea associated with these colitides.107 Elevated iNOS activity has been shown in colon adenomas, colon cancer, and metastases,115 and it is thought that NO may contribute to the progression of adenoma to carcinoma by damaging DNA, increasing gene expression of COX-2, or generating posttranslation modifications via nitrosylation of proteins. Indeed, it has been suggested that the increased cancer incidence in chronic ulcerative colitis may be related to excess NO generation.116 An area of mounting experimental interest is the putative role of NOS inhibitors in colon cancer chemoprotection, paralleling the role that COX-2 inhibitors appear to play in this process. Using an azoxymethaneinduced colon cancer model in rats, Rao et al.117 showed a reduction in the development of aberrant crypts, a precursor to colon cancer in animals treated with the preferential iNOS inhibitors, SC-51 and aminoguanidine, similar to that observed with the COX inhibitor, sulindac, and the specific COX-2 inhibitor, celecoxib. Further studies in this area appear warranted. Hepatotoxicity NO has been implicated in the processes of apoptosis and hepatotoxicity as well as hepatoprotection, depending on the specific experimental conditions and nature of the models.66,118 The models reflect a diverse array of diseases, including IRI, toxin-mediated injury, and others.66,119 –121 For example, the influence of pharmacologic supplementation of NO in these models is often difficult to interpret because models that use NO donors in isolated cell systems likely do not specifically duplicate either eNOS or iNOS function. Likewise, in vivo studies using broad NOS inhibitors that block both iNOS and eNOS function lead to complex effects, making interpretation of isoform selectivity difficult. For example, the effects of inhibiting eNOS and altering blood flow may be deleterious, whereas the effects of iNOS suppression may be beneficial. In addition, the redox context of the injurious model may also influence results. Models with superoxide generation may favor peroxynitrite formation, whereas disease states with little oxidative stress may allow NO generation without peroxynitrite generation. Peroxynitrite promotes apoptosis in some experimental conditions via inducing mitochondrial dysfunction with cytochrome c release, which in

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turn stimulates caspase activation and apoptosis.66 In contrast, NO can nitrosylate multiple caspases inhibiting their function and blocking apoptosis.122 In addition, NO may also inhibit apoptosis by activating PKG, though the mechanism by which this kinase blocks apoptosis remains obscure. Owing to the limitations in interpreting NOS isoform specificity through pharmacologic studies, in vivo models using isoform-specific NOS gene deletion are of particular utility. In this regard, iNOS has been implicated as a cause of immune-mediated liver injury in the murine concanavalin A model of T-cell–associated liver injury and also in acetaminophen hepatoxicity.123–126 However, iNOS has also been shown to be important in liver regeneration and recovery from liver injury.127 Thus, the role of NO in liver injury will also depend on the type of injury occurring and the importance of liver regeneration in recovering from the injury. These issues, and others, may account for the apparent disparities in the literature relating to the influence of iNOS-derived NO on cell injury. Hepatic Carcinogenesis Chronic injury and inflammation related to NO may also predispose to the promotion and development of neoplasia. Indeed, there is experimental evidence implicating NO in the development of cholangiocarcinoma and hepatocellular carcinoma. The procarcinogenic influence of NO may be related in part to the aforementioned influence of NO toward inhibiting apoptosis and thereby promoting dysregulated cell proliferation. However, other mechanisms may be relevant as well, including DNA and protein damage. For example, NO and downstream reactive nitrogen species can directly damage DNA by promoting base pair mutation, strand break, and cross-linking.128 Indeed, this event has been observed in the DNA of the key proliferative checkpoint protein, p53.128 NO may also regulate key proteins involved in carcinogenesis through posttranslational modifications including nitration and nitrosylation. For example, cysteine thiol S-nitrosylation and subsequent inhibition of the DNA repair enzyme hOgg1, has been shown in cholangiocarcinoma cell lines and implicated in the transformation process.129 Nitrosylation of caspases can also occur resulting in apoptosis, thereby contributing to the carcinogenic process.130 Lastly, NO may promote tumor growth through the promotion of tumor angiogenesis. Indeed, multiple steps required for tumor angiogenesis are promoted by NO, either downstream of growth factors or directly, most notably endothelial cell proliferation and vascular permeability.131 Importantly, these mechanisms of carcinogenesis may be relevant to

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the development of other GI epithelial malignancies as well.

Summary NO is implicated in multiple important processes in gastrohepatic biology and diseases, and the pathogenic role of NO in these processes remains an area of active investigation. Interestingly, some disease conditions are associated with excess NO generation; other disease conditions, such as achalasia, are associated with deficient NO generation, while in some syndromes, such as portal hypertension and liver injury, both deficiencies and excesses of NO generation appear to contribute, with the sum influence of NO being based on spatial factors and temporal kinetics. These paradoxical observations highlight the complexities of NO-based signaling in GI health and disease processes. Significant interest has now been focused toward modulating the NO system as a therapeutic option in several arenas, including motility disorders of the GI tract, GI cancer, vascular disorders, and modulation of inflammation and injury. Thus, with advances in NO targeting, delivery, and isoform selectivity, it is likely we will see more human trials using organ-selective NO supplementation and isoform-specific NOS inhibitors targeting a wide variety of diseases in the GI system.

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Received April 10, 2003. Accepted July 24, 2003. Address requests for reprints to: Vijay Shah, M.D., GI Research Unit, Alfred 2-435, Mayo Clinic Rochester, 200 First Street SW, Rochester, Minnesota 55905. e-mail: [email protected]; fax: (507) 255-6318.