Role of nitric oxide in gastrointestinal and hepatic function and disease

GASTROENTEROLOGY

SPECIAL REPORTS

1992;103:1928-1949

AND REVIEWS

Role of Nitric Oxide in Gastrointestinal Hepatic Function and Disease MARK E. STARK

and JOSEPH

H. SZURSZEWSKI

Division of Gastroenterology and Internal Medicine Clinic and Mayo Foundation, Rochester, Minnesota

A

lthough the early Roman physician Galen had a predominant influence on medical thought and practice for over 14 centuries, his conception of the vascular system has been described as a physiological mistake that prevented the advancement of medical science for centuries.‘,’ Galen based his description of the vascular system on the concept of “pneuma” or spirits, a vital principle consisting of matter in a finely divided or ethereal state that flowed through the vascular and nervous systems and animated the entire organism.lv’ Many diseases were thought to owe their origin to some disturbance of these ethereal spirits. Although these ideas were fanciful, recent evidence indicates that an ethereal substance is indeed formed in many organs and that it has important roles in physiology and pathophysiology. This ethereal substance is the gas nitric oxide. In 1987,it was shown that vascular endothelial cells could synthesize NO and that this gas acted as a labile humoral-like messenger that relaxed vascular smooth muscle.3 Studies during the last 5 years have shown that NO functions as a mediator, a messenger, or a regulator of cell function in a number of physiological systems and pathophysiological states.4 This article begins with an abbreviated background on the discovery and characteristics of NO, reviews recent evidence that indicates that NO may play a role in gastrointestinal and hepatic physiology and disease, and speculates on potential clinical implications. Background Discovery and Characterization Vascular Tissue

and

of NO in

In 1980, Furchgott and Zawadzki showed that the endothelial lining of blood vessels was essential for the vasorelaxant effect of acetylcholine and showed that this effect was mediated by release of an endothelial substance that relaxed vascular smooth muscle. This substance later became known as endothelium-derived relaxing factor (EDRF).’ Over the next several years, the pharmacological and chemi-

and Department

of Physiology

and Biophysics,

Mayo

cal properties of EDRF were characterized, but the identity of EDRF remained unknown. In 1987,two groups working independently using chemical assay and bioassay showed that NO was released from vascular endothelial cells and that the effects of NO and EDRF were indistinguishable.3re EDRF is now thought to be endothelium-derived NO. NO has properties that make it unlike any known biologic mediator.’ It is a colorless gas that is slightly soluble in water. In dilute solution, NO has a half-life of -40 seconds because of rapid oxidation to inorganic nitrite and nitrate. NO is also destroyed by superoxide anion. Superoxide dismutase protects NO from breakdown by superoxide anion.‘*’ NO binds to oxyhemoglobin and other heme-containing proteins; its biologic actions are rapidly terminated by binding to oxyhemoglobin. 3*6*10 In biologic systems, NO has a half-life of <5 seconds.3 Vascular endothelial cells synthesize NO enzymatically from the terminal guanidine nitrogen atom of L-arginine (Figure 1).‘1*‘2The activity of the enzyme NO synthase is dependent on NADPH, calcium, and calmodulin.‘2~13 The enzymatic synthesis of NO can be competitively inhibited by structural analogs of L-arginine, such as NG-monomethyl+arginine (LNMMA) and NC-nitro+arginine (L-NNA).14-‘” NO is very lipophilic and can readily permeate biologic membranese7 NO formed by endothelial cells is thought to diffuse into the cytosol of the adjacent vascular smooth muscle cells where it binds to soluble guanylate cyclase, a cytosolic heme-containing protein.17 Binding of NO to this enzyme increases production of S-cyclic guanosine monophosphate (cGMP) with subsequent relaxation of smooth muscle. 17-21 In humans, endothelium-derived NO is formed in both arteries and veins and contributes to the control of basal and stimulated regional blood flo~.~~*~~Endothelium-derived NO can also diffuse into nearby ad-

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the American Gastroenterological 0018-5085/92/$3.00

Association

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ible NO synthase generates NO for many hours without further stimulation.44 In the gastrointestinal system presently available, evidence indicates that NO mediates relaxation of the muscularis externa and may play an important role in gastrointestinal mucosal blood flow, mucosal protection, hemodynamic responses to liver disease, regulation of hepatocyte function, and mediation of hepatotoxicity.

Source Cell \ L-arginine NO synthase L-NMMA L-NNA

NO

NO and Nonadrenergic Inhibition

. Figure 1. Schematic representation of the synthesis and action of NO, showing inhibition of NO synthase by L-NMMA and LNNA, inactivation of NO by oxyhemoglobin, oxygen or superoxide anion, and activation of guanylate cyclase by NO. NO may also have effects in the source cell.

hering platelets in the lumen of the blood vessel to inhibit platelet adhesion and aggregation.24v25 The actions of endothelium-derived NO suggest that it may have important roles in hypertension, shock, and atherosclerosis. Role of Endogenous

NO in Nonvascular

Neural Inhibition Muscle

Noncholinergic

of Gastrointestinal

Smooth

The importance of inhibitory innervation of intestinal motility has been apparent since Bayliss and Starling showed neural reflexes intrinsic to the intestine.48 Nerve-mediated relaxation and membrane hyperpolarizations called inhibitory junctional potentials (IJPs) have been described in intestinal muscle of a number of mammals including humans, but the identity of the nonadrenergic, noncholinergic (NANC) inhibitory neurotransmitter that mediates these effects has remained unclear.4g*50

Tissue

NO is synthesized in many nonvascular tissues. Generation of NO from L-arginine in platelets appears to act as a negative feedback on platelet aggregation.26*27 NO is formed in neutrophils and macrophages where it may function as an effector molecule mediating cytotoxicity in immune reactions against tumor cells and intracellular parasites.28-31 In the brain, NO is produced in neurons in response to activation of excitatory amino acid receptors, and it activates soluble guanylate cyclase in adjacent presynaptic nerve terminals and astrocytes.32-34 NO is also formed in the adrenal gland,35 renal epithelial cells,36 and mast cells,37 but the functional significance in these tissues is not clear. A similar biochemical pathway involving the enzymatic conversion of L-arginine to NO plus L-citrulline is found in all of the cell types mentioned. However, the NO synthase enzymes in these various cell types appear to have differences, including differential sensitivity to inhibition by L-arginine analogs?g The most studied NO synthase in vascular endothelium is constitutive, which means that it is present and active without exposure to inducing agents.l’In contrast, synthesis of NO in macrophages occurs only when macrophages are exposed to inducing agents such as endotoxin or cytokines.4042 Vascular endothelial and vascular smooth muscle cells also express an inducible NO synthase similar to the inducible enzyme found in macrophages.4347 Once triggered by endotoxin or cytokines, this induc-

Table 1. NO and NANC Inhibition Evidence from in vitro animal studies supports NO role as NANC neurotransmitter NO released from ileocolonic junction and stomach during nerve stimulation69,‘1 Exogenous NO mimics NANC nerve evoked relaxation and hyperpolarization in GI smooth muscles71-81 Inhibition of NO synthesis or hemoglobin inactivation of NO attenuates effects of NANC nerve stimulation”~‘7~80-89 NO synthase localized to myenteric plexus and neuronal processes of intestineg2 NO may mediate inhibitory effects of other putative NANC neurotransmitters (e.g., VIP) NO may be second messenger formed in smooth muscleg3 NO may be released from final effector neuron in response to other neurotransmitter”~84~86~110 NO may be released from nonneural cells (e.g., interstitial cells of CajaQa6 NO mediates physiologic responses and reflexes in animal studies Descending inhibition of isolated intestine during peristaltic reflex78 Receptive relaxation in isolated stomach”6 Vagally mediated relaxation of stomach and LES in vivo97,ga~117 NO mediates NANC inhibition in human GI smooth muscle in vitro Exogenous NO mimics NANC nerve evoked relaxation and hyperpolarization in human jejunum8’ Inhibition of NO synthesis attenuates NANC nerve-evoked relaxation in human sigmoid colon and internal anal sphincter” Inhibition of NO synthesis or hemoglobin inactivation of NO attenuates NANC nerve-evoked hyperpolarization and relaxation in human jejunum”

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Of the putative NANC inhibitory neurotransmitters studied, adenosine triphosphate (ATP)51-54 and vasoactive intestinal peptide (VIP)55-5g are the substances for which the most evidence has been advanced, but in both cases the evidence is incomplete and, in some gastrointestinal smooth muscles, neither appear to be invo1ved.54-57*60-62Recent work has indicated that a nonpurinergic, nonpeptidergic system involving NO may mediate NANC neural inhibition of gastrointestinal smooth muscle (Figure 2). NO and NANC Neurotransmission Even before the identification of EDRF as NO, early studies by Gillespie et al. on the innervation of rodent and bovine genitalia-associated smooth muscles indicated that a nonpurinergic, nonpeptidergic neurotransmitter was involved in NANC neurotransmission. In these tissues, NANC inhibition could not be attributed to any known neurotransmitter, cGMP was the second messenger mediating relaxation, and the effect of NANC nerve stimulation was antagonized by oxyhemoglobin. 63-65It was recognized that the mediator of NANC inhibition in these tissues had chemical properties and actions similar to those of endothelium-derived NO, and later work indicated that NO is the mediator of NANC neurotransmission in these muscles.666* In 1990, Bult et al. provided evidence that NO is released on stimulation of enteric NANC nerves.6g Since then compelling evidence has emerged indicating that NO acts as a NANC neurotransmitter in the gut (Table 1).

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NO and Criteria for Neurotransmitter Identification Attempts to identify neurotransmitters have led investigators to define criteria that, if satisfied, would indicate that a substance is a neurotransmittera7’ For a substance to be considered a neurotransmitter its release from neurons needs to be detectable, and there must be identity of action between the effects of the substance when applied exogenously to the postjunctional receptors and the effect when released by nerve stimulation. Subsidiary conditions supporting the identification of a substance as a neurotransmitter include the demonstration that the effect of antagonists is the same on the response to exogenous application and nerve stimulation, identification of a mechanism of inactivation for the transmitter, and demonstration of mechanisms for neurotransmitter synthesis and storage in the neuron. Recent studies show that most of these criteria have been met for NO, supporting a role for NO as a neurotransmitter in the gastrointestinal tract. Release of NO. In in vitro experiments, electrical field stimulation of NANC nerves released a substance from the canine ileocolonic junction (ICJ)“” and rat gastric fundus, which when superfused over vascular smooth muscle caused relaxation. This substance was inactivated by supe.roxide anion and oxyhemoglobin, and its release was inhibited by an inhibitor of NO synthesis, indicating that it was NO or a closely related substance. Release of

NANC NWrOn

0 with NO Synthase

. . . = NANC neurotransmitter (VIP?, ATP?)

I

I

muscle

Figure 2. Schematic representation of possible mechanisms by which NO may mediate NANC inhibition of gastrointestinal smooth muscle. (A) Evidence supports this model with NO acting as a neurotransmitter from a final inhibitory neuron. NO binds to cytosolic guanylate cyclase and increases cGMP formation, leading to relaxation by unknown mechanisms. (B) Other possible mechanisms of NANC inhibition, including NO-dependent and NO-independent pathways. See text for details.

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this substance was stimulus frequency dependent and tetrodotoxin sensitive, indicating it was released from nerves. However, these experiments do not exclude the possibility that electrical stimulation released a neurotransmitter that in turn caused the release of NO from nonneuronal cells, such as from gastrointestinal smooth muscle itself (Figure ZB). Although the substance released during NANC nerve stimulation of the canine ICJ and rat gastric fundus had characteristics that indicate its identity was NO, a chemical assay showing that this substance is NO has not been reported. Identity of action. In a number of different gastrointestinal smooth muscles, the effects of exogenously applied NO and the effects of NANC nerve stimulation are similar. Exogenous NO-induced relaxation, grossly similar to that evoked by electrical stimulation of NANC nerves, has been observed in vitro in longitudinal and circular muscle strips from lower esophageal sphincter (LES), stomach, small intestine, ICJ, and internal anal sphincter from a number of different animal species.71-77 In isolated circular muscle from segments of rat colon, relaxation evoked by exogenous NO mimics the descending relaxation observed in response to intraluminal balloon distention.78 Intracellular recordings from smooth muscle cells from canine co1on7gv8ocanine ICJ”’ and canine jejunum75 show that exogenously applied NO evoked a membrane hyperpolarization that is similar to IJPs evoked by NANC nerve stimulation. In circular muscle of the normal human jejunum, exogenous NO evokes membrane hyperpolarization and inhibition of mechanical activity.** Although the latter effect of exogenously administered NO grossly mimics the effect of NANC nerve stimulation, analysis of the electrical responses shows distinct differences. The NANC IJP in the human jejunum consists of an initial large amplitude, rapid hyperpolarization followed by a smaller amplitude hyperpolarization that lasts from 10 to 15 seconds. The amplitude and rate of change of voltage of the initial rapid hyperpolarization are significantly greater than those of NO-evoked hyperpolarizations. However, the amplitude and time course of the later smaller amplitude hyperpolarization are similar to those of the NO-evoked hyperpolarizations. These observations suggest that NO may mediate the later sustained smaller-amplitude hyperpolarization of the IJP in circular muscle of the human jejunum but not the initial rapid hyperpolarization. Effect of antagonists. Although there are currently no known receptor antagonists to NO, inhibition of NO synthesis’4-‘6 by L-arginine analogs and inactivation of N03*6,10 by oxyhemoglobin have been used as pharmacological tools to investigate the possi-

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ble role of NO in NANC inhibition. Inhibition of NO synthesis or exogenous application of oxyhemoglobin in vitro attenuate the relaxing effects of electrical stimulation of NANC nerves in guinea pig co1on,B3 longitudinal muscle of canine duodenum and rat gastric fundus,71,84 and in circular muscle of canine LES,74 opossum LES,7”,85canine ileum,72 canine ICJ,‘” canine co1on,*6 opossum internal anal sphincter,77,67 human jejunum, ” human colon,*’ and human internal anal sphincter.“’ In circular muscle of opossum esophagus,” canine small intestine,75,8g canine ICJ,“’ canine co10n,80,go and human jejunum,*’ oxyhemoglobin and inhibitors of NO synthesis attenuate or block IJPs evoked by NANC nerve stimulation. A specific mechaMechanism of inactivation. nism of inactivation for NO in gastrointestinal tissue has not been identified. However, substances that rapidly inactivate NO, including oxygen and oxyhemoglobin, are present in gastrointestinal tissue and would be expected to limit the action of NO released from NANC nerves to a local region and for a brief period of time. Neural synthesis and storage ofNO. Although the results reviewed above provide strong evidence that NO plays an important role in mediating NANC inhibition of gastrointestinal smooth muscle, they do not definitively show that NO is a neurotransmitter. The data are also consistent with a second messenger role for NO within smooth muscle cells (Figure 2B). According to this alternative hypothesis, release of a neurotransmitter from NANC nerves stimulates the synthesis of NO within gastrointestinal smooth muscle cells.g* Two lines of evidence point toward a role for NO as a neurotransmitter. The strongest evidence comes from experiments in which NO synthase was localized by immunohistochemical staining in cells in the myenteric plexus and in neuronal processes in the circular muscle layer of the rat duodenum.g2 Additional evidence comes from the observed effects of oxyhemoglobin on inhibition evoked by stimulation of NANC nerves.72-75,7g Because the large hemoglobin molecule is not cell-permeant, its inhibitory effect on NANC neurotransmission requires NO to exist extracellularly at some point after its generation (Figure 2A). On the other hand there is also evidence that NO is produced in some gastrointestinal smooth muscle cells in response to the putative inhibitory neurotransmitter VIPag3At the present time, the possibility that release of a neurotransmitter from a NANC nerve triggers production of NO in a nonneural cell, such as an interstitial cell of Cajal, with subsequent action on the smooth muscle cells to cause relaxation (Figure 2B) cannot be ruled out. The possibility that NO may be synthesized in multiple sites in the muscularis of the gut, including gastrointestinal smooth muscle, should receive further con-

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sideration as NO synthase activity has been found in vascular smooth muscle.43994 A storage mechanism for NO in enteric nerves has not been shown. In fact, the details of synthesis and release of NO are not well understood even in vascular tissue, which has received more extensive study. It is unlikely that free NO is stored in vesicles because it readily permeates lipid membranes. It may be that NO is formed in nerves only when they are stimulated and that synthesis keeps pace with release, obviating the need for storage. Alternatively, an acid-stabilized S-nitrosothiol intermediate could be packaged and stored in vesicles, with nerve stimulation leading to exocytosis and spontaneous decomposition of the intermediate to form NO on exposure to the nonacidic extracellular space.g5 This mechanism would be comparable to storage and release of classical neurotransmitters. Evidence for storage of NO in thiol-bound intermediates has been found in vascular endothelium.g6 In the canine jejunum, the decrease in IJP amplitude caused by a NO synthesis inhibitor is maximal after a lo-minute exposure, whether or not the NANC nerves are repetitively stimulated during the exposure period.75 This suggests the absence of large pool of preformed vesicle-stored precursor that has to be depleted by repeated nerve stimulation before the effect of the NO synthesis inhibitor is seen. The data are more consistent with the notion that the NO synthetic pathway is activated only during nerve stimulation. However, S-nitrosocysteine, a nitrosothiol compound that liberates free NO, evokes a rapid hyperpolarization in circular muscle of the canine colon, which mimics the effects of exogenously applied NO and NANC nerve stimulation.7g Because control experiments indicated that the effects of Snitrosocysteine in the canine colon were caused by the liberation of free NO, these findings support the possibility that NO could be stored in vesicles in nerve terminals as a nitroso intermediate. It is possible, therefore, that both mechanisms of NO release may be operative and may vary depending on the tissue and type of physiological response. A related question is whether there is on-going synthesis and release of NO to affect tone in gastrointestinal smooth muscle in addition to the release of NO in response to electrical stimulation of NANC nerves. Inhibitors of NO synthesis increase basal tone in strips of longitudinal muscle of rat gastric fundus,71,84 in strips of circular muscle of canine ICJ,‘” and in the canine pylorus in vivo,g7 suggesting on-going release of NO in these tissues. In contrast, inhibition of NO synthesis had no effect on basal tone or on amplitude of spontaneous contractions in circular muscle of either the canine ileum72 or canine jejunum.75 Similarly, inhibition of NO synthase

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and addition of oxyhemoglobin has no effect on resting membrane potential, slow wave amplitude, or slow wave frequency recorded intracellularly from circular muscle of the canine jejunum.75 When considered together, the results of experiments described above would seem to suggest ongoing release of NO may occur in sphincters or smooth muscles that show predominately active tone or slow changes in tone like the gastric fundus, whereas ongoing release of NO does not occur in smooth muscles that show predominately phasic activity and little active tonic activity such as the ileum or jejunum. However, the recent finding that the force of spontaneous phasic contractions in the canine gastric antrum increases when synthesis of NO synthesis is inhibited’* does not support this hypothesis, as the antrum shows predominately phasic activity and little tonic activity. Also, the lack of effect of systemically administered NO synthesis inhibitors on resting pressures of the opossum LES in vivogg suggests an absence of ongoing synthesis and release of NO in this sphincter. The source of NO in smooth muscle in which there does appear to be ongoing release is not known. Some of the NO that is synthesized in enteric neurons might diffuse out of the neuron and into target smooth muscle cells. Ongoing release of NO may arise from nonneural cells in some regions of gastrointestinal tract. NO formation has been shown in isolated gastric muscle cells.g3 NO formation has also been shown in white blood cells,28-30 and in blood vessels of the gastrointestinal tract.g8*‘00Recently NO synthase activity was localized to the rat gastric mucosa,lol raising the possibility that NO formed in gastrointestinal mucosa may modulate the function of underlying gastrointestinal smooth muscle. Mechanism

of NO Action

Role of cGMP. Relaxation in gastrointestinal smooth muscle by NO may be mediated by increased levels of cGMP as described in vascular smooth mussmooth muscle cle (Figures 1 and 2). Gastrointestinal of a number of species including humans contains cGMP-dependent protein kinases.“’ Relaxation of the canine internal anal sphincter, opossum LES, and human LES in response to NANC nerve stimulation is associated with increases in cGMP in smooth muscle.‘03-‘o” NO-evoked relaxation of guinea pig taenia coli also is associated with an increase in cGMP.‘07 The mechanism by which NO-induced increases in cGMP lead to smooth muscle relaxation is not entirely known, although it may be linked to modulation of intracellular calcium concentration. In the rat anococcygeus, electrical stimulation of NANC nerves leads to release of NO, relaxation and a decrease in cytosolic calcium levels.‘08 In smooth

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muscle of the canine gastric antrum, sodium nitroprusside, which releases NO,log decreases the amplitude of the plateau potential of gastric action potentials and inhibits the increase in cytosolic Ca2+ and tension associated with the plateau potential.* These observations suggest that NO inhibits voltagedependent Ca2+ channels responsible for the plateau potential. Although sodium nitroprusside had no significant effect on either upstroke potential of the gastric slow wave or levels of free cytosolic calcium, there was nevertheless a large reduction in the force of contraction triggered by the upstroke potential suggesting that NO may also reduce the Ca2+ sensitivity of the contractile elements.” This effect of NO may be mediated by cGMP, as cGMP inhibits Ca2+induced contraction in this tissue.g8 Role of NO-evoked hyperpolarization. Exogenously administered NO evokes a membrane hyperpolarization in gastrointestinal smooth muscle which resembles the IJP seen following NANC nerve stimulation.75*7g~82It is not known if the hyperpolarization is caused by a direct effect of NO on membrane channels, or if it is mediated by a NO-induced increase in cGMP (Figure 2B). Methylene blue, which blocks the action of NO through the inhibition of guanylate cyclase, attenuates NO-mediated NANC inhibitory junction potentials in circular muscle of opossum esophagus and canine small intestine,8g indicating that increased cGMP is important in the NO-evoked hyperpolarization. In canine intestine, the membrane potential during NOevoked hyperpolarizations approaches the expected potassium equilibrium potential, suggesting that an increase in potassium conductance mediates the hyperpolarization. 75*7gIn patch clamp recordings from canine colonic myocytes in the cell-attached configuration, NO enhanced the open probability of Ca2+activated K+ channels.7g This evidence suggests that these channels mediate hyperpolarization in response to NANC neurotransmission and exogenous application of NO. Recordings of Ca2+-activated K+ channels in isolated patches of membrane during the presence of NO are needed to determine more definitively whether increases in potassium conductance associated with NO is due to a direct effect of NO on membrane ion channels specific for potassium or whether NO increases cGMP, which in turn increases potassium conductance. Physiological

Role of NO

in

NANC Inhibition

Relationship of NO to other putative NANC neurotransmitters. Although the evidence reviewed above provides strong support for the hypothesis that NO functions as an NANC neurotransmitter in several gastrointestinal smooth muscles, a role for other putative NANC neurotransmitters such as VIP and

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ATP cannot be ruled out. Indeed, much evidence supports a role for these substances in NANC neurotransmission.51-60 A number of different NANC neurotransmitters may be involved in initiating the same inhibitory response, and similar responses in different gastrointestinal smooth muscles and species may be due to different transmitters. Evidence supports the possibility that there may be more than one NANC inhibitory neurotransmitter in gastrointestinal smooth muscle. In some smooth muscles there appears to be two mechanisms of inhibitory transmission distinguished by sensitivity or resistance to blockade by the bee toxin apamin. In the guinea pig ileum, stimulation of NANC nerves evokes an IJP with two components: a rapid short latency hyperpolarization that is apamin-sensitive and a slower long-lasting hyperpolarization that is apamin-resistant61 This suggests that at least two neurotransmitters mediate the IJP or that a single inhibitory neurotransmitter may activate two different receptors linked to potassium channels with different kinetics or modes of activation. The relative contributions of apamin-sensitive and apamin-resistant mechanisms vary between regions of the gut.62 Other evidence for multiple mediators of inhibitory neurotransmission includes the finding that inhibitory neurotransmission in the rat duodenum is antagonized by the ATP antagonist nucleotide pyrophosphatase and by ATP desensitization, whereas neither the antagonist nor ATP desensitization affect inhibitory neurotransmission in the ileum.54 In a number of gastrointestinal smooth muscles, incubation with VIP antiserum blocks the response to exogenous VIP but only reduces the inhibitory response to NANC nerve stimulation supporting the hypothesis that a neurotransmitter in addition to VIP may be released from inhibitory NANC neurons.55-57 Experiments on the role of NO in NANC inhibitory neurotransmission also support the possibility that a number of different inhibitory neurotransmitters are released during NANC nerve stimulation. In the canine jejunum, inhibition of NO synthesis reduced but did not abolish the amplitude of the NANC IJP.75 In this tissue, oxyhemoglobin abolishes the hyperpolarizing response to exogenous NO but only partially reduces IJP amplitude evoked by NANC nerve stimulation,75 suggesting that the effects of NANC nerve stimulation were mediated by an additional neurotransmitter besides NO. Similar incomplete attenuation of the inhibitory effects of NANC neurotransmission by NO synthesis inhibitors and oxyhemoglobin has been shown in the canine LES,74 canine ICJ and ileum,72 canine co1on,go and rat gastric fundus.71*84 In the canine jejunum, although IJPs evoked by NANC nerve stimulation and hyperpolarizations evoked by exogenous NO

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had similar maximum amplitudes, the rates of hyperpolarization and repolarization were significantly faster for IJPs.~~ This may have been due to differences in the delivery of NO, but an alternate explanation may be that the initial rapid hyperpolarization of IJPs is not an NO-dependent mechanism but rather is due to the action of another inhibitory neurotransmitter. In some gastrointestinal smooth muscles, NO may serve as the final mediator for relaxations induced by other putative NANC neurotransmitters, such as VIP and ATP (Figure 2B). In the canine ileum and ICJ, ATP-induced relaxations are reduced by oxyhemoglobin and inhibitors of NO synthesis,“’ suggesting that NO is the final mediator for relaxation. Similar reductions of agonist-induced relaxations with NO synthesis inhibitors were noted with VIP in the rat gastric fundus and opossum internal anal sphincter. 77 NO could act as the final mediator of relaxations induced by these other putative NANC neurotransmitters in the following ways: as a neurotransmitter released from a final effector neuron in response to ATP or VIP, as a mediator released from a nonneuronal cell in response to ATP or VIP, or as a second messenger formed in smooth muscle in response to ATP or VIP (Figure 2B). In canine colonic smooth muscle, VIP acts presynaptically on noncholinergic intrinsic nerves leading to NO-mediated smooth muscle relaxation, possibly by inducing NO release from neurons.‘” In guinea pig gastric fundus, VIP release stimulates NO production in target smooth muscle cellsUg3In other tissues such as the canine LES,74 opossum LES,85 and rat duodenumll’ relaxations evoked by VIP or ATP were not affected by inhibitors of NO synthesis, indicating that VIP and ATP relaxed smooth muscle by a NO-independent mechanism and supporting the possibility of release of multiple inhibitory neurotransmitters that act through parallel pathways during NANC nerve stimulation. Recent experiments have shown that NANC IJPs in circular muscle of normal human jejunum have two components: an initial rapid, large amplitude hyperpolarization followed by slower, smaller amplitude hyperpolarization.” Inhibition of NO synthesis reduces the amplitude of the late sustained nerveevoked hyperpolarization, indicating that it is mediated by NO, but does not reduce the initial rapid hyperpolarization indicating that it is mediated by some other NANC neurotransmitter. In contrast, IJPs in the canine jejunum consist of only an initial rapid hyperpolarization, which is reduced in amplitude by inhibition of NO synthesis.75.82 These results suggest that NO mediates NANC inhibition in both the human and canine jejunum, but the apparent role of

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NO as a mediator of NANC inhibition is different in the two species. Although the evidence supports the possibility that NO is one of several NANC neurotransmitters, it is not known whether they are colocalized in the same neurons and if colocalized whether they are coreleased. The apparent corelease of multiple inhibitory neurotransmitters in in vitro muscle strip experiments may be an artifact of the experimental method, because electrical stimulation activates all enteric nerves simultaneously. The physiological role of multiple inhibitory neurotransmitters may be related to their half-life. A rapidly acting evanescent transmitter like NO would be expected to cause rapid transient relaxation, whereas other NANC neurotransmitters with longer half-lifes would be responsible for prolonged changes in smooth muscle tone. The relative activity of enteric neurons that release NO, neurons that release longer lived transmitters, and neurons that release both types would determine the type and duration of relaxation. Physiologic role ofN0 in gastrointestinal motility. Descending inhibition mediated by NANC enteric inhibitory neurons is an important component of the intestinal peristaltic reflex.*12p113The descending relaxation evoked in isolated segments of rat colon by balloon distension is mimicked by exogenous NO and is prevented by inhibitors of NO synthesis, suggesting that this reflex is mediated by N0.78 Entry of food or liquid into the stomach results in proximal stomach dilation and an increase in gastric capacity. Evidence suggests that this reflex is mediated by NANC neurons.114’“5 In the isolated guinea pig stomach, receptive relaxation is prevented by inhibitors of NO synthesis.‘l” In the canine gastric antrum, inhibitory modulation of phasic contractions appears to result from continuous spontaneous release of N0.g8Thus, it appears that inhibition of gastrointestinal smooth muscle mediated by NO plays a variety of important physiological roles. Because most of these studies were done in vitro, their applicability to the understanding of in vivo organ function remains uncertain. However, recent in vivo demonstration that vagally-mediated relaxation of the opossum LES,” rat stomach,l17 and canine stomachg7 is mediated by NO provides strong support for a physiological role for NO in NANC inhibition. Speculation

on Clinical Implications

Although a pathophysiological role for NO has not yet been identified, it is possible to speculate that changes in the density of NO-producing nerves, alterations in production of NO, or alterations in smooth muscle sensitivity to endogenous NO could play a role in some neuromuscular disorders. For example,

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decreases in neural NO production could lead to diseases in which there is sustained or vigorous nonperistaltic contractions such as in the aganglionic segment of Hirschsprung’s disease or the nonrelaxing LES of achalasia. Increased production of NO or increased sensitivity to NO in the LES could play a role in gastroesophageal reflux disease. An imbalance of normal excitatory and inhibitory nerve activity related to disorders of the enteric NO system could be involved in disease states in which there is abnormal intestinal motility, such as chronic idiopathic intestinal pseudo-obstruction or constipation. If alterations of the enteric NO system are found to be involved in disorders of intestinal motility in humans, pharmacological manipulation of the enteric NO system may provide effective therapy. It also seems likely that drugs will be used to manipulate the NO system in vascular tissue as therapy for disease states such as coronary disease and hypertension. Because NO appears to play an important role in the neural regulation of gastrointestinal motility, pharmacological manipulation of the vascular NO system should be expected to also affect the enteric NO system and have gastrointestinal side effects. NO and Gastrointestinal

Mucosal Protection

Gastric Mucosa Mucosal blood flow and gastric mucosal Iesions. Acute gastric mucosal erosions or ulcers that may be associated with serious gastrointestinal bleeding commonly occur in the settings of severe acute medical illness or ongoing sepsis, severe trauma or burns, CNS disease, and ingestion of gastrotoxic drugs.118-121The pathogenesis of acute gastric mucosal lesions is multifactorial, but alterations in gastric mucosal blood flow appear important in their developmenf.122-123 Mucosal blood flow is de-

Table 2. NO and Gastrointestinal

Mucosal

Protection

Endogenous NO may mediate gastric mucosal protection in animals Endogenous NO modulates vascular tone and gastric mucosal blood flo~‘~~-‘~~ Drugs that produce NO protect from acute mucosal damage’Z’,‘33 Inhibition of NO synthesis facilitates mucosal injury’” Endogenous NO may mediate intestinal mucosal protection in animals Inhibition of NO synthesis enhances endotoxin induced mucosal injury’40 Drugs that produce NO attenuate endotoxin induced mucosal injury14*

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pendent on the balance between the endothelial release of substances that enhance blood flow through vasodilator and/or antiaggregatory properties such as prostacyclin124 and vasodilator neuropeptides125 and those substances that reduce blood flow and platelet aggregation such as thromboxane126 and endothelin-1.“’ Evidence indicates that NO may be an endogenous vasodilator regulating gastric mucosal blood flow and maintaining mucosal integrity and defense (Table 2). NO and gastric mucosal blood flow. In the rat stomach, inhibition of NO synthesis reduces gastric mucosal blood flow as measured by a hydrogen gas clearing technique, indicating that endogenous NO modulates basal tone in the gastric vasculature.“’ Increases in gastric mucosal blood flow induced by acetylcholine or bradykinin in the rat is endothelium-dependent,12g~130 and NO synthase inhibitors attenuate pentagastrin-induced increases in gastric mucosal blood flo~,‘~~,l~~ indicating that endogenous NO plays a role in agonist-stimulated increases in mucosal blood flow as well. NO and gastric mucosal protection. Endogenous NO may contribute to mechanisms that protect against ulcerations of the gastric mucosa. Topical mucosal application of a NO solution or of glyceryl trinitrate or nitroprusside, which release NO,“’ reduces the severity of ethanol-induced hemorrhagic mucosal damage.*33 Intravenous administration of nitroprusside also inhibits mucosal damage.12’ Administration of inhibitors of NO synthesis alone did not lead to acute gastric mucosal damage in the rat.“’ However, inhibition of NO synthesis after depletion of vasodilator neuropeptides with chronic capsaicin pretreatment or after inhibition of prostacyclin synthesis with indomethacin did induce substantial mucosal injury. loo Another study suggests that the gastroprotective effect of sensory neuropeptides released by acute capsaicin exposure is mediated by NO.134 These results suggest a role for endogenous NO, interacting with prostacyclin and vasodilator neuropeptides in the regulation of gastric mucosal integrity. The mechanism underlying the mucosal protective actions of NO is not known. It seems likely that vasodilation or inhibition of platelet aggregation in the gastric microvasculature are involved, but it is possible that NO has other actions in the gastric mucosa that enhance or preserve epithelial cell function and continuity. The source of the gastroprotective endogenous NO is also not known. The vascular endothelium seems a likely source, but given the recent demonstrations of NO formation in many cell types, other sources including white blood cells, epithelial cells, or neurons should also be considered.

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Speculation on clinical implications. Damage to endothelial cells may be an important primary event that leads to acute gastric mucosal ulceration.‘35*‘36The development of mucosal ulceration after endothelial damage could be related to decreased endothelial production of vasoactive mediators with protective actions such as NO. NO production does decrease after endothelial damage in coronary blood vessels.‘37 Pharmacological interventions to increase gastric mucosal levels of NO by augmenting endogenous NO production or providing an exogenous source of NO-like nitroprusside could prove effective in the prevention of acute gastric mucosal injury in high-risk settings. The gastroprotective effects of the drug carbenoxolone may be mediated in part by NO. 13’However, the data regarding a mucosal protective role of NO are entirely from animal studies, and the role of NO in the human gastric mucosa has not been defined. Intestinal

Mucosa

Endotoxin-induced intestinal damage. The cardiovascular collapse and multiple metabolic derangements associated with septic shock are largely due to bacterial endotoxin, but the toxic effects of endotoxin are mediated by the production and release of biologic mediators by the host.13’ In the rat, intravenous injection of endotoxin induces acute intestinal vascular damage, characterized by vasocongestion, plasma leakage, and hemorrhage into the intestinal 1umen.‘40~‘41 Platelet activating factor and thromboxane A, are endogenous vasoactive mediators released in response to endotoxin in the rat. They appear to be important in the production of the gastrointestinal hemorrhagic lesions induced by endotoxin.‘42-144 Other vasoactive mediators released by endotoxin attenuate the harmful effects of endotoxin, and may be part of the host defense to endotoxic shock.‘45 Recent evidence suggests that endogenous formation of NO maintains microvascular integrity of the intestinal mucosa following acute endotoxin challenge, and endogenous NO may be one of the mediators that help protect against the harmful effects of endotoxic shock. NO and intestinal mucosal protection. In the rat, pretreatment with an inhibitor of NO synthesis enhances endotoxin-induced small intestinal damage and plasma leakage. 140The nitrovasodilator S-nitroso-N-acetyl-penicillamine, which generates NO, attenuates endotoxin-induced intestinal damage in this model.141 These results suggest that endogenous NO protects the intestinal mucosa and microvasculature against endotoxin-induced damage. The mechanisms are largely unknown. Endotoxin can stimulate the formation of oxygen radicals such as superoxide anion, which may mediate intestinal injury.146*147

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NO can interact with superoxide anion to produce a less toxic species’48 and may act as an antioxidant,‘4g which could explain how NO reduces the microvascular damage produced by endotoxin. Neutrophil adhesion to vascular endothelium and emigration from blood vessels can also be inhibited by N0,150 suggesting that endogenous NO may protect the intestine from endotoxin-induced damage by limiting the damaging effects of neutrophils. Speculation on clinical implications. The animal studies described above suggest that NO plays an important role in protecting the intestine from blood-borne toxins and tissue-destructive mediators. The role of NO in the protection of the human intestine from toxic mediators is unknown, but investigation into the role of NO in inflammatory conditions in which there is tissue damage or increased vascular permeability involving vasoactive mediators, toxic oxygen radicals, or neutrophils may provide important insights into the pathogenesis of idiopathic inflammatory bowel disease, infectious colitis, ischemic gastrointestinal injury, and gastrointestinal malignancy. NO and Hemodynamic Cirrhosis

Disturbances

NO and the Hyperdynamic Cirrhosis

Circulation

in of

Splanchnic and systemic hemodynamics in cirrhosis. Portal hypertension is associated with hemodynamic disturbances in both the splanchnic and systemic circulation. Chronic portal hypertension is associated with splanchnic hyperemia, which may

Table 3. NO and Hemodynamic

Disturbances

in Cirrhosis

Endogenous NO may mediate hyperdynamic circulation of cirrhosis Endogenous NO is an important modulator of vascular tone in animals and humanz2~23*“” Agonist-induced release of endogenous NO leads to hemodynamic responses similar to the hyperdynamic circulation of cirrhosis’76~“7 Endotoxin is found in high circulating levels in cirrhosis and may trigger endothelial NO synthesis leading to hyperdynamic circulation in animals and humans”s~‘8Q Urinary cGMP levels are increased in cirrhotic humans, possibly due to increased NO production leading to increased cGMP”” Endogenous NO production mediates systemic and splanchnic vasodilation in portal hypertensive rats200~202 Methylene blue, a blocker of NO action, elevated blood pressure in a patient with cirrhosis and severe hypotension’” NO producing drugs might be effective treatment of portal hypertension Molsidomine (NO donor) acutely reduces portal pressure in cirrhotic rats and humans2*3-225

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be important in maintaining chronic portal hypertension and may contribute to the development of the abnormalities of systemic hemodynamics seen in cirrhosis.‘51-154 These alterations in systemic hemodynamics include increased cardiac output and heart rate and decreased systemic vascular resistance and blood pressure.‘55-‘5g This hyperdynamic circulatory state develops before the onset of ascites and is found in 30%~50% of patients with cirrhosisl55~157 and in all animal models of portal hypertension. 152-154~180 In patients with cirrhosis and ascites, the severity of the hemodynamic derangements is an independent predicator of survival,“’ suggesting that the homeostatic responses to a decreased effective plasma volume may be harmful. The hyperdynamic circulatory state may be a reflection of a decrease in the “effective” plasma volume related to vasodilatation, which increases the vascular holding capacity.16’ Decreased effective plasma volume may also play a key role in the development of important complications of cirrhosis including ascites, edema, and hepatorenal syndrome (Figure 3).le3 According to the “underfill theory,” the primary event in the development of these complications is a decrease in effective plasma volume related to primary peripheral vasodilatation.‘63*164 This leads to activation of volume receptors that activate the renin-angiotensin system, increase sympathetic outflow, and may increase release of atria1 natriuretic factor and alter activity of the kallikrein-kinin system. These mediators increase renal sodium retention leading to ascites and edema and may alter renal blood flow leading to decreased glomerular filtration and the hepatorenal syndrome. This theory is

Cirrhosis Portal-Syste(mic Shunts Systemic Endotoxemia

t t “Effective” Volume

HYPERDYNAMIC CIRCULATION

t Sympathetic Outflow t AtrialNattiuretic Factor? +Kallikrein-kinin? A Other Mediators? Altered Renal Blood Flow ASCITES+,EDEMA

HEPATORENA: SYNDROME

Figure 3. A possible mechanism involving endotoxin induction of vascular NO synthase leading to decreased “effective” volume and complications of cirrhosis. See text for details.

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supported by evidence from studies of humans with cirrhosis.163 Evidence suggests that systemic and splanchnic vasodilatation seen in cirrhosis with portal hypertension is due to decreased sensitivity to endogenous vasoconstrictors,165 and/or increased activity of endogenous vasodilators.16B-1sa The list of putative vasodilators in cirrhosis is long’sg-174but no convincing evidence indicates a predominant role for any one of them. NO, endotoxin, and vasodilatation in cirrhosis. Recently it was proposed that NO acts as an endogenous vasodilator mediating the hyperdynamic circulation of cirrhosis.‘75 Endothelium-derived NO relaxes vascular smooth muscle and dilates arteries and veins contributing to the regulation of vascular tone in animals and humans.22*23*176In animals, release of endothelium-derived NO triggered by acetylcholine or other agonists leads to peripheral vasodilatation, hypotension, and increased heart rate.176v177If a substance that triggers endothelial NO formation is released into the circulation in cirrhosis, the resulting vasodilatation may produce the hyperdynamic circulatory state. There is evidence that supports a role for NO in the hyperdynamic circulation of cirrhosis and suggests that bacterial lipopolysaccharide endotoxin may be the trigger for increased endothelial NO formation (Figure 3, Table 3). When vascular tissue is exposed to endotoxin or cytokines in vitro, NO synthase is induced in the endothelium and smooth muscle, and increased NO production leads to vasorelaxation and decreased responsiveness to vasoconstrictors.43’46”78 In the dog and rat, in vivo studies show that the vasodilatation and decreased vascular responsiveness that occur in response to endotoxin or cytokines is mediated by NO synthesis.‘7g-‘83 The induction of NO synthase with increased production of NO may also explain the peripheral vasodilatation and decreased responsiveness to vasoconstrictors that occur in response to endotoxin infusion in humans.‘84~‘85 In humans who received a small bolus infusion of endotoxin, the peripheral vasodilatation was not immediate but gradually appeared during the 2 hours after infusion of the endotoxin and persisted for another 4-5 hours.lM These findings are consistent with the hypothesis that an inducible NO synthase is responsible for peripheral vasodilatation following endotoxin infusion in humans, because the lag time for NO production by the inducible NO synthase in animals is also 2 hours.44 Recently, inhibitors of NO synthase were shown to increase blood pressure in two patients with septic shock, providing clinical support that NO production mediates endotoxin-induced hypotension.la6

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High levels of endotoxin are detected in the systemic circulation of a large percentage of cirrhotic patients.‘87-‘*g Thus, endotoxin could serve as a trigger for the induction of vascular NO synthase in cirrhosis. This systemic endotoxemia occurs in cirrhotic patients even in the absence of obvious infection and is thought to result from abnormal portal-systemic shunting, which allows systemic spillover of gut-derived bacterial endotoxin that would normally be cleared by the liver.‘90-‘g2 Besides hemodynamic similarities between the effects of endotoxin infusion and the hyperdynamic circulation of cirrhosis, a number of lines of evidence support the hypothesis that endotoxin-triggered production of vascular NO causes the hyperdynamic circulation of cirrhosis. In a parabiotic model of portal hypertension, hyperdynamic circulation was found in the portal hypertensive animal, but not the normal parabiotic partner.lg3 These results suggest that the hyperdynamic circulatory state is not mediated by a transferable humoral factor, although they are consistent with the production of the hyperdynamic state by a locally acting mediator with an ultrashort half-life, such as NO. Because NO-induced relaxation of vascular smooth muscle involves an increase in cGMP production,17-lg increased production of cGMP would be expected in cirrhosis if NO mediates the hyperdynamic circulation. In the rat, urinary excretion of cGMP is a biologic marker of the level of vascular NO synthesis.1~~1g5 In cirrhotic humans, significantly elevated urinary cGMP levels were detected even before the onset of ascites.lg6 Although atria1 natriuretic peptide can increase cGMP production and may be elevated in cirrhosis, many of the cirrhotic patients with elevated cGMP did not have elevated levels of atria1 natriuretic peptide, suggesting that increased urinary cGMP may have been related to increased vascular production and action of N0.1g6 In animals, glucocorticoids prevent the induction of vascular NO synthase by endotoxin and prevent the consequent decrease in vascular tone.444s Information on the hemodynamic effects of glucocorticoids in human cirrhosis is limited, but they may promote a diuresis in some patients with cirrhosis.lg7 Glucocorticoid therapy may decrease mortality in selected patients,lg* but this is thought to be due to an effect on the activity of the underlying liver disease,“’ and the precise effect of glucocorticoids on hemodynamics in humans with cirrhosis is unknown. In rats with chronic portal hypertension induced by portal vein ligation, a hyperdynamic circulatory state develops manifested by hypotension, systemic and splanchnic vasodilatation, and increased cardiac oUtpUt.200*201 In this model, inhibition of NO synthe-

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sis, after induction of portal hypertension and the development of the hyperdynamic circulatory state, causes significant systemic and splanchnic vasoconstriction.200~201There is also evidence that NO mediates arterial hypotension in rats with portal hypertension due to a carbon tetrachloride induced cirrhosis.“’ These results support the hypothesis that endogenous NO synthesis mediates the systemic and splanchnic vasodilatation observed in portal hypertensive animals. A recent case report described an elevation in blood pressure in response to methylene blue infusion in a patient with liver failure due to alcoholic cirrhosis and severe hypotension resistant to pressor agents203 Methylene blue blocks the action of NO through the inhibition of guanylate cyclase,204 providing clinical evidence that the generation of NO is responsible for the hypotension of liver failure. Although the data described above support the hypothesis that endotoxin-mediated induction of vascular NO synthase leads to the hyperdynamic circulation of cirrhosis, other experimental results do not support this hypothesis. Although chronic endotoxin infusion in rats produced ascites, it did not change blood pressure, and the production of ascites appeared to be related to increased vascular permeability.205 In portal vein-ligated rats with a well-developed hyperdynamic circulatory state, no evidence of endotoxemia was found, production of tolerance to endotoxin by repeated exposure did not ameliorate the hyperdynamic state, and a reduction of intestinal bacteria with antibiotic therapy did not alter the splanchnic hemodynamic status.2os Speculation on clinical implications. If the endotoxemia of cirrhosis does lead to induction of vascular NO synthase, and if increased NO synthesis and release is responsible for the hyperdynamic circulation of cirrhosis, a number of potential therapeutic interventions are suggested. Blockade of this pathway might prevent or reverse some of the complications of cirrhosis that may be related to systemic vasodilatation, including ascites, edema, and the hepatorenal syndrome. Selective intestinal decontamination with quinoline antibiotics207 might effectively reduce the aerobic gram negative bowel flora, decrease endotoxemia, and prevent the induction of vascular NO synthase. Glucocorticoids or, preferably, agents with greater specificity and less toxicity might be used to prevent induction of NO synthase by endotoxin and prevent the development of the hyperdynamic circulatory state. Inhibitors of NO synthase, used as medications to stop the increased vascular production of NO in the hyperdynamic circulatory state, could prove to be effective therapy for cirrhotic ascites or hepatorenal syndrome.

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NO and Treatment

GASTROINTESTINAL

of Portal Hypertension

AND HEPATIC NITRIC OXIDE

Table 4. Role of NO in HepatoceJJuJar

Function

1939

and

Hepatotoxicity

Medical therapy to prevent variceal bleeding. Hemorrhage from esophageal varices in patients with portal hypertension is associated with high morbidity and mortality despite aggressive resuscitation and therapy.208 Because of this, attempts have been made to prevent variceal hemorrhage by lowering portal venous pressures in patients with cirrhosis. Beta-blockers may prevent variceal bleeding,209-211 but the long-term efficacy of this therapy remains unclear,212~213and they have the potential for adverse effects.2*4*215Preliminary evidence suggests that pharmacological modulation of NO activity may be a useful new mechanism to lower portal pressures. Organic nitrates cause vasodilatation by activating guanylate cyclase in vascular smooth muscle.20~21 The principal mode of activation of guanylate cyclase by these drugs may be through the liberation of NO from the organic nitrate.‘Og Organic nitrates reduce portal pressure, and their use in the treatment of cirrhotic patients has been advocated.216-21g However, organic nitrates also reduce systemic blood pressure, and chronic administration can induce tolerance,220-222 which would limit their effectiveness in the chronic therapy of portal hypertension. Molsidomine, another drug that acts through the generation of NO, has been investigated as therapy for portal hypertension. 223-226Molsidomine is one of a class of drugs called syndnonimines and is a longacting vasodilator that is an established antianginal agent. It is metabolized to form an active metabolite called SIN-l, which produces vasodilatation by acting as an NO donor, leading to the stimulation of cGMP production in vascular smooth muscle.‘0g~227 Unlike the organic nitrates, molsidomine has little or no effect on systemic blood pressure in patients with normal livers,228~22gand it does not induce tolerance 227,230 In rats with carbon tetrachloride-induced cirrhosis and portal hypertension, molsidomine causes a significant decrease in portal venous pressure, apparently by reducing portal-collateral resistances without affecting hepatic blood flo~.“~ However, molsidomine also reduces systemic arterial pressure, increases portal venous inflow, and may increase collateral blood flo~.“~ Although a decrease in portal venous pressure would be expected, to be beneficial in patients with cirrhosis, the other effects seen with molsidomine in this animal study could be detrimental by decreasing renal perfusion or increasing pressure in collateral veins such as esophageal varices. In patients with cirrhosis and portal hypertension, a single dose of molsidomine causes a significant and sustained decrease in portal venous pressure.224.225 Molsidomine also causes moderate decreases in he-

Endogenous NO may modulate hepatocellular function in cell cultures NO produced by Kupffer cells in response to endotoxin inhibits hepatocyte protein synthesisz38-z41 Activated Kupffer cell products (cytokines) trigger NO production by hepatocytes which inhibits hepatocyte protein synthesis242,245-24’ Mechanism of protein synthesis inhibition unknown Does not involve cell death243 NO may inhibit mitochondrial respiration2*-*5” NO may modulate enzymatic steps of messenger RNA processin$51~25z Endogenous NO may have a role in hepatotoxicity NO from activated macrophages may mediate endotoxin or drug induced hepatotoxicityZ8*25”25g NO binds to cytochrome P459 enzymes and may alter enzyme activity and affect drug-induced hepatoxicity’g-2’~26’-*~ NO may act as an antioxidant to protect liver from oxidative injUry’48.‘49.2”7

NO may produce

highly reactive species and accentuate oxidate liver injury2s’Z7’ NO production by hepatocytes may mediate allograft response to liver transplantationz78-z~

patic blood flow and systemic arterial pressure, but intrinsic hepatic clearance of indocyanine green was not reduced, indicating that molsidomine reduced portal pressure without impairing liver elimination function.224 These results suggest that molsidomine may be an effective and safe long-term treatment for portal hypertension, but its efficacy in preventing variceal bleeding will need to be determined. Role of NO in Hepatocellular Hepatotoxicity NO and Hepatocellular

Function

and

Protein Synthesis

Hepatic dysfunction in sepsis. Patients with multisystem failure due to sepsis often have evidence of hepatocellular dysfunction manifested as elevations of bilirubin and liver-related enzymes as well as decreased serum albumin.231-234 The hypoalbuminemia probably has multiple mechanisms, but is at least partly due to a decrease in hepatocellular synthetic function. 235The mechanism of the hepatocellular dysfunction is poorly understood, but one hypothesis is that bacterial products such as endotoxins cause hepatocellular dysfunction directly or through the action of mediators released by macrophages such as interleukin 1 (IL-l) and tumor necrosing factor (TNF).235*236 Kupffer cells activated by endotoxin or cytokines are known to release multiple biologically active products such as IL-l and TNF.237 These products of endotoxin-triggered Kupffer cells may mediate the decrease in hepatocyte protein synthesis in sepsis.238-240 Recent evi-

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STARK AND SZURSZEWSKI

KUPFFER CELL

HEPATOCYTE

ENDOTOXIN Figure 4. A possible mechanism of NO-mediated inhibition hepatocyte protein synthesis. See text for details.

of

dence indicates that NO may also mediate decreases in hepatocyte protein synthesis (Figure 4, Table 4).

239,241,242

NO and regulation of hepatocyte protein synthesis. Evidence supporting a role for NO as an inhibitor of hepatocyte protein synthesis in sepsis comes from a model that measures protein synthesis in rat hepatocytes that are cultured alone or in the presence of Kupffer cells. Coculture of macrophages or Kupffer cells with hepatocytes normally promotes hepatocyte protein synthesis.238,240 The addition of lipopolysaccharide endotoxin or killed Escherichia coli to cocultured hepatocytes and Kupffer cells but not to hepatocytes alone induces a profound decrease in hepatocyte protein synthesis without inducing hepatocellular death.238,240The Kupffer cellmediated inhibition of hepatocyte protein synthesis is associated with production of nitrates, nitrites, and citrulline and is blocked by L-arginine analogs which competitively inhibit nitric oxide synthesis, suggesting that production of NO from L-arginine is required.23g In fact, direct chemical measurement of NO in the supernatant of endotoxin-stimulated hepatocyte Kupffer cell cocultures shows that NO is released.241 The time course of NO release parallels the decrease in hepatocyte protein synthesis.241 Because exogenous authentic NO also inhibits hepatocyte protein synthesis, 243these experiments indicate that NO is a mediator of decreased hepatocyte protein synthesis. Although stimulated Kupffer cells are the source for some of the NO production in this mode1,24**244 it also appears that humoral Kupffer cell products induce the formation of NO by hepatocytes and that hepatocyte production of NO also mediates decreased hepatocyte protein synthesis (Figure 242,245-247 When hepatocytes in culture are exposed 4). to a cell-free supernatant from stimulated macrophages, hepatocyte protein synthesis is inhibited and the hepatocytes produce large quantities of NO.245 The inhibition of protein synthesis directly parallels

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NO production, and both are blocked by an inhibitor of NO synthesis, indicating that hepatocyte NO production mediates inhibition of hepatocyte protein synthesis.245 The production of NO in both Kupffer cells and hepatocytes appears to involve an inducible NO synthase as there is a delay of several hours between stimulation and the production of N0.241,245Induction of NO synthase and NO production can be stimulated by endotoxin alone in Kupffer cells, but not hepatocytes.241*244*246Induction of the hepatocyte NO synthase and the subsequent decrease in protein synthesis appear to require multiple cytokines produced by activated Kupffer cells, including TNF and IL-1.247 The mechanism by which NO inhibits hepatocyte protein synthesis is not known. It does not involve hepatocyte cell death because the effect is reversible, no hepatocellular enzymes are released, and the cells remain viable based on trypan blue exclusion.243 Although NO induces a rapid increase in cGMP levels in hepatocytes, a cGMP analog did not reproduce the inhibition of total protein synthesis, indicating that NO does not inhibit hepatocyte protein synthesis solely through the activation of guanylate cyclase.243 NO generated by macrophages inactivates complex I and complex II of the electron transport chain28,248 and the Krebs cycle enzyme aconitase.24g NO also inhibits these mitochondrial enzymes in hepatocytes, but the inhibition of mitochondrial respiration recovers within l-2 hours.250 Thus, it does not appear to explain the inhibition of protein synthesis, which persists for up to 12 hours.243 Northern blot analysis of hepatocyte albumin mRNA levels shows that NO has no effect on the relative abundance of hybridizable albumin mRNA,243 indicating that NO inhibits hepatocyte protein synthesis through an undefined translational or posttranslational mechanism. In platelets, NO can modulate the activity of an enzyme which may be involved in posttranslational steps of protein synthesis,251 raising the possibility that similar NO-sensitive enzymes may mediate the effects of NO on hepatocyte protein synthesis. In contrast, other work has shown that the inhibition of hepatocyte albumin synthesis by IL-1 and TNF was due to a pretranslational mechanism.236’252 The mechanism and role of NO in the modulation of hepatocyte protein synthesis during sepsis remain unclear. Physiological significance and speculation on clinical implications. The role of NO in hepatocellular dysfunction seen in humans with sepsis and endotoxemia is not known, and its potential therapeutic importance is unclear, because hepatocellular dysfunction and decreased albumin synthesis are not major determinants of survival in patients with

December

1992

multiorgan failure due to sepsis.231 However, it can be hypothesized that NO formation is involved in the decreased albumin synthesis seen in some patients with cirrhosis253 or in the impaired synthesis of coagulation factors found in other liver diseases.2” It is also possible that endogenous NO may modulate other hepatocellular functions such as the metabolism of glucose or lipids, although in the rat liver NO does not appear to mediate agonist-stimulated glycogenolysis. 255If NO is shown to have a role in human hepatocellular dysfunction, therapeutic interventions involving alterations in NO production may be possible. NO and Hepatotoxicity Endotoxin and drug-induced hepatotoxicity. Treatment of experimental animals with endotoxin or certain hepatotoxic drugs is associated with intrahepatic accumulation of inflammatory macrophages256-25” thought to promote hepatic damage through the release of toxic secretory products.25g Because NO is one product of activated macrophages and NO produced by activated macrophages was cytotoxic to a hepatoma cell line in vitro,28 it is possible that NO formed by activated macrophages may be a mediator of endotoxin or drug-induced hepatotoxicity. There is no direct evidence supporting this model. In fact, in an in vivo murine model of endotoxin-induced hepatic necrosis with known increased hepatocellular production of NO,24s increased NO production was found to protect the liver from damage. In this model, inhibition of NO synthesis with an L-arginine analog decreased the endotoxin-induced increase in NO synthesis and markedly increased hepatic injury.2s0 The cellular source of the NO and the mechanism of the apparent protective action are not known. Another possible way that endogenous NO might affect drug-induced hepatotoxicity is by alterations of the activity of the cytochrome P45O enzyme system, which plays important roles in drug metabolism and hepatotoxicity.26* NO binds to heme-containing proteins, including cytochrome P450 enzymes.262s2s3 The effect of NO binding to hepatic cytochrome P45O enzymes is not known, but it is possible that NO binding may influence drug-induced hepatotoxicity by altering the activity of these enzymes because NO binding has been shown to activate or inhibit the activity of related enzymes in other tissues.1g,21*264 NO and oxidative liver injury. Evidence indicates that reactive oxygen intermediates are pathogenic in several types of liver injurys2@j Reactive oxygen intermediates are substances with one or more unpaired electrons, and include superoxide anion, hydrogen peroxide, and hydroxyl radical. These substances have been linked to membrane and DNA

GASTROINTESTINAL

AND HEPATIC

NITRIC OXIDE

1941

damage, lipid peroxidation reactions, and to induction of hepatocyte killing.2e5*266 Some evidence suggests that NO may act as an antioxidant and may interact with superoxide anion and other radicals to produce less toxic species.‘4e*14g*267 Because reactive oxygen intermediates may mediate liver injury induced by macrophages and endotoxin,2m an antioxidant function of NO might explain apparent protective effect of NO synthesis in the murine model of endotoxin-induced hepatic necrosis discussed abovee2@’In contrast, other evidence suggests that NO may interact with reactive oxygen intermediates to form more toxic species. The reaction of NO with superoxide anion can produce the peroxynitrite anion, which can decompose to generate a strong oxidant with reactivity similar to hydroxyl radica1.26g Peroxynitrite can induce sulfhydryl oxidation270 and lipid peroxidation, suggesting that NO may have cytotoxic potential through its interaction with superoxide anion. Whether NO acts to attenuate or potentiate tissue injury from reactive oxygen intermediates, it may have an important role in conditions in which reactive oxygen intermediates may mediate hepatotoxicity, including ischemia-reperfusion injury in hepatic allografts, drug-induced hepatotoxicity, and immune-mediated liver damage.2e5 NO and immune-mediated hepatotoxicity. NO appears to play important roles in the function of the immune system as a cytotoxic macrophage effector mo1ecu1e,26~272’273 a modulator of neutrophil chemotaxis and adhesion,‘50p274 a mediator of tissue injury caused by deposition of immune complexes,275 and a regulator of lymphocyte proliferation.276*277 Thus, it would not be surprising to find a role for NO in liver diseases which may be mediated by the immune system, such as autoimmune hepatitis or viral hepatitis, although there is currently no direct evidence for this role. NO may be a potent inhibitory molecule in the in vitro rat splenocyte mixed-lymphocyte culture system, resulting in inhibition of allospecific proliferation and cytolytic T-cell induction.27” Recently NO production was shown by allograft-infiltrating macrophages in the rat sponge matrix allograft model of the allograft response,27g and the immunomodulatory drug FK506 was shown to inhibit NO production in these cells.280 These results suggest that NO may play a role in the immune response to hepatic transplantation. Conclusion The idea that an evanescent gas like NO acts as an important biologic mediator in diverse systems throughout the body at first seems as unbelievable as Galen’s concept of an ethereal “pneuma” flowing

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through the blood vessels and nerves, especially since a biologic function for NO was undiscovered until a few years ago. However, unlike Galen’s “pneuma,” there is considerable evidence supporting an important role for NO in normal physiological function and disease. This review has focused on the evidence that NO has important roles in normal function and diseases of the gastrointestinal tract and liver. Abundant evidence indicates that NO mediates NANC inhibition in gastrointestinal smooth muscle in animals and humans and suggests that NO may be a new and unusual neurotransmitter or neuromodulator. Preliminary evidence in animals indicates that endogenous NO may protect the gastrointestinal mucosal from injury. In animals and humans, endotoxin-stimulated vascular production of NO may mediate the hyperdynamic circulation of cirrhosis and may play a role in the development of ascites and hepatorenal syndrome. NO may play a role in the control of hepatic function because NO produced by Kupffer cells and hepatocytes inhibits protein synthesis in cultured hepatocytes. NO may mediate some forms of hepatotoxicity. It may be possible to treat some disorders by augmenting or inhibiting the activity of endogenous NO. However, NO appears to be a ubiquitous mediator. Nonspecific inhibition of NO synthase could lead to impairment of immune function, neurologic dysfunction, or other harmful effects. In intestinal mucosa and liver, endogenous NO appears to protect against the toxic effects of endotoxin.‘40”41~2s Because NO appears to modulate hepatic arterial blood flow 281*282 changes in endogenous NO could have harmful effects on hepatic function. In rats, inhibition of NO synthase prevents endotoxin-induced hypotension, but the use of a higher dose of the NO synthase inhibitor worsens hypotension, suggesting that complete inhibition of NO synthesis may be harmful.lB3 It may be possible to develop r_,-arginine analogs38*3g or other types of NO synthase inhibitors283 that inhibit only the NO synthase of a specific cell type or vascular region. It is likely that further studies of the role of NO will have important clinical implications in gastroenterology and hepatology. References

6.

7. 8.

9.

10.

11.

12.

13.

14.

3.

4.

5.

lial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 1980;288:373-376. lgnarro LJ, Buga GM, Wood KS, Byrns RE, Chaudhuri G. Endothelium-derived relaxing factor produced and released from artery and vein is nitric oxide. Proc Nat1 Acad Sci USA 1987;84:9265-9269, lgnarro LJ. Endothelium-derived nitric oxide: actions and properties. FASEB J 1989;3:31-36. Rubanyi GM, Vanhoutte PM. Superoxide anions and hyperoxia inactivate endothelium-derived relaxing factor. Am J Physiol 1986;250:H822-H827. Gryglewski RJ, Palmer RMJ, Moncada S. Superoxide anion is involved in the breakdown of endothelium-derived vascular relaxing factor. Nature 1986;320:454-456. Martin W, Smith JA, White DG. The mechanisms by which haemoglobin inhibits the relaxation of rabbit aorta induced by nitrovasodilators, nitric oxide or bovine retractor penis inhibitory factor. Br J Pharmacol 1986;89:563-571. Palmer RMJ, Ashton DS, Moncada S. Vascular endothelial cells synthesize nitric oxide from L-arginine. Nature 1988;333:664-666. Palmer RMJ, Moncada S. A novel citrulline-forming enzyme implicated in the formation of nitric oxide by vascular endothelial cells. Biochem Biophys Res Commun 1989;158:348352. Busse R, Mttlsch A. Calcium-dependent nitric oxide synthesis in endothelial cytosol is mediated by calmodulin. FEBS Lett 1990;265:133-136. Rees DD, Palmer RMJ, Hodson HF, Moncada S. A specific inhibitor of nitric oxide formation from L-arginine attenuates endothelium-dependent relaxation. Br J Pharmacol

1989;96:418-424. 15. Mtilsch A, Busse R. NG-nitro+arginine

16.

17.

18.

19.

20.

1. May MA. Galen: On the Usefulness 2.

of the Parts of the Body. Ithaca: Cornell University, 1968:44-64. Major RH. A History of Medicine. Springfield, IL: Charles C. Thomas, 1954:188-202. Palmer RMJ, Ferrige AG, Moncada S. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature 1987;327:524-526. Moncada S, Palmer RMJ, Higgs EA. Nitric oxide: Physiology, and phermacology. Pharmacol Rev pathophysiology, 1991;43:109-142. Furchgott RF, Zawadzki JV. The obligatory role of endothe-

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21.

22.

(INS-[imino(nitroamino)methyl]-L-ornithine) impairs endothelium-dependent dilations by inhibiting cytosolic nitric oxide synthesis from L-arginine. Naunyn Schmiedebergs Arch Pharmacol 1990;341:143-147, Rees DD, Palmer RMJ, Schulz R, Hodson HF, Moncada S. Characterization of three inhibitors of endothelial nitric oxide synthase in vitro and in vivo. Br J Pharmacol 1990;101:746-752. Wolin MS, Wood KS, lgnarro LJ. Guanylate cyclase from bovine lung. A kinetic analysis of the regulation of the purified soluble enzyme by protoporphyrin IX, heme, and nitrosylheme. J Biol Chem 1982;257:13312-13320. lgnarro LJ, Wood KS, Wolin MS. Regulation of purified soluble guanylate cyclase by porphyrins and metalloporphyrins: a unifying concept. Adv Cyclic Nucleotide Protein Phosphorylation Res 1984;17:267-274. Gruetter CA, Gruetter DY, Lyon JE, Kadowitz PJ, lgnarro LJ. Relationship between cyclic guanosine 3’:5’-monophosphate formation and relaxation of coronary arterial smooth muscle by glyceryl trinitrate, nitroprusside, nitrite and nitric oxide: Effects of methylene blue and methemoglobin. J Pharmacol Exp Ther 1981;219:181-186. Murad F, Mittal CK, Arnold WP, Katsuki S, Kimura H. Guanylate cyclase: activation by azide, nitro compounds, nitric oxide and hydroxyl radical and inhibition by hemoglobin and myoglobin. Adv Cyclic Nucleotide Res 1978;9:145-158. Katsuki S, Arnold W, Mittal C, Murad F. Stimulation of guanylate cyclase by sodium nitroprusside, nitroglycerin and nitric oxide in various tissue preparations and comparison to the effects of sodium azide and hydroxylamine. J Cyclic Nucleotide Res 1977;3:23-25. Valiance P, Collier J, Moncada S. Effects of endothelium-derived nitric oxide on peripheral arteriolar tone in man. Lancet 1989;2:997-1000.

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1992

23. Valiance P, Collier J, Moncada S. Nitric oxide synthesized from L-arginine mediates endothelium dependent dilatation in human veins in vivo. Cardiovasc Res 1989;23:1053-1057. 24.Radomski MW, Palmer RMJ, Moncada S. Comparative pharmacology of endothelium-derived relaxing factor, nitric oxBr J Pharmacol ide and prostacyclin in platelets. 1987;92:181-187. 25. Radomski MW, Palmer RMJ, Moncada S. Endogenous nitric oxide inhibits human platelet adhesion to vascular endothelium. Lancet 1987;2:1057-1058. 26. Radomski MW, Palmer RMJ, Moncada S. Characterization of the L-arginine: nitric oxide pathway in human platelets. Br J Pharmacol1990;101:325-328. 27. Radomski MW, Palmer RMJ, Moncada S. An L-arginine/nitric oxide pathway present in human platelets regulates aggregation. Proc Nat1 Acad Sci USA 1990;87:5193-5197. 28. Hibbs JB Jr., Taintor RR, Vavrin Z, Rachlin EM. Nitric oxide: A cytotoxic activated macrophage effector molecule. Biothem Biophy Res Commun 1988;157:87-94. 29. Stuehr DJ, Gross SS, Sakuma I, Levi R, Nathan CF. Activated murine macrophages secrete a metabolite of arginine with the bioactivity of endothelium-derived relaxing factor and the chemical reactivity of nitric oxide. J Exp Med 1989:169:1011-1020. 30. Salvemini D, DeNucci G, Gryglewski RJ, Vane JR. Human neutrophils and mononuclear cells inhibit platelet aggregation by releasing a nitric oxide-like factor. Proc Nat1 Acad Sci USA 1989;86:6328-6332, 31. Liew FY, Millott S, Parkinson C, Palmer RMJ, Moncada S. Macrophage killing of Leishmania parasite in vivo is mediated by nitric oxide from L-arginine. J Immunol 1990;144:4794-4797. 32. Garthwaite J, Charles SL, Chess-Williams R. Endotheliumderived relaxing factor release on activation of NMDA receptors suggests role as intercellular messenger in the brain. Nature 1988;336:385-388. 33. Knowles RG, Palacios M, Palmer RMJ, Moncada S. Formation of nitric oxide from L-arginine in the central nervous system: A transduction mechanism for stimulation of the soluble guanylate cyclase. Proc Nat1 Acad Sci USA 1989;86:5159-5162. 34. Bredt DS, Snyder SH. Nitric oxide mediates glutamatelinked enhancement of cGMP levels in the cerebellum. Proc Nat1 Acad Sci USA 1989;86:9030-9033. 35. Palacios M, Knowles RG, Palmer RMJ, Moncada S. Nitric oxide from L-arginine stimulates the soluble guanylate cyclase in adrenal glands. Biochem Biophys Res Commun 1989; 165:802-809. 36. Ishii K, Gorsky LD, Forstermann U, Murad F. Endotheliumderived relaxing factor (EDRF): the endogenous activator of soluble guanylate cyclase in various types of cells. J Appl Cardiol 1989;4:505-512. 37. Salvemini D, Masini E, Anggard E, Mannaioni PF, Vane J. Synthesis of a nitric oxide-like factor from L-arginine by rat serosal mast cells: Stimulation of guanylate cyclase and inhibition of platelet aggregation. Biochem Biophys Res Commun 1990;169:596-601. 38. Gross SS, Stuehr DJ, Aisaka K, Jaffe EA, Levi R, Griffith OW. Macrophage and endothelial cell nitric oxide synthesis: Celltype selective inhibition by NG-aminoarginine, NC-nitroarginine and No-methylarginine. Biochem Biophys Res Commun 1990;170:96-103. 39. Lambert LE, Whitten JP, Baron BM, Cheng HC, Doherty NS, McDonald IA. Nitric oxide synthesis in the CNS, endothelium and macrophages differs in it sensitivity to inhibition by arginine analogues. Life Sci 1991;48:69-75. 40. Hibbs JB, Jr., Taintor RR, Vavrin Z. Macrophage cytotoxicity:

GASTROINTESTINAL

AND HEPATIC NITRIC OXIDE

1943

role for L-arginine deiminase activity and imino nitrogen oxidation to nitrite. Science 1987;235:473-476. 41. Stuehr DJ, Marletta MA. Induction of nitrite/nitrate synthesis in murine macrophages by BCG infection, lymphokines or interferer+. J Immunol 1987;139:518-525, 42. Stuehr DJ, Marletta MA. Synthesis of nitrite and nitrate in murine macrophage cell lines. Cancer Res 1987;47:55905594. 43. Busse R, Miilsch A. Induction of nitric oxide synthase by cytokines in vascular smooth muscle cells. FEBS Lett 1990;275:87-90. 44. Radomski MW, Palmer RMJ, Moncada S. Glucocorticoids inhibit the expression of an inducible, but not constitutive, nitric oxide synthase in vascular endothelial cells. Proc Nat1 Acad Sci USA 1990;87:10043-10047. 45. Knowles RG, Salter M, Brooks SL, Moncada S. Anti-inflammatory glucocorticoids inhibit the induction by endotoxin of nitric oxide synthase in the lung, liver and aorta of the rat. Biochem Biophys Res Commun 1990;172:1042-1048, 46. Rees DD, Cellek S, Palmer RMJ, Moncada S. Dexamethasone prevents the induction by endotoxin of a nitric oxide synthase and the associated effects on vascular tone: An insight into endotoxin shock. Biochem Biophys Res Commun 1990;173:541-547. 47. Fleming I, Gray GA, Schott C, Stoclet JC. Inducible but not constitutive production of nitric oxide by vascular smooth muscle cells. Eur J Pharmacol 1991;200:375-376. 48. Bayliss WM, Starling EH. The movements and innervation of the small intestine. J Physiol (Lond) 1899;24:99-143. 49. Hoyle CHV, Burnstock G. Neuromuscular transmission in the gastrointestinal tract. In: Wood JD, ed. Handbook of Physiology. The Gastrointestinal System. Motility and Circulation. Bethesda: American Physiological Society, 1989:435464. 50. Bauer AJ, Sarr MG, Szurszewski JH. Opioids inhibit neuromuscular transmission in circular muscle of human and baboon jejunum. Gastroenterology 1991;101:970-976. 51. Burnstock G, Satchel1 DG, Smythe A. A comparison of the excitatory and inhibitory effects of non-adrenergic, noncholine@ nerve stimulation and exogenously applied ATP on a variety of smooth muscle preparations from different vertebrate species. Br J Pharmacol 1972;46:234-242. 52. Manzini S, Maggl CA, Meli A. Further evidence for involvement of adenosine-5’-triphosphate in non-adrenergic noncholinergic relaxation of the isolated rat duodenum. Eur J Pharmacol 1985;113:399-408. 53. Burnstock G, Cocks T, Kasakov L, Wong HK. Direct evidence for ATP release from non-adrenergic, non-cholinergic (“purinergic”) nerves in the guinea-pig taenia coli and bladder. Eur J Pharmacol1978;49:145-149, 54. Manzini S, Maggi CA, Meli A. Pharmacological evidence that at least two different non-adrenergic non-cholinergic inhibitory systems are present in the rat small intestine. Eur J Pharmacol 1986;123:229-236. 55. Goyal RK, Rattan S, Said S. VIP as a possible neurotransmitter of non-cholinergic non-adrenergic inhibitory neurons. Nature 1980;288:378-380. 56. Biancani P, Walch JH, Behar J. Vasoactive intestinal polypeptide: a neurotransmitter for lower esophageal sphincter relaxation. J Clin Invest 1984;73:963-967. 57. Behar J, Guenard V, Walsh JF, Biancani P. VIP and acetylcholine: neurotransmitters in esophageal circular smooth muscle. Am J Physiol 1989;257:G380-G385. 58. De Beurme FA, Lefebvre RA. Vasoactive intestinal polypeptide as possible mediator of relaxation in the rat gastric fundus. J Pharm Pharmacol1988;40:711-715. 59. Grider JR, Cable MB, Bitar KN, Said SI. Makhlouf GM. Va-

1944

STARK AND SZURSZEWSKI

soactive intestinal peptide; relaxant neurotransmitter in tenia coli of the guinea pig. Gastroenterology 1985;89:36-42. 60. Furness JB, Costa M. Influence of the enteric nervous system on motility. In: The enteric nervous system. Edinburgh: Churchill Livingston, 1987,137-189. 61. Niel JP, Bywater RAR, Taylor GS. Apamin-resistant poststimulus hyperpolarization in the circular muscle of the guinea-pig ileum. J Auton Nerv Syst 1983;9:565-569. 62. Costa M, Furness JB, Humphreys CMS. Apamin distinguishes two types of relaxation mediated by enteric nerves in the guinea-pig gastrointestinal tract. Naunyn Schmiedebergs Arch Pharmacol1986;332:79-88. 63. Gillespie JS. The rat anococcygeus and its response to nerve stimulation and to some drugs. Br J Pharmacol1972;45:404416. 64. Bowman A, Drummond AH. Cyclic GMP mediates neurogenie relaxation in the bovine retractor penis muscle. Br J Pharmacol1984;81:665-674. 65. Bowman A, Gillespie JS, Pollock D. Oxyhaemoglobin blocks non-adrenergic non-cholinergic inhibition in the bovine retractor penis muscle. Eur J Pharmacol 1982;85:221-224. 66. Gillespie JS, Liu X, Martin W. The effects of L-arginine and Nc-monomethyl L-arginine on the response of the rat anococcygeus muscle to NANC nerve stimulation. Br J Pharmacol 1989;98:1080-1082. 67. Gibson A, Mirzazadeh S, Hobbs AJ, Moore PK. L-Nc-monomethyl arginine and L-Nc-nitro arginine inhibit non-adrenergic, non-cholinergic relaxation of the mouse anococcygeus muscle. Br J Pharmacol 1990;99:602-606. 68. Gillespie JS, Sheng H. The effects of pyrogallol and hydroquinone on the response to NANC nerve stimulation in the rat anococcygeus and the bovine retractor penis muscle. Br J Pharmacol1990;99:194-196. 69. Bult H, Boeckxstaens GE, Pelckmans PA, Jordaens FH, Van Maercke YM, Herman AG. Nitric oxide as an inhibitory nonneurotransmitter. Nature adrenergic non-cholinergic 1990;345:346-347, 70. Furness JB, Costa M. Transmitter neurochemistry of enteric neurons. In: The enteric nervous system. Edinburgh: Churchill Livingston, 1987:55-89. 71. Boeckxstaens GE, Pelckmans PA, Bogers JJ, Bult H, De Man JG, Oosterbosch L, Herman AG, Van Maercke YM. Release of nitric oxide upon stimulation of nonadrenergic noncholinergic nerves in the rat gastric fundus. J Pharmacol Exp Ther 1991;256:441-447. 72. Boeckxstaens GE, Pelckmans PA, Bult H, De Man JG, Herman AG, Van Maercke YM. Non-adrenergic, non-cholinergic relaxation mediated by nitric oxide in the canine ileocoionic junction. Eur J Pharmacol1990;190:239-246. 73. Toda N, Baba H, Okamura T. Role of nitric oxide in nonadrenergic, non-cholinergic nerve-mediated relaxation in dog duodenal longitudinal muscle strips. Jpn J Pharmacol 1990;53:281-284. 74. De Man JG, Pelckmans PA, Boeckxstaens GE, Bult H, Oosterbosch L, Herman AG, Van Maercke YM. The role of nitric oxide in inhibitory non-adrenergic non-cholinergic neurotransmission in the canine lower oesophageal sphincter. Br J Pharmaco11991;103:1092-1096. 75. Stark ME, Bauer AJ, Szurszewski JH. Effect of nitric oxide on circular muscle of the canine small intestine. J Physiol (Lond) 1991;444:743-761. 76. Murray J, Du C, Ledlow A, Bates JN, Conklin JL. Nitric oxide: mediator of nonadrenergic noncholinergic responses of opossum esophageal muscle. Am J Physiol 1991;261:G401G406. 77.Rattan S, Chakder S. Role of nitric oxide as a mediator of

GASTROENTEROLOGY Vol. 103, No. 6

internal anal sphincter relaxation. Am J Physiol 1992; 262:G107-G112. 78. Hata F, Ishii T, Kanada A, Yamano N, Kataoka T, Takeuchi T, Yagasaki 0. Essential role of nitric oxide in descending inhibition in the rat proximal colon. Biochem Biophys Research Commun 1990;172:1400-1406. 79. Thornbury KD, Ward SM, Dalziel HH, Carl A, Westfall DP, Sanders KM. Nitric oxide and nitrosocysteine mimic nonadrenergic, noncholinergic hyperpolarization in canine proximal colon (rapid communication). Am J Physiol 1991; 261:G553-G557. 80. Ward SM, Dalziel HH, Thornbury KD, Westfall DP, Sanders KM. Nonadrenergic, noncholinergic inhibition and rebound excitation in canine colon depend on nitric oxide. Am J Physiol 1992;262:G237-G243. 81. Ward SM, McKeen ES, Sanders KM. Role of nitric oxide in non-adrenergic, non-cholinergic inhibitory junction potentials in canine ileocolonic sphincter. Br J Pharmacol 1992;105:776-782. 82. Stark ME, Bauer AJ, Sarr MG, Szurszewski JH. Nitric oxide mediates NANC inhibition by different mechanisms in human and canine jejunum. Gastroenterology (in press). 83. Shuttleworth CWR, Murphy R, Furness JB. Evidence that nitric oxide participates in non-adrenergic inhibitory transmission to intestinal muscle in the guinea-pig. Neuroscience Lett 1991;130:77-80. 84. Li CG, Rand MJ. Nitric oxide and vasoactive intestinal polypeptide mediate non-adrenergic, non-cholinergic inhibitory transmission to smooth muscle of the rat gastric fundus. Eur J Pharmacol1990;191:303-309. 85. Tettrup A, Svane D, Forman A. Nitric oxide mediating NANC inhibition in opossum lower esophageal sphincter. Am J Physiol 1991;260:G385-G389. 86. Huizinga JD, Tomlinson J, Pintin-Quezada J. Involvement of nitric oxide in nerve-mediated inhibition and action of vasoactive intestinal peptide in colonic smooth muscle. J Pharmat Exp Ter 1992;260:803-808. 87. Tettrup A, Glavind EB, Svane D. Involvement of the L-arginine-nitric oxide pathway in internal anal sphincter relaxation Gastroenterology 1992;102:409-415, 88. Burleigh DE. N’nitro-L-arginine reduces nonadrenergic, noncholinergic relaxations of the human gut. Gastroenterology 1992;102:679-683, 89. Christinck F, Jury J, Cayabyab F, Daniel EE. Nitric oxide may be the final mediator of nonadrenergic, noncholinergic inhibitory junction potentials in the gut. Can J Physiol Pharmacol 1991;69:1448-1458. 90. Dalziel HH, Thornbury KD, Ward SM, Sanders KM. Involvement of nitric oxide synthetic pathway in inhibitory junction potentials in canine proximal colon. Am J Physiol 1991;260:G789-G792. 91. Ignarro LJ, Bush PA, Buga GM, Rajfer J. Neurotransmitter identity doubt (letter). Nature 1990;347:131-132. 92. Bredt DS, Hwang PM, Snyder SH. Localization of nitric oxide synthase indicating a neural role for nitric oxide. Nature 1990;347:768-770. 93. Grider JR, Murthy KS, Jin JG, Makhlouf GM. Stimulation of nitric oxide from muscle cells by VIP: prejunctional enhancement of VIP release. Am J Physiol 1992;262:G774-G778. 94. Wood KS, Buga GM, Byrns RE, Ignarro LJ. Vascular smooth muscle-derived relaxing factor (MDRF) and its close similarity to nitric oxide. Biochem Biophys Res Commun 1990;170:80-88. 95. Ignarro LJ. Nitric oxide: A novel signal transduction mechanism for transcellular communication. Hypertension 1990; 16:477-483. 96. Mtilsch A, Mordvintcev P, Vanin AF, Busse R. The potent

December

97.

98.

99.

100.

101.

102.

103.

104.

105.

106.

107.

108.

109.

110.

111.

112. 113.

114.

1992

vasodilating and guanylyl cyclase activating dinitrosyliron(B) complex is stored in a protein-bound form in vascular tissue and is released by thiols. FEBS Lett 1991;294:252256. Allescher HD, Tougas G, Vergara P, Lu S, Daniel EE. Nitric oxide as a putative nonadrenergic, noncholinergic inhibitory transmitter in the canine pylorus in vivo. Am J Physiol 1992;262:G695-G702. Ozaki H, Blondfield DP, Hori M, Publicover NG, Kato I, Sanders KM. Spontaneous release of nitric oxide inhibits electrical Ca’+ and mechanical transients in canine gastric smooth muscle. J Physiol (Lond) 1991;445:231-247. Tettrup A, Knudsen MA, Gregersen H. The role of the L-arginine-nitric oxide pathway in relaxation of the opossum lower oesophageal sphincter. Br J Pharmacol 1991;104:113116. Whittle BRJ, Lopez-Belmonte J, Moncada S. Regulation of gastric mucosal integrity by endogenous nitric oxide: interactions with prostanoids and sensory neuropeptides in the rat. Br J Pharmacol 1990;99:607-611. Brown JF, Tepperman BL, Hanson PJ, Whittle BJR, Moncada S. Differential distribution of nitric oxide synthase between cell fractions isolated from the rat gastric mucosa. Biochem Biophys Res Commun 1992;184:680-685. Miller CA, Barnette MS, Ormsbee HS III, Torphy TJ. Cyclic nucleotide-dependent protein kinases in the lower esophageal sphincter. Am J Physiol 1986;251:G794-G803. Grous M, Joslyn AF, Thompson W, Barnette MS. Change in intracellular cyclic nucleotide content accompanies relaxation of the isolated canine internal anal sphincter. J Gastrointestinal Motility 1991;3:46-52. Barnette MS, Barone FC, Fowler PJ, Grous M, Price WJ, Ormsbee HS. Human lower oesophageal sphincter relaxation is associated with raised cyclic nucleotide content. Gut 1991:32:4-g. Barnette M, Torphy TJ, Grous M, Fine C, Ormsbee HS III. Cyclic GMP: a potential mediator of neurally- and drug-induced relaxation of opossum lower esophageal sphincter. J Pharmacol Exp Ther 1989;249:524-528. Torphy TJ, Fine CF, Burman M, Barnette MS, Ormsbee HS III. Lower esophageal sphincter relaxation is associated with increased cycle nucleotide content. Am J Physiol 1986; 251:G786-G793. Shikano K, Long CJ, Ohlstein EH, Berkowitz BA. Comparative pharmacology of endothelium-derived relaxing factor and nitric oxide. J Pharmacol Exp Ther 1988;247:873-881. Ramagopal MV, Leighton HJ. Effects of Nc-monomethyl-Larginine on field stimulation-induced decreases in cytosolic Ca’+ levels and relaxation in the rat anococcygeus. Eur J Pharmacol 1989;174:297-299. Feelisch M, Noack EA. Correlation between nitric oxide formation during degradation of organic nitrates and activation of guanylate cyclase. Eur J Pharmacol 1987;139:19-30. Boeckxstaens GE, Pelckmans PA, Bult H, De Man JG, Herman AG, Van Maercke YM. Evidence for nitric oxide as mediator of non-adrenergic, non-cholinergic relaxations induced by ATP and GABA in the canine gut. Br J Pharmacol 1991;102:434-438. Irie K, Muraki T, Furukawa K, Nomoto T. L-Nc-Nitro-arginine inhibits nicotine-induced relaxation of isolated rat duodenum. Eur J Pharmacol 1991;202:285-288. Jule Y. Nerve-mediated descending inhibition in the proximal colon of the rabbit. J Physiol (Lond) 1980;309:487-498. Costa M, Furness JB. The peristaltic reflex: an analysis of the nerve pathways and their pharmacology. Naunyn Schmiedebergs Arch Pharmacol 1976;294:47-60. Paton WDM, Vane JR. An analysis of the responses of the

GASTROINTESTINAL

115. 116.

117.

118.

119.

120.

121. 122.

123.

124.

125.

126.

127.

128.

129.

130.

131.

132.

133.

134.

AND HEPATIC NITRIC OXIDE

1945

isolated stomach to electrical stimulation and to drugs. J Physiol (Lond) 1963;165:10-46. Abrahamsson H. Studies on the inhibitory nervous control of gastric motility. Acta Physiol Stand Suppl 1973;390:1-38. Desai KM, Sessa WC, Vane JR. Involvement of nitric oxide in the reflex relaxation of the stomach to accommodate food or fluid. Nature 1991;351:477-479. Lefebvre RA, Hasrat J, Gobert A. Influence of Nc-nitro+arginine methyl ester on vagally induced gastric relaxation in the anaesthetized rat. Br J Pharmacol 1992;105:315-320. Czaja AJ, McAlhany JC, Pruitt BA Jr. Acute gastroduodenal disease after thermal injury: an endoscopic evaluation of incidence and natural history. N Engl J Med 1974;291:925-929. Kamada T, Fusamoto H, Kawano S, Hoguchi M, Hiramatsu K, Masuzawa M, Abe H, Fujii C, Sugimoto T. Gastrointestinal bleeding following head injury: a clinical study of 433 cases. J Trauma 1977;17:44-47. Peura DA, Johnson LF. Cimetidine for prevention and treatment of gastroduodenal mucosal lesions in patients in an intensive care unit. Ann Intern Med 1985;103:173-177. Sol1 AH. Nonsteroidal anti-inflammatory drugs and peptic ulcer disease. Ann Intern Med 1991;114:307-319. Robert A, Leung FW, Kaiser DG, Guth PH. Potentiation of aspirin-induced gastric lesions by exposure to cold in rats; role of acid secretion, mucosal blood flow and gastric mucosal prostanoid content. Gastroenterology 1989;97:1147-1158. Whittle BJR. The defensive role played by the gastric microcirculation. Methods Find Exp Clin Pharmacol 1989; ll(Supp1 1):35-38. Whittle BJR, Boughton-Smith NK, Moncada S, Vane JR. Actions of prostacyclin (PGI,) and its product, 6-oxo-PGF,, on the rat gastric mucosa in vivo and in vitro. Prostaglandins 1978;15:955-967. Esplugues JV, Whittle BJR, Moncada S. Local opioid-sensitive afferent sensory neurones in the modulation of gastric damage induced by Paf. Br J Pharmacol1989;97:579-585. Whittle BJR, Kauffman GL, Moncada S. Vasoconstriction with thromboxane A, induces ulceration of the gastric mucosa. Nature 1981;292:472-474. Wallace JL, Cirino G, De Nucci G, McKnight W, MacNaughton WK. Endothelin has potent ulcerogenic and vasoconstrictor actions in the stomach. Am J Physiol 1989; 256:G661-G666. Pique JM, Whittle BJR, Esplugues JV. The vasodilator role of endogenous nitric oxide in the rat gastric microcirculation. Eur J Pharmacol1989;174:293-296. Kitagawa H, Takeda F, Kohei H. Endothelium-dependent increases in rat gastric mucosal hemodynamics induced by acetylcholine and vagal stimulation. Eur J Pharmacol 1987;133:57-63. Thomas GR, Thiemermann C, Walder C, Vane JR. The effects of endothelium-dependent vasodilators on cardiac output and their distribution in the anaesthetized rat: a comparison with sodium nitroprusside. Br J Pharmacol 1988; 95:986-992. Walder CE, Thiemermann C, Vane JR. Endothelium-derived relaxing factor participates in the increased blood flow in response to pentagastrin in the rat stomach mucosa. Proc Roy Sot Lond (Biol) 1990;241:195-200, Pique JM, Esplugues JV, Whittle BJR. Endogenous nitric oxide as a mediator of gastric mucosal vasodilatation during acid secretion. Gastroenterology 1992;102:168-174. MacNaughton WK, Cirino G, Wallace JL. Endothelium-derived relaxing factor (nitric oxide) has protective actions in the stomach. Life Sci 1989;45:1869-1876. Peskar BM, Respondek M, Mtiller KM, Peskar BA. A role for

1946

135.

136.

137.

138.

139. 140.

141.

142.

143.

144.

145. 146.

147. 148.

149. 150.

151.

152.

153. 154.

STARK AND SZURSZEWSKI

nitric oxide in capsaicin-induced gastroprotection. Eur ) Pharmacol1991;198:113-114. Guth PH, Paulsen G, Nagata H. Histologic and microcirculatory changes in alcohol-induced gastric lesions in the rat: effect of prostaglandin cytoprotection. Gastroenterology 1984;87:1083-1090. Szabo S, Trier JS, Brown A, Schnoor J. Early vascular injury and increased vascular permeability in gastric mucosal injury caused by ethanol in the rat. Gastroenterology 1985;88:228-236. Lefer AM, Tsao PS, Lefer DJ, Ma X-L. Role of endothelial dysfunction in the pathogenesis of reperfusion injury after myocardial ischemia. FASEB J 1991;5:2029-2034. Dembinska-Kiec A, Pallapies D, Simmet T, Peskar BM, Peskar BA. Effect of carbenoxolone on the biological activity of nitric oxide: relation to gastroprotection. Br J Pharmacol 1991;104:811-816. Rackow EC, Astiz ME. Pathophysiology and treatment of septic shock. JAMA 1991;266:548-554. Hutcheson IR, Whittle BJR, Boughton-Smith NK. Role of nitric oxide in maintaining vascular integrity in endotoxin-induced acute intestinal damage in the rat. Br J Pharmacol 1990;101:815-820. Boughton-Smith NK, Hutcheson IR, Deakin AM, Whittle BJR, Moncada S. Protective effect of.%nitroso-N-acetyl-penicillamine in endotoxin-induced acute intestinal damage in the rat. Eur J Pharmacol1990;191:485-488. Wallace JL, Steel G, Whittle BJR, Lagente V, Vargaftig B. Evidence for platelet-activating factor as a mediator of endotoxin-induced gastrointestinal damage in the rat. Effects of three platelet-activating factor antagonists. Gastroenterology 1987;93:765-773. Whittle BJR, Boughton-Smith NK, Hutcheson IR, Esplugues JV, Wallace JL. Increased intestinal formation of Paf in endotoxin-induced damage in the rat. Br J Pharmacol 1987; 92:3-4. Boughton-Smith NK, Hutcheson I, Whittle BJR. Relationship between Pafacether and thromboxane A, biosynthesis in endotoxin-induced intestinal damage in the rat. Prostaglandins 1989;38:319-333. Lefer AM, Tabas J, Smith EF III. Salutary effects of prostacyclin in endotoxic shock. Pharmacology 1980;21:206-212. Pabst MJ, Johnston RB Jr. Increased production of superoxide anion by macrophages exposed in vitro to muramyl dipeptide or lipopolysaccharide. J Exp Med 1980;151:101-114. Granger DN, Rutili G, McCord JM. Superoxide radicals in feline intestinal ischemia. Gastroenterology 1981;81:22-29. McCall TB, Boughton-Smith NK, Palmer RMJ, Whittle BJR, Moncada S. Synthesis of nitric oxide from L-arginine by neutrophils. Release and interaction with superoxide anion. Biochem J 1989;261:293-296. Kanner J, Hare1 S, Granit R. Nitric oxide as an antioxidant. Arch Biochem Biophys 1991;289:130-138. Kubes P, Suzuki M, Granger DN. Nitric oxide: an endogenous modulator of leukocyte adhesion. Proc Nat1 Acad Sci USA 1991;88:4651-4655. Benoit JN, Granger DN. Splanchnic hemodynamics in chronic portal hypertension. Semin Liver Dis 1986;6:287298. Vorobioff J, Bredfeldt JE, Groszmann RJ. Increased blood flow through the portal system in cirrhotic rats. Gastroenterology 1984;87:1120-1126. Blanchet L, Lebrec D. Changes in splanchnic blood flow in portal hypertensive rats. Eur J Clin Invest 1982;12:327-330. Vorobioff J, Bredfeldt JE, Groszmann RJ. Hyperdynamic circulation in portal-hypertensive rat model: a primary factor

GASTROENTEROLOGY Vol. 103, No. 6

155. 156.

157. 158.

159.

160.

161.

162. 163.

164.

165.

166.

167.

168.

169.

170. 171.

172.

173.

174.

for maintenance of portal hypertension. Am J Physiol 1983;244:G52-G57. Kowalski HJ, Abelmann WH. The cardiac output at rest in Laennec’s cirrhosis. J Clin Invest 1953;32:1025-1033. Mashford ML, Mahon WA, Chalmers TC. Studies of the cardiovascular system in the hypotension of liver failure. N Engl J Med 1962;267:1071-1074. Murray JF, Dawson AM, Sherlock S. Circulatory changes in chronic liver disease. Am J Med 1958;24:358-367. Claypool JG, Delp M, Lin TK. Hemodynamic studies in patients with Laennec’s cirrhosis. Am J Med Sci 1957;234:4855. Kontos HA, Shapiro W, Mauck HP, Patterson JL Jr. General and regional circulatory alterations in cirrhosis of the liver. Am J Med 1964;37:526-535. Bosch J, Enriquez R, Groszmann RJ, Storer EH. Chronic bile duct ligation in the dog: hemodynamic characterization of a portal hypertension model. Hepatology 1983;3:1002-1007. Llach J, Gin&s P, Arroyo V, Rimola A, Tit6 L, Badalamenti S, Jimenez W, Gaya J, Rivera F, Rod& J. Prognostic value of arterial pressure, endogenous vasoactive systems, and renal function in cirrhotic patients admitted to the hospital for the treatment of ascites. Gastroenterology 1988;94:482-487. Papper S. The role of the kidney in Laennec’s cirrhosis of the liver. Medicine (Baltimore) 1958;37:299-316. Epstein M. Functional renal abnormalities in cirrhosis: pathophysiology and management. In: Zakim D, Boyer TD, eds. Hepatology. 2nd ed. Philadelphia: Saunders, 1990:493512. Bosch J, Gin& P, Arroyo V, Navasa M, Rod& J. Hepatic and systemic hemodynamics and the neurohumoral systems in cirrhosis. In: Epstein M, editor. The Kidney in Liver Disease. 3rd ed. Baltimore: Williams & Wilkins, 1988:286-305. Kiel JW, Pitts V, Benoit JN, Granger ND, Shepherd AP. Reduced vascular sensitivity to norepinephrine in portal hypertensive rats. Am J Physiol 1985;248:G192-G195. Benoit JN, Barrowman JA, Harper SL, Kvietys PR, Granger DN. Role of humoral factors in the intestinal hyperemia associated with chronic portal hypertension. Am J Physiol 1984;247:G486-G493. Korthuis RJ, Benoit JN, Kvietys PR, Townsley MI, Taylor AE, Granger DN. Humoral factors may mediate increased rat hindquarter blood flow in portal hypertension. Am J Physiol 1985;249:H827-H833. Kravetz D, Arderiu MT, Bosch J, Fuster J, Casamitjana R, Visa J, Rod& J. Hyperglucagonemia and hyperkinetic circulation after portocaval shunt in the rat. Am J Physiol 1987;252:G257-G261. Zipser RD, Hoefs JC, Speckart PF, Zia PK, Horton R. Prostaglandins: modulators of renal function and pressor resistance in chronic liver disease. J Clin Endocrinol Metab 1979;48:895-900. Fisher JE, Baldessarini RJ. False neurotransmitters and hepatic failure. Lancet 1971;2:75-80. Kitamura S, Yoshida T, Said SI. Vasoactive intestinal polypeptide: Inactivation in liver and potentiation in lung of anesthetized dogs. Proc Sot Exp Biol Med 1975;148:25-29. Fernandez-Cruz A, Marco J, Cuadrado LM, Gutkowski J, Rodriguez-Puyol D, Caramel0 C, Lopez-Novoa JM. Plasma levels of atria1 natriuretic peptide in cirrhotic patients (Letter). Lancet 1985;2:1439-1440. Perez-Ayuso RM, Arroyo V, Camps J, Rimola A, Costa J, Gaya J, Rivera F, Rod& J. Renal kallikrein excretion in cirrhotics with ascites: relationship to renal hemodynamics. Hepatology 1984;4:247-252. Caramel0 C, Fernandez-Gallardo S, Santo JC, Inarrea P, Sanchez-Crespo M, Lopez-Novoa JM, Hernando L. Increased lev-

December

1992

els of platelet-activating factor in blood from patients with cirrhosis of the liver. Eur J Clin Invest 1987;17:7-11. 175.Valiance P, Moncada S. Hyperdynamic circulation in cirrhosis: a role for nitric oxide? Lancet 1991;337:776-778. 176.Rees DD, Palmer RMJ, Moncada S. Role of endothelium-derived nitric oxide in the regulation of blood pressure. Proc Nat1 Acad Sci USA 1989;86:3375-3378. 177.Aisaka K, Gross SG, Griffith OW, Levi R. L-arginine availability determines the duration of acetylcholine-induced systemic vasodilation in vivo. Biochem Biophys Res Commun 1989;163:710-717. 178. Beasley D, Schwartz JH, Brenner BM. Interleukin 1 induces prolonged L-arginine-dependent cyclic guanosine monophosphate and nitrite production in rat vascular smooth muscle cells. J Clin Invest 1991;87:602-608. 179. Kilbourn RG, Jubran A, Gross SS, Griffith OW, Levi R, Adams J, Lodato RF. Reversal of endotoxin-mediated shock by NC-methyl-L-arginine, an inhibitor of nitric oxide synthesis. Biochem Biophys Res Commun 1990;172:1132-1138. 180.Gray GA, Schott C, Julou-Schaeffer G, Fleming I, Parratt JR, Stoclet J-C. The effect of inhibitors of the t.-arginine/nitric oxide pathway on endotoxin-induced loss of vascular responsiveness in anaesthetized rats. Br J Pharmacol 1991; 103:1218-1224. 181. Kilbourn RG, Gross SS, Jubran A, Adams J, Griffith OW, Levi R, Lodato RF. NG-methyl-L-arginine inhibits tumor necrosis factor-induced hypotension: Implications for the involvement of nitric oxide. Proc Nat1 Acad Sci USA 1990;87:36293632. 182. Fleming I, Julou-Schaeffer, Gray GA, Parratt JR, Stoclet J-C. Evidence that an L-arginine/nitric oxide dependent elevation of tissue cyclic GMP is involved in depression of vascular reactivity by endotoxin. Br J Pharmacol 1991;103:10471052. 183. Nava E, Palmer RMJ, Moncada S. Inhibition of nitric oxide synthesis in septic shock: how much is beneficial. Lancet 1991;338:1555-1557. 184. Suffredini AF, Fromm RF, Parker MM, Brenner M, Kovacs JA, Wesley RA, Parillo JE. The cardiovascular response of normal humans to the administration of endotoxin. N Engl J Med 1989;321:280-287. 185. Fong Y, Marano MA, Moldawer LL, Wei H, Calvano SE, Kenney JS, Allison AC, Cerami A, Shires GT, Lowry SF. The acute splanchnic and peripheral tissue metabolic response to endotoxin in humans. J Clin Invest 1990;85:1896-1904. 186. Petros A, Bennett D, Valiance P. Effect of nitric oxide synthase inhibitors on hypotension in patients with septic shock. Lancet 1991;338:1557-1558. 187. Yokota M, Kambayashi J, Tanaka T, Tsujinaka T, Sakon M, Mori T. A simple turbidimetric time assay of the endotoxin in plasma. J Biochem Biophys Methods 1989;18:97-104. 188. Lumsden AB, Henderson JM, Kutner MH. Endotoxin levels measured by a chromogenic assay in portal, hepatic and peripheral venous blood in patient with cirrhosis. Hepatology 1988;8:232-236. 189. Triger DR, Boyer TD, Levin J. Portal and systemic bacteremia and endotoxaemia in liver disease. Gut 1978;19:935939. 190. Groszmann R, Kotelanski B, Cohn JN, Khatri IM. Quantitation of portasystemic shunting from the splenic and mesenteric beds in alcoholic liver diseases. Am J Med 1972;53:715722. 191. Ueda H, Kitani K, Kameda H, Yamada H, Iio M. Detection of hepatic shunts by the use of macroaggregated ‘%albumin. Gastroenterology 1967;52:480-487. 192.Ohnishi K, Chin N, Sugita S, Saito M, Tanaka H, Terbayashi H, Saito M, Iida S, Nomura F, Okuda K. Quantitative aspects

GASTROINTESTINAL

AND HEPATIC NITRIC OXIDE

1947

of portal-systemic and arteriovenous shunts within the liver in cirrhosis. Gastroenterology 1987;93:129-134. 193.Sikuler E, Groszmann RJ. Hemodynamic studies in a parabiotic model of portal hypertension. Experientia 1985; 41:1323-1324. 194. Tolins JP, Palmer RMJ, Moncada S, Raij L. Role of endothehum-derived relaxing factor in regulation of renal hemodynamic responses. Am J Physiol1990;258:H655-H662. 195. Burton GA, MacNeil S, de Jonge A, Haylor J. Cyclic GMP release and vasodilatation induced by EDRF and atria1 natriuretic factor in the isolated perfused kidney of the rat. Br J Pharmacol 1990;99:364-368. 196. Miyase S, Fujiyama S, Chikazawa H, Sato T. Atria1 natriuretic peptide in liver cirrhosis with mild ascites. Gastroenterol Jpn 1990;25:356-362. 197. Lintrup J, Friis T, Nissen NI. Comparative studies on the diuretic and biochemical effects of prednisone and spironolactone in hepatic cirrhosis. Acta Med Stand 1966;179:13-21. 198. Copenhagen Study Group for Liver Diseases. Sex, ascites and alcoholism in survival of patients with cirrhosis: effect of prednisone. N Engl J Med 1974;291:271-273. 199. Schalm SW, Summerskill WHJ. Treatment of cirrhosis with prednisone. N Engl J Med 1975;292:1030-1031. 200. Pizcueta P, Pique JM, Bosch J, Fernandez M, Whittle BJR, Moncada S. Involvement of endogenous nitric oxide in the regulation of the rat splanchnic circulation (abstr). Gastroenterology 1991;100:A240. 201. Pizcueta P, Pique JM, Bosch J, Whittle BJR, Moncada S. Effects of endogenous nitric oxide inhibition on the hemodynamic changes of portal hypertensive rats (abstr). Gastroenterology 1991;100:A785. 202. Claria J, Jimenez W, Ros J, Asbert M, Castro A, Arroyo V, Rivera I, Rodes J. Pathogenesis of arterial hypotension in cirrhotic rats with ascites: role of endogenous nitric oxide. Hepatology 1992;15:343-349. 203. Midgley S, Gran IS, Haynes WG, Webb DJ. Nitric oxide in liver failure (letter). Lancet 1991;338:1590. 204. Martin W, Villani GM, Jothianandan D, Furchgott RF. Selective blockade of endothelium-dependent and glyceryltrinitrate-induced relaxation by hemoglobin and by methylene blue in the rabbit aorta. J Pharmacol Exp Ther 1984:232:708716. 205. Mastai R, Schiffrin E, Bandi JC, Sanchez S, De Paula JA, Spinedi E, Carter E. Chronic endotoxemia does not affect portal pressure but increases vascular permeability in an experimental model of ascites (abstr). Gastroenterology 1991; lOO:A772. 206. Mehta R, Gottstein J, Zeller WP, Lichtenberg R, Blei AT. Endotoxin and the hyperdynamic circulation of portal vein-ligated rats. Hepatology 1990;12:1152-1156. 207. Murray BE. Quinolines and the gastrointestinal tract. Eur J Clin Microbial Infect Dis 1989;8:1093-1101. 208. Graham DY, Smith JL. The course of patients after variceal hemorrhage. Gastroenterology 1981;80:800-809. 209. Lebrec D, Poynard T, Bernau J, Bercoff E, Nouel 0, Capron J-P, Poupon R, Bouvry M, Rueff B, Benhamou J-P. A randomized controlled study of propranolol for prevention of recurrent gastrointestinal bleeding in patients with cirrhosis: a final report. Hepatology 1984;4:355-358. 210. Pascal J-P, Cales P, Multicenter Study Group. Propranolol in the prevention of first upper gastrointestinal tract hemorrhage in patients with cirrhosis of the liver and esophageal varices. N Engl J Med 1987;317:856-861. 211. Poynard T, Cal&s P, Pasta L, Ideo G, Pascal J-P, Pagliaro L, Lebrec D, France-Italian Multicenter Study Group. Betaadrenergic-antagonist drugs in the prevention of gastrointes-

1948

212. 213.

214.

215.

216.

217.

218.

219.

220.

221.

222. 223.

224.

225.

226. 227. 228.

229.

230.

STARK AND SZURSZEWSKI

tinal bleeding in patients with cirrhosis and esophageal varices. N Engl J Med 1991;324:1532-1538. Bass NM. Preventing hemorrhage from esophageal varices. N Engl J Med 1987;317:893-895. Conn HO, Grace ND, Bosch J, Groszmann RJ, Rod& J, Wright SC, Matloff DS, Garcia-Tsao G, Fisher RL, Navasa M, Drewniak SJ, Atterbury CE, Bordas JM, Lerner E, Bramante C, Boston-New Haven-Barcelona Portal Hypertension Study Group. Propranolol in the prevention of the first hemorrhage from esophagogastric varices: a multicenter randomized clinical trial. Hepatology 1991;13:902-912. Braillon A, Jiron MI, Valla D, Cal&s P, Lebrec D. Effect of propranolol on hepatic blood flow in patients with cirrhosis. Clin Pharmacol Ther 1985;37:376-380. Vine1 JP, Caucanas JP, Cal& P, Suduca JM, Voigt JJ, Paschal JP. Effect of propranolol on metabolic activity of the liver in patients with alcoholic cirrhosis. J Hepatol 1988;7:186-192. Freeman JG, Barton JG, Record CO. Effect of isosorbide dinitrate, verapamil and labetalol on portal pressure in cirrhosis. Br Med J 1985;291:561-562. Kroeger RJ, Groszmann RJ. The effect of the combination of nitroglycerin and propranolol on splanchnic and systemic hemodynamics in a portal hypertensive rat model. Hepatology 1985;5:425-430. Blei AT, Garcia-Tsao G, Groszmann RJ, Kahrilas P, Ganger D, Morse S, Fung H-L. Hemodynamic evaluation of isosorbide dinitrate in alcoholic cirrhosis: pharmacokinetic-hemodynamic interactions. Gastroenterology 1987;93:576-583. Garcia-Tsao G, Groszmann RJ. Portal hemodynamics during nitroglycerin administration in cirrhotic patients. Hepatology 1987;7:805-809. Thadani U, Fung H-L, Darke AC, Parker JO. Oral isosorbide dinitrate in angina pectoris: comparison of duration of action and dose response relation during acute and sustained therapy. Am J Cardiol 1982;49:411-419. Parker JO, Fung H-L, Ruggirello D, Stone JA. Tolerance to isosorbide dinitrate: rate of development and reversal. Circulation 1983;68:1074-1080. Flaherty JR. Nitrate tolerance. A review of the evidence. Drugs 1989;37:523-550. Desmorat H, Vine1 J-P, Lahlou 0, Pipy B, Badia P, Cales P, Combis J-M, Souqual M-C, Pascal J-P. Systemic and splanchnit hemodynamic effects of molsidomine in rats with carbon tetrachloride-induced cirrhosis. Hepatology 1991;13:11811184. Vine1 JP, Monnin J-L, Combis J-M, Cal& P, Desmorat H, Pascal J-P. Hemodynamic evaluation of molsidomine: a vasodilator with antianginal properties in patients with alcoholic cirrhosis. Hepatology 1990;11:239-242. Ruiz de1 Arbol L, Garcia-Pagan JC, Feu F, Pizcueta P, Navasa M, Bosch J, Rod& J. Effects of molsidomine, a long acting venous dilator, on hepatic hemodynamics in patients with cirrhosis and portal hypertension (abstr). J Hepatology 1989;9(Suppl l):S79. Reden J. Molsidomine. Blood Vessels 1990;27:282-294, Kukovetz WR, Holzmann S. Mechanism of vasodilation by molsidomine. Am Heart J 1985;109:637-640. Takeshita A, Nakamura M, Tajimi T, Matsuguchi H, Kuroiwa A, Tanaka S, Kikuchi Y. Long lasting effect of oral molsidomine on exercise performance: a new antianginal agent. Circulation 1977;55:401-407. Majid PA, De Feyter PJF, Van Der Wall EE, Wardeh R, Roos JP. Molsidomine in the treatment of patients with angina pectoris: acute hemodynamic effects and clinical efficacy. N Engl J Med 1980;302:1-6. Acar J, Kulas A, Escudier B. Long-term clinical and hemodynamic results of molsidomine treatment in patients with refractory heart failure. Am Heart J 1985;109:685-687.

GASTROENTEROLOGY Vol. 103, No. 6

231. Cerra FB, Siegel JH, Border JR, Wiles J, McMenamy RR. The hepatic failure of sepsis: cellular versus substrate. Surgery 1979;86:409-422. 232. Zimmerman HJ. Jaundice due to bacterial infection. Gastroenterology 1979;77:362-374. 233. Neale G, Caughey DE, Mollin DL, Booth CC. Effects of intrahepatic and extrahepatic infection on liver function. Br Med J 1966;1:382-387. 234. Banks JG, Foulis AK, Ledingham IM, Macsween RNM. Liver function in septic shock. J Clin Path01 1982;35:1249-1252. 235. Moshage HJ, Janssen JAM, Franssen JH, Hafkenscheid JCM, Yap SH. Study of the molecular mechanism of decreased liver synthesis of albumin in inflammation, J Clin Invest 1987;79:1635-1641. 236. Ramadori G, Sipe JD, Dinarello CA, Mizel SB, Colten HR. Pretranslational modulation of acute phase hepatic protein synthesis by murine recombinant interleukin 1 (IL-l) and purified human IL-l. J Exp Med 1985;162:930-942. 237. Decker K. Biologically active products of stimulated liver macrophages (Kupffer cells). Eur J Biochem 1990;192:245261. 238. West MA, Keller GA, Cerra FB, Simmons RL. Killed Escherichia coli stimulates macrophage-mediated alterations in hepatocellular function during in vitro co-culture: a mechanism of altered liver function in sepsis. Infect Immun 1985;49:563-570. 239. Billiar TR, Curran RD, Stuehr DJ, West MA, Bentz BG, Simmons RL. An L-arginine-dependent mechanism mediates Kupffer cell inhibition of hepatocyte protein synthesis in vitro. J Exp Med 1989;169:1467-1472. 240. West MA, Billiar TR, Mazuski JE, Curran RJ, Cerra FB, Simmons RL. Endotoxin modulation of hepatocyte secretory and cellular protein synthesis is mediated by Kupffer cells. Arch Surg 1988;123:1400-1405. 241. Billiar TR, Curran RD, Ferrari FK, Williams DL, Simmons RL. Kupffer cell: hepatocyte cocultures release nitric oxide in response to bacterial endotoxin. J Surg Res 1990;48:349353. 242. Green SJ, Mellouk S, Hoffman SL, Meltzer MS, Nacy CA. Cellular mechanisms of nonspecific immunity to intracellular infection: cytokine-induced synthesis of toxic nitrogen oxides from L-arginine by macrophages and hepatocytes. Immunol Lett 1990;25:15-20. 243. Curran RD, Ferrari FK, Kispert PH, Stadler J, Stuehr DJ, Simmons RL, Billiar TR. Nitric oxide and nitric oxide-generating compounds inhibit hepatocyte protein synthesis. FASEB J 1991;5:2085-2092. 244. Gaillard T, Mtilsch A, Busse R, Klein H, Decker K. Regulation of nitric oxide production by stimulated rat Kupffer cells. Pathobiology 1991;59:280-283. 245. Curran RD, Billiar TR, Stuehr DJ, Hofmann K, Simmons RL. Hepatocytes produce nitrogen oxides from L-arginine in response to inflammatory products of Kupffer cells. J Exp Med 1989;170:1769-1774, 246. Billiar TR, Curran RD, Stuehr DJ, Stadler J, Simmons RL, Murray SA. Inducible cytosolic enzyme activity for the production of nitrogen oxides from L-arginine. Biochem Biophys Res Commun 1990;168:1034-1040. 247. Curran RD, Billiar TR, Stuehr DJ, Ochoa JB, Harbrecht BG, Flint SG, Simmons RL. Multiple cytokines are required to induce hepatocyte nitric oxide production and inhibit total protein synthesis. Ann Surg 1990;212:462-469. 248. Stuehr DJ, Nathan CF. Nitric oxide: a macrophage product responsible for cytostasis and respiratory inhibition in tumor target cells. J Exp Med 1989;169:1543-1555. 249. Hibbs JB Jr., Vavrin Z, Taintor RR. L-arginine is required for expression of the activated macrophage effector mechanism

December

1992

causing selective metabolic inhibition in target cells. J Immunol 1987;138:550-565. 250.Stadler J, Billiar TR, Curran RD, Stuehr DJ, Ochao JB, Simmons RL. Effect of exogenous and endogenous nitric oxide on mitochondrial respiration of rat hepatocytes. Am J Physiol 1991;26O:C910-C916. 251.Brtine B, Lapetina EG. Activation of a cytosolic ADP-ribosyltransferase by nitric oxide-generating agents. J Biol Chem 1989;264:8455-8458. 252.Perlmutter DH, Dinarello CA, Punsai PI, Colten HR. Cachectin/tumor necrosis factor regulates hepatic acute-phase gene expression. J Clin Invest 1986;78:1349-1354. 253.Rothschild MA, Oratz M, Zimmon D, Schreiber SS, Weiner I, Van Caneghem A. Albumin synthesis in cirrhotic subjects with ascites studied with carbonate-‘%. J Clin Invest 1969;48:344-350. 254.Blanchard RA, Furie BC, Jorgensen M, Kruger SF, Furie B. Acquired vitamin K-dependent carboxylation deficiency in liver disease. N Engl J Med 1981;305:242-248. 255. Moy JA, Bates JN, Fisher RA. Effects of nitric oxide on platelet-activating factor- and a-adrenergic-stimulated vasoconstriction and glycogenolysis in the perfused rat liver. J Biol Chem 1991;266:8092-8096. 256.Pilaro AM, Laskin DL. Accumulation of activated mononuclear phagocytes in the liver following lipopolysaccharide treatment of rats. J Leukoc Biol 1986;40:29-41. 257.Laskin DL, Pilaro AM. Potential role of activated macrophages in acetaminophen hepatotoxicity. I. Isolation and characterization of activated macrophages from rat liver. Toxic01 Appl Pharmacol1986;86:204-215. 258.Laskin DL, Robertson FM, Pilaro AM, Laskin JD. Activation of liver macrophages following phenobarbital treatment of rats. Hepatology 1988;8:1051-1055. 259.Laskin DL. Nonparenchymal cells and hepatotoxicity. Semin Liver Dis 1990;10:293-304. 260.Billiar TR, Curran RD, Harbrecht BG, Stuehr DJ, Demetris AJ, Simmons RL. Modulation of nitrogen oxide synthesis in vivo: Nc-monomethyl-t.-arginine inhibits endotoxin-induced nitrite/nitrate biosynthesis while promoting hepatic damage. J Leukoc Biol1990;48:565-569. 261.Watkins PB. Role of cytochromes P450 in drug metabolism and hepatotoxicity. Semin Liver Dis 1990;10:235-250. of ni262. Hu S, Kincaid JR. Resonance Raman characterization tric oxide adducts of cytochrome P45Ocam: The effect of substrate structure on the iron-ligand vibrations. J Am Chem Sot 1991;113:2843-2850, 263.Tsubaki M, Hiwatashi A, Ichikawa Y, Fujimoto Y, Ikekawa N, Hori H. Electron paramagnetic resonance study of ferrous cytochrome P-450,,-nitric oxide complexes: effects of 2O(R),22(R)-dihydroxycholesterol and reduced adrenodoxin. Biochemistry 1988;27:4856-4862. 264. Carr GJ, Ferguson SJ. Nitric oxide formed by nitrite reductase of Paracoccus denitrificans is sufficiently stable to inhibit cytochrome oxidase activity and is reduced by its reductase under aerobic conditions. Biochim Biophys Acta 1990;1017:57-62. 265.Arthur MJP. Reactive oxygen intermediates and liver injury. J Hepatol 1988;6:125-131. 266.Tribble DL, AW TY, Jones DP. The pathophysiological significance of lipid peroxidation in oxidative cell injury. Hepatology 1987;7:377-387. 267. Bruckdorfer KR, Dee G, Jacobs M, Rice-Evans CA. The protective action of nitric oxide against membrane damage induced by myoglobin radicals. Biochem Sot Trans 1990; 18:285-266. Arthur MJP, Bentley IS, Tanner AR, Kowalski Saunders P,

GASTROINTESTINAL

AND HEPATIC NITRIC OXIDE

1949

Millward-Sadler GH, Wright R. Oxygen-derived free radicals promote hepatic injury in the rat. Gastroenterology 1985;89:1114-1122. 269. Beckman JS, Beckman TW, Chen J, Marshall PA, Freeman BA. Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. Proc Nat1 Acad Sci USA 1990;87:1620-1624. 270.Radi R, Beckman JS, Bush KM, Freeman BA. Peroxynitrite oxidation of sulfhydryls: the cytotoxic potential of superoxide and nitric oxide. J Biol Chem 1991;266:4244-4250. 271.Radi R, Beckman JS, Bush KM, Freeman BA. Peroxynitriteinduced membrane lipid peroxydation: the cytotoxic potential of superoxide and nitric oxide. Arch Biochem Biophys 1991;288:481-487. 272.Lepoivre M, Fieschi F, Coves J, Thelander L, Fontecave M. Inactivation of ribonucleotide reductase by nitric oxide. Biothem Biophys Res Commun 1991;179:442-448. 273.Kwon NS, Stuehr D, Nathan CF. Inhibition of tumor cell ribonucleotide reductase by macrophage-derived nitric oxide. J Exp Med 1991;174:761-767. 274.Kaplan SS, Billiar T, Curran RD, Zdziarski UE, Simmons RL, Basford RE. Inhibition of chemotaxis with Nc-monomethylL-a&nine: a role for cyclic GMP. Blood 1989;74:1885-1887. 275.Mulligan MS, Hevel JM, Marletta MA, Ward PA. Tissue injury caused by deposition of immune complexes is L-arginine dependent. Proc Nat1 Acad Sci 1991;88:6338-6342. 276.Albina JE, Henry WL, Jr. Suppression of lymphocyte proliferation through the nitric oxide synthesizing pathway. J Surg Res 1991;50:403-409. 277.Efron DT, Kirk SJ, Regan MC, Wasserkrug HL, Barbul A. Nitric oxide generation from L-arginine is required for optimal human peripheral blood lymphocyte DNA synthesis. Surgery 1991;110:327-334. 278.Langrehr JM, Hoffman RA, Billiar TR, Lee KWW, Schraut WH, Simmons RL. Nitric oxide production regulates alloactivation in rat splenocyte mixed lymphocyte cultures. Transplant Proc 1991;23:183-184. 279.Langrehr JM, Hoffman RA, Billiar TR, Lee KKW, Schraut WH, Simmons RL. Nitric oxide synthesis in the in vivo allograft response: A possible regulatory mechanism. Surgery 1991;110:335-342. 280.Langrehr JM, Muller AR, Markus PM, Simmons RL, Hoffman RA. FK 506 inhibits nitric oxide production by cells infiltrating sponge matrix allografts. Transplant Proc 1991;23:32603261. 281.Brizzolara AL, Burnstock G. Endothelium-dependent and endothelium-independent vasodilatation of the hepatic artery in the rabbit. Br J Pharmaco11991;103:1206-1212. 282.Mathie RT, Ralevic V, Alexander B, Burnstock G. Nitric oxide is the mediator of ATP-induced dilatation of the rabbit hepatic arterial vascular bed. Br J Pharmaco11991;103:16021606. 283. Stuehr DJ, Fasehun OA, Kwon NS, Gross SS, Gonzalez JA, Levi R, Nathan CF. Inhibition of macrophage and endotheha1 cell nitric oxide synthase by diphenyleneiodonium and its analogs. FASEB J 1991;5:98-103. Received March 13,1992.Accepted July 28,1992. Address requests for reprints to: J. H. Szurszewski, Ph.D., 8 Guggenheim, Department of Physiology and Biophysics, Mayo Clinic, Rochester, Minnesota 55905. Supported by a grant from the National Institutes of Health (DK 17238). The authors express their appreciation to Dr. D. B. McGill for helpful comments and constructive criticisms of this review; to Jan Applequist for secretarial assistance; and to Philip Schmalz and Gary Stoltz for technical assistance.