Nitric oxide: An overview*

Nitric Oxide: An Overview David A. Rodeberg, MD, Mark S. Chaet, MD, Robert C. Bass, MD,

Marc S. Arkovitz, MD, Victor F. Garcia, MD, Cincinnati, Ohio

Nitric oxide (NO), a paracrine-acting gas enzy- matically synthesized from L-arginine, is a unique biologic mediator that has been implicated in a myriad of physiologic and pathophysiologic states. It is an important regulator of vascular tone and may be the mediator of the hemodynamic changes involved in sepsis and cirrhosis. In addition, there is increasing evidence that NO is involved in coagulation, immune function, inhibitory innervation of the gastrointestinal tract, protection of gastrointestinal mucosa, and the hepatotoxicity of cirrhosis. It has already been speculated that NO may represent a point of control or intervention in a number of disease states. The purpose of this paper is to provide the surgeon with a broad overview of the scientific and clinical aspects of this important molecule. Am J Surg. 1995;170:292-303.

S ince the initial identification of nitric oxide (NO) as a biologically active molecule, a tremendous number of basic science and clinical investigations about NO

have entered the literature. To underscore this point, NO was named "Molecule of the Year" by Science (1992). Nitric oxide should not be confused with the anesthetic nitrous oxide (N20). This point was made clear by Sir Humphrey Davy, who nearly succumbed after inhaling a large amount of NO. 1 Given the broad range of functions attributed to NO, we sought to review the NO literature that would be of value to surgeons. Many of the activities and mechanisms of action attributed to NO have been elucidated using animal models. For the purpose of this manuscript, these results have been generalized and combined to provide a clearer and more comprehensive picture of how NO probably functions in humans.

Nitric oxide, a gaseous, paramagnetic, free radical, is produced from the amino acid L-arginine (L-Arg) in nu- merous mammalian celt types (Figure 1). Nitric oxide possesses a half-life of 3 to 5 seconds and is rapidly neu-

From the Department of Surgery (DAR, RCB), University of Cincinnati Medical Center, and the Department of Pediatric Surgery (MSC, MSA, VFG), Childrens Hospital Medical Center, Cincinnati, Ohio.

Supported by Shriners of North America Research Fellowship and Surgical Infection Society Research Fellowship.

Requests for reprints should be addressed to David A. Rodeberg, MD, University of Cincinnati Medical Center, Department of Surgery, 231 Bethesda Avenue, Cincinnati, Ohio 45267.

Manuscript submitted April 15, 1994 and accepted in revised form November 15, 1994.

tralized by hemoglobin, methylene blue, and superoxide anion. The enzyme involved in NO production, NO synthase (NOS), converts the terminal guanidine group of t-Arg to NO with a m-hydroxyl-L-arginine intermediate compound. 2'3 The reaction requires nicotinamide adenine dinucleotide phosphate (NADPH) as a cofactor and uses molecular oxygen to oxidize the substrate to the final reaction products, NO and L-citrulline. 4 Two forms of NOS have been identified, each with unique basal activity levels and cofactor requirements. 4 The isoenzyme found in neuronal tissue and vascular endothelium is constitutively expressed and requires calcium/calmodulin as cofactors. The NOS initially identified in the macrophage is inducible, calcium independent, and produces NO in quan- tities several magnitudes greater than the constitutive form. Both forms ofNOS are competitively inhibited by L-Arg analogues such as L-N°-monomethyl-arginine (L- NMMA), NG-nitro-L-arginine-methyl ester (L-NAME), and NQnitro-L-arginine (L-NNA). 2,5 These compounds have been important for investigations of the biologic function of NO, as well as for creating an avenue for clinical investigation of NO blockade.

Initial investigations concentrated upon NO's involvement in cellular communication and vascular smooth-muscle relaxation. Further studies have revealed the ubiquitous nature of NO and its effects upon many other biological systems. Presented here is a review of the currently identified effects and mechanisms of mammalian NO production.

T H E V A S C U L A R SYSTEM Background

The vasodilatory property of acetylcholine has long been appreciated. It wasn't until 1980, however, that Furchgott and Zawadzki 6 demonstrated that the endothelium was required for acetylcholine-induced dilation. The acetylcholine molecule acts on a receptor present on the endothelial cell, resulting in the release of a diffusible substance, termed endothelium-derived relaxing factor (EDRF), causing the relaxation of adjacent smooth-muscle cells. Early investigations demonstrated that EDRF release is induced by many stimuli, including acetylcholine, bradykinin, histamine, adenine nucleotides, substance P, 5-hydroxytryptamine, endothelial stretch, shear stress, and hypoxia. 7 EDRF induces smooth-muscle relaxation by causing an increase of cyclic guanosine monophosphate (cGMP) within the smooth-muscle cell. s'11 Following a separate line of investigation, Murad 12 demonstrated that the effects of exogenously administered nitrates (azide, hydroxylamine, sodium nitrite, nitroglycerin, sodium nitroprusside, and nitrosamines) were mediated by the radical NO. Ignarro 13 demonstrated that endothelial cells were not required for exogenous-nitrate-mediated vasodilation. The nitrovasodilators were known to act

292 THE AMERICAN JOURNAL OF SURGERY ® VOLUME 170 SEPTEMBER 1995

through stimulation of soluble guanylate cyclase leading to the formation of cGMP, similar to EDRE s'9

In 1987, both Palmer and Ignarro independently proposed that EDRF and NO were the same molecule. 13'14 In a model using porcine endothelium cultured on microcarrier beads, they demonsn'ated many similarities between these two compounds. Both EDRF and NO were inhibited by superoxide anion, methylene blue, and hemoglobin, and were enhanced by superoxide dismutase. Both molecules possessed a half- life of only 3 to 5 seconds. Each caused an increase in intracellular cGMP levels in smooth-muscle cells by activating a soluble guanylate cyclase enzyme. Simultaneous EDRF bioassay and NO chemoluminescence assays illustrated that the amount of NO released was sufficient to account for all of EDRF's activity. 14

The release of NO from endothelial cells is primarily mediated by guanosine triphosphate (GTP)-associated proteins (G proteins), which link the receptors to NOS) 5 Different receptors stimulate different G proteins; with some being pertussis-toxin sensitive (serotonin) and others, toxin insensitive (bfadykinin). There is also the possibility that the pertussis-toxin-insensitive mechanisms ,nay not involve G proteins at all. The release of NO is dependent on pulsatile flow as well as shear stress, and is enhanced by other vasodilatory agents. ]6a7 Nitric oxide acts in a typical paracrine fashion. Diffusion into the vascular lumen affects platelets, while abluminal diffusion affects the surrounding smooth muscle. 7 It is possible that NO itself is not the diffusing agent but enters the extracellular domain as an S-nitrosylthiol intermediate compound that is formed in physiologic conditions when NO interacts with sulflaydryl groups. This hypothesis has been supported by the observation by Ignarro et al is that the activity of both exogenous and endogenous nitrates requires the presence of sulfhydryl groups. Further work demonstrated that EDRF has a longer half-life than

I OVERVIEW OF NITRIC OXIDE/RODEBERG ET AL

02

~ ' NO + L-citrulline L-arginine Nitric Oxide Synthase

nitrite (NO2-)

nitrate (NOa-)

Figure 1. Synthesis of nitric oxide (NO) in mammalian cells.

pure NO, which is more consistent with the need for an S- nitrosylthiol intermediate. 19'2° In addition, vascular Smooth- muscle cells and platelets respond to both S-nitrosylthiols and NO similarly. It has been postulated that, in the blood vessels of the central nervous system, NO is not the effector molecule at all, but that the responses are solely dependent on the S-nitrosylthiols. 21 However, the mo, re widely accepted opinion is that NO actually causes the smobth-muscle relaxation, and that the S-nitrosylthiols represent only intermediate compounds or reaction products. 22

The mechanism by which NO causes the relaxation of vascular smooth muscle was initially proposed after the observation that exogenous nitrates and NO caused an increase in cGMP, but not cyclic adenosine monophosphate (cAMP). 23 The cGMP-mediated relaxation of smooth muscle appears to occur by two mechanisms. First NO was observed to decrease intracellular calcium levels and increase the permeability of the potassium channels, leading to a hyperp01arization of the plasma membrane that prevents subsequent contracn0n . . 24 Second, cGMP activates protein kinases, which eventually lead to the dephosphorylation of the myosin light chains, preventing the myosin/actin interaction. 7

platelet aggregation

I 0 2 -

EDCF I

T B A 2

platelet disaggregation

EDRF I I ] ]

P G H 2 e n d o t h e l i n N O PGI 2

1 1

cGMP cAMP K* channel

contraction dilation

I endotheliUm

vascular smooth muscle

Figure 2. Interactions between vascular endothelium, smooth muscle, and platelets. Nitric oxide (NO) increases the permeability of the potassium channels, leading to a hyperpolarization of the plasma membrane that prevents subsequent contraction. NO and prostacyclin (PGI2) inhibit platelet aggregation and platelet or monocyte adhesion to the endothelium, and stimulate platelet dis - aggregation. PGH 2 = prostaglandin H2; TBA 2 = thromboxane; 02- = superoxide anion; EDRF = endothelium-derived relaxing factor; EDCF = endothelium-derived contracting factors; K + = potassium ion; cGMP = cyclic guanosine monophosphate; cAMP = cyclic adenosine monophosphate.

THE AMERICAN JOURNAL OF SURGERY ® VOLUME 170 SEPTEMBER 1995 29~

OVERVIEW OF NITRIC OXIDE/RODEBERG ET AL 1

• Vascular Tone Normal vascular tone is determined bythe interactions of

endothelium-derived vasodilators (ie, NO, prostacyclin [PGI2]) and vasoconstrictors (ie, endothelin) (Figure 2). Under baseline physiologic conditions, this balance is in fa- vor of vasodilation. 25"27 NO and PGI 2 inhibit smooth-muscle contraction and proliferation, platelet aggregation, and platelet or monocyte adhesion to the endothelium, and both stimulate platelet disaggregation. 2~,2s,29 These compounds op- erate synergistically in their vasodilatory and platelet-antiag- gregatory actions, but act through different second-messenger mechanisms; PGI 2 through cAMP, and NO through cGME Despite PGI2's significantly longer half-life, it appears that NO production is the major determinant of resting vascular tone. 3°'33 Using L-Arg analogues, researchers have demonstrated that the vasodilatory response to NO occurs predominantly in the arterial system, suggesting that arteries have a higher basal release of NO and are more dependent on NO for vascular tone regulation when compared to veins. Since veins have less NO release, they have a compensatory increased level of soluble guany!ate cyclase. This Would ex- plain the clinical observation that the venom capacitance vessels are more susceptible to the vasodilatory action of exogenous nitrovasodilators, which bypass NOS and directly stimulate the soluble guanylate cyclase. The development of tolerance to exogenously administered nitrovasodilators appears to be due to impaired metabolism to NO and desensitization of soluble guanylate cyclase to NO. 1s'34"36

Disease States Nitric oxide is an important physiologic regulator of blood

flow and perfusion pressure to isolated vascular beds in response to appropriate physiologic stimuli. For example, the renal, cardiac, pulmonary, and muscular vasculatums depend upon NO for basal regulation. 374: These observations have not only led to the improved understanding of vasomotor regulation, but also to many theories about various organ. based disease processes. Several investigations have presented evidence for the involvement of dysfunctional endothelium in hypertension, 42,43 atherosclerosis, 44 and vasospasm. 45

Decreased levels of activity of NO have been implicated as a mechanism of primary or essential hypertension. 46"49 The degree of endothelial dysfunction parallels the degree of hy. pertension. The inability of vessels to vasodilate appears to be related to decreased NO release and not to the reduced ability of smooth-muscle cells to respond to NO. 5°'5: This may be due to decreased t-Arg availability 52-54 or decreased G-protein function in atherosclerotic vessels secondary to the presence of Oxidized low-density lipoprotein (LDL). 55 A model has been proposed by Flavahan, 56 in which the endothelial dysfunction occurs in distinct phases. Early in the disease when there are low concentrations of oxidized LDL, the dysfunction would be due to impairment of the G-protein--dependent pathways. Later, as more oxidized LDL ac- cumulates, other signal transduction processes in the endothelial cell become dysfunctional, eventually inhibiting the release of NO by a nonspeeific action, such as decreased L-Arg availability, decreased sensitivity of the vascular smooth muscle to NO, and/or an increased degradation of

NO. Some studies suggest that a deficiency in NO may even exacerbate atherogenesis. This may occur since NO inhibits both the release of platelet granules containing mitogens and vascular smooth-muscle mitogenesis. 57'5s • Other pathologic conditions include NO inactivation by advanced glycosylation products found within endothelial and smooth-muscle cells of a diabetic animal model. 59"61 In addition, the hypotension of septic shock is dependent on NO and will be discussed in further detail in the immunologic section. Similarly, the vascular hyporeactivity to va- sopressor agents such as norepinephrine, seen with hemodynamic decompensation after prolonged hemorrhagic shock, is mediated by NO. 62 The vasospasm seen after subarachnoid hemorrhage is probably due to inactivation of NO by extravascular hemoglobin. 63 As more is learned about the mechanisms behind these abnormalities in NO metabolism, therapies based on NOS blockade or NO administration should become useful. Studies are currently under way looking at the therapeutic use of inhaled NO to improve the pulmonary hypertension and ventilation/perfusion mismatch that occurs during respiratory distress syndrome, and L-Arg analogue inhibitors are being used in models of septic shock.

Coagulation Nitric oxide can act in a paracrine fashion, and as such has

effects on both Sides of the endothelial cell. Within the lumen of the vessel, NO interacts with platelets, inhibiting both adhesion to endothelial cells and aggregation. 64,65 In 1986, Azuraa et a166 demonstrated that EDRF inhibited arachidonic-acid-induced platelet aggregation. Similarly, exogenous nitrovasodilators and NO were found to increase platelet intracellular cGMP, leading to a subsequent inhibition of aggregation. 67 Endothelial-dependent inhibition of platelet aggregation is controlled by two separate mechanisms. Endothelial-derived PGI 2 inhibits platelet aggregation by increasing intracellular cAMP, whereas NO works by increasing cGMP. 68 The two molecules act synergistically, but NO appears to be the major reguiat0rymolecule, since platelet adhesion to collagen and endothelial matrix can be completeiy blocked by NO, but only partially prevented by PGI2 .69"71 Nitric oxide also inhibits platelet and leukocyte adhesion to endothelial cells, as well as mediating platelet plug disaggregation. 26'28'29'7° Furthermore, nitrovasodilators have been shown to prolong the activity of tissue pla> minogen activator, thereby increasing thrombolytic activity and preventing vessel occlusion. 72

Platelets also release NO, which acts in an autocrine fashion to prevent aggregationY The platelets are stimulated to release NO by compounds such as bradykinin. 69 The platelet NOS enzyme is, like the endothelial cells, a constitutively expressed NADPH- and calcium/calmodulin-dependent enzyme. Platelet NOS activity is dependent on L-Arg, inhibited by L-Arg competitive analogues, and potentiated by cGMP phosphodiesterase inhibitors, similar to endothelial NOS.

T H E I M M U N E SYSTEM Background

Nitric oxide ha s emerged as a central participant in the immune response. The investigation of NO as an immunore-


I OVERVIEW oF NITRIC OXIDF-JRODEBERG ET AL

Figure 3. Proposed mechanism of macrophage cytotOxic effects. Macro- phages stimulated with interferon-gamma (IFN-7), lipopolysaccharide (LPS), and tumor necrosis factor (TNF) produce an ih- ducible form of nitric oxide (NO) syn- thetase, which in turn produces NO from L-arginine (L-ARG) in amounts several magnitudes greater than does the constitutive form found in neuronal and vascular tissue: Macrophage-derived NO causes an increase in target cell intracellular cyclic guanosine monophosphate (cGMP) levels and a decrease in DNA synthesis. It is thought that NO binds to the iron-sulfhydryl (Fe-S) prosthetic groups of aconitase and complexes I and II of the mitochondrial electron transport chain, disrupting Oxidative metabolism and causing target cell death.

IFN-~/

LPS

f L . . . . . . ! T N F A AA

TUMOR/BACTERIA

active Compound began with the observation that high levels of urinary nitrates were recovered from a patient with in- fectious diarrhea. 74 Later, the production of nitrates and nitrites was detected after immunostimulation of cell cultures, laboratory animals, and human subjects. 75"77 The source of these nitrogen compounds remained unclear until Stuehr and Marietta 7s demonstrated that serum and urinary nitrates were increased in normal mice after immunostimulation, but not in C3H/HeJ mice, which have a genetic alteration ren- dering their macrophages resistant to endotoxin. The rodent macrophage cell culture model has served as an invaluable tool for the investigation of immunostimulated nitrite and nitrate production. It was through the use of this model that Marietta et a179 identified NO as the intermediate compound in the L-Arg-to-nitrite/nitrate pathway.

The enzyme responsible for NO production in the mammalian macrophage differs from that in the endothelial cell. In contrast to the constitutively expressed enzyme found in endothelium, the macrophage NOS is inducible and not calcium dependent, but does require the cofact0rs NADPH and tetrahydrobiopterin for NO production, s° Immunologic stimulation increases the amount of inducible NOS by increasing its rate of transcription. 8~'84 Macrophages stimulated with interferon gamma (IFN-7) and !ipopolysaccharide (LPS) have NOS messenger ribonucleic acid (mRNA) present by 2 hours, and NOS protein, by 4 hours. The NOS protein may remain present for several days. s2 The inducible form of NOS produces NO in amounts several magnitudes greater than the constitutive form. Similar to the constitutive form, inducible NOS is inhibited by L-Arg analogues. The activity of the inducible form of NOS is preferentially inhibited by the L-Arg analog L-canavanine and by amino- guanidine, s5 The expression of the inducible form of NOS also is inhibited by glucocorticoids. 73'77'sl The mechanism through which steroids inhibit inducible NOS is unclear, since glucocorticoids affect the production of other cytokines that regulate NO production.

Although cultured macrophages can produce large amounts of NO, multiple cytokines are required to achieve

maximal induction. Laboratories have used various combinations of IFN- 7, tumor necrosis factor-alpha (TNF-cz), and interleukiml (IL-1), in addition to bacterial endotoxins and exotoxins, to induce macrophage NO production. IFN- 7 or LPS alone can induce noncytotoxic levels of NO. However, when cytokines work synergistically, they can induce cytotoxic levels of NO. s<s7 Priming cell cultures with IFN- 7 prior to exposure to other agents results in enhanced NO production. The synergistic activity between IFN- 7 and mi- crobial products is mediated by TNF-cz. 87 TNF-cz acts in an autocrine fashion to amplify the synthesis and release of NO by primed macrophages; however, like IL-1, it cannot stimulate NO synthesis by itself, s7 Antimicrobial activity and NO production parallel TNF-cz activity. One of the diffi- culties in this field of research has been the inability to induce human macrophage NO production with the methods used for rodent cell systems. There is a report, however, of the involvement of NO in human macrophage-mediated killing of an avirutent strain of Mycobacterium avium, ss In addition, human NO production has been demonstrated in hepatocytes after stimulation with various combinations of Escherichia coli LPS, IL-t6, TNF-a, and IFN-7. 89 Further work in developing an inducible human macrophage model is needed to fully understand the function of NO in the human immune response.

Inducible NO inhibits the viability and replication of many types of microbes. A strong correlation exists between antimicrobial activity and production of L-Arg-derived NO by cytokine-activated cells observed during in vitro studies. 87'9° The mechanism by which macrophage NO effects its cytotoxic activity remains unclear, although several hypothe- ses have been entertained (Figure 3). Superoxide anion and NO interact in vitro to form peroxynitrite, a precursor to two potent oxidants, hydroxide ion (OH-) and nitrite (NO2-).91,92 Although evidence for this reaction in living organisms has not been demonstrated, it is possible that the cytotoxicity of NO relies upon these and other reactive nitrite and oxygen intermediates. As previously noted, NO ac- tively binds heme groups. Some investigators postulate that

THE AMERICAN JOURNAL OF SURGERY ® VOLUME 170 SEPTEMBER 1995 295

OVERVIEW OF NITRIC OXIDE/RODEBERG ET AL I

NO binds to the iron-sul~ydryl prosthetic groups of aconitase and complexes I and II of the mitochondrial electron transport chain, resulting in cell death.

Nonspecific Immunity Nitric oxide production plays a role in the induction of non-

specific immunity and the antimicrobial response to a variety of extracellular parasites and some tumor cells. The importance of the macrophage and various cytokines such as TNF. cz and IFNq, in response to parasitic infection has been well documented. 93,94 Further investigation has implicated NO as the final mediator of parasitic defense. Nitric-oxide-dependent mechanisms have been identified in the killing of intracellular Trypanosoma, Mycobacterium, Schistosoma, and Leishmania organisms. 94"97 The importance of macrophage NO production in tumoricidal activity has also been described using in vitro models. 98'99 The precise mechanism of NO-mediated killing of these organisms is unknown. However, these observations suggest that macrophage NO production con- tributes significantly to nonspecific immunity.

Specific Immunity The role of NO in specific immunity has been another in-

teresting field of investigation. Human lymphocyte blasto- genic proliferation is augmented in the presence of t-Arg or exogenous NO during the synthesis phase of the cell cycle when deoxyribonucleic-acid (DNA) replication occurs, and is decreased when specific inhibitors of NOS are present, m° These observations suggest that NO production promotes DNA synthesis in immunologically activated lymphocytes. The mechanism may involve NO, since increases of cGMP have been demonstrated in proliferating lymphocytes. 1°1 Contradictory data have been reported using murine T-cell populations. In this system, T-cell proliferation is inhibited when co-cultured with NO-secreting, concanavalin-A-activated macrophages3 °2 Interestingly, in the murine system, it appears that the T-helper type 2 (Th2) cell production of IL-4 inhibits macrophage NOS, while IFN-~{ released by T- helper type 1 (Thl) cells induces NOS. m3 This represents a mechanism by which Thl and Th2 cells, mediators of de- layed-type hypersensitivity, reciprocally regulate macrophage NO synthesis. The differing effects of NO on lymphocyte proliferation may be due to quantitative differences in NO production, supporting a proposed theory that low concentrations of NO are required for lymphocyte replication, while excess amounts of NO are cytotoxic.

The involvement of NO in transplant rejection has been addressed from both diagnostic and therapeutic aspects. Langrehr and colleagues 1°4 using a rodent allograft model, demonstrated that NO production by mixed splenic lymphocytes and sponge matrix allograft-infihrating cells inhibit T-ceU antigen-specific proliferation. Further work using FK 506, an inhibitor of T-cell IL-2 production, suggests that NO production from sponge matrix graft-infiltrating cells is dependent upon IL-2 production3 °5 These observations support the theory that NO is involved in regulation of the response to allografts. The interaction between T-cell cytokine stimulation of macrophage NO and NO suppression of T- cell proliferation and cytolytic activity may be the mechanisms involved in this regulation.

In the above-described models, NO appears to contribute to the inhibition of cell-mediated rejection. In contrast to these observations, NO has been implicated as an important mediator of graft-versus-host reaction (GvHR). Significant amounts of NO are produced in the early course of GvHR and may serve as a useful marker to the onset of this process. 1°6a°7 Blockade of NO production with the L-Arg analogue L-NMMA abolishes the intestinal mucosal injury associated with GvHR in mice after intraperitoneal injection of splenocytes. 1°s These findings suggest a possible therapeutic role for specific NO-blocking agents in the man- agement of GvHR enteropathy. Using a rat heart allograft model, researchers have identified NO as an important part of vascularized allograft rejection. 1 Further studies on NO and solid organ transplantation are currently underway in several laboratories.

Sepsis and Multisystem Organ Failure The inducible form of NO is produced by macrophages re-

siding in various tissues in response to several noxious stimuli, including LPS. This response leads to overall NO levels that exceed the resting, nonstressed states. It is this relative overproduction of NO and the subsequent vasodilation that are thought to mediate N©'s physiologic role during sepsis and multiorgan failure. Clinical and experimental studies, however, failed to define a specific role for NO during sepsis or multiorgan failure. 1°9q15

Studies in patients who were septic from multiple causes (excluding trauma) have shown increased serum levels of NO, measured as the ratio of NOr/NO3-. 113 All trauma patients, including septic trauma patients, had NO levels that were lower than that of normal controls. The authors rea- soned that trauma patients had an inhibition of NO release secondary to hypovolemia. In this study, elevated NO levels were inversely related to systemic vascular resistance. Conversely, elevated NO levels correlated positively with increased serum endotoxi~ levels. Several subsequent clinical trials evaluated the efficacy of NOS blockade in treating the systemic effects of sepsis. 1~4,n5 Petros et al n5 demonstrated that the IV infusion of L-NMMA and L-NAME reversed the intractable hypotension and vascu!ar dilation observed in 2 septic patients. Similarly, Lorente et al u4 described a cohort of septic patients in whom an infusion of L-NMMA was associated with elevated blood pressure, systemic vascular resistance, and peripheral vascular resistance. These changes were transient, reversible with infusion of t-Arg, and paral- leled by a concomitant decrease in cardiac index and oxygen delivery. The size of the groups and the nature of the studies did not allow for evaluation of the effects of L- NMMA on survival, morbidity, and mortality.

Most of the experimental research of the role of NO during sepsis takes advantage of competitive inhibition of NOS. Inhibition of NOS has been looked at in various organ systems, as well as in the organism as a whole. It appears that NO mediates its effects during sepsis via its vasodilating properties.62,~16a 17 More important, however, is the fact that recent studies have shown both beneficial and harmful effects of endogenous NO on the stressed organism as a whole. Kilbourn et al l°9'11° demonstrated that blocking NOS with the L-Arg analogue L-NMMA reversed hypotension induced


OVERVIEW OF NITRIC OXIDF-JRODEBERG ET AL

by endotoxin and TNF infusion. The animals again became hypotensive when given L-Arg. Similarly, Lorente et al 112 described an increase in blood pressure, systemic vascular resistance, pulmonary artery pressure, and pulmonary vascular resistance when septic sheep where treated with L-NMMA. Pretreatment with L-Arg in this experiment abolished these changes. The conclusion to be drawn from these studies is that NO plays a pathologic role in the hemodynamic response to sepsis.

While these studies would tend to suggest that NOS blockade might be beneficial to the septic organism, and thus clinically important, other studies have drawn the opposite conclusion. Studies using LPS-injected rabbits concluded that L-NMMA injection prior to LPS administration had no ap- preciable effect on hypotension induced by LPS) Is Moreover, NOS blockade resulted in significantly higher mortality and coronary vasoconstriction. These effects were partially reversible with the addition of the NO donor, S- nitroso-N-acetyl penicillamine. Similarly, Klabunde and Helgren 119 showed that administration of L-NMMA in a canine sepsis model did little to abrogate endotoxin-induced hypotension.

The effects of NO on various organ systems during sepsis have also been explored. Nitric oxide is produced by inducible NOS present in the Kupffer cells of the liver. When stimulated in an in vitro model by LPS, however, hepatocytes required a combination ofTNE IL-1, OFN.?, and LPS for NO production3 2°'121 In addition, in vitro studies have found an association between NO production and inhibition of total protein synthesis in hepatocytes. 122 The conclusion from this latter study was that NO is harmful to the liver during sepsis. In vivo studies using LPS-challenged mice have, however, found that NOS blockade with L-NMMA was associated with significant liver injury, manifested by an elevation in liver enzymes, as well as in ornithine carbamoyltransferase. 12° The article suggests that NO improves or protects hepatic blood flow and perfusion during sepsis. The significance of the in vitro inhibition of protein synthesis is somewhat less clear in light of these in vivo findings.

In vitro studies have also demonstrated both beneficial as well as negative effects of NO on myocardium. Ballingand et a1123 described an elevated production of NO by myocytes when these cells were exposed to medium conditioned with LPS-stimulated macrophages. This elevated NO production was associated with reduced myocyte contractility, which was reversed with L-NMMA. Conversely, in an experimental model of feline splanchnic ischemia/reperfusion, serum myocardial depressant factor levels were reduced with the addition of sodium nitrite (NaNO2), an NO-generating molecule, n6

Studies in rats have used NOS blockade to investigate what role NO may play in the renal pathophysiology associated with hyp0volemic shock) 24 Specifically, the falls in mean arterial blood pressure, renal plasma flow, and glomerular fil- tration rate seen in hemorrhagic shock were reversed with the addition of L-NMMA. This sparing effect was reversed with the addition of h-Arg. The conclusion from this study was that NO plays an important role in the renal impairment seen during hemorrhagic shock. Similar studies in septic models have not been reported.

In summary, sepsis, hypovolemia, and hypoxia each play a significant role in the pathophysiology of multiorgan failure and macrophage activation. Inducible NO from activated macrophages may be responsible for the profound loss of vascular tone seen in septic patients. NO production by activated macrophages is complex and closely interrelated with the production of other mediators of inflammation, such as endotoxin, cytokines, prostaglandins, and interleukins. The problem with the experimental models that deal with NO synthesis is that NOS blockade is nonspecific, blocking both the inducible and constitutive forms, thus making it difficult to describe a specific role to the inducible form of NO. The role of NO during multiorgan failure most likely represents a complex interaction between the inducible and constitutive forms of NOS. The ability to reconcile these conflict- ing results will be determined by the ability to selectively block either the inducible or the constitutive form of NOS.

T H E G A S T R O I N T E S T I N A L SYSTEM Background

Nitric oxide has been implicated in a number of physiologic and pathophysiologic functions in the gut. The mechanisms by which NO is suspected of carrying out these actions fall into three major categories--neurotransmitter, vasorelaxant, and paracrine mediator. There is considerable evidence that NO is one of the nonadrenergic, noncholinergic (NANC) neurotransmitters, functions to protect gastric and intestinal mucosae, mediates the hemodynamic de- rangements seen in sepsis and cirrhosis, and acts as a paracrine mediator of hepatotoxicity. 125

NANC Neurotransmission An important class of neurons in the wall of the gut, the

NANC neurons mediate relaxation of the gastrointestinal smooth muscles, which may include many of the inhibitory reflexes of the gastrointestinal tract, such as relaxation of the lower esophageal sphincter (LES) during swallowing, proximal stomach relaxation during filling, and descending inhibition of small-intestine contraction due to disten- sion. 126'127 The neurotransmitter of NANC neurons has not been conclusively identified, although vasoactive intestinal peptide (VIP) and adenosine triphosphate (ATP) have been implicated. 127 However, there is now increasing evidence that NO, alone or in concert with other substances, is one of the primary neurotransmitters of the NANC neurons in many tissues.

Investigators have determined that stimulation of NANC neurons in canine ileocecal junction and rat gastric fundus causes the release of a compound with the characteristics of N O J 28'129 By using L-Arg analogues and oxyhemoglobin, it has been demonstrated that NO is involved in mediating NANC relaxation in the LES, 13° stomach, 13H33 small intestine, 134a35 sphincter of Oddi, 136 ileocecal junction and colon, ~37'138 and anal sphincters 139,14° in a variety of species. In human tissues, L-Arg analogues completely blocked NANC relaxation of the internal anal sphincter and partially blocked those in circular colonic muscles. TM These re- suits support the hypothesis that NO is the NANC neurotransmitter for the internal anal sphincter and that NO plays a role in colonic smooth-muscle relaxation. In circular mus-



TABLE

Criteria From the Literature for Nitric Oxide (NO) as a Neurotransmitter

Synthesis of NO in neurons 136,137

Release of a substance with characteristics of NO on stimulation of NANC neurons m,112

Application of exogenous NO mimics effects of NANC nerve stimulation130j 32

A mechanism for inactivation (NO rapidly and spontaneously breaks down) TM

Inhibition of NO synthesis with specific antagonists attenuates the effect of NANC stimulation m,~a2,1a4,~a5

NANC = nonadrenergic, noncholinergic neurotransmitters,

cle of human jejunum, exogenously administered NO grossly mimics the effects of NANC stimulation; however, there are differences in the characteristics of the electrical responses. In human jejunum, NANC stimulation elicits smooth-muscle-membrane hyperpolarizations known as inhibitory junctional potentials. Inhibitory junctional potentials are char- acterized by a rapid and Sustained component to the hyperpolarization. L-Arg analogues block the sustained component but not the initial rapid portion of the inhibitory junctional potentials. 142 These data suggest that more than one transmitter may be involved in mediating the NANC response in the human jejunum.

Nitric oxide has fulfilled most of the criteria classically used to define a neurotransmitter (Table) and may be the final NANC neurotransmitter. 143445 Several other neurotransmitters such as ATE y-aminobutyric acid, and VIP have been implicated in NANC neurotransmission, and the precise re- lationship of NO to other potential neurotransmitters remains to be defined. 14644s Nitric oxide may be co-released with other neurotransmitters, or its production may be stimulated by a neurotransmitter in the target muscle of some tissues. 149 tt is also possible that different NANC neurotransmitters may function in different parts of the gut, may vary between species, or may change within a specific organ with aging and development. 126

Gastrointestinal Mucosa In the stomach, a decrease in gastric mucosal blood flow

has been identified as a prerequisite to the development of acute erosions and stress ulcers) 5°,151 Nitric oxide synthase has been detected in the gastric mucosa, and NO appears to play a role in protecting the gastric mucosa during physiologic stress by acting as an endogenous vas0dilator and thus supporting mucosal blood f low. 152'125 Using hydrogen gas clearance to measure gastric mucosal blood flow, Pique et aP 53a54 have demonstrated that endogenous NO modulates basal tone in the gastric vasculature. In similar experiments Using L-Arg analogues to block NO production, NO was observed to have no effect on resting-acid output, but did make a substantial contribution to the vasodilation associated with the stimulation of acid secretion. 154 In other experiments, the use of L-NMMA alone did not result in injury to rat gastric mucosa. When used in conjunction with sensory neu- ropeptide depletion (capsaicin) or PGI 2 blockage (in- domethacin), however, L-NMMA resulted in a dose-dependent, acute mucosal injury. 155 Thus it appears that

NO, PGI2, and sensory neuropeptides interact to prevent gastric mucosal injury. In a rat model of ethanol-induced injury to gastric mucosa in ex vivo studies, topical application of NO reduced the severity of mucosal damage. 156 A brief delay in the application of NO, however, resulted in a loss of the protective effect. Since ethanol is associated with initial vasoconstriction and then vasodilation of the mucosal blood vessels, MacNaughton et a1156 concluded that NO may act by blocking the initial vasoconstriction associated with ethanol. The exact mechanism of NO's protective effect is unclear, but may relate to vasodilation, inhibition of platelet aggregation in the gastric microvasculature, or a protective effect on the epithelial cells themselves.- t25 No studies have been done in humans to date.

Loss of the intestinal mucosal barrier has been implicated in the cause of bacterial translocation and multisystem organ failure. 157J58 Recent work in animal models suggests that NO may help protect against some of the effects of endo- toxic shock by protecting the mucosa of the small intestine. In the small intestine of the rat', pretreatment with L-Arg analogues exacerbates acute intestinal vascular damage, plasma leakage, and hemorrhage, and is associated with endotoxin infusion3 59 S-nitroso-N-acetyl-penicillamine, which generates NO, moderated the damage in this model. 16° In an autoperfused segment of feline small intestine, infusion of L-NAME resulted in a sixfold increase in mucosal permeability, as assessed by leakage of chromium 51-labeled ethylenediaminetetraacetic acid ([SICr]EDTA) without gross mucosal damage)6 l This effect was reversed by treatment with L-Arg and appeared to be independent of blood flow or neutrophil adherence. This data led Kubes TM

to propose that NO has a direct effect on the epithelial cell barrier, and its inactivation under pathophysiologic conditions may contribute to loss of mucosal barrier function. In addition to supporting mucosal blood flow, NO may have an antioxidant effect by binding to and neutralizing superoxide anion, thereby limiting the damage induced by neutrophils during endotoxemia/25 Although these results suggest a role for NO in maintaining a mucosal barrier, the mechanism of this effect is unclear and may be multifactorial.

Hemodynamic Disturbances During Cirrhosis and Sepsis Portal hypertension secondary to cirrhosis and septic shock

involves a similar set of systemic hemodynamic disturbances. In both situations there is increased cardiac output, increased heart rate, decreased systemic vascular resistance, decreased blood pressure, and a resistance to vasopressors) 62a63 Tl~ere is now evidence that NO may be the mediator of these hemodynamic alterations.

Patients with cirrhosis have been noted to have high plasma levels of endotoxin despite the absence of clinically apparent infection/64 This finding, coupled with recognition of the similarity between the hemodynamic alterations of endotoxemia and cirrhosis, led Vallance and Moncada 165 to hypothesize that NO was the mediator of the hemodynamic alterations of cirrhosis. Methylene blue, an antagonist of NO, has been shown to increase blood pressure in patients with hypotension due to liver failureJ 66 In a rat model of cirrhosis, L-NMMA increased blood pressure, decreased cardiac output, and increased systemic vascular resistance. 167


Though these results are intriguing, it is by no means clear that endotoxin is the trigger for the hyperdynamic state seen in cirrhosis. It seems likely that NO has a role in the hemodynamic alterations of cirrhosis, but the exact nature of that role is yet unclear.

Hepatotoxicity Nitric oxide may be a potentially important paracrine me-

diator of function in the diseased liver. 168A69 West et aP 6s hy- pothesized that alterations in hepatocellular function may arise via interactions between hepatocytes and Kupffer cells, the specialized macrophages that line hepatic sinusoids. They demonstrated that when gentamicin-killed E coli or LPS was added to co-cultures of hepatocytes and Kupffer cells, but not hepatocytes alone, there was a marked decrease in hepatocyte protein synthesis, t6s,169 The Kupffer-cell mediated decrease in protein synthesis was dependent on metabolism of L.Arg and associated with the production of nitrates, nitrites, and t.-citrulline. 17° Kupffer cells were demonstrated to produce N O with a time course similar to that required for reduction of hepatocyte protein synthesis. 171'172 Finally, addition of N O or NO-generating compounds to hepatocytes inhibited protein synthesis in a time- dependent and reversible fashionJ 2z Total mRNA was not affected, indicating that NO acts at a translational or post- translational step.

A second source of NO that alters hepatocyte protein synthesis may be the hepatocyte itself. Curran et a1173 demonstrated that products from endotoxin-stimulated Kupffer cells were capable of stimulating the conversion of L-Arg to nitrites and nitrates within hepatocytes, and this was associated with a concurrent decrease in total protein synthesis. The mechanism by which N O modulates hepatocyte protein synthesis remains unclear. It does not involve cell death or increases in cGME ~z2 Although N O inhibits mitochondrial respiration, the effects last only I to 2 hours, and there- fore cannot be responsible for the 12-hour persistent inhibition of hepatocyte protein synthesis. 174

CONCLUSION Nitric oxide is a unique biologic mediator that has been im-

plicated in many physiologic and pathophysiologic states. It is an important regulator of vascular tone an d may be involved in sepsis, coagulation, immune function, inhibitory innervation of the gastrointestinal tract, protection of gastrointestinal mucosa, and the hepatotoxicity of cirrhosis. Because N O may represent a point of control or intervention in a number of disease states, surgeons should be aware of the scientific and clinical aspects of this important molecule.

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OVERVIEW OF NITRIC OXIDE/RODEBERG ET AL ]

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Nitric oxide: An overview*

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