INTRODUCTION

TOP ABSTRACT INTRODUCTION NITRIC OXIDE SYNTHESIS AND... INCREASED NO PRODUCTION IN... EXPERIMENTAL STUDIES WITH NO... EXPERIMENTAL STUDIES WITH NO... CLINICAL STUDIES WITH NO... REFERENCES

Nitric oxide (NO), a ubiquitous biological molecule produced by numerous cell types, is implicated in a wide range of disease processes, exerting both detrimental and beneficial effects at the cellular and vascular levels. Sepsis is one area where NO is thought to play a key role in pathogenesis. Importantly, most NO research has been conducted on animal models, and in attempting to understand the complexities of NO in sepsis, and interpret the many apparently conflicting results available, it is important to remember that the situation can vary according to the species being studied, the model of sepsis employed, the concentrations of NO involved, and the timing of measurements and observations made. An appreciation of the many differences in study design can help explain many of the controversial issues surrounding NO's involvement in the pathogenesis of sepsis and also the varying effects of NO inhibitors and donors.

There is a wealth of literature concerning the role and effects of NO in sepsis, and space limitations preclude detailed discussion of many articles. However, our review provides a brief but succint overview of NO synthesis and metabolism in animals and humans, followed by a more detailed analysis of its effects during sepsis and the results of blocking and enhancing its actions in experimental and clinical conditions.

	NITRIC OXIDE SYNTHESIS AND METABOLISM

TOP ABSTRACT INTRODUCTION NITRIC OXIDE SYNTHESIS AND... INCREASED NO PRODUCTION IN... EXPERIMENTAL STUDIES WITH NO... EXPERIMENTAL STUDIES WITH NO... CLINICAL STUDIES WITH NO... REFERENCES

NO synthesis requires the oxidation of a single guanidino nitrogen atom of L-arginine, a process involving the oxidation of nicotinamide adenosine dinucleotide phosphate (NADPH) and the reduction of molecular oxygen. The catalytic site for this process includes a heme group. Flavin adenine dinucleotide and flavin mononucleotide are both cofactors for the synthesis of NO.

Three major NO synthase (NOS) isoforms have so far been identified that can be broadly grouped together as constitutive NOS (cNOS) and inducible NOS (iNOS). The cDNA for these isoforms has been localized in several species, including humans. cNOS, either neural (nNOS or NOS1) or endothelial (ecNOS or NOS3), is always present, but relatively inactive until intracellular calcium levels rise. The small amounts of NO produced by the calcium-dependent cNOS are involved in various physiologic processes, including neurotransmission and vasodilation.

iNOS (or NOS2) is not normally active, although the enzyme may be detected in some cell types, including lung, small intestine, and platelets. However, when certain cells are activated by specific proinflammatory agents such as endotoxin, tumor necrosis factor (TNF), interferon-gamma (IFN), and interleukin-1 (IL-1), iNOS activity is induced. The activation of human iNOS involves transcription of messenger ribonucleic acid (mRNA), which is triggered by the binding of transcription factors, including nuclear factor-kappa B (NF-B), IFN-regulatory factor-1 (IRF-1), c-jun, c-fos, and signal transducer and activator of transcription (STAT)-1 to specific sites on the promoter of the iNOS gene. The activation of the newly synthesized iNOS requires post-transcriptional alterations and the presence of substrate and cofactors, including calmodulin. Calcium, although not required for calmodulin binding to iNOS, may also be important in the induction of iNOS mRNA. Once induced, large amounts of NO are produced by iNOS, provided that the availability of L-arginine is sufficient.

NO is a gaseous free radical that can be stored as nitrosothiol compounds. These are more stable than NO but retain NO-like properties. NO binds to heme-containing proteins such as guanylate cyclase (GC), which it activates to release guanosine 3'5'-cyclic monophosphate (cGMP). cGMP-mediated actions include smooth and myocardial muscle relaxation and inhibition of platelet aggregation. NO can also react with superoxide anion to form peroxynitrite, a potent oxidant with toxic effects on many molecules, including nucleic acids, lipids, and proteins. In particular, peroxynitrite impairs mitochondrial respiration and activates the poly-ADP ribose synthase (PARS) enzyme, resulting in reduced NAD, slowing the rate of glycolysis, electron transport, and ATP generation (1). NO may exert a negative feedback on its own production by inhibiting cNOS and iNOS expression (2).

	INCREASED NO PRODUCTION IN SEPSIS

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The finding of elevated circulating nitrite/nitrate, the stable byproducts of NO, in septic patients, combined with the reduction in vascular tone seen after endotoxin or proinflammatory cytokine administration, and the identification of endothelium-derived relaxing factor (EDRF) as NO, led to suggestions that NO was involved in the cardiovascular alterations of septic shock. The pathogenetic role of NO in sepsis and septic shock can, in fact, encompass both vascular alterations and the direct cellular toxic effects of NO or NO-related compounds. In several experimental models, endotoxin has been shown to increase the constitutive release of NO by the endothelium (3) and the activity of the iNOS enzyme (4). Mice lacking iNOS have been reported to be resistant to endotoxin-induced mortality (5) and vascular hypocontractility (6), supporting a key role for iNOS in endotoxin shock. In addition to endotoxin, cell wall components and enterotoxin from gram-positive organisms are also able to stimulate NO release.

Various inflammatory mediators have been implicated in the induction and activation of iNOS, particularly IFN, TNF, IL-1, and platelet activating factor (PAF), alone or synergistically. In addition to the activation of iNOS, cytokines and endotoxin may increase NO release by increasing arginine availability through the opening of the specific y⁺ channels and the expression of the cationic aminoacid transporter (CAT), or by increasing tetrahydrobiopterin levels, a key cofactor in NO synthesis.

The functional status of cNOS in endotoxemia and sepsis is not fully elucidated. Several experimental studies have demonstrated a decrease in cNOS activity resulting in an impairment in endothelial-dependent relaxation during endotoxemia and experimental sepsis (7, 8), possibly as the result of a cytokine- or hypoxia-induced shortened half-life of cNOS mRNA (9), or of altered calcium mobilization (10). Other investigators have shown increased endothelial NO release immediately after endotoxin administration either directly (3) or indirectly from the lack of effect of a specific iNOS inhibitor on the initial hypotension after endotoxin. The activity of endothelial NO in sepsis can actually transiently increase and then decrease later.

Cellular Effects

NO exerts in vitro toxic effects, including nuclear damage, protein and membrane phospholipid alterations, and the inhibition of mitochondrial respiration on several cell types. Mitochondrial dysfunction, a potentially toxic effect, could also be considered as an adaptive phenomenon, decreasing cellular metabolism when the energy supply is limited. The relevance of mitochondrial dysfunction is, however, questionable, as administration of SIN-1, a NO donor, in a canine model of endotoxic shock increased oxygen extraction capabilities (11). The toxicity of NO itself may be enhanced by the formation of peroxynitrite from the reaction of NO with superoxide (1). Therefore, the multiple organ dysfunction that often accompanies severe sepsis may be related to the cellular effects of excess NO or peroxynitrite. In contrast, NO may protect cells from the oxidative damage by scavenging oxygen free radicals (12) and inhibiting oxygen free radical production (13).

Hemodynamic Effects

With due consideration of the potential limitations related to aspects of experimental design, including the model used, the species, and the type and timing of the septic challenge, as well as the mode of action, selectivity, and dose and timing of administration of any pharmacologic intervention used, several reports support a critical role for NO or NO-related compounds in the regulation of vascular tone, platelet function, myocardial function, and hepato-splanchnic and renal blood flows.

Effects on vascular tone. During sepsis and endotoxemia, data support a pivotal role for NO in the endotoxin-induced (3) and TNF-induced (14) vasodilation and vascular hyporeactivity to vasoconstrictors. Both pharmacologic inhibition and iNOS gene deficiency are associated with a loss of endotoxin-induced vasodilation (6). The increased NO release has also been implicated in the diminished response to vasoconstrictors (14, 15).

Effects on myocardial function. NO may exert direct and indirect effects on cardiac function in sepsis. Myocardial iNOS activity has been reported in response to endotoxin and cytokines and inversely correlated with myocardial performance (16). The negative inotropic effects of NO are probably mediated by cGMP and the impairment in coronary autoregulation (17) and oxygen utilization.

Recently, several studies have added to the confusion surrounding the role of NO by demonstrating no effect of NO or NOS inhibitors on the myocardium or on -adrenergic responsiveness (18, 19). Some of these apparent inconsistencies may again be related to differences between the species of animal used or in the experimental preparation, the dose of NOS inhibitor, the dose of endotoxin, the type of tissue examined, the timing of treatment, harvesting, or the presence of calcium in the incubation medium prior to the experiment. Nevertheless, in most studies, low to moderate doses of iNOS inhibitors restore myocardial contractility in hearts exposed to proinflammatory cytokines, whereas at higher doses, the effect reverses itself. This finding may indicate that small amounts of NO produced by iNOS may be necessary to maintain contractility (20).

Effects on hepatosplanchnic blood flow and function. Endotoxin has been shown to induce iNOS synthesis in human intestinal and liver cells (21) with evidence of morphologic and functional damage, including increases in intestinal epithelial permeability (22) and, in animals, bacterial translocation and plasma concentrations of liver enzymes.

In addition to its effects on enterocytes and hepatocytes, NO also influences hepatosplanchnic blood flow during endotoxemia (23). Picomolar concentrations of NO (i.e., the amount of NO constitutively produced in the GI tract by nNOS and ecNOS) probably inhibit the vasoconstriction, mast cell activation, neutrophil and platelet adhesion, and capillary hyperpermeability observed during ischemia. Similarly, during acute endotoxemia, the vasorelaxant effects of NO are necessary to prevent hepatosplanchnic ischemia. In contrast, the large amount of NO produced during chronic gut inflammation, associated with an increased production of iNOS-derived NO, exerts deleterious effects on gut function and permeability. Small supplemental amounts of NO improve endotoxin-induced intestinal injury (24).

Effects on renal blood flow and function. NO is involved in the regulation of the renal microcirculation, and iNOS is produced in renal mesangial cells in response to endotoxin. Impaired renal function caused by vasoconstriction may occur in severe sepsis, and NO may be necessary to counterbalance this effect and maintain renal blood flow. The exact role of NO in the kidney remains to be fully defined and studies with NOS blockers have yielded conflicting results, some showing reduced renal blood flow and renal function (25), with others demonstrating improved creatinine clearance and glomerular filtration when atrial natriuretic peptide is chronically increased (26).

Effects on platelet function. Even though human platelets possess cNOS and iNOS, which can be activated by endotoxin and cytokines, and the involvement of NO in sepsis-induced platelet dysfunction is likely, it is not yet clearly defined. Indeed, in nonseptic conditions, endothelium-derived NO inhibits both platelet adhesion and platelet aggregation (27).

Respiratory Effects

Effects on pulmonary circulation. In normal lungs, basal NO release by cNOS probably contributes to the maintenance of a low resting pulmonary vascular tone. In sepsis, an imbalance between NO (a vasodilator) and the strong vasoconstricting endothelins and thromboxane results in pulmonary vasoconstriction, and pulmonary hypertension may be common. Systemic NO blockade can further increase pulmonary artery pressure (28). Importantly, L-monomethylarginine (L-NMMA), a NO antagonist, has greater effects on the pulmonary circulation in endotoxemic dogs than in control animals (29), suggesting a protective role for NO in the lungs by limiting pulmonary hypertension and hypoxic pulmonary vasoconstriction. However, this hypothesis was not confirmed in rats (30). On the contrary, NO inhalation produces selective pulmonary vasodilation with a reduction in pulmonary vascular resistance and pulmonary hypertension. Simultaneous administration of inhaled NO with systemic NO blockade can prevent the increase in pulmonary artery pressure seen with NOS blockers and improve gas exchange (31).

Effects on diaphragmatic function. Endotoxin activates iNOS in the diaphragm, but the reported effects of NOS activation on diaphragmatic function are variable, with some studies reporting impaired function (32) and others suggesting that iNOS may protect against endotoxin-induced diaphragmatic contractile dysfunction (33).

Immunologic Effects

NO can have important microbicidal effects against intracellular parasites, mycobacteria, and common ICU pathogens including Staphylococci. Indeed, these strains do not contain the thiol moieties able to scavenge NO.

NO may also affect the production of cytokines in response to bacterial products. NO increases endotoxin-induced TNF- release in human neutrophils (34), but has a down-regulating effect on TNF and IFN production to staphylococcal enterotoxin in mice (35). NO donors may reduce inflammation by limiting cytokine-induced endothelial activation, leukocyte adherence, and microvascular permeability alterations (36).

	EXPERIMENTAL STUDIES WITH NO ANTAGONISTS

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Many experimental agents have been employed to block the effects of NO (Table 1). The first agents to be used were competitive analogs of L-arginine. Interestingly, the uptake of these compounds by endothelial cells and macrophages can be influenced by cytokine or endotoxin administration, presumably via the y⁺ channels (37). L-canavanine, L-lysine, L-thiocitrulline, and their derivatives have been proposed as relatively selective iNOS inhibitors.

Non-aminoacid-based NOS competitive inhibitors share some similarities with the arginine analogs but their structure differs. Among these compounds, guanidines, isothioureas, and imidazoles have been used in septic shock. Aminoguanidine may selectively inhibit iNOS, and may also reduce the harmful effects of NO by reacting with peroxynitrite (38). Selective iNOS inhibitors may also inhibit prostaglandin production, thus having a dual anti-inflammatory action (39). Isothioureas are among the most potent inhibitors of NOS with variable isoform selectivity. Mercaptoethylguanidine represents a novel class of iNOS inhibitors with protective effects in various forms of shock and inflammation. Finally, corticosteroids have been used for their ability to prevent NOS induction, and methylene blue (MB) for its GC-inhibiting properties.

Effects of NO Blockade in Experimental Studies

There is no doubt that NO blockade results in a reversal of the hyperkinetic state associated with sepsis, causing a rise in arterial pressure and an increase in systemic vascular resistance. NO inhibition may restore the effects of catecholamines in vivo (15). Could NO blockade, therefore, replace norepinephrine or other strong vasoconstrictors? Norepinephrine may better maintain cardiac output through its -adrenergic effects, whereas NOS inhibitors usually decrease cardiac output, despite the potential increase in myocardial contractility. The use of dobutamine in combination with NOS inhibitors may help to maintain cardiac output and hence blood flow (40). A potential advantage of NOS inhibitors would be if they demonstrated a better blood flow distribution than norepinephrine, but this does not seem to be the case. Indeed, many studies have shown that NO inhibitors decrease blood flow and tissue oxygenation (11, 25). In contrast to nonselective NOS antagonists, the use of iNOS inhibitors is associated with increased survival (38).

Nonspecific NOS inhibition partially prevents endotoxin-induced left ventricular dysfunction (41), but may be associated with a decrease in cardiac output (29, 41), possibly because of increased right ventricular afterload as a result of increased pulmonary vascular resistance (42) or myocardial ischemia (17).

Discrepancies in the effects of nonselective NO blockade on mortality rates may be related to the model used, with the majority of investigators studying gram-negative infections using endotoxin. Other important issues are the dose of NOS inhibitor and the timing of the intervention. Indeed, in acute studies, iNOS may not have time to be expressed. For instance, in rats, induction of iNOS is maximum only at 6 h after endotoxin injection (4), and this may explain why NOS inhibitors administered earlier than this indeed appear to have no effect or to increase mortality. The relationship of time to NO activity is, however, not simple. Different stages of sepsis may be characterized by different degrees of cNOS and iNOS production and hence NO levels and the effectiveness of NO blockade may depend on the precise stage at which treatment is administered.

Overall, beneficial effects have been observed with inhibitors selective for iNOS, or with low doses of nonisoform selective inhibitors, whereas high doses of nonisoform selective NOS inhibitors have been detrimental. Regarding the beneficial effects of selective iNOS inhibitors, Wu and colleagues (43) showed that aminoguanidine attenuated the circulatory failure and the renal, hepatic, and pancreatic dysfunction caused by endotoxin in a rat model, and improved survival. Liaudet and colleagues (44), in a series of experiments in endotoxin-treated rats and mice, noted that another selective iNOS inhibitor, L-canavanine, produced beneficial hemodynamic and metabolic effects, reduced signs of liver and kidney dysfunction, and reduced mortality. Similarly, Szabó and colleagues (38) showed that S-methylisothiourea and mercaptoethylguanidine attenuated the rise in liver enzymes, bilirubin, and creatinine, prevented hypocalcemia, and improved survival in endotoxin-treated mice.

NO scavenging of NO already produced may be a valuable alternative to NOS inhibition. Cell-free hemoglobin is a NO scavenger that causes similar increases in arterial pressure, with less effect on pulmonary hypertension than L-NAME.

	EXPERIMENTAL STUDIES WITH NO DONORS

TOP ABSTRACT INTRODUCTION NITRIC OXIDE SYNTHESIS AND... INCREASED NO PRODUCTION IN... EXPERIMENTAL STUDIES WITH NO... EXPERIMENTAL STUDIES WITH NO... CLINICAL STUDIES WITH NO... REFERENCES

Although NO has many detrimental effects, small amounts are beneficial. NO donors have, therefore, been assessed in the treatment of experimental septic shock with improved systemic and hepatic perfusion (23). These observations await confirmation.

	CLINICAL STUDIES WITH NO ANTAGONISTS

TOP ABSTRACT INTRODUCTION NITRIC OXIDE SYNTHESIS AND... INCREASED NO PRODUCTION IN... EXPERIMENTAL STUDIES WITH NO... EXPERIMENTAL STUDIES WITH NO... CLINICAL STUDIES WITH NO... REFERENCES

In a study involving 15 patients with sepsis syndrome, Lorente and colleagues (45) studied the effects of a bolus injection of N-nitro-L-arginine and noted an increase in blood pressure and systemic vascular resistance accompanied by a decrease in cardiac index. Interestingly, in the same study (45), they gave L-arginine to another group of patients and noted an increase in cardiac index with a transient fall in blood pressure. In a randomized double-blind placebo-controlled trial, Petros and colleagues (28) investigated the effects of L-NMMA in 12 patients with severe sepsis. L-NMMA caused an increase in blood pressure and systemic vascular resistance with a fall in cardiac output, changes that were sustained during the infusion (28). A recent study by Avontuur and colleagues (46) investigated the effects of a 12-h continuous infusion of L-NAME in 11 patients with septic shock. Arterial pressure and arterial oxygenation were improved, enabling a reduction in catecholamine dosage. These effects were sustained throughout the infusion, although most apparent in the early stages. Seven of the 11 patients died, so despite its beneficial hemodynamic effects, L-NAME had no positive effects on mortality in this study (46).

Although many studies have reported beneficial effects on hemodynamic status, NOS inhibition results in raised pulmonary vascular resistance (28, 45, 46), and effects on outcome remain dubious (28, 46). In a recent series of multicenter clinical trials using L-NMMA, a first study (47) was designed to assess the effects of L-NMMA on patients with sepsis and to evaluate dose ranges required to restore mean arterial pressure. Treatment with L-NMMA enabled a 60 to 80% reduction in norepinephrine dosage. A phase II randomized placebo-controlled study demonstrated a higher percentage of patients with resolution of shock at 72 h in the L-NMMA group (unpublished data). However, a third study was recently discontinued because of an increased mortality rate in the treatment group (48).

MB has also been studied clinically in patients with septic shock. Studies (49, 50) have consistently reported that a short-term infusion of MB to patients with septic shock transiently increases arterial pressure, either related to an acute vasopressor effect (50) or to an improvement in myocardial function (49) and oxygen delivery.

Conclusions

Although many questions remain unanswered regarding the role of NO in septic shock, several facts are clear. First, there is an increase in NO release in septic shock that is well-documented in animals, although it has been less well-demonstrated in humans. This is not necessarily deleterious since some degree of vasodilation is necessary to increase oxygen delivery to the cells. Second, blocking NO formation, or the effects of NO, can increase arterial pressure and systemic vascular resistance but may also reduce cardiac index and raise pulmonary vascular resistance.

In our attempts to influence the effects of NO, we must aim to protect the positive while eradicating the negative. Selective NOS inhibitors may acheive this better than nonselective agents, but they have not yet undergone clinical testing. In addition, whereas NO blockade may indeed be beneficial in some clinical syndromes, in others NO donors may be more appropriate. It may be that different agents are needed at different times during the septic process, or that a combination of NO inhibitors with NO donors may be more effective. Our challenge is to correctly evaluate the NO balance and recognize those situations where NO is present in excess. The need for a better knowledge of the NO pathway in humans is crucial if we are to make further progress in this field.