The mediators responsible for maintenance of the hyperdynamic state and the low systemic vascular resistance (SVR) observed in sepsis have not been elucidated. Nitric oxide (.N = O) is a mediator with numerous functions, including regulation of vascular tone and a role in macrophage-mediated cytostasis and microbiostasis. Thirty-nine critically ill trauma and septic patients were studied to determine the relationship between .N = O production and the hyperdynamic state. high plasma levels of NO2-/NO3- (the stable end products of .N = O) were observed in septic patients (p less than 0.02). Low SVR and high endotoxin levels were associated with high NO2-/NO3- values (p = 0.029, p = 0.002). Changes in .N = O levels may mediate the vasodilation seen in sepsis. Low NO2-/NO3- levels were observed in trauma patients (p less than 0.001) and remained low even in the presence of sepsis (p = 0.001).

Although nitric oxide (.N = O) biosynthesis is inducible in rat hepatocytes (HC), the physiological significance of .N = O production by these cells is unknown. Short exposure of HC to authentic .N = O led to a concentration-dependent inhibition of mitochondrial aconitase, NADH-ubiquinone oxidoreductase, and succinate-ubiquinone oxidoreductase (complexes I and II of the mitochondrial electron transport chain). Most susceptible to .N = O inhibition was mitochondrial aconitase, in which a reduction in enzyme activity to 20.2 +/- 1.6% of control was observed. In contrast to mitochondrial aconitase, cytosolic aconitase activity was not inhibited by .N = O. After exposure to a maximal inhibitory concentration of .N = O, mitochondrial aconitase activity recovered completely within 6 h. Complex I did not fully recover within this incubation period. Endogenous .N = O biosynthesis was induced in HC by a specific combination of cytokines and lipopolysaccharide. After 18 h of incubation with these stimuli, a significant inhibition of mitochondrial aconitase activity to 70.8 +/- 2.4% of controls was detected. However, this was due only in part to the action of .N = O. A non- .N = O-dependent inhibition of mitochondrial function appeared to be mediated by tumor necrosis factor.

Attempts were made to promote or inhibit nitric oxide (. N = O) synthesis in a murine model of hepatic damage (Corynebacterium parvum followed by lipopolysaccharide; LPS) to determine the role of . N = O in the liver injury. Moderate hepatic damage and increases in circulating NO2-/NO3- levels were detectable after C. parvum alone. Administration of LPS to these mice resulted in severe hepatic damage and acute elevations in circulating nitrogen oxide levels. L-arg had no influence on the C. parvum or LPS-induced changes. NG-monomethyl-L-arginine (NMA) had no effect in the absence of LPS, but when given with LPS, a dose-dependent suppression in plasma NO2-/NO3- levels and an increase in liver injury were seen. The NMA-induced changes were partially reversed by the simultaneous administration of L-arg. These findings suggest a protective role for . N = O in this model.

Corynebacterium parvum-treated mice produce large amounts of circulating nitrogen oxides and develop a severe liver injury in response to lipopolysaccharide (LPS). Concurrent administration of NG-monomethyl-L-arginine not only suppresses nitric oxide synthesis in these animals but also profoundly increases the hepatic damage following LPS. In this report, we present evidence that the increased hepatic damage from inhibition of nitric oxide synthesis is mediated in part by superoxide and hydroxyl radicals. The hepatic damage induced by suppressing nitric oxide production during endotoxemia could be reduced by treating mice with superoxide dismutase and deferoxamine, scavengers of superoxide and hydroxyl radicals, respectively. This damage could also be prevented by treating mice with the anticoagulant heparin sodium. The results suggest that nitric oxide synthesis during endotoxemia is important in preventing hepatic damage by reducing oxygen radical-mediated hepatic injury and preventing intravascular thrombosis.

Recent findings suggest that nitric oxide (NO .) is an important biologic mediator produced by a number of cell types including endothelial cells (1, 2), macrophages , cerebellar neurons (6), and neutrophils (7) . L-Arginine (L-arg) serves as the substrate for NO-production (1-4), while L-citrulline and nitrite/nitrate (N02 -/NO3 -) are the stable endproducts of this metabolic pathway (8, 9). We have previously shown that cocultures of rat Kupffer cells (KC) and hepatocytes (HC) also metabolize L-arg to citrulline and N02 -/NO3 -in response to lipopolysaccharide (LPS) (10). This L-arg metabolism was associated with a profound suppression of coculture total protein synthesis . When KC and HC were cultured alone, only KC metabolized L-arg in response to LPS. Therefore, we originally hypothesized that KC, like other M0, were the sole site of L-arg metabolism. However, KC :HC cocultures exposed to LPS produced three to five times more N02-/NO3 -and citrulline than LPS-activated KC cultured alone. Investigation of this discrepancy led to the subject of this report, that transferable KC products induce the conversion of significant quantities of L-arg to NO . in HC and that this metabolism of L-arg is associated with a concurrent reduction in HC protein synthesis . Volume 170 November 1989 1769-1774 Materials and Methods Brief Definitive ReportCulture Medium . HC and KC cultures were performed in standard Williams medium E (0.24 MM L-arg) or L-arg-free Williams medium E (Gibco Laboratories, Grand Island, NY) supplemented with 10-6 M insulin, 15 mM Hepes, L-glutamine, penicillin, streptomycin, and 57o dialyzed calf serum . Additional culture reagents included L-arg HCl (Gibco Laboratories) and NG-monomethyl-L-arginine acetate (NMA), prepared by a modification of the method described by Corbin and Reporter (11).Cell Isolation . Liver cells were obtained from unfasted male Sprague-Dawley rats weighing 200-300 g (Harlan Sprague Dawley, Inc., Indianapolis, IN) . HC were harvested by an in situ collagenase (Type IV; Sigma Chemical Co., St. Louis, MO) perfusion and separated from nonparenchymal cells by differential sedimentation to >95% purity as previously described (12). Liver nonparenchymal cells were harvested by a pronase E (Sigma Chemical

The etiology and mechanisms by which severe trauma or sepsis induce hepatic failure are unknown. Previously we showed that Kupffer cells (KC), the fixed macrophages of the liver, induce a profound decrease in hepatocyte (HC) total-protein synthesis when exposed to endotoxin. Furthermore we demonstrated that endotoxin-activated KCs induce these changes in HC protein synthesis through the induction of a novel L-arginine-dependent biochemical pathway within the HC. In this pathway, the guanido nitrogen of L-arginine is converted to the highly reactive molecule nitric oxide (NO.). To identify the KC factors that act as signals for induction of HC NO. biosynthesis, recombinant cytokines were added to HC cultures and HC nitrogen oxide production and protein synthesis levels were determined. We found that no single cytokine, but rather a specific combination of tumor necrosis factor, interleukin-1, interferon-gamma, and endotoxin, were required for maximal induction of HC nitrogen oxide production. This specific combination of cytokines induced a 248.8 +/- 26.0 mumol/L (micromolar) increase in HC nitrogen oxide production and simultaneously inhibited HC total protein synthesis by 36.1% +/- 3.1%. These data demonstrate that multiple cytokines, produced by endotoxin-activated KC, induce the production of NO. within HC, which in turn leads to the inhibition of HC total-protein synthesis.

The hepatic failure associated with severe sepsis is characterized by specific, progressive, and often irreversible defects in hepatocellular metabolism (1). Although the etiologic microbe can often be identified, the direct causes and mechanisms of the hepatocellular dysfunction are poorly understood . We have hypothesized that Kupffer cells (KC), which interact with ambient septic stimuli, respond by providing signals to adjacent hepatocytes (HC) in sepsis . Furthermore, we have provided evidence (2, 3) that KC activated by LPS from Gram-negative bacteria can induce profound changes in the function of neighboring HC in coculture. In our model, coculture of either KC (2) or peritoneal macrophages (MO) (3) with HC normally promotes HC protein synthesis ([ 3 H]leucine incorporation) . The addition of LPS or killed Escherichia colt' to such cocultures induces a profound decrease in HC protein synthesis, as well as qualitative changes ([358]methionine, SDS-gel electrophoresis) in protein synthesis without inducing HC death (2, 3) . In this report we show that the inhibition in protein synthesis is mediated via an L-arginine-dependent mechanism.The metabolism of L-arginine by activated Mo to substances with cytostatic and even lethal effects on target cells is a relatively recent discovery. After the description by Stuehr and Marletta (4, 5) that LPS-triggered MO produced nitrite/nitrate (N02-/NO3-), Hibbs et al. (6,7) and Iyengar et al. (8) demonstrated that L-arginine was the substrate for the formation of both these nitrogen end products and citrulline. A role for the arginine-dependent mechanism in MO tumor cytotoxicity (6, 7) and microbiostatic activity (9) has been suggested. However, the in vivo functions of this novel MO mechanism have not yet been defined, but it is possible that there are both physiologic as well as pathologic roles. Our in vitro results raise the possibility that some metabolic responses to microbial invasion may be partially mediated by the L-arginine-dependent mechanism. What other metabolic responses are affected and the possible pathologic consequences remain to be studied.

Macrophage production of nitric oxide (.N = O) leads to considerable alterations of vital metabolic pathways in various target cells. The present study tested whether .N = O synthesis by Kupffer cells (KCs), the resident macrophages of the liver, interferes with the secretory function of these cells. As in other macrophage-type cells, the combination of lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) was a potent stimulus of .N = O synthesis by KC. Treatment with LPS and IFN-gamma also induced significant production of prostaglandin E2 (PGE2), thromboxane B2 (TBX2), tumor necrosis factor alpha (TNF-alpha), interleukin-1 (IL-1), and IL-6. Inhibition of .N = O synthesis by KC. Treatment with LPS and IFN-gamma also induced significant production of prostaglandin E2 (PGE2), thromboxane B2 (TBX2), tumor necrosis factor alpha (TNF-alpha), interleukin-1 (IL-1), and IL-6. Inhibition of .N = O synthesis by the L-arginine analogue of NG-monomethyl-L-arginine (NMA) resulted in a further increase of PGE2, TXB2, and IL-6 but not IL-1 and TNF-alpha production, indicating specific inhibitory effects of endogenous .N = O synthesis on the secretory activity of KCs. PGE2 production was most sensitive to the suppressive effect of .N = O and increased 24 h after stimulation with LPS and IFN-gamma from 16.3 +/- 4.9 ng/10(6) KCs without NMA to 94.3 +/- 17.9 ng/10(6) KCs with NMA. This effect of NMA was reversed by a 10-fold increase of the L-arginine concentration. No recovery of PGE2 production was seen when .N = O synthesis was blocked after 24 h. NMA treatment increased cyclooxygenase activity more than threefold, suggesting that .N = O inhibits PGE2 and TXB2 production through diminished PGH2 availability. .N = O synthesis did not significantly affect total protein synthesis or viability of the KCs. These results show that .N = O influences the production of specific inflammatory mediators by KCs.