Protective Role Of Nitric Oxide Research Articles

Protective role of nitric oxide in indomethacin-induced gastric ulceration by a mechanism independent of gastric acid secretion

Gastric ulceration was induced in rats by i.p. injection of the non-steroidal anti-inflammatory drug (NSAID), indomethacin (IND) (30 mg kg−1). Pyloric ligation was carried out in each animal before injection to enable collection of the gastric juice. Three hours later, the animals were killed and their stomachs were removed. In the gastric juice, the amounts of mucin, pepsin and HCl were assessed. Gastric mucosa were scrapped for the determination of nitric oxide (NO) (as nitrite) after evaluation of the gastric ulcer index. The influence of arginine (ARG) (300 mg kg−1), a NO precursor, NG-nitro- l -arginine methyl ester (l -NAME) (50 mg kg−1), a non-selective constitutive nitric oxide synthase/inducible nitric oxide synthase (cNOS/iNOS) inhibitor, and the selective iNOS inhibitor aminoguanidine (AMG) (50 mg kg−1) were studied. Each NO modulator was injected i.p. 30 min before IND administration. Results indicated that IND elevated gastric acidity by 80% of the normal group, decreased non-significantly mucosal nitrite by 22% and exhibited a remarkably high ulcer index (χ= 17). Neither mucin nor pepsin levels were significantly altered. In comparison with the IND group, pretreatment with l -NAME caused a significant decrease in gastric HCl, further decrease in mucosal nitrite (50% of normal) and a two-fold increase in the ulcer index score (χ= 34), despite the decrease in HCl. AMG did not alter gastric acidity, decreased mucosal nitrite by 38% of the normal value and failed to alter significantly the ulcer index of IND. On the other hand, pretreatment with ARG did not alter the gastric acidity and raised mucosal nitrite by 10% above normal. Surprisingly, ARG improved the gastric ulcer score (χ= 1) almost similar to the normal score (χ=zero). Therefore, this study creates a new pathway for the potential treatment of NSAID gastric ulceration through modulation of NO synthesis, regardless of the effect on gastric acidity.

EPR studies of nitric oxide interactions of alkoxyl and peroxyl radicals in in vitro and ex vivo model systems.

A model compound of lipid peroxidation, tert-butyl hydroperoxide (tBOOH), was used in vitro to investigate (i) the generation of tBOOH-derived free radicals by hematin or rat enterocytes and (ii) the modulation of cell-generated free radical production by a nitric oxide (NO) donor, or when these cells were primed to produce NO. In hematin-catalyzed decomposition of tBOOH, NO from nitrosoglutathione, or S-nitroso-N-acetylpenicillamine suppressed the generation of peroxyl radicals (measured by direct electron paramagnetic resonance) and tert-butylalkoxyl, methoxyl, and methyl radicals (measured by electron paramagnetic resonance spin trapping). Similarly, co-incubation of S-nitroso-N-acetylpenicillamine or nitrosoglutathione with tBOOH caused significant decreases in tBOOH-derived free radical generation catalyzed by enterocytes. Epithelial cells are the known source of the inducible form of NO synthase in the intestine of rats challenged with lipopolysaccharide (LPS). Enterocytes isolated from LPS-treated rats produced decreased levels of tBOOH-derived radicals. These decreases in free radical production were further decreased when these cells were treated with LPS in vitro. These findings demonstrated that exogenously added or endogenously produced NO could modulate the extent of tBOOH-derived free radical generation in enterocytes. These decreases in free radical production could, at least in part, describe the protective role of NO from hydroperoxide-induced injury.

Detrimental effects of nitric oxide inhibition on hepatic encephalopathy in rats with thioacetamide-induced fulminant hepatic failure.

Hepatic encephalopathy is a complex neuropsychiatric syndrome seen secondary to acute liver failure, chronic parenchymal liver disease, or portal-systemic anastomosis. Vasodilatation induced by nitric oxide (NO) may be involved in the development of hepatic coma. However, there are no comprehensive data concerning the effects of NO inhibition on the severity of hepatic encephalopathy. Male Sprague-Dawley rats weighing 300-350 g were used. Fulminant hepatic failure was induced by intraperitoneal injection of thioacetamide (TAA, 350 mg kg-1 day-1) for 3 days. Rats were divided into two groups to receive either NG-nitro-L-arginine methyl ester (L-NAME, 20 mg kg-1 day-1 via intragastric gavage) or normal saline (N/S) from 2 days prior to TAA administration for 5 days. Severity of encephalopathy was assessed by counts of motor activity and neurobehaviour test scores. Plasma levels of endotoxin, tumour necrosis factor-alpha and nitrate/nitrite were determined by the chromogenic Limulus assay, enzyme-linked immunosorbent assay and colorimetric assay, respectively. Compared with N/S-treated rats, the mortality rate was significantly higher in rats receiving L-NAME (59% vs. 18%, P < 0.01). Inhibition of NO had detrimental effects on the counts of motor activities (P < 0.05) and neurobehaviour score (P < 0.01). Rats treated with L-NAME had significantly higher plasma levels of endotoxin (26.7 +/- 3.8 pg mL-1) and tumour necrosis factor-alpha (29.4 +/- 6.5 pg mL-1) compared with rats treated with N/S (13.2 +/- 2.7 pg mL-1 and 11.2 +/- 2.6 pg mL-1, respectively, P < 0.01). Plasma levels of endotoxin and tumour necrosis factor-alpha, but not of nitrate/nitrite, were significantly correlated with the severity of hepatic encephalopathy (P < 0.05). Chronic L-NAME administration had detrimental effects on the severity of encephalopathy in TAA-treated rats, suggesting a protective role of NO in the development of fulminant hepatic failure.

Counter-protective role for interleukin-5 during acute Toxoplasma gondii infection.

The role of interleukin-5 (IL-5) during Toxoplasma gondii infection was investigated by comparing disease progression in IL-5 gene deficient (IL-5-/-) mice and their wild-type (WT) counterparts on a C57BL/6 background. IL-5-/- mice infected orally with T. gondii were less susceptible to infection than WT mice as demonstrated by reduced mortality rates. Consistent with this data, orally infected IL-5-/- mice had less severe pathological changes in their small intestines than WT mice at 8 days postinfection. At this time, splenocytes and mesenteric lymph node cells derived from IL-5-/- mice produced levels of IL-12, interferon-gamma (IFN-gamma), IL-4, IL-10, and nitric oxide (measured as nitrite) similar to those derived from WT mice when stimulated with Toxoplasma lysate antigen. However, peak serum IL-12 and IFN-gamma levels (at days 6 and 8, respectively) were significantly higher in IL-5-/- mice than in WT mice. In addition, WT mice but not IL-5-/- mice had raised levels of eosinophils in their peripheral blood between days 5 and 8 following infection. Oral administration of N omega-nitro-L-arginine methyl (from day 4 postinfection) increased mortality rates in both IL-5-/- and WT mice, indicating a protective role for nitric oxide during the early stages of oral T. gondii infection. In comparison with oral infection, no difference in mortality was observed between IL-5-/- and WT mice following intraperitoneal infection with T. gondii, with all mice surviving until 35 days postinfection. Similarly, no significant differences were observed in the severity of the meningitis, perivascular cuffing, or number of microglial nodules or parasites in the brains of intraperitoneally infected mice. Together, these results demonstrate a detrimental role for IL-5 during the early stage of oral infection with T. gondii which is associated with increased small-intestine pathology, eosinophilia, and reduced plasma IL-12 and IFN-gamma levels.

Nitric oxide synthase inhibition augments acute allergic reactions in the pig airways in vivo.

The aim of this study was to examine the effects of nitric oxide synthase inhibition on antigen- and histamine-induced acute airway reactions, in order to clarify the possible modulating role of NO. Twelve specific-pathogen-free pigs (sensitized with Ascaris suum antigen) were challenged with an antigen aerosol during mechanical ventilation and anaesthesia. Six pigs were pretreated with N(G)-nitro-L-arginine (L-NA, 10 mg x kg(-1)), a NO synthase inhibitor, 30 min before challenge. In separate experiments, seven sensitized pigs received histamine (5 mg) aerosols before and after L-NA treatment. It was found that pretreatment with L-NA resulted in an enhanced airways resistance response to antigen (areas under the curve 0-90 min were (mean+/-SEM) 1,119+/-160 versus 555+/-56 (cmH2O x L(-1) x s(-1) x min for controls, p<0.05 (Mann-Whitney U-test), whereas this response to histamine was not affected by L-NA. Moreover, L-NA pretreatment significantly enhanced total protein (1.85+/-0.43 versus 0.31+/-0.06 g x L(-1), p<0.01) and histamine levels (42.8+/-16.0 versus 2.6+/-0.8 nM, p<0.05) in bronchoalveolar lavage fluid 45 min after antigen challenge. In conclusion, this study showed that N(G)-nitro-L-arginine enhanced reactions occurring during the acute allergic reaction in pigs in vivo. This indicates a protective role of nitric oxide, which might occur through downregulation of histamine release from mast cells rather than a direct bronchodilating effect of nitric oxide.

Inhibition of nitric oxide synthesis augments pulmonary oedema in isolated perfused rabbit lung

The role of nitric oxide (NO) in precipitating pulmonary oedema in acute lung injury remains unclear. We have investigated the mechanism of involvement of NO in the maintenance of liquid balance in the isolated rabbit lung. Thirty pairs of lungs were perfused with colloid for up to 6 h, during which pulmonary vascular resistance (PVR) and capillary pressure (PCP) were measured frequently, and time to gain 5 g in weight (t5) was recorded. Four protocols with different perfusate additives were studied: (i) none (control, n = 11); (ii) 10 mmol NG-nitro-L-arginine methyl ester (L-NAME) (n = 6); (iii) 10 mmol L-NAME with 100 mumol lodoxamide, an inhibitor of mast cell degranulation (n = 7); (iv) 10 mmol L-NAME with 10 mumol 8-bromo-3',5'-cyclic guanosine monophosphate (8Br-cGMP), an analogue of cGMP that may reduce vascular permeability by relaxing contractile elements in endothelial cells (n = 6). Neither PVR nor PCP differed between protocols. L-NAME markedly reduced t5 from 248 (27) min (mean (SEM)) in protocol (i) to 144 (5) min in protocol (ii) (P < 0.05). Both lodoxamide (t5 = 178 (7) min) and 8Br-cGMP (t5 = 204 (10) min) substantially corrected the effect of L-NAME (P < 0.005). Results suggest that maintenance of a low permeability by NO may involve mast cell stabilization and endothelial cell relaxation.

Nitric oxide protects Cu,Zn-superoxide dismutase from hydrogen peroxide-induced inactivation

Reaction of Cu,Zn-superoxide dismutase (SOD1) and hydrogen peroxide generates a putative oxidant SOD-Cu 2+- OH that can inactivate the enzyme and oxidize 5,5′-dimethyl-1-pyrroline- N-oxide (DMPO) to DMPO- OH. In the presence of nitric oxide ( NO), the SOD1/H 2O 2 system is known to produce peroxynitrite (ONOO −). In contrast to the proposed cytotoxicity of NO conferred by ONOO −, we report here a protective role of NO in the H 2O 2-induced inactivation of SOD1. In a dose-dependent manner, NO suppressed formation of DMPO- OH and inactivation of the enzyme. Fragmentation of the enzyme was not affected by NO. Bicarbonate retarded formation of ONOO −, suggesting that NO competes with bicarbonate for the oxidant SOD-Cu 2+- OH. We propose that NO protects SOD1 from H 2O 2-induced inactivation by reducing SOD-Cu 2+- OH to the active SOD-Cu 2+ with concomitant production of NO + which reacts with H 2O 2 to give ONOO −.

Nitric Oxide Protects Venules Against Histamine-Induced Leaks

The goal of the present study was to investigate the prophylactic role of nitric oxide (NO) in the mesenteric microvasculature in preventing microvascular leakage subsequent to histamine application, and to evaluate the response of mast cells during these conditions. Regions of the rat mesenteric microcirculation were flushed free of blood and pretreated with either the nitric oxide donor sodium nitroprusside (SSP 10(-6) M) or Hepes-buffered saline containing 0.5% bovine serum albumin (HBS-BSA) for 15 minutes, then exposed to histamine (10(-3) M) for another three minutes. In another set of experiments, the microvasculature was treated with either histamine (10(-3) M) for three minutes or SNP (10(-6) M) for 15 minutes. A control group was treated with HBS-BSA for 15 minutes. The protective role of NO was evaluated by its ability to reduce or prevent histamine-induced venular leaks. Mesenteric microvessels pretreated with SNP before histamine suffusion showed a significant decrease in both area and number of venular leaks following the perfusion of fluorescein isothiocyanate-labeled bovine serum albumin (FITC-BSA). Although SNP pretreatment did not reduce the percentage of mast cells that degranulated in the presence of histamine, it did somewhat reduce the severity of the degranulation. This study demonstrated that nitric oxide availability protects mesenteric venules against histamine-induced leaks, but does not prevent degranulation of mast cells. Therefore, nitric oxide probably acts directly on venular endothelial cells to prevent leak formation.

Role of inducible nitric oxide synthase in dextran sulphate sodium-induced colitis.

Different authors have postulated both toxic and protective effects for nitric oxide (NO) in the pathophysiology of active inflammation. To examine the role of NO, especially that produced by the inducible form of nitric oxide synthase (iNOS), by investigating the effects of NOS inhibitors and NO donors on inflammation in experimental acute colitis. Acute colitis was induced in rats by dextran sulphate sodium (DSS). White blood cell counts and levels of thiobarbituric acid reactants in the portal blood were determined, as were histological changes in the colonic mucosa. We then evaluated the effects of N(G)-nitro-L-arginine methyl ester (L-NAME), aminoguanidine (AG) and an NO donor on DSS-induced changes in these inflammatory parameters. Inhibition of NO production by either L-NAME or AG worsened DSS-induced inflammation, suggesting a protective role for NO in acute colitis. On the other hand, a NO donor also exaggerated DSS-induced inflammatory parameters, suggesting that acute colitis may be aggravated by either too much or too little NO. These results suggest that medical treatment of ulcerative colitis must aim for maintenance of appropriate NO levels in the intestinal mucosa.

Inhibition of nitric oxide production exacerbates chronic ocular toxoplasmosis.

There is considerable controversy as to the roles of parasite proliferation and the inflammatory response in destruction of the retina during Toxoplasma gondii infection. A murine model was used to investigate the role of nitric oxide in pathogenesis of chronic ocular toxoplasmosis. Increased quantities of messenger RNA (mRNA) transcripts for iNOS were detected in the eyes of chronically infected C57BL/6 mice compared with noninfected control mice. Inhibition of nitric oxide (NO) by the addition of Lomega-nitro-L-arginine methyl ester (L-NAME) to the drinking water of infected mice between weeks 4-6 of infection, exacerbated ocular inflammation. The amount of inflammation was assessed semiquantitatively in histological sections of the eye. Eyes from L-NAME treated mice showed a significant increase in inflammation of the retina (P = 0.02), choroid (P = 0.03), and vitreous (P = 0.02) compared with control mice. These results demonstrate a protective role for NO in the control of chronic, ocular toxoplasmosis.

Glyceryl trinitrate, a nitric oxide donor, suppresses renal oxidant damage caused by potassium bromate

Nitric oxide (NO) is a short-lived, readily diffusible intracellular messenger molecule associated with multiple organ-specific regulatory functions. Endogenous stimulation or exogenous administration of NO have been shown to inhibit production of reactive oxygen species (ROS) and expression of oxidant-mediated molecular or tissue injury. Potassium bromate (KBrO3) is one such potent renal oxidant that acts through generation of ROS-mediated lipid peroxidation, and causes increased ornithine decarboxylase activity, enhanced rate of DNA synthesis and depletion of the antioxidant armoury of the tissue. In this study, we elucidate the effect of exogenous NO administration, using the NO donor glyceryl trinitrate (GTN), on KBrO3-induced nephrotoxicity, oxidative stress and cell proliferation. KBrO3 administration at a dose of 125 mg/kg body weight results in significant (P < 0.001) depletion in renal glutathione (GSH) content, and glutathione reductase (GR) activity with a concomitant increase in microsomal lipid peroxidation, and blood urea nitrogen (BUN) and creatinine levels. Parallel to these changes, we found significant enhancement in ornithine decarboxylase (ODC) activity and rate of renal DNA synthesis. Subsequent administration of GTN resulted in dose-dependent amelioration of GSH content and GR activity with concomitant inhibition of lipid peroxidation, and BUN and creatinine levels. In addition, GTN administration to KBrO3-intoxicated rats resulted in significant dose-dependent down regulation of enhanced ODC activity and rate of [3H]-thymidine incorporation in renal DNA, providing support for the protective role of NO in attenuation of KBrO3-induced oxidative stress and cell proliferation. Enhancement of oxidative tissue injury and cell proliferation on administration of the NO inhibitor, L-NAME, further demonstrates the protective efficacy of endogenous NO. These data suggest that NO inhibits KBrO3-induced tissue injury, oxidative stress and proliferative response in the rat kidney.

Protective role of nitric oxide in the TNFα-mediated hepatic microvascular inflammatory response following acute ethanol ingestion

Gut-drived endotoxin is thought to be involved in the pathogenesis of alcoholic liver disease. Nitric oxide (NO) has a protective function in attenuating hepatic injury elicited by endotoxin and is suspected to play a role in the hepatic microvascular responses induced following alcohol ingestion. As a result, the role of NO and the possible involvement of TNFα in these hepatic microcirculatory responses were investigated in mice using established in vivo microscopic methods. The livers were examined 3 h after acute ethanol (1 g/kg b.w.) ingestion alone or in combined with inhibition of NO synthesis by intravenous N G-monomethyl- l-arginine (L-NMMA) injection. Portal venous blood pressure and ethanol concentrations also were measured. Acute ethanol ingestion alone elicited 2-fold increase in leukocyte adherence (WBC) to the sinusoid lining but without alteration in blood flow when compared with maltose-dextrin treated controls. In contrast, in mice treated with the combination of L-NMMA and ethanol, WBC was increased an additional 3-fold above animals treated with ethanol alone. This was accompanied by a significant reduction in sinusoidal blood flow, no significant changes in portal blood pressure or concentrations of ethanol. Simultaneous administration of l-arginine eliminated these microcirculatory disturbances. Pretreatment with anti-mouse TNFα serum neutralized the microvascular responses elicited by the combined treatment of ethanol and L-NMMA. The results demonstrate that inhibition of NO production leads to hepatic microcirculatory dysfunction in response to a small dose of acute ethanol ingestion which by itself is scarcely stimulatory and that anti-TNFα neutralizes these responses. Thus, the results suggest that NO plays a significant role in stabilizing the TNFα mediated hepatic microvascular inflammatory responses elicited following acute ethanol ingestion.

Role of nitric oxide in intestinal ischaemia-reperfusion injury studied using electron paramagnetic resonance.

The role of nitric oxide in intestinal ischaemia-reperfusion (I/R) remains poorly defined, partly because of difficulty in detecting the nitric oxide free radical. In this study nitric oxide production was assessed during intestinal I/R by direct measurement using electron paramagnetic resonance (EPR), and the production of nitric oxide in jejunum and ileum was correlated with their different abilities to resist I/R injury. Rats were given an electron spin trapper (diethyldithiocarbamate/ferrous citrate) by intraperitoneal injection. Thirty-six segments each of jejunum and ileum were subjected to 15-90 min of ischaemia and 25 min of reperfusion. Tissue samples were analysed for EPR signals using a spectrometer. Mean(s.d.) basal nitric oxide level was significantly higher in ileum (3.39(1.42) units) than jejunum (0. 65(0.05) units) (P = 0.0005). Increasing ischaemic times in the ileum resulted in decreasing nitric oxide levels (85, 32 and 13 per cent of basal level at 30, 60 and 90 min respectively); reperfusion resulted in further nitric oxide reduction (mean decrease 26 per cent). Severe (grade 3) histological damage was observed in low nitric oxide states (after 15 min of I/R in jejunum, 60 min of I/R in ileum). Nitric oxide can be measured in intestinal tissues directly by EPR. The findings support a protective role for nitric oxide in I/R, and offer an explanation for the greater resistance to I/R of ileum.

Intracellular redox state determines whether nitric oxide is toxic or protective to rat oligodendrocytes in culture.

We found that several nitric oxide donors had similar potency in killing mature and immature forms of oligodendrocytes (OLs). Because of the possibility of interaction of nitric oxide with intracellular thiols, we tested the effect of the nitrosonium ion donor S-nitrosylglutathione (SNOG) in OL cultures in the setting of cystine deprivation, which has been shown to cause intracellular glutathione depletion. Surprisingly, the presence of 200 microM SNOG completely protected OLs against the toxicity of cystine depletion. This protection appeared to be due to nitric oxide, because it could be blocked by hemoglobin and potentiated by inclusion of superoxide dismutase. We tested the effect of three additional NO* donors and found that protection was not seen with diethylamine NONOate, a donor with a half-life measured in minutes, but was seen with dipropylenetriamine NONOate and diethylaminetriamine NONOate, donors with half-lives measured in hours. This need for donors with longer half-lives for the protective effect suggested that NO* was required when intracellular thiol concentrations were falling, a process evolving over hours in medium depleted of cystine. These studies suggest a novel protective role for nitric oxide in oxidative stress injury and raise the possibility that intracerebral nitric oxide production might be a mechanism of defense against oxidative stress injury in OLs.

The p75NTR-induced Apoptotic Program Develops through a Ceramide-Caspase Pathway Negatively Regulated by Nitric Oxide

SK-N-BE neuroblastoma cell clones transfected with p75(NTR) and lacking Trk neurotrophin receptors, previously reported to undergo extensive spontaneous apoptosis and to be protected by nerve growth factor (NGF) (Bunone, G., Mariotti, A., Compagni, A., Morandi, E., and Della Valle, G. (1997) Oncogene 14, 1463-1470), are shown to exhibit (i) increased levels of the pro-apoptotic lipid metabolite ceramide and (ii) high activity of caspases, the proteases of the cell death cascade. In the p75(NTR)-expressing cells, these parameters were partially normalized by prolonged NGF treatment, which, in addition, decreased apoptosis, similar to caspase blockers. Conversely, exogenous ceramide increased caspase activity and apoptosis in both wild-type and p75(NTR)-expressing cells. A new p75(NTR)-expressing clone characterized by low spontaneous apoptosis exhibited high endogenous ceramide and low caspase levels. A marked difference between the apoptotic and resistant clones concerned the very low and high activities of nitric-oxide (NO) synthase, respectively. Protection from apoptosis by NO was confirmed by results with the NO donor S-nitrosoacetylpenicillamine and the NO-trapping agent hemoglobin. We conclude that the p75(NTR) receptor, while free of NGF, triggers a cascade leading to apoptosis; the cascade includes generation of ceramide and increased caspase activity; and the protective role of NO occurs at step(s) in between the latter events.

Review article: the potential role of nitric oxide in chronic inflammatory bowel disorders.

The aetiology of the chronic inflammatory bowel diseases-ulcerative colitis and Crohn's disease-as well as 'microscopic colitis'-both collagenous (COC) and lymphocytic colitis (LC)-remains unknown. Autoimmune mechanisms, cytokine polymorphism, commensal bacteria, infectious agents and vascular impairment have all been proposed as playing important roles in the pathogenesis of this spectrum of diseases. A variety of proinflammatory mediators, including tumour necrosis factor alpha, interleukin-1beta, interferon gamma, leukotriene B4 and platelet activating factor, promote the adherence of phagocytes to the venular endothelium and extravasation of these cells into the colonic mucosa. In addition to large amounts of nitric oxide (NO), injurious peroxynitrite may be formed in the epithelium by the inducible nitric oxide synthase (iNOS), which is considered to elicit cytotoxicity by the generation of superoxide with reduced L-arginine availability. In active ulcerative colitis, and to a lesser extent in Crohn's disease, a greatly increased production of NO has been demonstrated by indirect and direct measurements. Surprisingly, even higher rates of production have been observed in COC-a condition which is never associated with injurious inflammation. The latter observation favours the notion that NO promotes mucosal integrity. Further evidence for a protective role of NO in chronic inflammatory bowel disorders is provided by the observation of increased susceptibility to the induction of experi mental colitis in 'knock-out' mice deficient in iNOS. Selective inhibitors of iNOS activity, as well as topical L-arginine, may therefore prove beneficial in inflammatory bowel disease by reducing the production of superoxide by iNOS, while only the former option may be expected to reduce diarrhoea in chronic inflammatory bowel disorders. Clearly, further experimental work needs to be done before testing topical L-arginine in human inflammatory bowel disease.

Protective role of nitric oxide in ischemia and reperfusion injury of the liver

Background: The suppressed production of nitric oxide (NO), associated with endothelial dysfunction, is thought to be a cause of ischemia and reperfusion injury of the liver. But findings of the salutary effects of NO enhancement on such injury have been conflicting. In this study, we tested our hypothesis that NO enhancement would attenuate ischemic liver injury. For this purpose, an NO precursor, L-arginine, and a novel NO donor, FK409, were applied to a 2-hour total hepatic vascular exclusion model in dogs. Study Design: L-arginine was administered IV at a dose of 100 mg/kg twice (n = 5), while 300 mg/kg twice of FK409 was infused continuously into the portal vein (n = 5). The drugs were given to the animals for 30 and 60 minutes before and after ischemia, respectively. Nontreated animals were used as the control (n = 10). Two-week survival, systemic and hepatic hemodynamics indices, liver function tests, energy metabolism, and histopathology were analyzed. Results: Both treatments comparably augmented hepatic tissue blood flow, decreased liver enzyme release, and increased high-energy phosphate restoration during the reperfusion period, all of which contributed to rescuing all of the treated animals from the 2-hour total hepatic ischemia. In contrast, ischemia caused 70% mortality in the control group. Histologically, structural abnormality and neutrophil infiltration were markedly attenuated by the treatments. Systemic hypotension was observed in the animals treated with FK409, however. Conclusions: Our data demonstrate that NO enhancement alleviates the liver injury caused by ischemia and reperfusion. The supplementation of L-arginine, rather than FK409, is considered more applicable to clinical use because of the absence of systemic adverse effects.

The amelioration of hepatocyte oxidative stress injury by nitric oxide released from S-nitroso-N-acetyl penicillamine: a study in immobilized perfused hepatocytes.

S-nitroso-N-acetyl penicillamine (SNAP, 0.1-0.5 mM) caused release of nitric oxide (NO) into the perfusion medium of immobilized hepatocytes. Oxidative injury of hepatocytes was evoked by tert-butyl hydroperoxide (TBH, 1 mM) and the functional and morphological ultrastructural integrity of the cells was monitored. At the end of a 270-min perfusion period, SNAP-induced NO reduced lactate dehydrogenase leakage in TBH-injured hepatocytes as compared to untreated TBH-injured cells (122% +/- 5 vs. 146% +/- 6 of control levels), lipid peroxides production (2.7 +/- 0.2 vs. 3.7 +/- 0.3 nmol/10(6) cells), increased O2 consumption (26 +/- 2 vs. 12 +/- 1 nmol/10(6) cells) although urea synthesis was reduced. SNAP improved the formation of granules in the Golgi complex as compared to untreated TBH-injured hepatocytes and preserved the ultrastructural architecture of mitochondria and the smooth endoplasmic reticulum. The present data support a possible protective role of NO in oxidative liver injury.

Protective roles of nitric oxide and testosterone in endotoxemia: evidence from NOS-2-deficient mice.

Lipopolysaccharide (LPS)-induced septic shock, which triggers nitric oxide (NO) overproduction, multiple organ dysfunction, and death, can be affected by gender and sex hormones. We hypothesized that NO is beneficial during endotoxemia and that this beneficial effect is influenced by sex hormones. C57BL/6 wild-type (WT) mice and congenic inducible NO synthase knockout (KO) mice were injected with LPS, and mortality was recorded for 4 days. After 5 mg/kg LPS, female KO mice had significantly higher mortality than WT. After 12.5 mg/kg LPS, both male and female KO mice had significantly higher mortality than WT. Ovariectomy did not alter mortality, but orchiectomy dramatically increased mortality in KO mice. After 5 mg/kg LPS, exogenous testosterone completely prevented the increased mortality in KO female and orchiectomized KO male mice. WT survival was not affected by exogenous testosterone. After 12.5 mg/kg LPS, exogenous testosterone significantly improved survival of female KO mice. Serum enzymes and organ edema, which may not correlate with mortality, were significantly and similarly increased in both WT and KO endotoxemic mice; however, edema was not observed in KO hearts. Thus, NO plays a protective role in endotoxemia while having differential effects on different organs. Importantly, testosterone is beneficial in endotoxemia when NO production is deficient, and may be therapeutic in certain septic patients.

Macrophage-induced inhibition of nitric oxide production in primary rat hepatocyte cultures via prostaglandin E2 release.

Kupffer cells and other macrophages play an important role in pathogenesis of toxicants in the liver. The aim of this study was to evaluate the effect of macrophages on hepatocyte production of nitric oxide (NO), which has been previously reported to be protective toward oxidative stress induced in primary rat hepatocytes. For this purpose, RAW 264.7 macrophages were added to primary rat hepatocytes at various ratios between macrophages and hepatocytes. These cocultures were supplemented with lipopolysaccharide (LPS) and interferon gamma (IFN-gamma) for 23 hours to induce NO synthase and trigger NO production. NO production was followed by quantification of nitrites in culture medium and dinitrosyl iron complexes (DNIC) in intact hepatocytes after separation from macrophages. In cocultured hepatocytes incubated with LPS and IFN-gamma, DNIC and nitrite levels decreased compared with those observed in hepatocytes cultured without macrophages in the same conditions. Moreover, inhibition of NO production in hepatocyte cocultures was macrophage-number-dependent. Macrophage-conditioned medium also inhibited NO production in hepatocytes, suggesting that the effect of macrophages was mediated by soluble factors. Among the soluble factors known to decrease NO levels are some cytokines, growth factors, reactive oxygen species, and prostaglandins. Ultrafiltration of macrophage-conditioned medium through a 500-d membrane to rule out higher-molecular-weight molecules, such as anti-inflammatory cytokines and growth factors, failed to restore NO production. In the same way, the use of superoxide dismutase (SOD) and catalase (CAT) to eliminate reactive oxygen species produced by macrophages did not lead to recovery of NO levels in hepatocytes. However, when NO synthesis was inhibited in macrophages by NG-monomethyl-L-arginine (L-NMMA), hepatocytes recovered the capacity to produce NO. A net decrease of prostaglandin E2 (PGE2) release by macrophages was concomitantly observed. Moreover, inhibition of PGE2 production in macrophages by indomethacin led to restoration of NO levels. Taken together, our observations suggest that NO synthesized by macrophages can decrease NO production in hepatocytes via PGE2 release. Because of the protective role of NO toward many liver injuries, it may be postulated that macrophages contribute through this mechanism to liver damage.