The Enhancing Effect of Tumour Necrosis Factor-α on Oxidative Stress in Endotoxemia

Abstract: The enhancing effect of tumour necrosis factor-α (TNF-α) on oxidative stress with or without a sublethal dose of endotoxin was examined. The mortality of mice treated with recombinant human TNF-α (1X10⁴ units/mouse, intravenously) and endotoxin (0.01-1 mg/kg, intraperitoneally) was dependent on the dose of endotoxin. The liver lipid peroxide level, superoxide anion generation and serum lactate dehydrogenase activity, especially serum lactate dehydrogenase-5 isozyme leakage, in mice 2-4 hr after administration of recombinant human TNF to endotoxin-pretreated mice (0.5 mg/kg, intraperitoneally) were markedly higher than in those without endotoxin, whereas the administration of recombinant human TNF significantly decreased the non-protein sulfhydryl level, superoxide dismutase and glutathione peroxide activities in the liver of endotoxin-injected mice compared with those in mice treated with recombinant human TNF or endotoxin alone. Furthermore, findings clearly demonstrated that J774A.1 cells stimulated with recombinant human TNF (1X10⁴ units/ml) can effectively produce nitric oxide in the presence of endotoxin, and the production was dependent on the dose of endotoxin (0.01-10 μg/ml). The level of lipid peroxide in mice 4 hr after administration of recombinant human TNF and lead acetate (50 mg/kg, intravenously) was markedly higher than that in the mice treated with recombinant human TNF alone. By contrast, injection of polymyxin-B (20 mg/kg, intraperitoneally, an anti-endotoxin drug) markedly decreased the lipid peroxide level in the liver of the mice treated with recombinant human TNF and lead acetate. These findings suggest that the oxidative stress caused by TNF occurs as a enhancing effect of endotoxion or by bacterial translocation from the intestinal gut under reduction of reticuloendothelial system function in various disease states, and that the effect of TNF may cause a marked increase of toxicity of oxidative stress by endotoxin.     

Nitric oxide reverses endotoxin-induced inflammatory hyperalgesia via inhibition of prostacyclin production in mice.

We examined whether nitric oxide (NO), derived from constitutive NO synthase (NOS) and/or inducible NOS (iNOS), could contribute to endotoxin-induced inflammatory hyperalgesia via interacting with nuclear factor-kappaB (NF-kappaB), inducible cyclooxygenase (COX-2) and/or polyADP-ribose synthase (PARS). Injection of endotoxin (10 mg kg(-1), i.p.) to mice elicited hyperalgesia, determined by hot plate test, which is prevented by NO precursor (L-arginine), cNOS/iNOS inhibitor (N(G)-nitro-L-arginine methyl ester; L-NAME), NF-kappaB inhibitor (N-acetylserotonin), COX inhibitor (indomethacin), COX-2 inhibitor (DFU) and PARS inhibitor (3-aminobenzamide). Endotoxin caused a decrease in serum nitrite levels prevented by N-acetylserotonin, L-arginine, indomethacin, DFU or 3-aminobenzamide. Endotoxin increased serum 6-keto-PGF(1alpha) levels diminished by L-arginine or aminoguanidine (iNOS inhibitor). L-Arginine, L-NAME, aminoguanidine, DFU or 3-aminobenzamide prevented endotoxin-induced decrease in heart, lungs, kidneys and brain nitrite and malonedialdehyde levels and myeloperoxidase activity. In conclusion, NO reverses endotoxin-induced inflammatory hyperalgesia via inhibition of prostacyclin production, and also contributes to the analgesic effect of NF-kappaB, COX or PARS inhibitors.     

Role of nitric oxide in systemic effect of theophylline on mouse body temperature

In the present study, the interaction of nitric oxide synthase (NOS) inhibitors, L-NAME (N(G)-nitro-L-arginine methyl ester HCl) and L-NA (N(omega)-nitro-L-arginine), and its precursor, L-arginine (2-(S)-2-amino-5-[(aminoiminomethyl)amino] pentatonic acid), with theophylline on mouse body temperature was studied. Intraperitoneal (i.p.) injection of different doses of theophylline altered body temperature. Lower doses of theophylline (12.5 and 25 mg/kg) increased, but a higher dose (100 mg/kg) reduced, the animals' body temperature. The combination of L-arginine (20 and 40 mg/kg) with the highest dose of theophylline potentiated the hypothermic effect induced by the latter drug, while L-arginine by itself did not alter body temperature. L-NAME (10-80 mg/kg) or L-NA (10 mg/kg) plus a lower dose of theophylline (12.5 mg/kg) reduced the theophylline-induced hyperthermic response. L-NA (1, 5, and 10 mg/kg) in combination with the high dose of theophylline (100 mg/kg) also induced greater hypothermia. Both L-NAME and L-NA by themselves reduced body temperature. It is concluded that nitric oxide (NO) may be involved in the effects of theophylline on body temperature in mice.     

COMPARISON OF RESPONSES TO AMINOGUANIDINE AND Nω-NITRO-L-ARGININE METHYL ESTER IN THE RAT AORTA

1. We have compared the effect of aminoguanidine with that of N^ω-nitro-L-arginine methyl ester on isolated thoracic aortic rings obtained either from endotoxin (lipopolysaccharide, 10 mg/kg, i.v. for 3 h) or vehicle (saline) treated rats. 2. Administration of endotoxin for 3h resulted in a hypo tension and a significant reduction of pressor responses to nor-epinephrine (1 μg/kg, i.v.) in the anaesthetized rat. 3. In intact rings obtained from vehicle treated rats, amino guanidine (0.3 and 1mmol/L) had no significant effect on acetylcholine-induced relaxation (10^-9–10^-5mol/L), whereas N^ω-nitro-L-arginine methyl ester (0.3mmol/L and 1mmol/L) abolished that response, suggesting that aminoguanidine does not inhibit the activity of constitutive nitric oxide synthase. 4. Relaxation induced by L-arginine (10^-6–10^-2mol/L) was competitively inhibited by both aminoguanidine (0.3 mmol/L) and N^ω-nitro-L-arginine methyl ester (0.3 mmol/L) in endo thelium-denuded aortic rings obtained from endotoxin treated rats. 5. Three hours of endotoxaemia was associated with an impairment of contraction to norepinephrine (10^-9–10^-6 mol/L) in the endothelium-denuded aorta ex vivo. This hyporeactivity to norepinephrine was partially restored by treatment of the vessels either with aminoguanidine (0.3 mmol/L) or with N^ω-nitro-l-arginine methyl ester (0.3 mmol/L) in vitro. 6. These results in isolated thoracic aortae of the rat reinforce that aminoguanidine is a selective inhibitor of the inducible nitric oxide synthase, whereas N^ω-nitro-L-arginine methyl ester is a non-selective inhibitor of both the inducible and constitutive nitric oxide synthase.     

Role of Zn(2+) in oxidative stress caused by endotoxin challenge

The role of Zn(2+) in oxidative stress during endotoxemia was investigated. In rats fed a Zn(2+)-deficient diet (Zn(2+) concentration of less than 1.5 mg/kg) for 8 weeks, the Zn(2+) level in the serum was about 62% lower than that in rats fed a Zn(2+)-adequate diet (Zn(2+) concentration, 50 mg/kg). The Zn(2+) level in serum 18 h after administration of endotoxin (6 mg/kg, i.p.) to Zn(2+)-deficient diet rats was markedly lower than that of the endotoxin/Zn(2+)-adequate diet group. Lipid peroxide formation in the liver of Zn(2+)-deficient diet rats was markedly increased 18 h after endotoxin injection compared with that in the endotoxin/Zn(2+)-adequate diet group. Metallothionein in the liver of endotoxin/Zn(2+)-adequate diet rats was increased more than 17-fold by endotoxin administration, while a markedly lower level of metallothionein was observed in the endotoxin/Zn(2+)-deficient diet group. On the other hand, treatment with ZnSO(4) (100 microM) significantly increased endotoxin (1 microg/ml)-induced tumor necrosis factor-alpha (TNF-alpha) production in J774A.1 cells. Our results clearly demonstrated that treatment with ZnSO(4) significantly inhibited the endotoxin-induced increase in intracellular Ca(2+) level in J774A.1 cells. However, a cell membrane-permeable Zn(2+) chelator, N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN, 1 microM), did not affect the endotoxin-induced TNF-alpha production or Ca(2+) level in J774A.1 cells. In addition, we investigated whether Zn(2+) can suppress nitric oxide (NO) generation and cytotoxicity in endotoxin-treated cells. Treatment with ZnSO(4) (50 microM) significantly inhibited endotoxin-induced NO production in J774A.1 cells, but did not affect endotoxin-induced cytotoxicity. These findings suggest that zinc may play an important role, at least in part, in the oxidative stress during endotoxemia.     

Role of nitric oxide in maintaining vascular integrity in endotoxin-induced acute intestinal damage in the rat.

1. The role of endogenous nitric oxide (NO) in maintaining intestinal vascular integrity following acute endotoxin (E. coli. lipopolysaccharide) challenge was investigated in the anaesthetized rat by use of NG-monomethyl-L-arginine (L-NMMA), a selective inhibitor of NO synthesis. 2. L-NMMA (10-50 mg kg-1, i.v.) pretreatment enhanced both the macroscopic and histological intestinal damage and the increases in vascular permeability, measured as the leakage of [125I]-labelled human serum albumen, induced after 15 min by endotoxin (50 mg kg-1, i.v.). 3. The effects of L-NMMA (50 mg kg-1, i.v.) were enantiomer specific, as D-NMMA had no effect. Furthermore, these effects were reversed by L-arginine (300 mg kg-1, i.v.), the precursor of NO synthesis but not by D-arginine (300 mg kg-1, i.v.). 4. L-NMMA (10-50 mg kg-1, i.v.) increased mean systemic arterial blood pressure but this does not appear to be the mechanism by which endotoxin-induced intestinal damage was enhanced, since similar systemic pressor responses induced by phenylephrine (10 micrograms kg-1 min-1, i.v.), had no such effect. 5. The results suggest that synthesis of NO from L-arginine has a role in maintaining the microvascular integrity of the intestinal mucosa following acute endotoxin challenge.     

Role of Ca2+ on Endotoxin-Sensitivity by Galactosamine Challenge: Lipid Peroxide Formation and Hepatotoxicity in Zymosan-Primed Mice

Abstract: This study was investigated to clarify the role of intracellular Ca²⁺ following endotoxin treatment (1 mg/kg, intraperitoneally) to D-galactosamine-sensitized mice (400 mg/kg, intraperitoneally), and to observe lipid peroxide levels, an index of hepatotoxicity, in endotoxin/galactosamine (Ga1N)-challenged mice under activation of macrophages, especially Kupffer cells, by zymosan. The liver lipid peroxide level and serum glutamic pyruvic transminase activity in mice 18 hr after administration of endotoxin/Ga1N were markedly higher than those in mice treated only with endotoxin. In spite of an increase in lipid peroxide formation, there was little or no effect of Ga1N administration on xanthine oxidase and superoxide dismutase activities in mice given endotoxin. However, the injection of verapamil (10 mg/kg, subcutaneously) markedly decreased lipid peroxide levels in liver of endotoxin/Ga1N-injected mice. In the mice given a Ca²⁺-deficient diet, lipid peroxide level in liver after endotoxin/Ga1N injection was markedly decreased compared to that in mice fed a normal diet. Administration of dexamethasone (200 μg/kg, intraperitoneally) in mice 1 hr before treatment with endotoxin/Ga1N did not induce lipid peroxide formation. Administration of endotoxin to Ga1N-treated mice resulted in a higher level of liver cytosolic free Ca²⁺ ([Ca²⁺]_i) than that in endotoxin-treated mice. On the other hand, Ca²⁺-ATPase activity in liver plasma membrane in the endotoxin/Ga1N-treated mice was markedly decreased as compared with endotoxin alone. On the contrary, the Ca²⁺-ATPase activity in liver mitochondria was higher in endotoxaemic mice treated with Ga1N than in mice given endotoxin alone. State 3 respiration and respiratory control index, which are parameters of mitochondrial function, were decreased more in the liver of mice treated with endotoxin/Ga1N than in the endotoxin-treated group. These findings suggest that [Ca²⁺]_i may participate in the lipid peroxide formation which results from endotoxin/Ga1N-induced hepatotoxicity under conditions of zymosan-activated macrophages, and that the increases of endotoxin-sensitivity caused by Ga1N challenge may greatly contribute to Ca²⁺-mobilization in the hepatocyte.     

Mediation of nitric oxide in inhibitory effect of morphine against electroshock-induced convulsions in mice

Nitric oxide (NO) and morphine have been coupled in many physiological as well as pathological processes. The present study examined the involvement of the L-arginine/NO pathway in the anticonvulsant properties of systemic morphine (2-30 mg/kg) against electroshock seizures (ECS) in mice. Morphine decreased the intensity of maximal electroshock seizures (MES) and increased the threshold for ECS. Neither the NOS substrate L-arginine (30, 60, and 100 mg/kg), the reversible nonspecific NOS inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME; 3, 10, and 30 mg/kg), the irreversible specific inducible NOS inhibitor aminoguanidine (20, 50, and 100 mg/kg), nor the opioid receptor antagonist naloxone (0.1, 0.3, and 1 mg/kg) did alter per se the ECS threshold or the intensity of MES at doses used. However, both naloxone and L-NAME, but not aminoguanidine, inhibited the anticonvulsant effects of morphine (30 mg/kg) against ECS, while L-arginine potentiated the anticonvulsant effects of lower doses of morphine (2 or 10 mg/kg). Low doses of naloxone (0.1 or 0.3 mg/kg) or L-NAME (3 mg/kg), which did not alter morphine effect per se, showed additive anticonvulsant effects against MES. Thus, the L-arginine/NO pathway seems to play a role in the anticonvulsant properties of morphine against ECS and this mediation involves the constitutive, but not the inducible, form of nitric oxide synthase.     

Pressor effect of NG-monomethyl-L-arginine in SHRSP

Pressor effects of NG-monomethyl-L-arginine (L-NMMA), a selective inhibitor of nitric oxide production from L-arginine, on mean blood pressure (MBP) were investigated in conscious Wistar Kyoto rats (WKY) and stroke-prone spontaneously hypertensive rats (SHRSP). L-NMMA (0.1-10 mg/kg, i.v.) elicited a dose-dependent increase in the MBP of WKY and SHRSP. The pressor response to L-NMMA was more marked in SHRSP than in WKY. These results suggest that nitric oxide may play an important role in the blood pressure regulation in the conscious SHRSP.     

NG-monomethyl-L-arginine and NG-nitro-L-arginine inhibit endothelium-dependent relaxations in human isolated omental arteries.

The L-arginine analogues NG-monomethyl-L-arginine (L-NMMA, 10(-4) M) and NG-nitro-L-arginine methyl ester (L-NAME, 10(-4) M), which specifically inhibit the synthesis of nitric oxide from L-arginine, significantly reduced acetylcholine-induced endothelium-dependent relaxations in rings of human omental arteries. The inhibitory potency of L-NMMA and L-NAME was similar. Addition of L-NMMA or L-NAME to the organ bath did not induce any significant changes in the resting tension of the tissues. The effects of L-NMMA were reversed by L-arginine (3 x 10(-4) M). The L-NMMA enantiomer, D-NMMA (10(-4) M), did not influence either the basal tone of the preparation or the relaxing effects of acetylcholine. Arterial relaxations induced by sodium nitroprusside (10(-6) M) were not influenced by incubation with L-NMMA or L-NAME. These results suggest that endothelium-dependent relaxations in human omental arteries are mediated by the endogenous and substrate-specific generation of nitric oxide from L-arginine.     

Modification of antinociceptive action of Ukrain by endogenous nitric oxide in the writhing syndrome test in mice

The effects of L-arginine, the physiological precursor of nitric oxide (NO), and inhibitors of NO-synthase on the antinociceptive action of Ukrain (4.75, 9.5, and 19.0 mg/kg i.p.) were investigated using the writhing syndrome test in mice. It was found that L-arginine (0.1 or 1.0 mg/kg i.p.) significantly decreased or enhanced the antinociceptive effect of Ukrain, depending on the combination administered. In addition, the inhibitors of NO-synthase NG-nitro-L-arginine methyl ester (L-NAME) (1.0 and 10 mg/kg i.p.), 7-nitroindazole (1.0 mg/kg i.p.) and NG-monomethyl-L-arginine acetate (L-NMMA) (1.0 mg/kg i.p.) significantly enhanced Ukrain-induced antinociception. These results suggest that endogenous NO can modify the antinociceptive effect of Ukrain.     

An L-arginine-derived factor mediates endotoxin-induced vascular hyposensitivity to calcium

Aortas removed from rats treated with bacterial endotoxin displayed a reduced sensitivity to calcium (CaCl2, 10 microM-10 mM) in depolarizing medium (100 mM K+). Sensitivity was reduced further in the presence of L-arginine (1 mM) but restored to control by N omega-nitroarginine methyl ester (L-NAME, 300 microM) or NG-monomethyl-L-arginine (L-NMMA, 300 microM), inhibitors of nitric oxide synthesis from L-arginine. Furthermore, addition of methylene blue (10 microM), an inhibitor of soluble guanylate cyclase, restored the contractile response to 10 mM CaCl2. The results suggest that vascular hyposensitivity to calcium involves stimulation of guanylate cyclase subsequent to activation of the L-arginine pathway by endotoxin.     

Inhibitory effects of endogenous L-arginine analogues on nitric oxide synthesis in platelets: role in platelet hyperaggregability in hypertension

1. An increase in plasma concentrations of endogenous L-arginine analogues, which are inhibitors of nitric oxide (NO) synthesis, may be involved in platelet activation and the increased risk of thrombosis in essential hypertension. Nitric oxide is synthesised in platelets from the amino acid L-arginine by inducible and constitutive isoforms of NO synthase (NOS), which leads to increased levels of cGMP. 2. In the present study, we investigated basal intraplatelet cGMP levels, platelet aggregation and pro-inflammatory biomarkers in hypertensive patients. The effects of endogenous (N(G)-monomethyl-L-arginine (L-NMMA) and asymmetric dimethylarginine (ADMA); both at 1 mmol/L) and exogenous (aminoguanidine and N(G)-nitro-L-arginine; both at 1 mmol/L) L-arginine analogues and the neutral amino acid L-leucine (1 mmol/L) in inhibiting NOS activity in platelets were also investigated. 3. Twelve healthy controls and 18 hypertensive patients participated in the study. Platelet aggregation induced by collagen was increased in hypertensive patients (95 +/- 5%) compared with controls (72 +/- 5%). Basal NOS activity and intraplatelet cGMP levels were reduced in hypertensive platelets. Moreover, ADMA, L-NMMA and L-leucine were effective inhibitors of NO synthesis in both hypertensive and control platelets. Essential hypertension led to an inflammatory response, with increased plasma concentrations of fibrinogen, C-reactive protein and cytokines. 4. These findings provide evidence that, in essential arterial hypertension, the enhanced plasma levels of endogenous L-arginine analogues ADMA and L-NMMA, potent inhibitors of L-arginine transport and NO synthesis in platelets, may play a role in increased platelet aggregation via a cGMP-dependent mechanism.     

Nitric oxide involvement in consolidation, but not retrieval phase of cognitive performance enhanced by atorvastatin in mice

Atorvastatin, a widely-used medication in treatment of hypercholesterolemia, has shown some benefits in treating cognition impairment in Alzheimer's disease. In this study, effects of atorvastatin on spatial recognition memory and the involvement of nitric oxide (NO) has been determined on consolidation and retrieval of memory in a two-trial recognition Y-maze test. Memory was impaired using scopolamine (1mg/kg, i.p.); atorvastatin (1, 5mg/kg, p.o.) was administered, either in presence or in absence of a non-specific NO synthase inhibitor, L-NAME (3, 10mg/kg, i.p.); a specific inducible NO synthase inhibitor, aminoguanidine (100mg/kg, i.p.); and a NO precursor, L-arginine (750 mg/kg, i.p.). Results: 1) atorvastatin (5mg/kg) significantly improved memory performance in a dose-dependent manner on consolidation and retrieval stage of memory in scopolamine-treated mice; 2) the beneficial effects of atorvastatin on memory consolidation was significantly reversed by L-NAME (10mg/kg) and aminoguanidine; 3) L-arginine slightly potentiated the effects of sub-effective dose of atorvastatin (1mg/kg) on memory consolidation; 4) either L-NAME (up to 10mg/kg), or aminoguanidine did not affect the memory improvement by atorvastatin on retrieval stage; 5) the effects of sub-effective dose of atorvastatin (1mg/kg) on retrieval of memory were not potentiated by L-arginine. The present study demonstrates that atorvastatin improves both consolidation and retrieval phases of memory. This effect is affected by NO synthase inhibitors and NO precursor, L-arginine, only in memory consolidation phase, but not in retrieval phase. It is concluded that NO might be involved in consolidation of spatial memory improvement by atorvastatin.     

L-arginine-induced relaxation of the rat isolated penile bulb.

The effects of L-arginine, the precursor in the synthesis of nitric oxide (NO), were investigated in the penile bulb isolated from saline (control) or lipopolysaccharide (20 mg/kg, i.p.)-treated rats. Four consecutive concentration-response curves for L-arginine were made at hourly intervals with the penile bulb. L-arginine (10(7)-10(-3) M) elicited a concentration- and time-dependent relaxation response in the control group. The NO synthase (NOS) inhibitors, N(G)-methyl-L-arginine (L-NMMA) and aminoguanidine, guanylate cyclase inhibitor, 1-H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ) and protein synthesis inhibitor, cycloheximide, inhibited L-arginine-induced relaxation. In the lipopolysaccharide-group, L-arginine produced a pronounced non-time-dependent relaxation at the first concentration-response curve, which was not different from the fourth response of the control group. This response was also inhibited by aminoguanidine. These results show that L-arginine induced NO-mediated relaxation and suggest the presence of a biochemical pathway converting L-arginine to NO, which is probably an inducible type in the penile bulb.     

The effect of cyclosporin A on morphine tolerance and dependence: involvement of L-arginine/nitric oxide pathway

Cyclosporin A is known to decrease nitric oxide (NO) production in nervous tissues. The effects of systemic cyclosporine A on the induction and expression of morphine tolerance and dependence, acute morphine-induced antinociception, and the probable involvement of the L-arginine/nitric oxide pathway in these effects were assessed in mice. Cyclosporin A (20 mg/kg), N(G)-nitro-L-arginine methyl ester (L-NAME) (10 mg/kg) and a combination of the two at lower and per se non-effective doses (5 and 3 mg/kg, respectively) showed a similar pattern of action, inhibiting the induction of tolerance to morphine-induced antinociception and increasing the antinociception threshold in the expression phase of morphine tolerance. These agents also inhibited the expression of morphine dependence as assessed by naloxone-precipitated withdrawal signs, while having no effect on the induction of morphine dependence. L-Arginine, at a per se non-effective dose (60 mg/kg), inhibited the effects of Cyclosporin A. Moreover, acute administration of Cyclosporin A (20 mg/kg) or L-NAME (10 mg/kg) enhanced the antinociception induced by acute administration of morphine (5 mg/kg), while chronic pretreatment with Cyclosporin A (20 mg/kg) or L-NAME (10 mg/kg) for 2 days (twice daily) did not affect morphine-induced antinociception. The inducible nitric oxide synthase inhibitor, aminoguanidine (100 mg/kg), did not alter morphine antinociception, tolerance or dependence. In conclusion, decreasing NO production through constitutive nitric oxide synthase may be a mechanism through which cyclosporin A differentially modulates morphine tolerance, dependence and antinociception.     

Activation of multiple mitogen-activated protein kinases by recombinant calcitonin gene-related peptide receptor

Aminoguanidine is an inhibitor of the inducible form of nitric oxide synthase (iNOS). In the present study, the effect of aminoguanidine on concanavalin A-induced hepatitis was examined. Treatment of mice with concanavalin A (10 mg/kg, i.v.) induced interferon-gamma and iNOS mRNA expression in the liver before the elevation of plasma alanine aminotransferase activity. Immunohistochemical study showed the induction of iNOS protein expression in the area of necrosis. Aminoguanidine (1, 3 and 10 mg/kg, i.p.) inhibited the concanavalin A-induced elevation of alanine aminotransferase activity. Aminoguanidine (10 mg/kg, i.p.) did not inhibit concanavalin A-induced interleukin-2, interferon-gamma, tumor necrosis factor-alpha or iNOS mRNA expression in the liver. The plasma nitrite/nitrate level was elevated at 6 and 24 h after concanavalin A treatment. The elevation of nitrite/nitrate was inhibited by aminoguanidine (10 mg/kg, i.p.). From these results, we conclude that nitric oxide formed by iNOS may be involved in the development of concanavalin A-induced hepatitis.     

Interaction of inducible nitric oxide synthase and cyclooxygenase-2 inhibitors in formalin-induced nociception in mice

Studies with inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 inhibitor were conducted to assess their synergistic antinociceptive effect and possible therapeutic advantage. The antinociceptive interaction of rofecoxib, a selective cyclooxygenase-2 inhibitor, with aminoguanidine hydrochloride, a selective iNOS inhibitor, was examined in the formalin-induced paw-licking model in mice. Analysis of variance (ANOVA) and the isobolographic method were used to identify the nature of the antinociceptive interaction. Different doses of rofecoxib (1, 3, 10 and 30 mg/kg) and aminoguanidine hydrochloride (10, 30, 100 and 300 mg/kg) alone were administered orally to adult male albino mice (20-30 g). Only high doses of rofecoxib (10 and 30 mg/kg) and aminoguanidine hydrochloride (100 and 300 mg/kg) showed a statistically significant antinociceptive effect. Combination of a subthreshold dose of rofecoxib (1 mg/kg) with increasing doses of aminoguanidine hydrochloride (30, 100 and 300 mg/kg) resulted in potentiated antinociception (P<0.05). Combined therapy with a subthreshold dose of aminoguanidine hydrochloride (30 mg/kg) with increasing doses of rofecoxib (1, 3, 10 and 30 mg/kg) also resulted in significant antinociception (P<0.05). These results suggest that rofecoxib and aminoguanidine hydrochloride act synergistically in their antinociceptive action in mice. A possible mechanism of interaction is that nitric oxide (NO) stimulates the activity of cyclooxygenase-2 by combining with its heme component. Furthermore, the present results suggest that combination therapy with rofecoxib and aminoguanidine hydrochloride may provide an alternative for the clinical control of pain.     

Reduction by NG-nitro-L-arginine of H2O2-induced endothelial cell injury.

1. The effects of three analogues of NG-nitro-L-arginine (L-NOARG) and NG-monomethyl-L-arginine (L-NMMA), inhibitors of nitric oxide (NO) synthase, on hydrogen peroxide (H2O2)-induced endothelial cell injury were studied. 2. Endothelial cell injury was assessed by measuring the release of intracellular lactate dehydrogenase (LDH) and 51Cr. 3. Addition of H2O2 (250-1,000 microM) to endothelial cells induced the release of LDH dose-dependently. The release of LDH was reduced by pretreatment with NG-nitro-L-arginine methyl ester (L-NAME, 10(-4)-4 x 10(-3) M), L-NOARG (10(-4)-4 x 10(-3) M) and NG-nitro-L-arginine benzyl ester (L-NABE, 10(-4)-4 x 10(-3) M), inhibitors of NO synthase. 4. L-NOARG analogues also reduced H2O2-induced 51Cr release from endothelial cells, while L-NMMA had no effect. 5. The protective effect of L-NAME was not reversed by addition of L-arginine (L-Arg, 1-10 mM). 6. Both L-NAME and L-NMMA completely inhibited L-Arg metabolism to L-citrulline coupled with NO synthesis. 7. These findings suggest that L-NOARG analogues but not L-NMMA reduced H2O2-induced endothelial cell injury, and that these effects may not be related to inhibition of NO production.     

L-arginine protects against stress-induced gastric mucosal lesions by preserving gastric mucus

1. We have shown that exogenously administered L-arginine protects against water immersion restraint (WIR) stress-induced gastric mucosal lesions in rats through preservation of nitric oxide (NO) generation via constitutive nitric oxide synthase (cNOS), but not inducible nitric oxide synthase (iNOS), in the gastric mucosa. We have also indicated that impaired gastric mucus synthesis and secretion occur through a decrease in gastric cNOS activity in WIR-stressed rats. Therefore, in the presesnt study, we examined whether exogenously administered L-arginine exerts a protective effect against WIR stress-induced gastric mucosal lesions in rats through preservation of gastric mucus synthesis and secretion by NO generated from the administered amino acid via cNOS in the gastric mucosa. 2. Rats were subjected to WIR stress for 3 and 6 h. Either L-arginine (150-600 mg/kg) or D-arginine (600 mg/kg) was injected intraperitoneally 0.5 h prior to WIR stress. Either N(G)-monomethyl L-arginine (L-NMMA; 100 mg/kg) or N(G)-monomethyl D-arginine (D-NMMA; 100 mg/kg) was injected subcutaneously 0.5 h prior to WIR stress. Total NOS, cNOS, iNOS, nitrite and nitrate (breakdown products of NO), hexosamine (an index of gastric mucin) and adherent mucus were assayed in the gastric mucosa. 3. Pretreatment with L-arginine, but not D-arginine, protected against gastric mucosal lesions in rats subjected to WIR stress for 3 and 6 h in a dose-dependent manner. Pretreatment with L-arginine, but not D-arginine, attenuated decreases in hexosamine and adherent mucus concentrations and cNOS activity and increases in total NOS and iNOS activities and nitrite/nitrate concentration in the gastric mucosal tissue of rats subjected to WIR stress for 3 and 6 h in a dose-dependent manner. Both the protective effect of L-arginine against gastric mucosal lesions and the attenuating effect of the amino acid on the decreases in gastric mucosal hexosamine and adherent mucus concentrations and cNOS activity in rats subjected to WIR stress for 6 h were counteracted by cotreatment with L-NMMA, a nitric oxide synthase inhibitor, but not D-NMMA. 4. These results suggest that exogenously administered L-arginine exerts a protective effect against stress-induced gastric mucosal lesions in rats at least partly through preservation of gastric mucus synthesis and secretion by NO produced from the administered amino acid via cNOS in gastric mucosal tissue.