In vitro application of endotoxin enhances nitric oxide production in thoracic aortas from Mg-deficient rats.

Since endotoxin-induced vascular hyporeactivity to phenylephrine is enhanced in Mg-deficient rats, this study was designed to determine whether endotoxin directly enhances nitric oxide (NO) production in thoracic aortas isolated from Mg-deficient rats in vitro. Thoracic aortas isolated from Mg-deficient and control rats were cultured for 6 h with or without endotoxin (LPS). LPS (0.01-1.0 microg) increased NO production in a concentration-dependent manner. NO production in the presence of 0.1 and 1.0 microg/mL LPS was significantly higher in Mg-deficient rat aortas compared to aortas from control rats. The enhanced NO production was not significantly affected by endothelium-denudation. LPS-stimulated NO production was fully inhibited by a selective iNOS inhibitor, 1400W (0.1, 1.0 microM), in control rat aortas, but in Mg-deficient rat aortas inhibition by 1400W was only partial. A similar inhibitory effect was observed with anti-CD14 and anti-TLR4 antibodies. These results suggest that endotoxin enhances NO production in Mg-deficient rat aortas directly, and that endotoxin receptors might, at least in part, contribute to this enhancement.     

The role of interferon-gamma,nitric oxide and lipopolysaccharide in intestinal graft-versus-host disease developing in F1-hybrid mice

(C57BL/6 x DBA/2)F1-hybrid mice injected with lymphoid cells from wild-type, C57BL/6 donors develop acute, lethal graft-versus-host disease (GVHD) in which the intestine is a major target. In its destructive phase intestinal GVHD is characterized by apoptosis of intestinal crypt epithelial cells and the development of endotoxaemia. Injection of as little as 10 microg endotoxin is lethal in mice with acute GVHD, and associated with the release of large amounts of tumour necrosis factor-alpha (TNF-alpha) into the serum. To explore the role of interferon-gamma (IFN-gamma) in the pathogenesis of intestinal GVHD we used IFN-gamma gene knockout (gko) mice as donors. Recipients of grafts from these donors did not develop intestinal GVHD and, unlike recipients of wild-type grafts, did not die when injected with lipopolysaccharide (LPS). We also found that injection 10 microg LPS into recipients of wild-type grafts induced apoptosis of intestinal epithelial crypt cells and was associated with a burst of nitric oxide production in the intestine. Administration of N(omega)nitro L-arginine methyl ester blocked this response. In contrast, LPS did not induce either intestinal epithelial cell apoptosis or increased nitric oxide production in recipients of IFN-gamma gko grafts. These findings indicate that donor-derived IFN-gamma is instrumental for the development of intestinal GVHD. In a previous study we showed that recipients of IFN-gamma gko grafts develop high levels of LPS-induced TNF-alpha release. When our current data are viewed in the context of this observation, they suggest that intestinal epithelial cell apoptosis in the parent-->F1-hybrid model of acute GVHD is mediated primarily by nitric oxide rather than TNF-alpha, and that this depends on donor-derived IFN-gamma.     

Paradoxical facilitation of pentylenetetrazole-induced convulsion susceptibility in mice lacking neuronal nitric oxide synthase

The major aim of this study was to elucidate the relationship between nitric oxide (NO) and generalized epilepsy. Mice lacking the neuronal nitric oxide synthase (nNOS) gene (nNOS^−/−) were used in this study to determine the relationship between nNOS α and NO in pentylentetrazole (PTZ)-induced convulsions. nNOS^−/− mice exhibited severe convulsions following injection with a subconvulsive dose of PTZ (40 mg/kg i.p.) and convulsive doses were lethal in all of the mice (60 mg/kg i.p.) following tonic convulsions.     

L-arginine-dependent nitric oxide formation and nitrite release in bone marrow-derived macrophages stimulated with bacterial lipopeptide and lipopolysaccharide.

This study shows that stimulating bone marrow-derived macrophages with either lipopolysaccharide (LPS) or the lipopeptide N-palmitoyl-S-(2,3-bis(palmitoyloxy)-(2RS)-propyl)-(R)- cysteinyl-alanyl-glycine (Pam3Cys-Ala-Gly), a synthetic analogue of the N-terminal part of bacterial lipoprotein, leads to the formation of nitric oxide (NO) and nitrite (NO2-), a stable analogue of NO. NO was detected by applying the chemiluminescence method and by measuring the activity of exogenously added soluble guanylate cyclase (GC), which is strongly and selectively activated by NO. Synthesis of NO and NO2- occurs via activation of the L-arginine and NADPH-dependent enzyme(s) present in the cytosol of bone marrow-derived macrophages. No produced by this non-constitutive L-arginine pathway is thought to be responsible for the cytostatic and killing properties of macrophages (Stuehr & Nathan, 1989). Macrophages stimulated either with LPS or Pam3Cys-Ala-Gly exhibited a 6-hr lag time before engaging in nitrite synthesis, a time at which expression of the NO-forming enzyme had already reached its maximum. The regulation of NO and NO2- synthesis during macrophage development seems to differ from that of cytokine synthesis. Whereas cytokine release varies during a culture period up to 20 days, NO synthesis and expression of the NO-forming enzyme remain unaltered. These studies show that, similar to LPS, Pam3Cys-Ala-Gly is a potent activator of 'the oxidative L-arginine pathway' in bone marrow-derived macrophages. Whether both stimuli use the same signal transfer mechanism to induce this pathway and whether NO synthesized by this pathway is involved in the activation of the enzyme guanylate cyclase in macrophages requires clarification.     

Further analysis of lethal factors in cycloheximide-treated mice given low doses of bacterial lipopolysaccharide

Endotoxin challenge (0.2 micrograms per mouse) 6 h after a standard dose of cycloheximide was compared with the previously-reported effects of simultaneous injection of cycloheximide and endotoxin. With the submicrogram dose of endotoxin given 6 h after cycloheximide, fatalities occurred without evidence of thrombogenic bilateral renal cortical necrosis which characterized mice dying after the two agents were given together. Anticoagulation with heparin or with ancrod is life-saving, indicating that cycloheximide-treated mice were fatally susceptible to fibrinogen-to-fibrin conversion, and that, in contrast to the situation where cycloheximide and endotoxin are given simultaneously, there was no essential demand for supplementary glycocorticosteroid. A dose of 5.0 micrograms of endotoxin given 6 h after cycloheximide was fatal; again no renal cortical necrosis occurred, but both ancrod and hydrocortisone were essential to ensure survival.     

Preexposure of mouse peritoneal macrophages to lipopolysaccharide and other stimuli enhances the nitric oxide response to secondary stimuli

The aim of this study was to compare the regulation of the production of tumor necrosis factor- (TNF-) and secondary nitric oxide (NO) in macrophages submitted to a sequence of two stimulations. Pre-exposure for 18h of mouse thioglycollate-elicited peritoneal macrophages to low doses (1–10 ng/ml) of lipopolysaccharide (LPS), in the presence or absence of serum, induces on one hand a desensitization (endotoxin tolerance) for secondary TNF- reponses to LPS and, on the other hand, a 4 fold increase (priming) of serondary NO responses. Preexposure to components from Gram-positive bacteria (lipoteichoic acid, peptidoglycan) and to a synthetic lipid structurally related to lipid A (compound M4), induced similar effects. In contrast to the desensitization for TNF- secretion, the priming for NO production was not mimicked by sodium nitroprusside, a generator of NO. The results suggest that concomitant but distinct activation pathways induced by LPS and other agents can be dissociated by serum-independent modulation processes elicited by preexposure of the cells to LPS itself, or to other stimuli.accepted by M. J. Parnham     

Expression of neuronal and inducible nitric oxide synthases in the thymus under normal conditions and after administration of bacterial endotoxin

The topography of thymocytes expressing neuronal and inducible nitric oxide synthases and changes in the content of luminescent immunoreactive products in these cells after intraperitoneal injection of bacterial lipopolysaccharide were studied by double immunohistochemical labeling. Under normal conditions neuronal nitric oxide synthase-immunopositive cells formed a wide network in thymus medulla (except for perivascular regions). Inducible nitric oxide synthase was expressed in single cells at the corticomedullary boundary. Lipopolysaccharide markedly increased the intensity of luminescence and number of inducible nitric oxide synthase-immunoreactive cells. However, this agent sharply decreased the intensity of luminescence in neuronal nitric oxide synthase-immunopositive cells of the stroma. Our results indicate that neuronal and inducible nitric oxide synthases are synthesized in various stromal cells of the thymus. Expression of these enzyme isoforms undergoes opposite changes during inflammation.     

Haemodynamic responses to angiotensin II in conscious lambs: role of nitric oxide and prostaglandins

It was hypothesized that nitric oxide (NO) and prostaglandins (PGs) play a synergistic role in modulating haemodynamic responses to angiotensin II (ANG II) in an age-dependent manner. To this end, experiments were carried out in conscious, chronically instrumented lambs aged ∼1 week (N = 9) and ∼6 weeks (N = 10) to evaluate the haemodynamic responses to ANG II, before and after treatment with the l-arginine analogue, N-nitro-l-arginine methyl ester (l-NAME), as well as the cyclooxygenase inhibitor, indomethacin (INDO). Pressor and renal blood flow responses to ANG II were measured before (control) and after administration of l-NAME (20 mg kg⁻¹), following pretreatment with either vehicle (VEH) (experiment 1) or INDO (1 mg kg⁻¹, experiment 2). The two experiments were carried out at minimum intervals of 48 h. In both age groups, the pressor and renal vasoconstrictor responses to ANG II were augmented by pretreatment with INDO, the effects being similar at 1 and 6 weeks. The haemodynamic responses to ANG II were, however, not altered after l-NAME following pretreatment with either VEH or INDO. These data provide new evidence that soon after birth, endogenously produced PGs, but not endogenously produced NO, balance the vasoconstrictor actions of ANG II. There is, however, no apparent interaction between PGs and NO in modulating the responses to ANG II postnatally.     

Induction of proinflammatory cytokines and nitric oxide by Trypanosoma cruzi in renal cells

Chagas disease is typically associated with cardiac involvement. During the acute phase of murine infection with Trypanosoma cruzi, severe acute myocarditis can develop. Prior to cardiac alteration, however, infected mice present with renal inflammatory infiltration causing acute kidney injury due to an ischemia/reperfusion lesion. Thus, the present study was undertaken in order to evaluate whether the parasites or some of their components would directly affect renal cells. As such, this study employed kidney cell lines (mesangial, epithelial, and proximal tubular) that mimic different regions of the renal system. Mesangial cells are more resistant to infection, showing reduced parasite internalization relative to epithelial and proximal tubular cells. Upon infection, mesangial cells produced more nitric oxide, tumor factor necrosis-α, and interferon-γ and showed decreased viability when compared to the other cell lines. These results indicate that the resistance of mesangial cells to infection may be related to the increased expression of nitric oxide and proinflammatory cytokines. Conversely, the high levels of nitric oxide produced by these cells caused impairment of cell integrity and viability. Higher nitric oxide concentrations promote cellular injury and can be involved in the genesis of ischemia/reperfusion lesions in acute kidney injury.     

Induction of rheumatoid factors in mice by immune complexes of bacterial lipopolysaccharide with mouse IgG antibody

Administration of 2,4,6-trinitrophenylated E. coli lipopolysaccharide (TNP-LPS) complexed with mouse IgG antibody to TNP specifically gave rise to a marked production of rheumatoid-like factors (RF) in the recipient mice, in contrast to the low and nonspecific RF production via polyclonal B cell activation by the same dosage of LPS or TNP-LPS alone. The RF activity induced by the LPS immune complexes was associated with both IgG and IgM and directed primarily to the C gamma 2 region as judged by the heterophilic reactivity toward fragments of rabbit IgG. The results suggest that antibody molecules attached to LPS constitute novel epitopic groups on the mitogenic carrier and stimulate B cells in a specific manner to induce the autoantibodies.     

Developmental patterns of seizure susceptibility in inbred strains of mice.

Samples of mice from each of 6 inbred strains were tested for audiogenic and electroconvulsive seizures at 5 ages (14, 21, 28, 35, and 42 days). A moderately large within-strain correlation (.67) was found, indicating that developmental patterns of susceptibility to audiogenic and electroconvulsive seizures are similar within each strain. The finding of an even larger between-strain correlation (.91) indicated that strains which are highly susceptible to audiogenic seizures are also likely to be highly susceptible to electroconvulsive seizures. In a 2nd experiment, whole brain norepinephrine and serotonin were assayed in each of 5 inbred strains at 21 and 42 days of age. Results were consistent with the hypothesis that levels of these amines are inversely related to seizure susceptibility. Mice from strains which were susceptible to seizures at 21 days of age had significantly lower levels of norepinephrine and serotonin in brain than did 42-day-old, seizure-resistant animals.     

The involvement of nitric oxide in corpus luteum regression in the rat: feedback mechanism between prostaglandin F(2alpha) and nitric oxide.

In the corpus luteum (CL), prostaglandin F(2alpha) (PGF(2alpha)) is a physiological agent with luteolytic actions. Nitric oxide (NO) is a messenger molecule capable of modulating diverse pathophysiological processes. The aim of the present study was to investigate the role of ovarian NO in PGE (a luteotrophic prostanoid) and PGF(2alpha) (a luteolytic prostanoid) production and in progesterone synthesis during CL regression in the rat. To obtain a longer functional CL, we used a pseudopregnant (PSP) rat model. By means of intrabursa ovarian sac treatment of two competitive nitric oxide synthase (NOS) inhibitors, N(G)-monomethyl-L-arginine (L-NMMA, 1 mg/kg) and N(W)-nitro-L-arginine methyl ester (L-NAME; 3 mg/kg), and sodium nitroprusside (SNP, 0.05 mg/kg) as a NO generator, we found that NO, produced by the ovarian tissue during the last 2 days of CL development (days 8 and 9), increased PGF(2alpha) production in the ovary and diminished serum progesterone concentrations leading to CL involution. We also proposed a positive feedback mechanism between PGF(2alpha) and NO, to ensure luteal regression. Thus, we injected intraperitoneally a luteolytic dose (3 microg/kg) of a synthetic PGF(2alpha) during the mid and late phase of CL development. Ovarian NOS activity was evaluated. The results confirmed our hypothesis; we did not see any effect in the mid-stage of CL development, but increased ovarian NOS activity was found in PGF(2alpha)-injected late pseudopregnant rats.     

Role of prostaglandins and nitric oxide in gastric damage induced by metamizol in rats

OBJECTIVE AND DESIGN: In addition to the depletion of prostaglandins (PGs), oxygen free radicals generation and nitrogen species haven been implicated in non-steroidal anti-inflammatory drugs (NSAIDs)-induced gastric injury. The aim of the present study was to examine changes in PGE2 generation and its relationship with proinflammatory parameters and nitric oxide (NO) production in the comparative pathogenesis of gastric injury induced by metamizol vs. diclofenac, NSAIDs that present different gastric tolerability and cyclooxygenase (COX) inhibition profiles. MATERIAL: Studies were performed in Wistar-Han rats. TREATMENTS: Metamizol (120, 500 and 1,000 mg/kg body weight) and diclofenac (50 mg/kg body weight) were given by oral administration. METHODS: Determinations were made of macroscopic and histological evaluation of gastric mucosal injury, gastric prostaglandin synthesis (PGE2 levels), myeloperoxidase activity (MPO), tumor necrosis factor-alpha levels (TNF-a), cyclic guanosine monophosphate (cGMP), nitric oxide synthase activity (NOS) and NOS mRNA expression. RESULTS: Metamizol, only at the highest doses assayed, provoked weak lesions in the gastric mucosa. To the contrary, diclofenac treatment presented the highest grade of lesion. All treatments decreased PGE2 gastric generation. Treatment of the animals with metamizol neither modified the MPO activity nor TNF-alpha levels. In contrast, statistically significant increases in both parameters were observed after diclofenac administration. cGMP levels were not influenced with diclofenac treatment, nevertheless metamizol reduced the nucleotide levels, which was accompanied by an inhibition of constitutive NOS (cNOS) activity without modifying the mRNA expression of the enzyme. CONCLUSIONS: In addition to inhibition of PG synthesis, damage induced by metamizol was associated with an inhibition of the NO/cGMP pathway and cNOS activity. In contrast, diclofenac-induced gastric damage was associated with an increase of the inflammatory response.     

Interplay between DtxR and nitric oxide reductase activities: a functional genomics approach indicating involvement of homologous protein domains in bacterial pathogenesis

Corynebacterium diphtheriae pathogenesis depends on the production of toxin (Dtx), which in turn depends on a micromolar concentration of nitric oxide (NO)-mediated deactivation of DtxR (an iron-dependent regulator). Inside a host, the pathogen often encounters excess of NO that acts as an oxidative toxicant. Therefore a critical level of NO needs to be maintained by the pathogen. This necessitates reduction of excess NO by the presence of a reductase, namely nitric oxide reductase (NOR). Similar to the expression of toxin, the expression of NOR is possibly regulated by another regulator NorR, as has been found in other gram positive and gram-negative bacteria. Therefore, a correlation between concentration of NO on the deactivation of DtxR and transactivation of NorR becomes apparent. However, unlike many other pathogens the presence of NOR and NorR in C. diphtheriae has not been established. We applied a combination of bioinformatics and comparative genomics approach on C. diphtheriae genome using Escherichia coli as a model organism to find some structural and functional homologoues for the two genes in question. The various domain characteristics for the two proteins (NOR and NorR) have been taken into account in this analysis. Through extensive genome and proteome search we have been able to identify key regulatory genes, which are possibly involved in coordination and control of NO stress in C. diphtheriae. Our finding will progress the understanding of the complete regulatory mechanism for evasion and maintenance of pathogenesis by this and other pathogenic organisms.     

Role of galectin-3 in breast cancer metastasis: involvement of nitric oxide

We investigated the role of galectin-3 in metastasis of human breast carcinoma BT549 cells using the experimental liver metastasis model. Underlying mechanisms were then elucidated using the liver/tumor co-culture and cell culture systems. After intrasplenic injection, galectin-3 cDNA transfected BT549 cells (BT549(gal-3 wt)) formed metastatic colonies in the liver, while galectin-3 null BT549 cells (BT549(par)) did not, demonstrating that galectin-3 enhances metastatic potential. More than 90% of BT549(gal-3 wt) cells survived after 24 hours-co-culture with the liver fragments isolated following ischemia treatment. In contrast, more than half of BT549(par) cells showed metabolic death following co-culture with the liver fragments. When the liver from inducible nitric oxide synthase (iNOS) knockout mice was used, no cytotoxicity to BT549(par) cells was observed. Thus, iNOS exerts cytotoxicity on BT549(par) cells and galectin-3 can protect against iNOS-induced cytotoxicity. BT549(gal-3 wt) also exhibited enhanced survival against peroxynitrite (up to 400 micromol/L) in vitro. A single mutation in the NWGR motif of galectin-3 obliterated both metastatic capability and cell survival, indicating that the antiapoptotic function of galectin-3 is involved in enhanced metastasis. In conclusion, galectin-3 enhances the metastatic potential of BT549 cells through resistance to the products of iNOS, possibly through its bcl-2-like antiapoptotic function.