Endothelin enhances lipopolysaccharide-induced expression of inducible nitric oxide synthase in rat glial cells

Lipopolysaccharide is known to stimulate production of nitrite via expression of inducible nitric oxide (NO) synthase in not only macrophages but also glial cells. We found that in glial cell cultures lipopolysaccharide-stimulated inducible NO synthase expression and nitrite accumulation were synergistically enhanced by pretreatment with endothelin, whereas endothelin itself did not induce these responses. Pretreatment with endothelin-1, endothelin-3, and the selective endothelin type B (ETB) receptor agonist IRL 1620 caused the same effect with similar potencies, suggesting that the synergism was mediated via the endothelin ETB receptor. A protein kinase C inhibitor, calphostin C, suppressed endothelin-3-enhanced inducible NO synthase expression. Pretreatment with either endothelin-3 or phorbol ester enhanced lipopolysaccharide-induced production of tumor necrosis factor-alpha (TNF-alpha). Simultaneous addition of TNF-alpha increased lipopolysaccharide-stimulated inducible NO synthase expression. These results suggest that the increase in inducible NO synthase expression by endothelin was due to the elevated TNF-alpha production via protein kinase C. Our findings present the possibility that endothelin is implicated in neurotoxicity via enhancement of inducible NO synthase expression.     

Cell-specific effects of pentoxifylline on nitric oxide production and inducible nitric oxide synthase mRNA expression

Cytokine-stimulated astrocytes and macrophages are potent producers of nitric oxide (NO), a free radical proposed to play an important role in organ-specific autoimmunity, including demyelinating diseases of the central nervous system. The aim of this study was to investigate effects of pentoxifylline (PTX), a phosphodiesterase inhibitor with immunomodulatory properties, on NO production and inducible NO synthase (iNOS) mRNA expression in rat astrocytes and macrophages. We have shown that PTX affects cytokine (interferon-gamma, IFN-gamma; interleukin-1, IL-1; tumour-necrosis factor-alpha, TNF-alpha)-induced NO production in both cell types, but in the opposite manner--enhancing in astrocytes and suppressive in macrophages. While PTX did not have any effect on enzymatic activity of iNOS in activated cells, expression of iNOS mRNA was elevated in astrocytes and decreased in macrophages treated with cytokines and PTX. Treatment with PTX alone affected neither NO production nor iNOS mRNA levels in astrocytes or macrophages. This study indicates involvement of different signalling pathways associated with iNOS induction in astrocytes and macrophages, thus emphasizing complexity of regulation of NO synthesis in different cell types.     

Selective expression of inducible nitric oxide synthase in human prostate carcinoma

BACKGROUND: Nitric oxide (NO) is synthesized by inducible nitric oxide synthase (iNOS) and plays an important role in tumor growth and angiogenesis. NO generation by iNOS also influences the cytotoxicity of macrophages and tumor-induced immunosuppression. Before now, the expression of iNOS in prostate carcinoma tissue had not been determined. METHODS: In this study, tissue sections from 16 patients with prostate carcinoma were studied immunohistochemically and compared with tissue specimens from 10 patients with benign hyperplasia. RESULTS: Positive iNOS immunostaining was detected in all sections from patients with prostate carcinoma. The malignant epithelial cells were highly positive. The antibody against iNOS also marked round cells, which had the same cell shape as that observed for macrophages. These cells were located in stroma and epithelium adjacent to tumor islets. However, round cells in benign tissue stained negative for iNOS. None of the benign hyperplasia specimens stained positive for iNOS immunohistochemically. CONCLUSIONS: Prostate carcinoma tissue had a high iNOS content, whereas benign tissue did not. The authors suggest that epithelial iNOS expression can be used as a specific immunohistochemical marker for prostate carcinoma. NO generation by iNOS may play multiple roles in the development of this disease.     

Neuronal expression of inducible nitric oxide synthase after oxygen and glucose deprivation in rat forebrain slices

We apply a simple method for aligning protein sequences on the basis of a 3D structure, on a large scale, to the proteins in the scop classification of fold families. This allows us to assess, understand, and improve our automatic method against an objective, manually derived standard, a type of comprehensive evaluation that has not yet been possible for other structural alignment algorithms. Our basic approach directly matches the backbones of two structures, using repeated cycles of dynamic programming and least-squares fitting to determine an alignment minimizing coordinate difference. Because of simplicity, our method can be readily modified to take into account additional features of protein structure such as the orientation of side chains or the location-dependent cost of opening a gap. Our basic method, augmented by such modifications, can find reasonable alignments for all but 1.5% of the known structural similarities in scop, i.e., all but 32 of the 2,107 superfamily pairs. We discuss the specific protein structural features that make these 32 pairs so difficult to align and show how our procedure effectively partitions the relationships in scop into different categories, depending on what aspects of protein structure are involved (e.g., depending on whether or not consideration of side-chain orientation is necessary for proper alignment). We also show how our pairwise alignment procedure can be extended to generate a multiple alignment for a group of related structures. We have compared these alignments in detail with corresponding manual ones culled from the literature. We find good agreement (to within 95% for the core regions), and detailed comparison highlights how particular protein structural features (such as certain strands) are problematical to align, giving somewhat ambiguous results. With these improvements and systematic tests, our procedure should be useful for the development of scop and the future classification of protein folds.     

Role of nitric oxide in rat nephrotoxic nephritis: comparison between inducible and constitutive nitric oxide synthase

Nitric oxide (NO), generated by inducible NO synthase (iNOS) in migrating macrophages, is increased in glomerulonephritis. This study investigates the effect of NO inhibition on rat nephrotoxic nephritis (NTN) to clarify the role of NO production in glomerular damage. NTN was induced in Sprague Dawley rats by an injection of an anti-glomerular basement membrane (GBM) antibody. Urinary nitrite excretion and nitrite release from kidney slices (5.47 +/- 1.19 versus 2.15 +/- 0.73 nmol/mg protein, NTN versus Control, P < 0.05) were increased in NTN on day 2. Glomerular macrophage infiltration and intercellular adhesion molecule (ICAM)-1 expression increased from day 2. iNOS expression was increased in interstitial macrophages. Glomerular endothelial cell NOS (ecNOS) expression evaluated by counting immunogold particles along GBM was suppressed (0.06 +/- 0.02 versus 0.35 +/- 0.04 gold/micron GBM, P < 0.0001). Glomerular damage developed progressively. NG-nitro-L-arginine methyl ester (L-NAME), which inhibits both iNOS and ecNOS and aminoguanidine (AG), a relatively selective inhibitor for iNOS, equally suppressed nitrite in urine and renal tissue. Glomerular ICAM-1 expression and macrophage infiltration were reduced by L-NAME, but not by AG. Expression of ecNOS was significantly increased by L-NAME (0.91 +/- 0.08, P < 0.0001 versus NTN), but slightly by AG (0.18 +/- 0.04). AG significantly and L-NAME slightly attenuated the glomerular damage at day 4. In conclusion, suppression of iNOS prevents glomerular damage in the early stage of NTN. Treatment by L-NAME reduces macrophage infiltration by suppression of ICAM-1 expression, which may be explained by an increase in ecNOS expression.     

Adrenomedullin augments inducible nitric oxide synthase expression in cytokine-stimulated cardiac myocytes

BACKGROUND: Plasma levels of adrenomedullin are increased in patients with congestive heart failure, but there has been no report concerning the effects of adrenomedullin on the heart. We investigated the effects of adrenomedullin on NO synthase activity in cardiac myocytes. METHODS AND RESULTS: We measured the production of nitrite, a stable metabolite of NO, in cultured neonatal rat cardiac myocytes with the Griess reagent. Inducible NO synthase mRNA and protein expression were assayed by Northern and Western blotting, respectively. Incubation of the cultures with interleukin-1 beta (10 ng/mL) for 24 hours caused a significant increase in nitrite accumulation. Adrenomedullin significantly augmented nitrite production by interleukin-1 beta-stimulated but not by unstimulated cardiac myocytes in a dose-dependent manner (10(-10) to 10(-6) mol/L). The adrenomedullin-induced nitrite production by interleukin-1 beta-stimulated cells was accompanied by increased inducible NO synthase mRNA and protein expression. In the presence of dibutyryl cAMP, the interleukin-1 beta-induced nitrite accumulation was increased further, but the stimulatory effect of adrenomedullin on nitrite production was abolished. Adrenomedullin dose-dependently increased intracellular cAMP levels in cardiac myocytes. Addition of the calcitonin gene-related peptide (CGRP) receptor antagonist CGRP[8-37] to the culture dose-dependently inhibited both cAMP and NO generation stimulated by adrenomedullin. CONCLUSIONS: These results indicate that adrenomedullin acts on cardiac myocytes and augments NO synthesis in these cells under cytokine-stimulated conditions, at least partially through a cAMP-dependent pathway.     

Up-regulation of inducible nitric oxide synthase and production of nitric oxide by the Swarm rat and human chondrosarcoma

Phospholipids are the major constituents of cell membranes, and have numerous structural and functional roles in the nervous system. Although the metabolic pathways responsible for the syntheses of the phosphatides phosphatidylcholine (PtdCho), phosphatidylethanolamine (PtdEtn), and phosphatidylserine (PtdSer) are well understood, the mechanisms controlling these pathways in neural tissue have not been fully characterized. Recent studies have suggested that the main factors controlling PtdCho and PtdEtn synthesis by the Kennedy cycle tend to be the intracellular levels of key substrates for the biosynthetic enzymes, or changes in the activities of the rate-limiting enzymes. Moreover, different control mechanisms may operate, depending upon the functional state of the tissue.     

Endogenous opioids regulate the expression of inducible nitric oxide synthase by splenocytes

In the present study, we tested the hypothesis that lipopolysaccharide (LPS)-induced expression of nitric oxide synthase (iNOS) by splenocytes is modulated through the activation of endogenous opioids in the central nervous system. The initial studies determined the parameters of LPS-induced expression of iNOS by splenocytes. Rats were injected with LPS at doses of 0, 1, 10, 100, and 1000 microg/kg, and measures of both iNOS mRNA and protein showed a dose-dependent increase in expression. In a time course study, rats received 100 microg/kg LPS and were killed at 0, 2, 4, 8, and 16 h postinjection. Both iNOS mRNA and protein expression was detectable at the 2-h time point, with peak expression occurring at 8 h. To evaluate the involvement of endogenous opioids, the opioid receptor antagonist naltrexone was administered at 0, 0.1, 1, or 10 mg/kg s.c. in combination with LPS (100 microg/kg), with a second injection of naltrexone at the same dose 4 h after the injection of LPS. Naltrexone induced a pronounced dose-dependent reduction in iNOS mRNA and protein expression by splenocytes. The modulation of iNOS expression occurs via central opioid receptors as intracerebroventricular administration but not peripheral administration of N-methylnaltrexone, the quaternary form of naltrexone that does not readily cross the blood-brain barrier, reduced the expression of iNOS. For all of the manipulations, nitrite/nitrate levels in the plasma showed effects similar to those for iNOS mRNA and protein. Collectively, these findings indicate that central opioid receptors are involved in the in vivo regulation of splenic nitric oxide production.     

Nitric oxide is overproduced by peritoneal macrophages in rat taurocholate pancreatitis: the mechanism of inducible nitric oxide synthase expression

To investigate the pathobiology of severe acute pancreatitis, we studied the expression of inducible nitric oxide synthase (iNOS) in peritoneal macrophages of experimental pancreatitis. Taurocholate (TCA) pancreatitis and cerulein (CE) pancreatitis were used as models of lethal and self-limited pancreatitis, respectively, and the mechanism of iNOS expression in peritoneal macrophages was studied. Serum nitrate and nitrite (NOx) concentrations increased during the course of TCA pancreatitis, and iNOS-immunoreactivity was detected in the peritoneal macrophages 12 h after the induction of TCA pancreatitis, but these phenomena were not observed in CE pancreatitis. Despite the difference in the iNOS expression, the iNOS messenger RNA (mRNA) and the activation of nuclear factor-kappa B (NF-kappa B) were detected in the peritoneal macrophages of both pancreatitis models. The supernatant of TCA pancreatitis ascites could induce iNOS in the peritoneal macrophages of normal rats in vitro, but the peritoneal lavage fluid of CE pancreatitis rats could not. The results indicated that there may be qualitative or quantitative differences in the macrophage activation between the two types of experimental pancreatitis and suggested that the ascites of rats with lethal acute pancreatitis contains some soluble factors that activate the macrophage/monocyte system and cause an overproduction of NO by the iNOS expression.     

Localization of inducible nitric oxide synthase in acute renal allograft rejection in the rat

There is increasing evidence for a role for nitric oxide (NO) in the alloimmune response and induction of NO synthesis occurs during allograft rejection. The aim of this study was to investigate the source of NO synthesis in rejecting allografts. Localization of inducible nitric oxide synthase (iNOS) was studied by immunohistochemistry, in a rat model of acute renal allograft rejection, in unmodified Lewis recipients in which rejection is complete 7 days after transplantation of F1 hybrid Lewis-Brown Norway kidneys. High levels of iNOS expression were found in infiltrating mononuclear cells in glomeruli and interstitium of rejecting kidneys; there was no expression in parenchymal renal cells, or in control isografts of either rat strain. Expression of iNOS in the cortex was present from 4 to 6 days posttransplantation, and had declined by the 7th day, where expression was principally in the medulla. The pattern of iNOS staining was similar to ED1 staining, a marker for rat macrophages. These findings suggest that infiltrating macrophages in the graft reaction are a prominent source of NO; this iNOS expression supports a role for NO in the modulation of local allogeneic responses, and possibly as a mediator of cytotoxic graft damage.     

Inhibition of nitric oxide synthesis enhances the expression of inducible nitric oxide synthase mRNA and protein in a model of chronic liver inflammation

In septic shock the inhibition of inducible nitric oxide synthase (iNOS) could be of therapeutic value. However, side effects have to be investigated. Therefore we studied the effects of chronic NOS inhibition on the level of iNOS expression in a model of chronic liver inflammation induced by Corynebacterium parvum (C. parvum) which causes sustained iNOS expression in the liver. NOS inhibitors decreased the rise in plasma levels and urinary excretion of nitrite/nitrate by about 50%; however, iNOS mRNA and protein were increased to 200% and 150%, respectively. Thus chronic inhibition of NOS can result in an increase in iNOS mRNA level and protein under conditions when iNOS is expressed. This could result in an overproduction of NO upon removal of the NOS-inhibitor.     

Protective effects of mercaptoethylguanidine, a selective inhibitor of inducible nitric oxide synthase, in ligature-induced periodontitis in the rat

We have demonstrated previously that photomodification permeabilizes cardiac cells as evidenced by activation of a whole-cell leak current. In this paper we report that photomodification induces in cell-attached and inside-out cardiac membrane patches a chaotic current. Unlike current recordings from many protein ion channels that show stepwise amplitude changes associated with open and closed states of the channel, the chaotic current consists of variable amplitude spike-like transitions. The amplitudes of these spikes can vary from tenths to tens of picoamperes at a constant transmembrane potential. We provide evidence that the chaotic current is transmembrane rather than trans-seal and has a voltage dependency expected for current flow through nonspecific conductance pathways. Photomodification can also induce high conductance states (greater than 500 pS) in cell-attached and inside-out patches. We present evidence that the high conductance state is also not related to seal breakdown. Our results suggest that both the chaotic current activity in and high conductance state of photomodified cardiac membrane patches result from the opening of many small conductance, nonspecific pathways through the membrane.     

Enhanced expression of inducible nitric oxide synthase in chronic gastritis with intestinal metaplasia

Twelve Standardbred foals (age 3-6 months), with little previous exposure to parasites, were allocated to 2 groups and put onto pasture with low (Group L) or high (Group H) levels of larval contamination of large strongyles and cyathostomes. After 4 weeks grazing in September, the foals were housed indoors until necropsy 15 weeks later. Foals in Group H became clinically more affected than those of Group L in that they showed loss of vigour, weight gain depression, intermittent soft faeces and inappetence. One foal of Group H had persistent diarrhoea and was subjected to euthanasia 12 weeks after housing. Signs of colic were not observed. Faecal egg counts were significantly higher in Group H than in Group L (P<0.05). At necropsy, the mean number of S. vulgaris and cyathostomes was 20 and 18,000, respectively, in Group L, and 167 and 25,000 in Group H. Routine blood chemistry did not specifically reveal presence of S.vulgaris in pre-patency. A transient neutrophilia and eosinophilia, most prominent in Group H, was seen 2-8 weeks after start of exposure and anaemia was observed later in Group H. Serum albumin and albumin/globulin ratio were reduced, particularly in Group H, and a marked hyperbetaglobulinaemia was observed at 16-20 weeks in Group H. In conclusion, heavy infections with strongyles including S. vulgaris may become established in weaned foals after a brief period on pasture. Infections may be expressed clinically as debilitation, inappetence and intermittent diarrhoea without colic, and the need for control is imperative.     

Molecular cloning and expression of inducible nitric oxide synthase in chick embryonic ventricular myocytes

In order to determine the effect of kappa-opioid receptor agonist on the beta1-adrenoceptor stimulation in the heart, the effects of norepinephrine (NE), a beta1-adrenoceptor agonist, on contraction and electrically induced intracellular calcium ([Ca2+]i) transient in the single rat ventricular myocyte pretreated with a kappa-opioid receptor agonist, trans-(+/-)-3, 4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl]cyclohexyl)-benzeneacetamide (U50,488H), at 0.01-1 microM were studied with a video edge tracker method and a spectrofluorometric method using fura-2 as calcium indicator, respectively. NE at 0.01-10 microM augmented both twitch amplitude and electrically induced [Ca2+]i transient dose-dependently, which were abolished by propranolol at 1 microM, a beta-adrenoceptor antagonist. The effects of NE on both contraction and [Ca2+]i transient were attenuated in a dose-dependent manner by U50,488H at 0.01-1 microM, which itself had no effect at all. The maximum response ( Emax) was decreased, while the concentration that produces 50% of the maximum response (EC50) was enhanced, by U50, 488H. The inhibitory effects of U50,488H on beta-adrenoceptor stimulation were completely blocked by pretreatment with norbinaltorphimine, a specific kappa-opioid receptor antagonist at 1 microM, or preincubation with pertussis toxin (PTX) at 200 ng/ml for 6 h. On the other hand, the inhibition on NE-induced augmentation in electrically induced [Ca2+]i transient by U50,488H was not affected by pretreatment with U73122, a specific inhibitor of phospholipase C (PLC), at 10 microM for 30 min. U50,488H attenuated the augmentation of the electrically stimulated [Ca2+]i transient induced by forskolin at 0.1 and 0.5 microM. It did not, however, affect the augmentation of the electrically induced [Ca2+]i transient by N6, 2'-O-dibutyryl adenosine cyclic monophosphate (DB-cAMP). The results suggest that kappa-opioid receptor stimulation by U50,488H at 10(-6 )M or lower may inhibit the effects of beta-adrenoceptor stimulation by acting at a PTX-sensitive G-protein and AC, but not via the phosphoinositol pathway.     

Macrophage-derived nitric oxide induces apoptosis of rat hepatoma cells in vivo

Although nitric oxide (NO) has been postulated to play important roles in host defense mechanisms against tumor cells, a direct evidence supporting this hypothesis is lacking. To obtain molecular insights into the antitumor action of NO, its metabolism and effect on ascites hepatoma (AH-130) cells were investigated in tumor-bearing rats. Kinetic analysis revealed that substantial amounts of nitrite and nitrate, metabolites of NO, appeared in plasma and ascites of AH-130-inoculated rats. Western blot analysis revealed that a large number of macrophages that expressed inducible type of NO synthase (iNOS) appeared in cancerous ascites, particularly during 1 to 2 weeks after inoculation of AH-130 cells. When NO generation by peritoneal macrophages increased, a significant fraction of AH-130 in ascites fluid underwent apoptosis as judged from the fragmentation of their nuclear DNA. Kinetic analysis revealed that NO strongly inhibited mitochondrial electron transport and changed calcium status in AH-130 cells, particularly under low oxygen tensions such as in cancerous ascites. Intraperitoneal injection of NO donor strongly enhanced DNA fragmentation of AH-130 cells. Antimycin A, a specific inhibitor for mitochondrial electron transport, also induced DNA fragmentation of AH-130 cells by a mechanism that was inhibited by adding ascorbate and tetramethyl-p-phenylene diamine (TMPD) as electron donors. These results indicate that NO derived from peritoneal macrophages inhibits mitochondrial electron transport and disturbs calcium homeostasis in ascites hepatoma AH-130 cells, thereby inducing their apoptosis in vivo.     

Pneumococci stimulate the production of the inducible nitric oxide synthase and nitric oxide by murine macrophages

The role of nitric oxide (NO) in the pathophysiology of gram-positive sepsis is uncertain. In inflammatory conditions, high-output NO production is catalyzed by the enzyme inducible nitric oxide synthase (iNOS). The ability of 2 strains of pneumococci, pneumococcal cell wall preparations, and purified pneumococcal capsule (Pnu-Imune 23) to trigger the production of iNOS protein and NO in RAW 264.7 murine macrophages was tested. Live pneumococci, oxacillin-killed pneumococci, and pneumococcal cell wall preparations stimulated the production of iNOS and NO by RAW 264.7 cells in the presence, but not the absence, of low concentrations of recombinant murine interferon-gamma. In contrast, purified pneumococcal capsule induced little or no iNOS or NO production by these cells. Thus, pneumococci stimulate high-output NO production by murine macrophages. The potential role of NO in the pathogenesis of pneumococcal sepsis deserves further study.