Biomedical subjects

A L Salzman

Publications and source records attributed to A L Salzman.

At least 37 records · Page 2Linked to original sources

PubMed2000

Immunohistochemical localization of protein 3-nitrotyrosine and S-nitrosocysteine in a murine model of inhaled nitric oxide therapy.

Inhaled nitric oxide (INO) therapy is currently used clinically to selectively dilate the pulmonary vasculature and to help treat persistent pulmonary hypertension and bronchopulmonary dysplasia in the neonate. However, in the presence of oxygen or superoxide, nitric oxide forms potentially harmful reactive nitrogen species. Using an experimental mice model, we examined the effects of concurrent hyperoxia and INO on protein tyrosine nitration and cysteine S-nitrosylation in pulmonary tissue. Data showed enhanced 3-nitrotyrosine staining within the airway epithelium and alveolar interstitium of mice lungs treated with hyperoxia, which did not increase significantly with INO administration. Within the alveolar interstitium, 3-nitrotyrosine staining was localized to macrophages. S-Nitrosocysteine staining in airway epithelium was significantly enhanced with INO administration regardless of oxygen content. These data suggest that the formation of protein S-nitrosocysteine is the major protein modification during administration of INO.

Administration, Inhalation↗

PubMed2000

Progesterone inhibits inducible nitric oxide synthase mRNA expression in human intestinal epithelial cells.

Progesterone inhibits the transcription of inducible nitric oxide (NO) synthase (iNOS) in murine macrophages. The effect of female sex steroids on the regulation of the human iNOS gene, which shares no identity in the 5' and 3' non-coding regions with its murine homolog, is unknown. Pretreatment of the human enterocytic cells DLD-1 and Caco-2BBe with estradiol or dexamethasone had no effect on NO production induced by IL-1beta, LPS, and IFN-gamma. In contrast, NO production was inhibited by progesterone when administered as a pre-treatment or as a post-treatment 6 h after cytokine exposure (IC50 in DLD-1 and Caco-2BBe cells = 66 and 45 microM). Progesterone pre-treatment inhibited cytokine-induced iNOS mRNA expression by 66% and 58% in DLD-1 and Caco-2BBe cells, respectively. Nuclear run-on analysis demonstrated that progesterone did not inhibit cytokine-induced iNOS transcription. These data imply that progesterone inhibits iNOS mRNA expression at a post-transcriptional level, which is the dominant mode of iNOS regulation in human enterocytes. Since iNOS-derived NO production has been related to the inflammatory and tumorigenic response of progesterone-receptor bearing tissues, the repression of iNOS mRNA expression by a female sex steroid could play an important role in the regulation of a broad range of physiologic processes.

Caco-2 Cells↗

PubMed2000

Mercaptoethylguanidine inhibits the inflammatory response in a murine model of chronic infection with Pseudomonas aeruginosa.

Chronic airway inflammation induced by Pseudomonas aeruginosa is the eventual cause of respiratory failure in most people affected by cystic fibrosis. Recent evidence implicates the involvement of free radical and oxidant stress in the pathogenesis of the inflammatory injury. Here we report the efficacy of a novel experimental therapeutic, mercaptoethylguanidine (MEG), which has combined actions as a selective inhibitor of the inducible nitric oxide synthase and as a scavenger of peroxynitrite, a potent oxidant formed in the reaction of nitric oxide and superoxide radical. Chronic pulmonary infection was established in FVB/N mice by intratracheal administration of 10(5) colony-forming units of P. aeruginosa in agar beads. Treatment with MEG (10 mg/kg/dose every 8 h i.p.) inhibited weight loss in the first 3 days and reduced histologic injury at 8 days postinfection. MEG also reduced myeloperoxidase activity, a marker of neutrophil infiltration, at 8 days and concentrations of the proinflammatory cytokines interleukin-1beta, tumor necrosis factor-alpha, and macrophage inflammatory protein 2 in whole lung homogenates. MEG-treated animals and controls had similar perioperative mortality and comparable colony counts of P. aeruginosa at 8 days, indicating that MEG did not exacerbate infection. Our data suggest that MEG may be an effective immunomodulatory therapy of pulmonary inflammation induced by chronic infection.

Anesthesia↗

PubMed1999

Complement component C3 production in IL-1beta-stimulated human intestinal epithelial cells is blocked by NF-kappaB inhibitors and by transfection with ser 32/36 mutant IkappaBalpha.

BACKGROUND: Recent studies suggest that interleukin-1beta (IL-1beta) stimulates the production of the acute phase protein complement component C3 in human intestinal epithelial cells. The transcription factor NF-kappaB activates different genes involved in the response to cytokines. It is not known if IL-1beta-induced C3 production in the enterocyte is regulated by NF-kappaB. MATERIALS AND METHODS: Cultured Caco-2 cells, a human intestinal epithelial cell line, were treated with one of the NF-kappaB inhibitors, tosyl-lys-chloromethylketone (TLCK), genistein, or pyrrolidine dithiocarbamate (PDTC), or with N-acetyl-leu-leu-norleucinal (LLnL), a proteasome inhibitor known to block the degradation of Ikappabeta, the cytosolic inhibitor of NF-kappaB. Following this treatment, the Caco-2 cells were stimulated with IL-1beta, and C3 levels in the culture medium were measured after 24 h by ELISA. C3 mRNA levels were determined after 4 h by Northern blot analysis. In other experiments, Caco-2 cells were transfected with a mutant IkappaBalpha in which serines 32 and 36 were substituted by alanine. This mutation prevents IkBalpha phosphorylation and subsequent NF-kappaB nuclear translocation. After transfection, the cells were stimulated with IL-1beta, and C3 levels in the culture medium were measured after 24 h. Cytosolic IkappaBalpha was determined by Western blot analysis. RESULTS: TLCK, genistein, and LLnL each inhibited IL-1beta-induced C3 production in a dose-dependent fashion. These responses were associated with decreased C3 mRNA levels. In contrast, PDTC did not influence C3 production or C3 mRNA in the Caco-2 cells. Transfection of the Caco-2 cells with the Ser 32/36 mutant IkBalpha resulted in maintained IkappaBalpha levels and decreased IL-beta-induced C3 production. CONCLUSIONS: IL-1beta-stimulated C3 production in the enterocyte may be regulated by NF-kappaB.

Base Sequence↗

PubMed1999

Blockade of Poly(ADP-ribose) synthetase inhibits neutrophil recruitment, oxidant generation, and mucosal injury in murine colitis.

BACKGROUND & AIMS: Inflammatory bowel disease is characterized by oxidative and nitrosative stress, leukocyte infiltration, and up-regulation of intercellular adhesion molecule 1 (ICAM-1) expression in the colon. Recent data show that oxidative and nitrosative stress in isolated enterocytes produces DNA single-strand breaks that activate the nuclear enzyme poly(ADP-ribose) synthetase (PARS), resulting in depletion of intracellular energetics and increased paracellular permeability. The aim of the present study was to examine the in vivo relevance of this injury pathway. METHODS: Colitis was induced by rectal instillation of trinitrobenzenesulfonic acid (TNBS) in mice with a genetic deficiency of PARS (PARS-/-) and in wild-type littermates. RESULTS: In wild-type mice, TNBS treatment resulted in colonic erosion and ulceration that was maintained up to 7 days. Neutrophil infiltration (indicated by myeloperoxidase activity in the mucosa) was associated with up-regulation of ICAM-1 and high levels of malondialdehyde and nitrotyrosine. TNBS-treated PARS-/- mice experienced a similar colonic injury that was, however, completely resolved by 6 days. Resolution of the damage was associated with absence of ICAM-1 up-regulation, reduction of neutrophil infiltration, lipid peroxidation, and nitrosative damage. CONCLUSIONS: These data show that PARS plays a critical role in colonic inflammation possibly by regulating ICAM-1 expression, neutrophil recruitment, and the subsequent oxidant generation.

Animals↗

PubMed1999

Protein kinase C-dependent activation of NF-kappaB in enterocytes is independent of IkappaB degradation.

BACKGROUND & AIMS: Nuclear translocation of the NF-kappaB family of transcription factors is a proximal step in the signal transduction of a pleiotropic group of proinflammatory genes. Activation of RelA is under the negative control of IkappaB, a family of proteins degraded in response to immunologic and oxidant stimuli. The aim of this study was to examine this mechanism of NF-kappaB activation in intestinal epithelial cells. METHODS: DLD-1 cell monolayers stimulated by interleukin (IL)-1beta or phorbol myristate acetate (PMA) were assayed for the nuclear translocation of NF-kappaB and immunoreactivity of various IkappaB isoforms that regulate NF-kappaB1/RelA activation. RESULTS: NF-kappaB activation triggered by PMA was not associated with the disappearance of immunoreactive IkappaBalpha, IkappaBbeta, IkappaBgamma, or IkappaBepsilon or with the dissociation of intact IkappaB from RelA. NF-kappaB activation induced by PMA was blocked by the protein kinase C inhibitor staurosporine but not by the proteasomal inhibitor N-acetyl-leucine leucine norleucinal (ALLN). In contrast, IL-1beta-induced NF-kappaB activation was associated with the disappearance of IkappaBalpha and was inhibited by ALLN but not staurosporine. CONCLUSIONS: Our data imply the existence of a novel pathway of NF-kappaB activation mediated by protein kinase C that does not require proteosomal degradation or the loss of IkappaB.

Biological Transport↗

PubMed1999

Beneficial effects of mercaptoethylguanidine, an inhibitor of the inducible isoform of nitric oxide synthase and a scavenger of peroxynitrite, in a porcine model of delayed hemorrhagic shock.

OBJECTIVE: In rodent models, enhanced formation of nitric oxide and formation of peroxynitrite have been implicated in the pathogenesis of various forms of shock. Here we examined the effect of mercaptoethylguanidine (MEG), an inducible nitric oxide synthase inhibitor and peroxynitrite scavenger, in a severe hemorrhagic shock model. DESIGN: Randomized, placebo-controlled trial. SETTING: Animal laboratory. SUBJECTS: Twenty-one anesthetized immature Yorkshire pigs. INTERVENTIONS: Mechanical ventilation, sternotomy, continuous cardiac output (pulmonary artery flowmetry), and systemic and intracardial pressure measurements were taken. Pigs were bled to a cardiac index of 40 mL/kg/min for 2 hrs, which was followed by saline resuscitation (20 mL/kg). MEG was administered in the resuscitation fluid (15 mg/kg bolus plus 15 mg/kg/hr infusion). MEASUREMENTS AND MAIN RESULTS: Hemodynamic variables, systemic and mixed venous blood gas tensions and oxygenation, arterial lactate concentration, myeloperoxidase activity, malondialdehyde content, and histologic injury in the lung and intestine were measured. Reduction of cardiac output to 40 mL/kg/min led to the following changes during hypovolemia: decreases in mean arterial blood pressure (to 30-35 mm Hg), both atrial pressures, systemic oxygen consumption (by 35%), mixed venous saturation (by 65%), and lactic acidosis (5.5-6.0 mM). Fluid replacement failed to restore blood pressure and cardiac output during resuscitation and was followed by gradual hemodynamic decompensation. Hemorrhagic shock induced lipid peroxidation, neutrophil deposition, and severe histologic alterations in the lung and intestine. MEG significantly ameliorated the decrease in blood pressure and cardiac output during resuscitation, improved survival rate, reduced lipid peroxidation in the intestine, and ameliorated neutrophil accumulation in the lung and intestine. MEG prevented the reduction in oxygen consumption during resuscitation. CONCLUSIONS: When given during resuscitation, MEG exerted beneficial effects in a porcine model of severe hemorrhagic shock. We propose that the mode of MEG's action is related to improved cardiac contractility.

Analysis of Variance↗

PubMed1999

Effects of a novel guanylyl cyclase inhibitor on the vascular actions of nitric oxide and peroxynitrite in immunostimulated smooth muscle cells and in endotoxic shock.

OBJECTIVE: Nitric oxide (NO), produced by the inducible isoform of NO synthase (NOS) in circulatory shock exerts cytotoxic and vasodilator effects. Part of these effects are mediated by formation of peroxynitrite, a toxic oxidant produced by the rapid reaction of NO and superoxide. Other parts of the vascular actions of NO in shock are thought to be mediated by the action of NO on the soluble guanylyl cyclase (GC) in the smooth muscle and subsequent decrease in the intracellular calcium levels. Using 1H-(1,2,4)oxadiazolo(4,3-alpha)quinoxalin-1 -one (ODQ), a potent inhibitor of GC, we studied the role of GC activation in the NO- and peroxynitrite-related vascular alterations. DESIGN: In vitro: Controlled experiment using cultured rat aortic smooth muscle cells. In vivo: Prospective, randomized, controlled animal study. SETTING: Experimental laboratory. SUBJECTS: Male Wistar rats and male Swiss mice. INTERVENTIONS: In vitro: a) Stimulation of rat aortic smooth muscle cells with bacterial lipopolysaccharide (LPS) and gamma-interferon, measurement of the production of nitrite and nitrate (breakdown products of NO), and suppression of mitochondrial respiration for 24 to 48 hrs, in the presence or absence of ODQ; and b) in norepinephrine-precontracted endothelium-denuded thoracic aortic rings, exposure to LPS (10 ng/mL) in the presence or absence of ODQ. In vivo: Rats treated in vivo with LPS (10 mg/kg iv for 3 hrs) and mice challenged with 60 mg/kg LPS ip, in the presence or absence of ODQ. MEASUREMENTS AND MAIN RESULTS: Stimulation of rat aortic smooth muscle cells with bacterial LPS and gamma-interferon induced the production of nitrite and nitrate (breakdown products of NO) and suppression of mitochondrial respiration for 24 to 48 hrs. The amount of NO produced was slightly enhanced with ODQ (10-100 EM), whereas the suppression of mitochondrial respiration was not affected by ODQ (1-100 microM). ODQ did not affect the degree of suppression of mitochondrial respiration in response to NO donor agents or to peroxynitrite. Exposure to LPS (10 ng/mL) for 6 hrs caused a time-dependent relaxation of norepinephrine-precontracted endothelium-denuded thoracic aortic rings. This response was caused by the expression of inducible NOS and could be blocked by pharmacologic inhibitors of NOS such as N(G)-methylL-arginine. ODQ (1 microM) prevented the LPS-induced loss of vascular tone in this experimental system. Similar to the in vitro responses, there was a significant suppression of the norepinephrine-induced contractions in ex vivo experiments, in which rings were taken from animals treated in vivo with LPS (10 mg/kg for 3 hrs). ODQ treatment in vitro (1 microM) caused a complete restoration of the contractile responses. In mice challenged with 60 mg/kg LPS ip, ODQ (20 mg/kg), given either as a pretreatment or as a 4-hr posttreatment, improved survival at 24-144 hrs. CONCLUSION: These studies indicate that GC activation does not contribute to NO- or peroxynitrite-induced cytotoxicity but does contribute to the vascular hyporeactivity induced by endotoxin in vitro and in vivo. GC inhibition alone is sufficient to influence survival in a murine model of severe sepsis.

Animals↗

PubMed1999

Bacterial invasion is not required for activation of NF-kappaB in enterocytes.

Pathogenic enteric microorganisms induce the NF-kappaB-dependent expression of proinflammatory genes in intestinal epithelial cells. The purpose of the present study was to clarify the contribution of microbial invasion to the degradation of the regulatory protein Ikappa Balpha and the subsequent activation of NF-kappaB in cultured intestinal epithelial cells. Caco-2BBe cells were incubated with Salmonella dublin, Salmonella typhimurium, or a weakly invasive strain of E. coli. S. dublin and S. typhimurium (10(7) organisms/ml) induced equivalent concentration-dependent gel mobility shifts of an NF-kappaB consensus sequence that was preceded by Ikappa Balpha degradation. E. coli (10(7) organisms/ml) did not induce Ikappa Balpha degradation or NF-kappaB translocation. Pretreatment with cytochalasin D blocked invasion of all three strains but had no effect on Ikappa Balpha degradation or NF-kappaB activation. S. dublin and S. typhimurium adhered to Caco-2BBe cells 3- to 10-fold more than E. coli. NF-kappaB activation was prevented by physical separation of S. dublin from Caco-2BBe cells by a 0. 4-micrometers-pore-size filter. Our results imply that bacterial adhesion, rather than invasion or release of a secreted factor, is sufficient to induce IkappaBalpha degradation and NF-kappaB activation in intestinal epithelial cells. Our data suggest that strategies to reduce enteric inflammation should be directed to the reduction of bacterial enterocyte adhesion.

Caco-2 Cells↗

PubMed1999

Reduced oxidative and nitrosative damage in murine experimental colitis in the absence of inducible nitric oxide synthase.

BACKGROUND: Oxidative and nitrosative stress have been implicated in the pathogenesis of inflammatory bowel diseases. AIMS: To study the role of nitric oxide (NO) derived from inducible NO synthase (iNOS) in an experimental model of murine enterocolitis. METHODS: Trinitrobenzene sulphonic acid (TNBS) was instilled per rectum to induce a lethal colitis in iNOS deficient mice and in wild type controls. The distal colon was evaluated for histological evidence of inflammation, iNOS expression and activity, tyrosine nitration and malondialdehyde formation (as indexes of nitrosative and oxidative stress), myeloperoxidase activity (as index of neutrophil infiltration), and tissue localisation of intercellular adhesion molecule 1 (ICAM-1). RESULTS: TNBS administration induced a high mortality and weight loss associated with a severe colonic mucosal erosion and ulceration, increased myeloperoxidase activity, increased concentrations of malondialdehyde, and an intense staining for nitrotyrosine and ICAM-1 in wild type mice. Genetic ablation of iNOS gene conferred to mice a significant resistance to TNBS induced lethality and colonic damage, and notably reduced nitrotyrosine formation and concentrations of malondialdehyde; it did not, however, affect neutrophil infiltration and intestinal ICAM-1 expression in the injured tissue. CONCLUSION: Data show that activation of iNOS is required for nitrosative and oxidative damage in experimental colitis.

Animals↗

PubMed1999

5-aminosalicylic acid inhibits iNOS transcription in human intestinal epithelial cells.

The mechanism by which 5-aminosalicylic acid (5-ASA) reduces mucosal injury in colitis is undefined. In murine cells, 5-ASA modulates the expression of the inducible nitric oxide (NO) synthase, a potential mediator of colitic injury, but its effect on the human isoform is unknown. Given the lack of conserved regulation of iNOS expression in rodent and human systems, we sought to test the effect of 5-ASA on the expression of human iNOS in cultured enterocytes. Transformed human intestinal epithelial cells, DLD-1 and Caco-2BBe, were stimulated by IL-1beta and IFN-gamma and analyzed for iNOS upregulation and NO production in the presence of various aminosalicylates. 5-ASA, but not 4-ASA, dose-dependently inhibited NO production by both cell lines [IC50 (mM) DLD-1 =4.5, Caco-2BBe =2. 5]. 5-ASA also inhibited the expression of iNOS protein and mRNA and blocked cytokine-induced transcriptional upregulation of the iNOS gene. 5-ASA (1-5 mM) had no effect on cytokine-induced nuclear translocation of NF-kappaB or expression of IRF-1, transactivating factors which regulates the human iNOS enhancer. We conclude that 5-ASA inhibits iNOS expression and NO production at therapeutically relevant concentrations. The inhibition occurs at the level of transcriptional activation and is independent of IRF-1 and NF-kappaB. Since NO is an important final effector of mucosal injury in inflammatory bowel disease, these findings may have implications for the clinical efficacy of 5-ASA.

Anti-Inflammatory Agents, Non-Steroidal↗

PubMed1999

Stress response decreases the interleukin-1beta-induced production of complement component C3 in human intestinal epithelial cells.

Interleukin-1beta (IL-1beta) increases the production of complement component C3 in enterocytes. Heat shock regulates the response to cytokines and other inflammatory mediators in various cell types. We tested the hypothesis that the heat-shock response regulates IL-1beta-induced C3 production in the enterocyte. Cultured Caco-2 cells, a human intestinal epithelial cell line, were treated with sodium arsenite (10-500 microM) for 1 h or subjected to hyperthermia (43 degrees C) for 1-4 h, and allowed to recover for 1 h. The cells were then treated with IL-1beta (0.5 ng/ml) for up to 24 h, whereafter C3 levels were measured by ELISA and C3 mRNA by Northern blot analysis. Heat-shock protein of 72 kDa (hsp72) was determined by Western blot analysis. Treatment of the cells with sodium arsenite or subjecting them to hyperthermia induced the expression of hsp72. The IL-1beta-induced expression of C3 mRNA and C3 production were down-regulated by hyperthermia and sodium arsenite in a dose-dependent fashion. The results suggest that the stress response induced by hyperthermia or sodium arsenite decreases IL-1beta-induced C3 production in human enterocytes.

Arsenites↗

PubMed1998

Hydroxyguanidines inhibit peroxynitrite-induced oxidation.

Hydroxyguanidines (OHGs), including the endogenously formed NG-hydroxy-L-arginine (OH-arg), can react with nitric oxide (NO) and nitrogen oxides (NOx) in vitro. Therefore, we have tested OHGs and related compounds for their ability to scavenge peroxynitrite and to protect against peroxynitrite-induced oxidative processes in cells. Hydroxyguanidine, NG-hydroxy-L-arginine and other N-substituted OHGs, dose-dependently inhibited the in vitro oxidation of dihydrorhodamine (DHR) by peroxynitrite (PN), with similar or better efficacy than glutathione or cysteine. Amidoximes, aminoguanidines and O-substituted OHGs were less effective, and guanidines were without effect. In contrast to their effects on DHR oxidation, OHGs exerted only minimal inhibitory effects on the hydroxylation of benzoate by PN, suggesting that OHGs do not react with the activated isomer of peroxynitrous acid. Selected compounds were tested for protection against PN-induced suppression of mitochondrial respiration and protein oxidation in cultured J774 murine macrophages. Aminoguanidines afforded some protection against the effects of PN, but substituted-phenyl OHGs were considerably more effective. Analysis of the products of the reaction of 4-methoxybenzyl-OHG with PN showed rapid formation of nitrosated derivatives, as well as 4-methoxybenzylcyanamide and a small amount of 4-methoxybenzylurea. Nitric oxide and nitrous oxide were also evolved, but indirectly, arising from the decomposition of one of the nitrosation products. The current results demonstrate that hydroxyguanidines react with PN to protect cells against PN-mediated injury and may be more effective than the endogenous antioxidants cysteine and glutathione.

Arginine↗

PubMed1998

An agonist of adenosine A3 receptors decreases interleukin-12 and interferon-gamma production and prevents lethality in endotoxemic mice.

We have recently observed that the selective adenosine A3 receptor agonist N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (IB-MECA) augments interleukin-10 and inhibits tumor necrosis factor-alpha production in endotoxemic mice. In the present study, we extended our investigations into the effect of this compound on the bacterial lipopolysaccharide (endotoxin)-induced inflammatory response in the BALB/c, as well as in the C57BL/6 interleukin-10+/+ and the interleukin-10 deficient C57BL/6 interleukin-10(0)/0 mice strains. In the BALB/c mice, i.p. pre-treatment with IB-MECA (0.2 and 0.5 mg/kg) decreased lipopolysaccharide (60 mg/kg i.p.)-induced plasma levels of interleukin-12 (p40 and p70), interferon-gamma, and nitrite/nitrate (breakdown products of nitric oxide (NO)). On the other hand, pre-treatment with this compound failed to influence lipopolysaccharide-induced plasma interleukin-1 alpha, interleukin-6, and corticosterone concentrations. Similar to its effect in BALB/c mice, IB-MECA enhanced the release of interleukin-10 in the C57BL/6 interleukin-10+/+ mice. Furthermore, IB-MECA inhibited the production of interleukin-12, interferon-gamma, and NO in both the C57BL/6 interleukin-10+/+ and C57BL/6 interleukin-10(0)/0 mice, suggesting that the inhibition of pro-inflammatory cytokine production by this compound is independent of the increased release of interleukin-10. Finally, pre-treatment with this compound protected mice against lipopolysaccharide (60 mg/kg i.p.)-induced lethality. These results indicate that stimulation of adenosine A3 receptors has potent anti-inflammatory effects and may represent a potential strategy in the treatment of septic shock and other inflammatory diseases.

Adenosine↗

PubMed1998

Constitutive and adaptive detoxification of nitric oxide in Escherichia coli. Role of nitric-oxide dioxygenase in the protection of aconitase.

Nitric oxide (NO.) is a naturally occurring toxin that some organisms adaptively resist. In aerobic or anaerobic Escherichia coli, low levels of NO. exposure inactivated the NO.-sensitive citric acid cycle enzyme aconitase, and inactivation was more effective when the adaptive synthesis of NO.-defensive proteins was blocked with chloramphenicol. Protection of aconitase in aerobically grown E. coli was dependent upon O2, was potently inhibited by cyanide, and was correlated with an induced rate of cellular NO. consumption. Constitutive and adaptive cellular NO. consumption in aerobic cells was also dependent upon O2 and inhibited by cyanide. Exposure of aerobic cells to NO. accordingly elevated the activity of the O2-dependent and cyanide-sensitive NO. dioxygenase (NOD). Anaerobic E. coli exposed to NO. or nitrate induced a modest O2-independent and cyanide-resistant NO.-metabolizing activity and a more robust O2-stimulated cyanide-sensitive activity. The latter activity was attributed to NOD. The results support a role for NOD in the aerobic detoxification of NO. and suggest functions for NOD and a cyanide-resistant NO. scavenging activity in anaerobic cells.

Aconitate Hydratase↗

PubMed1998

Poly(ADP-ribose) synthetase activation mediates mitochondrial injury during oxidant-induced cell death.

Reactive oxidant species are important mediators of tissue injury in shock, inflammation, and reperfusion injury. The actions of a number of these oxidants (e.g., hydroxyl radical and peroxynitrite, a reactive oxidant produced by the reaction of nitric oxide and superoxide) are mediated in part by the activation of the nuclear nick sensor enzyme, poly(ADP)-ribose synthetase (PARS), with consequent cellular energy depletion. Here we investigated whether PARS activation contributes to the mitochondrial alterations in cells exposed to oxidants. Authentic peroxynitrite (20 microM), the peroxynitrite-generating compound 3-morpholinosidnonimine, the combination of pyrogallol and S-nitroso-N-acetyl-D,L-penicillamine, as well as hydrogen peroxide induced a time- and dose-dependent decrease in mitochondrial transmembrane potential (delta psi(m)) in thymocytes, as determined by flow cytometry using the mitochondrial potential sensitive dyes DiOC6(3) and JC-1. A time- and dose-dependent increase in secondary reactive oxygen intermediate production and loss of cardiolipin, an indicator of mitochondrial membrane damage, were also observed, as measured by flow cytometry using the fluorescent dyes dihydroethidine and nonyl-acridine orange, respectively. Inhibition of PARS by 3-aminobenzamide or 5-iodo-6-amino-1,2-benzopyrone attenuated peroxynitrite-induced delta psi(m) reduction, secondary reactive oxygen intermediate generation, cardiolipin degradation, and intracellular calcium mobilization. Furthermore, thymocytes from PARS-deficient animals were protected against the peroxynitrite- and hydrogen peroxide-induced functional and ultrastructural mitochondrial alterations. In conclusion, mitochondrial perturbations during oxidant-mediated cytotoxicity are, to a significant degree, related to PARS activation rather than to direct effects of the oxidants on the mitochondria.

Animals↗

PubMed1998

Nitric oxide dioxygenase: an enzymic function for flavohemoglobin.

Nitric oxide (NO*) is a toxin, and various life forms appear to have evolved strategies for its detoxification. NO*-resistant mutants of Escherichia coli were isolated that rapidly consumed NO*. An NO*-converting activity was reconstituted in extracts that required NADPH, FAD, and O2, was cyanide-sensitive, and produced NO3-. This nitric oxide dioxygenase (NOD) contained 19 of 20 N-terminal amino acids identical to those of the E. coli flavohemoglobin. Furthermore, NOD activity was produced by the flavohemoglobin gene and was inducible by NO*. Flavohemoglobin/NOD-deficient mutants were also sensitive to growth inhibition by gaseous NO*. The results identify a function for the evolutionarily conserved flavohemoglobins and, moreover, suggest that NO* detoxification may be a more ancient function for the widely distributed hemoglobins, and associated methemoglobin reductases, than dioxygen transport and storage.

Amino Acid Sequence↗

PubMed1998

Stimulation of beta-adrenoceptors inhibits endotoxin-induced IL-12 production in normal and IL-10 deficient mice.

Stimulation of beta-adrenoceptors has been shown to regulate the production of various inflammatory mediators. In the present study, we investigated in mice whether ligation of beta-adrenoceptors, modulates lipopolysaccharide (LPS)-induced plasma levels of interleukin (IL)-12, interferon-gamma (IFN-gamma), and IL-10. In BALB/c mice, isoproterenol (1-10 mg kg(-1) , i.p.), a selective agonist of beta-adrenoceptors and also dexamethasone (10 mg kg(-1), i.p.) pretreatment 30 min before the administration of LPS suppressed plasma IL-12 (p40 and p70) concentrations as determined at various time points after the LPS challenge. The inhibition of IL-12 release by isoproterenol was prevented by the beta-adrenoceptor antagonist propranolol confirming the involvement of beta-adrenoceptors in the effect of isoproterenol. Furthermore, pretreatment of the animals with propranolol alone enhanced LPS-induced plasma IL-12, suggesting that endogenous catecholamines inhibit IL-12 production via the beta-adrenoceptors. In IL-10 deficient C57BL/6 IL-10(-/-) mice, plasma levels of IL-12 and IFN-gamma were significantly higher than in their counterparts, with more than 70-fold increase in IL-12. Furthermore, while augmenting the IL-10 response in C57BL/6 IL-10(+/+), isoproterenol inhibited the production of IL-12 in both the C57BL/6 IL-10(+/+) and C57BL/6 IL-10(-/-) mice, suggesting that the inhibition of IL-12 production by this compound is independent of the increased release of IL-10. Our results demonstrate, for the first time, that stimulation of beta-adrenoceptors by isoproterenol or endogenous catecholamines suppresses IL-12 production in vivo.

Adrenergic beta-Agonists↗