S-nitroso-acetyl-penicillamine Research Articles

Synthesis and Characterization of a Sustained Nitric Oxide-Releasing Orthodontic Elastomeric Chain for Antimicrobial Action.

The acidic byproducts of bacteria in plaque around orthodontic brackets contribute to white spot lesion (WSL) formation. Nitric oxide (NO) has antibacterial properties, hindering biofilm formation and inhibiting the growth of oral microbes. Materials that mimic NO release could prevent oral bacteria-related pathologies. This study aims to integrate S-nitroso-acetylpenicillamine (SNAP), a promising NO donor, into orthodontic elastomeric ligatures, apply an additional polymer coating, and evaluate the NO-release kinetics and antimicrobial activity against Streptococus mutans. SNAP was added to clear elastomeric chains (8 loops, 23 mm long) at three concentrations (50, 75, 100 mg/mL, and a control). Chains were then coated, via electrospinning, with additional polymer (Elastollan®) to aid in extending the NO release. NO flux was measured daily for 30 days. Samples with 75 mg/mL SNAP + Elastollan® were tested against S. mutans for inhibition of biofilm formation on and around the chain. SNAP was successfully integrated into ligatures at each concentration. Only the 75 mg/mL SNAP chains maintained their elasticity. After polymer coating, samples exhibited a significant burst of NO on the first day, exceeding the machine's reading capacity, which gradually decreased over 29 days. Ligatures also inhibited S. mutans growth and biofilm formation. Future research will assess their mechanical properties and cytotoxicity. This study presents a novel strategy to address white spot lesion (WSL) formation and bacterial-related pathologies by utilizing nitric oxide-releasing materials. Manufactured chains with antimicrobial properties provide a promising solution for orthodontic challenges, showing significant potential for academic-industrial collaboration and commercial viability.

Exogenous nitric oxide generated from SNAP blocks porcine circovirus type 2 replication and regulates NF-κB activity in PK-15 cells

ObjectivesPCV2-associated disease (PCVAD), caused by porcine circovirus type 2 (PCV2) infection, is one of the major infectious diseases in the global swine industry. Nitric oxide (NO), as an important signalling molecule, has antiviral activities against a variety of viruses. To date, limited knowledge is available on the role of NO during PCV2 infection. MethodsThis study was conducted to investigate the effects of exogenous NO on PCV2 replication in vitro. To exclude the possibility that the detected antiviral effects were due to cell toxicity, maximum non-cytotoxic concentrations of the drugs were determined. Kinetics of NO production were assessed after drug treatment. The antiviral activities of NO at different concentrations and at different time points were carefully assessed by measuring the virus titers, viral DNA copies and percentage of PCV2-infected cells. Regulation of NF-κB activity by exogenous NO was also investigated. ResultsKinetics of NO production indicated that S-nitroso-acetylpenicillamine (SNAP) produced NO in a dose-dependent manner, while NO was scavenged by its scavenger haemoglobin (Hb). An in vitro antiviral assay demonstrated that exogenous NO strongly inhibited PCV2 replication in a time-dependent and dose-dependent manner, whereas the inhibitory effects could be reversed by Hb. Furthermore, inhibition of NF-κB activity induced by NO contributed to a notable decrease in PCV2 replication. ConclusionThese findings provide a new potential antiviral therapy against PCV2 infection, and the antiviral effects of exogenous NO may be partly achieved by regulating NF-κB activity.

Inhibition of SOS Response by Nitric Oxide Donors in Escherichia coli Blocks Toxin Production and Hypermutation.

BackgroundPrevious reports have differed as to whether nitric oxide inhibits or stimulates the SOS response, a bacterial stress response that is often triggered by DNA damage. The SOS response is an important regulator of production of Shiga toxins (Stx) in Shiga-toxigenic E. coli (STEC). In addition, the SOS response is accompanied by hypermutation, which can lead to de novo emergence of antibiotic resistance. We studied these effects in vitro as well as in vivo.ResultsNitric oxide donors inhibited induction of the SOS response by classical inducers such as mitomycin C, ciprofloxacin, and zidovudine, as measured by assays for E. coli RecA. Nitric oxide donors also inhibited Stx toxin protein production as well as stx2 RNA in vitro and in vivo. In vivo experiments were performed with ligated ileal segments in the rabbit using a 20 h infection. The NO donor S-nitroso-acetylpenicillamine (SNAP) reduced hypermutation in vitro and in vivo, as measured by emergence of rifampin resistance. SNAP blocked the ability of the RecA protein to bind to single-stranded DNA in an electrophoretic mobility shift assay (EMSA) in vitro, an early event in the SOS response. The inhibitory effects of SNAP were additive with those of zinc acetate.ConclusionsNitric oxide donors blocked the initiation step of the SOS response. Downstream effects of this blockade included inhibition of Stx production and of hypermutation. Infection of rabbit loops with STEC resulted in a downregulation, rather than stimulation, of nitric oxide host defenses at 20 h of infection.

Liquid-infused nitric oxide-releasing (LINORel) silicone for decreased fouling, thrombosis, and infection of medical devices

Recent reports on liquid-infused materials have shown promise in creating ultra-low fouling surfaces, but are limited in their ability to prevent bacterial proliferation and prevent platelet activation in blood-contacting applications. In this work, a liquid-infused nitric oxide-releasing (LINORel) material is created by incorporating the nitric oxide (NO) donor S-nitroso-acetylpenicillamine (SNAP) and silicone oil in commercial medical grade silicone rubber tubing through a solvent swelling process. This combination provides several key advantages over previous NO-releasing materials, including decreased leaching of NO donor, controlled release of NO, and maintenance of ultra-low fouling property of liquid-infused materials. The LINORel tubing reduces protein adhesion as observed using fluorescence imaging, and platelet adhesion (81.7 ± 2.5%) in vitro over a 2 h period. The LINORel combination greatly reduces bacterial adhesion and biofilm formation of two most common pathogens responsible for hospital acquired infections: gram-positive Staphylococcus aureus and gram-negative Pseudomonas aeruginosa (99.3 ± 1.9% and 88.5 ± 3.3% respectively) over a 7-day period in a CDC bioreactor environment. Overall, the LINORel approach provides a synergistic combination of active and passive non-fouling approaches to increase biocompatibility and reduce infection associated with medical devices.

Development of nitric oxide catalytic coatings by conjugating 3,3-disulfodipropionic acid and 3,3-diselenodipropionic acid for improving hemocompatibility.

Nitric oxide (NO), discovered as an endothelium-derived relaxing factor, has been found to have multiple intracellular effects in vascular diseases including vasorelaxation regulation, endothelial regeneration, inhibition of leukocyte chemotaxis, and platelet activation. In the work described here, the authors have developed a NO-catalytic bioactive coating for improving hemocompatibility. The authors first prepared a dopamine and hexamethylendiamine (PDAM/HD) amine-rich adherent copolymer coating to introduce amine groups onto 316L stainless steel, followed by covalently conjugating 3,3-disulfodipropionic acid (S-S) and 3,3-diselenodipropionic acid (Se-Se), which mimic glutathione peroxidase-like catalytic production of NO. S-S and Se-Se were immobilized on the PDAM/HD surface via carbodiimide coupling chemistry. X-ray photoelectron spectroscopy analysis revealed clear S2p and Se3d signals, confirming the immobilization of S-S and Se-Se on the PDAM/HD surface. The NO release behavior of different samples was investigated. In detail, two species of thionitrites (RSNO), S-nitrosoglutathione (GSNO, endogenous NO donors) and S-nitrosoacetylpenicillamine (SNAP) were chosen as NO donors to investigate the NO catalytic properties of S-S and Se-Se modified PDAM/HD surfaces. Not only Se-Se@PDAM/HD but also S-S@PDAM/HD coatings showed the ability to continuously catalyze RSNO to generate NO in the presence of proper thiol reducing agent. For the Se-Se@PDAM/HD coating, the NO release amount and rate were greater than S-S@PDAM/HD in both GSNO and SNAP conditions. The results showed that organosulfide species possesses NO catalytic ability as well as organoselenium species. The authors demonstrated that both S-S@PDAM/HD and Se-Se@PDAM/HD coatings exhibited outstanding inhibition effect on platelet adhesion, aggregation and activation via the cyclic guanylate monophosphate signal pathway. Thus these results suggested that NO catalytic coatings based on organoselenium and organosulfide species immobilization can help to improve hemocompatibility. NO-catalytic strategies possess huge potential applications in blood-contacting devices.

NO donors subunit and state‐dependently increase desensitization of acid sensitive ion channels

Nitric oxide donors were studied on acid‐sensing channels (ASICs) of rat striatal neurons and CHO cells expressing recombinant receptors. S‐nitroso‐acetyl‐penicillamine (SNAP) potentiate or inhibit ASICs depending on their functional state. It potentiated ASICs after pretreatment with extracellular pH 7.4. When ASICs were partially desensitized by pretreatment with pH 7.0, application of SNAP led to increase of their desensitisization. The potentiating effect of NO on ASICs is explained by an increase of ASIC proton sensitivity, whereas inhibition of ASICs by NO is due to an increase of their steady‐state desensitization. Potentiation and inhibition produced by SNAP had different subunit dependence. The most sensitive to potentiation were ASIC1a/2a and ASIC2a channels while ASIC2a and ASIC3 were the most sensitive to inhibition. Potentiation, produced by SNAP, was prevented by sulphhydryl reagent mercaptoethanol, but it did not affect inhibition produced by SNAP. Thus potentiation and inhibition seems to be mediated by different binding sites. We suppose both effects of SNAP are most probably due to nitrosylation, but not to soluble guanylate cyclase activation, because they were unaffected by ODQ, inhibitor of NO‐sensitive guanylate cyclase, and by 8‐Br‐cGMP, an activator of cGMP‐dependent PKG cascades. Work was supported by grant RFBR/STGI 09‐04‐92662‐Ind‐a.

Administration of a nitric oxide donor inhibits mglA expression by intracellular Francisella tularensis and counteracts phagosomal escape and subversion of TNF-α secretion

Francisella tularensis is a highly virulent intracellular bacterium capable of rapid multiplication in phagocytic cells. Previous studies have revealed that activation of F. tularensis-infected macrophages leads to control of infection and reactive nitrogen and oxygen species make important contributions to the bacterial killing. We investigated the effects of adding S-nitroso-acetyl-penicillamine (SNAP), which generates nitric oxide, or 3-morpholinosydnonimine hydrochloride, which indirectly leads to formation of peroxynitrite, to J774 murine macrophage-like cell cultures infected with F. tularensis LVS. Addition of SNAP led to significantly increased colocalization between LAMP-1 and bacteria, indicating containment of F. tularensis in the phagosome within 2 h, although no killing occurred within 4 h. A specific inhibitory effect on bacterial transcription was observed since the gene encoding the global regulator MglA was inhibited 50-100-fold. F. tularensis-infected J774 cells were incapable of secreting TNF-α in response to Escherichia coli LPS but addition of SNAP almost completely reversed the suppression. Similarly, infection with an MglA mutant did not inhibit LPS-induced TNF-α secretion of J774 cells. Strong staining of nitrotyrosine was observed in SNAP-treated bacteria, and MS identified nitration of two ribosomal 50S proteins, a CBS domain pair protein and bacterioferritin. The results demonstrated that addition of SNAP initially did not affect the viability of intracellular F. tularensis LVS but led to containment of the bacteria in the phagosome. Moreover, the treatment resulted in modification by nitration of several F. tularensis proteins.

The endothelial nitric oxide synthase/nitric oxide system is involved in the defective quality of bovine oocytes from low mid-antral follicle count ovaries1

In a previous survey concerning cows of reproductive age, we demonstrated that oocytes isolated from ovaries with <10 medium antral follicles of 2 to 6 mm in diameter (low ovaries; Lo) show less developmental competence than oocytes collected from ovaries with >10 medium antral follicles (high ovaries; Hi). The aim of the present study was to evaluate whether a defective endothelial nitric oxide synthase/nitric oxide (eNOS/NO) system and vasculature in healthy medium antral follicles is likely to reduce oocyte competence from Lo ovaries. Thus, experiments were conducted to 1) immunolocalize eNOS protein during folliculogenesis; 2) quantify eNOS protein/vasculature in the follicle wall; and 3) verify if NO donor, S-nitroso acetyl penicillamine (SNAP) administration during in vitro maturation affects developmental competence of oocytes isolated from Lo ovaries. Endothelial nitric oxide synthase protein was detected in granulosa and theca cells, as well as in blood vessels from primordial to antral follicles. Quantitative analysis indicated that in medium antral follicles from Lo ovaries, eNOS protein expression and vasculature were reduced (P < 0.05). The addition of SNAP improved blastocyst and hatching rates of oocytes from Lo ovaries, promoting a percentage similar to oocytes from Hi ovaries, and reduced the percentage of apoptotic nuclei in in vitro-produced blastocysts (P < 0.05). Results from our study suggest that in bovine ovaries with small mid antral follicle number, a defective eNOS/NO system is related to a reduced follicle vasculature and may affect oocyte quality, thus inducing a premature decline of fertility.

Direct effect of mild hypothermia on the coronary vasodilation induced by an ATP-sensitive K channel opener, a nitric oxide donor and isoflurane in isolated rat hearts

Deliberate mild hypothermia (MHT) is applied for cerebroprotection after cardiopulmonary resuscitation and during cardiac surgery. MHT has been shown to alter both contractility and relaxation of blood vessels in the brain. However, the effects of MHT on drug-induced vasodilation are not fully understood. The aim of this study was to clarify the effects of MHT on the coronary vasodilation induced by cromakalim (an ATP-sensitive K channel opener), S-nitroso acetyl-penicillamine (SNAP; a nitric oxide donor), and isoflurane in isolated rat hearts. Male SD rat hearts were isolated and perfused with Krebs-Henseleit buffer. Coronary flow was measured with the coronary perfusion pressure kept at 60 mmHg, and coronary vascular resistance (CVR) was calculated. After cardiac arrest was induced by tetrodotoxin, the hearts were allocated to one of three temperature groups: 37, 34, and 31 degrees C (n = 7 for each). All groups received 0.01, 0.1, and 1.0 microM of either cromakalim or SNAP or were exposed to isoflurane at 1MAC and 2MAC. Finally, 50 mM of adenosine was administered to obtain maximal coronary vasodilation. CVR significantly increased after cardiac arrest, but did not change after the application of each temperature. Cromakalim, SNAP and isoflurane significantly decreased CVR in each temperature group. There were no significant differences in CVR among the three temperature groups with any of the test drugs. These results indicate that cromakalim-, SNAP-, and isoflurane-induced coronary vasodilation are not affected by MHT.

Enrichment of Hepatocyte-like Cells with Upregulated Metabolic and Differentiated Function Derived from Embryonic Stem Cells Using S-NitrosoAcetylPenicillamine

The generation of a large number of fully functional hepatocytes from a renewable cell source can provide an unlimited resource for bioartificial liver devices and cell replacement therapies. We have established a directed differentiation system using sodium butyrate treatment to generate an enriched population of hepatocyte-like cells from embryonic stem cells. A metabolic analysis of the hepatocyte populations revealed glycolytic and mitochondrial phenotypes similar to mouse hepatoma cells, implying that these cells represent an immature hepatocyte phenotype. To mediate further differentiation, S-NitrosoAcetylPenicillamine (SNAP), a nitric oxide donor, was utilized to induce mitochondrial development in the precursor populations. A comparative analysis of the different treated populations showed that 500microM SNAP treatment resulted in the generation of an enriched population of metabolically mature hepatocyte-like cells with increased differentiated function. Specifically, 500microM SNAP treatment increased glucose consumption, lactate production rates, mitochondrial mass, and potential as compared to untreated populations. In addition, functional analysis revealed that intracellular albumin content, urea secretion rates, and cytochrome P450 7a1 promoter activity were increased in the treated population. The methodology described here to generate an enriched population of metabolically and functionally mature hepatocyte-like cells may have potential implications in drug discovery and regenerative medicine.

Effect of exogenous nitric oxide on the proliferation of a human osteoblast (HOS) cell line induced by hydroxyapatite.

The aim of this study was to test the hypothesis that the proliferation of hydroxyapatite (HA)-induced human osteoblast cell line (HOS cells) may be up-regulated by exogenous nitric oxide (NO). HOS cells were cultured on the surface of HA with or without the presence of a NO donor, S-nitroso acetyl penicillamine (SNAP) or nitroso acetyl penicillamine (NAP). 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide) known as carboxy PTIO, a NO scavenger, was added in the cell cultures with or without the presence of SNAP. The cells were also pre-treated with L-N5-(1-iminoethyl)orthinine hydrochloride (L-NIO), an endothelial nitric oxide synthase (eNOS) inhibitor, or anti-integrin alphaV antibody before culturing on HA surfaces with or without the presence of SNAP. Medium, cells alone or cells pretreated with these inhibitors or antibodies was used as the controls. Cell proliferation was assessed by colorimetric assay. The results showed that SNAP, but not NAP, augmented HA-induced HOS cell proliferation. This modulatory effect of SNAP on HA-induced HOS cell proliferation was abolished by carboxy PTIO or anti-integrin alphaV antibody, but only partially reduced by L-NIO. Therefore, the results of this study suggest that exogenous NO alone may up-regulate the proliferation of HOS cells attached on HA surfaces via integrin alphaV molecules.

Effect of exogenous nitric oxide on murine splenic immune response induced by Aggregatibacter actinomycetemcomitans lipopolysaccharide

The aim of this study was to determine the effect of exogenous nitric oxide (NO) on the induction of murine splenic immune response to Aggregatibacter actinomycetemcomitans lipopolysaccharide (LPS) in vitro. BALB/c mice were immunized with A. actinomycetemcomitans LPS and a control group was sham-immunized. Spleen cells were obtained, cultured and stimulated with A. actinomycetemcomitans LPS with or without the presence of S-nitroso acetyl-penicillamine (SNAP), a NO donor, and carboxy-PTIO, an NO scavenger. Culture supernatants were assessed for inducible nitric oxide synthase (iNOS) activity, specific IgG subclass levels, and both IFN-γ and IL-4 levels. The results showed that in A. actinomycetemcomitans LPS-stimulated cells, SNAP enhances iNOS activity but inhibits the levels of specific IgG2a and IFN-γ suggesting a Th1 response. The effect of SNAP on these immune parameters was ablated by carboxy-PTIO. These results suggest that exogenous NO may suppress the Th1-like immune response of A. actinomycetemcomitans LPS-stimulated murine spleen cells.

Nitric Oxide Delays Oocyte Aging

Nitric oxide (NO) is a ubiquitous signaling molecule that plays a crucial role in oocyte maturation and embryo development. However, its role in oocyte aging is unclear. To examine how NO affects oocyte aging, we retrieved young and relatively old mouse oocytes and exposed them to increasing concentrations of NO donor S-nitroso acetyl penicillamine (SNAP). Aging related phenomena of ooplasmic microtubule dynamics (OMD), cortical granule (CG) exocytosis, zona pellucida (ZP) hardening, and spindle/chromatin integrity were studied at each SNAP concentration using fluorescence immunocytochemistry and confocal microscopy and compared with respective unexposed controls. Exposure of both young and old oocytes to NO resulted in a significant diminution in OMD and ZP dissolution time, whereas spontaneous CG loss decreased in old NO exposed oocytes compared to controls (P < 0.001 for all). Furthermore, NO exposure decreased the rate of spindle abnormalities in oocytes compared to unexposed controls. Interestingly, in old oocytes, the positive influence of NO was attenuated beyond 0.23 microM/min and disappeared at 0.46 microM/min NO. Overall, a significant dose-response relationship was noted between NO exposure and markers of aging with between 50 and 100 microM SNAP (0.11-0.23 microM/min NO, P < 0.0001). Collectively, our results demonstrate for the first time that exposure to NO delays oocyte aging and improves the integrity of the microtubular spindle apparatus in young and old oocytes.

The contribution of reactive nitrogen and oxygen species to the killing of Francisella tularensis LVS by murine macrophages

Intracellular killing of Francisella tularensis by macrophages depends on interferon-gamma (IFN-γ)-induced activation of the cells. The importance of inducible nitric oxide synthase (iNOS) or NADPH phagocyte oxidase (phox) for the cidal activity was studied. Murine IFN-γ-activated peritoneal exudate cells (PEC) produced nitric oxide (NO), measured as nitrite plus nitrate, and superoxide. When PEC were infected with the live vaccine strain, LVS, of F. tularensis, the number of viable bacteria was at least 1000-fold lower in the presence than in the absence of IFN-γ after 48 h of incubation. PEC from iNOS-gene-deficient (iNOS –/–) mice killed F. tularensis LVS less effectively than did PEC from wild-type mice. PEC from phox gene-deficient (p47 phox –/–) mice were capable of killing the bacteria, but killing was less efficient, although still significant, in the presence of NG-monomethyl- l-arginine (NMMLA), an inhibitor of iNOS. A decomposition catalyst of ONOO –, FeTPPS, completely reversed the IFN-γ-induced killing of F. tularensis LVS. Under host cell-free conditions, F. tularensis LVS was exposed to S-nitroso-acetyl-penicillamine (SNAP), which generates NO, or 3-morpholinosydnonimine hydrochloride (SIN-1), which generates NO and superoxide, leading to formation of ONOO –. During 6 h of incubation, SNAP caused no killing of F. tularensis LVS, whereas effective killing occurred in the presence of equimolar concentrations of SIN-1. The results suggest that mechanisms dependent on iNOS and to a minor degree, phox, contribute to the IFN-γ-induced macrophage killing of F. tularensis LVS. ONOO – is likely to be a major mediator of the killing.

Kinetics of parasite cysteine proteinase inactivation by NO-donors

NO-donors block Plasmodium, Trypanosoma, and Leishmania life cycle inactivating parasite cysteine proteinases. In this study, the inactivation of falcipain, cruzipain, and Leishmania infantum cysteine proteinase by S-nitroso-5-dimethylaminonaphthalene-1-sulphonyl (dansyl-SNO), S-nitrosoglutathione (GSNO), (±)-( E)-4-ethyl-2-[( E)-hydroxyimino]-5-nitro-3-hexenamide (NOR-3), and S-nitrosoacetylpenicillamine (SNAP) is reported. With NO-donors in excess over the parasite cysteine proteinase, the time course of enzyme inactivation corresponds to a pseudo-first-order reaction for more than 90% of its course. The concentration dependence of the pseudo-first-order rate constant is second-order at low NO-donor concentrations but tends to first-order at high NO-donor concentrations. This behavior may be explained by a relatively fast pre-equilibrium followed by a limiting pseudo-first-order process. Kinetic parameters of cruzipain inactivation by GSNO were affected by the acidic p K shift of one ionizing group (from p K unl=5.7 to p K lig=4.8) upon GSNO-induced enzyme inactivation. Falcipain, cruzipain, and L. infantum cysteine proteinase inactivation by dansyl-SNO, GSNO, NOR-3, and SNAP is prevented and reversed by dithionite and l-ascorbic acid. However, the incubation of L. infantum cysteine proteinase with dansyl-SNO does not result in the appearance of fluorescence of the enzyme. More than 90% of the S-transnitrosylation product GSH existed in the inactivation reaction, suggesting that S-transnitrosylation is the favorite process for parasite cysteine proteinase inactivation. Furthermore, the fluorogenic substrate N-α-benzyloxycarbonyl- l-phenylalanyl- l-arginine-(7-amino-4-methylcoumarin) protects L. infantum cysteine proteinase from inactivation by SNAP. These results indicate that parasite cysteine proteinase inactivation by NO-donors occurs via NO-mediated S-nitrosylation of the Cys25 catalytic residue.

Regulation of rat pial arteriolar smooth muscle relaxation in vivo through multidrug resistance protein 5-mediated cGMP efflux.

Multidrug resistance protein 5 (MRP5) has been linked to cGMP cellular export in peripheral vascular smooth muscle cells (VSMCs) and is widely expressed in brain vascular tissue. In the present study, we examined whether knockdown of MRP5 in pial arterioles [via antisense oligodeoxynucleotide (ODN) applications] affected nitric oxide (NO)/cGMP-induced dilations. The antisense or (as a control) missense ODN was applied to the cortical surface approximately 24 h before study via closed cranial windows. The efficacy of the antisense vs. missense ODN in eliciting selective reductions in MRP5 expression was confirmed by analysis of MRP5 mRNA in pial tissue. Unexpectedly, in initial studies, a significantly lower maximal pial arteriolar diameter increase in the presence of the NO donor S-nitrosoacetylpenicillamine (SNAP) was seen in the antisense vs. missense ODN-treated rats (35 vs. 48% diameter increase, respectively). It was suspected that this related to a reduced vascular smooth muscle cell sensitivity to cGMP due to prolonged exposure to increased intracellular cGMP levels elevated by overnight restriction of cGMP efflux. That postulate was supported by a finding of a diminished vasodilating response to the cGMP-dependent protein kinase-activating cGMP analog 8-p-chlorophenylthio-cGMP in antisense vs. missense ODN-treated rats. To prevent desensitization, additional rats were studied in the presence of chronic NOS inhibition via Nomega-nitro-L-arginine. In the NO synthase (NOS)-inhibited rats, the maximal SNAP response was much higher in the antisense (62% increase) vs. the missense ODN (40% increase) group. A similar result was obtained when monitoring responses to the soluble guanylyl cyclase-activating drugs YC-1 and BAY 41-2272. Moreover, in the presence of NOS inhibition, the normal SNAP-induced rise in periarachnoid cerebrospinal fluid cGMP levels, which reflects cGMP efflux, was absent in the antisense ODN-treated rats, a finding consistent with loss of MRP5 function. In conclusion, if one minimizes the confounding effects of basal cGMP production, a clearer picture emerges, one that indicates an important role for MRP5-mediated cGMP efflux in the regulation of NO-induced cerebral arteriolar relaxation.

ERK1/2 and JNKs, but not p38 kinase, are involved in reactive oxygen species-mediated induction of osteopontin gene expression by angiotensin II and interleukin-1beta in adult rat cardiac fibroblasts.

Osteopontin (OPN), also called cytokine Eta-1, expressed in the myocardium co-incident with heart failure plays an important role in post myocardial infarction (MI) remodeling by promoting collagen synthesis and accumulation. Angiotensin II (Ang II) and inflammatory cytokines are increased in the heart following MI. We studied the involvement of mitogen-activated protein kinases (ERK1/2, JNKs, p38 kinase) and reactive oxygen species (ROS) in Ang II- and cytokine-induced OPN gene expression in adult rat cardiac fibroblasts. Ang II alone increased OPN mRNA (3.3 +/- 0.3-folds; P < 0.05; n = 7), while interleukin-1beta (IL-1beta), tumor necrosis factor (TNF-alpha), and interferon-gamma (IFN-gamma) had no effect. A combination of Ang II with IL-1beta or TNF-alpha, not IFN-gamma, increased OPN mRNA more than Ang II alone. Nitric oxide donor, S-nitrosoacetylpenicillamine (SNAP), alone or in combination with Ang II had no effect. Diphenylene iodonium (DPI), inhibitor of NAD(P)H oxidase, and tiron, superoxide scavenger, inhibited Ang II- and Ang II+ IL-1beta-stimulated increases in OPN mRNA. Ang II activated ERK1/2 within 5 min of treatment, not JNKs. IL-1beta activated ERK1/2 and JNKs within 15 min of treatment. A combination of Ang II and IL-1beta activated ERK1/2 within 5 min of treatment. None of these stimuli activated p38 kinase. DPI almost completely inhibited Ang II + IL-1beta-stimulated activation of ERK1/2, while partially inhibiting JNKs. PD98059, ERK1/2 pathway inhibitor, and SP600125, JNKs inhibitor, partially inhibited Ang II + IL-1beta-stimulated increases in OPN mRNA. A combination of PD98059 and SP600125 almost completely inhibited Ang II + IL-1beta-stimulated increases in OPN mRNA. Thus, Ang II alone increases OPN expression, while IL-1beta and TNF-alpha act synergistically with Ang II to increase OPN mRNA possibly via NO independent mechanisms. The synergistic increase in OPN mRNA involves ROS-mediated activation of ERK1/2 and JNKs, not P38 kinase, pathways in cardiac fibroblasts.

Non-specific immune response of turbot, Scophthalmus maximus (L.), experimentally infected with a pathogenic Vibrio pelagius.

The effect of a pathogenic Vibrio pelagius, isolated during a mass mortality of turbot larvae, on the non-specific immune response of turbot, Scophthalmus maximus (L.), macrophages was studied both in vitro and in vivo. The in vitro treatment of head kidney (HK) macrophages with viable V. pelagius caused a significant inhibition of the chemiluminescence (CL) response in comparison with untreated macrophages, while incubation with heat-killed bacteria did not affect this response. In vivo, the intraperitoneal injection of V. pelagius resulted in a significant inhibition of the CL response in infected fish at days 1 and 4 post-infection compared with the control fish response. The HK macrophage nitric oxide (NO) production was enhanced by in vitro incubation with intermediate doses of viable V. pelagius (5 x 10(3) and 5 x 10(4) bacteria mL(-1)) and higher doses of the heat-killed bacteria (5 x 10(4)-5 x 10(6) bacteria mL(-1)). In both cases, the NO inhibitor N-omega -nitro-L-arginine was capable of down-regulating the specific NO induction caused by incubation with the bacterial treatments. In contrast, incubation with ECPs at higher doses caused a reduction in NO production. In vivo, a significant enhancement in NO production was also observed in macrophage supernatants at day 10 post-infection. Lysozyme concentration in the serum was also significantly increased in the experimentally infected fish at days 4 and 10 post-injection. In addition, viable V. pelagius and its ECPs significantly reduced HK macrophage viability in vitro, whereas no significant differences in viability were observed during the incubation with heat-killed bacteria. As NO production was enhanced in the experimentally infected fish, the inhibitory effect of the NO donor, S-nitroso-acetyl-penicillamine (SNAP), was tested in vitro in a cell-free assay. The results showed that growth of V. pelagius was significantly inhibited using SNAP at a high concentration (1 mM).

Toxicity of nitric oxide and peroxynitrite to Photobacterium damselae subsp. piscicida

Virulent strains of Photobacterium damselae subsp. piscicida ( Pdp) were grown in media with or without glucose supplementation (to enhance polysaccharide capsule formation) and the bactericidal action of nitric oxide (NO) and peroxynitrites was evaluated in a cell-free assay. Treatment with the NO-donor S-nitroso-acetyl-penicillamine (SNAP) induced a dose-and time-dependent decrease in Pdp survival. This effect was greater for strains grown without glucose supplementation (C forms) than for their counterparts grown with glucose supplementation (C+ forms). Addition of superoxide anion (O 2 −) generating systems (Xanthine/Xanthine oxidase, glucose/glucose oxidase) to the culture media further enhanced the bactericidal effect of NO. A similar bactericidal effect, with the same pattern of sensitivity, was observed when C+ and C forms of the bacteria were treated with 3-morpholino-sydonimide hydrochloride (SIN-1), a compound which simultaneously generates NO and O 2 −. Addition of superoxide dismutase (SOD) or SOD plus catalase (CAT) did not fully reverse the toxic action of SIN-1 and the bactericidal effect was similar for both C and C+ forms suggesting that while NO alone is sufficient to cause damage in all strains of the pathogen tested, growth in glucose supplemented medium enhanced protection to reactive oxygen intermediates rather than NO.

Suppression of Pro-Inflammatory Cytokine Release by Selective Inhibition of Inducible Nitric Oxide Synthase in Mucosal Explants from Patients with Ulcerative Colitis

Background: In ulcerative colitis (UC), inflammatory damage is associated with increased production of pro-inflammatory cytokines and nitric oxide through the inducible nitric oxide synthase (iNOS) pathway. In an animal model of acute experimental colitis we have previously shown amelioration of inflammation with the highly selective iNOS inhibitor 1400 W. The aim of the present study was to investigate the effects of selective iNOS inhibition on the production of pro-inflammatory cytokines by the colon mucosa in UC. Methods: Inflamed and uninflamed mucosa from patients with severe UC were incubated with a highly selective iNOS inhibitor N-[3-(aminomethyl)benzyl]acetamidine (1400 W), with a relatively selective cNOS inhibitor N(G)-nitro-L-arginine-methyl-esther (L-NAME), or with an NO-donor, S-nitroso-acetylpenicillamine (SNAP). Cytokine concentrations in the incubation medium were quantitated with ELISA. Results: Compared to uninflamed mucosa there was an increase in iNOS protein and nitrotyrosine levels in inflamed mucosal samples. Immunolocalization of iNOS and nitrotyrosine showed their expression in inflammatory cells in the lamina propria. Expression of iNOS was also found in the epithelial brush border. Selective inhibition of iNOS suppressed the release of tumour necrosis factor alpha (TNF-α, by 66%) and interleukin-6 (IL-6, by 27%). The NO-donor, SNAP, augmented the secretion of TNF-α, IL-6 and IL-1-β (by 62%, 52% and 175%, respectively) and decreased the release of IL-1 receptor antagonist (IL-1Ra, by 34%) by the inflamed mucosa. Moreover, in uninflamed samples, 1400 W suppressed the production of TNF-α (by 69%) and incubation with SNAP decreased IL-6 concentrations by 48%. The cNOS over iNOS selective inhibitor L-NAME had no significant effects on the accumulation of cytokines. Conclusion: Selective inhibition of iNOS suppresses mucosal TNF-α and IL-6 release in active UC, whereas NO seems to exacerbate the inflammatory response. These results suggest that selective iNOS inhibition may have therapeutic promise in the treatment of UC.