Amyloid-beta (25-35) increases activity of neuronal NO-synthase in rat brain

Nitric oxide (NO) is a free radical with multiple functions in the nervous system. NO plays an important role in the mechanisms of neurodegenerative diseases including Alzheimer's disease. The main source of NO in the brain is an enzymatic activity of nitric oxide synthase (NOS). The aim of the present study was to analyze the expression and activity of both neuronal (nNOS) and inducible (iNOS) isoenzymes in the cerebral cortex and hippocampus of rats after intracerebroventricular administration of amyloid-beta (A beta) peptide fragment A beta(25-35). NADPHd histochemistry as well as immunohistochemistry were also used to investigate nNOS and iNOS expression in rat brain. The data presented here show that A beta(25-35) did not influence levels of nNOS or iNOS mRNA or protein expression in both structures studied. A beta(25-35) activated nNOS in the cerebral cortex and hippocampus without effect on iNOS activity. A beta(25-35) decreased the number of NADPHd-expressing neurons in the neocortex, but it did not significantly influence the number NADPHd-positive cells in the hippocampus. The peptide had no effect on the number of nNOS containing cells. We hypothesize that increased synthesis of NO induced by A beta(25-35) is related to qualitative alterations of nNOS molecule, but not to changes in NOS protein expression.     

S-Nitrosation and regulation of inducible nitric oxide synthase

The inducible isoform of nitric oxide synthase (iNOS) and three zinc tetrathiolate mutants (C104A, C109A, and C104A/C109A) were expressed in Escherichia coli and purified. The mutants were found by ICP-AES and the zinc-specific PAR colorimetric assay to be zinc free, whereas the wild-type iNOS zinc content was 0.38 +/- 0.01 mol of Zn/mol of iNOS dimer. The cysteine mutants (C104A and C109A) had an activity within error of wild-type iNOS (2.24 +/- 0.12 micromol of NO min(-1) mg(-1)), but the double cysteine mutant had a modestly decreased activity (1.75 +/- 0.14 micromol of NO min(-1) mg(-1)). To determine if NO could stimulate release of zinc and dimer dissociation, wild-type protein was allowed to react with an NO donor, DEA/NO, followed by buffer exchange. ICP-AES of samples treated with 10 microM DEA/NO showed a decrease in zinc content (0.23 +/- 0.01 to 0.09 +/- 0.01 mol of Zn/mol of iNOS dimer) with no loss of heme iron. Gel filtration of wild-type iNOS treated similarly resulted in approximately 20% more monomeric iNOS compared to a DEA-treated sample. Only wild-type iNOS had decreased activity (42 +/- 2%) after reaction with 50 microM DEA/NO compared to a control sample. Using the biotin switch method under the same conditions, only wild-type iNOS had increased levels of S-biotinylation. S-Biotinylation was mapped to C104 and C109 on wild-type iNOS using LysC digestion and MALDI-TOF/TOF MS. Immunoprecipitation of iNOS from the mouse macrophage cell line, RAW-264.7, and the biotin switch method were used to confirm endogenous S-nitrosation of iNOS. The data show that S-nitrosation of the zinc tetrathiolate cysteine results in zinc release from the dimer interface and formation of inactive monomers, suggesting that this mode of inhibition might occur in vivo.     

2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potentiates nitrosation of a heterocyclic amine carcinogen by nitric oxide

Although nitrosation plays an important role in initiation of carcinogenesis, the reactive nitrogen oxygen species (RNOS) mediating this reaction by multiple pathways have not been determined. The heterocyclic amine carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) was used as a target to investigate RNOS and pathways for potentiation of nitric oxide (NO)-mediated nitrosation. 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (CPTIO) oxidizes NO to NO(2)(.) and was used as a tool to investigate NO(2)(.) potentiation of nitrosation. The IQ nitrosation product, 2-nitrosoamino-3-methylimidazo[4,5-f]quinoline ((14)C-N-NO-IQ), was monitored by HPLC. Autoxidation of NO, generated by spermine NONOate (2.4 microM NO/min) for 7.5 min, did not convert 10 microM (14)C-IQ to N-NO-IQ. However, the presence of 15 muM CPTIO resulted in 3 microM N-NO-IQ formation. Potentiation by CPTIO occurred at low and high fluxes of NO, 0.075 to 1.2 microM/min, and over a range of IQ to CPTIO ratios of 0.5 to 10. A significant portion of N-NO-IQ formation was insensitive to azide (10 mM) inhibition, suggesting oxidative nitrosylation. NADH (0.02 mM) did not alter nitrosation by autoxidation, but effectively inhibited potentiation by CPTIO. Ascorbic acid (0.2 mM) and 5,5-dimethyl-1-pyrroline N-oxide (30 mM) inhibited nitrosation with or without CPTIO, while superoxide dismutase was not inhibitory. The RNOS produced by CPTIO had a 27-fold greater affinity for IQ than those produced by autoxidation. Results are consistent with NO(2)(.) or a RNOS like NO(2)(.) potentiating IQ oxidative nitrosylation. Nitrosation occurring at both low and high fluxes of NO can contribute to carcinogenesis.     

Lipopolysaccharide (LPS) Stimulates the Production of Tumor Necrosis Factor (TNF)-α and Expression of Inducible Nitric Oxide Synthase (iNOS) by Osteoclasts (OCL) in Murine Bone Marrow Cell Culture

Osteoclasts (OCL) resorb bone. They are essential for the development of normal bones and the repair of impaired bones. The function of OCL is presumed to be supported by cytokines and other biological mediators, including tumor necrosis factor (TNF)-α and nitric oxide (NO). Bacterial lipopolysaccharide (LPS) is a potent inducer of TNF-α and inducible nitric oxide synthase (iNOS), which is the specific enzyme for synthesizing NO from L-arginine. To obtain direct evidence on LPS-induced TNF-α production and iNOS expression by OCL, OCL-enriched cultures were prepared by 7-day cocultures of bone marrow cells of adult BALB/c mice and osteoblastic cells (OBs) derived from calvaria of newborn BALB/c mice, and the generation of TNF-α and iNOS in OCL stimulated with LPS was examined immunocytochemically. When the cultured cells were stimulated with 100 ng/ml of LPS, OCL clearly showed TNF-α and iNOS expression. Without LPS-stimulation, no expression was observed. TNF activity in the culture supernatants of the OCL-enriched cultures in the presence of LPS was also detected by cytotoxic assay that used TNF-sensitive L929 cells. The dentin resorption activity of OCL was estimated by area and number of pits formed on dentin slices, which were covered by the OCL fraction and cultured in the presence or absence of LPS, sodium nitroprusside (SNP; a NO generating compound), N^G-monomethyl L-arginine acetate (L-NMMA; a competitive inhibitor of NO synthase (NOS)), or LPS plus L-NMMA. Pit formation was obviously inhibited in the presence of SNP and slightly inhibited in the presence of L-NMMA, but it was not affected in the presence of LPS or LPS plus L-NMMA. These findings indicate that OCL produces TNF and expresses iNOS in response to LPS, but the LPS-activation of OCL scarcely affects pit formation by them.     

A possible photosensitizer: Tobacco-specific nitrosamine, 4-(N-methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), induced mutations, DNA strand breaks and oxidative and methylative damage with UVA

We discovered the directly acting mutagenicity of the tobacco-specific nitrosamine, 4-(N-methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), with UVA light (320-400nm) in Ames bacteria and phage M13mp2 in the absence of metabolic activation. We have investigated the spectrum of mutations caused by UVA-activated NNK. The majority (57%) of induced sequence changes were comprised of GC to CG, GC to TA and GC to AT. This suggested that modification of guanine residues was responsible for these mutations. Hence, we explored the formation of 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodG) and O(6)-methylguanine (O(6)meG) in the DNA. When calf thymus DNA was treated with NNK and UVA, the amount of 8-oxodG/dG and O(6)meG/G in the DNA increased up to 20-fold and 100-fold, respectively, compared with the untreated control. DNA strand breaks were observed following NNK and UVA treatment, and the strand breaks were suppressed in the presence of scavengers for oxygen and NO radical. The formation of NO was also observed in NNK solutions irradiated with UVA. We analyzed the photodynamic spectrum of mutation induction, 8-oxodG formation and NO formation using monochromatic radiation. The patterns of the action spectra were comparable to the absorption spectrum of NNK. We conclude that NNK may act as a photosensitizer in response to UVA to produce NO and other oxidative and alkylative intermediates following the formation of 8-oxodG and O(6)meG in DNA, which may lead to mutations and DNA strand breaks.     

Angiotensin-(1-7) increases neuronal potassium current via a nitric oxide-dependent mechanism

Actions of angiotensin-(1-7) [Ang-(1-7)], a heptapeptide of the renin-angiotensin system, in the periphery are mediated, at least in part, by activation of nitric oxide (NO) synthase (NOS) and generation NO(·). Studies of the central nervous system have shown that NO(·) acts as a sympathoinhibitory molecule and thus may play a protective role in neurocardiovascular diseases associated with sympathoexcitation, such as hypertension and heart failure. However, the contribution of NO in the intraneuronal signaling pathway of Ang-(1-7) and the subsequent modulation of neuronal activity remains unclear. Here, we tested the hypothesis that neuronal NOS (nNOS)-derived NO(·) mediates changes in neuronal activity following Ang-(1-7) stimulation. For these studies, we used differentiated catecholaminergic (CATH.a) neurons, which we show express the Ang-(1-7) receptor (Mas R) and nNOS. Stimulation of CATH.a neurons with Ang-(1-7) (100 nM) increased intracellular NO levels, as measured by 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM) fluorescence and confocal microscopy. This response was significantly attenuated in neurons pretreated with the Mas R antagonist (A-779), a nonspecific NOS inhibitor (nitro-L-arginine methyl ester), or an nNOS inhibitor (S-methyl-L-thiocitrulline, SMTC), but not by endothelial NOS (eNOS) or inhibitory NOS (iNOS) inhibition {L-N-5-(1-iminoethyl)ornithine (L-NIO) and 1400W, respectively}. To examine the effect of Ang-(1-7)-NO(·) signaling on neuronal activity, we recorded voltage-gated outward K(+) current (I(Kv)) in CATH.a neurons using the whole cell configuration of the patch-clamp technique. Ang-(1-7) significantly increased I(Kv), and this response was inhibited by A-779 or S-methyl-L-thiocitrulline, but not L-NIO or 1400W. These findings indicate that Ang-(1-7) is capable of increasing nNOS-derived NO(·) levels, which in turn, activates hyperpolarizing I(Kv) in catecholaminergic neurons.     

Regulation of inducible nitric oxide synthase by self-generated NO

A ferric heme-nitric oxide (NO) complex can build up in mouse inducible nitric oxide synthase (iNOS) during NO synthesis from L-arginine. We investigated its formation kinetics, effect on catalytic activity, dependence on solution NO concentration, and effect on enzyme oxygen response (apparent KmO2). Heme-NO complex formation was biphasic and was linked kinetically to an inhibition of electron flux and catalysis in iNOS. Experiments that utilized a superoxide generating system to scavenge NO showed that the magnitude of heme-NO complex formation directly depended on the NO concentration achieved in the reaction solution. However, a minor portion of heme-NO complex (20%) still formed during NO synthesis even when solution NO was completely scavenged. Formation of the intrinsic heme-NO complex, and the heme-NO complex related to buildup of solution NO, increased the apparent KmO2 of iNOS by 10- and 4-fold, respectively. Together, the data show heme-NO complex buildup in iNOS is due to both intrinsic NO binding and to equilibrium binding of solution NO, with the latter predominating when NO reaches high nanomolar to low micromolar concentrations. This behavior distinguishes iNOS from the other NOS isoforms and indicates a more complex regulation is possible for its activity and oxygen response in biologic settings.     

Induction of iNOS restricts functional activity of both eNOS and nNOS in pig cerebral artery

The aim of the study was to investigate the effect of iNOS expression on eNOS and nNOS functional activity in porcine cerebral arteries. iNOS was induced in pig basilar arteries using lipopolysaccharide (LPS). Arteries expressing iNOS generated NO and relaxed when challenged with L-arginine (30 microM), an effect that was reduced by treatment with dexamethasone (coincubated with LPS) and prevented by the iNOS inhibitor 1400 W (administered 10 min prior to precontraction). eNOS was activated by A23187 and was found to be impaired in arteries that had iNOS induced (A23187 1 microM relaxation: control 110+/-8%, LPS-treated 50+/-16% ; p<0.05, N=5-6). This was due mainly to reduced formation of NO by A23187 (NO concentration in response to A23187 1 microM: control 25+/-6 nM, LPS-treated 0.8+/-1.2 nM; p<0.001, N=5-6), in addition to a small reduction in the vasodilator response to the NO-donors NOC-22 and SIN-1. Cerebral vasodilation produced by stimulation of intramural nitrergic nerves was impaired in arteries that had iNOS induced, and this was reversed by 1400 W (control 23+/-4% relaxation, LPS-treated 11+/-1% relaxation, LPS plus 1400 W 10 microM treated 25+/-2% relaxation; p<0.01 for control versus LPS, N=6). It is concluded that the induction of iNOS in cerebral arteries reduces NO-mediated vasodilation initiated by eNOS and by nNOS, primarily by modulation of NO formation.     

(-)-Linalool inhibits in vitro NO formation: Probable involvement in the antinociceptive activity of this monoterpene compound

Recent studies performed in our laboratory have shown that (-)-linalool, the natural occurring enantiomer in essential oils, possesses anti-inflammatory, antihyperalgesic and antinociceptive effects in different animal models. The antinociceptive and antihyperalgesic effect of (-)-linalool has been ascribed to the stimulation of the cholinergic, opioidergic and dopaminergic systems, to its local anaesthetic activity and to the blockade of N-Methyl-d-aspartate receptors (NMDA). Since nitric oxide (NO) and prostaglandin E(2) (PGE(2)) play an important role in oedema formation and hyperalgesia and nociception development, to investigate the mechanism of these actions of the (-)-linalool, we examined the effects of this compound on lipopolysaccharide (LPS)-induced responses in macrophage cell line J774.A1. Exposure of LPS-stimulated cells to (-)-linalool significantly inhibited nitrite accumulation in the culture medium without inhibiting the LPS-stimulated increase of inducible nitric oxide synthase (iNOS) expression, suggesting that the inhibitory activity of (-)-linalool is mainly due to the iNOS enzyme activity. In contrast, exposure of LPS-stimulated cells to (-)-linalool failed, if not at the highest concentration, both in inhibiting PGE(2) release and in inhibiting increase of inducible cyclooxygenase-2 (COX(2)) expression in the culture medium. Collectively, these results indicate that the reduction of NO production/release is responsible, at least partially, for the molecular mechanisms of (-)-linalool antinociceptive effect, probably through mechanisms where cholinergic and glutamatergic systems are involved.     

Late preconditioning induced by NO donors, adenosine A1 receptor agonists, and delta1-opioid receptor agonists is mediated by iNOS

Although ischemia-induced late preconditioning (PC) is known to be mediated by inducible nitric oxide (NO) synthase (iNOS), the role of this enzyme in pharmacologically induced late PC remains unclear. We tested whether targeted disruption of the iNOS gene abrogates late PC elicited by three structurally different NO donors [diethylenetriamine/NO (DETA/NO), nitroglycerin (NTG), and S-nitroso-N-acetyl-penicillamine (SNAP)], an adenosine A1 receptor agonist [2-chloro-N6-cyclopentyladenosine (CCPA)], and a delta1-opioid receptor agonist (TAN-670). The mice were subjected to a 30-min coronary occlusion followed by 24 h of reperfusion. In iNOS knockout (iNOS-/-) mice, infarct size was similar to wild-type (WT) controls, indicating that iNOS does not modulate infarct size in the absence of PC. Pretreatment of WT mice with DETA/NO, NTG, SNAP, TAN-670, or CCPA 24 h before coronary occlusion markedly reduced infarct size. In iNOS-/- mice, however, the late PC effect elicited by DETA/NO, NTG, SNAP, TAN-670, and CCPA was completely abrogated. Furthermore, in WT mice pretreated with TAN-670 or CCPA, the selective iNOS inhibitor 1400W also abolished the delayed PC properties of these drugs; 1400W had no effect in WT mice. These data demonstrate that iNOS plays an obligatory role in NO donor-induced, adenosine A1 receptor agonist-induced, and delta1-opioid receptor agonist-induced late PC, underscoring the critical role of this enzyme as a common mediator of cardiac adaptations to stress.     

Quercetin tetraacetyl derivative inhibits LPS-induced nitric oxide synthase (iNOS) expression in J774A.1 cells

In inflammation, nitric oxide (NO) acts as a pro-inflammatory mediator, which is synthesized by inducible nitric oxide synthase (iNOS) in response to pro-inflammatory agents such as lipopolysaccharide (LPS). Quercetin (Qt) has anti-inflammatory properties through its ability to inhibits nitric oxide production and iNOS expression in different cellular types. In the present study, we evaluated the effect of a semi-synthetic acetyl (quercetin-3,5,7,3′-tetraacetyl: TAQt) Qt derivative and two natural sulphated (quercetin-3-acetyl-7,3′,4′-trisulphate: ATS and quercetin-3,7,3′,4′-tetrasulphate: QTS) Qt derivatives on the LPS-induced NO production and iNOS expression in J774A.1 cells. Our results demonstrate that only TAQt inhibited the NO production by decreasing the iNOS mRNA and protein levels. In addition, we showed that TAQt blocked the LPS-induced nuclear NF-κB translocation by inhibiting the IκB-α degradation. Hence, as TAQt inhibited the LPS-induced iNOS expression and NO production, it could therefore be considered as a potential therapeutic agent for the treatment of inflammatory diseases related with the NO system.     

Strain-dependent difference in inducible nitric oxide synthesis (iNOS) expression in rat pancreatic islets correlates with interferon regulating factor 1 (IRF-1) and heat shock protein 70 (HSP70) expression

Nitric oxide (NO) may be a necessary but not sufficient mediator of cytokine-mediated, selective beta-cell destruction. Previously, we have described a difference in NO-dependent IL-1beta sensitivity in vivo and in vitro of pancreatic islets from two rat strains, Brown Norway (BN) and Wistar Kyoto (WK), the latter being the more sensitive strain. Here we investigated whether strain-dependent, differential islet iNOS expression was associated with differences in islet expression of the IL-1 receptor type 1(IL-1RI) or interferon regulating factor 1 (IRF-1), and/or caused differences in HSP70 expression, a marker of cell defence against oxidative stress. Methods: isolated islets from both rat strains were exposed to increasing concentrations of IL-1beta (0-150 pg/ml) for 24 hours or for varying culture periods (4-48 hours) to 15 pg/ml of IL-1beta. Measurements: accumulated insulin and nitrite release into incubation medium, and islet mRNA and protein expression of iNOS, IL-1RI, IRF-1 and HSP70 by semi-quantitative RT-PCR and Western blotting. Results: Higher insulin and lower nitrite release into the incubation medium were seen for BN compared to WK rats islets in both dose- and time-response experiments. IRF-1 expression preceded iNOS expression and was more pronounced in WK than in BN islets. No strain differences were observed for islet expression of IL-1RI. A strain-dependent HSP70 expression in response to IL-1beta with the highest levels in WK rat islets following iNOS expression was seen. Conclusion: There was a strain-dependent difference in iNOS expression which was associated with IRF-1 and HSP70 expression.     

Oxidative stress induces vascular heme oxygenase-1 expression in ovariectomized rats

Heme oxygenase-1 (HO-1), an inducible stress protein, has been implicated in cytoprotection against oxidative stress in vitro and in vivo. Estrogens also have antioxidant effects. This study investigated the time course of HO-1 and inducible nitric oxide synthase (iNOS) expression in the aortas of ovariectomized rats, and the regulatory relationship between the NO/NOS and the carbon monoxide/HO systems. HO-1 and iNOS protein expression was induced by ovariectomy (Ovx) and was extremely high 2-6 weeks after Ovx compared with the sham-operated group. Expression of the constitutive enzymes HO-2 and endothelial NOS did not differ significantly between sham-operated and Ovx rats. 17beta-Estradiol (E(2)) replacement reversed these changes in rats after Ovx. Long-term treatment with the antioxidant tempol significantly inhibited HO-1 and iNOS expression. The iNOS inhibitor aminoguanidine significantly suppressed the induction of HO-1. Oxidized glutathione in the hearts of Ovx rats increased gradually, with significant elevation at 3-6 weeks after Ovx compared with the sham-operated group, whereas plasma levels of NO metabolites were significantly reduced 4-6 weeks after Ovx. Treatment with the HO inhibitor zinc protoporphyrin IX blocked HO-1 induction, but significantly increased the plasma levels of NO metabolites. In conclusion, HO-1 is induced by oxidative stress resulting from E(2) depletion. The NO/iNOS system contributes to the induction of HO-1, which may subsequently suppress iNOS activity to modulate vasculoprotective effects after menopause.