Oltipraz inhibits inducible nitric oxide synthase in vitro and inhibits nitric oxide production in activated microglial cells

The clinically relevant drug oltipraz (OPZ) has previously been shown to inhibit cytochrome P450 enzymes [Chem. Res. Toxicol. 13 (2000) 245]. The current study reveals that OPZ is also able to inhibit *NO formation by purified inducible nitric oxide synthase (iNOS) but not by neuronal nitric oxide synthase in hemoglobin assays. The inhibition of iNOS by OPZ is reversible and competitive with an IC(50) of 5.9 microM and Ki of 0.6 microM. In murine BV-2 microglial cells, an immortalized cell line that produces *NO in response to lipopolysaccharide (LPS), OPZ is able to block the formation of nitrite in LPS treated cells. The inhibitory effect of OPZ on LPS treated cells is not due to cell toxicity. Finally, treatment of cells with OPZ does not induce or suppress expression of iNOS protein as shown by Western blot analysis.     

Inducible nitric oxide synthase is key to peroxynitrite-mediated,LPS-induced protein radical formation in murine microglial BV2 cells

Microglia are the resident immune cells in the brain. Microglial activation is characteristic of several inflammatory and neurodegenerative diseases including Alzheimer's disease, multiple sclerosis, and Parkinson's disease. Though lipopolysaccharide (LPS)-induced microglial activation in models of Parkinson's disease is well documented, the free radical-mediated protein radical formation and its underlying mechanism during LPS-induced microglial activation are not known. Here we have used immuno-spin trapping and RNA interference to investigate the role of inducible nitric oxide synthase (iNOS) in peroxynitrite-mediated protein radical formation in murine microglial BV2 cells treated with LPS. Treatment of BV2 cells with LPS resulted in morphological changes, induction of iNOS, and increased protein radical formation. Pretreatments with FeTPPS (a peroxynitrite decomposition catalyst), L-NAME (total NOS inhibitor), 1400W (iNOS inhibitor), and apocynin significantly attenuated LPS-induced protein radical formation and tyrosine nitration. Results obtained with coumarin-7-boronic acid, a highly specific probe for peroxynitrite detection, correlated with LPS-induced tyrosine nitration, which demonstrated involvement of peroxynitrite in protein radical formation. A similar degree of protection conferred by 1400W and L-NAME led us to conclude that only iNOS, and no other forms of NOS, is involved in LPS-induced peroxynitrite formation. Subsequently, siRNA for iNOS, the iNOS-specific inhibitor 1400W, the NF-κB inhibitor PDTC, and the p38 MAPK inhibitor SB202190 was used to inhibit iNOS directly or indirectly. Inhibition of iNOS precisely correlated with decreased protein radical formation in LPS-treated BV2 cells. The time course of protein radical formation also matched the time course of iNOS expression. Taken together, these results prove the role of iNOS in peroxynitrite-mediated protein radical formation in LPS-treated microglial BV2 cells.     

Lipopolysaccharide down-regulates inducible nitric oxide synthase expression in swine heart in vivo

Studies of the regulation of iNOS expression have provided many contradictory results. Comparing iNOS expression profile between cell types or organs of the same animal under the same experimental conditions may provide an explanation for these conflicting results. We have examined iNOS mRNA and protein expression in heart and liver of the same group of pigs. We found that there is a sharp difference in iNOS expression between heart and liver. The iNOS mRNA and protein was constitutively expressed in the heart at high level, but was not detectable in the liver of the same control animal. Lipopolysaccharide (LPS, 100 microg/kg, i.v.) caused a marked iNOS induction in the liver, but significantly down-regulated iNOS expression in the heart. This differential iNOS expression appears to be physiologically relevant, since LPS and the iNOS inhibitor, S-methylisothiourea, exerted different effects on hepatic and myocardial blood flow. Our data demonstrate a fundamental difference in iNOS regulation in the heart and liver of swine, and may explain the contradictory data on the regulation of iNOS expression.     

Deficiency of inducible nitric oxide synthase protects against MPTP toxicity in vivo

MPTP produces clinical, biochemical, and neuropathologic changes reminiscent of those that occur in idiopathic Parkinson's disease (PD). In the present study we show that MPTP treatment led to activation of microglia in the substantia nigra pars compacta (SNpc), which was associated and colocalized with an increase in inducible nitric oxide synthase (iNOS) expression. In iNOS-deficient mice the increase of iNOS expression but not the activation of microglia was blocked. Dopaminergic SNpc neurons of iNOS-deficient mice were almost completely protected from MPTP toxicity in a chronic paradigm of MPTP toxicity. Because the MPTP-induced decrease in striatal concentrations of dopamine and its metabolites did not differ between iNOS-deficient mice and their wild-type littermates, this protection was not associated with a preservation of nigrostriatal terminals. Our results suggest that iNOS-derived nitric oxide produced in microglia plays an important role in the death of dopaminergic neurons but that other mechanisms contribute to the loss of dopaminergic terminals in MPTP neurotoxicity. We conclude that inhibition of iNOS may be a promising target for the treatment of PD.     

Phosphoinositol 3 kinase inhibitor, LY294002 increases bcl-2 protein and inhibits okadaic acid-induced apoptosis in Bcl-2 expressing renal epithelial cells

Protein phosphorylation plays an indispensable role in cellular regulation of mitosis, metabolism, differentiation, and death. We previously reported that the protein phosphatase inhibitor okadaic acid (OKA) induces apoptosis in renal epithelial cells in culture. In the present study, we examined the role of phosphotidylinositol 3 (PI3) kinase signaling in okadaic acid-induced apoptosis by pre-treating normal rat kidney renal epithelial cells expressing human bcl-2 with the PI3 kinase inhibitors, LY294002 and wortmannin, followed by apoptosis-inducing concentrations of okadaic acid. Given the reported cell survival activity of PI3 kinase signaling mostly attributed to Akt kinase activation, we hypothesized that inhibition of PI3 kinase would enhance okadaic-induced apoptosis. Surprisingly, our data show that pretreatment with LY294002, but not wortmannin, attenuated okadaic acid-induced apoptosis. In contrast, to LY294002, wortmannin enhanced apoptosis. Interestingly, we also found that LY294002 treatment increased bcl-2 protein levels in normal rat kidney epithelial cells expressing bcl-2 (NRK-bcl-2). In untreated cells, bcl-2 appeared to be mainly perinuclear, coincident with the nuclear membrane, or in the cytosol. In OKA treated cells that were pre-treated with Ly294002, bcl-2 was highly co-localized with mitochondria, but in cells treated with okadaic acid alone, bcl-2 was associated with fragmented chromatin. In this model, it appears that LY294002 may exert anti-apoptotic effects by a previously unreported treatment related increase in bcl-2. Although it is widely accepted that bcl-2 protein can inhibit apoptosis, we propose that the subcellular location of bcl-2 is an important determinant in whether bcl-2 effectively inhibits apoptosis.     

Oxalomalate affects the inducible nitric oxide synthase expression and activity

Inducible nitric oxide synthase (iNOS) is an homodimeric enzyme which produces large amounts of nitric oxide (NO) in response to inflammatory stimuli. Several factors affect the synthesis and catalytic activity of iNOS. Particularly, dimerization of NOS monomers is promoted by heme, whereas an intracellular depletion of heme and/or L-arginine considerably decreases NOS resistance to proteolysis. In this study, we found that oxalomalate (OMA, oxalomalic acid, alpha-hydroxy-beta-oxalosuccinic acid), an inhibitor of both aconitase and NADP-dependent isocitrate dehydrogenase, inhibited nitrite production and iNOS protein expression in lipopolysaccharide (LPS)-activated J774 macrophages, without affecting iNOS mRNA content. Furthermore, injection of OMA precursors to LPS-stimulated rats also decreased nitrite production and iNOS expression in isolated peritoneal macrophages. Interestingly, alpha-ketoglutarate or succinyl-CoA administration reversed OMA effect on NO production, thus correlating NO biosynthesis with the anabolic capacity of Krebs cycle. When protein synthesis was blocked by cycloheximide in LPS-activated J774 cells treated with OMA, iNOS protein levels, evaluated by Western blot analysis and (35)S-metabolic labelling, were decreased, suggesting that OMA reduces iNOS biosynthesis and induces an increase in the degradation rate of iNOS protein. Moreover, we showed that OMA inhibits the activity of the iNOS from lung of LPS-treated rats by enzymatic assay. Our results, demonstrating that OMA acts regulating synthesis, catalytic activity and degradation of iNOS, suggest that this compound might have a potential role in reducing the NO overproduction occurring in some pathological conditions.     

Triptolide inhibits cyclooxygenase-2 and inducible nitric oxide synthase expression in human colon cancer and leukemia cells

Triptolide (TP),a traditional Chinese medicine,has been reported to be effective in thetreatment of autoimmune diseases and exerting antineoplastic activity in several human tumor cell lines.Thisstudy investigates the antitumor effect of TP in human colon cancer cells (SW114) and myelocytic leukemia(K562),and elucidates the possible molecular mechanism involved.SW114 and K562 cells were treatedwith different doses of TP (0,5,10,20,or 50 ng/ml).The cell viability was assessed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT). Results demonstrated that TP inhibited the proliferation ofboth tumor cell lines in a dose-dependent manner.To further investigate its mechanisms,the productsprostaglandin E_2 (PGE2) and nitric oxide (NO) were measured by enzyme-linked immunosorbent assay(ELISA).Our data showed that TP strongly inhibited the production of NO and PGE_2. Consistent with theseresults,the expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) was up-regulatedboth at the mRNA level and the protein expression level,as shown by real-time RT-PCR and Westernblotting.These results indicated that the inhibition of the inflammatory factor COX-2 and iNOS activitycould be involved in the antitumor mechanisms of TP.     

Intraprotein electron transfer in inducible nitric oxide synthase holoenzyme

Intraprotein electron transfer (IET) from flavin mononucleotide (FMN) to heme is essential in NO synthesis by NO synthase (NOS). Our previous laser flash photolysis studies provided a direct determination of the kinetics of the FMN–heme IET in a truncated two-domain construct (oxyFMN) of murine inducible NOS (iNOS), in which only the oxygenase and FMN domains along with the calmodulin (CaM) binding site are present (Feng et al. J. Am. Chem. Soc. 128, 3808–3811, 2006). Here we report the kinetics of the IET in a human iNOS oxyFMN construct, a human iNOS holoenzyme, and a murine iNOS holoenzyme, using CO photolysis in comparative studies on partially reduced NOS and a NOS oxygenase construct that lacks the FMN domain. The IET rate constants for the human and murine iNOS holoenzymes are 34 ± 5 and 35 ± 3 s⁻¹, respectively, thereby providing a direct measurement of this IET between the catalytically significant redox couples of FMN and heme in the iNOS holoenzyme. These values are approximately an order of magnitude smaller than that in the corresponding iNOS oxyFMN construct, suggesting that in the holoenzyme the rate-limiting step in the IET is the conversion of the shielded electron-accepting (input) state to a new electron-donating (output) state. The fact that there is no rapid IET component in the kinetic traces obtained with the iNOS holoenzyme implies that the enzyme remains mainly in the input state. The IET rate constant value for the iNOS holoenzyme is similar to that obtained for a CaM-bound neuronal NOS holoenzyme, suggesting that CaM activation effectively removes the inhibitory effect of the unique autoregulatory insert in neuronal NOS. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Levodopa modulating effects of inducible nitric oxide synthase and reactive oxygen species in glioma cells

Neurological injury and Parkinson disease (PD) are often associated with the increase of nitric oxide (NO) and free radicals from resident glial cells in the brain. In vitro, exposure to L-3-4-dihydroxyphenylalanine (L-DOPA), one of the main therapeutic agents for the treatment of PD, can lead to neurotoxicity. In this study, lipopolysaccharide (LPS) and interferon-gamma (IFN-g) were used to stimulate C6 glioma cells in the presence of varying concentrations of L-DOPA (1 microM-1 mM). The results indicated a slight augmentation of NO(2)(-) production at low concentrations of L-DOPA (<100 microM) and complete inhibition of NO(2)(-) at higher concentrations (500 microM, 1 mM), (p < 0.001). Western blot analysis corroborated that L-DOPA effects on iNOS was at the level of its protein expression. Total reactive oxygen species (ROS) were detected using 2', 7'-dichlorofluorescein diacetate fluorescence dye (2', 7'-DCFC) and there was an increase of intensity with the increasing concentrations of L-DOPA. Furthermore, large amounts of superoxide (O(2)(-)) and hydrogen peroxide (H(2)O(2)) were generated from the autoxidation of L-DOPA. C6 cells contain high levels of catalase, with inadequate levels of superoxide dismutase (SOD); therefore, there was an accumulation of O(2)(-), tantamount to elevation in 2'7'-DCFC intensity. Simultaneous accumulation of O(2)(-) and NO(2)(-) would propel formation of peroxynitrite (ONOO-). SOD completely attenuated the autoxidation of L-DOPA and significantly reversed the inhibitory effects on iNOS at high concentrations. The data obtained confirmed that the observed effects on iNOS were not due to the activation of the D(1) or beta1 adrenergic receptors by L-DOPA. It was concluded from this study that L-DOPA contributed to the modulation of iNOS and to the increase of O(2)(-) production in the stimulated glioma cells in vitro.     

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.