Proteolytic cleavage of inducible nitric oxide synthase (iNOS) by calpain I.

Proteolytic degradation of inducible nitric oxide synthase (iNOS or NOS2; EC 1.14.13.39) is one of the key steps by which the synthetic glucocorticoid dexamethasone controls the amount of iNOS protein and thus the production of nitric oxide (NO) in interferon-gamma-stimulated RAW 264.7 cells. In the present study we examined the role of the calmodulin (CaM)-binding site present within iNOS protein for the proteolytic degradation by the calcium-dependent neutral cysteine protease calpain I (EC 3.4.22.17). Using pulse chase experiments as well as cell-free degradation assays we show that the iNOS monomer is a direct substrate for cleavage by calpain I. Two structural determinants are involved in proteolytic cleavage, the canonical CaM-binding domain present at amino acids 501-532 and a conformational determinant located within iNOS. The access of the CaM-binding region appears to be critical for substrate cleavage as incubation of in vitro synthesized iNOS with purified CaM inhibits iNOS degradation by calpain I. Moreover, cytosolic CaM levels are decreased upon treatment of RAW 264.7 cells with dexamethasone as assessed by immunoprecipitation. The data shown herein provide novel insights into the underlying mechanisms involved in the anti-inflammatory actions of glucocorticoids.     

Proteolytic cleavage of inducible nitric oxide synthase (iNOS) by calpain I

Proteolytic degradation of inducible nitric oxide synthase (iNOS or NOS2; EC 1.14.13.39) is one of the key steps by which the synthetic glucocorticoid dexamethasone controls the amount of iNOS protein and thus the production of nitric oxide (NO) in interferon-γ-stimulated RAW 264.7 cells. In the present study we examined the role of the calmodulin (CaM)-binding site present within iNOS protein for the proteolytic degradation by the calcium-dependent neutral cysteine protease calpain I (EC 3.4.22.17). Using pulse chase experiments as well as cell-free degradation assays we show that the iNOS monomer is a direct substrate for cleavage by calpain I. Two structural determinants are involved in proteolytic cleavage, the canonical CaM-binding domain present at amino acids 501–532 and a conformational determinant located within iNOS. The access of the CaM-binding region appears to be critical for substrate cleavage as incubation of in vitro synthesized iNOS with purified CaM inhibits iNOS degradation by calpain I. Moreover, cytosolic CaM levels are decreased upon treatment of RAW 264.7 cells with dexamethasone as assessed by immunoprecipitation. The data shown herein provide novel insights into the underlying mechanisms involved in the anti-inflammatory actions of glucocorticoids.     

Inducible nitric oxide synthase inhibitors of Chinese herbs. Part 2: naturally occurring furanocoumarins

Inducible nitric oxide synthase (iNOS)-dependent production of nitric oxide (NO) plays an important role in inflammation. The effects of various naturally occurring furanocoumarins on NO production in lipopolysaccharide (LPS)-activated RAW 264.7 macrophage cells were evaluated in vitro. The results showed that angelicin, pimpinellin, sphondin, byakangelicol, oxypeucedanin, oxypeucedanin hydrate, xanthotoxin, and cnidilin are potential NO production inhibitors, and their IC50 values for inhibition of nitrite production were 19.5, 15.6, 9.8, 16.9, 16.8, 15.8, 16.6, and 17.7 microg/mL, respectively. Distinct structure-activity relationships were also revealed for the NO production inhibitory activities of these furanocoumarins. Activities of the angelicin type such as pimpinellin and sphondin were more potent than those of the psoralen type. Presence of a methoxy at the C6 position in the angelicin type seemed to be essential to augment the activity. Western blot analysis demonstrated that only sphondin dose-dependently inhibited the expression of the iNOS protein at 2.5-20 microg/mL. However, iNOS enzyme activity was stimulated with LPS for 12 h and sphondin was administered (20 microg/mL) for 24 h, which did not reasonably inhibit iNOS enzyme activity. L-NAME (100 microM), a known specific inhibitor of iNOS, was employed as a positive control with the same protocol and showed more than 50% inhibition activity. The results demonstrate that the NO production inhibitory activity of sphondin is due to the effect of iNOS expression, but not by direct inhibition of iNOS enzyme activity. Thus, sphondin may act as a potent inhibitor of NO production under tissue-damaging inflammatory conditions.     

Synthesis of azaisoflavones and their inhibitory activities of NO production in activated microglia

A series of azaisoflavones were synthesized and their biological activities were evaluated for nitric oxide (NO) production and inducible NO synthase (iNOS) expression in BV-2 microglia cell lines. Among these compounds, compound 8d was the most potent with IC₅₀ 7.83 μM for inhibition of NO production. Also, compound 8d inhibited expression of iNOS in LPS-induced BV2 cells. This result suggests that compound 8d inhibited the production of NO by suppressing the expression of iNOS.     

Role of Nitric Oxide in the Progression of Pneumoconiosis

Conflicting evidence has been reported as to whether nitric oxide (NO) possesses anti-inflammatory or inflammatory properties. Data are presented indicating that in vitro or in vivo exposure to selected occupational dusts, i.e., crystalline silica, organic dust contaminated with endotoxin, or asbestos, results in upregulation of inducible nitric oxide synthase (iNOS) and the production of NO by alveolar macrophages and pulmonary epithelial cells. Nitric oxide production is associated temporally and anatomically with pulmonary damage, inflammation, and disease progression in response to occupational dusts. Blockage of inducible nitric oxide synthase by administration of NOS inhibitors or in iNOS knockout mice decreases the magnitude of injury and inflammation following in vivo exposure to silica, endotoxin, or asbestos. Therefore, NO may play an important role in the initiation and progression of pneumoconiosis.     

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.     

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.     

Mesenchymal stem cells: a double-edged sword in regulating immune responses

Mesenchymal stem cells (MSCs) have been employed successfully to treat various immune disorders in animal models and clinical settings. Our previous studies have shown that MSCs can become highly immunosuppressive upon stimulation by inflammatory cytokines, an effect exerted through the concerted action of chemokines and nitric oxide (NO). Here, we show that MSCs can also enhance immune responses. This immune-promoting effect occurred when proinflammatory cytokines were inadequate to elicit sufficient NO production. When inducible nitric oxide synthase (iNOS) production was inhibited or genetically ablated, MSCs strongly enhance T-cell proliferation in vitro and the delayed-type hypersensitivity response in vivo. Furthermore, iNOS(-/-) MSCs significantly inhibited melanoma growth. It is likely that in the absence of NO, chemokines act to promote immune responses. Indeed, in CCR5(-/-)CXCR3(-/-) mice, the immune-promoting effect of iNOS(-/-) MSCs is greatly diminished. Thus, NO acts as a switch in MSC-mediated immunomodulation. More importantly, the dual effect on immune reactions was also observed in human MSCs, in which indoleamine 2,3-dioxygenase (IDO) acts as a switch. This study provides novel information about the pathophysiological roles of MSCs.     

Leishmania donovani: inhibition of phagosomal maturation is rescued by nitric oxide in macrophages

Leishmania donovani promastigotes, the causative agent of visceral leishmaniasis, survive inside macrophages by inhibiting phagosomal maturation. The main surface glycoconjugate on promastigotes, lipophosphoglycan (LPG), is crucial for parasite survival. LPG has several detrimental effects on macrophage function, including inhibition of periphagosomal filamentous actin (F-actin) breakdown during phagosomal maturation. However, in RAW 264.7 macrophages pre-stimulated with lipopolysaccharide (LPS) and interferon gamma (IFNgamma), known to up-regulate inducible nitric oxide synthase (iNOS) and nitric oxide (NO) production, L. donovani promastigotes are unable to inhibit periphagosomal F-actin breakdown and phagosomal maturation proceeds normally. Moreover, the iNOS inhibitor aminoguanidine, blocked the positive effects of LPS/IFNgamma suggesting that NO is a key player in F-actin remodeling. In conclusion, production of NO by stimulated macrophages seems to allow phagosomal maturation following uptake of L. donovani promastigotes, suggesting a novel mechanism whereby NO facilitates killing of an intracellular pathogen.     

Kinase suppressor of Ras-1 protects against pulmonary Pseudomonas aeruginosa infections

Pseudomonas aeruginosa is a Gram-negative pathogen that causes severe infections in immunocompromised individuals and individuals with cystic fibrosis or chronic obstructive pulmonary disease (COPD). Here we show that kinase suppressor of Ras-1 (Ksr1)-deficient mice are highly susceptible to pulmonary P. aeruginosa infection accompanied by uncontrolled pulmonary cytokine release, sepsis and death, whereas wild-type mice clear the infection. Ksr1 recruits and assembles inducible nitric oxide (NO) synthase (iNOS) and heat shock protein-90 (Hsp90) to enhance iNOS activity and to release NO upon infection. Ksr1 deficiency prevents lung alveolar macrophages and neutrophils from activating iNOS, producing NO and killing bacteria. Restoring NO production restores the bactericidal capability of Ksr1-deficient lung alveolar macrophages and neutrophils and rescues Ksr1-deficient mice from P. aeruginosa infection. Our findings suggest that Ksr1 functions as a previously unknown scaffold that enhances iNOS activity and is therefore crucial for the pulmonary response to P. aeruginosa infections.     

Cell-specific inhibition of inducible nitric oxide synthase activation by leflunomide

The influence of a novel immunomodulating drug, leflunomide, on iNOS-dependent nitric oxide (NO) production in rodent macrophages and fibroblasts was investigated. Leflunomide's active metabolite A77 1726 caused a dose-dependent decrease of NO production in IFN-gamma-treated L929 fibroblasts. The observed effect was cell-specific, as well as stimulus-specific, since A77 1726 did not affect NO production in IFN-gamma-stimulated murine peritoneal macrophages or db-cAMP-treated L929 cells. A77 1726 reduced expression of IFN-gamma-induced iNOS and IRF-1 mRNA in L929 cells, while iNOS enzymatic activity remained unchanged. Specific inhibitor of MAP kinase kinase (MEK), PD98059, but not unselective protein kinase inhibitor genistein, completely mimicked cell-type-specific and stimulus-specific NO-inhibitory action of leflunomide. Therefore, the recently described inhibition of MEK/MAP pathway by leflunomide could present a possible mechanism for its suppression of iNOS activation in L929 fibroblasts. Finally, a similar inhibitory effect of A77 1726 on both NO production and iNOS mRNA expression was observed also in IFN-gamma + LPS-activated murine and rat primary fibroblasts.     

Lipid hydroperoxides inhibit nitric oxide production in RAW264.7 macrophages

The effects of oxidatively modified low density lipoprotein (oxLDL) on atherogenesis may be partly mediated by alterations in the production of nitric oxide (NO) by vascular cells. Lipid hydroperoxides (LOOH) and lysophosphatidylcholine (lysoPC) are the major primary products of LDL oxidation. The purpose of this study was to characterize the effects of oxLDL, LOOH and lysoPC on NO production and the expression of inducible nitric oxide synthase (iNOS) gene in lipopolysaccharide (LPS) stimulated macrophages. LDL was oxidized using an azo-initiator 2,2'-azobis (2-amidinopropane) HCl (ABAP) and octadecadienoic acid was oxidized by lipoxygenase to generate 13-hydroperoxyl octadecadienoic acid (13-HPODE). Our study showed that oxLDL markedly decreased the production of NO, the levels of iNOS protein and iNOS mRNA in LPS stimulated macrophages. The inhibition potential of oxLDL on NO production and iNOS gene expression depended on the levels of LOOH formed in oxLDL and was not due to oxLDL cytotoxicity. Furthermore, 13-HPODE markedly reduced NO production and iNOS protein levels, whereas lysoPC showed only slight reduction. The effects of 13-HPODE and lysoPC did not require an acetylated LDL carrier. Our results suggest that 13-HPODE is a much more potent inhibitor of NO production and iNOS gene expression than lysoPC in LPS stimulated RAW264.7 macrophages.     

Synthesis, in vitro anti-inflammatory and cytotoxic evaluation, and mechanism of action studies of 1-benzoyl-β-carboline and 1-benzoyl-3-carboxy-β-carboline derivatives

In the present study, various 1-substituted and 1,3-disubstituted β-carboline derivatives were synthesized by a modified single-step Pictet-Spengler reaction. The compounds were examined for cytotoxicity and anti-inflammatory activity, as measured by the inhibition of prostaglandin E(2) (PGE(2)) production and nitric oxide (NO) production. While only two compounds (28 and 31) showed marginal cytotoxicity against four human cancer cell lines, most of the tested compounds exhibited potent inhibitory activity of both NO and PGE(2) production. Moreover, compounds 6 and 16 significantly reduced the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX2), suggesting that β-carboline analogs can inhibit NO and PGE(2) production at the translational level. In addition, several of the β-carboline derivatives (1, 2, 4-8, 11, 13, 22, 25, 27, 31, and 41-43) displayed significant inhibitory activity of superoxide anion (O(2)(·-)) generation or elastase release compared to the reference compound, with 6 being the most potent. N-Formyl-L-methionyl-phenylalanine (FMLP)-induced phosphorylation of c-JunN-terminal kinase (JNK) and protein kinase B (AKT) were also inhibited by 6, suggesting that it suppresses human neutrophil functions by inhibiting the activation of JNK and AKT signaling pathways. Therefore, the synthetic 1-benzoyl-3-carboxy β-carboline analogs may have great potential to be developed as anti-inflammatory agents.     

Increased nitric oxide production accompanies blunted hypoxic pulmonary vasoconstriction in hyperoxic rat lung

Hyperoxia may affect lung physiology in different ways. We investigated the effect of hyperoxia on the protein expression of endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS), nitric oxide (NO) production, and hypoxic pulmonary vasoconstriction (HPV) in rat lung. Twenty-four male rats were divided into hyperoxic and normoxic groups. Hyperoxic rats were placed in > 90% F1O2 for 60 h prior to experiments. After baseline in vitro analysis, the rats underwent isolated, perfused lung experiments. Two consecutive hypoxic challenges (10 min each) were administered with the administration of a non-specific NOS inhibitor, N-nitro-L-arginine methyl ester (L-NAME), in between. We measured intravascular NO production, pulmonary arterial pressure, and protein expression of eNOS and iNOS by immunohistochemistry. We found that hyperoxia rats exhibited increased baseline NO production (P < 0.001) and blunted HPV response (P < 0.001) during hypoxic challenges compared to normoxia rats. We also detected a temporal association between the attenuation in HPV and increased NO production level with a negative pre-L-NAME correlation between HPV and NO (R = 0.52, P < 0.05). After L-NAME administration, a second hypoxic challenge restored the HPV response in the hyperoxic group. There were increased protein expression of eNOS (12.6 +/- 3.1-fold, n = 3) (X200) and iNOS (8.1 +/- 2.6-fold, n = 3) (X200) in the hyperoxia group. We conclude that hyperoxia increases the protein expression of eNOS and iNOS with a subsequent increased release of endogenous NO, which attenuates the HPV response.     

Nitric oxide produced by iNOS is associated with collagen synthesis in keloid scar formation

Nitric oxide (NO) has emerged as an important mediator of many physiological functions. Recent reports have shown that NO participates in the wound healing process, however, its role in keloid formation remains unclear. This study aimed to investigate the effect of NO on keloid fibroblasts (KF) and to determine the levels of inducible nitric oxide synthase (iNOS) expression in clinical specimens of keloid. Scar tissue from seven keloid patients with matched perilesion skin tissue controls was studied for inducible nitric oxide synthase expression and location. In addition, primary keloid and normal scar skin fibroblast cultures were set up to investigate the effects of NO in inducing collagen type I expression. Inducible nitric oxide synthase expression, and NO production were elevated in keloid scar tissues but not in matched perilesion skin tissues. Furthermore, exposure of KF to exogenous NO resulted in increased expression of collagen type I in a dose-dependent manner. NO exposure also induced time-course dependent collagen I expression that peaked at 24h in KF. Taken together, these results indicate that excess collagen formations in keloid lesion may be attributed to iNOS overexpression.