21-氨基类固醇对脑外伤鼠一氧化氮合酶的影响

目的探讨21-氨基类固醇U-74389G对鼠脑外伤后一氧化氮合酶的影响。方法用一氧化氮合酶(NOS)试剂盒测定中度脑损伤后脑组织及血清中NOS的活性。结果对照组脑组织及血清中NOS活性较假外伤组增高,而治疗组比对照组下降。结论U-74389G能抑制NOS活性,可能是其脑保护作用的机制之一。     

The inducible nitric oxide synthase in vascular and cardiac tissue.

Expression of the inducible form of nitric oxide synthase (iNOS) has been reported in a variety of cardiovascular diseases. The resulting high output nitric oxide (NO) formation, besides the level of iNOS expression, depends also on the expression of the metabolic pathways providing the enzyme with substrate and cofactor. NO may trigger short and long term effects which are either beneficial or deleterious, depending on the molecular targets with which it interacts. These interactions are governed by local factors (like the redox state). In the cardiovascular system, the major targets involve not only guanylyl cyclase, but also other haem proteins, protein thiols, iron-non-haem complexes, and superoxide anion (forming peroxynitrite). The latter has several intracellular targets and may be cytotoxic, despite the existence of endogenous defence mechanisms. These interactions may either trigger NO effects or represent releasable NO stores, able to buffer NO and prolong its effects in blood vessels and in the heart. Besides selectively inhibiting iNOS, a number of other therapeutic strategies are conceivable to alleviate deleterious effects of excessive NO formation, including peroxynitrite (ONOO-) scavenging and inhibition of metabolic pathways triggered by ONOO-. When available, these approaches might have the advantage to preserve beneficial effects of iNOS induction. Counteracting vascular hyper-responsiveness to endogenous vasoconstrictor agonists in septic shock, or inducing cardiac protection against ischaemia-reperfusion injury are examples of such beneficial effects of iNOS induction.     

Adrenocorticotropin reverses vascular dysfunction and protects against splanchnic artery occlusion shock.

1. Tumour necrosis factor (TNF-alpha) is involved in the pathogenesis of splanchnic artery occlusion (SAO) shock. On the other hand, inhibition of TNF-alpha is an important component of the mechanism of action of melanocortins in reversing haemorrhagic shock. We therefore investigated the effects of the melanocortin peptide ACTH-(1 - 24) (adrenocorticotropin fragment 1 - 24) on the vascular failure induced by SAO shock. 2. SAO-shocked rats had a decreased survival rate (0% at 4 h of reperfusion, while sham-shocked rats survived for more than 4 h), enhanced serum TNF-alpha concentrations (755+/-81 U ml-1), decreased mean arterial blood pressure, leukopenia, and increased ileal leukocyte accumulation, as revealed by means of myeloperoxidase activity (MPO=9.4+/-1 U g-1 tissue). Moreover, aortic rings from shocked rats showed a marked hyporeactivity to phenylephrine (PE, 1 nM - 10 microM) (Emax and ED50 in shocked rats=7.16 mN mg-1 tissue and 120 nM, respectively; Emax and ED50 in sham-shocked rats=16.31 mN mg-1 tissue and 100 nM, respectively), reduced responsiveness to acetylcholine (ACh, 10 nM-10 microM) (Emax and ED50 in shocked rats=30% relaxation and 520 nM, respectively; Emax and ED50 in sham-shocked rats=82% relaxation and 510 nM, respectively) and increased staining for intercellular adhesion molecule-1 (ICAM-1). 3. ACTH-(1 - 24) [160 microg kg-1 intravenously (i.v.), 5 min after SAO] increased survival rate [SAO+ACTH-(1 - 24)=80% at 4 h of reperfusion], reversed hypotension, reduced serum TNF-alpha (55+/-13 U ml-1), ameliorated leukopenia, reduced ileal MPO (1.2+/-0.2 U g-1 tissue), restored the reactivity to PE, improved the responsiveness to ACh and blunted the enhanced immunostaining for ICAM-1 in the aorta. 4. Adrenalectomy only in part - but not significantly - reduced the ACTH-induced shock reversal, the survival rate of SAO+ACTH-(1 - 24) adrenalectomized rats being 60% at 4 h of reperfusion; and methylprednisolone (80 mg-1 i.v., 5 min after SAO) had a non-significant effect (10% survival) at 4 h of reperfusion. 5. The present data show that melanocortins are effective also in SAO shock, their effect being, at least in part, mediated by reduced production of TNF-alpha. Furthermore, they demonstrate, for the first time, that this inhibition is responsible for the adrenocorticotropin-induced reversal of vascular failure and leukocyte accumulation.     

蛋白激酶C对内毒素休克大鼠肝损伤诱导型一氧化氮合成酶表达的调控

目的　探讨蛋白激酶C(PKC)对内毒素休克大鼠肝损伤诱导型一氧化氮合成酶 (iNOS)表达的调控。方法　将大鼠随机分为 6组 ,分别为正常对照组、脂多糖 (LPS)组、LPS +琼脂糖 (AG) (10 0mg/kg)组、LPS +H7小剂量 (12 .5mg/kg)组、LPS +1(5 异喹啉磺酰基 ) 2 甲基哌嗪 (H7)大剂量 (2 5mg/kg)组、LPS +H7+佛波醇乙酯 (PMA) (3mg/kg)组。注射药物后观察其血压及血清学、苏木素 伊红 (HE)染色、免疫组织化学染色、iNOS蛋白活性检测、WesternBlotting免疫印迹法检测iNOS等指标。 结果　①血压及血清学 :AG升压迅速且持续 ;H7也有升压作用。②组织学 :LPS可致肝组织呈弥漫性病变 ,H7能改善LPS所致的肝损伤。③iNOS免疫组织化学 :正常组iNOS染色阴性 ,LPS组iNOS强阳性表达 ;H7能抑制iNOS表达 ,但不如AG明显。④iNOS蛋白活性检测 :LPS可致iNOS活性显著增强 (P <0 .0 1) ,H7可抑制iNOS ,与剂量相关 ,并可被PMA逆转 ,但AG抑制作用最强。⑤iNOSWesternBlotting免疫印迹观察 :正常组iNOS条带缺如 ,H7对iNOS表达有抑制作用 ,而AG组对iNOS抑制作用最强。结论　PKC可能参与内毒素诱导的iNOS表达 ,但不同于AG的是 ,H7能改善肝功能。故抑制PKC活性可更广泛地减轻内毒素条件下炎症所致的肝损伤 ,从而对内毒素所致的脏器衰?     

Equol inhibits nitric oxide production and inducible nitric oxide synthase gene expression through down-regulating the activation of Akt

In the present study, we report the inhibitory effect of equol on nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) gene expression in murine macrophages. In vivo administration of equol (i.p.) attenuated NO production by peritoneal adherent cells isolated from lipopolysaccharide (LPS)-treated mice. Equol dose-dependently inhibited the LPS-induced production of NO in isolated peritoneal adherent cells and RAW 264.7 cells. The mRNA expression of iNOS was also blocked by equol in LPS-stimulated RAW 264.7 cells. Further study demonstrated that the LPS-induced activation of Akt was suppressed by equol in RAW 264.7 cells while the activation of ERK, SAPK/JNK and p38 MAP kinase was not affected. Equol also blocked LPS-induced NF-kappaB activation. Moreover, the LPS-induced NO production and NF-kappaB activation was inhibited by LY294002, a specific inhibitor of phosphatidylinositol 3-kinase/Akt pathway, in RAW 264.7 cells. These results suggest that equol might inhibit NO production and iNOS gene expression, at least in part, by blocking Akt activation and subsequent down-regulation of NF-kappaB activity.     

The induction of nitric oxide synthase and intestinal vascular permeability by endotoxin in the rat.

1. The effect of endotoxin (E. coli lipopolysaccharide) on the induction of nitric oxide synthase (NOS) and the changes in vascular permeability in the colon and jejunum over a 5 h period have been investigated in the rat. 2. Under resting conditions, a calcium-dependent constitutive NOS, determined by the conversion of radiolabelled L-arginine to citrulline, was detected in homogenates of both colonic and jejunal tissue. 3. Administration of endotoxin (3 mg kg-1, i.v.) led, after a 2 h lag period, to the appearance of calcium-independent NOS activity in the colon and jejunum ex vivo, characteristic of the inducible NOS enzyme. 4. Administration of endotoxin led to an increase in colonic and jejunal vascular permeability after a lag period of 3 h, determined by the leakage of radiolabelled albumin. 5. Pretreatment with dexamethasone (1 mg kg-1 s.c., 2 h prior to challenge) inhibited both the induction of NOS and the vascular leakage induced by endotoxin. 6. Administration of the NO synthase inhibitor NG-monomethyl-L-arginine (12.5-50 mg kg-1, s.c.) 3 h after endotoxin injection, dose-dependently reduced the subsequent increase in vascular permeability in jejunum and colon, an effect reversed by L-arginine (300 mg kg-1, s.c.). 7. These findings suggest that induction of NOS is associated with the vascular injury induced by endotoxin in the rat colon and jejunum.     

Platelet activation, oxidative stress and overexpression of inducible nitric oxide synthase in moderate heart failure

1. Chronic heart failure (CHF) is a common disabling disorder associated with thromboembolic events, the genesis of which is not yet fully understood. Nitric oxide (NO), derived from the vascular endothelium and platelets, has an important role in the physiological regulation of blood flow. It is generated from the amino acid L-arginine via NO synthase (NOS). 2. The main objective of the present study was to investigate NO production and its relationship with platelet aggregation, oxidative stress, inflammation and related amino acids in patients with moderate CHF. The expression and activity of NOS isoforms were analysed by western blotting and conversion of L-[(3)H]-arginine to L-[(3)H]-citrulline, respectively, in CHF patients (n = 12) and healthy controls (n = 15). Collagen- and ADP-induced platelet aggregation, oxidative stress (thiobarbituric acid-reactive substances (TBARS) formation and superoxide dismutase (SOD) activity) and plasma levels of amino acids and inflammatory markers (fibrinogen and C-reactive protein (CRP)) were also determined. 3. Both collagen- and ADP-induced platelet aggregation were increased in CHF patients compared with controls. Platelets from CHF patients did not show any changes in NOS activity in the presence of overexpression of inducible NOS. Systemic and intraplatelet TBARS production was elevated, whereas SOD activity was decreased in CHF patients. l-arginine plasma concentrations were lower in CHF patients than in controls. Systemic levels of CRP and fibrinogen were increased in CHF patients. 4. The results show that, in patients with moderate CHF, there is platelet activation and reduced intraplatelet NO bioavailability due to oxidative stress, which suggests a role for platelets in the prothrombotic state.     

Rengyolone inhibits inducible nitric oxide synthase expression and nitric oxide production by down-regulation of NF-kappaB and p38 MAP kinase activity in LPS-stimulated RAW 264.7 cells

Nitric oxide (NO) is recognized as a mediator and regulator of inflammatory responses. Rengyolone, a cyclohexylethanoid isolated from the fruits of Forsythia koreana, exhibits anti-inflammatory activity with unknown mechanism. In this study, we found that rengyolone has a strong inhibitory effect on the production of nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha). Rengyolone also inhibited inducible nitric oxide synthase (iNOS) gene expression and cyclooxygenase 2 (COX-2) by lipopolysaccharide (LPS). In order to explore the mechanism responsible for the inhibition of iNOS gene expression by rengyolone, we investigated its effect on LPS-induced nuclear factor-kappaB (NF-kappaB) activation. The LPS-induced DNA binding activity of NF-kappaB was significantly inhibited by rengyolone, and this effect was mediated through inhibition of the degradation of inhibitory factor-kappaBalpha and phosphorylation of p38 MAP kinase. Furthermore, rengyolone suppressed the expression of ICE protein in IL-1beta-treated D10S cells. Taken together, these results suggest that rengyolone attenuates the inflammation through inhibition of NO production and iNOS expression by blockade of NF-kappaB and p38 MAPK activation in LPS-stimulated RAW 264.7 cells.     

A sesquiterpene, dehydrocostus lactone, inhibits the expression of inducible nitric oxide synthase and TNF-alpha in LPS-activated macrophages

Nitric oxide (NO) and tumor necrosis factor alpha (TNF-alpha) are the major mediators produced in activated macrophages which contribute to the circulatory failure associated with septic shock. A sesquiterpene lactone compound (dehydrocostus lactone) isolated from the medicinal plant, Saussurea lappa, inhibited the production of NO in lipopolysaccharide (LPS)-activated RAW 264.7 cells by suppressing inducible nitric oxide synthase enzyme expression. This compound also decreased the TNF-alpha level in LPS-activated systems in vitro and in vivo. Thus, dehydrocostus lactone may be a possible candidate for the development of new drugs to treat endotoxemia accompanied by the overproduction of NO and TNF-alpha.     

Dexamethasone inhibits inducible nitric-oxide synthase expression and nitric oxide production by destabilizing mRNA in lipopolysaccharide-treated macrophages

Nitric oxide (NO) production through the inducible nitric-oxide synthase (iNOS) pathway is increased in inflammatory diseases and leads to cellular injury. Anti-inflammatory steroids inhibit the expression of various inflammatory genes, including iNOS. In the present study, we investigated the mechanism how dexamethasone decreased NO production in murine J774 macrophages. Dexamethasone (0.1-10 microM) inhibited the production of NO and iNOS protein in a dose-dependent manner in cells stimulated with lipopolysaccharides (LPS). In contrast, in cells treated with a combination of LPS and interferon-gamma (IFN-gamma), dexamethasone did not reduce iNOS expression and NO formation. Dissociated glucocorticoid RU24858 inhibited iNOS expression and NO production to levels comparable with that of dexamethasone, suggesting that the reduced iNOS expression by dexamethasone is not a GRE-mediated event. In further studies, the effect of dexamethasone on iNOS mRNA levels was tested by actinomycin assay. The half-life of iNOS mRNA after LPS treatment was 5 h 40 min, and dexamethasone reduced it to 3 h. The increased degradation of iNOS mRNA was reversed by a protein synthesis inhibitor cycloheximide. iNOS mRNA was more stabile in cells treated with a combination of LPS plus IFN-gamma (half-life = 8 h 20 min), and dexamethasone had a minor effect in these conditions. In conclusion, dexamethasone decreases iNOS-dependent NO production by destabilizing iNOS mRNA in LPS-treated cells by a mechanism that requires de novo protein synthesis. Also, decreased iNOS mRNA and protein expression and NO formation by dexamethasone was not found in cells treated with a combination of LPS plus IFN-gamma, suggesting that the effect of dexamethasone is stimulus-dependent.     

Ethanol consumption increases the expression of endothelial nitric oxide synthase, inducible nitric oxide synthase and metalloproteinases in the rat kidney

Objectives The effects of longterm ethanol consumption on the levels of nitric oxide (NO) and the expression of endothelial NO synthase (eNOS), inducible NO synthase (iNOS) and metalloproteinase‐2 (MMP‐2) were studied in rat kidney. Methods Male Wistar rats were treated with 20% ethanol (v/v) for 6 weeks. Nitrite and nitrate generation was measured by chemiluminescence. Protein and mRNA levels of eNOS and iNOS were assessed by immunohistochemistry and quantitative real‐time polymerase chain reaction, respectively. MMP‐2 activity was determined by gelatin zymography. Histopathological changes in kidneys and indices of renal function (creatinine and urea) and tissue injury (mitochondrial respiration) were also investigated. Results Chronic ethanol consumption did not alter malondialdehyde levels in the kidney. Ethanol consumption induced a significant increase in renal nitrite and nitrate levels. Treatment with ethanol increased mRNA expression of both eNOS and iNOS. Immunohistochemical assays showed increased immunostaining for eNOS and iNOS after treatment with ethanol. Kidneys from ethanol‐treated rats showed increased activity of MMP‐2. Histopathological investigation of kidneys from ethanol‐treated animals revealed tubular necrosis. Indices of renal function and tissue injury were not altered in ethanol‐treated rats. Conclusions Ethanol consumption increased renal metalloproteinase expression/activity, which was accompanied by histopathological changes in the kidney and elevated NO generation. Since iNOS‐derived NO and MMPs contribute to progressive renal injury, the increased levels of NO and MMPs observed in ethanol‐treated rats might contribute to progressive renal damage.     

高糖抑制牛主动脉内皮细胞诱导型和结构型一氧化氮合酶的表达

目的:研究高糖对新生小牛主动脉内皮细胞(BAEC)一氧化氮合酶(NOS)表达的影响。方法:BAEC培养并传代于含正常葡萄糖(5.5mmol·L~(-1),NGBAEC),高糖(25 mmol·L~(-1),HG-BAEC)或高渗(葡萄糖5.5 甘露醇19.5 mmol·L~(-1),Mann-BAEC)的无酚红M1640培养基.Griess反应检测脂多糖(LPS)诱导的一氧化氮(NO)产生.Westem blot法检测结构型NOS(ecNOS)及诱导型NOS(iNOS)表达。结果:LPS(0.25-2 mg·L~(-1))剂量依赖性刺激BAEC产生NO,并在LPS 1 mg·L~(-1)达峰值。高糖显著抑制LPS诱导的NO产生(亚硝酸μmol·L~(-1):HG-BAEC 43±8,vs NG-BAEC 71±11,Mann-BAEC 70±9,n=4,P<0.01)。同样,与NG-和Mann-BAEC相比,HG-BAEC iNOS表达下降39.9％和39.3％,ecNOS表达下降28％和24％,而NG-与Mann-BAEC之间,LPS诱导的NO产量和iNOS和ecNOS的表达无差别。结论:高糖抑制BAEC NO的释放,与NOS的低表达有关。     

NO-donating aspirin inhibits both the expression and catalytic activity of inducible nitric oxide synthase in HT-29 human colon cancer cells

Nitric oxide-releasing aspirin (NO-ASA) is emerging as a potentially important chemopreventive agent against colon cancer. We examined in HT-29 human colon adenocarcinoma cells the effect of NO-ASA on the inducible form of nitric oxide synthase (NOS2), an enzyme implicated in colon carcinogenesis. NO-ASA inhibited in a time- and concentration-dependent manner the expression of NOS2 up to 70% compared to control (IC50 for this effect = 46 microM). NO-ASA also decreased the corresponding steady-state mRNA levels and this reduction preceded the reduction of protein levels by at least 6 h. NO-ASA also reduced the enzymatic activity of NOS2, as determined by a direct enzyme assay (maximal reduction = 80%) and by determining the accumulation of NO in the culture medium (IC50 for this effect = 36 microM). These effects of NO-ASA on NOS2 were paralleled by inhibition in cell growth (IC50 = 8.5 microM). These findings indicate that NO-ASA profoundly inhibits both the expression and enzymatic activity of NOS2 and suggest that these effects may represent an important mechanism for the colon cancer chemopreventive effect of NO-ASA.     

Design, synthesis, and activity of 2-imidazol-1-ylpyrimidine derived inducible nitric oxide synthase dimerization inhibitors

By the screening of a combinatorial library for inhibitors of nitric oxide (NO) formation by the inducible isoform of nitric oxide synthase (iNOS) using a whole-cell assay, 2-(imidazol-1-yl)pyrimidines were identified. Compounds were found to inhibit the dimerization of iNOS monomers, thus preventing the formation of the dimeric, active form of the enzyme. Optimization led to the selection of the potent, selective, and orally available iNOS dimerization inhibitor, 21b, which significantly ameliorated adjuvant-induced arthritis in a rat model. Analysis of the crystal structure of the 21b--iNOS monomer complex provided a rationalization for both the SAR and the mechanism by which 21b blocks the formation of the protein--protein interaction present in the dimeric form of iNOS.     

Down-regulation of phenobarbital-induced cytochrome P4502B mRNAs and proteins by endotoxin in mice: independence from nitric oxide production by inducible nitric oxide synthase

Multiple hepatic cytochrome P450 enzymes are down-regulated at the mRNA and protein levels during inflammation and infection. A body of evidence suggests that nitric oxide (NO) produced from inducible NO synthase (NOS2) is responsible for some of these effects. The current study was designed to examine the NO dependencies of the down-regulation of phenobarbital-induced CYP2B mRNAs and proteins by bacterial endotoxin (lipopolysaccharide, LPS) treatment in vivo, using an NOS2-null mouse model. Treatment of C57/BL6 mice with 0.3 mg/kg of LPS maximally suppressed phenobarbital-induced CYP2B9 and 2B10 mRNAs measured 12 hr after injection, whereas 1-10 mg/kg of LPS was required to elevate NO production. Down-regulation of CYP2B mRNAs by 1 mg/kg of LPS was equivalent in wild-type and NOS2-null mice. No effect of LPS in the dose range of 0.3 to 10 mg/kg was observed on microsomal CYP2B protein levels measured 12 hr after treatment, whereas 1 mg/kg of LPS suppressed CYP2B proteins 24 hr after treatment in both wild-type and NOS2-null mice. We conclude that the main mechanism for the down-regulation of CYP2B proteins in mouse liver following moderate- or high-dose LPS treatment is via NO-independent suppression of CYP2B9 and 2B10 mRNAs. Unlike rat hepatocytes, the contribution of a rapid, NO-dependent mechanism of CYP2B protein suppression in mouse liver appears to be minor or non-existent.     

Wogonin, baicalin, and baicalein inhibition of inducible nitric oxide synthase and cyclooxygenase-2 gene expressions induced by nitric oxide synthase inhibitors and lipopolysaccharide

We previously reported that oroxylin A, a polyphenolic compound, was a potent inhibitor of lipopolysaccharide (LPS)-induced expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). In the present study, three oroxylin A structurally related polyphenols isolated from the Chinese herb Huang Qui, namely baicalin, baicalein, and wogonin, were examined for their effects on LPS-induced nitric oxide (NO) production and iNOS and COX-2 gene expressions in RAW 264.7 macrophages. The results indicated that these three polyphenolic compounds inhibited LPS-induced NO production in a concentration-dependent manner without a notable cytotoxic effect on these cells. The decrease in NO production was in parallel with the inhibition by these polyphenolic compounds of LPS-induced iNOS gene expression. However, these three compounds did not directly affect iNOS enzyme activity. In addition, wogonin, but not baicalin or baicalein, inhibited LPS-induced prostaglandin E2 (PGE2) production and COX-2 gene expression without affecting COX-2 enzyme activity. Furthermore, N-nitro-L-arginine (NLA) and N-nitro-L-arginine methyl ester (L-NAME) pretreatment enhanced LPS-induced iNOS (but not COX-2) protein expression, which was inhibited by these three polyphenolic compounds. Wogonin, but not baicalin or baicalein, similarly inhibited PGE2 production and COX-2 protein expression in NLA/LPS or L-NAME/LPS-co-treated RAW 264.7 cells. These results indicated that co-treatment with NOS inhibitors and polyphenolic compounds such as wogonin effectively blocks acute production of NO and, at the same time, inhibits expression of iNOS and COX-2 genes.     

2,4,6-Trinitrotoluene inhibits endothelial nitric oxide synthase activity and elevates blood pressure in rats

2,4,6-Trinitrotoluene (TNT), which is widely used in explosives, is an important occupational and environmental pollutant. Human exposure to TNT has been reported to be associated with cardiovascular dysfunction, but the mechanism is not well understood. In this study, we examine the endothelial nitric oxide synthase (eNOS) activity and blood pressure value following TNT exposure. With a crude enzyme preparation, we found that TNT inhibited the enzyme activity of eNOS in a concentration-dependent manner (IC₅₀ value=49.4 μM). With an intraperitoneal administration of TNT (10 and 30 mg/kg) to rats, systolic blood pressure was significantly elevated 1 h after TNT exposure (1.2- and 1.3-fold of that of the control, respectively). Under the conditions, however, experiments with the inducible NOS inhibitor aminoguanidine revealed that an adaptive response against hypertension caused by TNT occurs. These results suggest that TNT is an environmental chemical that acts as an uncoupler of constitutive NOS isozymes, resulting in decreased nitric oxide formation associated with hypertension in rats.     

Effect of anti-fungal imidazoles on mRNA levels and enzyme activity of inducible nitric oxide synthase.

1. Experiments were performed to examine the effects of anti-fungal imidazole compounds (clotrimazole, econazole and miconazole) on the induction of nitric oxide (NO) synthase and subsequent production of NO in the cultured murine monocyte/macrophage cell line J774 using a specific cDNA probe for inducible NO synthase mRNA and by monitoring nitrite production. 2. Stimulation of J774 cells with lipopolysaccharide (LPS, 10 micrograms ml-1) resulted in the induction of NO synthase activity as determined by nitrite accumulation in the culture medium (48 +/- 3 nmol per 10(6) cells over 24 h). Production of nitrite was inhibited by co-incubation of cells with LPS (10 micrograms ml-1) and either dexamethasone (10 microM) or NG-monomethyl-L-arginine (L-NMMA; 0.1 mM), however, only L-NMMA was an effective inhibitor of nitrite production when added after induction of NO synthase had occurred. 3. Co-incubation of J774 cells with LPS (10 micrograms ml-1) and either clotrimazole, econazole or miconazole (1-10 microM) resulted in a concentration-dependent inhibition of nitrite production over the subsequent 24 h without any evidence for a cytotoxic effect. However, addition of these imidazoles after induction of NO synthase did not inhibit nitrite production. 4. Messenger RNA for inducible NO synthase was not detected in unstimulated J774 cells. Treatment with LPS (10 micrograms ml-1) for 4 h resulted in significant expression of mRNA for inducible NO synthase which was not altered in the presence of econazole (10 microM) but was reduced significantly by dexamethasone (10 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Nitric oxide inhalation inhibits inducible nitric oxide synthase but not nitrotyrosine formation and cell apoptosis in rat lungs with meconium-induced injury

AIM: To investigate the effects of inhaled nitric oxide (NO) on pulmonary inflammation, apoptosis, peroxidation and protein nitration in a rat model of acute lung injury (ALI) induced by meconium. METHODS: Twenty-four healthy male Sprague-Dawley rats were randomly devided into 3 groups (n=8): meconium-induced ALI with intratracheal instillation of 1 mL/kg saline (Mec/saline group), continuous inhalation of NO at 20 muL/L. (Mec/iNO), and the control group (control). Electromicroscopic examination was used to determine the extent of epithelial apoptosis. TUNEL was used to detect DNA fragmentation in pulmonary apoptotic cells, expressed as the apoptosis index (AI). Western blotting was used to detect pulmonary inducible NO synthase (iNOS) expression. RT-PCR was used to detect interleukin (IL)-1beta mRNA expression. Cell count in bronchoalveolar lavage (BAL), myeloperoxidase (MPO) activity, as well as malondialdehyde (MDA) and nitrotyrosine formation, the markers of toxic NO-superoxide pathway in rat lung parenchyma specimens, were also examined. RESULTS: Expression of iNOS protein and IL-1beta mRNA were increased significantly in the Mec/saline group (both P<0.01) compared with the control group. BAL cell count, MPO activity, lung injury score, pulmonary AI, MDA level and nitrotyrosine formation were also increased significantly (all P<0.01). The meconium-induced iNOS protein and IL-1beta mRNA expression were inhibited significantly by NO inhalation when compared with the Mec/saline group (both P<0.01). BAL cell count, MPO activity and lung injury score were also decreased significantly (P<0.01 or P<0.05). However, there were no statistical differences in MDA level, nitrotyrosine formation or pulmonary AI between the Mec/saline and Mec/iNO groups. Electromicroscopic examination revealed a significant degree of epithelial apoptosis in both the Mec/saline and Mec/iNO groups. CONCLUSIONS: Early continuous inhalation of NO 20 muL/L may protect the lungs from inflammatory injury, but does not decrease epithelial apoptosis or lung nitrotyrosine formation. Inhalation of NO alone is not associated with a detectable increase in oxidant stress.