The lethal effects of cytokine-induced nitric oxide on cardiac myocytes are blocked by nitric oxide synthase antagonism or transforming growth factor beta.

Inducible nitric oxide (NO) produced by macrophages is cytotoxic to invading organisms and has an important role in host defense. Recent studies have demonstrated inducible NO production within the heart, and that cytokine-induced NO mediates alterations in cardiac contractility, but the cytotoxic potential of nitric oxide with respect to the heart has not been defined. To evaluate the role of inducible nitric oxide synthase (iNOS) on cardiac myocyte cytotoxicity, we exposed adult rat cardiac myocytes to either cytokines alone or to activated J774 macrophages in coculture. Increased expression of both iNOS message and protein was seen in J774 macrophages treated with IFN gamma and LPS and cardiac myocytes treated with TNF-alpha, IL-1 beta, and IFN gamma. Increased NO synthesis was confirmed in both the coculture and isolated myocyte preparations by increased nitrite production. Increased NO synthesis was associated with a parallel increase in myocyte death as measured by CPK release into the culture medium as well as by loss of membrane integrity, visualized by trypan blue staining. Addition of the competitive NO synthase inhibitor L-NMMA to the culture medium prevented both the increased nitrite production and the cytotoxicity observed after cytokine treatment in both the isolated myocyte and the coculture experiments. Because transforming growth-factor beta modulates iNOS expression in other cell types, we evaluated its effects on cardiac myocyte iNOS expression and NO-mediated myocyte cytotoxicity. TGF-beta reduced expression of cardiac myocyte iNOS message and protein, reduced nitrite production, and reduced NO-mediated cytotoxicity in parallel. Taken together, these experiments show the cytotoxic potential of endogenous NO production within the heart, and suggest a role for TGF-beta or NO synthase antagonists to mute these lethal effects. These findings may help explain the cardiac response to sepsis or allograft rejection, as well as the progression of dilated cardiomyopathies of diverse etiologies.     

八肽缩胆囊素对脂多糖诱导血管内皮细胞诱生型一氧化氮合酶表达变化的抑制作用

目的探讨八肽缩胆囊素（CCK-8）对脂多糖（LPS）诱导血管内皮细胞诱生型一氧化氯合酶（iNOS）表达变化的影响。方法培养人脐静脉内皮细胞株ECV-304细胞。用0．01、0．1和1mg／L LPS处理2～24h，用生理盐水、10mol／LCCK-8和0．1mg／L LPS＋10^-8、10^-7、10^-8mol／L CCK-8处理16h；用比色法检测培养液中一氧化氮（NO）含量、细胞NOS活性，免疫细胞化学及蛋白质免疫印迹法检测iNOS蛋白表达。结果与生理盐水处理的对照组比较，LPS诱导培养液NO含量增多、细胞NOS活性增高、iNOS蛋白表达上调；CCK-8剂量依赣性抑制LPS的上述效应。而单独作用对iNOS蛋白表达、NOS活性和NO含量均无明显影响。结论CCK-8可以明显抑制LPS引起ECV-304细胞iNOS蛋白表达上调。减少NO生成。     

一氧化氮合成酶在人肾小球系膜细胞中表达的研究

目的探讨肾小球系膜细胞 (MC)诱生型一氧化氮合成酶 (iNOS)的表达。方法采用 4个月龄水囊引产的胎儿 ,取胎肾 ,用不锈钢网筛分离技术 ,培养原代系膜细胞 ,用胰酶传代 ,取Ⅲ代细胞用于实验。设实验组和对照组 ,观察脂多糖 (LPS)刺激后不同时间 (4、6、2 4、4 8小时 )及一氧化氮合成酶抑制剂 (L NAME)刺激后同一时间 (2 4小时 )对诱生型一氧化氮合成酶表达的影响。结果细胞为梭形、星形或树枝状 ,核居中央 ,呈圆形或椭圆形 ;免疫荧光结果为抗结蛋白抗体、抗Ⅳ型胶原、抗纤维连接蛋白 (FN)、抗层粘连蛋白 (LN)阳性 ;抗角蛋白、抗Ⅷ因子抗体阴性 ;在未刺激的MC中无iNOSmRNA的表达 ;LPS刺激后 4小时 ,即可检测到iNOS ;6小时后开始明显增加 ;2 4小时达到高峰 ;至 4 8小时iNOS仍较高 ;L NAME为iNOS特异性的抑制剂。结论肾小球MC确含有iNOS ,且在生理状态下呈稳定状态 ,病理状态下 (如LPS刺激后 )表达上调。     

Inhibition of extracellular signal-regulated kinase suppresses endotoxin-induced nitric oxide synthesis in mouse macrophages and in human colon epithelial cells

Macrophages produce large amounts of nitric oxide (NO) in response to proinflammatory cytokines and lipopolysaccharide (LPS) by expressing inducible isoform of NO synthase (iNOS). We examined the role of extracellular signal-regulated kinase p42/44(MAPK) (Erk1/2) in signal transduction pathways leading to induction of NO synthesis in response to LPS in J774 mouse macrophages and T-84 human colon epithelial cells. LPS activated Erk1/2 and induced iNOS and subsequent NO production. Erk1/2 activation was inhibited by PD 98059, a specific inhibitor of mitogen-activated protein kinase kinase (Mek) that is an upstream activator of Erk1/2. At corresponding concentrations PD 98059 reduced LPS-induced NO formation by 40 to 50% by inhibiting iNOS expression in J774 and T-84 cells. Inhibition of iNOS expression was not mediated by nuclear factor-kappaB because PD 98059 had no effect on nuclear factor-kappaB activity in J774 macrophages. In addition, PD 98059 reduced LPS-induced L-arginine transport into the cells as measured in J774 macrophages, whereas the availability of tetrahydrobiopterin was not a limiting factor in NO production after PD 98059. Our results indicate that Erk1/2 activation mediates up-regulation but is not essential for LPS-induced iNOS expression.     

Aspirin inhibits inducible nitric oxide synthase expression and tumour necrosis factor-alpha release by cultured smooth muscle cells

BACKGROUND: Inflammatory related cardiovascular disease, i.e. cardiac allograft rejection, myocarditis, septic shock, are accompanied by cytokine production, which stimulates the expression of inducible nitric oxide (iNOS). MATERIALS AND METHODS: The aim of the present study was to examine whether anti-inflammatory doses of acetylsalicylic acid (aspirin) could regulate iNOS protein expression in bovine vascular smooth muscle cells (BVSMCs) in culture. RESULTS: Interleukin 1 beta (IL-1 beta, 0.03 U mL-1) induced nitric oxide release by BVSMCs. Aspirin inhibited nitric oxide release from IL-1 beta-stimulated BVSMCs in a dose-dependent manner. In addition, aspirin significantly inhibited iNOS protein expression in BVSMCs and reduced the translocation of the nuclear factor-kappa B (NF-kappa B). Furthermore, aspirin and the blockade of NO generation by BVSMCs reduced the production of tumour necrosis factor alpha (TNF-alpha) by these cells. CONCLUSION: High doses of aspirin inhibited iNOS protein expression in BVSMCs and decreased NF-kappa B mobilization. The inhibition of iNOS expression by aspirin was further associated with a reduced ability of BVSMCs to produce TNF-alpha. This study could provide new mechanisms of action for aspirin in the treatment of the inflammation-related cardiovascular diseases.     

Dual inhibition of nitric oxide and prostaglandin production contributes to the antiinflammatory properties of nitric oxide synthase inhibitors.

We have recently put forward the hypothesis that the dual inhibition of proinflammatory nitric oxide (NO) and prostaglandins (PG) may contribute to the antiinflammatory properties of nitric oxide synthase (NOS) inhibitors. This hypothesis was tested in the present study. A rapid inflammatory response characterized by edema, high levels of nitrites (NO2-, a breakdown product of NO), PG, and cellular infiltration into a fluid exudate was induced by the administration of carrageenan into the subcutaneous rat air pouch. The time course of the induction of inducible nitric oxide synthase (iNOS) protein in the pouch tissue was found to coincide with the production of NO2-. Dexamethasone inhibited both iNOS protein expression and NO2- synthesis in the fluid exudate (IC50 = 0.16 mg/kg). Oral administration of N-iminoethyl-L-lysine (L-NIL) or NG-nitro-L-arginine methyl ester (NO2Arg) not only blocked nitrite accumulation in the pouch fluid in a dose-dependent fashion but also attenuated the elevated release of PG. Finally, carrageenan administration produced a time-dependent increase in cellular infiltration into the pouch exudate that was inhibited by dexamethasone and NOS inhibitors. At early times, i.e., 6 h, the cellular infiltrate is composed primarily of neutrophils (98%). Pretreatment with colchicine reduced both neutrophil infiltration and leukotriene B4 accumulation in the air pouch by 98% but did not affect either NO2- or PG levels. In conclusion, the major findings of this paper are that (a) selective inhibitors of iNOS are clearly antiinflammatory agents by inhibiting not only NO but also PG and cellular infiltration and (b) that neutrophils are not responsible for high levels of NO and PG produced.     

1-alpha,25-Dihydroxyvitamin D3 regulates inducible nitric oxide synthase messenger RNA expression and nitric oxide release in macrophage-like RAW 264.7 cells

The expression of inducible nitric oxide synthase (iNOS) expression and release of nitric oxide (NO) from macrophages are markedly increased in granulomatous infections. Activation of macrophages 1alpha-hydroxylase results in an increase of 1alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)]. However, the significance of this increased production is not completely understood. In this study, we analyzed 1,25(OH)(2)D(3) and NO production in patients with tuberculosis infection and hypercalcemia and used lipopolysaccharide (LPS) to stimulate RAW 264.7 cells in an attempt to assess iNOS expression and gaseous NO production regulated by 1,25(OH)(2)D(3). Peroxynitrite (OONO(-)) production and lactate dehydrogenase activity were also examined. Without additional stimulation, peripheral-blood mononuclear cells (PBMCs) from patients with tuberculosis converted more 25-hydroxyvitamin D(3) to 1,25(OH)(2)D(3) than did those from normal controls. These PBMCs released less NO than did those from control subjects, at baseline and in the stimulated state. We found that 1,25(OH)(2)D(3) dose-dependently inhibited iNOS messenger RNA expression of the LPS-stimulated RAW 264.7 cells and also significantly reduced the gaseous NO release and OONO(-) production. Paralleling the 1,25(OH)(2)D(3)-induced inhibition of NO release were reductions in OONO(-) and LDH production. In conclusion, 1,25(OH)(2)D(3) inhibited iNOS expression and reduced NO production by LPS-stimulated macrophages in the range of physiological doses. Inhibition of the NO surge was coupled with a reduction in OONO(-) and LDH production. Increased 1,25(OH)(2)D(3) production and decreased release of NO from the PBMCs of patients with tuberculosis and hypercalcemia were also noted. We propose that 1,25(OH)(2)D(3) production by macrophages may protect themselves against oxidative injuries caused by the NO burst. In the case of tuberculosis infection, increased 1,25(OH)(2)D(3) synthesis may further contribute to the development of an unwanted phenomenon-hypercalcemia.     

Spontaneously increased production of nitric oxide and aberrant expression of the inducible nitric oxide synthase in vivo in the transforming growth factor beta 1 null mouse

Transforming growth factor beta 1 null mice (TGF-beta 1-/-) suffer from multifocal inflammation and die by 3-4 wk of age. In these mice, levels of nitric oxide (NO) reaction products in serum are elevated approximately fourfold over levels in controls, peaking at 15-17 d of life. Shortterm treatment of TGF-beta 1-/- mice with NG-monomethyl-L- arginine suppressed this elevated production of NO. Expression of inducible NO synthase (iNOS) mRNA and protein is increased in the kidney and heart of TGF-beta 1-/- mice. These findings demonstrate that TGF-beta 1 negatively regulates iNOS expression in vivo, as had been inferred from mechanistic studies on the control of iNOS expression by TGF-beta 1 in vitro.     

Cytokine-induced intestinal epithelial hyperpermeability: role of nitric oxide.

OBJECTIVE: Incubation of enterocytic monolayers with interferon (IFN)-gamma increases nitric oxide (NO) production and permeability, but NO synthesis inhibitors ameliorate the development of IFN-gamma-induced hyperpermeability. Induction of inducible nitric oxide synthase (iNOS), an isoform of the enzyme responsible for NO biosynthesis, is often enhanced by the synergistic effects of multiple cytokines. Moreover, many of the cytopathic effects of NO are mediated by peroxynitrite, which is produced by the reaction of NO with superoxide radical anion. In the present study, we sought to determine whether combinations of several proinflammatory cytokines, including IFN-gamma, interleukin-1beta, and tumor necrosis factor-alpha, have synergistic effects on the induction of iNOS expression and/or hyperpermeability to hydrophilic solutes in cultured enterocytic monolayers. We also assessed the effects of aminoguanidine (a relatively selective iNOS inhibitor), L-N(G)-monomethyl arginine (an isoform-nonselective NO synthase inhibitor), and Tiron (a superoxide radical anion scavenger) on the development of cytokine-induced hyperpermeability. DESIGN: Caco-2 monolayers were incubated under control conditions or with IFN-gamma, interleukin-1beta, or tumor necrosis factor-alpha alone, pairwise combinations of these cytokines, or all three cytokines together (cytomix; CM). iNOS messenger RNA (mRNA) expression was assessed using Northern blot analysis. The permeability of Caco-2 monolayers growing on permeable supports in bicameral chambers was assessed by measuring the apical-to-basolateral flux of fluorescein disulfonic acid. The concentration of nitrate plus nitrite in culture supernatants, an indirect measure of NO production, was determined using the Griess reaction. RESULTS: After 24 hrs of incubation, up-regulation of iNOS mRNA expression was evident only in cells exposed to IFN-gamma-containing formulations. Expression of iNOS mRNA was far greater in cells incubated with CM than in cells treated with IFN-gamma alone or either of the two-component IFN-gamma-containing cytokine combinations. Compared with IFN-gamma, CM resulted in a higher rate of NO production over 48 hrs of incubation. The permeability of Caco-2 monolayers increased by approximately six-fold and approximately 20-fold after incubation for 48 hrs with IFN-gamma alone and CM, respectively. The increase in permeability induced by incubation with CM was significantly ameliorated by the addition of aminoguanidine, L-N(G)-monomethyl arginine, or Tiron. CONCLUSIONS: IFN-gamma-containing combinations of cytokines are potent inducers of iNOS in cultured enterocytic monolayers. Peroxynitrite may be an important mediator of cytokine-induced gut epithelial hyperpermeability.     

Sphingosine 1-phosphate inhibits nitric oxide production induced by interleukin-1beta in rat vascular smooth muscle cells

Sphingosine 1-phosphate (S1P) is a lipid mediator that exerts potent and diverse biological effects on several cardiovascular cells. We investigated the effect of S1P on interleukin (IL)-1beta-induced nitric oxide (NO) production and inducible NO synthase (iNOS) expression in rat vascular smooth muscle cells (VSMCs). S1P inhibited NO production at concentrations higher than 0.1 muM; this was associated with the inhibition of iNOS protein and mRNA expression. S1P also inhibited IL-1beta-induced GTP cyclohydrolase I (GTPCH) mRNA expression. Pertussis toxin (PTX) partially attenuated the inhibitory effects of S1P on NO production and iNOS protein induction, whereas it completely blocked the inhibitory effects on iNOS and GTPCH mRNA expression. S1P inhibited iNOS expression in Ca(2+)-depleted conditions; PTX did not modify this effect. The Rho kinase inhibitor Y 27632 partially but significantly attenuated the inhibitory effect of S1P on iNOS expression in Ca(2+)-depleted condition but did not affect it in the presence of Ca(2+). S1P significantly inhibited IL-1beta-induced persistent activation of extracellular signal-regulated kinase (ERK) but had no effect in Ca(2+)-depleted conditions. Thus, S1P inhibits IL-1beta induction of NO production and iNOS expression in rat VSMCs through multiple mechanisms involving both PTX-sensitive and -insensitive G proteins coupled to S1P receptors. Furthermore, Ca(2+)-dependent ERK inhibition and Ca(2+)-independent Rho kinase activation might be involved in the inhibitory mechanism of iNOS expression. Through its action on NO production by VSMCs, S1P may play an important role in the progression of local vascular injury associated with thrombosis, atherosclerosis, and hypertension.     

Inhibition of interleukin-1-induced proteoglycan degradation and nitric oxide production in bovine articular cartilage/chondrocyte cultures by the natural product, hymenialdisine.

The effects of hymenialdisine (SK&F 108752) were evaluated on interleukin-1 (IL-1)-induced proteoglycan (PG) degradation, PG synthesis, nitric oxide (NO) production, and inducible nitric oxide synthase (iNOS) gene expression in bovine articular cartilage (BAC) and/or cartilage-derived chondrocytes. Cartilage disks from 0- to 3-month-old calves were treated with IL-1alpha or retinoic acid. PG release was determined by measuring glycosaminoglycan release, and nitrite production was measured as a readout for NO. Inhibition of iNOS gene expression was measured by Northern blot analysis in IL-1alpha-stimulated, cartilage-derived chondrocytes. To measure PG synthesis, chondrocytes were established in alginate beads and treated with hymenialdisine, and then [(35)S]sulfate incorporation into PGs was determined. Hymenialdisine inhibited IL-1alpha-stimulated PG breakdown in BAC in a dose-related manner with an IC(50) of approximately 0.6 microM. Herbimycin, a protein tyrosine kinase inhibitor, also inhibited PG breakdown, whereas RO 32-0432, a protein kinase C inhibitor, had no effect. Both hymenialdisine and herbimycin also were able to inhibit retinoic acid-stimulated PG release. IL-1alpha-stimulated NO production in BAC was inhibited by hymenialdisine and herbimycin at similar concentrations. The effect on iNOS gene expression was determined by Northern blot analysis in chondrocytes grown in monolayer, and inhibition by hymenialdisine was observed with an IC(50) of approximately 0.8 microM. In chondrocytes cultured in alginate beads, IL-1alpha inhibited PG synthesis, whereas hymenialdisine stimulated synthesis at low concentrations (0.6 and 1.25 microM), and higher doses (2.5 microM) were not stimulatory. Compounds with this profile may have utility in the treatment of osteoarthritis.     

Endogenously synthesized nitric oxide prevents endotoxin-induced glomerular thrombosis.

Escherichia coli endotoxin (LPS) can induce the clinical syndrome of septic shock and renal cortical necrosis and can stimulate nitric oxide (NO) production from macrophages, vascular smooth muscle, and glomerular mesangial cells in vitro. NO is an endogenous vasodilator, which also inhibits platelet aggregation and adhesion. We therefore sought to determine whether LPS would stimulate NO production in vivo and, if so, whether this NO would modulate endotoxin-induced glomerular thrombosis. The stable NO end-products, NO2 and NO3, were measured in serum and urine collections from rats during baseline and after injection of LPS, with or without substances that modulate NO synthesis. The urinary excretion of NO2/NO3 was 1,964 +/- 311 nm/8 h during the baseline and increased to 6,833 +/- 776 nm/8 h after a single intraperitoneal injection of 0.1 mg/kg LPS (P < 0.05). The serum concentration of NO2/NO3 also significantly increased after LPS injection. Both the urine and serum stimulation was significantly prevented by the NO synthesis inhibitor, Nw-nitro-L-arginine methyl ester (L-NAME). L-Arginine, given with LPS+L-NAME significantly restored the NO2/NO3 levels in the urine. Ex vivo incubation of tissues from rats treated with LPS demonstrated NO production by the aorta, whole kidney, and glomeruli, but not cortical tubules. Histological examination of kidneys from rats given either LPS or L-NAME alone revealed that 2 and 4.5% of the glomeruli contained capillary thrombosis, respectively. In contrast, rats given LPS+L-NAME developed thrombosis in 55% of glomeruli (P < 0.001), which was significantly prevented when L-arginine was given concomitantly. We conclude that LPS stimulates endogenous production of NO in vivo and that this NO is critical in preventing LPS-induced renal thrombosis.     

In vitro modulation of inducible nitric oxide synthase gene expression and nitric oxide synthesis by procalcitonin

OBJECTIVE: Serum procalcitonin (PCT) concentration was recently introduced as valuable diagnostic marker for systemic bacterial infection and sepsis. At present, the cellular sources and biological properties of PCT are unclear. During sepsis and septic shock, inducible nitric oxide synthase (iNOS) gene expression is stimulated followed by the release of large amounts of nitric oxide (NO). We investigated the possible association between PCT and iNOS gene expression in an in vitro cell culture model. DESIGN: Prospective, controlled in vitro cell culture study. SETTING: University research laboratories. INTERVENTIONS: Confluent rat vascular smooth muscle cells (VSMC) were incubated for 24 hrs and 48 hrs with PCT (1 ng/mL, 10 ng/mL, 100 ng/mL, 1,000 ng/mL, 5,000 ng/mL) alone or with the combination of tumor necrosis factor-alpha (TNF-alpha, 500 U/mL) plus interferon-gamma (IFN-gamma, 100 U/mL). iNOS gene expression was measured by qualitative as well as quantitative polymerase chain reaction analysis, NO release was estimated by the modified Griess method. MEASUREMENTS AND MAIN RESULTS: PCT in increasing concentrations had no effect on iNOS gene expression and nitrite/nitrate release for 24 hrs and 48 hrs, respectively. However, PCT ameliorated TNF-alpha/IFN-gamma-induced iNOS gene expression in a dose-dependent manner (maximal inhibition at PCT 100 ng/mL by -66% for 24 hrs and -80% for 48 hrs). This was accompanied by a significantly reduced release of nitrite/nitrate into the cell culture supernatant (maximal reduction at PCT 100 ng/mL by -56% and -45% for 24 hrs and 48 hrs, respectively). CONCLUSIONS: We conclude that recombinant PCT inhibits the iNOS-inducing effects of the proinflammatory cytokines TNF-alpha/ IFN-gamma in a dose-dependent manner. This might be a counter-regulatory mechanism directed against the large production of NO and the concomitant systemic hypotension in severe sepsis and septic shock.     

Up-regulation of nitric oxide production by interferon-gamma in cultured peritoneal macrophages from patients with cirrhosis.

We previously described a long-lasting overproduction of nitric oxide (NO) in cirrhotic patients with spontaneous bacterial peritonitis. The aim of the present study was to investigate the presence of the inducible NO pathway in peritoneal macrophages. Ascitic fluids were collected from 29 patients with cirrhosis, aged between 35 and 82 years. Peritoneal macrophages were isolated and cultured in the presence or absence of 1 microg/ml lipopolysaccharide and/or 500 units/ml interferon-gamma (IFN-gamma) for 6 days. NO production was measured as nitrate+nitrite (NO(x)), inducible NO synthase (iNOS) protein expression was analysed by immunocytochemistry and Western blot analysis using a specific anti-(human iNOS) antibody, and the catalytic activity of NOS was revealed by cytochemical staining for NADPH-dependent diaphorase. Cultured macrophages spontaneously released small amounts of NO(x) [median (10-90th percentile) of 18 separate experiments: 3.3 (0-8) micromol/l]. Addition of lipopolysaccharide alone or in combination with IFN-gamma to the culture medium did not change the levels of NO(x), while IFN-gamma alone dramatically increased NO production [13.4 (3.5-28.3) micromol/l; P<0.001]. Macrophages were stimulated by IFN-gamma to a greater extent in patients with recent spontaneous bacterial peritonitis (n=13) than in those in a stable clinical condition (n=18) [19.8 (10.5-30.1) and 10.0 (3.2-14.5) micromol/l respectively; P<0.001]. Macrophages freshly isolated or stimulated with IFN-gamma expressed iNOS protein, as shown by Western blot and immunocytochemical analysis, and stained for NADPH diaphorase. Our findings demonstrate the presence of iNOS protein in peritoneal macrophages from cirrhotic patients. The role of IFN-gamma appears to be a determinant for the up-regulation of NO production, particularly under conditions of infection. Therefore peritoneal macrophages producing large amounts of NO at the site of infection may contribute to maintaining splanchnic vasodilation in these patients.     

肾血管性高血压对诱导型一氧化氮合酶表达及活性的影响

目的通过测定肾血管性高血压大鼠血管及肾组织诱导型一氧化氮合酶（iNOS）活性及表达的变化情况,探讨血压与iNOS间的关系。方法运用肾动脉不全结扎方法制备SD大鼠肾血管性高血压模型,并应用Greiss反应、L-精氨酸同位素标记法及Westernblot等分别测定一氧化氮的终产物——尿中NO