Inducible nitric oxide synthase expression and nuclear factor-kappaB activation in alveolar type II cells in lung injury

Alveolar type II cells (type II cells) play a crucial role in the progression and repair of lung inflammation and injury. We investigated whether inducible nitric oxide synthase (iNOS) was expressed and nuclear factor-kappaB (NF-kappaB) was activated in type II cells in lung injury. After injecting lipopolysaccharide (LPS) or saline in the rat, the lungs were excised and type II cells were isolated. iNOS and its mRNA were expressed both in lung tissue and isolated type II cells in response to LPS. The lungs from saline-treated rats showed only minimal expression of iNOS. Electrophoretic mobility shift assay revealed that expression of NF-kappaB in the nuclear extracts was augmented by LPS, and p5O/NFkappaB was expressed in type II cells in LPS-treated rats. Intraperitoneal dexamethasone almost completely inhibited the iNOS expression and attenuated the activation of NF-kappaB in the LPS-treated lung. These findings suggest that type II cells can be a source of NO production in lung injury,and that the effects of corticosteroids may be in part through inhibition of both iNOS expression and NF-kappaB activation.     

Increased hepatic expression of nitric oxide synthase type Ⅱ in cirrhotic rats

AIM: To determine the role and effect of nitric oxide synthase type Ⅱ (NOSII) in cirrhotic rats.METHODS: Expression of NOSⅡ mRNA was detected by real time RT-PCR. The activity of nitric oxide synthase and serum levels of NO, systemic and portal hemodynamics and degrees of cirrhosis were measured with high sensitive methods. Chinese traditional medicine tetrandrine was used to treat cirrhotic rats and to evaluate the function of NO.Double-blind method was applied during the experiment.RESULTS: The concentration of NO and the activity of NOS were increased markedly at all stages of cirrhosis, and iNOSmRNA was greatly expressed. Meanwhile the portalvenous-pressure (PVP), and portal-venous-flow (PVF) were significantly increased. NO, NOS and iNOSmRNA were positively correlated to the quantity of hepatic fibrosis.Tetrandrine significantly inhibited NO production and the expression of iNOSmRNA.CONCLUSION: Increased hepatic expression of NOSII is one of the important causes of hepatic cirrhosis and portal hypertension.     

Inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) expression in fulminant hepatic failure

BACKGROUND/AIMS: Inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) have important functions in inflammation and vasoregulation but their role in fulminant hepatic failure (FHF) is not well understood. METHODS: Intrahepatic in situ staining and semi-quantification of iNOS and eNOS by immunohistochemistry in 25 patients with FHF, in 40 patients with chronic liver diseases (CLD) and in ten normal controls (NC). RESULTS: Expression patterns of iNOS and eNOS differed. While in NC only faint iNOS expression was found in some Kupffer cells/macrophages and hepatocytes, eNOS was expressed constitutively in sinusoidal and vascular endothelial cells. In CLD, iNOS expression was induced in Kupffer cells/macrophages and hepatocytes, representing the main iNOS expressing cell types. Additionally, bile ducts, vascular endothelial cells and lymphocytes also expressed iNOS (P = 0.001). In contrast, no differences were found between eNOS expression in CLD and NC (P = 0.64). The same cell types expressed eNOS and iNOS in FHF but numbers of both were significantly enhanced, exceeding the levels seen in CLD (P < 0.001, P = 0.017). CONCLUSIONS: Our data demonstrate that iNOS and eNOS are differently regulated in physiologic conditions and in liver disease. While eNOS seems to be involved in the physiological regulation of hepatic perfusion, strong upregulation of iNOS might contribute to inflammatory processes in FHF.     

Phosphorylation of Thr(495) regulates Ca(2+)/calmodulin-dependent endothelial nitric oxide synthase activity

The activity of the endothelial nitric oxide synthase (eNOS) can be regulated independently of an increase in Ca(2+) by the phosphorylation of Ser(1177) but results only in a low nitric oxide (NO) output. In the present study, we assessed whether the agonist-induced (Ca(2+)-dependent, high-output) activation of eNOS is associated with changes in the phosphorylation of Thr(495) in the calmodulin (CaM)-binding domain. eNOS Thr(495) was constitutively phosphorylated in porcine aortic endothelial cells and was rapidly dephosphorylated after bradykinin stimulation. In the same cells, bradykinin enhanced the phosphorylation of Ser(1177), which was maximal after 5 minutes, and abolished by the CaM-dependent kinase II (CaMKII) inhibitor KN-93. Bradykinin also enhanced the association of CaMKII with eNOS. Phosphorylation of Thr(495) was attenuated by the protein kinase C (PKC) inhibitor Ro 31-8220 and after PKC downregulation using phorbol 12-myristate 13-acetate. The agonist-induced dephosphorylation of Thr(495) was completely Ca(2+)-dependent and inhibited by the PP1 inhibitor calyculin A. Little CaM was bound to eNOS immunoprecipitated from unstimulated cells, but the agonist-induced dephosphorylation of Thr(495) enhanced the association of CaM. Mutation of Thr(495) to alanine increased CaM binding to eNOS in the absence of cell stimulation, whereas the corresponding Asp(495) mutant bound almost no CaM. Accordingly, NO production by the Ala(495) mutant was more sensitive to Ca(2+)/CaM than the aspartate mutant. These results suggest that the dual phosphorylation of Ser(1177) and Thr(495) determines the activity of eNOS in agonist-stimulated endothelial cells. Moreover, the dephosphorylation of Thr(495) by PP1 precedes the phosphorylation of Ser(1177) by CaMKII. The full text of this article is available at http://www.circresaha.org.     

利拉鲁肽通过抑制核因子κB p65磷酸化调控人脐静脉内皮细胞一氧化氮合酶的表达

目的 探讨利拉鲁肽在高糖环境下对人脐静脉内皮细胞（HUVECs）功能的影响及其可能机制.方法 高糖环境下（25 mmol/L葡萄糖）培养人脐静脉内皮细胞,分别给予10、100、1 000 μg/L利拉鲁肽进行干预,采用实时定量聚合酶链反应（RT-PCR）和Western blotting法分别检测内皮型一氧化氮合酶（eNOS）、诱导型一氧化氮合酶（iNOS）以及核因子κB p65 （NF-κB p65）的mRNA、蛋白表达水平;应用肿瘤坏死因子-α（TNF-α）干预利拉鲁肽处理后的HUVECs,观察eNOS、iNOS、NF-κB p65的mRNA、蛋白表达水平的变化.研究结果多组间采用单因素方差分析,两组间比较采用SLD分析.结果 与普通培养基（7 mmol/L葡萄糖）组相比,高糖环境下HUVECs的eNOS mRNA和蛋白表达均降低,iNOS mRNA和蛋白表达及磷酸化NF-κB p65蛋白表达均增高（t=2.79、5.75、4.32、4.85、7.12,均P＜0.05）.与0μg/L组相比,1 000μg/L利拉鲁肽干预组HUVECs的eNOS mRNA、eNOS蛋白表达均上调,iNOS mRNA、iNOS蛋白表达及NF-κB p65磷酸化蛋白表达均下调（t=5.12、9.34、6.70、5.50、8.94,均P＜0.05）.与单独使用利拉鲁肽组相比,TNF-α联合利拉鲁肽组HUVECs的eNOSmRNA和蛋白表达均下调,iNOS mRNA和蛋白表达及磷酸化NF-κB p65蛋白表达均上调（t=3.33～7.87,均P＜0.05）.结论 利拉鲁肽可通过抑制NF-κB p65磷酸化在转录和翻译水平上增加eNOS表达、降低iNOS的表达,从而改善内皮细胞功能,预防糖尿病动脉粥样硬化.     

Discrepant effects of inducible nitric oxide synthase modulation on systemic and splanchnic endothelial nitric oxide synthase activity and expression in cirrhotic rats

BACKGROUND: Arterial vasodilatation, which is a major factor in the pathogenesis of the hyperkinetic circulatory state and portal hypertension in cirrhosis, is due to arterial nitric oxide (NO) overproduction secondary to endothelial NO synthase (eNOS) and inducible NOS (iNOS) upregulation. However, in cirrhosis, the respective roles of eNOS and iNOS isoforms in NO overproduction are still unknown and the effect of iNOS modulation on eNOS activity and expression has not been evaluated in the systemic or splanchnic vessels. The aim of this study was to evaluate the effects of modulating aortic and superior mesenteric arteries (SMA) iNOS on arterial eNOS activity and expression in rats with cirrhosis. METHODS: eNOS and iNOS protein expression and eNOS activity (assessed by its phosphorylation at serine 1177) were measured in the aortas and SMA in untreated and treated cirrhotic rats with lipopolysaccharide (LPS), N-iminoethyl-L-lysine (L-NIL), a selective iNOS inhibitor, and LPS plus L-NIL. RESULTS: LPS administration significantly increased eNOS and iNOS protein expression and eNOS activity in the aortas of both sham-operated and cirrhotic rats. However, in SMA, LPS administration induced a decrease in eNOS protein expression and activity and an increase in iNOS protein expression. CONCLUSION: The results of this study may explain the worsening of the hyperdynamic state in cirrhosis during septic shock by direct LPS-induced eNOS activation in large systemic vessels, and its inhibition in concomitant small splanchnic vasculature by iNOS synthesized NO.     

辛伐他汀对脓毒症大鼠心肌细胞核因子-κB、诱导型一氧化氮合酶表达的影响及意义

目的 观察核因子(NF)-κB和诱导型一氧比氮合酶(iNOS)在脓毒症大鼠心肌细胞的表达,同时探讨辛伐他汀对其表达的影响,以及他汀类药物的心肌保护性作用.方法 雌性Wistar大鼠30只随机分为三组:正常组(N组),盲肠结扎穿孔术组(CLP组)和辛伐他汀组(S组),处理方法:①S组:喂养辛伐他汀20 mg/kg每天一次共2w.②2w后S组与CLP组大鼠行盲肠结扎穿孔术.③术后48 h取各组大鼠心肌组织制成石蜡切片,采用HE染色和免疫组织化学染色的方法检测心肌纤维iNOS和NF-κB的蛋白表达.结果 HE染色显示CLP组大鼠心肌纤维水肿,疏松,间质充血水肿,而S组心肌的组织病理学改变较CLP组轻.免疫组织化学染色显示CLP组大鼠心肌细胞iNOS与NF-κB蛋白表达于术后48 h较N组明显增加(P＜0.05),S组大鼠心肌细胞iNOS与NF-κB蛋白表达与CLP组相比明显减弱(P＜0.05),与N组相比无明显差异(P＞0.05).结论 辛伐他汀可以减弱脓毒症大鼠心肌细胞iNOS和NF-κB的蛋白表达,改善脓毒症时的心脏功能.     

Neuronal nitric oxide synthase is up-regulated by angiotensin II and attenuates NADPH oxidase activity and facilitates relaxation in murine left ventricular myocytes

Angiotensin II (Ang II) is critical in myocardial pathogenesis, mostly via stimulating NADPH oxidase. Neuronal nitric oxide synthase (nNOS) has recently been shown to play important roles in modulating myocardial oxidative stress and contractility. Here, we examine whether nNOS is regulated by Ang II and affects NADPH oxidase production of intracellular reactive oxygen species (ROS(i)) and contractile function in left ventricular (LV) myocytes. Our results showed that Ang II induced biphasic effects on ROS(i) and LV myocyte relaxation (TR(50)) without affecting the amplitude of sarcomere shortening and L-type Ca(2+) current density: TR(50) was prolonged at 30 min but was shortened after 3h (or after Ang II treatment in vivo). Correspondingly, ROS(i) was increased, followed by a reduction to control level. Quantitative RT-PCR and immunoblotting experiments showed that Ang II (3h) increased the mRNA and protein expression of nNOS and increased NO production (nitrite assay) in LV myocyte homogenates, suggesting that nNOS activity may be enhanced and involved in mediating the effects of Ang II. Indeed, n(omega)-nitro-l-arginine methyl ester (l-NAME) or a selective nNOS inhibitor, S-methyl-l-thiocitrulline (SMTC) increased NADPH oxidase production of superoxide/ROS(i) and abolished faster myocyte relaxation induced by Ang II. The positive lusitropic effect of Ang II was not mediated by PKA-, CaMKII-dependent signaling or peroxynitrite. Conversely, inhibition of cGMP/PKG pathway abolished the Ang II-induced faster relaxation by reducing phospholamban (PLN) Ser(16) phosphorylation. Taken together, these results clearly demonstrate that myocardial nNOS is up-regulated by Ang II and functions as an early adaptive mechanism to attenuate NADPH oxidase activity and facilitate myocardial relaxation.     

Steatosis correlates with hepatic expression of death receptors and activation of nuclear factor‐κB in chronic hepatitis C

Background: Steatosis is recognized as a predictor of the severity as well as the progression of fibrosis in chronic hepatitis C. The mechanisms that cause increased hepatocellular injury associated with steatosis remain largely unknown. Methods: We studied the correlation of hepatic expression of death receptors: Fas and tumour necrosis factor‐α receptor 1 (TNF‐R1), and downstream caspase (caspase‐3) with hepatic steatosis by immunohistochemical study in chronic hepatitis C and determined the role of nuclear factor‐κB (NF‐κB). Results: Ninety patients (49 males and 41 females, mean age of 50.5 ± 10.4 years, genotype 1 or 2) with chronic hepatitis C virus infection were recruited. The factors associated with steatosis grade were body mass index (P=0.004) and fibrosis stage (P=0.034). Moderate/severe steatosis was an independent variable associated with advanced fibrosis stage by stepwise logistic regression analysis. The expression of immunoreactivity for Fas, TNF‐R1 and active caspases‐3 in liver tissues was significantly correlated with the steatosis grade (P<0.001, P<0.001 and P<0.001 respectively). The extent of active caspases‐3 correlated significantly with the expression of Fas (r=0.659, P<0.001) and TNF‐R1 (r=0.617, P<0.001). NF‐κB p65 expression correlated significantly with the extent of Fas (r=0.405, P<0.001), TNF‐R1 (r=0.448, P=0.002) and active caspase‐3 (r=0.313, P=0.003), and correlated with steatosis grade (P<0.001) but not with inflammatory and fibrosis scores. Conclusion: Our observations suggest a mechanism whereby steatosis contributes to the progression of liver injury in chronic hepatitis C through upregulation of death receptors and activation of NF‐κB.     

溃疡性结肠炎组织中NF-κB,OX-2和iNOS表达的意义

目的:转录因子NF-кB的诱导,调控着免疫和炎症反应中众多基因的表达.溃疡性结肠炎(ulcerative colitis,C)存在上皮细胞、淋巴细胞、巨噬细胞的异常激活及细胞因子的表达失调.我们检测了UC组织中NF-кBp65,及两种启动子含NF-кB结合位点的蛋白COX-2和iNOS的表达和分布,探讨他们在UC发病机制中的作用,及三者之间的关系.方法:用免疫组化SP法检测39例活动期溃疡性结肠炎内镜活检标本及30例正常对照石蜡包埋组织中NF-κBp65,OX-2和iNOS的表达情况.结果:UC组p65,OX-2和iNOS均为阳性表达,主要分布于上皮细胞.固有层炎性细胞及血管内皮细胞也有程度不同的表达对照组均为阴性或弱阳性表达.三者在UC组的表达与对照组相比,差异均有非常显著性(P＜0.01).p65的表达与内镜及病理分级有关.内镜Ⅱ级与Ⅰ级比较(5.8±2.6 vs 3.6±1.9),差异显著(P＜0.05).病理Ⅱ,Ⅲ级与Ⅰ级比较(6.1±2.4,.3±2.5 vs 4.0±2 3),差异显著(P＜0.05,＜0.01).iNOS的表达与病理分级有关,Ⅲ级与Ⅰ,Ⅱ级比较(7.8±2.5 vs 4.6±2.3,5.0±1.6),差异显著(P＜0.01,＜0.05).COX-2的表达在病情轻重,内镜及病理分级间差异无显著性(P＞0.05).p65与COX-2,NOS表达显著相关(rs1分别为0.713,.706;P＜0.01),COX-2与iNOS表达显著相关(rs1=0.854,＜0.01).结论:NF-кB的诱导参与UC的发生、发展.COX-2,NOS也参与UC的炎症及损伤过程,可能iNOS发挥的作用更显著.COX-2,NOS表达的调控机制可能相似,且与NF-κBp65的诱导有直接关系.