Which comes first: Renal inflammation or oxidative stress in spontaneously hypertensive rats?

The present study was undertaken to identify whether inflammation or oxidative stress is the primary abnormality in the kidney in spontaneously hypertensive rats (SHR). Renal inflammation and oxidative stress were evaluated in 2- and 3-week-old prehypertensive SHR and age-matched genetically normotensive control Wistar-Kyoto (WKY) rats. Blood pressure was similar in WKY and SHR rats at 2 and 3 weeks, of age. Renal inflammation (macrophage and nuclear factor-κB) was elevated in SHR at 3 weeks, but not at 2 weeks, of age compared with age-matched WKY rats. Renal oxidative stress (nitrotyrosine, 8-hydroxy-2′-deoxyguanosine and p47phox) was also clearly elevated in 3-week-old SHR compared with age-matched WKY rats. Additionally, NADPH oxidase subunit p47phox was found elevated in 2-week-old SHR compared to age-matched WKY rats. Moreover, antioxidant (N-acetyl-l-cysteine and Tempol) treatment reduced renal inflammation in prehypertensive SHR. We therefore conclude that the oxidative stress appears before inflammation as a primary abnormality in the kidney in prehypertensive SHR.     

Oxidative damage of the gastric mucosa in Helicobacter pylori positive chronic atrophic and nonatrophic gastritis, before and after eradication

Background. Helicobacter pylori is the main cause of gastritis and a primary carcinogen. The aim of this study was to assess oxidative damage in mucosal compartments of gastric mucosa in H. pylori positive and negative atrophic and nonatrophic gastritis. Materials and methods. Five groups of 10 patients each were identified according to H. pylori positive or negative chronic atrophic (Hp‐CAG and CAG, respectively) and nonatrophic gastritis (Hp‐CG and CG, respectively), and H. pylori negative normal mucosa (controls). Oxidative damage was evaluated by nitrotyrosine immunohistochemistry in the whole mucosa and in each compartment at baseline and at 2 and 12 months after eradication. Types of intestinal metaplasia were classified by histochemistry. Results. Total nitrotyrosine levels appeared significantly higher in H. pylori positive than in negative patients, and in Hp‐CAG than in Hp‐CG (p < .001); no differences were found between H. pylori negative gastritis and normal mucosa. Nitrotyrosine were found in foveolae and intestinal metaplasia only in Hp‐CAG. At 12 months after H. pylori eradication, total nitrotyrosine levels showed a trend toward a decrease in Hp‐CG and decreased significantly in Hp‐CAG (p = .002), disappearing from the foveolae (p = .002), but remaining unchanged in intestinal metaplasia. Type I and II of intestinal metaplasia were present with the same prevalence in Hp‐CAG and CAG, and did not change after H. pylori eradication. Conclusions. Oxidative damage of the gastric mucosa increases from Hp‐CG to Hp‐CAG, involving the foveolae and intestinal metaplasia. H. pylori eradication induces a complete healing of foveolae but not of intestinal metaplasia, reducing the overall oxidative damage in the mucosa.     

Systemic Biochemical Effects of Inhaled NO in Rats: Increased Expressions of NOS III, Nitrotyrosine-, and Phosphotyrosine-Immunoreactive Proteins in Liver and Kidney Tissues

Inhaled nitric oxide (iNO) has been shown to reduce pulmonary hypertension associated with several disease states. The effects of iNO are thought to be restricted to the pulmonary vasculature because of its rapid inactivation by hemoglobin. Recent data have suggested, however, that iNO can form nitrosothiols, which can be carried throughout the circulation, thus increasing the half life and bioactivity on NO. Other studies have shown that iNO can affect intestinal ischemia and renal hemodynamics. In this study, rats were exposed to 49 +/- 4 ppm or 107 +/- 13 ppm NO for 4 h and the lung, spleen, liver, and kidney tissues were removed and measured for NOS II and NOS III protein, nitrotyrosine (NT), and phosphotyrosine (PT) immunoreactivity. Following 107 ppm iNO, increases in NOS III protein expression, NT, and PT were observed in the liver and kidney, but not in the lung or spleen. No such increases were noted after the lower dose of iNO. These results paralleled those shown for isobutyl nitrite that we reported earlier and indicated that iNO can cause changes in protein chemistry in organs and tissues beyond the lungs. Since iNO produced little systemic hemodynamic effects, it is unlikely that the observed biochemical alterations were derived secondarily from physiological changes.     

内毒诱导肺动脉内皮细胞生成过氧亚硝基阴离子的意义

目的：探讨牛肺动脉内皮细胞（BPAEC）产生过氧亚硝基阴离子（ONOO^-）的能力及其作用。方法：用流式细胞免疫荧光技术,定量检测内毒素主要成分脂多糖（LPS）诱导培养的BPAEC中ONOO^-生成标志物硝基酪氨酸（NT）的含量,观察ONOO^-对BPAEC形态变化的影响。结果：LPS可剂量依赖性诱导BPAEC产生ONOO^-明显增多,并为氮基胍部分翻转;ONOO^-可导致BPAEC明显回缩,胞体变小,细胞间隙增宽。结论：LPS诱导BPAEC产生增多的ONOO^-可能参与介导LPS对BPAEC本身的损伤效应。     

Vanadate protects human neuroblastoma SH-SY5Y cells against peroxynitrite-induced cell death

We investigated the effect of vanadate, a tyrosine phosphatase inhibitor, on cell death induced by peroxynitrite in human neuroblastoma SH-SY5Y cells. Vanadate prevented cell death induced by 3-morpholinosydnonimine (SIN-1), a peroxynitrite donor; whereas SIN-1-induced cell death was not prevented by neither okadaic acid, an inhibitor of serine/threonine phosphatases 1 and 2A, nor cyclosporin A, an inhibitor of serine/threonine phosphatase 2B. Vanadate did not prevent cell death induced by N-ethyl-2-(1-ethyl-hydroxy-2-nitrosohydrazino)-ethanamine, a nitric oxide donor. Wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3-kinase), did not block the protective effect of vanadate, suggesting that the protective effect of vanadate is independent on PI3-kinase. Vanadate increased tyrosine phosphorylation of several proteins including the focal adhesion protein p130 Crk-associated substrate (p130(cas)). By the treatment with SIN-1, the endogenous association of p130(cas) and Crk was disrupted, and the association was restored by vanadate treatment. These results suggest that disruption of tyrosine phosphorylation signaling may be critical for peroxynitrite-induced cell death, and that vanadate prevents cell death at least in part through the enhancement in tyrosine phosphorylation of the proteins including p130(cas).     

Protein nitration

Various proteins/enzymes obtained commercially were tested for the presence of endogenously nitrated tyrosine by Western blot analysis omitting reducing agent in the step of SDS-PAGE. Histones II-S and VIII-S, IgG, cAMP-dependent protein kinase (PKA), phosphorylase b, and phosphorylase kinase exhibited strong immunoreactive bands. Histone VI-S, glycogen synthase, lactate dehydrogenase, actin, thyroglobulin, and macroglobulin exhibited moderate immunoreactivity. Histone III-S, casein, acetyl cholinesterase, DNase I, and lipase had only traceable immunoreactivity. Whereas histone VII-S, pyruvate kinase, trypsin, pepsin, chymotrypsin, protease IV, and protease XIII, and glutathione S-transferase lacked immunoreactivity. A variation of immunoreactivity between hypertensive and normaltensive rat hearts was found in the histone-agarose fractions of crude extracts. Additionally, nitrotyrosine immunoreactivity was observed in non-mammalian organisms including Eschericia coli, Saccharomyces cerevisiae and Triticum vulgaris. Upon the treatment of 15 M peroxynitrite (PN), strong oxidant derived from nitric oxide (NO), the apparent K_m of PKA for cAMP increased from approximately 10^-8 to 10^-6 M. The results imply that the varied nitration of tyrosine residues in proteins/enzymes may occur as a post-translational modification in vivo, and such discriminative nitration may be vital in PN/NO-regulated signal transduction cascade.     

Protein changes in macrophages induced by plasma from rats exposed to 35 GHz millimeter waves

A macrophage assay and proteomic screening were used to investigate the biological activity of soluble factors in the plasma of millimeter wave‐exposed rats. NR8383 rat macrophages were incubated for 24 h with 10% plasma from male Sprague–Dawley rats that had been exposed to sham conditions, or exposed to 42 °C environmental heat or 35 GHz millimeter waves at 75 mW/cm² until core temperature reached 41.0 °C. Two‐dimensional polyacrylamide gel electrophoresis, image analysis, and Western blotting were used to analyze approximately 600 protein spots in the cell lysates for changes in protein abundance and levels of 3‐nitrotyrosine, a marker of macrophage stimulation. Proteins of interest were identified using peptide mass fingerprinting. Compared to plasma from sham‐exposed rats, plasma from environmental heat‐ or millimeter wave‐exposed rats increased the expression of 11 proteins, and levels of 3‐nitrotyrosine in seven proteins, in the NR8383 cells. These altered proteins are associated with inflammation, oxidative stress, and energy metabolism. Findings of this study indicate both environmental heat and 35 GHz millimeter wave exposure elicit the release of macrophage‐activating mediators into the plasma of rats. Bioelectromagnetics 31:656–663, 2010. © 2010 Wiley‐Liss, Inc.     

Ischemia induced peroxynitrite dependent modifications of cardiomyocyte MLC1 increases its degradation by MMP‐2 leading to contractile dysfunction

Damage to cardiac contractile proteins during ischemia followed by reperfusion is mediated by reactive oxygen species such as peroxynitrite (ONOO⁻), resulting in impairment of cardiac systolic function. However, the pathophysiology of systolic dysfunction during ischemia only, before reperfusion, remains unclear. We suggest that increased ONOO⁻ generation during ischemia leads to nitration/nitrosylation of myosin light chain 1 (MLC1) and its increased degradation by matrix metalloproteinase-2 (MMP-2), which leads to impairment of cardiomyocyte contractility. We also postulate that inhibition of ONOO⁻ action by use of a ONOO⁻ scavenger results in improved recovery from ischemic injury. Isolated rat cardiomyocytes were subjected to 15 and 60 min. of simulated ischemia. Intact MLC1 levels, measured by 2D gel electrophoresis and immunoblot, were shown to decrease with increasing duration of ischemia, which correlated with increasing levels of nitrotyrosine and nitrite/nitrate. In vitro degradation of human recombinant MLC1 by MMP-2 increased after ONOO⁻ exposure of MLC1 in a concentration-dependent manner. Mass spectrometry analysis of ischemic rat cardiomyocyte MLC1 showed nitration of tyrosines 78 and 190, as well as of corresponding tyrosines 73 and 185 within recombinant human cardiac MLC1 treated with ONOO⁻. Recombinant human cardiac MLC1 was additionally nitrosylated at cysteine 67 and 76 corresponding to cysteine 81 of rat MLC1. Here we show that increased ONOO⁻ production during ischemia induces MLC1 nitration/nitrosylation leading to its increased degradation by MMP-2. Inhibition of MLC1 nitration/nitrosylation during ischemia by the ONOO⁻ scavenger FeTPPS (5,10,15,20-tetrakis-[4-sulfonatophenyl]-porphyrinato-iron[III]), or inhition of MMP-2 activity with phenanthroline, provides an effective protection of cardiomyocyte contractility.     

Effect of partial outlet obstruction on nitrotyrosine content and distribution within the rabbit bladder

Purpose: Evidence indicates that free radicals are etiological factors in obstructive bladder disease. However, it is not clear which species of reactive oxygen or nitrogen species mediate the damage. The current studies were designed to determine if partial outlet obstruction in rabbits results in the generation of nitrotyrosine (NT). Materials and methods: Sixteen rabbits were separated into four groups of four. The rabbits in groups 1 and 2 underwent sham operation while rabbits in groups 3 and 4 underwent partial outlet obstruction. The rabbits in groups 1 and 3 were evaluated after 1 week of obstruction and the rabbits in groups 2 and 4 were evaluated after 2 weeks of obstruction. A separate group of four controls were evaluated simultaneously with the sham and obstructed rabbits. Four rabbits from each group were evaluated after 1 and 2 weeks of obstruction. Four control rabbits were also evaluated. Isolated strips were evaluated for contractile responses and NT content of the mucosa and muscle were quantitated by Western blot analysis. Results: (1) The mucosa contains both 42 and 62 kD proteins exhibiting a strong nitrotyrosine signal; the muscle presents a signal only at 62 kD. (2) The sham operations had no effect on nitrotyrosine distribution or content. (3) The nitrotyrosine of both mucosal proteins and the muscle protein are increased in the 1 week obstructed bladder; whereas, only the 62 kD signal is increased in the two week obstructed bladder mucosa. (4) The contractile response to FS are reduced to a significantly greater degree than the responses to carbachol, KCl, or ATP. Conclusions: These studies clearly demonstrated that partial outlet obstruction in rabbits results in significant increases in nitrotyrosine within the bladder and may contribute to the contractile dysfunctions mediated by partial outlet obstruction. (Mol Cell Biochem 276: 143–148, 2005)     

微管蛋白酪氨酸连接酶类似物12通过干扰微管蛋白酪氨酸硝基化促进Hep-2细胞生长

硝基化酪氨酸与酪氨酸在结构上相似,它在病理情况下会出现,并在细胞内与微管蛋白结合,从而阻碍微管的正常功能. 硝基化酪氨酸在肿瘤中的作用,目前研究甚少.本文利用头颈鳞癌Hep-2细胞株,研究微管蛋白酪氨酸连接酶类似物12（tubulin tyrosine ligase like 12,TTLL12）和硝基化酪氨酸对头颈鳞癌Hep-2生长的影响,通过Western 印迹试验和MTT试验发现,随着硝基化酪氨酸的浓度升高,细胞内生成的硝基化酪氨酸微管蛋白含量也增高,同时细胞生长受抑制的程度显著增高; 对建立的TTLL12高表达细胞株加入硝基化酪氨酸培养,结果显示,TTLL12高表达细胞株内的硝基化酪氨酸微管蛋白含量明显低于对照组细胞;对照组细胞的生长明显受到抑制,而高表达细胞株的生长无明显改变,两者的细胞生长有显著性差异（P＜0.05）.本研究结果提示,TTLL12可通过阻碍硝基化酪氨酸与微管蛋白的结合,使头颈鳞癌Hep-2细胞逃避硝基化酪氨酸的打击. 对这一调控机制的进一步研究,必将有助于控制肿瘤细胞的生长,为治疗肿瘤寻找到新的治疗靶点.