Differential nitric oxide synthase expression during hepatic ischemia-reperfusion

BACKGROUND: In recent years the important role of nitric oxide in hepatic ischemia-reperfusion injury has been increasingly recognised. The prevailing consensus is that reperfusion injury may be partly the result of decreased production of nitric oxide from endothelial nitric oxide synthase and excessive production of nitric oxide from the inducible isoform. We therefore undertook this study to characterize the expression of different nitric oxide synthase isoforms during hepatic reperfusion. METHODS: Male Wistar rats (n = 6) were subjected to 45 minutes of partial hepatic ischemia (left lateral and median lobes) followed by 6 hours of reperfusion. Control animals (n = 6) were subjected to sham laparotomy. The expression of endothelial and inducible nitric oxide synthase was examined using immunohistochemistry and Western blotting. Liver sections were also stained with nitrotyrosine antibody, a specific marker of protein damage induced by peroxynitrite (a highly reactive free radical formed from nitric oxide). RESULTS: Liver sections from all the control animals showed normal expression of the endothelial isoform and no expression of inducible nitric oxide synthase. Livers from all the animals subjected to hepatic ischemia showed decreased expression of endothelial nitric oxide synthase, and all but one animal from this group showed expression of the inducible isoform both in inflammatory cells and in hepatocytes. Western blotting confirmed these findings. Staining with the antinitrotyrosine antibody was also confined to five liver sections from animals subjected to hepatic ischemia. CONCLUSIONS: During the reperfusion period after hepatic ischemia, endothelial nitric oxide synthase is downregulated while inducible nitric oxide synthase is expressed in both hepatocytes and inflammatory cells. The presence of nitrotyrosine in livers subjected to hepatic ischemia-reperfusion suggests that the expression of inducible nitric oxide synthase plays an important role in mediating reperfusion injury in this model.     

Caspase-3-dependent apoptosis in Escherichia coli-infected urothelium: involvement of inducible nitric oxide synthase

OBJECTIVE: To investigate if nitric oxide (NO) derived from inducible NO synthase (iNOS) regulates apoptosis and/or cell shedding in uroepithelial cells in vivo and in vitro, as bladder epithelial cells undergo shedding during urinary tract infection, which is considered a part of the host's defence and is thought to take place via an apoptotic pathway. MATERIALS AND METHODS: Bladders and kidneys of mice infected with Escherichia coli were used to study caspase-3 immunoreactivity at different times after infection. Wild-type (E. coli 1177) and type-1 recombinant (E. coli PKL4) bacteria were used. iNOS-deficient mice were used to study the association of caspase-3 with iNOS. Isolated human uroepithelial cells were used to examine the effect of the NO donor DETA/NO and the peroxynitrite generator SIN-1 on caspase-3 activity and cell shedding in vitro. RESULTS: Many caspase-3 immunoreactive neutrophils were found soon after infection and some superficial bladder epithelial cells were also immunoreactive for caspase-3. CONCLUSIONS: Caspase-3 immunoreactivity was also detected in neutrophils and bladder epithelial cells of infected iNOS-deficient mice. There was no co-expression between iNOS- and caspase-3 in bladder epithelial cells. DETA/NO and SIN-1 did not stimulate caspase-3 activity or cell shedding in isolated human uroepithelial cells. Caspase-3 and iNOS are not co-expressed in uroepithelial cells and apoptosis is evident in the absence of iNOS. Exogenous NO did not induce apoptosis or cell shedding in isolated human uroepithelial cells.     

The preventive effects of thiopental and propofol on testicular ischemia-reperfusion injury

BACKGROUND: Testicular torsion is a urological emergency that requires immediate surgical intervention to prevent testicular damage. The aim of the study was to investigate the preventive effects of thiopental and propofol as anesthetics on testicular ischemia-reperfusion injury. METHODS: Forty male Wistar Albino rats were randomly assigned to four groups of 10 rats each. During 5 h, anesthesia was induced and maintained with thiopental in groups 1 and 2 and with propofol in groups 3 and 4. Groups 2 and 4 received left testicular ischemia (torsion) during 1 h and reperfusion (detorsion) during 4 h. Groups 1 and 3 (control groups) had no testicular torsion and detorsion. At the end of 5 h, animals were killed and both ipsilateral and contralateral testes were removed for histopathologic examination and measurement of tissue MDA (malondialdehyde) and NO (nitric oxide) levels. RESULTS: In the contralateral testes of all the groups, MDA and NO measurements were not different from ipsilateral testes of the control groups. Between the groups 1 and 3, there were no differences in MDA and NO levels. Although torsion/detorsion of testes in group 4 caused significantly increased levels of tissue MDA and NO values compared with group 3, ischemia-reperfusion in group 2 caused a further increase in these levels compared with group 4. The ipsilateral testes in the control groups did not show any morphological changes. Testicular torsion/detorsion in rats with thiopental anesthesia (group 2) caused significantly greater histopathologic injury levels than rats with propofol anesthesia (group 4). CONCLUSION: Our results suggest that propofol as an anesthetic agent may prevent testicular damage by scavenging reactive oxygen and nitrogen species and inhibiting lipid peroxidation in an animal model of testicular torsion and detorsion.     

Inhibition of nitric oxide synthase reduces renal ischemia/reperfusion injury

BACKGROUND: The role of nitric oxide (NO) production because of inducible nitric oxide synthase (iNOS) in the pathogenesis of renal ischemia/reperfusion (I/R) injury is unclear. In this study the roles of both iNOS and NO were characterized in a rat model of renal I/R injury. In addition, the effect of iNOS inhibition on renal function was evaluated. METHODS: Sprague-Dawley rats underwent 45 min of left renal ischemia and contralateral nephrectomy followed by various periods of reperfusion and renal function analysis [plasma creatinine, fractional excretion of sodium (FENa), creatinine clearance (CrCl), and measurement of plasma and urine NO levels]. In addition, the effect of treatment with 1400W, a highly selective iNOS inhibitor, was evaluated. RESULTS: Renal dysfunction peaked at 48 h after reperfusion and immunohistochemistry studies revealed iNOS expression in the vasculature (3 h) and renal tubules (48 h) after reperfusion. Renal function improved significantly in treated animals compared to controls [creatinine of 1.1 v. 1.9 mg/dl (P < 0.05) and CrCl of 0.54 v. 0.31 ml/min (P < 0.05), respectively]. In addition, FENa was decreased by 50%, plasma NO levels were significantly lower (32.7 v. 45.7 micromol/L, P < 0.01), and deposition of nitrotyosine in the tubules of treated rats was less than in control animals. CONCLUSIONS: These data support the hypothesis that iNOS and NO are involved in the pathogenesis of renal I/R injury and suggests that use of iNOS inhibitors may be a valuable therapeutic strategy clinical situations where renal I/R may be prevalent.     

N(G)-nitro-L-arginine methylester,a nitric oxide synthase inhibitor,diminishes apoptosis induced by ischemia-reperfusion in the rat bladder

AIMS: To evaluated the effects of N(G)-nitro-L-arginine methylester (L-NAME), a nitric oxide synthase (NOS) inhibitor, on apoptosis induced by ischemia-reperfusion injury in the rat through a muscle bath and TUNEL staining. METHODS: The rat abdominal aorta was clamped to induce ischemia-reperfusion (I-R) injury in the rat bladder dome with or without L-NAME (30 mg/kg, i.p. 30 minutes before the ischemia) according to our previous reports [Saito et al., 1998 Life Sci. 62:PL149-56; Saito and Miyagawa, 1999 J. Urol. 162:1490-5]. Some rats were exposed to 30 minutes of ischemia only, whereas others also received 30 minutes, 3 days, or 7 days of reperfusion after the ischemia. Muscle bath studies with carbachol and 100 mM KCl were performed to confirm the bladder function. TUNEL and hematoxylin and eosin staining were performed in the experimental rat bladders to detect apoptosis. RESULTS: The contractile responses of the rat bladder dome after 30 minutes of ischemia differed slightly, not significantly, from those of controls. Reperfusion (30 minutes) produced significant reductions in the contractile responses to carbachol and KCl in the rat bladders. The treatment with L-NAME significantly reduced the extent of reperfusion injury, as judged by pharmacologic experiments. At 3 and 7 days after the induction of ischemia-reperfusion, the contractile responses were improved compared with the 30-minute reperfusion group. For each duration group, treatment with L-NAME significantly increased the contractile responses compared with the I-R group without L-NAME. Ischemia-reperfusion induced apoptosis, and the peak in TUNEL-positive cells was observed 3 days after the insult. Pretreatment with L-NAME reduced the induction of apoptosis. CONCLUSION: Our data indicated treatment with L-NAME can reduce apoptosis induced by ischemia-reperfusion in the bladder.     

诱导型一氧化氮合酶在舒芬太尼预处理减轻大鼠心肌缺血再灌注损伤中的作用

目的 探讨诱导型一氧化氮合酶(iNOS)在舒芬太尼预处理减轻大鼠心肌缺血再灌注损伤中的作用.方法 成年雄性SD大鼠30只,体重250～330 g,采用随机数字表法,将大鼠随机分为5组(n=6):假手术组(S组)只穿线,不结扎;心肌缺血再灌注组(I/R组)采用结扎左冠状动脉前降支30min,再灌注120 min的方法制备大鼠心肌缺血再灌注损伤模型;舒芬太尼预处理组(SF组)缺血前24 h经尾静脉输注舒芬太尼120μg/kg,输注时间30 min;舒芬太尼预处理+iNOS特异性抑制剂S-甲硫脲组(SF+SMT组)缺血前24 h经尾静脉输注舒芬太尼120μg/kg,缺血前10 min静脉注射SMT 10 mg/kg;SMT组缺血前10 min静脉注射SMT 10 mg/kg.于缺血前30 min、缺血30 min、再灌注120 min时记录HR和MAP,计算RPP(SP× HR).于再灌注120 min时取颈动脉血样2 ml,测定血浆NO浓度,随后取心脏制病理切片,测定缺血危险区(AAR)和梗死区(IS)体积,计算心肌梗死体积(IS/AAR),测定心肌iNOS表达.结果 与S组比较,余4组再灌注120 min时MAP和RPP降低,IS/AAR升高,I/R组和SMT组缺血30 min时MAP和RPP降低(P＜0.05);与I/R组比较,SF组、SF+SMT组和SMT组HR、MAP和RPP差异无统计学意义,SF+SMT组和SMT组IS/AAR和血浆NO浓度差异无统计学意义(P＞0.05),SF组IS/AAR降低,血浆NO浓度和心肌iNOS表达升高(P＜0.05).结论 iNOS参与了舒芬太尼预处理减轻大鼠心肌缺血再灌注损伤的过程.

Abstract：

Objective To investigate the role of inducible nitric oxide synthase (iNOS) in reduction of myocardial ischemia-reperfusion (I/R) injury by sufentanil preconditioning in rats. Methods Thirty adult male SD rats, weighing 250-330 g, were randomly divided into 5 groups ( n =6 each): sham operation group (group S),I/R group, sufentanil preconditioning group (group SF), sufentanil preconditioning + a specific inhibitor of iNOS S-methyl thiourea (SMT) group (group SF+ SMT) and S-methyl thiourea group (group SMT). In I/R,SF,SF+SMT and SMT groups, myocardial I/R was produced by occlusion of left anterior descending coronary artery for 30 min followed by 120 min reperfusion. Group SF received 30 min infusion of sufentanil 120 μg/kg via caudal vein 24 h before ischemia. Group SF + SMT received infusion of sufentanil 120 μg/kg via caudal vein 24 h before ischemia and then SMT 10 mg/kg was injected 10 min before ischemia. In group SMT, SMT 10 mg/kg was injected 10min before ischemia. MAP and HR were recorded at 30 min before ischemia, at 30 min of ischemia and at the end of reperfusion. The rate-pressure product (RPP) was calculated. Arterial blood samples were obtained immediately at the end of reperfusion to determine the plasma concentration of NO. Then the animals were sacrificed and myo cardial tissues were obtained to determine the area at risk (AAR), infarct size (IS) and iNOS expression. IS/AAR was calculated. Results Compared with group S, MAP and RPP were significantly decreased, while IS/AAR was significantly increased at 120 min of reperfusion in the other four groups, and MAP and RPP were significantly decreased at 30 min of ischemia in I/R and SMT groups ( P ＜ 0.05). Compared with group I/R, no significant change was found in HR, MAP and RPP in SF, SF + SMT and SMT groups, and in IS/AAR and plasma NO concentrations in SF + SMT and SMT groups ( P ＞ 0.05), but IS/AAR was significantly decreased, and the plasma NO concentration and iNOS expression were significantly increased in group SF ( P ＜ 0. 05). Conclusion iNOS is involved in reduction of myocardial I/R injury by sufentanil preconditioning in rats.     

Role of inducible nitric oxide synthase in pig liver transplantation

BACKGROUND: Previously, we clarified the role of inducible nitric oxide synthase (iNOS) and the protective effect of an iNOS inhibitor in warm ischemia and reperfusion model. In this study, we investigated whether the same effects would be obtained by iNOS inhibitor in liver transplantation model. MATERIAL AND METHODS: Orthotopic liver transplantation was performed in pigs in the usual manner after about 6 h of cold preservation in University of Wisconsin solution. Aminoguanidine hemisulfate (AG) was used as the iNOS inhibitor and AG was administered intraportally at the dose of 10 mg/kg just after reperfusion. Two experimental groups were subjected, control group (n = 10), and AG group (n = 10). We investigated changes of serum nitrite/nitrate (NOx) and aspartate aminotransferase (AST). Expression of iNOS was examined by immunohistochemistry, including a double immunofluorescnce technique in combination with cofocal laser scanning microscopy. RESULTS: Serum NOx and AST were significantly lower in the AG group. Histological hepatic damage and thrombocyte thrombi were attenuated in the AG group. Expression of iNOS was recognized strongly at Kupffer cells and neutrophils in the centrilobular region of liver after reperfusion by cofocal laser scanning microscopy. Moreover, iNOS staining was attenuated in AG group compared with control group. CONCLUSIONS: These results indicate that hepatic ischemia and reperfusion injury in liver transplantation might be triggered by iNOS expression of Kupffer cells and neutrophils, and attenuated by administration of an iNOS inhibitor. Moreover, AG showed down regulation of iNOS expression after reperfusion.     

Upregulated expression of inducible nitric oxide synthase plays a key role in early apoptosis after anterior cruciate ligament injury.

The reason that the anterior cruciate ligament (ACL) has a very poor healing potential after injury is not well understood. In this study, we investigated the role of nitric oxide (NO) in the apoptotic cell death of ACL cells using a rabbit model and in vitro cell culture. The apoptosis of ACL cells in vivo was analyzed by TUNEL assay and electron microscopy. NO synthase (NOS) expression was observed by immunohistochemical analysis. ACL cells were cultured and the susceptibility to NO-induced apoptosis was tested. Inducible NOS (iNOS) expression after treatment with cytokines was examined by immunohistochemical and RT-PCR analyses. Mitogen-activated protein kinase (MAPK) inhibitors were used for the analysis of downstream signals. A significant number of apoptotic cells were observed on days 1 to 3 after injury; the apoptotic rate returned to the control level by day 7. Upregulation of iNOS in the ACL remnant was observed at day 1. Intraarticular injection of NOS inhibitor suppressed the apoptotic rate. Isolated ACL cells showed much higher susceptibility to NO-induced apoptosis than did medial collateral ligament cells. IL-1beta stimulated ACL cells to upregulate iNOS mRNA and increase NO production. p38 MAPK inhibitor decreased NO-induced apoptosis. Rapid iNOS induction after injury contributes to the high apoptotic rate of ACL cells, and this may partly account for the poor healing capacity of this ligament. iNOS and NO production is suggested to be stimulated by IL-1beta, and NO activates the p38 MAPK pathway and triggers an apoptotic signal in ACL cells.     

诱导性一氧化氮合酶在大鼠肝缺血再灌注损伤中的作用

目的 探讨诱导性一氧化氮合酶（iNOS）在大鼠部分肝缺血再灌注损伤中的作用.方法 选取雄性Sprague-Dawley大鼠30只,体重225～250 g,随机分成氨基胍（AG）组、脂多糖（LPS）组、对照组.每组均按相同方法建立70%的肝缺血再灌注损伤模型（缺血1 h,再灌注6 h）,取再灌注大鼠肝组织及血清样本.氨基胍（AG）组（n=10）：术前30 min尾静脉注射AG 100 mg/kg（质量浓度10 kg/L）;脂多糖（LPS）组（n=10）：术前30 min尾静脉注射LPS 10 mg/kg（质量浓度1 kg/L）;对照组（n=10）：术前30 min尾静脉注射生理盐水（10 μL/kg）.检测血清谷氨酸转氨酶（ALT）水平,实时荧光定量PCR测定肝组织iNOS mRNA的表达,Western blot测定肝组织iNOS蛋白的表达,考马斯法测定肝组织匀浆丙二醛（MDA）含量、超氧化物歧化酶（SOD）活性,HE染色光镜下组织学观察等.结果 AG组与对照组相比,肝组织中iNOS mRNA表达量明显下降（P〈0.05）,iNOS蛋白表达量明显下降（P〈0.05）;ALT和MDA明显下降（P〈0.05）;SOD明显升高（P〈0.05）;肝细胞水肿较轻,排列相对整齐.LPS组与对照组相比,肝组织中iNOS mRNA表达量明显升高（P〈0.05）,iNOS蛋白表达量明显升高（P〈0.01）;ALT和MDA明显升高（P〈0.05）;SOD则明显降低（P〈0.05）;肝细胞水肿,排列紊乱,并且出现水样变性.结论 iNOS 升高会加重缺血再灌注损伤,这一过程可能通过改变氧化还原状态实现.     

Gene expression of inducible nitric oxide synthase in injured spinal cord tissue

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异丙酚对高血压大鼠胸主动脉内皮型一氧化氮合酶和诱导型一氧化氮合酶表达的影响

目的 评价异丙酚对高血压大鼠胸主动脉内皮型一氧化氮合酶(eNOS)和诱导型一氧化氮合酶(iNOS)表达的影响.方法 SD大鼠,雌雄各半,体重240～ 280 g,采用皮下注射去氧皮质酮的方法制备高血压模型,采用随机数字表法,将64只造模成功的大鼠随机分为4组(n＝16):高血压组(H组)、小剂量异丙酚组(P1组)、中剂量异丙酚组(P2组)和大剂量异丙酚组(P3组).P1组、P2组和P3组分别静脉输注异丙酚20、30、40 mg·kg-1·h-13 h,H组给予等容量生理盐水.分别于给药前、给药1h、3h时记录平均动脉压(MAP).给药3h时处死大鼠,摘眼球法采集血样,硝酸还原酶法测定血清一氧化氮(N0)浓度,取胸主动脉,采用RT-PCR和Western blot法测定eNOS mRNA、iNOS mRNA及其蛋白表达水平.结果 与H组比较,P1组、P2组和P3组给药3h时MAP降低,血清NO浓度升高,主动脉eNOS mRNA及其蛋白表达上调,主动脉iNOS mRNA及其蛋白表达下调,且呈剂量依赖性(P<0.05或0.01).结论 异丙酚降低高血压大鼠血压的机制与下调iNOS表达,上调血管内皮细胞eNOS表达,促进NO释放有关.     

Endothelial nitric oxide synthase expression in ischemia-reperfusion injury after living related-donor renal transplantation

Ischemia-reperfusion injury during renal transplantation has been linked to early graft dysfunction and late graft failure. Nitric oxide (NO), produced by NO synthase (NOS), participates in the recovery from ischemia. We correlated the intensity of graft immunoreactivity for the endothelial NOS isoform (eNOS) during early reperfusion with graft function in 25 children receiving grafts from related donors. Renal allograft biopsy specimens were obtained before transplantation, 1 h after renal artery reperfusion, and 1 year after transplantation. Immunohistochemical staining for eNOS occurred mainly within the endothelium of glomerular capillaries and peritubular capillaries as well as in tubule cells. The mean intensity score for eNOS staining (0-9) was 3.0+/-1.4 before transplantation, 4.5+/-1.9 at 1 h, and 3.3+/-1.9 at 1 year (baseline vs 1 h, P<0.05). Creatinine clearance (ml/min) in patients with a 1-h eNOS score of below 5 and of at least 5, respectively, was 77.1+/-28.4 vs 104.3+/-25.3 at 1 month, 78.7+/-33.4 vs 105.2+/-24.4 at 3 months, 64.7+/-30.1 vs 100.1+/-25.3 at 1 year, 58.2+/-31.3 vs 84.7+/-18.8 at 3 years, and 71.2+/-19.7 vs 78.3+/-23.1 at 5 years ( P<0.05 for 1 month, 1 year, and 3 years). We concluded that elevated eNOS expression after reperfusion in living related-donor renal transplantation enhances the recovery from renal ischemia and, consequently, reduces late graft deterioration.     

米非司酮对早孕胎盘绒毛诱导型一氧化氮合酶和细胞凋亡的影响

目的了解米非司酮对早孕绒毛诱导型一氧化氮合酶（iNOS）及滋养层细胞凋亡的影响,进一步探讨米非司酢早孕药物流产机理。方法选择40例早孕药物流产患者（实验组）,采用原位杂交及免疫组化方法检测绒毛中iNOS的表达,细胞凋亡采用3＇-OH末端缺口标记法（TUNEL）显示绒毛滋养细胞中原位凋亡细胞的表达。利用计算机图象分析系纺（CMIAS）,iNOS表达指标以数密度（N／S）及阳性单位（Pu）计算,细胞凋亡以每个统计场凋亡细胞个数（N）、凋亡细胞平均噘光度（OD）、凋亡细胞面密度（凋亡细胞面积／统计场面积,DS／TS）表示,并与40例正常人工流产的绒毛和蜕膜组织（对照组：比较。结果实验组绒毛组织iNOS原位杂交结果显示N／S、Pu分别为0．12±0．010、15．3±2．6,对照组分别为0．02±0．003、3．1±0．5,两组有极显著性差异（P〈0．01）;免疫组化结果显示实验组和对照组N／s、Pu分别为0．09±0．01、10．24±1．55及0．016±0．002、1．26±0．33,差异有显著性。实验组绒毛滋养细胞凋亡指数显著增高,各指标两组比较差异显著。结论米非司酮早孕药物流产与绒毛组织iNOS活性及滋养细胞凋亡有关。     

诱导型一氧化氮合成酶在迟发性血管痉挛中的作用

目的　以大鼠迟发性脑血管痉挛模型为基础研究诱导型一氧化氮合成酶 (iNOS)在迟发性血管痉挛发展中的作用。方法　 3 2只雄性SD大鼠随机分为实验组和对照组 ,实验组枕大池二次注血诱导迟发性脑血管痉挛 ,对照组枕大池注射生理盐水。第 8天行脑血管造影 ,枕大池抽取脑脊液测一氧化氮 (NO)浓度。逆转录 聚合酶链反应 (RT PCR )法和免疫组织化学法测定并评价iNOSmRNA和蛋白质在基底动脉、大脑中动脉和皮质中的表达。结果　颅内动脉血管减影提示对照组颈内动脉颅内段、大脑中动脉 (MCA)明显变细 ,大脑中动脉中段直径 (MD)与镫骨动脉中段直径 (SD)之比衡量大脑中动脉的管径显示实验组MCA管径较对照组MCA管径减少 3 0 %。对照组脑脊液中NO浓度为 (11.70± 2 .62 ) μmol/L ,实验组脑脊液中NO的浓度为(5 5 .67± 12 .84)μmol/L。iNOSmRNA和蛋白质表达于基底动脉、大脑中动脉和皮质 ,其中基底动脉表达最强。 结论　iNOS作为迟发性脑血管痉挛发展中的关键因素参与血管壁的迟发性损伤。     

The effect of nitric oxide in testicular ischemia-reperfusion injury

This experiment was carried out to investigate the effect of endogenous nitric oxide (NO) on the ischemia-reperfusion injury of testis.Testicular ischemia was achieved by twisting the right testis and spermatic cord 1080 counter-clockwise for 30 minutes and reperfusion was allowed for 30 minutes after detorsion of 33 rats. Animals were treated with normal saline in controls just before detorsion,NG-nitro-L-arginine methyl ester (L-NAME), and L-arginine (L-arg) in others.The tissue damage was evaluated with light microscopy, malondialdehyde (MDA) level in tissue, and the blood flow measurement using ¹³³xenon (Xe) clearence technique.MDA indicator of reperfusion injury increased 25% after detorsion when only normal saline was given, L-NAME further increased MDA, L-arginin decreased MDA to control level.Conclusion: L-arginin infusion during the detorsion reduced the reperfusion injury of testis and improved the testicular blood flow after the detorsion.     

Expression of nitric oxide and inducible nitric oxide synthase in acute renal allograft rejection in the rat

BACKGROUND: Recent studies have shown that nitric oxide (NO) synthases, particularly inducible nitric oxide synthase (i-NOS), are induced in acute rejection episodes following heart, liver, pancreas and kidney allotransplantation. Furthermore, tissue and cellular injury has been demonstrated to be mediated by peroxynitrite (ONOO-), a metabolite of NO as well as a potent oxidant. However, a detailed relationship between NO, i-NOS and graft injury in transplantation remains elusive. METHODS: The present study used the following models of renal transplantation in rats: allografts (n = 5, Brown-Norway to Lewis [LEW] rats), isografts (n = 5, LEW to LEW) and allografts treated with aminoguanidine (AG), an i-NOS inhibitor (n = 5). Blood urea nitrogen (BUN), serum creatinine (SCr) and urinary and serum nitrosocompounds (NOx) were measured on days 2, 4 and 7 post-transplant. Western blot analysis of i-NOS protein expression and measurement of i-NOS activity were carried out in grafts harvested on Day 7, along with immunohistochemical and histopathological examinations. RESULTS: In the allograft group, both BUN and SCr levels increased markedly on Day 7, in parallel with a sharp increase in NOx. A band stained by anti-i-NOS antibody was detected at approximately 130 kDa, along with high levels of i-NOS activity and diffusely distributed i-NOS-positive cells (macrophages). Histologically, an acute rejection episode was confirmed (Grade 3 according to Banff classifications). In the AG group, reduced renal function and graft injury were significantly less severe than in the allograft group. CONCLUSIONS: In rat renal allograft acute rejection, markedly increased levels of serum NOx were observed, along with enhanced tissue i-NOS activity, together resulting in graft injury. AG administration suppressed the increase of serum NOx levels, with concomitant mitigation of tissue injury and renal function impairment.     

Hepatic and mesenteric nitric oxide synthase expression in a rat model of CCl(4)-induced cirrhosis

BACKGROUND: Cirrhosis and portal hypertension are frequently linked with changes in expression of nitric oxide synthase (NOS) and/or endotoxaemia. AIMS: This study tested the following hypothesis: that inducible (i)NOS activity is increased within the visceral circulation concurrently with decreased constitutive (c)NOS activity in the hepatic sinusoids and that the concentration of NO metabolites in portal blood is consequent on endotoxin concentration. MATERIALS AND METHODS: Plasma concentrations of (nitrite + nitrate) and endotoxin, together with hepatic and mesenteric NOS activity (arginine/citrulline method) and protein expression (histochemistry) plus portal and arterial blood pressure, were determined in rats made severely cirrhotic by intragastric CCl(4) over 14 weeks (n = 6) compared with age-matched controls (n = 5). The concentrations of [nitrite + nitrate] and endotoxin in portal plasma were also directly compared in rats made cirrhotic for a period of 8-14 weeks (n = 10). RESULTS: In rats with advanced cirrhosis, arterial [nitrite + nitrate] was 93.1 (22.4) micromol/L (mean, SEM) compared with 29.1 (6.1) micromol/L in controls (P < 0.05); portal plasma [NO(2)(-) + NO3(-)] was 127.1 (27.2) compared with 24.7 (4.7) micromol/L in controls (P < 0.05). Cirrhotic rats had higher endotoxin concentration in plasma compared with controls (systemic: 85.0 (24.5) versus 1.7 (0.2) EU/ml, P < 0.05; portal: 180.3 (47.9) versus 1.7 (0.2) EU/ml, P < 0.05). The same severely cirrhotic rats possessed decreased cNOS activity in liver (2.95 [0.40] versus 5.29 [0.85] pmol/min/g; P < 0.05) and increased iNOS activity in mesentery (4.83 [1.23] versus 1.47 [0.15] pmol/min/g; P < 0.05) compared with controls. Histochemical observations confirmed these findings. Rats given CCl(4) for a period of 8-14 weeks possessed high endotoxin concentration in portal plasma, with correspondingly high [nitrite + nitrate] (r(2) = 0.954; P < 0.001). CONCLUSIONS: An endotoxin-induced increase in mesenteric iNOS activity and a decrease in hepatic cNOS activity may account for, respectively, the hyperdynamic visceral circulation and the increased intrahepatic resistance of cirrhosis.     

异丙酚对缺氧复氧后大鼠海马神经元诱生型一氧化氮合酶表达的影响

目的 观察异丙酚对原代培养海马神经元缺氧复氧后诱生型一氧化氮合酶(iNOS)表达及存活率的影响。方法 原代培养SD大鼠海马神经元随机分为四组:正常对照组、缺氧组、缺氧 异丙酚4μg/ml组、缺氧 异丙酚12μg/ml组。MTT法测定体外缺氧4h复氧24h后海马神经元的存活率,免疫细胞化学法测定iNOS表达的程度。结果 与缺氧组比较,两异丙酚组缺氧复氧后海马神经元的存活率提高(P<0.05),iNOS表达的灰度值降低(P<0.05或0.01),iNOS阳性表达率降低,并呈剂量依赖性(P<0.01)。结论 异丙酚可降低大鼠海马神经元缺氧复氧后iNOS的表达,提高存活率。