急性心肌梗死大鼠心脏一氧化氮合酶表达的改变

目的 通过建立大鼠心肌梗死模型，观察急性心肌梗死对大鼠心脏内皮型一氧化氮合酶mRNA和诱导型一氧化氮合酶蛋白表达的影响。方法48只健康成年SD大鼠（体重200～250g）随机分为假手术组和缺血组，取1、2、8和24h四个不同时间点观察。采用开胸结扎冠状动脉左前降支建立心肌缺血模型，逆转录聚合酶链反应检测大鼠心肌梗死后1、2及24h三个时段缺血心肌内皮型一氧化氮合酶mRNA的表达；免疫组织化学染色检测冠状动脉结扎后8h缺血心肌诱导型一氧化氮合酶蛋白的表达。结果冠状动脉结扎后2h，缺血组大鼠缺血心肌组织内皮型一氧化氮合酶mRNA表达下降（P〈0．05），并持续至结扎后24h；结扎后24h组内皮型一氧化氮mRNA的表达与结扎后2h组相比无显著性差异（P〉0．05）。冠状动脉结扎后8h，梗死区存活心肌组织细胞诱导型一氧化氮合酶蛋白大量表达，而假手术组未见诱导型一氧化氮合酶蛋白表达。结论正常大鼠心肌组织有内皮型一氧化氮合酶基因表达，无诱导型一氧化氮合酶蛋白表达。在心肌梗死早期缺血心肌内皮型一氧化氮合酶mRNA表达减少。心肌急性缺血刺激早期诱导大鼠缺血心肌组织诱导型一氧化氮合酶蛋白大量表达。     

家兔腹主动脉球囊导管损伤后血管平滑肌细胞凋亡和一氧化氮合成酶基因表达

采用脱氧核苷酸转移酶介导的缺口末端标记法，观察兔腹主动脉经球囊导管损伤后0、8、24h，3天和5天时血管中膜平行肌细胞和外膜细胞凋亡的变化．提取血管组织RNA，采用反转录一多聚酶链反应测定一氧化氮合成酶基因表达。结果表明，血管损伤后8h可见中膜平滑肌细胞凋亡增加，24h达高峰，3天和5天仍维持在较高水平，外膜细胞凋亡无明显改变。而诱导型一氧化氮合成酶基因表达亦于8h明显增加，在24h达最高，与细胞凋亡存在平行关系。提示中膜平滑肌细胞凋亡是球囊导管损伤血管的一个早期反应，诱导型一氧化氮合成酸可能参与了此过程。     

尾加压素Ⅱ对大鼠胸主动脉及肠系膜动脉各部分产生一氧化氮的影响

目的取离体大鼠胸主动脉和肠系膜动脉,将UⅡ与血管各层(内、中、外膜)组织共同孵育,观察UⅡ对NOS活性及NO生成的影响,以探讨UⅡ的血管活性效应的机理.材料与方法分离雄性SD大鼠胸主动脉和肠系膜动脉,将胸主动脉各层分离,上述组织分别加入不同浓度尾加压素Ⅱ在37 ℃、通以95%O2～5%CO2气体条件下进行血管组织孵育,孵育时间为2小时和4小时.测定孵育液中亚硝酸盐含量及组织中一氧化氮合酶活性.取最适浓度及最佳时间点,孵育血管后进行诱导型一氧化氮合酶免疫组织化学染色进行表达定位.结果尾加压素Ⅱ(10-9、10-8 mol/L)可以引起胸主动脉外膜一氧化氮生成增加,一氧化氮合酶活性增强,P<0.05,免疫组化证实血管外膜诱导型一氧化氮合酶表达呈强阳性,孵育2小时和4小时没有显著性差异,P>0.05;10-10～10-8 mol/L浓度尾加压素Ⅱ可以浓度依赖性、时间依赖性刺激肠系膜动脉一氧化氮生成,免疫组化证实诱导型一氧化氮合酶在血管外膜和中膜表达增加,但NOS活性没有明显变化.结论 UⅡ可以刺激胸主动脉和肠系膜动脉血管外膜产生NO.UⅡ对胸主动脉及肠系膜动脉NO/NOS系统影响不同,可能是UⅡ作用于血管引起不同生物学效应的机制之一.     

老年大鼠主动脉对胰岛素敏感性的改变及其机制

目的观察大鼠主动脉对胰岛素敏感性的增龄改变并分析其可能机制。方法老年组采用老年（18月龄）SD大鼠20只,随机选取成年（15周龄）SD大鼠20只为对照组。采用离体血管灌流技术,观察胸主动脉对胰岛素反应性的变化,并同时测定两组主动脉血管一氧化氮（NO）释放量及血管一氧化氮合酶（eNOS）活性;免疫组织化学法及Western Blot法检测老年组及成年组胸主动脉eNOS的蛋白表达变化。结果胰岛素可以浓度依赖性舒张成年大鼠胸主动脉,舒血管作用具有内皮依赖性。与之相比,老年大鼠主动脉对胰岛素的舒张反应显著下降（P<0.05）。同时发现,与成年组相比,老年组NO释放量以及eNOS活性显著降低（P<0.05）,免疫组织化学染色显示老年组主动脉eNOS的蛋白表达显著降低;而Western Blot检测发现老年组血管eNOS磷酸化水平显著降低（P<0.05）。结论血管组织内源性eNOS-NO系统活性下降可能是衰老导致的血管胰岛素敏感性下降的重要机制。     

糖尿病肾病大鼠肾组织中神经胶质瘤相关蛋白-2表达及意义的观察

目的 观察神经胶质瘤相关蛋白-2(GLIPR-2)在DN大鼠肾组织中的表达及意义. 方法 将Wistar大鼠30只分为正常对照(NC)组和DN组.造模成功16周末检测两组血肌酐(Scr)、24 hUAlb水平.HE染色观察肾组织病理变化.采用免疫组织化学方法检测肾间质小管GLIPR-2表达水平.结果 DN组Scr、24 hUAlb较NC组升高[(37.05±1.41) vs (1.22±0.83)μmol/L;(13.31±0.67)vs(3.10±0.49)mg/24 h,P＜0.05].GLIPR-2主要表达在DN组肾间质小管上皮细胞中,GLIPR-2蛋白表达水平与Scr、24 hUAlb呈正相关(r=0.905、0.913,P＜0.05). 结论 DN大鼠肾间质小管GLIPR-2蛋白表达水平升高,与Scr、24 hUAlb呈正相关.     

大鼠内毒素性急性肝损伤后肝细胞凋亡与炎性因子的表达

目的 探讨脂多糖诱导D-氨基半乳糖胺致敏大鼠急性肝损伤肝细胞凋亡、炎性因子表达情况及其发生机制.方法 56只大鼠分为0 h对照组与1、2、4、6、24和48 h脂多糖+D-氨基半乳糖胺处理组.在相应时间点处死大鼠后收集肝组织及血清,肝组织苏木精-伊红染色后光学显微镜下观察ELISA法检测血清细胞因子表达;反转录(RT)-PCR法检测TNF-α、IL-β、诱导型一氧化氮合酶(iNOS)和p53基因表达;收集24 h肝组织用底物显色法检测Caspase-3、8、9,12活性.组间比较用方差分析.结果 经药物处理后,肝组织出现碎片状坏死、大量炎性细胞浸润等表现,从6 h开始,24 h和48 h显著加重.血清TNF-α浓度在1 h处理组为(727.8±261.3)ng/L,显著高于对照组及其他处理组(F=49.82,P<0.01),2 h处理组为(156.4±52.2)ng/L,显著低于1 h组,但高于对照组(F:30.23,P<0.01);血清IL-β浓度逐渐上升,24 h处理组最高,为(360.5±121.6)ng/L(F=18.61,P<0.01).24 h处理组肝组织Caspase-3、8、9、12活性明显高于对照组(F=84.96,P<0.01).iNOS基因在对照组无表达,药物作用后6 h达最高,24 h和48 h则显著下降(F=34.07,P<0.01);p53基因在24 h和48 h处理组表达明显增高(F=37.43,P<0.01);TNF-α和IL-1β基因表达均较对照组升高(F=2.94,P<0.05),其峰值均出现在1 h处理组.结论 小剂量脂多糖可诱导D-氨基半乳糖胺致敏大鼠发生急性肝损伤;Caspase-3、8、9、12活性明显增强是其特征性改变之一;肝损伤的发生与TNF-α、iNOS和p53基因早期高水平表达有密切关系.     

低剂量促红细胞生成素对大鼠肾移植物急性炎症反应的影响

目的探讨促红细胞生成素(EPO)对大鼠肾移植物急性炎症反应的影响及可能机制。方法建立单侧大鼠同系基因肾移植模型,随机分为假手术组、肾移植模型组、EPO给药组。PAS染色观察肾脏病理学变化,免疫组织化学检测移植肾组织中单核/巨噬细胞表面标志物(ED)-1、诱导型一氧化氮合成酶(i NOS)、半胱氨酸-天冬氨酸蛋白酶(Caspase)-3表达水平。结果与肾移植模型组相比,EPO给药组急性炎症反应明显减轻,肾组织病理学损伤明显改善,ED-1、i NOS、Caspase-3的表达水平也明显减低。结论 EPO可通过减轻大鼠肾移植后炎症细胞浸润、细胞因子释放,抗氧化、抑制凋亡等作用,减轻大鼠肾移植后的急性炎症反应,从而保护肾移植后肾移植物的功能。     

尾加压素Ⅱ对大鼠胸主动脉及肠系膜动脉各部分产生一氧化氮的影响

目的　取离体大鼠胸主动脉和肠系膜动脉 ,将UⅡ与血管各层 (内、中、外膜 )组织共同孵育 ,观察UⅡ对NOS活性及NO生成的影响 ,以探讨UⅡ的血管活性效应的机理。材料与方法　分离雄性SD大鼠胸主动脉和肠系膜动脉 ,将胸主动脉各层分离 ,上述组织分别加入不同浓度尾加压素Ⅱ在 37℃、通以 95 %O2 ～ 5 %CO2 气体条件下进行血管组织孵育 ,孵育时间为 2小时和 4小时。测定孵育液中亚硝酸盐含量及组织中一氧化氮合酶活性。取最适浓度及最佳时间点 ,孵育血管后进行诱导型一氧化氮合酶免疫组织化学染色进行表达定位。结果　尾加压素Ⅱ (1 0 - 9、1 0 - 8mol/L)可以引起胸主动脉外膜一氧化氮生成增加 ,一氧化氮合酶活性增强 ,P <0 0 5 ,免疫组化证实血管外膜诱导型一氧化氮合酶表达呈强阳性 ,孵育 2小时和 4小时没有显著性差异 ,P >0 0 5 ;1 0 - 1 0 ～ 1 0 - 8mol/L浓度尾加压素Ⅱ可以浓度依赖性、时间依赖性刺激肠系膜动脉一氧化氮生成 ,免疫组化证实诱导型一氧化氮合酶在血管外膜和中膜表达增加 ,但NOS活性没有明显变化。结论　UⅡ可以刺激胸主动脉和肠系膜动脉血管外膜产生NO。UⅡ对胸主动脉及肠系膜动脉NO/NOS系统影响不同 ,可能是UⅡ作用于血管引起不同生物学效应的机制之一。     

骨化三醇对阿霉素肾病大鼠肾组织nephrin表达的影响

目的研究骨化三醇对阿霉素(ADR)肾病大鼠肾组织损伤及足细胞裂隙隔膜分子(nephrin)表达的影响。方法6周龄雄性SD大鼠分为:正常对照组(Control组),ADR肾病组,骨化三醇治疗组(ADR+VD组)。大鼠模型以单次尾静脉注射阿霉素(7.5 mg/kg),治疗组大鼠灌胃法服用0.25μg/(kg·d)骨化三醇。所有大鼠在处死前称重、留尿检测24 h尿蛋白(UP),留血检测血清白蛋白(Alb)、尿素氮(BUN)、肌酐(Cre);苏木素-伊红(HE)染色、马松三色染色(Masson)、透射电镜(TEM)观察肾病理学结构;免疫荧光观察nephrin的定位;免疫组织化学染色半定量检测nephrin的表达;Western印迹检测nephrin蛋白表达。结果与ADR肾病组比,ADR+VD组24 h UP、BUN、Cre、肾脏指数显著降低(P<0.05),Alb水平显著升高(P<0.05);组织化学染色显示骨化三醇减轻肾组织系膜基质增生及纤维化;TEM显示骨化三醇修复足细胞损伤;免疫组化与Western印迹检测结果显示高度一致性,骨化三醇升高ADR引起的nephrin表达降低(P<0.05)。结论骨化三醇能够减轻ADR引起的肾损伤和大量蛋白尿,这与改善肾足细胞标记分子nephrin低表达密切相关。     

褪黑素对内毒素血症大鼠胸主动脉诱导型一氧化氮合酶活性的影响

目的探讨诱导型一氧化氮合酶在褪黑素改善脂多糖诱导的体循环血管反应性失调中的作用。方法实验分为溶剂对照组、脂多糖组、脂多糖+褪黑素组和褪黑素组。制备离体胸主动脉环,应用血管张力检测技术检测各组血管环在诱导型一氧化氮合酶抑制剂氨基胍、或非特异性一氧化氮合酶抑制剂L-硝基精氨酸孵育前后对苯肾上腺素和乙酰胆碱的反应性变化;应用扫描电镜技术观察血管内皮超微形态学改变;酶法检测血管组织匀浆中诱导型一氧化氮合酶活性。结果褪黑素可显著改善脂多糖诱导的胸主动脉对缩血管剂苯肾上腺素的低反应性;氨基胍孵育后,脂多糖组和脂多糖+褪黑素组对苯肾上腺素的收缩反应分别提高了51.43%和24.53%,差异有显著性(P<0.01);与脂多糖组比较,脂多糖+褪黑素组的诱导型一氧化氮合酶活性显著降低(P<0.05)。结论褪黑素对诱导型一氧化氮合酶有一定的抑制作用,这可能是褪黑素改善内毒素血症大鼠的血管反应性失调的机制之一。     

脓毒症时各型一氧化氮合酶在大鼠心脏中的表达及其可能机制

目的研究脓毒症时各型一氧化氮合酶(NOS)在心脏中的损伤作用及其机制。方法成年雄性Wistar大鼠腹腔注射脂多糖(LPS)制备脓毒症模型。应用多导生理仪监测大鼠心功能变化;用分光光度计法测定大鼠心肌组织NOS的活性;用RT-PCR和Western blot对大鼠心肌组织各型NOS的表达进行半定量分析。结果给予LPS后6h大鼠心肌收缩和舒张功能受损下降,心肌中iNOS的活性明显升高,eNOS和nNOS(合称cNOS)活性减弱;RT-PCR和Western blot结果显示,给予LPS后cNOS的表达减少,给予LPS后iNOS表达量明显增加。结论脓毒症时,iNOS、nNOS和eNOS的表达和活性发生改变;心肌细胞上iNOS表达及活性升高,这些变化可能在心功能降低中发挥作用。     

Regulation of the urea cycle enzyme genes in nitric oxide synthesis

Nitric oxide (NO) is synthesized from arginine by nitric-oxide synthase (NOS), and citrulline that is generated can be recycled to arginine by argininosuccinate synthase (AS) and argininosuccinate lyase (AL). Rats were injected with bacterial lipopolysaccharide (LPS) and expression of the inducible isoform of NOS (iNOS), AS and AL was analysed. In RNA blot analysis, iNOS mRNA was induced by LPS in the lung, heart, liver and spleen, and less strongly in the skeletal muscle and testis. AS and AL mRNAs were induced in the lung and spleen. Kinetic studies showed that iNOS mRNA increased rapidly in both spleen and lung, reached a maximum 2–5 h after the treatment, and decreased thereafter. On the other hand, AS mRNA increased more slowly and reached a maximum in 6–12 h (by about 10-fold in the spleen and 2-fold in the lung). AL mRNA in the spleen and lung increased slowly and remained high upto 24 h. In immunohistochemical analysis, macrophages in the spleen that were negative for iNOS and AS before LPS treatment were strongly positive for both iNOS and AS after this treatment. As iNOS, AS and AL were co-induced in rat tissues and cells, citrulline–arginine recycling seems to be important in NO synthesis under the conditions of stimulation.Arginine is a common substrate of NOS and arginase. Rat peritoneal macrophages were cultured in the presence of LPS and expression of iNOS and liver-type arginase (arginase I) was analysed. mRNAs for iNOS and arginase I were induced by LPS in a dose-dependent manner. iNOS mRNA appeared 2 h after LPS treatment and increased up to a near-maximum at 8–12 h. On the other hand, arginase I mRNA began to increase after 4 h with a lag time and reached a maximum at 12 h. Immunoblot analysis showed that iNOS and arginase I proteins were also induced. Induction of iNOS and arginase I mRNAs were also observed in LPS-injected rats in vivo. Thus, arginase I appears to have an important role in downregulating NO synthesis in murine macrophages by decreasing the availability of arginine.A cDNA for human arginase II, an arginase isozyme, was isolated. A polypeptide of 354 amino acid residues including the putative NH₂-terminal presequence for mitochondrial import was predicted. It was 59% identical with arginase I. mRNA for human arginase II was present in the kidney and other tissues but was not detected in the liver. Arginase II mRNA was co-induced with iNOS mRNA in murine macrophage-like RAW 264.7 cells by LPS. This induction was enhanced by dexamethasone and dibutyrul cAMP, and was prevented by interferon-γ.These results indicate that NO synthesis is regulated by arginine-synthesizing and -degrading enzymes in a complicated manner.     

Role of neuronal nitric oxide synthase and inducible nitric oxide synthase in intestinal injury in neonatal rats

AIM: To investigate the dynamic change and role of neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS) in neonatal rat with intestinal injury and to define whether necrotizing enterocolitis (NEC) is associated with the levels of nitric oxide synthase (NOS) in the mucosa of the affected intestine tissue. METHODS: Wistar rats less than 24 h in age received an intraperitoneal injection with 5 mg/kg lipopolysaccharide (IPS). Ileum tissues were collected at 1, 3, 6, 12 and 24 h following LPS challenge for histological evaluation of NEC and for measurements of nNOS and iNOS. The correlation between the degree of intestinal injury and levels of NOS was determined. RESULTS: The LPS-injected pups showed a significant increase in injury scores versus the control. The expression of nNOS protein and mRNA was diminished after LPS injection. There was a negative significant correlation between the nNOS protein and the grade of median intestinal injury within 24 h. The expression of iNOS protein and mRNA was significantly increased in the peak of intestinal injury. CONCLUSION: nNOS and iNOS play different roles in LPS-induced intestinal injury. Caution should be exerted concerning potential therapeutic uses of NOS inhibitors in NEC.     

The role of nitric oxide in endotoxin-induced cardiodepression

OBJECTIVE:

To determine the participation of inducible nitric oxide synthase (iNOS) in cardiodepressive phenomena during late preconditioning caused by subtoxic doses of lipopolysaccharide (LPS).

METHODS:

Spontaneously beating hearts isolated from male Wistar rats (350 g to 400 g), intact or preconditioned with LPS (0.25 mg/kg given intraperitoneally 18 h before heart excision), were used to measure contractile performance during 30 min of ischemia and 40 min of reperfusion in the Langendorff mode. For selective iNOS blockade, hearts were perfused with phenylene-1,3-bis(ethane-2-isothiourea) (50 nmol/L). Expression of iNOS (determined using Western blotting) and NOS activities were determined in frozen myocardial tissues.

RESULTS:

Subtoxic doses of LPS caused iNOS induction in the heart and depression of contractile function, but improved heart postischemic recovery. In all groups of animals, expression of iNOS was higher in the right than left ventricles. Ischemia and postischemic reperfusion of intact heart intensified production of nitric oxide (NO), predominantly by iNOS. The preconditioning led to iNOS activation during ischemia in the left ventricle and iNOS depression in the right ventricle, owing to feedback caused by the initially higher iNOS expression and activity in the right ventricle. Postischemic reperfusion diminished NOS activities in preconditioned myocardial tissues. Blockade of iNOS significantly slowed preconditioned heart recovery and partially restored left ventricular developed pressure, but only after 20 min of reperfusion.

CONCLUSIONS:

iNOS-produced NO plays a role in the development of delayed cardioprotection and cardiodepressive effects (in part) after extravasal administration of a minimal dose of endotoxin.     

Cytokines, endotoxin, and glucocorticoids regulate the expression of inducible nitric oxide synthase in hepatocytes.

Nitric oxide (NO.) is a short-lived mediator which can be induced in a variety of cell types and produces many physiologic and metabolic changes in target cells. The inducible or high-output NO. synthase (NOS) pathway was first characterized in macrophages activated by lipopolysaccharide (LPS) and interferon gamma (IFN-gamma). Hepatocytes also express an inducible NOS following exposure to the combination of endotoxin (LPS) and tumor necrosis factor (TNF), interleukin 1 (IL-1), and IFN-gamma. In this study, to identify which of these cytokines, if any, was acting to induce the gene expression for hepatocyte NOS, we measured the levels of rat hepatocyte NOS mRNA by Northern blot analysis after stimulation by various combinations of endotoxin and cytokines in vitro. We found the mRNA for hepatocyte NOS to be a single band at approximately 4.5 kilobases which was maximally up-regulated (approximately 70-fold) by the combination of TNF, IL-1, IFN-gamma, and LPS. Abundance of NOS mRNA peaked 6-8 hr after stimulation and then declined by 25% at 24 hr. Unstimulated hepatocytes in vitro showed only a trace mRNA band after prolonged autoradiographic exposure. As single agents, TNF and IL-1 were the most effective inducers of hepatocyte NOS mRNA. Combinations of two or three stimuli revealed strong synergy between TNF, IL-1, and IFN-gamma. The increased mRNA levels correlated with elevated nitrogen oxide release and cGMP levels in the culture supernatants. Dexamethasone and cycloheximide inhibited induction of mRNA for hepatocyte NOS in a dose-dependent fashion. The addition of NG-monomethyl-L-arginine had no effect on mRNA levels but effectively blocked NO. formation. The inducible hepatocyte NOS mRNA was also detected in rat hepatocytes following chronic hepatic inflammation triggered by Corynebacterium parvum injection in vivo. These data demonstrate that the inducible NOS is functional in rat hepatocytes both in vitro and in vivo and that this pathway is under complex control. Endotoxin and inflammatory cytokines act synergistically to up-regulate gene expression for hepatocyte NOS, whereas glucocorticoids down-regulate the mRNA.     

Rat liver slices as a tool to study LPS-induced inflammatory response in the liver

BACKGROUND/AIMS: Inflammation in the liver is a complex interaction between parenchymal and non-parenchymal cells, and therefore can not be studied in vitro in pure cultures of these cells. METHODS: We investigated whether Kupffer cells in the liver slice are still responsive to an inflammatory stimulus of lipopolysaccharide (LPS), and evoke an inflammatory response in the hepatocytes. RESULTS: TNFalpha, IL-1beta and IL-10 were significantly elevated in culture medium of LPS-stimulated rat liver slices. Nitric oxide (NO) production of LPS-treated slices gradually increased from 5 to 24 h (24 h: 81+/-5 microM vs. 14+/-2 microM in control P < 0.05), paralleled by inducible nitric oxide synthase (iNOS) in the hepatocytes, iNOS mRNA was induced after 3 h. NO production but not iNOS induction was significantly inhibited by NOS inhibitors S-methylisothiourea and N(G)-nitro-L-arginine methylester. Both pentoxifylline and dexamethasone inhibited TNFalpha and IL-1beta production, albeit to a different extent, iNOS induction and, as a result thereof, NO production. CONCLUSIONS: These results imply that non-parenchymal cells in liver slices are viable and can be activated by LPS. In addition, it is concluded that the upregulation of iNOS in hepatocytes by LPS is caused by cytokines produced by Kupffer cells because inhibition of TNFalpha and IL-1beta production attenuated iNOS induction.     

Mechanisms underlying growth hormone effects in augmenting nitric oxide production and protein tyrosine nitration during endotoxin challenge

The present study defined the effects of GH administration on components of the nitric oxide (NO)-generating cascade to account for observed increases in NO production and protein nitration after an immune challenge. Calves were assigned to groups with or without GH treatment (100 microg GH/kg body weight or placebo im, daily for 12 d) and with or without low-level endotoxin [lipopolysaccharide (LPS), 2.5 microg/kg, or placebo, iv]. Plasma was obtained for estimation of NO changes as [NO(2)(-) + NO(3)(-)] (NO(x)). Transcutaneous liver biopsies were collected for measurement of protein tyrosine nitration, cationic amino acid transporter (CAT)-2 mRNA transporter, and constitutive NO synthase (cNOS), inducible NOS (iNOS), and arginase activity. Liver protein nitration increased more than 10-fold 24 h after LPS and an additional 2-fold in animals treated with GH before LPS. GH increased plasma NO(x) after LPS to levels 27% greater than those measured in non-GH-treated calves. LPS increased CAT-2 mRNA after LPS; GH was associated with a 24% reduction in CAT-2 mRNA content at the peak time response. cNOS activity was 3-fold greater than iNOS after LPS. NOS activities were increased 140% (cNOS) at 3 h and 169% (iNOS) at 6 h, respectively, after LPS; GH treatment increased cNOS activity and the phosphorylation of endothelial NOS after LPS more than 2-fold over that measured in non-GH-treated calves. The data suggest that an increased production of nitrated protein develops in the liver during low-level, proinflammatory stress, and nitration is increased by GH administration through a direct effect on the competing activities of NOS and arginase, modulatable critical control points in the proinflammatory cascade.     

Lipopolysaccharide-induced diaphragmatic contractile dysfunction in mice lacking the inducible nitric oxide synthase.

The goal of this study was to evaluate the importance of the inducible nitric oxide synthase (iNOS) in lipopolysaccharide (LPS)-induced diaphragmatic contractile dysfunction. Many investigators have proposed that iNOS induction in the ventilatory and limb muscles of animals injected with Escherichia coli LPS leads to impaired muscle contractility and increased fatigability. We tested this proposal by examining wild-type mice and iNOS-deficient (iNOS knockout) mice. Both types of mice were injected with either saline (control) or E. coli LPS and killed after 12 h. Diaphragm nitric oxide synthase (NOS) activity, NOS expression, and muscle contractility were assessed with L-citrulline assay, immunoblotting, and in vitro bath preparation, respectively. LPS injection in wild-type mice induced iNOS protein expression and augmented total diaphragmatic NOS activity, which coincided with impaired muscle force generated at frequencies higher than 30 Hz. In iNOS knockout mice, injection of LPS augmented constitutive muscle NOS activity, upregulated the expression of the neuronal NOS (nNOS), but elicited a significantly greater decline in force generated in response to high frequency of stimulation compared with wild-type animals. We conclude that iNOS may play a protective role in attenuating the inhibitory influence of LPS on muscle contractility.     

肝细胞一氧化氮合酶的诱导及动力学研究

目的对内毒素和几种细胞因子诱导肝细胞一氧化氮合酶的协同效应及酶动力学参数进行研究.方法原位预灌流和段原酶循环灌流大鼠肝脏、分离肝实质细胞,观察内毒素、IFN-Y、IFN-α、TNFα、IL-lβ、IL-6及不同组合对肝细胞一氧化氮合酶活性、cGMP及NO2-+NO3的影响,分析酶动力学特征及皮质甾与酶诱导的量效关系.结果内毒素+IFN-v+TNFα+IL-lβ(IL-6)组合诱导酶表达效应最显著;酶参数分析显示Km、Vmax分别为108μmol/L和2632pmol@min1mg-1蛋白质,竞争性抑制剂L-NMMA、L-NNA作用的Ki分别为056μmolL及094μmol/L;诱导时间进程显示iNOS活性表达在9h达到峰值,但cGMP及NO2-NO3-的释放持续增加可维持至l8h;地塞米松和氢化可的松抑制肝细胞酶诱导的IC50分别为35×10-8mol/L和26×10-0mol/L.结论肝细胞诱导性一氧化氮合酶的表达依赖特异多细胞因子协同作用,这种可诱导性特征可能在内毒素血症和败血症休克发病机制中具有重要意义.