Roles of Neuronal Nitric Oxide Synthase and Inducible Nitric Oxide Synthase in Intestinal Transplantation of Rats

Objective

The study was designed to evaluate the role of neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS) in ischemia-reperfusion injury (IRI) and acute rejection (AR) in rat intestinal transplantation, by administration of nitric oxide inhibitor N^G-nitro-L-arginine methyl ester (LNAME).

Materials and methods

Rats that underwent orthotopic intestinal transplantation were assigned to 2 sets of groups: (1) iso-geneic group (Lewis-Lewis), L-NAME 0 mg/kg/d group (1-1), 4 mg/kg/d (group 1-2), or 8 mg/kg/d (group 1-3) injected intraperitoneally or (2) allogeneic group (Dark Agouti-Lewis), L-NAME 0 mg/kg/d (group 2-1) or 8 mg/kg/d (group 2-2) injected intraperitoneally. We examined survival times, light microscopy as well as maltose absorption tests. The nNOS and iNOS activities were measured by immunohistochemical methods.

Results

Histologic examination showed inhibited iNOS activity compared with group l-l, and Park scores decreased significantly in group 1-2 at 30 minutes after reperfusion (1.42 ± 0.38 vs 2.58 ± 0.49, P < .01). Both iNOS and nNOS activities were inhibited and Park scores increased significantly in group 1-3 from 30 minutes to day 3 after reperfusion (P < .0l). nNOS activity decreased and iNOS activity increased among group 2-1 during AR. Compared with group 2-1, iNOS activity was inhibited, progression of AR delayed, and survival significantly prolonged in group 2-2 (10.17 ± 0.98 vs 6.83 ± 0.75, P < .01).

Conclusion

This study suggested that decreased nNOS and increased iNOS activity both contributed to IRI and AR. More importantly, nNOS more importantly than iNOS activity was closely related to graft structure and function.     

Inducible Nitric Oxide Synthase: From Cloning to Therapeutic Applications

Elucidation of the many roles of nitric oxide (NO) in homeostasis and disease states has uncovered many areas where manipulation of NO production would be of therapeutic benefit. Recent advances in gene transfer technology and the cloning of the inducible nitric oxide synthase (iNOS) gene have led to the development of strategies for gene therapy to increase NO production for the treatment of disorders ranging from vascular restenosis to impaired wound healing. This review summarizes the current status of iNOS gene therapy research.     

Expression of Inducible Nitric Oxide Synthase in Human Lungs

The aim of this study was to investigate the expression of inducible nitric oxide synthase (iNOS) in lungs of patients with or without adult respiratory distress syndrome (ARDS). We compared the expression of iNOS by immunohistochemical analysis and polymerase chain reaction in the human lungs collected during open-lung biopsy or at autopsy. The expression of iNOS mRNA was present in all lung samples; however, only 3 out of 11 lung samples showed weak staining for iNOS. Although the involvement of nitric oxide in animal models of ARDS is reported, production of nitric oxide in human lungs is still controversial. The data presented here suggest that human lungs express iNOS mRNA but that the production of iNOS protein may be tightly regulated and is expressed in pulmonary inflammation.     

Nitric Oxide Produced by Inducible Nitric Oxide Synthase Delays Gastric Emptying in Lipopolysaccharide-treated Rats

Background: Endotoxin induces nitric oxide synthase (NOS), resulting in relaxation of gastric smooth muscle. The authors examined the effect of NO produced in response to lipopolysaccharide (LPS) treatment on gastric emptying in rats, and they also examined the effects of a selective inhibitor of inducible NOS (iNOS), aminoguanidine, and a suppressor of iNOS gene expression, dexamethasone.

Methods: Male Wistar rats weighing 200-250 g were used. LPS-treated rats received LPS (0.2-10 mg/kg) diluted in physiologic saline intraperitoneally. Before and at different intervals up to 8 h after administration of LPS, measurements of gastric emptying were performed in groups of 3-5 rats, by determining the amount of phenol red remaining in the stomach 20 min after intragastric instillation. In additional group of LPS (2 mg/kg)-treated rats, the gastric fundus was isolated 6 h after administration, and the tension changes in response to L-arginine, a substrate for NOS, and electrical transmural stimulation (3 Hz, 5 s) were recorded isometrically.

Results: (1) Gastric emptying was delayed by pretreatment with LPS in a dose- and time-dependent fashion (reduction from 68 +/- 12% to 22 +/- 7% with a dose of 2 mg/kg for 6 h). Aminoguanidine (50 mg/kg) or dexamethasone (5 mg/kg) partially inhibited the delay (to 39 +/- 4% or to 40 +/- 10%, respectively). (2) L-arginine (0.1 mM) produced a relaxation (28 +/- 2% reduction in active tension) in the gastric fundus strips isolated from LPS-treated rats but not from LPS-untreated rats. The relaxation was inhibited by aminoguanidine (1 mM). In contrast, the relaxation response to the electrical stimulation was not affected by aminoguanidine (0.1-1 mM).     

iNOS及eNOS mRNA表达在急性坏死性胰腺炎肺损伤中的作用

目的通过观察急性坏死性胰腺炎(acute necrotizing pancreatitis,ANP)大鼠肺组织中诱导型一氧化氮合酶(inducible nitric oxide synthase,i NOS)mRNA和内皮型一氧化氮合酶(endothelial nitric oxide synthase,eNOS)mRNA的表达,探讨内源性NOS在ANP肺损伤中的作用。方法 40只Wistar大鼠随机分为ANP组(n=30)和假手术组(SO组,n=10),采用5%牛磺胆酸钠逆行胆胰管注射法建立大鼠ANP模型。光镜下观察ANP造模后3 h、6 h及12 h时大鼠肺组织病理变化,并应用RT-PCR法检测相应时相肺组织中i NOS及eNOS mRNA的表达水平。结果随着病程延长,ANP组肺组织可见不同程度的充血、水肿、炎性细胞浸润、出血、坏死等病理损害,各时间点其肺损伤评分均明显高于SO组(P0.05),且呈逐渐升高趋势(P0.05)。ANP组3 h、6 h及12 hi NOS和eNOS mRNA的表达水平较SO组均有明显升高(P0.05)。结论肺组织中i NOS及eNOS mRNA的过度表达可能是大鼠ANP肺损伤发生的重要原因之一,这为采用抑制i NOS及eNOS mRNA的表达减轻ANP肺损伤的治疗手段提供了理论依据。     

Nitric Oxide Synthase Inhibitors Alter Ventilation in Isoflurane Anesthetized Rats

Background: Nitric oxide (NO) is present in medullary structures and can modulate respiratory rhythm. The authors determined if spontaneous ventilation at rest and in response to increased carbon dioxide is altered by selective neuronal NO synthase (NOS; 7-nitro-indazole, 7-NI) or nonselective (neuronal plus endothelial) NOS (NG -L-arginine methyl ester [L-NAME] and NG -monomethyl L-arginine [L-NMMA]) inhibitors in rats anesthetized with isoflurane.

Methods: Fifty-four rats received either L-NAME or L-NMMA (1, 10, and 30 mg/kg) or 7-NI (20, 80, and 400 mg/kg) and were compared with time controls (isoflurane = 1.4%), with isoflurane concentrations (1.6%, 1.8%, and 2%) increased consistent with the increased anesthetic depth caused by NOS inhibitors, or with L-arginine (300 mg/kg). Tidal volume (VT), respiratory frequency (f), minute ventilation (V with dotE), and ventilatory responses to increasing carbon dioxide were determined.

Results: L-NAME and L-NMMA decreased resting VT and V with dot (E), whereas 7-NI had no effect. Increasing concentrations of isoflurane decreased resting f, VT, and V with dotE. L-NAME and L-NMMA decreased VT and V with dotE, whereas 7-NI had no effect at 8%, 9%, and 10% end-tidal carbon dioxide (ETCO2). Increasing concentrations of isoflurane decreased f, VT, and V with dotE at 8%, 9%, and 10% ETCO2. The slope of V with dotE versus ETCO2 was decreased by isoflurane but was unaffected by L-NAME, L-NMMA, or 7-NI. L-arginine alone had no effect on ventilation.     

AMT, an Inducible Nitric Oxide Synthase Inhibitor, Enhances Islet Engraftment

We co-transplanted silica gel-entrapping 4H-1,3-Thiazin-2-amine,5,6-dihydro-6-methyl monohydrochloride (AMT) with islets to evaluate the effects of AMT on early graft dysfunction in a syngeneic mouse model. The mean diameter of AMT-embedding silica gel particles was 595 ± 275 nm. The cumulative release of AMT was 29% at 1 hour and 45% at 72 hours. Sixteen streptozotocin-induced diabetic mice were separated into 3 groups. Group A received 50 islets (n = 4). Group B received 50 islets and blank silica gel (n = 6). Group C received 50 islets plus silica-gel containing 6.4 μg AMT (n = 6). Mice in group C required significantly less time for temporary posttransplantation hyperglycemia than those in groups A and B (A, 39 ± 7 vs B, 40 ± 5 vs C, 24 ± 2 days; P < .05). The insulin contents of grafts retrieved at 13 weeks were 1.17 ± 0.11 (n = 4), 1.01 ± 0.16 (n = 6), and 1.68 ± 0.30 μg (n = 6) for mice in groups A, B, and C, respectively. Pancreatic remnant insulin did not differ significantly between the 3 groups (A, 0.32 ± 0.04 [n = 4] vs B, 0.29 ± 0.06 [n = 6] vs C, 0.40 ± 0.05 μg [n = 6]; P > .05). In vitro study revealed that 4 and 20 nmol/L of sol-gel-embedded AMT protected 87% and 96% RIN-m5F cells from 1 ng/mL interleukin-1β-mediated destruction, respectively. Silica-gel-entrapped AMT protects islet graft from a nonspecific inflammatory destruction, which is partly mediated via interleukin-1β.     

Opioid-induced Hyperalgesia in a Murine Model of Postoperative Pain: Role of Nitric Oxide Generated from the Inducible Nitric Oxide Synthase

Background: Opioid-induced delayed hyperalgesia and allodynia have been reported in human and animal models. The authors evaluated the influence of different opioids used during clinical anesthesia on nociceptive sensitivity and incisional pain in mice. The role of the inducible nitric oxide synthase on surgical pain and opioid-induced pronociception also was investigated.

Methods: CD1 mice were used to study the efficacy of opioids inducing pronociception and enhancing incisional pain. The implication of nitric oxide generated from the inducible nitric oxide synthase was investigated using knockout mice (C57/BL6) for its gene. Mice underwent right hind paw surgery under sevoflurane anesthesia combined with subcutaneous administration of saline or the opioids fentanyl (0.05 mg/kg), alfentanil (1 mg/kg), and remifentanil (0.04 mg/kg). Nociception was evaluated daily for 7 days using paw-pressure, plantar, and von Frey tests.

Results: The antinociceptive effect of opioids was followed by long-lasting thermal hyperalgesia and mechanical allodynia (each lasting between 2 and 7 days), but not mechanical hyperalgesia. Intraoperative infusion of opioids significantly enhanced incisional pain in all tests. The most prominent effects were observed with remifentanil. The inducible nitric oxide synthase gene deletion attenuated both remifentanil- and incision-induced pronociceptive effects. In mutant mice for the inducible nitric oxide synthase gene, remifentanil was still efficient in enhancing incisional pain, but the global pronociceptive effect was attenuated significantly as compared with wild-type mice.     

Volatile Anesthetics Differentially Affect Immunostimulated Expression of Inducible Nitric Oxide Synthase: Role of Intracellular Calcium

Background: Nitric oxide released by inducible nitric oxide synthase (iNOS) plays an important role in immune responses and systemic vasodilation in septic shock. Volatile anesthetics have been reported to interfere with signal transduction and gene expression. We studied the effect of volatile anesthetics on activity and expression of iNOS and potential mechanisms of action.

Methods: Nitrite release and iNOS expression were determined using the Griess reaction and Western and Northern blot techniques, respectively, in J774 murine macrophages stimulated with lipopolysaccharide and [gamma]-interferon in the absence and presence of various concentrations (0.25-2.0 minimum alveolar concentration [MAC]) of volatile anesthetics (i.e., halothane, enflurane, isoflurane, desflurane). Furthermore, potential interference of volatile anesthetics with specific signal transduction pathways was investigated.

Results: All volatile anesthetics, studied in a time- and dose-dependent manner, suppressed nitrite production and iNOS expression in J774 macrophages stimulated by lipopolysaccharide or [gamma]-interferon at clinically relevant concentrations. The inhibition was completely antagonized by ionomycin but unaffected by diacylglycerol, phorbol myristate acetate, and C2-ceramide. In contrast, in cells costimulated by lipopolysaccharide plus [gamma]-interferon, volatile anesthetics significantly increased nitrite production and iNOS expression independent of ionomycin and other mediators studied.     

Reinforcement Therapy Using Nitric Oxide Synthase Inhibitors Against Endotoxin Shock in Dogs

Purpose Nitric oxide synthase (NOS) inhibitors were confirmed to correct the hypotension associated with septic shock, but the overall prognosis is often pessimistic. The histological findings failed to show any improvement. In fact, some patients even exhibited signs of exacerbation. The purpose of this study was to investigate the therapeutic effects of NOS inhibitors and catecholamines in dogs suffering from endotoxin shock. The histological changes produced by these agents were also evaluated. Methods Mongrel dogs were used under midazolam anesthesia. A PiCCO continuous cardiac output monitoring catheter was placed in the femoral artery, and a central venous monitoring catheter was placed in the external carotid artery. Results Endotoxin (0.5 mg/kg, i.v.) was administered to cause shock. After this shock state was observed, the NOS inhibitors and catecholamines raised the blood pressure, and norepinephrine (NA, 2 mg/kg/h) was found to be more potent than S-methylisothiourea (SMT, 20 mg/kg/h). The combined effects of SMT-NA or SMT-DOB were greater than those of NA or dobutamine (DOB) alone. The histological changes induced by endotoxin shock were not ameliorated by the administration of NOS inhibitors but instead appeared to be exacerbated to some degree. Conclusion NOS inhibitors combined with cathecholamines were thus suggested to be able to reduce the cathecolamine dosage in patients suffering from septic shock; They are thus considered to be hemodynamically effective agents.     

大鼠脊髓损伤后诱导型一氧化氮合酶基因表达变化的实验研究

目的探讨大鼠脊髓损伤后诱导型一氧化氮合酶(iNOS)mRNA表达的变化规律.方法SD大鼠48只,随机分为8组,采用Allen's脊髓损伤打击模型,以逆转录-聚合酶链反应(RT-PCR)法测定伤段脊髓组织iNOS mRNA表达情况.结果iNOS mRNA在脊髓损伤前即有表达,损伤后早期无明显变化,伤后72h开始升高,1周时达到高峰.结论脊髓继发性损害的持续时间可能大于传统观念,针对继发性损害所作的治疗应持续至脊髓损伤后较长的一段时间.     

一氧化氮、一氧化氮合酶与伴精索静脉曲张不育患者精液参数的关系

目的:探讨一氧化氮(NO)、一氧化氮合酶(NOS)与伴精索静脉曲张(VC)不育患者精液参数之间的关系。方法:根据体格检查和彩色多普勒超声检查选择伴VC的不育患者(组1,n=53),其中临床型和亚临床型分别为21例和32例;同时选择非VC少弱精子症患者(组2,n=29)和正常生育者(组3,n=28)作对照组。采用硝酸还原法分别测定外周血和精浆中NO含量和NOS活性。用计算机辅助精液分析仪测定VC组患者精子密度、活动精子(a+b级精子)和快速前向运动精子百分率。结果:①组1外周血清NO含量和NOS活性与组2及组3相比差异无显著性(P>0.05),但精浆中NO含量和NOS活性组1明显高于其他两组,差异有显著性(P<0.01和P<0.05)。②组1中,随着曲张的精索静脉内径的增加,外周血清和精浆中NO含量和NOS活性均有所上升,但只有精浆中临床型和亚临床型之间相比差异有显著性(P<0.05)。③组1中,随着精子密度和精子活力的下降,外周血清和精浆中NO含量和NOS活性均有上升趋势,且精子密度≥20×106/ml和≤10×106/ml之间,精子活力≥50%和≤25%之间差异有显著性(P均<0.05)。结论:在VC诊断中精浆中NO含量和NOS活性测定较外周血清中更有意义。早期测定精浆NO含量和NOS活性对VC的诊断和治疗具有重要的临床价值。     

衰老对大鼠阴茎组织诱导型一氧化氮合酶的表达和细胞凋亡的影响

目的:探讨年龄因素对大鼠阴茎组织诱导型一氧化氮合酶(iNOS)表达和细胞凋亡的影响。方法:雄性Wistar大鼠随机分为衰老组(10只,>25月龄)、老年组(8只,12～15月龄)和青年组(6只,3～4月龄),分别用免疫组化法检测阴茎组织iNOS的表达和原位缺口末端标记法(TUNEL)检测细胞凋亡率。结果:衰老组、老年组及青年组iNOS的表达用相应的吸光度(A)表示分别为:0.24±0.03、0.17±0.02和0.12±0.03;细胞凋亡率分别为:(1.41±0.78)%、(0.94±0.43)%和(0.50±0.23)%,组间比较差异均有显著性(P均<0.05)。结论:衰老大鼠阴茎组织iNOS表达增多,细胞凋亡现象明显增加,衰老可能是老年性阴茎勃起功能障碍的机制之一。     

大鼠诱导型一氧化氮合酶短发夹环RNA质粒的构建及鉴定

目的：构建编码诱导型一氧化氮合酶（iNOS）Jfl动子的shRNA质粒表达载体。为利用基因激活技术治疗勃起功能障碍的研究做准备。方法：根据大鼠iNOS启动子序列设计并合成shRNA寡核苷酸片段．退火形成双链并克隆进入载体pDC316-EGFP-U6,构建重组质粒,并进行PCR鉴定以及测序分析。结果：PCR鉴定以及测序证实重组质粒构建成功。结论：成功构建了靶向iNOS基因的shRNA质粒表达载体．为进一步探索勃起功能障碍基因治疗奠定了基础。     

Proteinuria Is Reduced by Inhibition of Inducible Nitric Oxide Synthase in Rat Renal Ischemia-Reperfusion Injury

Background

Ischemia-reperfusion (IR)-induced nephrotoxicity is associated with proteinuria. There are reports on the involvement of inducible nitric oxide synthase (iNOS) in proteinuria in conjunction with renal disease. This study was designed to investigate the effect of N6-(1-iminoethyl)-L-lysine hydrochloride (L-Nil), a selective inhibitor of iNOS, to prevent proteinuria in IR injury.

Methods

Ischemia was induced by 40-minute clamping of the renal arteries followed by 6-hour reperfusion. Rats were administered either L-Nil (3 mg/kg intravenous bolus followed by infusion of 1 mg/kg/h) or saline. To monitor glomerular and tubular functional changes before and after treatment, we measured blood urea nitrogen, plasma creatinine, and urinary N-acetyl-β-D-glucosaminidase activity. Total protein (TP), albumin, and low- (LMW) and high-molecular-weight (HMW) protein excretion rates were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis of urine samples. Kidney ultrastructure was examined through a transmission electron microscope (TEM).

Results

IR resulted in significant LMW and HMW proteinuria. L-Nil significantly prevented the IR-induced increases in TP, albumin, and α1-microglobulin excretion. TEM showed loss of microvilli of the proximal tubule cells, injured mitochondria, and foamy changes in the structure of nuclear and cytoplasm in IR group. L-Nil reduced IR-mediated renal ultrastructural changes and tubular proteinuria.

Discussion

This study suggested possible differences in the mechanism(s) of nephrotoxicity induced by iNOS in the glomeruli and tubular cells. The types of proteins excreted in the urine should be considered in the treatment strategy. In conclusion, this study suggested the involvement of iNOS in IR-induced tubular proteinuria.     

肝细胞癌中iNOS和P53蛋白的表达及其与肿瘤血管形成的关系

目的　研究诱导型一氧化氮合酶 (iNOS)和p5 3蛋白在肝细胞癌中的表达及其与肿瘤血管形成的关系。方法　采用免疫组化和图像分析技术检测 5 9例肝细胞癌患者肿瘤组织中iNOS和p5 3蛋白的表达 ,CD34单克隆抗体免疫组化染色检测肿瘤组织微血管密度 (MVD)。结果　①iNOS和p5 3蛋白在肝细胞癌组织中表达阳性率分别为 81.4 % ( 4 8/ 5 9)和 6 4 .4 % ( 38/ 5 9) ,表达强度 (IOD值 )分别为 5 6 35± 12 87和 335 2± 873。②MVD为 32 .5± 2 .73,以肿瘤边缘和癌旁组织微血管较密集。③iNOS的表达与p5 3蛋白表达呈显著正相关 (r=0 .6 5 ,P<0 .0 5 ) ;iNOS的表达与MVD呈显著正相关 (r=0 .75 ,P<0 .0 5 ) ;p5 3蛋白的表达与MVD呈显著正相关 (r=0 .72 ,P<0 .0 5 )。结论　肝细胞癌组织中存在iNOS和p5 3蛋白的高表达 ;iNOS和p5 3可促进肝癌血管形成