小鼠急性自体血栓性肺栓塞后内皮型一氧化氮合酶及一氧化氮表达变化

目的:建立操作简便、成功率高、耗资低廉的急性血栓性肺栓塞(acute pulmonary thrombo-embolism,APE)小鼠动物模型,并观察肺内内皮型一氧化氮合酶(endothelial nitric oxide synthase,eNOS)及血清一氧化氮(nitric oxide,NO)的表达变化情况。方法:雄性C57BL/6小鼠(n=234),随机分为三组,即正常对照组(n=39)、假手术组(n=66)和模型组(n=129),各组又随机分为1 h、1d和2d三个亚组。模型组自右颈静脉注入自体血栓栓子,假手术组以0.9%氨化钠偏代替自体血栓栓子注入。到指定时间点处死小鼠,取肺组织进行大体观察,苏木素-伊红(HE)染色观察肺组织和肺血管结构的变化,Mallory氏磷钨酸苏木精(PTAH)染色显示血管内血栓中的纤维素。以试剂盒测定外周血NO水平,免疫组化观察肺组织中eNOS蛋白的表达情况。结果:模型组HE染色光镜下可见注入的血栓栓子,PTAH染色光镜下栓子中可见蓝紫色纤维素网状结构,且大多数栓子栓塞于肺动脉段水平。与1 h模型组相比,1 d和2 d模型组的栓子数目逐渐减少(分别约为1 h模型组的90%和30%)。另外,部分远端肺动脉中发现有大量红细胞聚集,可能是由于继发血栓的形成。注栓1 d后,模型组与正常让外周血NO水平(22.666±2.937)μmol/L与正常对照组[(50.596±5.799)μmol/L,P<0.01]和假手术组[(37.797±4.880)μmol/L,P<0.05]比较差异有统计学意义。与正常组及假手术组相比,1 d模型组肺动脉内皮eNOS蛋白表达有所增高。结论:用自体血栓栓子可成功诱导小鼠APE形成,APE后血清NO生成减少,肺动脉内皮eNOS表达升高。     

Type I nitric oxide synthase is decreased in the fetal pulmonary circulation of hypertensive lambs

The nitric oxide (NO)/guanosine 3',5'-cyclic monophosphate (cGMP) pathway plays an essential role in mediating pulmonary vasodilatation during transition of the pulmonary circulation at birth. We used immunoblot analysis (Western) and semiquantitative immunohistochemistry to study the presence, distribution, and relative amounts of type I nitric oxide synthase (NOS-I). Immunoblots were performed on normal fetal sheep lungs, whereas immunohistochemistry for NOS-I was compared between lungs from normal fetal lambs vs. fetal lambs with persistent pulmonary hypertension of the newborn (PPHN) induced by ligation of the ductus arteriosus.Western blot analysis using a polyclonal antibody detected NOS-I protein in homogenates of normal fetal sheep lungs. Abundant NOS-I immunoreactivity was observed exclusively in the precapillary resistance vessels, i.e., terminal bronchiole-associated arteries (TA) and respiratory bronchiole-associated arteries (RA) in normal fetal lung. In marked contrast, immunoreactivity for NOS-I was significantly reduced in the TA and RA of hypertensive lungs.We conclude that there is a heterogeneous distribution of NOS-I in the normal fetal sheep lung, but that NOS-I staining is significantly reduced in lambs with PPHN.     

梅毒患者血清一氧化氮和一氧化氮合酶水平测定

目的　检测梅毒螺旋体感染者血清中一氧化氮 (NO)和一氧化氮合酶 (NOS)的水平。方法　用分光光度法测定血清中NO水平和NOS活性 ,血清中NO3 和NO2 总量代表体内NO水平 ,NOS催化L 精氨酸和氧的反应生成NO的多少代表血清NOS活性。结果　梅毒患者NO浓度为 115± 36 3nmol/L ,NOS活性为 35 8± 7 3U/ml,二者均远远高于正常对照组。结论　梅毒螺旋体的感染引起患者体内NO水平和NOS活性升高 ,NO在梅毒感染中可能发挥重要的作用。     

Effects of nitric oxide inhalation after pulmonary thromboendarterectomy for chronic pulmonary thromboembolism

STUDY OBJECTIVES: To examine the hypothesis that nitric oxide (NO) inhalation improves hemodynamics and gas exchange in patients with chronic pulmonary thromboembolism after pulmonary thromboendarterectomy. DESIGN: Prospective crossover clinical study. SETTING:: Surgical ICU in a national education and research hospital. PATIENTS:: Seven patients (mean age +/- SD, 54 +/- 11 years) who underwent elective pulmonary thromboendarterectomy for chronic pulmonary thromboembolism. INTERVENTIONS: Patients breathed 20 parts per million of NO gas for 30 min at 12-h intervals until extubation of the trachea. MEASUREMENTS AND RESULTS: Hemodynamics and arterial blood gas levels were analyzed before, during, and after NO inhalation. Waveform of pulmonary artery pressure (PAP) was evaluated using fractional pulse pressure (PPf): (systolic PAP - diastolic PAP)/mean PAP. After surgery, pulmonary vascular resistance decreased, PPf decreased, and cardiac index increased significantly. At the first trial, NO inhalation resulted in a slight improvement in arterial oxygen tension (from 173 +/- 33 to 196 +/- 44 mm Hg; p < 0.05), while hemodynamics did not change significantly. Twelve hours later, NO inhalation decreased pulmonary vascular resistance index (from 312 +/- 98 to 277 +/- 93 dyne.s. cm(-5)/m(2); p < 0.01), while the change in oxygenation was not significant. CONCLUSIONS: Immediately after pulmonary thromboendarterectomy for chronic pulmonary thromboembolism, NO inhalation improved oxygenation; at 12 h after surgery, NO inhalation resulted in decreased pulmonary vascular resistance, although both changes were small.     

一氧化氮对缺氧性肺动脉高压大鼠血浆降钙素基因相关肽含量的影响

目的 探讨一氧化氮(NO)对缺氧性肺动脉高压(HPH)大鼠血浆降钙素基因相关肽(CGRP)含量的影响。方法 将Wistar大鼠40只分为四组:对照组(n=10),缺氧组(n=10),缺氧 L-NAME组(n=10),缺氧 L-Arg组(n=10)。通过P50压力传感器法测量定四组大鼠肺动脉平均压(PAMP),用放射免疫方法测定各组大鼠血浆CGRP的含量。结果 缺氧组的PAMP显著高于对照组(P<0.05),缺氧 L-Arg组的PAMP显著低于缺氧组(P<0.05);缺氧组的有室(RV)千重/左室 室间隔(LV S)干重比值显著高于对照组(P<0.01),缺氧 L-NAME组的RV/LV S比值显著高于缺氧组(P<0.05)及缺氧 L-Arg组(P<0.01),缺氧 L-Arg组的CGRP含量最高,与缺氧 L-NAME组和缺氧组有高度显著性差异(P<0.01,P<0.05),PAMP与血浆CGRP含量呈明显负相关(r=-0.426,P<0.05)。结论 通过上述试验推测NO可能通过影响CGRP的释放调节HPH。     

一氧化氮在砷中毒中作用的研究

目的探讨砷对机体ＮＯ的影响及ＮＯ在砷中毒发病中的作用。方法采用动物实验和人群调查方法。结果慢性染砷实验中,小鼠血清、肾脏ＮＯ２－／ＮＯ３－含量中、高剂量组显著降低,肝脏、心脏ＮＯ２－／ＮＯ３－含量各剂量组显著降低,且呈剂量—效应关系。全血ＧＳＨ含量低剂量组显著升高,中、高剂量组显著降低,肝脏、心脏ＧＳＨ含量各剂量组均显著下降,肾脏ＧＳＨ含量中、高剂量组显著降低。红细胞、心脏ＳＯＤ活性中、高剂量组显著下降,肝脏ＳＯＤ活性低剂量组显著升高,中、高剂量组显著下降,肾脏各剂量组未见显著差异。人群调查,砷病区病人血中ＮＯ２－／ＮＯ３－、ＧＳＨ含量显著低于非病区健康人,且二者呈正相关,红细胞ＳＯＤ活性未见显著差异。结论砷可导致ＮＯ含量的下降。砷可导致ＧＳＨ含量和ＳＯＤ活性的变化,进而影响ＮＯ合成和代谢     

吸入NO对婴幼儿体外循环中肺表面活性物质的影响

目的: 探讨吸入一氧化氮(NO)对婴幼儿体外循环（cardiopulmonary bypass,CPB）中肺表面活性物质的影响。 方法: 将30例患室间隔缺损的婴幼儿随机分为对照组和NO组,NO组在CPB期间吸入40 μl/L NO直至关胸。CPB前、主动脉开放后1,5,10 min以少量生理盐水灌洗气道,分别测定气道吸出物(BAL)中总磷脂(TPL)、饱和卵磷脂(SatPC)、总蛋白(TP)值,并计算SatPC/TPL和SatPC/TP。结果: CPB后两组SatPC/TPL、SatPC/TP较CPB前明显降低(P<0.01)。NO组SatPC/TPL和SatPC/TP下降的幅度明显小于对照组(P<0.01)。结论: 婴幼儿CPB术中存在明显的肺损害,表现为一些亚临床性肺功能损伤。吸入40 μl/L的NO对CPB期间肺功能有明显的保护作用。     

Effect of inhaled nitric oxide on pulmonary function in cystic fibrosis.

Concentrations of nitric oxide (NO) have been found to be reduced in both the upper and lower airway of patients with cystic fibrosis (CF). As NO modulates bronchomuscular tone, low NO levels may contribute to the obstructive lung disease in these patients. To assess whether increasing inspiratory NO concentrations has any impact on lung function, we have studied 13 CF patients aged 14-38 years in a clinically stable condition and nine healthy controls. NO was applied via a mixing chamber for 5 min with NO concentrations of 100 parts per billion, 1 and 40 parts per million. Spirometry was performed at baseline and after inhalation on each occasion. There were no clinical side-effects at any NO concentration and no changes in oxygen saturation were observed. Lung function remained unchanged in all subjects throughout the study period. Sputum nitrate and nitrite concentrations before and after inhalation of high NO concentrations (40 ppm) in eight CF patients did not show any significant changes, even though a tendency to higher nitrate levels was observed (399 +/- 231 vs. 556 +/- 474 mumol l-1). Therefore, inhaled NO at either the physiological levels present in the upper airway of normal individuals or those used therapeutically to treat pulmonary hypertension has no immediate effect on bronchomuscular tone in patients with cystic fibrosis.     

Reactivity of pulmonary circulation and right ventricle function to inhaled nitric oxide in systemic sclerosis patients

Systemic sclerosis (SSc) is complicated by pulmonary hypertension and right ventricle (RV) failure in approximately 10% of the patients. Factors influencing the reactivity of pulmonary circulation to vasodilators are not established, while the examination of vasoreactivity is important in determining the treatment, because systemic administration of oral vasodilators can induce severe adverse events in nonresponders. The mechanism of RV failure in SSc is unclear and may result either from increased RV afterload or intrinsic myocardial disease. The aim of the study was to assess the reactivity of pulmonary circulation to inhaled nitric oxide (iNO) and to evaluate its influence on RV function in SSc patients with elevated right ventricle systolic pressure (RVSP). In 60 SSc patients aged 24–73 (58 females, two males; 33 patients with limited SSc and 27 with diffuse SSc), echocardiographic examination with tissue Doppler echocardiography (TDE) was performed. RV function was measured by systolic (S) and early diastolic (E) velocity of tricuspid annulus by TDE. In patients with RVSP >45 mmHg, the reactivity of pulmonary circulation was assessed by iNO test. High-resolution computerized tomography (HRCT) was performed to assess the extent of pulmonary fibrosis. Of 14 SSc subjects with elevated RVSP (13 females, one male; RVSP 47–62 mmHg), positive reaction to iNO was observed in five (RVSP decreased from 51.6 ± 3.7 to 32.24 ± 2.3 mmHg); nine patients were not reactive (RVSP 53.5 ± 5.7 mmHg before iNO vs. 49.6 ± 6.7 mmHg). RV systolic function was decreased in patients with elevated RVSP as compared to the patients with normal pulmonary pressure (S velocity 13.2 ± 1.3 vs. 14.4 ± 1.6 cm/s, respectively, p < 0.05). Significant increase of RV systolic function during iNO test was found in reactive patients only (S velocity before iNO 12.8 ± 1.2 cm/s, during iNO 14.5 ± 1.5 cm/s, p < 0.01). RVSP decrease strongly correlated with S velocity increase (r = 0.95, p < 0.0001). Response to iNO was found only in limited form of SSc; diffuse SSc patients showed no response. Pulmonary fibrosis on HRCT was more frequent in subjects nonreactive to iNO (67% of patients) than in the reactive group (40% of patients). The reactivity of pulmonary circulation to iNO in SSc patients with elevated RVSP was found predominantly in limited form of the disease. Pulmonary fibrosis typical for diffuse SSc was more frequent in nonreactive subjects. Elevated pulmonary pressure plays an important role in RV systolic dysfunction. Pulmonary pressure decrease during iNO test leads to the improvement of RV systolic function. Therapy for right-heart failure in reactive SSc patients should be directed, if possible, at the decrease in pulmonary resistance.     

Effects of S-nitrosation of hemoglobin on hypoxic pulmonary vasoconstriction and nitric oxide flux

Free hemoglobin (Hb) augments hypoxic pulmonary vasoconstriction (HPV), ostensibly by scavenging nitric oxide (NO). However, recent evidence suggests that Hb that is S-nitrosated may act as an NO donor and vasodilator. We studied the effects of oxyHb, Hb that is chemically modified to prevent heme binding or oxidation of NO (cyanometHb), and Hb that is S-nitrosated (SNO-Hb and SNO-cyanometHb) on HPV, expired NO (eNO), and perfusate S-nitrosothiol (SNO) concentration in isolated, perfused rabbit lungs. Perfusate containing either 4 microM oxyHb or SNO-Hb increased normoxic pulmonary artery pressure (Ppa), augmented HPV dramatically, and resulted in an 80% fall in eNO in comparison to perfusion with buffer, whereas 4 microM cyanometHb or SNO-cynanometHb had no effect on these variables. Excess glutathione (GSH) added to perfusate containing SNO-Hb resulted in a 20 to 40% fall in the perfusate SNO concentration, with a concomitant increase in metHb content, without affecting Ppa, HPV, or eNO. In conclusion, free Hb augments HPV by scavenging NO, an effect that is not prevented by S-nitrosation. NO released from SNO-Hb in the presence of GSH does not produce measurable vascular effects in the lung or changes in eNO because of immediate oxidation and metHb formation.     

肺血栓栓塞症患者一氧化氮和血小板功能的变化

目的研究肺血栓栓塞症患者溶栓抗凝治疗前后一氧化氮(NO)和血小板功能的变化。方法流式细胞仪检测肺血栓栓塞患者和健康者血小板P-选择素(Ps)的表达,同时测定血浆中NO、血管性血友病因子(vWF)、血栓素B2(TXB2)和6酮--前列腺素F1α(6-keto-PGF1α),比较治疗前后一周各项指标的变化。应用NO的前体L精-氨酸(L-Arg)处理后,检测其血小板上Ps表达及N-硝基-L精氨甲基酯(L-NANE)预处理后L-Arg对血小板上Ps的表达的影响。结果PTE组Ps、vWF、TXB2的水平显著高于对照组,而治疗后明显下降(P均<0.01);NO和6-keto-PGF1α显著低于对照组,治疗后明显升高(P均<0.01)。应用L-Arg后,血小板Ps表达减少,L-NAME预处理可明显阻断这一效应。结论PTE患者存在明显的血管内皮损伤,NO的水平降低,这可能是PTE患者血小板Ps表达增加,促进血小板活化,导致PTE进一步发展的原因之一。     

Modulation of the hyperdynamic circulation of cirrhotic rats by nitric oxide inhibition.

The effects of NG-monomethyl-L-arginine (L-NMMA), an inhibitor of nitric oxide (NO) biosynthesis on the splanchnic and systemic circulation, were investigated in rats with cirrhosis induced by carbon tetrachloride. Portal hypertension in these rats was accompanied by decreased arterial blood pressure and peripheral vascular resistance as well as by splanchnic vasodilation with increased portal venous inflow and decreased splanchnic resistance. Intravenous bolus administration of L-NMMA (25 mg/kg) significantly increased systemic blood pressure and decreased cardiac output. L-NMMA also significantly increased systemic and splanchnic vascular resistance; whereas blood flow to the stomach, small intestine, colon, pancreas, mesentery, spleen, and kidney was decreased significantly. L-NMMA did not alter the portal pressure or portosystemic shunting in these cirrhotic rats, yet portal vascular resistance increased, suggesting effects on the intrahepatic and collateral circulation. Pretreatment with L-arginine (300 mg/kg) prevented the hemodynamic changes induced by L-NMMA. These findings support the concept that local excess formation of NO contributes to changes in splanchnic circulation associated with portal hypertension in cirrhosis.     

Higher level of plasma nitric oxide in spontaneously hypertensive rats.

We had detected a slightly, but significantly, higher level of plasma nitrite/nitrate in the spontaneously hypertensive rat (SHR) by using the nitric oxide (NO) analyzer (Sievers 280 NOA), which converts nitrate (including nitrate converted from nitrite) to NO. Here, we examined whether the release of NO from protein-bound dinitrosyl nonheme iron complexes (DNIC) contributes to the elevated plasma nitrate level in the SHR. The SHR and their genetic normotensive controls, Wistar-Kyoto rats (WKY), were anesthestized and cannulized for monitoring blood pressure, collecting a blood sample, and the administration of endotoxin (lipopolysaccharide [LPS]). The nitrate levels (an indicator of NO formation) in the plasma and the aorta were measured by an NO analyzer. In addition, the relaxation of acetylcholine (ACh) in the presence or absence of N(omega)-nitro-L-arginine methyl ester (L-NAME) was also examined in thoracic aortae obtained from both strains. The slight, but significant, increase of basal nitrate levels in the plasma and aorta were observed, and the former was further enhanced in SHR treated with LPS for 3 h. In vitro, the ACh-induced relaxation was attenuated in the aortae obtained from SHR. However, this difference between SHR and WKY (without LPS treatment) was abolished by treatment of rings with L-NAME (30 micromol/L), suggesting that an impairment of NO formation was observed in the SHR. After rats were treated with LPS for 3 h, the ACh-induced relaxation was reduced in the WKY, but not in the SHR. In addition, a 10-fold increase of L-NAME was needed to abolish the difference in ACh-induced relaxation between SHR and WKY, indicating an expression of inducible NO synthase in both strains treated with LPS. We suggest that the elevated plasma NO level in SHR may be due to the release of NO from DNIC in the vascular bed to combat the hypertensive state.     

Endogenous endothelins and nitric oxide in hypoxic pulmonary vasoconstriction.

The effects of endothelin receptor blockade on the pulmonary circulation have been reported variably, possibly in relation to a more or less important associated release of endogenous nitric oxide (NO). The aim of this study was to test whether endothelin antagonism would inhibit hypoxic pulmonary vasoconstriction, and if it would not, then would it do so after NO synthase inhibition. Hypoxic pulmonary vasoconstriction (HPV) was evaluated in anesthetised dogs by the increase in the mean pulmonary artery pressure (Ppa) minus occluded Ppa (Ppao) gradient in response to hypoxia (inspiratory oxygen fraction of 0.1) at constant pulmonary blood flow. Bosentan, an endothelin A and B receptor antagonist, did not affect baseline Ppa, Ppao or systemic arterial pressure (Psa) and did not alter HPV (n=8). The NO synthase inhibitor N(G)-nitro-L-arginine (L-NA) did not affect baseline Ppa and Ppao, but increased Psa and enhanced HPV (n=12). The addition of bosentan in these dogs did not affect baseline Ppa or Ppao, but decreased Psa and inhibited HPV. Exhaled NO was decreased by L-NA and by bosentan and abolished by L-NA+bosentan (n=9). The authors conclude that endogenous nitric oxide is released by, and opposes the vasoconstricting effects of, endothelins in vivo, reducing systemic blood pressure and limiting hypoxic pulmonary vasoconstriction.     

Hyperkinetic circulation and decreased sensitivity to vasoconstrictors following portacaval shunt in the rat. Effects of chronic nitric oxide inhibition.

BACKGROUND/AIMS: This study aimed to investigate: (i) whether the hyperkinetic circulation that develops after portacaval shunt is associated with decreased vascular sensitivity to vasoconstrictors and (ii) the role of nitric oxide on its pathogenesis. METHODS: Portacaval-shunted and sham-operated rats received long-term treatment with the nitric oxide inhibitor L-NAME (osmotic minipump) or its inactive enantiomer D-NAME. Measurements of arterial pressure, cardiac output and superior mesenteric artery blood flow (transit-time flow probe) were done 4 days later in baseline conditions and after increasing doses of methoxamine. Peripheral and superior mesenteric vascular resistance were calculated. RESULTS: Portacaval shunted rats showed a significantly lower peripheral and superior mesenteric vascular resistance and a significant reduction in their response to incremental doses of methoxamine than sham-operated controls. Chronic nitric oxide inhibition attenuated the systemic but not the splanchnic vasodilatation and totally corrected the hyposensitivity to methoxamine of portacaval-shunted rats. However, they still had a significantly lower peripheral and superior mesenteric vascular resistance than sham-operated rats. CONCLUSIONS: This study shows that the splanchnic and systemic hyporesponsiveness to methoxamine observed in portacaval-shunted rats could be explained by an excess of nitric oxide. However, other factors may be involved in maintaining splanchnic and systemic vasodilatation despite NO-inhibition.     

Effects of HIV co-infection and chemotherapy on the urinary levels of nitric oxide metabolites in patients with pulmonary tuberculosis.

The presence of nitric oxide (NO) and its role as a factor in host defence against intracellular pathogens in human macrophages is controversial. We measured the metabolites of NO (nitrite (NO2-) and nitrate (NO3-)) in urine from Ethiopian patients suffering from tuberculosis. The urinary level of NO2-/NO3- in a group of healthy Ethiopians was 1020+/-471 microM (n = 22). Untreated HIV negative patients with active pulmonary tuberculosis (1574+/-588 microM, p<0.01, n = 12) and household contacts to tuberculosis patients (1949+/-812 microM, p = 0.006, n = 7) had significantly higher levels of urinary NO2-/NO3- than the control group. Untreated HIV positive patients with pulmonary tuberculosis did not have increased levels of urinary NO2-/NO3- (1101+/-614 microM, n = 6). Some of the HIV negative untreated patients with pulmonary tuberculosis (1710+/-519 microM, n = 6) were followed up after treatment and showed a reduction in the levels of urinary NO2-/NO3- 1 week after treatment (945+/-599 microM, p<0.05). We conclude that HIV negative patients with active pulmonary tuberculosis have increased urinary levels of nitric oxide metabolites with a reduction following specific anti-tuberculous chemotherapy.     

Evaluation of bioavailability of nitric oxide in coronary circulation by direct measurement of plasma nitric oxide concentration

Although bioavailability of NO in the coronary circulation is commonly evaluated by acetylcholine (ACh)-induced vasodilation, a change in plasma NO concentration and its relation to the flow response after injection of ACh are still unknown. Thus, we directly measured the concentration of NO in the coronary sinus by using a catheter-type NO sensor for coronary sinus. An NO-sensitive sensor was located and fixed in a 4-Fr catheter with a soft tip for protection of vascular wall. After calibration with an NO-saturated pure water, the catheter-type NO sensor was located in the coronary sinus in anesthetized dogs. The coronary flow velocity (CFV) was measured with a Doppler guide wire. Intracoronary injection of ACh (0.4 and 1.0 microg/kg) increased plasma NO concentration in a dose-dependent manner (3-10 nM). Although ACh increased CFV by 95%, there was no significant difference between the two ACh doses. After ACh, the peak value of plasma NO concentration was observed significantly later than CFV. N(G)-methyl-L-arginine (NO synthase inhibitor) decreased basal NO concentration by 3 nM and suppressed the ACh-induced NO synthesis with no significant change in average peak velocity. We conclude that production of NO in the coronary circulation can be evaluated in the coronary sinus. Although ACh increases both CFV and NO concentration, CFV dose not reflect NO concentration in terms of magnitude and time course. Direct measurement of plasma NO concentration by the catheter-type NO sensor is useful to evaluate bioavailability of NO in the coronary circulation.