Immunolocalization of the Inducible Nitric Oxide Synthase Isoform in Human Fetal Membranes

PROBLEM: Nitric oxide (NO) synthesized by fetal membranes may protect the fetus from maternal infection or immune challenge or have a tocolytic effect on myometrium. The sites of synthesis and enzymes responsible for NO production in human fetal membranes remain unidentified. METHOD OF STUDY: Fetal membranes were obtained from four groups of patients: term (>37 weeks gestation) or preterm (<37 weeks gestation), both either in labor or not in labor. Frozen sections of membrane rolls were immunostained for inducible (iNOS) and endothelial (eNOS) nitric oxide synthase isoforms and the monocyte/macrophage marker CD14. RESULTS: Positive iNOS immunostaining was found in fibroblasts of amnionic and chorionic mesenchyme and in decidual macrophages identified by CD14 from all four groups of tissues. No iNOS immunostaining was seen in amnion epithelium or chorion trophoblast. Very intense iNOS staining was seen with evidence of monocyte/macrophage invasion of membranes. eNOS immunostaining was only found in decidual vascular endothelium. CONCLUSIONS: Constitutive expression of iNOS in decidual macrophages and fetal membrane fibroblasts may form an immune barrier against maternal insult. In chorioamnionitis, macrophage recruitment and NO expression may be part of the maternal immune response.     

Analysis of factors that determine the compliance of rat jejunum to distension in vivo

Distension of the intestine is commonly used to elicit reflex responses at other sites in the gastrointestinal tract, and also to evaluate pain of intestinal origin. The sensory neurones, that initiate the reflexes or pain responses, react to the forces generated in the wall of the intestine. Thus, the responses of the intestine at the site of distension, particularly changes in contractile activity, influence the signals from the gut. In the present work we have analysed the relationship between distension and pressure changes in the jejunum of the rat, in vivo. Isovolumic distension for 5 min caused an initial pressure increase which declined quickly in the first 30 s, and then declined more slowly. Phasic pressure increases were superimposed on the baseline pressure change. Hexamethonium blocked the phasic pressure increases, whereas the initial rapid and subsequent slower pressure decline during distension persisted. Inhibition of nitric oxide synthase (NOS) increased intraluminal pressure and caused increased frequency and irregularity of phasic pressure increases. However, the decline in jejunal pressure during distension was not changed by inhibition of NOS. The pressure decline during isovolumic distension was similar whether saline or paraffin oil were used to distend the intestine, indicating that the decline was not due to increased hydrostatic pressure causing water and electrolyte to cross the mucosal epithelium from the lumen to the intestinal interstitium. Hyoscine had no significant effect on the pressure profile when the intestine was distended. However, when the systemic or the local circulation of the jejunum was infused with nicardipine, the pressure that was achieved during isovolumic distension was less, although the rate of change in pressure during the slow decline was similar. It is concluded that distension evokes phasic pressure increases in the jejunum, that are nerve-mediated, and increases the tension in the wall through a stretch-activated increase in contractile force generated by the circular muscle. The decline in pressure during maintained distension is primarily a consequence of visco-elastic properties of the wall of the intestine.     

EFFECTS OF SARAFOTOXIN S6c ON RENAL HAEMODYNAMICS AND URINE FORMATION IN ANAESTHETIZED DOGS

1. The effects of sarafotoxin S6c (S6c), a selective endothelin ETB receptor agonist, on renal haemodynamics and urine formation were examined in anaesthetized dogs. 2. Intrarenal arterial infusion of S6c at a rate of 1 or 5 ng/kg per min produced a transient increase in renal blood flow (RBF), with no change in systemic blood pressure and heart rate; RBF then decreased gradually to below the basal value. There were significant and dose-dependent increases in urine flow and free water clearance and decreases in urine osmolality during S6c infusion, whereas urinary excretion of sodium and glomerular filtration rate (GFR) remained unchanged. Simultaneously, S6c administration elicited a marked increase in urinary excretion of nitric oxide (NO) metabolites, N0₂^? and N0₃^? (UNO*V). 3. In dogs simultaneously administered S6c (5 ng/kg per min) and iV^G-nitro-L-arginine (NOARG; 40 (jig/kg per min), a NO synthase inhibitor, the renal vasodilator effect of S6c was abolished and marked reductions in RBF and GFR were observed. The S6c-induced diuretic action was not affected by NOARG. In the presence of NOARG, there was a small amount of UNO_xV at the basal level and the administration of S6c did not increase UNOxV. 4. These results suggest that an intrarenal arterial infusion of S6c enhances the production of NO in the kidney and that this enhancement contributes to the peptide-induced renal vasodilation. In contrast, it is unlikely that S6c-induced water diuresis is related to NO production stimulated by this peptide.     

Neurotransmitter release evoked by α-latrotoxin in the smooth muscle of the female pig urethra

Neuronal regulation of smooth muscle tone in the female pig urethra has mainly been studied in vitro using electrical field stimulation (EFS) of nerves. Excitatory control is considered to be exerted by released noradrenaline, whereas inhibitory control is non-adrenergic non-cholinergic (NANC), and mediated by nitric oxide (NO), and an as yet unidentified agent. We investigated the functional and morphological effects of α-latrotoxin (αLTX), a spider neurotoxin believed to cause massive release of vesicle-stored neurotransmitters, on spontaneously developed urethral smooth muscle tone. The effects were compared to those of EFS and high potassium. In the presence of the NO-synthesis inhibitor N^ω-nitro-L-arginine (L-NOARG: 0.3 mM) both αLTX and EFS evoked contractions. After treatment with scopolamine and phentolamine, no contraction was observed, and under these conditions αLTX and EFS induced relaxation. At low frequencies (<12 Hz), the EFS-induced relaxations were rapid, whereas at higher frequencies (>12 Hz), they were biphasic, consisting of a rapid first phase followed by a more long-lasting second phase. L-NOARG abolished the relaxations at low frequencies, as well as the first phase of the biphasic relaxation. The second phase was not affected by treatment with L-NOARG, but 0.1 μM ω-conotoxin GVIA, blocker of N-type voltage-operated calcium- channels (VOCCs), markedly reduced or abolished the response. In the presence of L-NOARG or ω-conotoxin GVIA, the αLTX-induced relaxation was significantly decreased, and the combination of L-NOARG and ω-conotoxin GVIA further reduced or abolished the relaxation. In preparationstreated with tetrodotoxin or scorpion venom, believed to inactivate nerves by acting on sodium channels, αLTX and EFS had no effects. αLTX-induced relaxation was not associated with changes in cyclic GMP or cyclic AMP content. High (80 mM) potassium solution induced a triphasic response of the preparation. A transient relaxation was followed by a restoration of tone, and then by a persistent relaxation. The persistent relaxation was slightly reduced by scorpion venom or L-NOARG, but reduced by 50% by a combination of L-NOARG and ω-conotoxin GVIA. Ultrastructural analysis of the urethral circular smooth muscle layer revealed a moderate amount of nerve profiles supplying the smooth muscle. In control preparations, the nerve profiles contained both small synaptic vesicles and large dense core vesicles. αLTX caused a major loss of both types of vesicle. The present data suggest that αLTX has the ability to release not only adrenergic and cholinergic transmitters, but also NANC mediators of relaxation, including NO, from nerve terminals in the urethra. Received: 13 January 1997 / Accepted: 17 April 1997     

缺氧缺血性脑病新生鼠胃壁内一氧化氮的改变

目的探讨缺氧缺血性脑病新生鼠胃壁局部一氧化氮（ＮＯ）的改变及窒息对消化系统的影响。方法采用二氢硫辛酰胺脱氢酶ＮＡＤＰＨ组织化学方法，检测２４只正常或缺氧新生鼠胃壁各层一氧化氮合成酶（ＮＯＳ）的分布变化。结果急性缺氧组与正常对照组相比，ＮＯＳ阳性产物无论在分布、染色深浅、纤维密度及ＮＯＳ阳性胞体数目上，差异均无显著意义（Ｐ＞０．０５）。但在缺氧缺血性脑病组，其肌层的ＮＯＳ阳性纤维无论是密度还是染色深浅，均明显强于正常对照组，ＮＯＳ阳性胞体亦明显多于正常对照组，其差异有非常显著意义（Ｐ＜０．０１）；而粘膜和粘膜下层的ＮＯＳ分布与正常对照组相比，差异无显著意义（Ｐ＞０．０５）。结论窒息时胃动力降低及胃粘膜病变与一氧化氮在胃壁内的改变有关     

Clinical studies of asthma phenotypes focusing on the role of leukotrienes

Introduction: Inflammation in the airways in connection to asthma is complex and the mechanisms underlying the associated clinical symptoms involve the interaction of many different kinds of cells and mediators, giving rise to different phenotypes. Objective: The objective of the present thesis was to investigate the molecular and cellular mechanisms that result in two of these phenotypes, i.e. aspirin‐intolerant asthma (AIA) and allergic asthma. The main focus was on leukotrienes. Materials and Methods: (i) Thirty‐three subjects with diagnosed AIA were challenged with celecoxib, a selective inhibitor of cyclooxygenase (COX)‐2. (ii) With the ultimate objective of finding a marker that could be used to identify patients with leukotriene‐associated asthma, the capacity to produce leukotrienes and the responsiveness to inhaled leukotrienes were determined in 20 subjects with mild asthma and in 10 healthy control individuals. (iii) Eight individuals with mild allergic asthma were challenged repeatedly with low doses of allergen in an experimental model aimed at mimicking the natural exposure to allergen. Exhaled nitric oxide was measured throughout the study. (iv) Thirteen patients with allergic asthma were subjected to bronchial challenges with methacholine and leukotriene D₄ (LTD₄) prior to and after administration of 500‐µg fluticasone twice daily for 2 weeks, and their levels of exhaled nitric oxide and urinary leukotriene E₄ (LTE₄) were determined. Results: (i) Both escalating doses from 5–100 mg (administered in a blinded, placebo‐controlled study) and an open‐label challenge with 200 + 200 mg celecoxib were tolerated well by AIA individuals. (ii) Neither group exhibited a correlation between the formation of leukotriene B₄ by their whole blood in response to ex vivo stimulation or urinary levels of LTE₄ and airway responsiveness to LTD₄. (iii) The level of nitric oxide in the air that they exhaled rose significantly. At the same time, these subjects did not report any symptoms of asthma, did not require rescue by bronchodilator medication, and did not display any change in the calibre of their airways. (iv) Inhalation of glucocorticoid attenuated the responsiveness to methacholine and reduced the level of exhaled nitric oxide, but neither the responsiveness to LTD₄ nor urinary excretion of LTE₄ was affected. Conclusions: (i) This finding indicates that the intolerance reaction leading to broncho‐constriction in patients with AIA is caused by inhibition of COX‐1 and, furthermore, provides a scientific basis for administration of selective inhibitors of COX‐2 to alleviate prostaglandin‐mediated pain and inflammation in these patients. (ii) In further attempts to predict which asthmatic patients will respond well to anti‐leukotriene treatment, investigations on the capacity for leukotriene synthesis, responsiveness to these agents and expression of their specific receptors in the lungs are being performed. (iii) Monitoring of exhaled nitric oxide on a daily basis may allow for early detection of exacerbation in subjects with allergic asthma. (iv) Neither the release nor the actions of leukotrienes appear to be sensitive to inhaled glucocorticoids, strengthening the rationale for using a combination of glucocorticosteroids and anti‐leukotrienes to treat allergic asthma.     

慢性低O2高CO2时NO-sGC-cGMP细胞信号转导通路变化及与肺动脉高压的关系

目的 :探讨慢性低O2 高CO2 性肺动脉高压发生与发展中NO sGC cGMP细胞信号转导通路的变化和作用。方法 :雄性Sprague Dawley大鼠随机分为对照组与低O2 高CO2 肺动脉高压 1w、2w及 4w组。用比色法测定血浆NO含量 ,酶动力学法测定肺组织sGC活性 ,12 5 I 放射免疫法检测肺组织cGMP含量。结果 :低O2 高CO2 1w、2w、4w组mPAP较对照组均明显升高 (P均 <0 .0 1)。而血浆NO含量、肺组织sGC活性和肺组织cGMP含量均显著降低 (分别P <0 .0 5,P <0 .0 1或P <0 .0 0 1)。mPAP与血浆NO含量 (r =-0 .80 7,P <0 .0 1)、与肺组织sGC活性 (r=-0 .754,P <0 .0 1)、与肺组织cGMP含量 (r=-0 .62 1,P <0 .0 1)之间均存在显著负相关。结论 :低O2 高CO2 引起的NO sGC cGMP转导通路的遏制性变化导致肺动脉舒张性降低是形成肺动脉高压的重要机制     

AUGMENTED ENDOGENOUS NITRIC OXIDE PRODUCTION IN PARTIAL PORTAL VEIN-LIGATED RATS

1. Endothelium-derived nitric oxide (NO) is a potent vasodilator. Because the body oxidizes it to nitrate ions, NO₃-, measurement of the serum concentration and the urinary excretion of NO₃- may be an index for endogenous NO. We investigated the role of NO on hyperdynamic circulation in cirrhotic and partial portal vein-ligated rats by measuring NO₃. 2. Liver cirrhosis was induced by administration of thioacetamide. Systemic and splanchnic haemodynamics and splenic-systemic shunting were determined by tracer microspheres. The concentration of NO₃- was measured by using high-performance liquid chromatography with an anion-column. 3. We found that systemic and splanchnic hyperdynamic circulation existed to almost the same extent in cirrhotic and in portal vein-ligated rats as compared to the controls and sham-operated rats, respectively. Splenic-systemic shunting was markedly greater in portal vein-ligated rats than in cirrhotic rats. 4. Serum NO₃- levels and urinary excretion of NO₃- in cirrhotic rats tended to increase as compared to the controls. On the other hand, the levels in portal vein-ligated rats were significantly increased as compared to those of the sham-operated rats, and were significantly and negatively correlated to the splanchnic arterial resistance and total vascular resistance. The amount of urinary excretion of NO₃- significantly correlated to splenic-systemic shunting (r = 0.61, P<0.05) only in portal vein-ligated rats. 5. We suggest that these high levels of NO₃- in portal vein-ligated rats relate to the extensive formation of porto-collateral vasculature or acute changes in systemic and splanchnic haemodynamics due to portal vein-ligation.     

再生障碍性贫血患者血清一氧化氮与细胞因子水平分析

生物体内的一氧化氮 (NO)作为一种反应极强的效应分子 ,不仅参与免疫调控 ,而且也是造血祖细胞生长和分化不可缺少的调节因子 [1 ]。本文通过对再生障碍性贫血 (AA)患者血清 NO与白细胞介素 - 2 (IL- 2 )、肿瘤坏死子 (TNF)、血小板生成素 (TPO)、红细胞生成素 (EPO)、GM- CSF、5种细胞因子水平测定 ,分析其与外周血象及各细胞因子间的相互关系 ,并探讨 NO及 IL- 2等细胞因子在 AA发生发展过程中的作用。1　材料和方法1.1　材料1.1.1　标本来源　 AA患者 5 0例 ,以 2 0 0 2年 3月至 2 0 0 4年4月我院确诊为 AA的患者为研究对象…     

Handling of asymmetrical dimethylarginine and symmetrical dimethylarginine by the rat kidney under basal conditions and during endotoxaemia.

BACKGROUND: Asymmetrical dimethylarginine (ADMA) is capable of inhibiting nitric oxide synthase enzymes, whereas symmetrical dimethylarginine (SDMA) competes with arginine transport. The potential role of inflammation in the metabolism of ADMA has been elucidated in an in vitro model using tumour necrosis factor-alpha, resulting in a decreased activity of the ADMA-degrading enzyme dimethylarginine dimethylaminohydrolase (DDAH). The kidney probably plays a crucial role in the metabolism of ADMA by both urinary excretion and degradation by DDAH. We aimed to further elucidate the role of the kidney in a rat model under basal conditions and during endotoxaemia. METHODS: Twenty-five male Wistar rats weighing 275-300 g were used for this study. The combination of arteriovenous concentration differences and kidney blood flow allowed calculation of net organ fluxes. Blood flow was measured using radiolabelled microspheres according to the reference sample method. Concentrations of ADMA, SDMA and arginine were measured by high-performance liquid chromatography. RESULTS: The kidney showed net uptake of both ADMA and SDMA and fractional extraction rates were 35% and 31%, respectively. Endotoxaemia resulted in a lower systemic ADMA concentration (P = 0.01), which was not explained by an increased net renal uptake. Systemic SDMA concentrations increased during endotoxaemia (P = 0.007), which was accompanied by increased creatinine concentrations. CONCLUSIONS: The rat kidney plays a crucial role in the regulation of concentrations of dimethylarginines, as both ADMA and SDMA were eliminated from the systemic circulation in substantial amounts. Furthermore, evidence for the role of endotoxaemia in the metabolism of dimethylarginines was obtained as plasma levels of ADMA were significantly lower in endotoxaemic rats.