Role of nitric oxide in implantation and menstruation

Nitric oxide (NO) is a major paracrine mediator of various biological processes, including vascular functions and inflammation. In blood vessels, NO is produced by the low-input constitutive endothelial NO synthase (eNOS) and is a potent vasodilator and platelet aggregation inhibitor. The inducible NOS isoform (iNOS) is capable of producing NO at high concentrations which have pro-inflammatory properties. Immunohistochemical and molecular studies of endometrial NOS expression, as well as animal experiments with NOS inhibitors, indicate that NO plays an important role in endometrial functions such as endometrial receptivity, implantation and menstruation. In rodents, both iNOS and eNOS are highly up-regulated in the implantation sites, and NOS inhibitors show synergistic effects with antiprogestins in inhibiting the establishment of pregnancy. In the human endometrium, eNOS have been localized in the glandular epithelium and in endometrial microvascular endothelium, primarily during the luteal phase. iNOS has been found in the endometrial epithelium during menstruation, in immunocompetent endometrial cells, and in decidualized stromal cells. In primates, NO may be involved in the initiation and maintenance of menstrual bleeding by inducing tissue breakdown and vascular relaxation as well as by inhibiting platelet aggregation. Endometrium-derived NO may also play a role in myometrial relaxation during menstruation. These studies open up new applications for NO-donating and -inhibiting agents in uterine disorders. NO donors may be useful in the treatment of dysmenorrhoea and for promoting fertility. Antiprogestins, progesterone receptor modulators and iNOS inhibitors may find applications in the treatment and prevention of abnormal uterine bleeding.     

Myometrial constitutive nitric oxide synthase expression is increased during human pregnancy

Nitric oxide (NO), derived from L-arginine by the action of nitric oxide synthase (NOS), is a mediator of many diverse biological activities, including vasodilation, neurotransmission and inhibition of platelet adhesion. A role for NO in the maintenance of rat and rabbit pregnancy is supported by a variety of studies. A recent study in women demonstrated that myometrial inducible NOS (iNOS) expression was greater in the early third trimester than either the late third trimester or in the non-pregnant condition, suggesting that increased iNOS expression is involved in the maintenance of human pregnancy. Constitutive NOS (cNOS) expression was not determined. The aim of this study was to compare constitutive NOS (both eNOS and bNOS) expression in the human non-pregnant uterus, preterm pregnant uterus (25-34 weeks gestation) and term pregnant uterus (>37 weeks gestation) using immunohistochemistry and Western blotting. Preterm pregnant samples were taken from women with a variety of pathologies necessitating early delivery. We found that eNOS and bNOS protein concentrations were greater in the preterm pregnant myometrium than non-pregnant myometrium. eNOS, but not bNOS, protein concentration was lower in myometrial samples obtained at term compared with those obtained preterm. We conclude that the constitutive isoforms of NOS are also up-regulated in human pregnancy and may play a role in the maintenance of myometrial quiescence.     

GnRH agonist-suppressed expression of nitric oxide synthases and generation of peroxynitrite in adenomyosis

Because overproduction of nitric oxide (NO) and peroxynitrite is known to cause tissue injury, the expression of NO synthases (NOS) and generation of peroxynitrite were investigated in adenomyosis. Immunoreactivities to endothelial and inducible NOS demonstrated phase-dependent changes in normal endometrium, and in eutopic endometrium of adenomyosis. However, NOS were expressed throughout the menstrual cycle in ectopic endometrium from the majority of patients with adenomyosis. Nitrotyrosine, a footprint of peroxynitrite, was detected concomitantly with NOS protein. This suggested that high doses of NO and superoxide are produced in the ectopic endometrium, presumably by stimulation with bioactive molecules such as cytokines and growth factors. The expression of NOS and generation of peroxynitrite were markedly reduced by administration of gonadotrophin-releasing hormone agonists (GnRHa). The suppression of serum concentrations of nitrite/nitrate, stable metabolites of NO, by long-term administration of GnRHa was also demonstrated. The suppression of synthesis of NO and/or peroxynitrite may be part of both the therapeutic and adverse effects of GnRHa therapy.     

Roles of endogenous nitric oxide synthase inhibitors and endothelin-1 for regulating myometrial contractions during gestation in the rat

This study investigated the roles of endogenous nitric oxide synthase (NOS) inhibitors and endothelin-1 (ET-1) for regulating myometrial contractions during gestation in the rat. Basal and stimulated cyclic GMP production with L-arginine as a NOS substrate or sodium nitroprusside (SNP) as a NO donor were significantly enhanced at the middle of gestation (14th day), while these were greatly decreased at term (22nd day), suggesting the accelerated NO production and/or up-regulation of guanylate cyclase at the middle of gestation. NOS within the myometrium was mainly Ca(2+)dependent and partly Ca(2 + )independent and remained unaffected by aminoguanidine as an inhibitor of inducible NOS in non-pregnant and gestational myometrium. NOS activity per se and endothelial NOS (eNOS) protein expression remained unchanged at the middle and term gestation. Neuronal NOS (nNOS) and inducible NOS (iNOS) proteins were undetectable. SNP at a high concentration of 100 micromol/l failed to modify the spontaneous and ET-1-induced rhythmic contractions in non-pregnant and gestational myometrium. Contents of N(G)-monomethyl-L-arginine (L-NMMA) plus asymmetric N(G),N(G)-dimethyl-L-arginine (ADMA) as endogenous NOS inhibitors and ET-1 within the myometrium were significantly decreased at 14th and 20th days of gestation, whereas these were significantly increased at term gestation (22nd day) and after delivery. There was a significant and positive correlation between endogenous NOS inhibitor content and ET-1. ET-1 within the myometrium was significantly increased with a concomitant decrease in cyclic GMP production after the intraperitoneal application of authentic L-NMMA for 2 weeks, suggesting that the impaired NO production with endogenous NOS inhibitors would result in increased ET-1 content. These results suggest that endogenous NOS inhibitors such as L-NMMA and ADMA play an important role for regulating NO production in rat myometrium. The impaired NO production due to accumulated endogenous NOS inhibitors possibly results in increased ET-1 content within the myometrium, thereby increasing myometrial contractions at term gestation and after delivery.     

Identification of nitric oxide synthase in human uterus

The aim of this study was to investigate the presence of nitricoxide synthase (NOS) in human uterus. Tissues were obtainedat operation from 10 women undergoing hysterectomy for benigndisease. In-situ hybridization was used to determine the distributionof mRNA for NOS with a 483 bp digoxigenin-labelled antisenseriboprobe. Localization of NOS was detected by (i) immunocytochemistryusing a monoclonal antibody raised against bovine constitutiveendothelial NOS, and (ii) NADPH diaphorase, which has been suggestedto co-localize with brain NOS. Messenger RNA for NOS was detectedin endometrium and myometrium from nine of 10 women, predominantlyin endometrial glandular epithelium and stroma and myometrialblood vessels. NOS-like immunoreactivity was seen in endometrialstroma and myometrial blood vessels, whereas NADPH diaphoraseactivity was localized mainly to endometrial glandular epitheliumand myometrial blood vessels. These studies suggest that differentforms of constitutive NOS are present in human endometrium andmyometrium, and that nitric oxide may play a role in the paracrinecontrol of the uterine vascular bed.     

Modulation of autoimmune diseases by nitric oxide

Nitric oxide (NO) is an intercellular messenger that performs a number of functions, including neurotransmission, vasodilatation, inhibition of platelet aggregation, and modulation of leukocyte adhesion. NO has recently been shown to act as a potent cytotoxic effector molecule as well as to play an important role in the pathogenesis of organ-specific autoimmunity. NO may also modulate the immune response by interfering with Th1/Th2 balance in autoimmune diseases. This review will discuss the role of NO and nitric oxide synthase (NOS) in pathophysiologic and therapeutic implications in various autoimmune diseases with particular reference to T helper-1 (Th1) and T helper-2 (Th2) cytokines.     

一氧化氮、一氧化氮合酶和心脏功能

一氧化氮合酶(nitric　oxide synthase,NOS)催化L-精氨酸的氧化反应生成L-胍氨酸和一氧化氮(nitric oxide,NO)。其产物NO可通过依赖cGMP(环磷酸鸟苷)途径与非依赖cGMP途径发挥其复杂的生理学功能,如在心血管系统具有维持血管张力、调节血压,抑制血管平滑肌细胞迁移、增生,抑制血小板聚集与白细胞对血管壁的粘附以及调节影响心肌收缩与舒张功能的作用,并参与心率变异调节功能。本文就3种NOS同工酶的基因及其基因表达调节及影响因素进行简要综述。着重介绍nNOS1的心脏自主神经调节机制,iNOS对心脏收缩抑制以及心脏保护与损伤的双重作用,并对eNOS参与心功能调节的机制及其它生理、病理学等方面研究进展进行综述。     

Central control mechanisms of circulation in the medulla oblongata by nitric oxide

Nitric oxide (NO) is involved in numerous physiological functions. Besides its role as an endothelium-dependent relaxing factor (EDRF), NO inhibits platelet aggregation, contributes to cytotoxicity against bacteria, is active in synaptic transmission within the brain, etc. NO synthase (NOS) is distributed in brain regions related to the regulation of cardiovascular functions. NO has been inferred not only to act directly on vascular vessels, but also to regulate circulation within the brain. In this review paper, we mainly consider the functions of NO in the cardiovascular center of the medulla oblongata. That is, we describe the anatomical distribution of NOS in the brain, effects of intravenous and intracerebroventricular administration of NOS inhibitors on the circulation, effects of microinjection of NO donors and NOS inhibitors into the nucleus tractus solitarius (NTS) and ventrolateral medulla (VLM), the results of electrophysiological studies on these areas, and finally, the data obtained by new molecular biological techniques.     

Changes in the expression of nitric oxide synthase in the human uterine cervix during pregnancy and parturition

Nitric oxide (NO) has been proposed as a mediator of cervical ripening. We investigated the expression, using Western blotting, and localization, using immunohistochemistry, of the nitric oxide synthase (NOS) enzymes, inducible NOS (iNOS), endothelial NOS (eNOS) and neuronal NOS (bNOS) in the human cervix during pregnancy and parturition. Cervical biopsies were obtained from non-pregnant women, women in the first trimester of pregnancy, and pregnant women at term before and after the onset of labour. Each of the NOS isoforms was localized in the cervices of both non-pregnant and pregnant subjects using immunohistochemistry. iNOS expression was significantly greater in early pregnancy compared with the non-pregnant state (P: < 0.005). iNOS expression was up-regulated further in samples obtained in the third trimester compared with the first trimester. bNOS expression was greater in samples from the first trimester of pregnancy than in non-pregnant samples (P: < 0. 005), but showed no additional increase in late pregnancy or with the onset of labour. eNOS expression was increased in samples obtained in the third trimester both before (P: = 0.002) and after the onset of labour (P: < 0.002) when compared with non-pregnant samples. The increased expression of NOS isoforms in late pregnancy supports the hypothesis that NO is involved in the process of cervical ripening.     

Augmented endothelial nitric oxide synthase (eNOS) protein expression in human pregnant myometrium: possible involvement of eNOS promoter activation by estrogen via both estrogen receptor (ER)alpha and ERbeta

The aim of the present study was to investigate the possible contribution of estrogen to pregnancy-associated modulation of nitric oxide production in the human myometrium during pregnancy. Both endothelial nitric oxide synthase (eNOS) and inducible NOS (iNOS) proteins were clearly expressed in the non-pregnant myometrium and were elevated in the first trimester of pregnancy. Oral contraceptive pills augmented eNOS, but not iNOS, protein expression in the non-pregnant human myometrium. In cultured human myometrial cells, estrogen receptor (ER)alpha and ERbeta expression was extremely low. Therefore, we used either ERalpha or ERbeta expression vector to investigate the effect of 17beta-estradiol treatment on eNOS promoter activity using eNOS promoter/luciferase vector in cultured human myometrial cells. 17beta-estradiol treatment significantly augmented eNOS promoter activity in cells co-transfected with either ERalpha or ERbeta, and this augmentation was dose-dependently suppressed by ICI 182780, an estrogen antagonist. These data suggest the possibility that both ERalpha and ERbeta are involved in the estrogen-associated regulation of eNOS gene expression in the human myometrium.     

NO在脂联素抑制高脂血症大鼠血小板聚集中的作用

目的: 探讨一氧化氮(NO)信号转导通路在脂联素抑制高脂血症血小板聚集机制中的作用。方法: 采用成年大鼠饲以高脂饲料14周,分离其血小板并以重组脂联素(rAPN)孵育。采用免疫荧光、Western blotting等方法观察检测血小板聚集、NO含量、超氧化物含量、内皮型一氧化氮合酶(eNOS)/诱导型一氧化氮合酶(iNOS)的表达和抗氧化物活性。结果: 采用rAPN处理能抑制高脂血症诱导的血小板聚集(P<0.05),并导致血小板NO的生成显著减少。同时,在高脂血症血小板中,采用rAPN处理还能显著减少超氧化物的生成(降低62%, P<0.05) 并增强其抗氧化能力(增加38%, P<0.05)。此外,高脂血症诱导的eNOS磷酸化的降低和iNOS表达的增加在rAPN处理后被显著逆转(P<0.05, P<0.01)。结论: 脂联素是一种抑制高脂血症血小板聚集的脂肪细胞因子,其机制与减少超氧化物水平、增加抗氧化物活性和阻断iNOS的表达有关。     

Pharmacological manipulation of nitric oxide levels in mouse follicle cultures demonstrates key role of extrafollicular control of ovulation

BACKGROUND: Nitric oxide (NO) is involved in ovulation, but it is uncertain whether the mechanisms responsible are systemic or local. In vivo and in perfused ovaries, inhibition of nitric oxide synthase (NOS) reduces ovulation. This study used a well-characterized system of isolated follicle culture to assess which isoforms of NOS might be involved at the follicular level. METHODS: NOS inhibitors, stimulators and NO donors were used to manipulate the environment of mouse cultured follicles, selectively targeting different isoforms of NOS. Follicle survival and ovulation in vitro were monitored using established markers. RESULTS: Inhibition of endothelial NOS did not affect survival or ovulation of cultured follicles. Inhibition of inducible nitric oxide synthase (iNOS) had mild effects in this system, generally inhibitory of ovulation. NO donors had variable inhibitory effects, including toxic effects. Stimulators of iNOS appeared to show mixed, mostly inhibitory, effects that possibly involved different mechanisms. CONCLUSIONS: The results suggest that relatively minor effects on survival and ovulation in vitro are achieved by modulating the NO pathway, and the marked effects of NOS inhibitors in vivo do not occur in vitro. It is therefore probable that the effects of NOS inhibitors in vivo are mediated via effects outside the follicle.     

Rat myometrial smooth muscle cells express endothelial nitric oxide synthase

We examined if rat myometrial cells in culture generate nitric oxide (NO) and express various isoforms of NO synthase (NOS). Myometrial cells isolated from rats on day 18 of gestation were incubated with various stimulators and inhibitors of NOS for 24 and 48 h, and NO production was evaluated by measuring nitrites in the media and NOS proteins in the cell lysates. NO was produced by myometrial cells and its production inhibited by N(G)-methyl-L-arginine (L-NMMA). This inhibition was reversed by L-arginine (3 mM). Interleukin-1beta (IL- 1beta) significantly stimulated NO production, in a dose-dependent manner. The IL-1beta-stimulated NO production was inhibited by the NOS inhibitor, L-NMMA, whose effects were reversed by L-arginine. Abundant NOS III protein was detectable in freshly isolated myometrial cells, and this was maintained in culture in the presence of fetal bovine serum (FBS; 10%). In the absence of FBS, NOS III levels decreased significantly (by 90%) within 24 h. In contrast, NOS I and NOS II proteins were undetectable in freshly isolated muscle cells and in cells cultured without IL-1beta. However, NOS II protein in these cells was induced by IL-1beta. Thus, NO is produced by myometrial cells through the NOS III isoform, and the myometrial NO may be important in maintaining uterine quiescence during pregnancy.      

Neuronal and inducible nitric oxide synthase expressions and activities in the hippocampi and cortices of young adult,aged cognitively unimpaired,and impaired Long-Evans rats

Nitric oxide (NO) is a neurosignaling molecule that appears to play a significant role in learning and memory. This molecule has also been implicated in neurotoxicity due to its oxidative properties. Previous experiments from our laboratories have demonstrated elevated hippocampal and cortical neuronal nitric oxide synthase (NOS) mRNA levels in aged cognitively unimpaired and impaired Long-Evans rats, which could represent either increased neuronal NOS activity thereby leading to NO-mediated neurotoxicity, or a compensatory response by aged neurones to maintain physiological nitric oxide output. The current study measured the protein expression and activity levels of neuronal and inducible NOS in young adult (6 months) and aged (24-26 months) Long-Evans rats by means of western blotting and NOS activity assay. Aged animals were assigned as either cognitively unimpaired or aged with moderate cognitive impairments based on their performances in the Morris water maze behavioural task. Our results showed that hippocampal and cortical neuronal NOS expressions were significantly decreased in aged animals. These aged animals also exhibited increased hippocampal and cortical inducible NOS expressions. Between the two aged animal groups, cognitively impaired rats showed significantly lower hippocampal and cortical neuronal but higher hippocampal inducible NOS expressions. Young adult rats exhibited significantly higher hippocampal and cortical NOS activities than the aged animals. Aged animals with cognitive deficits showed significantly lower hippocampal NOS activity than cognitively unimpaired aged rats.Our data indicate that aging is associated with a decline in neuronal but elevated inducible NOS functioning in brain areas involved in learning and memory. These phenomena could contribute to the cognitive deficits observed in a sub-population of aged animals.     

Intra- and inter-hemispheric coupling of electroencephalographic 8-13 Hz rhythm in humans and force of static finger extension

Information on equipment and subcellular distribution of nitric oxide synthase (NOS) isoforms in myenteric neurons and pacemaker cells (ICC) might help to identify nitric oxide (NO) pathway(s) acting on gastrointestinal motility. In sections of mouse colon labelled with neuronal (n)NOS, endothelial (e)NOS and inducible (i)NOS antibodies, all myenteric neurons co-expressed eNOS and iNOS and a subpopulation of them co-expressed nNOS. ICC co-expressed nNOS and eNOS. In the neurons, nNOS-labeling was intracytoplasmatic, in the ICC at cell periphery. In both cell types, eNOS-labeling was on intracytoplasmatic granules, likely mitochondria. In conclusion, myenteric neurons and ICC co-express several NOS isoforms with specific subcellular distribution. Different nNOS splice variants are presumably present: intracytoplasmatic nNOSbeta and nNOSalpha producing neurogenic NO, plasma membrane-bound nNOSalpha producing ICCgenic NO. eNOS might be implicated in mitochondrial respiration and, in ICC, also in pacemaker activity. Neurons express iNOS also in basal condition.     

Rhinovirus infection induces expression of type 2 nitric oxide synthase in human respiratory epithelial cells in vitro and in vivo

BACKGROUND: Human rhinovirus (HRV) infections are the predominant cause of the common cold and are associated with exacerbations of asthma. Nitric oxide (NO) may play an important role in host defense by means of its potent antiviral properties. OBJECTIVE: We sought to determine whether epithelial expression of type 2 nitric oxide synthase (NOS 2), which produces NO, is induced on rhinovirus infection in vitro and in vivo. METHODS: Primary cultures of human airway epithelial cells were infected with HRV-16, and NOS 2 mRNA expression was assessed by conventional and real-time RT-PCR and NOS 2 protein by using Western blot analysis. Human subjects were also infected with HRV-16 in vivo, and mRNA for NOS 2 was assessed in nasal epithelial scrapings obtained before and after infection. RESULTS: NOS 2 mRNA levels increased within 8 hours after HRV-16 infection of cultured cells and remained elevated up to 48 hours after infection. NOS 2 protein was elevated at 24 hours. Induction of NOS 2 did not occur with UV-inactivated HRV-16 but could be reproduced by using double-stranded RNA, indicating that induction was dependent on viral replication. Increased NOS 2 expression was also observed in nasal epithelial scrapings during symptomatic colds. CONCLUSION: Increased epithelial expression of NOS 2 mRNA occurs as part of the host response to HRV infection in vitro and in vivo. Given the antiviral effects of NO, we speculate that increased host production of NO may play an important role in host defense during HRV infections.