Nitric oxide synthase expression and nitric oxide toxicity in oligodendrocytes

Oligodendrocytes (OLG) have more complex interactions with nitric oxide (NO) than initially suspected. Historically, OLG were seen only as targets of high NO levels released from other cells. Expression of nitric oxide synthase type II (NOS-2) in primary cultures of OLGs stimulated by cytokines led to controversy due to the presence of small numbers of microglia, cells also inducible for NOS-2 expression. The present review summarizes the findings that immature OLG express NOS-2, but that they do not in their most mature stage in culture as membrane sheet-bearing cells. This raises questions about the regulation of NOS-2 expression in OLG. Additionally, novel data are presented on NOS-3 expression in cultured OLG. If confirmed in vivo, this finding suggests that constitutive NOS-3 expression may play a key role in OLG injury due to its activation by calcium, in interaction with pathways mediating glutamate toxicity. The authors discuss in vivo NO levels to place in vitro findings in context, and compare OLG sensitivity to NO with that of other brain cells. Lastly, the multiple interactions of NO are considered with regard to glutamate cytotoxicity, the antioxidant glutathione, mitochondrial function, and myelin architecture.     

Nitric oxide synthase expression in lungs of pulmonary hypertensive patients with heart disease.

Since little is known about the contribution of endothelial nitric oxide synthase (e-NOS) to the mechanism of pulmonary vasospasm and the development of pulmonary vascular occlusive disease, we elucidate how e-NOS is expressed in lung biopsy specimens obtained from operative patients with pulmonary hypertension. Lung biopsy specimens were obtained from 17 patients who underwent open-heart operations for various heart diseases. A piece of normal lung specimen was also obtained from the resected lungs of three lung cancer patients as a control. e-NOS expression was visualized with a monoclonal antibody against e-NOS, and the level of expression was partially quantified. Significantly high levels of e-NOS expression were seen in adult patients, whose preoperative mean pulmonary arterial pressures were greater than 20 mm Hg. In contrast, e-NOS expression in pediatric patients with the same levels of mean pulmonary arterial pressure was the same as that in the controls and in low pulmonary arterial pressure. There was a statistically significant positive correlation between the level of e-NOS expression and Heath--Edwards grading. These data suggest that the e-NOS expression in lung tissue is induced when pulmonary vascular obstructive diseases progress.     

Nitric oxide synthase in the human pituitary gland.

Nitric oxide (NO) is generated by the NO synthase family of isozymes, which is present in many mammalian cells. The constitutive NO synthase isozymes generate NO, which acts via signal transduction mechanisms in the regulation of many functions including vascular tone and blood pressure, and the inducible isozymes mediate immunological mechanisms by cytotoxic and cytostatic effects. To determine whether NO has a role in anterior pituitary cell function, immunohistochemistry and in situ hybridization analyses were used to study NO synthase expression in normal and neoplastic human pituitary tissues. Brain NO synthase was localized in the anterior pituitary in secretory and in folliculo-stellate cells and in the posterior pituitary. Pituitary adenomas had higher levels of brain NO synthase protein and mRNA compared with normal pituitaries. Endothelial NO synthase was also present in anterior and posterior pituitary cells and in endothelial cells of the pituitary. Immunoblotting studies with brain NO synthase antibodies detected a slowly migrating approximately 155-kd band and more rapidly migrating approximately 90-kd and approximately 60-kd bands. Endothelial NO synthase, but not macrophage NO synthase, was also detected in the pituitary by immunoblotting studies, confirming the immunohistochemical observations. These findings indicate that NO synthase is expressed in normal and neoplastic human pituitary tissues with increased levels of brain NO synthase protein and mRNA in adenomas compared with non-neoplastic pituitary cells and suggest that NO may play a regulatory role in hormone secretion in anterior pituitary cells.     

Nitric oxide synthase activity and localization do not change in uterus and placenta during human parturition

Animal studies have suggested that nitric oxide, a smooth muscle relaxant, is a fundamental mediator in the initiation of parturition. The purpose of this study was to test the hypothesis that the onset of human labour is associated with a reduction in the activity of the enzyme nitric oxide synthase (NOS), within the uterus. Samples of myometrium, placenta, decidua and fetal membranes were collected during Caesarean section from 11 women before and 11 women after the onset of labour at term. Immunocytochemistry was used to localize each of the three isoforms of NOS (endothelial NOS, brain NOS, and inducible NOS) in each of these tissues and the intensity of staining was qualitatively assessed. NOS enzyme activity was determined in homogenates of frozen myometrium, placenta and fetal membranes (with attached decidua), by measuring conversion of radio-labelled L-arginine to L-citrulline. Each of the three isoforms of NOS was localized in each of the tissues. We found no difference in either the expression or enzyme activity of NOS in myometrium, placenta or fetal membranes before and during labour at term. These results suggest that, in contrast to animal studies, a decrease in NOS enzyme activity may not be involved in the onset of parturition at term in the human.      

Nitric oxide synthase distribution during implantation in the mouse.

The peri-implantation period is a critical time during murine development. Although the importance of nitric oxide has been demonstrated during gestation, its role in implantation has not been fully defined. The aim of this study was to quantify (by Western blotting) two prominent nitric oxide synthase (NOS) isoforms, inducible (iNOS) and endothelial (eNOS) and localize all three forms [iNOS, eNOS, and neuronal (nNOS)] by immunohistochemistry in uterine tissue from days 4 through 8 of pregnancy. By day 6, iNOS values were significantly elevated in implantation sites compared with interimplantation regions and continued to rise through day 8. Analysis of eNOS was similar, but implantation site values peaked by days 6 and 7. Labelled iNOS cells were within the decidua, around myometrial vessels, and within the ectoplacental cone. At implantation, eNOS was conspicuous, displaying label adjacent to the embryo in vessels of the primary decidual zone. nNOS was localized mainly in the mesometrium and myometrium and did not appear to change throughout the peri-implantation period. The increased iNOS and eNOS values following implantation in the embryonic site may imply roles in tissue remodelling, immunosuppression and vasoregulation. Nitric oxide may play an important role in the mechanisms of implantation where these factors are keys to successful pregnancy.     

慢性乙型肝炎患者体内一氧化氮和一氧化氮合酶水平的研究

目的　探讨慢性乙型肝炎(慢乙肝)患者体内一氧化氮(NO)和一氧化氮合酶(NOS)水平及其意义。方法　检测37例慢乙肝患者NO、NOS[诱生型NOS(iNOS)和结构型NOS(cNOS) ]、肝功能、乙型肝炎病毒(HBV)DNA和HBV基因型(GT)并做出统计分析。结果　慢乙肝患者组与正常对照组比较,NO和iNOS的浓度均明显升高(P <0 0 5 ) ;丙氨酸转氨酶(ALT)异常组与正常对照组及ALT正常组比较:NO和iNOS的浓度均明显升高(P <0 0 5 ) ;ALT正常组与正常对照组比较:NO的浓度明显升高(P <0 0 5 ) ;cNOS在各组间比较差异无统计学意义。在慢乙肝患者中:NO和iNOS浓度与ALT水平呈明显正相关(r=0 36 7,r=0 4 74 )。NO和NOS与HBVDNA指标间均无明显相关关系。不同基因型组之间,NO和NOS的浓度差异无统计学意义(P >0 0 5 )。结论　在慢乙肝患者中,存在NO和iNOS水平升高的现象。慢乙肝患者ALT升高时,NO浓度高,对机体损伤重;慢乙肝患者ALT正常者,NO对机体无明显的损伤。NO和NOS与HBVDNA是相对独立的检测指标。NO水平与不同HBVGT患者病情及预后不同无明显关系。     

Nitric oxide synthase immunoreactivity in rat spinal cord.

Immunoreactivity to nitric oxide synthase (NOS-IR) and choline acetyltransferase (ChAT-IR) was detected in the adult rat spinal cord using the avidin-biotin-peroxidase technique. Intensely stained NOS-positive neurons with cell processes were observed in the intermediolateral cell column of the thoracic and sacral segments and around the central canal of all segments. These areas also contained ChAT-IR neurons. A number of small- to medium-sized NOS-IR cells were noted in the superficial and deeper laminae throughout the entire cord. NOS-IR was not detected in the ventral horn motoneurons, which were, however, ChAT-IR. The results indicate that NOS-IR is present in autonomic preganglionic neurons and in selected neurons in the dorsal horn and lamina X, but appears to be absent in motoneurons.     

Nitric oxide synthase immunoreactivity in human bladder carcinoma.

AIMS: To study the expression of the endothelial and inducible isoforms of nitric oxide synthase (eNOS and iNOS, respectively) in human bladder carcinoma and schistosomal bladder disease, and to compare it with normal adult and fetal urothelium. Nitric oxide is thought to play a complex role in human carcinogenesis, but has only recently been investigated in bladder cancer. METHODS: Immunohistochemistry was performed on paraffin wax embedded sections of 33 human bladder carcinomas and five bladder carcinoma cell lines; in addition, seven schistosomal bladder cases and normal and fetal urothelium were investigated. In the cell lines enzymatic activity was examined by the NADPH diaphorase reaction. RESULTS: Immunoreactivity for eNOS was present in most cells of all 31 cases examined. Immunoreactivity for iNOS was less abundant and was seen in 23 of 25 cases. Similar findings were noted in schistosomal bladder cancer. In the normal bladder mucosa, eNOS immunoreactivity was found only in the superficial cell layer and iNOS was not expressed, whereas in the fetal urothelium immunoreactivity for both isoforms was seen in all cell layers. Enzymatic activity and immunoreactivity for eNOS and iNOS were evident in the five bladder carcinoma cell lines. CONCLUSIONS: It is possible that NOS plays a role in the differentiation of the transitional epithelium in fetal life, has a biological function in the adult bladder mucosa, and is involved in bladder carcinogenesis. eNOS and iNOS immunoreactivity do not differ in schistosomal and non-schistosomal bladder carcinoma, but resemble the pattern of expression typical of fetal urothelium.     

Distinct regulation of nitric oxide and cyclic guanosine monophosphate production by steroid hormones in the rat uterus

It has previously been reported that uterine nitric oxide (NO) production is enhanced during rat pregnancy compared to non-pregnant, labouring or postpartum states. The present hypothesis is that these changes in uterine NO production during pregnancy are caused by the interplay of oestrogen and progesterone. It is further postulated that changes in cyclic guanosine monophosphate (cGMP) production closely follow the changes in uterine NO synthesis. To test these hypotheses a variety of hormonal regimens (17beta-oestradiol, progesterone and combinations) were applied to different rat models (prepubertal, non-pregnant intact and ovariectomized as well as pregnant rats). The production of nitric oxide (NO) as well as basal and in-vitro NO-stimulated cGMP tissue content were measured in parallel. NO production was measured by the accumulation of nitrites and nitrates in a 24 h incubation medium as analysed by Greiss reaction. cGMP content was measured by radioimmunoassay. Diethylenetriamine/NO (DETA/NO) was used as NO donor. NO production in the rat uterus was markedly increased by pregnancy compared to other physiological (prepubertal, or cycling dioestrus) and experimentally induced (OVX) states. In contrast, uterine cGMP was significantly decreased in pregnancy. Pregnancy also inhibited the elevation in uterine cGMP after in-vitro NO challenge. Chronic 17beta-oestradiol treatment in prepubertal and/or OVX models increased NO production and also mimicked the effect of pregnancy on cGMP. Administration of progesterone in prepubertal rats induced a parallel decrease in both uterine NO and cGMP. In conclusion, sex steroid hormones distinctly regulate uterine NO and cGMP production depending upon the dose and regimen used, as well as the animal's reproductive state.     

Nitric oxide synthase expression in the transient ischemic rat retina: neuroprotection of betaxolol

Betaxolol is a β-adrenergic blocker but its neuroprotective action is generally thought to be due to its calcium channel blocking properties. In this study, we investigated neuronal cell damage and changes in the expression of neuronal nitric oxide synthase (nNOS) immunoreactivity in the ischemic retina and its relationship to the neuroprotection of betaxolol treatment after ischemic injury. Using the retina after ischemia, the expression of nNOS was studied by immunocytochemistry. In control retinas, two types of amacrine cells and a class of displaced amacrine cells were nNOS-labeled. After ischemia/reperfusion, the number of nNOS immunoreactive cells increased in both the ganglion cell layer and the inner nuclear layer compared to the control retinas. However, when experiments were carried out on animals that had been treated with betaxolol twice daily after ischemia/reperfusion, the number of nNOS immunoreactive cells decreased compared to the untreated ischemic retinas. These results suggest that an increase in nNOS expression could be associated with the degenerative changes in the ischemic retina, and that betaxolol treatment appears to play a role in protecting retinal tissue from ischemic damage.     

Nitric oxide-mediated mechanism of neuronal nitric oxide synthase and inducible nitric oxide synthase expression during hypoxia in the cerebral cortex of newborn piglets

Previously, we have shown that hypoxia results in increased generation of nitric oxide free radicals in the cerebral cortex of newborn piglets that may be due to up-regulation of nitric oxide synthases, neuronal nitric oxide synthase and inducible nitric oxide synthase. The present study tests the hypothesis that hypoxia results in increased expression of neuronal nitric oxide synthase and inducible nitric oxide synthase in the cerebral cortex of newborn piglets and that the increased expression is nitric oxide-mediated. Newborn piglets, 2-4 days old, were divided to normoxic (n=4), hypoxic (n=4) and hypoxic-treated with 7-nitro-indazole-sodium salt, a selective neuronal nitric oxide synthase inhibitor (hypoxic-7-nitro-indazole-sodium salt, n=6, 1 mg/kg, 60 min prior to hypoxia). Piglets were anesthetized, ventilated and exposed to an FiO2 of 0.21 or 0.07 for 60 min. Cerebral tissue hypoxia was documented biochemically by determining ATP and phosphocreatine. The expression of neuronal nitric oxide synthase and inducible nitric oxide synthase was determined by Western blot using specific antibodies for neuronal nitric oxide synthase and inducible nitric oxide synthase. Protein bands were detected by enhanced chemiluminescence, analyzed by imaging densitometry and the protein band density expressed as absorbance (OD x mm(2)). The density of neuronal nitric oxide synthase in the normoxic, hypoxic and hypoxic-7-nitro-indazole-sodium salt groups was: 41.56+/-4.27 in normoxic, 61.82+/-3.57 in hypoxic (P<0.05) and 47.80+/-1.56 in hypoxic-7-nitro-indazole-sodium salt groups (P=NS vs normoxic), respectively. Similarly, the density of inducible nitric oxide synthase in the normoxic, hypoxic and hypoxic-7-nitro-indazole-sodium salt groups was: 105.21+/-9.09, 157.71+/-13.33 (P<0.05 vx normoxic), 117.84+/-10.32 (p=NS vx normoxic), respectively. The data show that hypoxia results in increased expression of neuronal nitric oxide synthase and inducible nitric oxide synthase proteins in the cerebral cortex of newborn piglets and that the hypoxia-induced increased expression is prevented by the administration of 7-nitro-indazole-sodium salt. Furthermore, the neuronal nitric oxide synthase inhibition prevented the inducible nitric oxide synthase expression for a period of 7 days after hypoxia. Since administration of 7-nitro-indazole-sodium salt prevents nitric oxide generation by inhibiting neuronal nitric oxide synthase, we conclude that the hypoxia-induced increased expression of neuronal nitric oxide synthase and inducible nitric oxide synthase is mediated by neuronal nitric oxide synthase derived nitric oxide. We speculate that during hypoxia nitric oxide-mediated up-regulation of nitric oxide synthases will continue the perpetual cycle of nitric oxide generation-->NOS up-regulation-->nitric oxide generation resulting in hypoxic neuronal death.     

Nitric oxide synthase in damaged sensory neuron

NADPH diaphorase colocalized with nitric oxide synthase was studied in damaged protoneurons of the nodose ganglion of rabbit vagus nerve. The number and activity of nitric oxide synthase-expressing neurons increased. Nitric oxide is involved in neuron remodeling. Translated fromByulleten' Eksperimental'noi biologii i Meditsiny, Vol. 128, No. 10, pp. 463–465, October, 1999     

Nitric oxide synthase activity in infantile hypertrophic pyloric stenosis.

BACKGROUND. Hypertrophic pyloric stenosis is a common infantile disorder characterized by enlarged pyloric musculature and gastric-outlet obstruction. Its physiopathologic mechanism is not known, but a defect in pyloric relaxation (pylorospasm) has been postulated. Nitric oxide is a mediator of relaxation in the mammalian digestive tract, raising the possibility that pylorospasm could be caused by a defect in nitric oxide production. Since neuronal nitric oxide synthase and NADPH diaphorase are identical, we used the NADPH diaphorase histochemical reaction to study the distribution of nitric oxide synthase in pyloric tissue from patients with infantile hypertrophic pyloric stenosis. METHODS. We studied pyloric tissue from nine infants with infantile hypertrophic pyloric stenosis and seven control infants and children. Cryostat sections were processed for NADPH diaphorase histochemical analysis. A polyclonal tau antiserum was used to identify the enteric nervous system by immunohistochemical methods. RESULTS. NADPH diaphorase activity was restricted to the enteric nervous system and blood vessels. In the pyloric tissues from the control patients, intense diaphorase activity was present in the nerve fibers of the circular musculature, in the neurons and nerve bundles of the myenteric plexus, and in some nerve fibers of the longitudinal musculature. In the pyloric tissues from patients with infantile hypertrophic pyloric stenosis, the enteric nerve fibers in the hypertrophied circular musculature were enlarged and distorted and did not contain diaphorase activity, whereas the activity in the myenteric plexus and the longitudinal musculature was preserved. CONCLUSIONS. We suggest that a lack of nitric oxide synthase in pyloric tissue is responsible for pylorospasm in infantile hypertrophic pyloric stenosis.     

Nitric oxide and nitric oxide synthase in the early phase of perinatal asphyxia of the rat.

The role of nitric oxide, a compound involved in neurotransmission and regulation of cerebral blood flow, in cerebral ischemia is still not fully elucidated yet. Although well studied in adult systems of cerebral ischemia/hypoxia, information on nitric oxide in perinatal asphyxia is limited and, in particular, no direct evidence for its generation has been provided. We therefore decided to study nitric oxide generation in brain of asphyctic rat pups by biophysical and biochemical methods. We used a simple, non-invasive rat model resembling the clinical situation in perinatal asphyxia: rat pups delivered by Caesarean section were placed into a water bath at 37 degrees C still in patent membranes for various asphyctic periods (up to 20 min). Brain pH, cerebral blood flow, neuronal nitrix oxide synthase messenger RNA (by northern and dot blot analysis), immunoreactive protein (by western blot analysis) and nitric oxide synthase activity were determined; generation of nitric oxide was evaluated directly by electron paramagnetic resonance spectroscopy. Neuronal nitric oxide synthase messenger RNA activity and nitric oxide generation were unaffected, whereas neuronal nitric oxide synthase-immunoreactive protein of 150,000 mol. wt was decreased and of 136,000 mol. wt was increased with the length of the asphyctic period. This is the first report on direct evidence for the generation of nitric oxide in perinatal asphyxia and we demonstrate that nitric oxide production remains unaffected even by 20 min of asphyxia, at a time-point when cerebral blood flow was increased four-fold and severe acidosis was present. However, it was found that levels of immunoreactive neuronal nitric oxide synthase of 136,000 mol. wt were increased paralleling the length of asphyxia. Levels of the 150,000 mol. wt immunoreactive neuronal nitric oxide synthase protein decreased, suggesting a different regulation pattern. Thus, the present biochemical and biophysical results form the basis for further investigations on nitric oxide in perinatal asphyxia.     

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.     

巨噬细胞诱导型一氧化氮合酶的表达调节机制

一氧化氮是一种重要的巨噬细胞免疫效应分子,它参与免疫调节和宿主防御反应.一氧化氮的生成主要由诱导型一氧化氮合酶调节,然而诱导型一氧化氮合酶表达的调节机制及信号通路尚不完全清楚.