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.     

mRNA and protein expression and activities of nitric oxide synthases in the lumbar spinal cord of neonatal rats after sciatic nerve transection and melatonin administration

Sciatic axotomy in 2-day-old rats (P2) causes lumbar motoneuron loss, which could be associated with nitric oxide (NO) production. NO may be produced by three isoforms of synthase (NOS): neuronal (nNOS), endothelial (eNOS) and inducible (iNOS). We investigated NOS expression and NO synthesis in the lumbar enlargement of rats after sciatic nerve transection at P2 and treatment with the antioxidant melatonin (sc; 1 mg/kg). At time points ranging from P2 to P7, expression of each isoform was assessed by RT-PCR and immunohistochemistry; catalytic rates of calcium-dependent (nNOS, eNOS) and independent (iNOS) NOS were measured by the conversion of [3H]L-arginine to [3H]L-citrulline. All NOS isoforms were expressed and active in unlesioned animals. nNOS and iNOS were detected in some small cells in the parenchyma. Only endothelial cells were positive for eNOS. No NOS isoform was detected in motoneurons. Axotomy did not change these immunohistochemical findings, nNOS and iNOS mRNA expression and calcium-independent activity at all survival times. However, sciatic nerve transection reduced eNOS mRNA levels at P7 and increased calcium-dependent activity at 1 and 6 h. Melatonin did not alter NOS expression. Despite having no action on NOS activity in unlesioned controls the neurohormone enhanced calcium-dependent activity at 1 and 72 h and reduced calcium-independent catalysis at 72 h in lesioned rats. These results suggest that NOS isoforms are constitutive in the neonatal lumbar enlargement and are not overexpressed after sciatic axotomy. Changes in NO synthesis induced by axotomy and melatonin administration in the current model are discussed considering some beneficial and deleterious effects that NO may have.     

Nitric oxide in health and disease of the respiratory system

During the past decade a plethora of studies have unravelled the multiple roles of nitric oxide (NO) in airway physiology and pathophysiology. In the respiratory tract, NO is produced by a wide variety of cell types and is generated via oxidation of l-arginine that is catalyzed by the enzyme NO synthase (NOS). NOS exists in three distinct isoforms: neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS). NO derived from the constitutive isoforms of NOS (nNOS and eNOS) and other NO-adduct molecules (nitrosothiols) have been shown to be modulators of bronchomotor tone. On the other hand, NO derived from iNOS seems to be a proinflammatory mediator with immunomodulatory effects. The concentration of this molecule in exhaled air is abnormal in activated states of different inflammatory airway diseases, and its monitoring is potentially a major advance in the management of, e.g., asthma. Finally, the production of NO under oxidative stress conditions secondarily generates strong oxidizing agents (reactive nitrogen species) that may modulate the development of chronic inflammatory airway diseases and/or amplify the inflammatory response. The fundamental mechanisms driving the altered NO bioactivity under pathological conditions still need to be fully clarified, because their regulation provides a novel target in the prevention and treatment of chronic inflammatory diseases of the airways.     

Pseudorabies virus-induced expression of nitric oxide synthase isoforms

In addition to its role as a neurotransmitter, studies have postulated both neuroprotective and neurotoxic roles for nitric oxide (NO) generated in response to infections with neurotropic viruses. This study examined the expression of neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS) isoforms of NOS induced by neuronal infection with virulent and attenuated strains of pseudorabies virus (PRV). Caudal brainstem neurons infected by peripheral inoculation of the viscera served as the model system. Neuronal infection induced the expression of nNOS and iNOS, but the timing and the apparent magnitude of NOS expression varied according to the virulence of the infecting strain of virus. Expression of nNOS was observed in infected neurons that did not express this enzyme in control animals, and the onset of expression was earlier in animals infected with virulent PRV. Expression of iNOS was largely restricted to monocytes and macrophages that invaded the brain in response to PRV infection. These iNOS-expressing cells were observed earlier in animals infected with the virulent virus, and were differentially concentrated in areas exhibiting virus-induced neuropathology. Collectively, these data suggest functionally diverse roles for NO in the brain response to PRV neuronal infection.     

睾丸一氧化氮合酶与雄性生殖

一氧化氮合酶 (nitricoxidesynthase ,NOS)在NADPH(还原型辅酶Ⅱ )存在下催化L -精氨酸分解生成一氧化氮(nitricoxide,NO)。NOS有以下几种形式 :神经型NOS(nNOS) ,诱导型NOS(iNOS) ,内皮型NOS(eNOS)。NO是目前研究最多的体内信息分子和效应分子 ,广泛存在于人体的心血管系统、神经系统、消化系统、免疫系统、生殖系统等。NOS和NO对生殖活动的作用已被广泛研究 ,如对睾丸微循环的调解 ,参与睾酮分泌 ,调解精子活动等。本文对睾丸NOS/NO与雄性哺乳动物生殖关系的研究进展作一概述。     

大鼠严重烧伤后体内一氧化氮水平变化与预后的关系

目的:研究一氧化氮(NO)及一氧化氮合酶(NOS)在严重烧伤早期大鼠体内的变化规律及其与预后的可能联系。方法:检测严重烧伤前后大鼠血液中NO代谢产物NO^-₂/NO^-₃及脑、肺脏和十二指肠组织中神经型(nNOS)和诱生型一氧化氮合酶(iNOS)蛋白的水平,同时统计各组大鼠的存活率。结果:烧伤后大鼠血液中NO^-₂/NO^-₃水平显著增高,非选择性NOS抑制剂L-NAME和选择性iNOS抑制剂氨基胍(AG)对其均有抑制作用,以L-NAME为甚;nNOS蛋白在伤后部分升高,L-NAME和AG均轻度上调nNOS水平;iNOS在正常组织中不表达,烧伤后表达异常增高,L-NAME和AG对此均无影响;与对照组比较,AG组大鼠存活时间延长,L-NAME组存活时间缩短。结论:严重烧伤后的血管扩张、血压降低和血管反应性低下与iNOS蛋白水平过度增高及其释放的大量NO关系密切。     

Non-lethal Sepsis and Nitric Oxide Inhibition in the Guinea Pig

Nitric oxide (NO) is synthesized from arginine by three distinct isoforms of nitric oxide synthase (NOS). Two of these isoforms, endothelial NOS (eNOS, type III NOS) and neuronal NOS (nNOS, type I NOS) are expressed in a constitutive manner and are responsible for regulating physiological functions. The induction of the third isoform of nitric oxide synthase (iNOS) by inflammatory processes and the subsequent overproduction of NO is thought to contribute to the tissue damage that occurs in a number of diseases having an inflammatory component, such as sepsis. As a model of sepsis, non-lethal endotoxemia in Hartley guinea pigs was induced using three serotypes of lipopolysaccharide (LPS, 30 mg/kg, i.p.). Each stimulated an increase in total plasma nitrites 6 h following their administration. Treatment orally with two inhibitors of NOS, L-nitroarginine methyl ester (L-NAME), a non-selective inhibitor and N-iminoethyl-L-lysine (L-NIL), a selective iNOS inhibitor, 30 min after the induction of sepsis, inhibited (p < 0.05) the increase in plasma nitrites with ED₅₀ values of 7 ± 1 and 0.4 ± 0.03 mg/kg, respectively, suggesting that NOS inhibitors may be useful in the treatment of human sepsis, or other diseases where excess NO levels have been implicated, such as asthma, arthritis and inflammatory bowel disease.     

肢体缺血预适应的小肠保护作用及与一氧化氮/内皮素-1的关系

目的：观察一氧化氮/内皮素-1(NO/ET-1)失衡与肢体缺血再灌注(I/R)后小肠损伤的关系，以及缺血预适应(IPC)对NO/ET-1系统的调节作用。 方法： 雄性Wistar大鼠18只，随机分为对照（control）组，缺血再灌注（IR）组和缺血预适应（IPC+IR）组，每组6只，分别测定血浆和小肠组织二氨氧化酶(DAO)、一氧化氮(NO)、内皮素-1(ET-1)、NO/ET-1比值的含量变化及小肠组织的髓过氧化物酶(MPO)、DNA双链百分率(ratio of DNA chain %)、总一氧化氮合酶（tNOS）、诱导型一氧化氮合酶（iNOS）、结构型一氧化氮合酶（cNOS）的水平；免疫组化法检测小肠组织的诱导型一氧化氮合酶（iNOS）、内皮型一氧化氮合酶（eNOS）的表达。 结果： IR组血浆和小肠组织NO、ET-1，血浆DAO及组织MPO均明显高于对照组，而 NO/ET-1的比值，组织DAO及DNA双链百分率明显少于对照组；小肠粘膜iNOS的表达及总NOS活性高于对照组，cNOS（主要为eNOS）的表达少于对照组。IPC+IR组血浆和小肠组织NO、ET-1，血浆DAO及组织MPO均明显少于IR组，而 NO/ET-1的比值，组织DAO及DNA双链百分率却明显高于IR组；小肠粘膜iNOS的表达及总NOS活性少于IR组，cNOS（主要为eNOS）的表达高于IR组。 结论： 肢体I/R后小肠损伤与NO/ET-1失衡有关，IPC对肢体IR继发的小肠粘膜损伤的拮抗作用可能通过对NO/ET-1系统的调节作用而介导，此时内皮源的NO产生增加，非内皮源的NO产生减少。     

Mice with an inactivation of the inducible nitric oxide synthase gene are susceptible to experimental autoimmune encephalomyelitis

Nitric oxide (NO) generated by the inducible nitric oxide synthase (iNOS) has been implicated in the pathogenesis of experimental autoimmune encephalomyelitis (EAE). In this study mice genetically deficient for iNOS are shown to be susceptible to EAE induced by immunization with myelin oligodendrocyte glycoprotein (MOG). In iNOS (–/–) mice the course of disease was earlier in onset and more aggressive compared to control animals. A disease-relevant compensatory up-regulation of neuronal (n)NOS and endothelial (e)NOS with increased production of NO in iNOS (–/–) mice is excluded by 1) the failure to detect increased nNOS and eNOS mRNA, 2) the absence of detection of nitrosylated tyrosine residues in EAE tissue indicating absence of NO-derived peroxynitrite, and 3) the lack of disease-preventing effects of N^G-nitro-L -arginine methylester. In conclusion, these results do not support the hypothesis that NO is crucial for the development of EAE.     

Localization of nitric oxide synthase in human trophoblast cells: role of nitric oxide in trophoblast proliferation and differentiation

PROBLEM: There are conflicting reports about the isoform of nitric oxide synthase (NOS) present in trophoblast cells. In this study, we have examined the presence of different NOS isoforms in trophoblast cells. In addition, the role of nitric oxide (NO) in trophoblast function has also been studied by investigating the possible role of nitric oxide in trophoblast proliferation and differentiation. METHOD OF STUDY: NOS isoforms in primary-term trophoblast and JEG-3 cells were identified by immunocytochemistry. The intracellular localization of this enzyme was determined by confocal laser scanning microscopy. Trophoblast proliferation was studied by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasolium bromide (MTT) conversion assay and cellular differentiation was monitored by human chorionic gonodotropin (hCG) and progesterone secretion, measured by radioimmunoassay. RESULTS: The immunoreactive NOS was present in human trophoblast cells of normal term placenta and JEG-3 cells (a choriocarcinoma cell line) maintained in culture. Nicotinamide adenine dinucleotide phosphate (NADPH)-dependent diaphorase activity overlapped with the immunostaining of NOS. Specific antibodies against the different isoforms of NOS detected the presence of neuronal-type NOS (nNOS) only. The other two isoforms, i.e., eNOS (endothelial) and iNOS (macrophage specific) were completely absent. The nNOS was localized in cell cytoplasm. In culture, JEG-3 cells normally undergo proliferation and cytotrophoblast cells in primary culture differentiate to form hormone-secreting syncytial cells. Sodium nitroprusside (SNP), a nitric oxide donor, when added to the culture, significantly increased proliferation of JEG-3 cells and inhibited the differentiation of cytotrophoblast cells. The arrest by SNP in the formation of syncytial cells was further evidenced by the low secretion profile of hCG and progesterone. CONCLUSIONS: Our findings suggest for the first time the presence of nNOS in the human trophoblast cells and a previously unrecognized role of NO in trophoblast proliferation and differentiation.     

Protein tyrosine nitration in the ventilatory muscles: role of nitric oxide synthases

Modification of tyrosine residues and formation of 3-nitrotyrosine is one of the most commonly identified effects of reactive nitrogen species on proteins. In this study we evaluated the presence and localization of tyrosine nitration in various ventilatory and limb muscles. We also assessed the contribution of the neuronal (nNOS), the endothelial (eNOS), and the inducible (iNOS) isoforms of nitric oxide synthase (NOS) to tyrosine nitration in skeletal muscles both under normal conditions and in response to severe sepsis. In normal rats and mice, muscle tyrosine nitration was detected at 52, 48, 40, 30, 18, and 10 kD protein bands. Tyrosine nitration of the majority of these protein bands was significantly reduced within 1 h of in vivo NOS inhibition in rats. Diaphragmatic protein tyrosine nitration in mice deficient in the inducible NOS (iNOS-/-) averaged ~ 50% of that detected in wild-type (iNOS+/+) mice. Injection of bacterial lipopolysaccharides (LPS) in rats produced a significant rise in protein tyrosine nitration in the mitochondrial and membrane fractions but not in the cytosol of ventilatory muscles. Absence of iNOS expression (iNOS-/-), but not nNOS (nNOS-/-) or eNOS (eNOS-/-), in genetically altered mice resulted in a significant reduction in LPS-mediated rise in diaphragmatic nitrotyrosine. We conclude that tyrosine nitration of proteins occurs in normal muscle fibers and is dependent mainly on the activity of the iNOS isoform. Sepsis-mediated increase in protein tyrosine nitration is limited to the mitochondria and cell membrane and is highly dependent on the activity of the iNOS but not the nNOS or eNOS isoforms.     

Nitric oxide, iNOS, and inflammation in cystic fibrosis

Nitric oxide (NO) is produced from three isoforms of nitric oxide synthase (NOS), neuronal (nNOS), endothelial (eNOS) and inducible (iNOS). Cystic fibrosis (CF) patients have an increased bacterial load in the airways which stimulates iNOS and therefore NO production. Upregulation of iNOS in normal epithelial cells protects the lung from damage, but in CF cells, iNOS is not upregulated and NO production is reduced. Reduced iNOS expression is associated with neutrophil sequestration in the lung, thus increasing the potential damage from neutrophil proteases and reactive oxygen species. In contrast, high concentrations of NO may augment the inflammatory process in acute lung injury from sepsis. Meng et al. have shown that cystic fibrosis epithelial cells, when stimulated by a cytokine mix and co-cultured with activated neutrophils, have reduced iNOS expression compared to normal epithelial cells. Although iNOS expression may not accurately reflect activity and NO production may arise from elsewhere, this study suggests that reduced iNOS expression may play a part in the pathophysiological processes in cystic fibrosis.     

Overexpression of P104L mutant caveolin-3 in mice develops hypertrophic cardiomyopathy with enhanced contractility in association with increased endothelial nitric oxide synthase activity

The effect of endogenous nitric oxide synthase (NOS) on cardiac contractility and architecture has been a matter of debate. A role for NOS in cardiac hypertrophy has recently been demonstrated by studies which have shown hypertrophic cardiomyopathy (HCM) with altered contractility in constitutive NOS (cNOS) knockout mice. Caveolin-3, a strong inhibitor of all NOS isoforms, is expressed in sarcolemmal caveolae microdomains and binds to cNOS in vivo: endothelial nitric oxide synthase (eNOS) in cardiac myocytes and neuronal nitric oxide synthase (nNOS) in skeletal myocytes. The current study characterized the biochemical and cardiac parameters of P104L mutant caveolin-3 transgenic mice, a model of an autosomal dominant limb-girdle muscular dystrophy (LGMD1C). Transgenic mouse hearts demonstrated HCM, enhanced basal contractility, decreased left ventricular end diastolic diameter, and loss and cytoplasmic mislocalization of caveolin-3 protein. Surprisingly, cardiac muscle showed activation of eNOS catalytic activity without increased expression of all NOS isoforms. These data suggest that a moderate increase in eNOS activity associated with loss of caveolin-3 results in HCM.     

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.     

iNOS expression in rat aorta is increased after spinal cord transection: a possible cause of orthostatic hypotension in man

Orthostatic hypotension commonly occurs in persons with spinal cord injury (SCI), limiting rehabilitation and independence. Findings of increased production of nitric oxide (NO) by inducible nitric oxide synthase (iNOS) after exposure to simulated microgravity suggest that increased iNOS expression contributes to OH in persons with SCI. To test this possibility, male Wistar rats underwent surgical transection of the spinal cord (T10) or sham-SCI surgery followed by euthanasia 3, 7 or 14 days later. Expression in thoracic aortic of inducible (iNOS), endothelial (eNOS) and neuronal (nNOS) NOS was then determined. In SCI rats, expression of iNOS mRNA was decreased at 3 days, had returned to normal levels of expression at 7 days and was increased at 14 days post-SCI (1.8-fold). In contrast, levels of eNOS mRNA were increased at 3 days (1.4-fold), then declined over time reaching levels by day 14 that were reduced compared to sham-SCI (0.23-fold). There were no significant effects of SCI on nNOS expression. These findings suggest a possible role for increased iNOS expression in the pathogenesis of OH in persons with SCI.     

Neurochemistry within ventrolateral medulla and cardiovascular effects during static exercise following eNOS antagonism

Nitric oxide synthase (NOS), necessary for the production of nitric oxide from l-arginine, exists in three isoforms: neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS). We have previously demonstrated that blockade of nNOS within the rostral (RVLM) and caudal ventrolateral medulla (CVLM) differentially modulated cardiovascular responses to static exercise [Ishide, T., Nauli, S.M., Maher, T.J., Ally, A., 2003. Cardiovascular responses and neurotransmitter changes following blockade of nNOS within the ventrolateral medulla during static muscle contraction. Brain Res. 977, 80-89]. In this study, we have examined the effects of bilaterally microdialyzing a specific eNOS antagonist into the RVLM and CVLM on cardiovascular responses and glutamatergic/GABAergic neurotransmission during the exercise pressor reflex in anesthetized rats. Bilateral microdialysis of a selective eNOS antagonist, l-N(5)-(1-iminoethyl)ornithine (l-NIO; 10.0 microM) into the RVLM potentiated cardiovascular responses and increased extracellular fluid glutamate levels during a static muscle contraction. At the same time, levels of GABA within the RVLM were decreased. The cardiovascular responses and neurochemical changes to muscle contraction recovered after discontinuation of the drug. In contrast, bilateral application of the eNOS antagonist into the CVLM attenuated cardiovascular responses and glutamate concentrations during a static muscle contraction, but augmented levels of GABA. These results demonstrate that eNOS within the ventrolateral medulla plays an important role in modulating glutamate/GABAergic neurotransmission, that in turn regulates the exercise pressor reflex. The present study provides further evidence of simultaneous sympathoexcitatory and sympathoinhibitory effects of nitric oxide within the RVLM and CVLM involved in the neural control of circulation during static exercise.     

Role of shear stress on nitrite and NOS protein content in different size conduit arteries of swine

Aim: Inherent fundamental difference exists among arteries of different sizes. The purpose of this study was to evaluate the relation between regional difference of wall shear stress (WSS) in various sizes arteries and contents of nitrite and NO synthase (NOS) isoforms. Methods: Five different conduit arteries in a wide range of diameter (1–8 mm) were examined in the hind limbs of 13 pigs. Blood flow rate and outer diameter were measured in vivo to determine WSS. Arterial tissues were harvested for the measurement of nitrite and NOS protein contents. The concentration of nitrite, a product of NO synthesis, was determined by high‐performance liquid chromatography method. Western blot analysis was used to assess the protein contents of endothelial NOS (eNOS), inducible NOS (iNOS) and neuronal NOS (nNOS). Results: Our data show that WSS increases with a decrease in artery diameter. Nitrite level increases with increasing WSS and hence decreases with artery diameter. The eNOS protein contents decrease with an increase in diameter. No significant difference for iNOS and nNOS protein contents was found with different artery diameter. A significant positive correlation between tissue nitrite and eNOS protein contents was also observed. Finally, the WSS‐normalized eNOS is not significantly different in various size vessels. Conclusion: Regional difference in blood flow has no effect on iNOS and nNOS protein contents in these conduit arteries. Regional difference in eNOS expression and nitrite contents may be related to the WSS‐induced NO by the endothelium under normal physiological conditions.