Nitric oxide regulates neurogenesis in adult olfactory epithelium in vitro

Nitric oxide regulates neurogenesis in the developing and adult brain. The olfactory epithelium is a site of neurogenesis in the adult and previous studies suggest a role for nitric oxide in this tissue during development. We investigated whether neuronal precursor proliferation and differentiation is regulated by nitric oxide using primary cultures of olfactory epithelial cells and an immortalized, clonal, neuronal precursor cell line derived from adult olfactory epithelium. In these cultures NOS inhibition reduced cell proliferation and stimulated neuronal differentiation, including expression of a voltage-dependent potassium conductance of the delayed rectifier type. In the neuronal precursor cell line, differentiation was associated with a significant decrease in nitric oxide release. In contrast, addition of nitric oxide stimulated proliferation and reduced neuronal differentiation. Nitric oxide regulated olfactory neurogenesis independently of added growth factors. Taken together these results indicate that nitric oxide levels can regulate cell proliferation and neuronal differentiation of olfactory precursor cells.     

Reduced neuronal nitric oxide synthase is involved in ischemia-induced hippocampal neurogenesis by up-regulating inducible nitric oxide synthase expression

Nitric oxide (NO), a free radical with signaling functions in the CNS, is implicated in some developmental processes, including neuronal survival, precursor proliferation, and differentiation. However, neuronal nitric oxide synthase (nNOS) -derived NO and inducible nitric oxide synthase (iNOS) -derived NO play opposite role in regulating neurogenesis in the dentate gyrus after cerebral ischemia. In this study, we show that focal cerebral ischemia reduced nNOS expression and enzymatic activity in the hippocampus. Ischemia-induced cell proliferation in the dentate gyrus was augmented in the null mutant mice lacking nNOS gene (nNOS−/−) and in the rats receiving 7-nitroindazole, a selective nNOS inhibitor, after stroke. Inhibition of nNOS ameliorated ischemic injury, up-regulated iNOS expression, and enzymatic activity in the ischemic hippocampus. Inhibition of nNOS increased and iNOS inhibitor decreased cAMP response element-binding protein phosphorylation in the ipsilateral hippocampus in the late stage of stroke. Moreover, the effects of 7-nitroindazole on neurogenesis after ischemia disappeared in the null mutant mice lacking iNOS gene (iNOS−/−). These results suggest that reduced nNOS is involved in ischemia-induced hippocampal neurogenesis by up-regulating iNOS expression and cAMP response element-binding protein phosphorylation.     

The protective effect of aminoguanidine on cerebral ischemic damage in the rat brain

The NADPH-diaphorase (NADPH-d) histochemical technique is commonly used to localize the nitric oxide (NO) produced by the enzyme nitric oxide synthase (NOS) in neural tissue. The expression of inducible nitric oxide synthase (iNOS) is induced in the late stage of cerebral ischemia, and NO produced by iNOS contributes to the delay in recovery from brain neuronal damage. The present study was performed to investigate whether the increase in nitric oxide production via inducible nitric oxide synthase was suppressed by the administration of aminoguanidine, a selective iNOS inhibitor, as it follows a decrease of NADPH-diaphorase activity (a marker for NOS) after four-vessel occlusion used as an ischemic model. The administration of aminoguanidine (100 mg/kg i.p., twice per day up to 3 days immediately after the ischemic insult) reduced the number of NADPH-diaphorase positive cells to control levels. Our results indicated that aminoguanidine suppressed NADPH-diaphorase activity, and also decreased the number of NADPH-diaphorase positive cells in the CA1 region of the hippocampus following ischemic brain injury.     

Attenuation of cardiac mitochondrial dysfunction by melatonin in septic mice

The existence of an inducible mitochondrial nitric oxide synthase has been recently related to the nitrosative/oxidative damage and mitochondrial dysfunction that occurs during endotoxemia. Melatonin inhibits both inducible nitric oxide synthase and inducible mitochondrial nitric oxide synthase activities, a finding related to the antiseptic properties of the indoleamine. Hence, we examined the changes in inducible nitric oxide synthase/inducible mitochondrial nitric oxide synthase expression and activity, bioenergetics and oxidative stress in heart mitochondria following cecal ligation and puncture-induced sepsis in wild-type (iNOS(+/+)) and inducible nitric oxide synthase-deficient (iNOS(-/-)) mice. We also evaluated whether melatonin reduces the expression of inducible nitric oxide synthase/inducible mitochondrial nitric oxide synthase, and whether this inhibition improves mitochondrial function in this experimental paradigm. The results show that cecal ligation and puncture induced an increase of inducible mitochondrial nitric oxide synthase in iNOS(+/+) mice that was accompanied by oxidative stress, respiratory chain impairment, and reduced ATP production, although the ATPase activity remained unchanged. Real-time PCR analysis showed that induction of inducible nitric oxide synthase during sepsis was related to the increase of inducible mitochondrial nitric oxide synthase activity, as both inducible nitric oxide synthase and inducible mitochondrial nitric oxide synthase were absent in iNOS(-/-) mice. The induction of inducible mitochondrial nitric oxide synthase was associated with mitochondrial dysfunction, because heart mitochondria from iNOS(-/-) mice were unaffected during sepsis. Melatonin treatment blunted sepsis-induced inducible nitric oxide synthase/inducible mitochondrial nitric oxide synthase isoforms, prevented the impairment of mitochondrial homeostasis under sepsis, and restored ATP production. These properties of melatonin should be considered in clinical sepsis.     

The localization of neuronal nitric oxide synthase may influence its role in neuronal precursor proliferation and synaptic maintenance

Neuronal nitric oxide synthase (nNOS) is implicated in some developmental processes, including neuronal survival, differentiation, and precursor proliferation. To define the roles of nNOS in neuronal development, we utilized the olfactory system as a model. We hypothesized that the role of nNOS may be influenced by its localization. nNOS expression was developmentally regulated in the olfactory system. During early postnatal development, nNOS was expressed in developing neurons in the olfactory epithelium (OE), while in the adult its expression was restricted to periglomerular (PG) cells in the olfactory bulb (OB). At postnatal week 1 (P1W), loss of nNOS due to targeted gene deletion resulted in a decrease in immature neurons in the OE due to decreased proliferation of neuronal precursors. While the pool of neuronal precursors and neurogenesis normalized in the nNOS null mouse by P6W, there was an overgrowth of mitral or tufted cells dendrites and a decreased number of active synapses in the OB. Cyclic GMP (cGMP) immunostaining was reduced in the OE and in the glomeruli of the OB at early postnatal and adult ages, respectively. Our results suggest that nNOS appears necessary for neurogenesis in the OE during early postnatal development and for glomerular organization in the OB in the adult. Thus, the location of nNOS, either within cell bodies or perisynaptically, may influence its developmental role.     

Oltipraz inhibits inducible nitric oxide synthase in vitro and inhibits nitric oxide production in activated microglial cells

The clinically relevant drug oltipraz (OPZ) has previously been shown to inhibit cytochrome P450 enzymes [Chem. Res. Toxicol. 13 (2000) 245]. The current study reveals that OPZ is also able to inhibit *NO formation by purified inducible nitric oxide synthase (iNOS) but not by neuronal nitric oxide synthase in hemoglobin assays. The inhibition of iNOS by OPZ is reversible and competitive with an IC(50) of 5.9 microM and Ki of 0.6 microM. In murine BV-2 microglial cells, an immortalized cell line that produces *NO in response to lipopolysaccharide (LPS), OPZ is able to block the formation of nitrite in LPS treated cells. The inhibitory effect of OPZ on LPS treated cells is not due to cell toxicity. Finally, treatment of cells with OPZ does not induce or suppress expression of iNOS protein as shown by Western blot analysis.     

Inhibition of macrophage nitric oxide production by gangliosides derived from a spontaneous T cell lymphoma: the involved mechanisms.

Gangliosides (DLG) derived from a spontaneous T cell lymphoma (Dalton's lymphoma) have been shown to impair the ability of lipopolysaccharide-activated macrophages to produce nitric oxide (NO). However, the mechanism and nature of this effect is not known. In this investigation, we sought to (1) determine whether the inhibitory action of DLG on macrophages is through the modulation of inducible nitric oxide synthase (iNOS) expression and (2) identify the possible mechanisms and signal transduction events underlying the inhibitory action of DLG. Immunoblot analysis of DLG-treated macrophages showed a decrease in iNOS expression. DLG also inhibited the production of monokines interleukin-1 and tumor necrosis factor by macrophages. However, the DLG-induced inhibition was reversible in nature. Studies showed that DLG-induced inhibition of macrophage activation could be blocked by sodium orthovanadate, indicating a role of phosphatase activity in ganglioside-induced inhibition.     

Protective effect of radicicol against LPS/IFN-gamma-induced neuronal cell death in rat cortical neuron-glia cultures

We investigated the protective activity of radicicol, an antifungal antibiotic, against inflammation-induced neurotoxicity in neuron-glia cultures. Radicicol potently prevented the loss of neuronal cell bodies and neurites from LPS/IFN-gamma-induced neurotoxicity in rat cortical neuron-glia cultures with an EC(50) value of 0.09 microM. Radicicol inhibited the LPS/IFN-gamma-induced expression of inducible nitric oxide synthase (iNOS) and production of nitric oxide (NO) in microglia. Additionally, radicicol decreased the LPS/IFN-gamma-induced release of tumor necrosis factor-alpha (TNF-alpha) in the cultures. The inhibitory potency of radicicol against the production of NO and TNF-alpha was well correlated with the protection of neurons. These results suggest that the protective effect of radicicol against LPS/IFN-gamma-induced neuronal cell death in neuron-glia cultures is mediated via the inhibition of TNF-alpha release, as well as the suppression of iNOS expression in microglia.     

红花黄色素对新生鼠缺氧后一氧化氮合酶表达的影响

目的:观察红花黄色素对缺氧后脑内诱生型一氧化氮合酶(iNOS)、神经原型一氧化氮合酶(nNOS)及内皮型一氧化氮合酶(eNOS)基因表达的影响,探讨红花黄色素抗缺氧脑损伤的作用.方法:采用SD新生鼠缺氧模型,于缺氧前30 min腹腔注射红花黄色素生药7g/kg,缺氧40 min后复氧48 h,提取脑组织总RNA,应用RT-PCR技术检测三种NOS mRNA的表达量.结果:新生鼠缺氧再复氧48 h,脑内iNOS、nNOS基因表达上升(P＜0.05),预先给予红花黄色素能抑制iNOS、nNOS基因的表达(P＜0.05),但eNOS基因表达不受影响.结论:红花黄色素对缺氧脑损伤的保护作用与NOS基因表达有关.     

一氧化氮对心肌的抑制性保护作用

内皮型与神经型一氧化氮合酶(eNOS,nNOS)在心肌细胞内持续表达,而细胞应激可引起诱导型NOS(iNOS)表达.心肌细胞结构型eNOS与nNOS源性NO,在生理条件下对心肌主要发挥4方面的抑制作用:减缓心肌细胞搏动频率,轻度抑制心肌细胞收缩功能,加速心肌细胞舒张并增加顺应性,以及轻度抑制线粒体电子传递而增强氧利用效...     

Penehyclidine hydrochloride attenuates LPS-induced iNOS production by inhibiting p38 MAPK activation in endothelial cells

The aim of this study was to investigate the inhibitory effect of penehyclidine hydrochloride (PHC) on lipopolysaccharide (LPS)-induced nitric oxide (NO) and inducible nitric oxide synthase (iNOS) production in human endothelial cell. Cultured endothelial cells were pretreated with PHC, followed by LPS treatment. NO activity were determined. iNOS expression and p38 mitogen-activated protein kinase (p38 MAPK) protein expression were measured by Western blot analysis. LPS treatment significantly induced p38 MAPK activation, iNOS expression, and NO production, which could be attenuated by 2 μg/ml PHC pretreatment. Furthermore, our study showed LPS-induced NO production and iNOS expression were suppressed by p38 MAPK inhibitor SB203580 pretreatment. We concluded that PHC attenuates NO production and iNOS expression by suppressing the activation of p38 MAPK pathway, thereby implicating a mechanism by which PHC may exert its protective effects against LPS-induced endothelial cell injury.     

Induction of Nitric Oxide Synthase and Nitric Oxide-Mediated Apoptosis in Neuronal PC12 Cells After Stimulation with Tumor Necrosis FActor-α/Lipopolysaccharide

Abstract: Exposure of neuronal PC12 cells, differentiated by nerve growth factor, to tumor necrosis factor-α (TNF-α) and bacterial lipopolysaccharide (LPS) resulted in de novo synthesis of inducible nitric oxide synthase (iNOS) mRNA and protein with an increase up to 24 h. Brain NOS expression was unaffected. The induction of iNOS in differntiated PC12 cells was associated with cell death characterized by features of apoptosis, The NOS inhibitors N -monomethylarginine, aminoguanidine, and 2-amino-5,6-dihydro-6-methyl-4 H -1,3-thiazine HCl prevented TNF-α/LPS-induced cell death and DNA fragmentation, suggesting that the TNF-α/LPS-induced cell death is mediated by iNOS-derived NO. This hypothesis is supported by the finding that addition of l -arginine, which serves as a precursor and limiting factor of enzyme-derived NO production, potentiated TNF-α/LPS-induced loss of viability.     

缺血再灌注对小鼠肠神经丛nNOS 和iNOS表达的影响

目的观察缺血再灌注后小鼠回肠神经型一氧化氮合酶(neuron alnitric oxide synthase,nNOS)和诱导型一氧化氮合酶(induciblenitric oxide synthase,iNOS)的表达,探讨肠缺血再灌注损伤(ischemia-reperfusion injury,IRI)的发生机制。方法采用小鼠肠系膜上动脉缺血再灌注模型,根据不同再灌注时间对小鼠随机分1d组、3d组、5d组、7d组、对照组和假手术组,用SP法检测小鼠回肠nNOS和iNOS的表达情况。结果与对照组和假手术组相比较,nNOS在再灌注1d后开始在肌间神经丛持续高表达(P<0.01);而iNOS在再灌注3d后开始在肌间神经丛持续高表达(P<0.05)。结论nNOS和iNOS在肠缺血再灌注后的表达增强,提示一氧化氮及一氧化氮合酶与肠神经节细胞在缺血再灌注中的损伤有着密切关系。     

Regulation of neuronal proliferation and differentiation by nitric oxide

Many studies have revealed the free radical nitric oxide (NO) to be an important modulator of vascular and neuronal physiology. It also plays a developmental role in regulating synapse formation and patterning. Recent studies suggest that NO may also mediate the switch from proliferation to differentiation during neurogenesis. Many mechanisms of this response are conserved between neuronal precursor cells and the cells of the vascular system, where NO can inhibit the proliferative response of endothelial and smooth-muscle cells to injury. In cultured neuroblastoma cells, NO synthase (NOS) expression is increased in the presence of various growth factors and mitogens. Subsequent production of NO leads to cessation of cell division and the acquisition of a differentiated phenotype. The inhibitory action of NO on neuroblast proliferation has also been demonstrated in vivo for vertebrate and invertebrate nervous systems, as well as in the adult brain. Potential downstream effectors of NO include the second messenger cyclic GMP, activation of the tumor-suppressor genes p53 and Rb, and the cyclin-dependent kinase inhibitor p21. These studies highlight a new role for NO in the nervous system, as a coordinator of proliferation and patterning during neural development and adult neurogenesis.     

The Effect of a Spinal Cord Hemisection on Changes in Nitric Oxide Synthase Pools in the Site of Injury and in Regions Located Far Away from the Injured Site

1. The present study was designed to examine the nitric oxide synthase activities (constitutive and inducible) in the site of injury in response to Th10-Th11 spinal cord hemisection and, to determine whether unilateral disconnection of the spinal cord influences the NOS pools on the contra- and ipsilateral sides in segments located far away from the epicentre of injury.2. A radioassay detection was used to determine Ca²⁺-dependent and inducible nitric oxide synthase activities. Somal, axonal and neuropil neuronal nitric oxide synthase was assessed by immunocytochemical study. A quantitative assessment of neuronal nitric oxide synthase immunoreactivity was made by an image analyser. The level of neuronal nitric oxide synthase protein was measured by the Western blot analysis.3. Our data show the increase of inducible nitric oxide synthase activity and a decrease of Ca²⁺-dependent nitric oxide synthase activity in the injured site analysed 1 and 7 days after surgery. In segments remote from the epicentre of injury the inducible nitric oxide synthase activity was increased at both time points. Ca²⁺-dependent nitric oxide synthase activity had decreased in L5-S1 segments in a group of animals surviving for 7 days. A hemisection performed at thoracic level did not cause significant difference in the nitric oxide synthase activities and in the level of neuronal nitric oxide synthase protein between the contra- and ipsilateral sides in C6-Th1 and L5-S1 segments taken as a whole. Significant differences were observed, but only when the spinal cord was analysed segment by segment, and/or was divided into dorsal and ventral parts. The cell counts in the cervicothoracic (C7-Th1) and lumbosacral (L5-S1) enlargements revealed changes in neuronal nitric oxide synthase immunoreactivity on the ipsilateral side of the injury. The densitometric area measurements confirmed the reduction of somal, neuropil and axonal neuronal nitric oxide synthase immunoreactive staining in the ventral part of rostrally oriented segments.4. Our findings provide evidence that the changes in nitric oxide synthase pools are limited not only to impact zone, but spread outside the original lesion. The regional distribution of nitric oxide synthase activity and neuronal nitric oxide synthase immunoreactivity, measured segment by segment shows that nitric oxide may play a significant role in the stepping cycle in the quadrupeds.     

Inhibition of glycogen synthase kinase-3 enhances the differentiation and reduces the proliferation of adult human olfactory epithelium neural precursors

The olfactory epithelium (OE) contains neural precursor cells which can be easily harvested from a minimally invasive nasal biopsy, making them a valuable cell source to study human neural cell lineages in health and disease. Glycogen synthase kinase-3 (GSK-3) has been implicated in the etiology and treatment of neuropsychiatric disorders and also in the regulation of murine neural precursor cell fate in vitro and in vivo. In this study, we examined the impact of decreased GSK-3 activity on the fate of adult human OE neural precursors in vitro. GSK-3 inhibition was achieved using ATP-competitive (6-bromoindirubin-3′-oxime and CHIR99021) or substrate-competitive (TAT-eIF2B) inhibitors to eliminate potential confounding effects on cell fate due to off-target kinase inhibition. GSK-3 inhibitors decreased the number of neural precursor cells in OE cell cultures through a reduction in proliferation. Decreased proliferation was not associated with a reduction in cell survival but was accompanied by a reduction in nestin expression and a substantial increase in the expression of the neuronal differentiation markers MAP1B and neurofilament (NF-M) after 10 days in culture. Taken together, these results suggest that GSK-3 inhibition promotes the early stages of neuronal differentiation in cultures of adult human neural precursors and provide insights into the mechanisms by which alterations in GSK-3 signaling affect adult human neurogenesis, a cellular process strongly suspected to play a role in the etiology of neuropsychiatric disorders.     

Phenylhomophthalimide-type NOS inhibitors derived from thalidomide

Thalidomide shows moderate inhibitory activity toward neuronal nitric oxide synthase (nNOS) and inducible NOS (iNOS), but not toward endothelial NOS (eNOS). Structural development studies of thalidomide yielded novel phenylhomophthalimide-type NOS inhibitors with enhanced activity and different subtype selectivity.     

iNOS expression inhibits hypoxia-inducible factor-1 activity

Hypoxia-inducible factor-1 (HIF-1) activates genes important in vascular function such as vascular endothelial growth factor (VEGF), erythropoietin (EPO), and inducible nitric oxide synthase (iNOS). iNOS catalyzes the synthesis of nitric oxide (NO), a free radical gas that mediates a number of cellular processes, including regulation of gene expression, vasodilatation, and neurotransmission. Here we demonstrate that iNOS expression inhibits HIF-1 activity under hypoxia in C6 glioma cells transfected with an iNOS gene and a VEGF promoter-driven luciferase gene. HIF-1 induction of VEGF-luciferase activity in C6 cell is also inhibited by sodium nitroprusside (SNP). Furthermore, pretreatment of C6 cells with N-acetyl-l-cysteine (NAC), an antioxidant, nullified the inhibitory effect of iNOS on HIF-1 binding. These results demonstrate that NO generated by iNOS expression inhibits HIF-1 activity in hypoxic C6 cells and suggest a negative feedback loop in the HIF-1 --> iNOS cascade.     

Role of nitric oxide during neurogenesis in the olfactory epithelium

In mammals, neurogenesis continues during adulthood in restricted places of the nervous system, namely the subventricular zone, the dentate gyms and the olfactory epithelium. A dual role of the second messenger nitric oxide has been reported in such places, either promoting or inhibiting proliferation of neuronal precursors depending on the cellular signal implicated. In this review the regulation of adult olfactory epithelium neurogenesis by nitric oxide is discussed.