藏鸡诱导型一氧化氮合酶基因低氧适应功能分析

低氧刺激诱导型一氧化氮合酶(Inducible nitric-oxide synthase, iNOS)催化产生NO, 增加血流, 改善组织供氧。文章采用测序和PCR-RFLP技术检测藏鸡及低地鸡iNOS基因编码区、5′侧翼区(2.0 kb片段)序列和3′侧翼序列SNP, 并测定低氧和常氧孵化时鸡胚尿囊绒毛膜组织iNOS基因表达量和酶活力。结果在iNOS基因 5′侧翼区发现一个与低氧适应相关的藏鸡高频率突变SNP位点(-870C→T), 藏鸡该突变的等位基因T频率高于低地鸡种。藏鸡iNOS基因表达量和酶活力在低氧孵化环境中多高于矮小鸡。结果表明藏鸡群体iNOS基因的突变及其低氧表达量的增加是其适应低氧环境的重要基础。     

Chronic iron overload enhances inducible nitric oxide synthase expression in rat liver.

Iron is an essential micronutrient promoting oxidative stress in the liver of overloaded animals and human, which may trigger the expression of redox-sensitive genes. We have tested the hypothesis that chronic iron overload (CIO) enhances inducible nitric oxide synthase (iNOS) expression in rat liver by extracellular signal-regulated kinase (ERK1/2) and NF-kappaB activation. CIO (diet enriched with 3%(wt/wt) carbonyl-iron for 12 weeks) increased liver protein carbonylation and decreased reduced glutathione (GSH) content and the GSH/GSSG ratio after 6 weeks, parameters that are normalized after 8-12 weeks of treatment. These changes are paralleled by higher phosphorylated-ERK1/2 to non-phosphorylated-ERK1/2 ratios at 6 and 8 weeks, increased NF-kappaB DNA binding to the iNOS gene promoter at 8-12 weeks, and higher iNOS mRNA expression and activity at 8 and 12 weeks. It is concluded that CIO triggers liver oxidative stress at early times, with upregulation of iNOS expression involving the ERK/NF-kappaB pathway at later times, a finding that may represent a hepatoprotective mechanism against CIO toxicity in addition to the recovery of GSH homeostasis.     

Effect of subchronic acrylamide exposure on the expression of neuronal and inducible nitric oxide synthase in rat brain

Acrylamide (ACR) is a known industrial neurotoxic chemical. Evidence suggests that ACR neurotoxic effect is related to brain neurotransmission disturbances. Since nitric oxide (NO) acts as a neurotransmission modulator and is produced by nitric oxide synthase (NOS), the neuronal NOS (nNOS) and inducible NOS (iNOS) expression pattern were determined in rat cerebral cortex and striatum after subchronic exposure to ACR. Using immunocytochemistry, the neuronal count of nNOS or optical density of iNOS from sections at three coronal levels, bregma 1.0, -0.4, and -2.3 mm, were compared between ACR-treated and control rats. At all three levels, nNOS expressions were uniformly decreased in most of the neocortical subregions following the treatment of ACR. At bregma level 1.0 mm, total numbers of nNOS expressing neurons were significantly decreased to 58.7% and 64.7% of the control in the cortex and striatum of ACR-treated rats, respectively. However, at the bregma level -2.3 mm, ACR treatment did not produce a significant difference in the numbers of nNOS expressing neurons both in the cortex and striatum. Contrary to nNOS, iNOS expressions were consistently increased to approximately 32% in the neocortex and 25% in the striatum, following the subchronic ACR treatment. These data suggest that subchronic ACR exposure involves compensatory mechanism on nNOS and iNOS expression to maintain the homeostasis of NO at the rostral part of the neocortex and the striatum. However, in the caudal brain, increased iNOS expression did not suppress nNOS expression. Therefore, the present study is consistent with the hypothesis that ACR toxicity is mediated through the disturbance to the NO signaling pathway and exhibits a rostrocaudal difference through the differential expressions of nNOS and iNOS in the neocortex and the striatum.     

RNA interference-produced autoregulation of inducible nitric oxide synthase expression

Vector-mediated delivery of short-hairpin RNA (shRNA) to regulate gene expression holds a great therapeutic promise. We hypothesize that gene expression can be autoregulated with RNA interference. We used inducible nitric oxide synthase (iNOS) as a gene model to test this hypothesis. Lipopolysaccharide dose-dependently increased iNOS in rat aortic smooth muscle cells and the nitrite production from these cells. These increases were attenuated in cells transfected with plasmids containing code for iNOS shRNA whose expression was controlled by an iNOS promoter. The production of shRNA was lipopolysaccharide dose-dependent. The lipopolysaccharide-induced iNOS expression in rat C6 glioma cells also was attenuated by transfection with plasmids containing the iNOS shRNA code. These results provide proof-of-concept evidence for using RNA interference technique to achieve autoregulation of gene expression.     

Chronic stress induces the expression of inducible nitric oxide synthase in rat brain cortex

Long-term exposure to stress has detrimental effects on several brain functions in many species, including humans, and leads to neurodegenerative changes. However, the underlying neural mechanisms by which stress causes neurodegeneration are still unknown. We have investigated the role of endogenously released nitric oxide (NO) in this phenomenon and the possible induction of the inducible NO synthase (iNOS) isoform. In adult male rats, stress (immobilization for 6 h during 21 days) increases the activity of a calcium-independent NO synthase and induces the expression of iNOS in cortical neurons as seen by immunohistochemical and western blot analysis. Three weeks of repeated immobilization increases immunoreactivity for nitrotyrosine, a nitration product of peroxynitrite. Repeated stress causes accumulation of the NO metabolites NO2+ NO3- (NOx-) accumulation in cortex, and these changes occur in parallel with lactate dehydrogenase (LDH) release and impairment of glutamate uptake in synaptosomes. Administration of the selective iNOS inhibitor aminoguanidine (400 mg/kg i.p. daily from days 7 to 21 of stress) prevents NOx- accumulation in cortex, LDH release, and impairment of glutamate uptake in synaptosomes. Taken together, these findings indicate that a sustained overproduction of NO via iNOS expression may be responsible, at least in part, for some of the neurodegenerative changes caused by stress and support a possible neuroprotective role for specific iNOS inhibitors in this situation.     

Age-related decline of inducible nitric oxide synthase gene expression in primary cultured rat hepatocytes

Hepatocytes exhibit a non-specific immune response by expressing the enzyme inducible nitric oxide (NO) synthase (iNOS, NOS2) through the stimulation of a mixture of cytokines, or a single cytokine such as interleukin-1beta. We examined the age-dependent inducibility of the iNOS gene expression and the capacity of NO production in response to lipopolysaccharide (LPS) or interleukin-1beta (IL-1beta) in primary cultured rat hepatocytes that were isolated from the livers of rats, 3 (young) and 24 (aging) months of age. NO production (NO2-), indicating iNOS activity, was much higher in the young rat hepatocytes following stimulation with LPS or IL-1beta. Likewise, in the young hepatocytes, Western blot analyses showed much higher protein levels in the iNOS expression; it was also a little higher in mRNA levels that were analyzed by RT-PCR. Furthermore, after stimulation with IL-1beta, the levels of transactivation of nuclear factor-KB (NF-kappaB) that were involved in the induction of the iNOS gene were reduced without a significant difference in the aged cells. Therefore, the decrease of NO formation in the aged hepatocytes was due to the belated and incomplete inducibility of the iNOS protein expression, together with a minor contribution of the reduced-transactivation of NF-kappaB. These results suggest that the age-related decline of the iNOS gene expression in primary rat hepatocytes may be associated with the increased incidence of many infective diseases with aging.     

17Beta-estradiol inhibits ovariectomy-induced expression of inducible nitric oxide synthase in rat aorta in vivo

The objective of this study was to elucidate a role of ovarian steroid hormones in the production of immunologic nitric oxide (NO) synthases in the female rat aorta in vivo. Aortic homogenates were analyzed by using western blot with isoform-specific antibodies against endothelial NOS (eNOS) and inducible NOS (iNOS). Two weeks after ovariectomy (OVX), rats (10-week-old) were treated with 17beta-estradiol (E2) and/or progesterone (P4) for 5 days, and aortae were obtained from these rats on the following day. OVX markedly increased the levels of iNOS protein in abdominal aorta, whereas treatment with E2 or a combination of E2 and P4 inhibited the induction of iNOS in the aorta. The present findings indicate that endogeneous estrogen negatively regulates the expression of iNOS in abdominal aorta, and suggest that changes in the levels of circulating estrogen may affect vascular function.     

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.     

Regulation of inducible nitric oxide synthase activity/expression in rat hearts from ghrelin-treated rats

The purpose of this study was to examine the effects of ghrelin on protein kinase B (Akt) and mitogen-activated protein kinase p42/44 (ERK1/2) activation as well as ghrelin effects on inducible nitric oxide (NO) synthase (iNOS; for gene Nos2) activity/expression in rat hearts. Male Wistar rats were treated with ghrelin (0.3 nmol/5 μl) or an equal volume of phosphate-buffered saline, injected every 24 h into the lateral cerebral ventricle for 5 days and 2 h after the last treatment the animals were sacrificed. Serum NO, L-arginine (L-Arg), and arginase activity were measured spectrophotometrically. For phosphorylation of Akt, ERK1/2, and iNOS protein expression, Western blot method was used. The expression of Nos2 mRNA was measured by the quantitative real-time polymerase chain reaction (qRT-PCR). Treatment with ghrelin significantly increased NO production in serum by 1.4-fold compared with control. The concentration of L-Arg was significantly higher in ghrelin-treated rats than in control while arginase activity was significantly lower in ghrelin-treated than in control hearts. Ghrelin treatment increased phosphorylation of Akt by 1.9-fold and ERK1/2 by 1.6-fold and increased iNOS expression by 2.5-fold compared with control. In addition, ghrelin treatment increased Nos2 gene expression by 2.2-fold as determined by qRT-PCR. These results indicate that ghrelin regulation of iNOS expression/activity is mediated via Akt/ERK1/2 signaling pathway. These results may be relevant to understanding molecular mechanisms underlying direct cardiovascular actions of ghrelin.     

Testosterone inhibits expression of inducible nitric oxide synthase in murine macrophages

We investigated the effect of testosterone, the main sexual steroid hormone in men, upon inducible nitric oxide synthesis in murine macrophages. Incubation of murine macrophages (RAW 264.7 cells) stimulated by bacterial lipopolysaccharide (2 microg/ml) with increasing amounts of testosterone (0.1-40 microM) showed a dose dependent inhibition of inducible nitric oxide synthesis. Inducible nitric oxide synthase protein expression was reduced in a dose dependent manner as revealed by immunoblotting when cells were incubated with increasing amounts of testosterone. This was associated with a decline in iNOS mRNA-levels as determined by competitive semiquantitative PCR. As nitric oxide plays an important role in immune defense and atherosclerosis prevention, testosterone-induced iNOS inhibition could lead to an elevated risk of infection as well as to the development of atherosclerotic lesions.     

Ferulic acid and its water-soluble derivatives inhibit nitric oxide production and inducible nitric oxide synthase expression in rat primary astrocytes

We recently reported that two water-soluble derivatives of ferulic acid (1-feruloyl glycerol, 1-feruloyl diglycerol) previously developed by our group exhibited protective effects against amyloid-β–induced neurodegeneration in vitro and in vivo. In the current study, we aimed to further understand this process by examining the derivatives’ ability to suppress abnormal activation of astrocytes, the key event of neurodegeneration. We investigated the effects of ferulic acid (FA) derivatives on nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in rat primary astrocytes. The results showed that these compounds inhibited NO production and iNOS expression in a concentration-dependent manner and that the mechanism underlying these effects was the suppression of the nuclear factor-κB pathway. This evidence suggests that FA and its derivatives may be effective neuroprotective agents and could be useful in the treatment of neurodegenerative diseases, such as Alzheimer’s disease and Parkinson’s disease.