Regional variations and age-related changes in nitric oxide synthase and arginase in the sub-regions of the hippocampus

L-arginine can be metabolised by nitric oxide synthase (NOS) with the formation of L-citrulline and nitric oxide (NO), or arginase with the production of L-ornithine and urea. In contrast to studies showing a potential involvement of NOS/NO in the aging process, the role of arginase has not been well documented. The present study investigates for the first time the regional variations and age-related changes in both NOS and arginase in sub-regions of the hippocampus. In young adult rats, although the total NOS activity was not significantly different across the hippocampal CA1, CA2/3 and the dentate gyrus (DG) sub-regions, the total arginase activity showed a clear regional variation with the highest level in DG. Western blotting revealed that the highest levels of neuronal NOS (nNOS) and endothelial NOS (eNOS) proteins were located in CA1. Arginase I is expressed at a very low level in the brain (the whole hippocampus) as compared with the liver. By contrast, arginase II protein shows an extremely high expression in the brain with little or no expression in the liver. There was no regional variation in arginase I or arginase II protein expression across the sub-regions of the hippocampus. When a comparison was made between young (4-month-old) and aged (24-month-old) rats, a significant increase in total NOS activity was found in DG and significant decreases in arginase activity were observed in the CA1 and CA2/3 regions in the aged animals. Western blotting further revealed a dramatic decrease in eNOS protein expression in aged CA2/3 with no age-associated changes in nNOS, arginase I and II protein expression in any region examined. Interestingly, evidence of activity or protein expression of the inducible isoform of NOS (iNOS) was not detected in any tissue from either group. The present results, in conjunction with previous findings, support the contribution of NOS/NO to aging but question the involvement of iNOS in the normal aging process. Region-specific changes in arginase suggest that this enzyme may also contribute to aging.     

Mutual regulation between serine and nitric oxide metabolism in human glioblastoma cells

D-Serine indirectly caused dose- and time-dependent inhibition of neuronal nitric oxide synthase (nNOS) without affecting endothelial nitric oxide synthase (eNOS) in human glioblastoma cell line U87. Activity of D-amino acid oxidase (DAAO), catalyzing the oxidative deamination of d-amino acid, was enhanced by NO in a dose-dependent manner. Recently, we have reported that serine racemase (SR) is inhibited by NO and activated by D-serine through nitrosylation and denitrosylation, respectively [K. Shoji, S. Mariotto, A.R. Ciampa, H. Suzuki, Regulation of serine racemase activity by D-serine and nitric oxide in human glioblastoma cells, Neurosci. Lett., in press]. Thus, the metabolism of both d-serine and NO in U87 cells is functionally correlated in a complex manner. Suppression of NO production by d-serine in U87 cells contrasts its known action in enhancing nNOS in neurons.     

Neuronal nitric oxide synthase is the dominant nitric oxide supplier for the survival of dorsal root ganglia after peripheral nerve axotomy

This study was designed to determine whether nitric oxide supply may be a major factor in the survival of dorsal root ganglia in a sciatic nerve injury model. Wild-type (WT) mice were compared with knockout (KO) mice lacking neuronal nitric oxide synthase (nNOS) or endothelial (eNOS). The NO-generating capacities were analysed by NOS immunohistochemistry and NADPH-diaphorase staining 1, 2, 6, and 12 weeks after nerve transection. The occurrence and morphological type of neuronal death were determined by TUNEL reaction and ultrastructural examination. Cell loss following nerve section, whist dependent on the availability of NO, as shown by its marked elevation in nNOS KO mice, did not correlate well with nNOS expression in WT animals. Whereas a lack of eNOS was tolerated, deficiency of nNOS led to an enhanced cell loss. The results suggest a crucial role of NO supply after transection of peripheral nerves with a particular significance of the nNOS isoform.     

Complementary inhibition of cerebral aneurysm formation by eNOS and nNOS

The rupture of cerebral aneurysm (CA) and subsequent subarachnoid hemorrhage can cause fatal results. Recent experimental findings have suggested that the mechanism of CA formation is based on chronic inflammation in arterial walls by hemodynamic force. Endothelial nitric oxide synthase (eNOS) protects arterial walls from vascular inflammation by relieving hemodynamic force through nitric oxide (NO) production. Thus, the expression and protective role of eNOS in CA formation have been investigated in this study. In this study, experimental induced rodent CA models by carotid ligation and systemic hypertension were used. The expression of eNOS was examined in rat CA models and revealed that it was decreased at the site of CA formation. Next, CA was induced in eNOS(-/-) mice to clarify the role of eNOS in CA formation. In eNOS(-/-) mice, the incidence of CA formation was similar to that found in wild-type mice. In CA walls of eNOS(-/-) mice, the expression of neuronal nitric oxide synthase (nNOS) was upregulated compared with that in wild-type mice, suggesting the compensatory effect of nNOS. Hence, eNOS(-/-) nNOS(-/-) mice were generated, underwent CA induction and confirmed that eNOS(-/-) nNOS(-/-) mice exhibited an increased incidence of CA formation accompanied by accelerated macrophage infiltration. These results suggested that the deficiency of eNOS could be compensated by nNOS upregulation in cerebral arteries and that the eNOS and nNOS complementarily had the protective role in CA formation. The results of this study will provide us with new insight about the mechanisms of CA formation and the functional redundancy between eNOS and nNOS in cerebral arteries.     

Acid-sensing ion channel 1 and nitric oxide synthase are in adjacent layers in the wall of rat and human cerebral arteries

Extracellular acidification activates a family of proteins known as acid-sensing ion channels (ASICs). One ASIC subtype, ASIC type 1 (ASIC1), may play an important role in synaptic plasticity, memory, fear conditioning and ischemic brain injury. ASIC1 is found primarily in neurons, but one report showed its expression in isolated mouse cerebrovascular cells. In this study, we sought to determine if ASIC1 is present in intact rat and human major cerebral arteries. A potential physiological significance of such a finding is suggested by studies showing that nitric oxide (NO), which acts as a powerful vasodilator, may modulate proton-gated currents in cultured cells expressing ASIC1s. Because both constitutive NO synthesizing enzymes, neuronal nitric oxide synthase (nNOS) and endothelial NOS (eNOS), are expressed in cerebral arteries we also studied the anatomical relationship between ASIC1 and nNOS or eNOS in both rat and human cerebral arteries. Western blot analysis demonstrated ASIC1 in cerebral arteries from both species. Immunofluorescent histochemistry and confocal microscopy also showed that ASIC1-immunoreactivity (IR), colocalized with the smooth muscle marker alpha-smooth muscle actin (SMA), was present in the anterior cerebral artery (ACA), middle cerebral artery (MCA), posterior cerebral artery (PCA) and basilar artery (BA) of rat and human. Expression of ASIC1 in cerebral arteries is consistent with a role for ASIC1 in modulating cerebrovascular tone both in rat and human. Potential interactions between smooth muscle ASIC1 and nNOS or eNOS were supported by the presence of nNOS-IR in the neighboring adventitial layer and the presence of nNOS-IR and eNOS-IR in the adjacent endothelial layer of the cerebral arteries.     

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.     

Alteration of rat hippocampal neurogenesis and neuronal nitric oxide synthase expression upon prenatal exposure to tamoxifen

The present study delineates the effect of tamoxifen on neuronal density and expression of neuronal nitric oxide synthase (nNOS) in hippocampal nerve cells during prenatal and postnatal periods in rats. Pregnant rats were administered with tamoxifen one day prior to labor (E21) and on the childbirth day (E22). Hippocampi of embryos at E22 and newborns at postnatal days of 1, 7, and 21 (P1, P7, and P21) were investigated. Density of the neurons in areas of the developing hippocampus including cornu ammonis (CA1, CA3), dentate gyrus, and subiculum were studied. Our findings show that the number of pyramidal neurons was significantly decreased in CA1 and subiculum of tamoxifen-treated rats in E22, P1, and P7. We found that cellular density was lower in early stages of development, however, cellular density and thickness gradually increased during the development particularly in the third week. We found that nNOS expression was decreased in E22, P1, and P7 in animals treated with tamoxifen. The present study shows that tamoxifen affects development and differentiation of postnatal rat hippocampus, CA1 neurons, and nNOS expression.     

LPS对大鼠海马CA2～3区nNOS和FOS蛋白表达的影响

目的 探讨NO和c-fos在SD大鼠海马CA2～3区免疫调节中的作用。方法 腹腔注射LPS600μg／kg建立免疫激发模型，对照组注射等量的生理盐水，用免疫组化方法和图像分析技术，观察两组大鼠海马CA2～3区nNOS和FOS蛋白的表达，检测OD值并进行统计学分析。结果 nNOS和FOS蛋白在大鼠海马各区均有散在分布，LPS刺激组海马CA2～3区nNOS和c-fos免疫阳性产物的OD值较对照组高，差异具有统计学意义。结论海马CA2～3区可能通过NO和(或)c-fos途径参与调节LPS诱导的免疫反应过程。     

尿酸抑制人脐静脉内皮细胞合成一氧化氮

目的 研究尿酸(UA)对人脐静脉内皮细胞(HUVEC)表达内皮型一氧化氮合酶(eNOS)及分泌一氧化氮(NO)的影响.方法 不同浓度UA(0、0.5、1、1.5及2 mg/L)及50 mg/L ox-LDL(阳性对照)分别作用HUVEC 24、48及72 h,用real-time PCR法测定HUVEC eNOS mRNA;Western blot法检测细胞eNOS蛋白;酶法检测上清液NO的含量.结果 UA 0.5 mg/L组eNOS mRNA表达水平明显高于对照组(P＜0.05);随着UA浓度升高(1、1.5及2 mg/L组),及其作用时间延长,HUVEC eNOS mRNA及蛋白表达水平及上清液NO分泌最相比对照均明显下降(72 h N02-/N03-2 mg/L组与对照组分别为0.52±0.18与1.00±0.10,P＜0.05),且趋势与ox-LDL组相同.结论 0.5 mg/L以上浓度的UA呈浓度及时间依赖性抑制HUVEC eNOS表达及NO合成,提示高浓度的UA可能损伤血管内皮功能.     

Inducible and endothelial nitric oxide synthase expression during development of transplant arteriosclerosis in rat aortic grafts.

In the vascular system, distinct isoforms of nitric oxide synthase (NOS) generate nitric oxide (NO), which acts as a biological messenger. Its role in the development of transplant arteriosclerosis (TA) is still unclear. To investigate whether NO is involved in TA, we studied the expression of NOS isoforms, inducible NOS (iNOS) and endothelial NOS (eNOS), by immunohistochemistry and in situ hybridization during the first two post-transplantation months and their relation with cold ischemia (1 to 24 hours) and reperfusion injury using an aortic transplantation model in the rat. We found an increased iNOS expression in the intima and adventitia and a decreased expression in the media, whereas eNOS expression was not significantly altered during the development of TA. Co-localization studies suggested that iNOS-positive cells were vascular smooth muscle cells, monocyte-derived macrophages, and endothelial cells. Prolonged ischemic storage time resulted in an increase in eNOS expression in the neointima. In situ hybridization showed iNOS mRNA expression by vascular cells in the neointima and media. NO produced by iNOS and eNOS may be involved, at least in part, in the pathogenesis of TA in aortic grafts. Additional studies are needed to confirm the modulatory mechanism of NO during the development of TA.     

Vascular endothelial growth factor and nitric oxide production in response to hypoxia in the choroid plexus in neonatal brain

Damage to the choroid plexus in 1-day-old Wistar rats subjected to hypoxia was investigated. The mRNA and protein expression of hypoxia-inducible factor-1alpha (HIF-1alpha), endothelial, neuronal, inducible nitric oxide synthase (eNOS, nNOS, iNOS), and vascular endothelial growth factor (VEGF) along with nitric oxide (NO) production and VEGF concentration was up-regulated significantly in hypoxic rats. Ultrastructurally, the choroid plexus epithelial cells showed massive accumulation of glycogen. A striking feature was the extrusion of cytoplasmic fragments from the apical cell surfaces into the ventricular lumen following the hypoxic insult. Intraventricular macrophages showed increased expression of complement type 3 receptors, major histocompatibility complex class I and II antigens, and ED1 antigens. Following an intravenous injection of horseradish peroxidase (HRP), a large number of intraventricular macrophages were labeled suggesting enhanced leakage of the tracer from the blood vessels in the choroid plexus connective tissue stroma into the ventricular lumen. We suggest that increased production of NO in hypoxia is linked to the structural alteration of the choroid plexus, and along with VEGF, may lead to increased vascular permeability. Melatonin treatment reduced VEGF and NO levels as well as leakage of HRP suggesting its potential value in ameliorating damage in choroid plexus pathologies.     

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.     

长时间应用L-精氨酸增强大鼠学习记忆功能和大脑皮质、海马nNOS或c-fos的表达及神经元数目的增加(英文)

为探讨长时间应用一氧化氮(NO)对学习记忆功能和神经系统可塑性的影响,以及NO与c-fos基因表达的关系,分别给初断乳的大鼠灌胃NO前体左旋-精氨酸(L-Arg)或一氧化氮合酶抑制剂L-NAME,用Morris水迷宫检测大鼠的学习记忆功能,用免疫组化技术和HE染色检测大脑皮质和海马CA1、CA3、DG区的神经元型一氧化氮合酶(nNOS)和c-fos基因的表达以及神经细胞数目的变化。将大鼠随机分成L-Arg组、L-NAME组和对照组。大鼠断乳后分别每天用L-Arg[200mg/(kg.d)]、L-NAME[50mg/(kg.d)]和等剂量的蒸馏水灌胃,持续3个月。结果显示:长期应用NO前体L-Arg可明显缩短大鼠的寻台潜伏期,促进nNOS和c-fos基因的表达,同时大脑皮质和海马CA1、CA3、DG区的神经元数目增加;而长期应用一氧化氮合酶抑制剂L-NAME可抑制大鼠寻台潜伏期的缩短和nNOS、c-fos基因的表达,同时大脑皮质和海马CA1、CA3、DG区的神经元数目减少。因此我们认为长时间应用NO可促进神经系统c-fos基因的表达和幼鼠神经系统的发育,即可塑性的变化,继而影响大鼠的学习记忆功能。     

Corticosterone and phenytoin reduce neuronal nitric oxide synthase messenger RNA expression in rat hippocampus.

The production and release of the corticosteroids, namely the glucocorticoids and the mineralocorticoids, are regulated by various stimuli, including stress. Previous studies from our laboratory have shown that chronic exposure to stress or to stress levels of glucocorticoids produces atrophy of the apical dendrites of CA3 pyramidal neurons in the hippocampus. This stress-induced dendritic remodeling is blocked by the anti-epileptic drug phenytoin, which suppresses glutamate release, and also by N-methyl-D-aspartate receptor antagonists. These results suggest an interaction between glucocorticoids and excitatory amino acids in the development of stress-induced atrophy of CA3 pyramidal neurons. Since nitric oxide is proposed to play an important role in mediating both the physiological and pathophysiological actions of excitatory amino acids, we examined the regulation of neuronal nitric oxide synthase messenger RNA expression by corticosterone and phenytoin in the rat hippocampus. The expression of neuronal nitric oxide synthase messenger RNA in hippocampal pyramidal neurons and granule neurons of the dentate gyrus was unaffected by 21-day administration of corticosterone (40 mg/kg), phenytoin (40 mg/kg) or the combination of corticosterone and phenytoin. However, in hippocampal interneurons, corticosterone/ phenytoin co-administration led to a significant reduction in neuronal nitric oxide synthase messenger RNA levels when compared with vehicle controls. These results suggest that, during exposure to stress levels of corticosterone, phenytoin inhibits glucocorticoid-induced atrophy of CA3 pyramidal neurons by reducing neuronal nitric oxide synthase expression in hippocampal interneurons. Moreover, these results may provide another example of synaptic plasticity in the hippocampus mediated by nitric oxide synthase.     

Impaired vasomodulation is associated with reduced neuronal nitric oxide synthase in skeletal muscle of ovariectomized rats

In exercising skeletal muscle, vasoconstrictor responses to α-adrenoceptor activation are attenuated in part by nitric oxide (NO) produced by the neuronal isoform of NO synthase (nNOS), which is expressed constitutively in skeletal muscle cells. In skeletal muscle of pregnant animals, nNOS mRNA is upregulated, suggesting that muscle nNOS expression is modulated by the steroid hormone oestrogen. Whether oestrogen-induced changes in nNOS expression have measurable effects on vasoregulation in skeletal muscle is unknown. In this study, we hypothesized that oestrogen deficiency would reduce muscle nNOS expression, resulting in impaired modulation of sympathetic vasoconstriction in exercising skeletal muscle. Compared to gonadally intact rats, we found that ovariectomized (OVX) rats were characterized by greater sympathetic vasoconstriction in contracting hindlimb and reduced nNOS, but not eNOS, in skeletal muscle. In addition, NOS inhibition resulted in a greater enhancement of sympathetic vasoconstriction in contracting hindlimbs of intact compared to OVX rats. These effects of oestrogen deficiency were prevented by chronic treatment of OVX rats with 17β-oestradiol, but not with chronic progesterone or acute oestradiol. Further analysis revealed that skeletal muscle nNOS correlated directly with plasma 17β-oestradiol and inversely with the magnitude of sympathetic vasoconstrictor responses in contracting hindlimbs. These data indicate that NO-dependent attenuation of sympathetic vasoconstriction in contracting skeletal muscle is impaired in oestrogen-deficient female rats, and suggest that this impairment may be mediated by reduced skeletal muscle nNOS expression.     

Rapid changes on nitrinergic system in female mouse hippocampus during the ovarian cycle

Fluctuating levels of estradiol and progesterone during the estrous cycle may induce structural changes in several brain nuclei including the hippocampus, where some neurons express estrogen receptors. Nitric oxide plays a wide range of functions in the nervous system generally by acting as a neurotransmitter-like molecule. It has been demonstrated that long-term treatments with estradiol in ovariectomized females and with testosterone in castrated males induce neuronal nitric oxide synthase (nNOS) expression in rat hypothalamus, whereas changes in nNOS immunoreactivity or in associated NADPH-diaphorase activity were observed both in hypothalamus and in amygdala during different phases of estrous cycle. Estradiol could induce nNOS expression in several brain regions in rodents. Therefore, to clarify if the hippocampal NO producing system is a target for gonadal hormones in physiological conditions, we have investigated the effects of estrous cycle in the expression of nNOS immunoreactivity on two-month-old intact female mice.Immunoreactive cells were observed in all hippocampal subregions: the higher number was detected in the pyramidal layer of CA1 region and in polymorph layer of dentate gyrus. The number of nNOS positive neurons fluctuates during the estrous cycle, reaching its peak during proestrus and metaestrus, and these variations were statistically significant in CA1, CA2 and CA3 regions. These results suggest that the nitrinergic system is a target for estrogen action in the hippocampus, and that this action may take place in physiological conditions according to the short-term variations of gonadal hormones during the estrous cycle.     

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.