Antenatal insults modify newborn olfactory function by nitric oxide produced from neuronal nitric oxide synthase

Newborn feeding, maternal, bonding, growth and wellbeing depend upon intact odor recognition in the early postnatal period. Antenatal stress may affect postnatal odor recognition. We investigated the exact role of a neurotransmitter, nitric oxide (NO), in newborn olfactory function. We hypothesized that olfactory neuron activity depended on NO generated by neuronal NO synthase (NOS). Utilizing in vivo functional manganese enhanced MRI (MEMRI) in a rabbit model of cerebral palsy we had shown previously that in utero hypoxia-ischemia (H-I) at E22 (70% gestation) resulted in impaired postnatal response to odorants and poor feeding. With the same antenatal insult, we manipulated NO levels in the olfactory neuron in postnatal day 1 (P1) kits by administration of intranasal NO donors or a highly selective nNOS inhibitor. Olfactory function was quantitatively measured by the response to amyl acetate stimulation by MEMRI. The relevance of nNOS to normal olfactory development was confirmed by the increase of nNOS gene expression from fetal ages to P1 in olfactory epithelium and bulbs. In control kits, nNOS inhibition decreased NO production in the olfactory system and increased MEMRI slope enhancement. In H-I kits the MEMRI slope did not increase, implicating modification of endogenous NO-mediated olfactory function by the antenatal insult. NO donors as a source of exogenous NO did not significantly change function in either group. In conclusion, olfactory epithelium nNOS in newborn rabbits probably modulates olfactory signal transduction. Antenatal H-I injury remote from delivery may affect early functional development of the olfactory system by decreasing NO-dependent signal transduction.     

Induction of neuronal nitric oxide synthase by methylmercury in the cerebellum.

A free radical, nitric oxide (NO), besides being a messenger molecule in the brain, becomes a neurotoxin if overproduced. We recently reported that methylmercury (MeHg) induces neuronal NO synthase (nNOS) in Purkinje cells. In the present study, we examined the distribution and the mechanism of nNOS induction by MeHg. Subcutaneous administration of MeHg chloride to mice, 10 mg/kg/day for 9 days, increased calcium-dependent NOS activity to 60% more than the controls only in the cerebellum but not in other brain regions. The Western blots showed a comparable increase in nNOS protein in the cerebellum. A N-methyl-D-aspartate (NMDA) receptor antagonist, MK-801, did not block, but rather enhanced, the increase in the nNOS activity. Another NMDA antagonist, 3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP), did not affect the nNOS activity. The Western blots of protein kinase C (PKC), which is an important cofactor regulating nNOS, did not change after the administration of MeHg. These results show that MeHg induces biologically active nNOS selectively in the cerebellum. The induction is independent of PKC and is not reduced by the blockade of the NMDA receptor.     

Functional importance of neuronal nitric oxide synthase in the endothelium of rat basilar arteries

The function of the neuronal isoform of nitric oxide synthase (nNOS) was studied by comparing the effects of the specific nNOS blocker 7-nitro indazole monosodium salt (7-NINA) with that of the general NOS inhibitor N(G)-nitro-L-arginine (L-NA) in isolated rat basilar arteries (BAs). 7-NINA had no significant effect on the resting tone of the vessels, while both L-NA and 1H-[1,2,4]oxadiazolo[4, 3-a]quinoxalin-1-one (ODQ), a selective inhibitor of the soluble guanylyl cyclase, induced contraction. The relaxant effect of bradykinin was attenuated in the presence of L-NA but was not changed by 7-NINA. In contrast, 7-NINA markedly reduced the acetylcholine-induced, endothelium-dependent relaxation. These results demonstrate that nNOS contributes significantly to the relaxant effect of acetylcholine, indicating the functional importance of this isoenzyme in the cerebrovascular endothelium.     

Inhibition of nitric oxide synthase protects against hypoxic neuronal injury.

We investigated the action of nitric oxide in hypoxic neuronal injury, using the hippocampal slice. Inhibition of nitric oxide synthase with the competitive inhibitor, NG-monomethyl-L-arginine, provided significant protection against hypoxia for population spike, EPSP and fiber volley responses, with an EC50 of 30 microM for protection of antidromic population spike amplitude. Confirming a stereo-specific site of action, this protection was reversed by the addition of L-arginine, but not D-arginine. These results indicate that nitric oxide generation may mediate acute CA1 neuronal injury during hypoxia, and that inhibition of nitric oxide synthase may be a useful neuroprotective strategy.     

Inhibition of neuronal nitric oxide synthase increases dopamine efflux from rat striatum

Summary. The physiological role of nitric oxide in the control of striatal dopamine release has not been fully established, therefore, the effect of neuronally produced nitric oxide (NO) on striatal dopamine (DA) efflux were investigated using in vivo microdialysis in anaesthetised and conscious rats. In the anaesthetised rat, the nitric oxide synthase inhibitors N^G-nitro-L-arginine methylester (L-NAME) and 7-nitroindazole monosodium salt (7-NINA) produced concentration-dependent increases in DA efflux. The L-NAME (1 mM)- and 7-NINA (1 mM)-induced increase was reduced by co-administration with the NO precursor, L-arginine (L-ARG; 1 mM) by 37% and 54% respectively, and was prevented by tetrodotoxin (TTX, 1 μM). Similarly, in conscious rats, L-NAME (1 mM) and 7-NINA (1 mM) increased DA efflux to 161% and 166% of basal efflux respectively. These data suggest that neuronally produced NO inhibits striatal DA efflux through an indirect mechanism. Received February 28, 2002; accepted September 24, 2002 Published online December 9, 2002 Acknowledgements This study was supported by the Medical Research Council, the Parkinson's Disease Society and the National Parkinson Foundation, Miami. MTS held a MRC Training Fellowship, SR is a Fellow of the National Parkinson Foundation, Miami. Authors' address: Prof. P. Jenner, Neurodegenerative Diseases Research Centre, Guy's, King's and St Thomas' School of Biomedical Sciences, King's College London, London, SE1 1UL, United Kingdom, e-mail: div.pharm@kcl.ac.uk Abbreviations DA dopamine; DOPAC 3,4-dihydroxyphenylacetic acid; HVA homovanillic acid; NO nitric oxide; D-NAME N^G-nitro-D-arginine methyl ester; L-NAME N^G-nitro-L-arginine methyl ester; NO nitric oxide; MAO-B monoamine oxidase-B; 7-NINA monosodium salt of 7-nitroindazole; NOS nitric oxide synthase     

Tetrahydrobiopterin depletion and ubiquitylation of neuronal nitric oxide synthase

Tetrahydrobiopterin is a necessary cofactor for the synthesis of nitric oxide by the hemeprotein enzyme, NO-synthase (NOS). It is widely thought that inadequate levels of tetrahydrobiopterin lead to tissue injury and organ dysfunction due, in part, to formation of superoxide from pterin-deficient NOS. In the course of studies on the ubiquitylation of neuronal NOS (nNOS), we have found that certain substrate analogs, such as N(G)-nitro-L-arginine, stabilize the dimeric form of nNOS and protect the enzyme from ubiquitylation. Since tetrahydrobiopterin is known to bind near heme and confers stability to the active dimeric structure of nNOS, we wondered if the loss of tetrahydrobiopterin could be an endogenous signal for nNOS ubiquitylation and degradation. We show here in HEK293 cells stably transfected with nNOS that depletion of tetrahydrobiopterin by treatment with 2,4-diamino-6-hydroxypyrimidine leads to destabilization of the dimeric form and enhances ubiquitylation of nNOS. Sepiapterin, a precursor to tetrahydrobiopterin in the salvage pathway, completely reverses the effect of 2,4-diamino-6-hydroxypyrimidine on nNOS ubiquitylation. Consistent with that found in cells, the in vitro ubiquitylation of nNOS by reticulocyte proteins decreases when tetrahydrobiopterin is present. Thus, inadequate amounts of tetrahydrobiopterin may lead to a sustained decrease in the steady state level of nNOS that is not readily reversed.     

Effects of selective neuronal nitric oxide synthase inhibition on sleep and wakefulness in the rat

The role played by the unconventional messenger Nitric Oxide (NO) upon the sleep-wake cycle remains controversial. Evidence suggests a positive role of NO on Slow Wave Sleep (SWS) and Paradoxical Sleep (PS) regulation, favoring sleep. However, other studies have found a role of NO upon wakefulness and alertness, inhibiting sleep. Divergences have been explained in part because of the use of different inhibitors of nitric oxide synthases (NOS). The aim of this study is to analyse the effects of a highly selective neuronal NOS inhibitor (3-Bromo7-Nitroindazole) on sleep-wake states in rats. Male Wistar rats were stereotaxically prepared for polysomnography. 3-Bromo-7-Nitroindazole (10, 20, 40 mg/kg, i.p.) dissolved in DMSO 10% filled with saline, or vehicle (DMSO 10% in saline) was administered at the beginning of the light period. Three hours of polygraphic recordings were evaluated for stages of vigilance. Results show dose-dependent effects of 3-Bromo7-Nitroindazole upon sleep: 10 mg/kg decreases duration and number of episodes of deep SWS, increasing duration of light SWS. 20 mg/kg decreased duration of light and deep SWS, while active and quiet wake increased. Deep SWS and PS latency increased. Number of episodes of PS decreased, as well as number of cycles of sleep and time spent asleep. 40 mg/kg reduced duration of deep SWS and increased mean episode duration of light SWS. Therefore, sleep states are affected by selective inhibition of nNOS, reducing in all cases deep SWS. These results support the hypothesis that nitric oxide, produced by nNOS, is involved in sleep processes, favoring sleep.     

神经元型一氧化氮合酶的神经生物学研究进展(英文)

神经元型一氧化氮合酶(neuronal nitric oxide synthase,nNOS)主要表达于神经元,在星形胶质细胞和神经干细胞中也有一定水平的表达。不同的mRNA拼接形式产生了nNOS蛋白的5种亚型,包括nNOS- 、nNOS- 、nNOS- 、nNOS- 和nNOS-2。nNOS单体不具催化活性,二聚体是其活性形式。nNOS单体发生二聚化需要四氢生物蝶呤、血红素以及L-精氨酸的结合。nNOS的表达在很大程度上依赖于cAMP反应元件结合蛋白的活化,其催化活性的调节与热休克蛋白90/ 热休克蛋白70、钙调节蛋白、PIN 蛋白,以及自身Ser847和Ser1412位点的磷酸化和脱磷酸化相关。能与nNOS相互作用的蛋白主要有9种,包括突触后密度蛋白95(post-synaptic density protein 95, PSD95)、CALM、CAMKIIA、DLG4、DLG2、PFK-M、CAPON、syntrophin和dynein轻链。其中 PSD95、CAPON和PFK-M是神经元中最重要的 nNOS 调节蛋白。PSD95与nNOS 的相互作用能介导突触形成,并参与N-甲基-D-天冬氨酸诱导的神...     

The cerebral metabolic consequences of nitric oxide synthase deficiency: glucose utilization in endothelial and neuronal nitric oxide synthase null mice.

Nitric oxide has multiple physiologic roles in the CNS. Inhibiting nitric oxide synthesis might therefore alter functional activity within the brain. We used [14C]-2-deoxyglucose in vivo autoradiography to measure local CMRglc in "knockout" mice lacking the genes for either the endothelial (eNOS) or neuronal (nNOS) isoforms of nitric oxide synthase, and in the progenitor strains (SV129, C57B1/6). Glucose utilization levels did not significantly differ between nNOS and eNOS knockout mice and C57B1/6 mice in any of the 48 brain regions examined, but were relatively lower in some subcortical regions in SV129 mice.     

Inhibition of neuronal nitric oxide synthase protects against MPTP toxicity

Previous work showed that several relatively specific inhibitors of neuronal nitric oxide synthase (nNOS) produce protection against MPTP induced dopaminergic toxicity. We examined whether a highly specific novel inhibitor of nNOS, ARRI 7338, could also protect against MPTP toxicity. ARR17338 produced dose-dependent significant protection against MPTP induced depletion of dopamine and protected against MPTP induced depletions of tyrosine hydroxylase immunostained neurons in the substantia nigra. These results provide further evidence that inhibitors of nNOS may be useful for the treatment of Parkinson's disease.     

Association analysis for neuronal nitric oxide synthase gene polymorphism with major depression and fluoxetine response

Nitric oxide (NO) is produced from its precursor L-arginine by the enzyme NO synthase (NOS), which includes at least three distinct isoforms - neuronal (nNOS), endothelial, and inducible NOS. Recent studies have implicated NOS in the mechanism that underlies the therapeutic efficacy of antidepressant medication. In addition, major depressive disorder (MDD) patients were found to have significantly higher plasma nitrate concentrations than normal subjects, an index of NO production, in comparison to normal subjects. In a population-based association study, we tested the hypothesis that the nNOS C276T polymorphism confers susceptibility to MDD. We also examined the association between this polymorphism and therapeutic fluoxetine response in 114 MDD patients who underwent a 4-week fluoxetine treatment. The results demonstrate that the nNOS variants are found at similar frequencies in MDD patients and healthy control subjects. Further, we did not discover any genetic variants that influenced the fluoxetine response in MDD patients treated with fluoxetine. Our findings suggest that this nNOS C276T polymorphism does not play a major role in the susceptibility to, or fluoxetine response in, MDD. However, the association between other NOS variants and MDD or antidepressant response, including sexual dysfunction, may warrant further investigation.     

Purkinje cells express neuronal nitric oxide synthase after methylmercury administration

To study the effects of chemical injury on the cerebella nitric oxide synthase (NOS), we administered methylmercury chloride subcutaneously to mice, 10 mg/kg/day for 9 days. In the methylmercury-treated cerebellum, Purkinje cells were positive both for NADPH-diaphorase and for neuronal NOS. Calcium-dependent NOS activity was increased to 160% of the controls. The present study suggests the ability of Purkinje cells to produce NO through the expression of neuronal NOS.     

Increased nitrotyrosine immunoreactivity in substantia nigra neurons in MPTP treated baboons is blocked by inhibition of neuronal nitric oxide synthase

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) produces clinical, biochemical and neuropathologic changes reminiscent of those which occur in idiopathic Parkinson's disease. 7-Nitroindazole (7-NI) is a relatively selective inhibitor of the neuronal isoform of nitric oxide synthase. We previously demonstrated that administration of 7-NI is effective in blocking MPTP toxicity in both mice and baboons. This was suggested to be due to inhibition of the generation of peroxynitrite which can nitrate tyrosines. In the present study we found increased 3-nitrotyrosine immunoreactivity in the substantia nigra of MPTP treated baboons, which was blocked by coadministration of 7-NI. These findings provide further evidence that peroxynitrite may play a role in MPTP induced parkinsonism in baboons.     

Inducible nitric oxide synthase and nitric oxide production by oligodendrocytes

It has been previously demonstrated that microglia and astrocytes produce micromolar amounts of nitric oxide in vitro. In this study, we demonstrate that primary rat oligodendrocytes can be stimulated to produce iNOS mRNA as detected by Northern blot and in situ hybridization analysis and a 131-kDa iNOS protein by Western blot analysis; protein was also detected in cells by single- and double-label immunohistochemistry for iNOS and the oligodendrocyte-specific marker CNPase. NO/NOS are produced as a consequence of activation of the gene encoding the inducible nitric oxide synthase as determined by inhibition with actinomycin D and cyclohexamide. The iNOS is functional, leading to calcium/calmodulin-independent NO production in these in vitro cultures. J. Neurosci. Res. 48:372–384, 1997. © 1997 Wiley-Liss, Inc.     

Effects of testosterone on neuronal nitric oxide synthase and tyrosine hydroxylase

Male rat copulatory ability decreases dramatically following castration. This may be due in part to the impairment of medial preoptic area (MPOA) dopamine (DA) release. Previous studies showed that extracellular DA levels in the MPOA of castrates were lower than in intact males, both during basal conditions and in the presence of a receptive female. However, tissue levels of DA in the MPOA were higher in castrates than in intact males, suggesting that DA synthesis may be normal or increased in castrates, but that release may be compromised. The current study found that neither long term (2 months) nor short term (2 weeks) castration had any effect on the number of neurons in the DA A₁₄ area that were immunoreactive (ir) for tyrosine hydroxylase (TH), the rate limiting enzyme for DA synthesis. Therefore, castration may not affect DA synthesis in the MPOA. Tissue levels of neurotransmitter reflect release, as well as synthesis. We previously reported that nitric oxide (NO) may increase DA release in the MPOA. The present study tested whether castration affected the number of NO producing cells in the MPOA. Long term, but not short term, castration significantly decreased the number of NADPH-d (nicotinamide adenine dinucleotide phosphate diaphorase) positive neurons and brain nitric oxide synthase immunoreactive (bNOS-ir) neurons in the medial preoptic nucleus (MPN). This suggests that in gonadally intact animals testosterone may activate NOS, which increases the production of NO. Long or short term castration had no effect on the numbers of bNOS-ir neurons in the paraventricular nucleus (PVN) or medial amygdala. However, short term castration decreased bNOS-ir neurons in the bed nucleus of stria terminalis (BNST). Thus, one means by which testosterone promotes male sexual behavior may be by increasing production of NO in the MPOA, which increases local DA release.     

神经源型一氧化氮合酶C276T基因多态性与抑郁症相关分析

目的测定抑郁症患者抗抑郁剂治疗前后血浆一氧化氮（NO）水平变化,旨在探讨神经源型一氧化氮合酶（nNOS）基因C276T多态性与血浆NO浓度及抑郁症发病相关性。方法采用硝酸盐还原酶法测定正常对照组及抑郁症患者治疗前后血浆NO水平;全部受试者取全血标本提取基因组DNA,并采用PCR-RFLP方法对nNOS基因C276T多态性进行基因分型。结果患者组疗前血浆NO水平为（76.8±31.6）μmol/L显著高于疗后[（66.9±25.7）μmol/L,P=0.044]及正常对照组[（64.2±33.3）μmol/L,P=0.02],两组疗后血浆NO水平相比差异无显著性（P=0.588）;根据PCR-RFLP结果,nNOS基因可见两种等位基因条带C、T,组成三种基因型CC、CT、TT,两组等位基因及基因型分布频率差异无显著性（均P〉0.05）,且携带不同基因型者之间血浆NO水平差异亦无显著性（均P〉0.05）。结论血浆NO浓度增高可能是抑郁症发病的影响因素;nNOS基因C276T多态性可能不直接影响血浆NO浓度,也不是抑郁症发病的主要基因因素。     

Antisense knockdown of neuronal nitric oxide synthase antagonizes nitrous oxide-induced behavior

The behavioral effects of nitrous oxide (N(2)O) were antagonized by non-specific inhibitors of nitric oxide synthase (NOS). To identify the isoform of NOS involved in this response, mice were pretreated with an antisense oligodeoxynucleotide (AS-ODN) against neuronal NOS, and then tested in a light/dark exploration paradigm. The AS-ODN but not the mismatch ODN significantly antagonized N(2)O induced behavior and also reduced NOS activity in the cerebellum and hippocampus. These results implicate neuronal NOS in the N(2)O response.     

Neuroprotection via inhibition of nitric oxide synthase by bis(7)-tacrine

Here we report that bis(7)-tacrine, a novel acetylcholinesterase inhibitor, exerts neuroprotective effects by inhibition of nitric oxide synthase. In cortical neurons at 12 days in vitro, bis(7)-tacrine concentration-dependently reduced cell death induced by glutamate, beta-amyloid and L-arginine, but not by nitric sodium nitroprusside. N-monomethyl-L-arginine, a nitric oxide synthase inhibitor, also prevented the former three types but not the last type of the cytotoxicity; however, nitric oxide scavengers blocked all of these insults, indicating that nitric oxide mediated these neuronal injuries. Furthermore, with nitric oxide synthase activity assays, it was found that bis(7)-tacrine not only suppressed the activation of nitric oxide synthase caused by glutamate in cortical neurons, but also directly inhibited the activity of nitric oxide synthase in vitro.     

Up-regulated expression of neuronal nitric oxide synthase in experimental diabetic retina

Nitric oxide (NO) can play either a neuroprotective or a neurotoxic role in diverse neurodegenerative conditions. This study investigated the differential expression of neuronal nitric oxide synthase (nNOS) in the streptozotocin-induced diabetic rat retina to clarify the involvement of NO produced from neurons in the early pathogenesis of diabetic retinopathy. A decrease in thickness of the outer retina was evident at 12 and 24 weeks after onset of diabetes. nNOS was immunolocalized in two subtypes of amacrine cells, displaced amacrine cells and in some bipolar cells in the normal retinas. The densities of each type of nNOS-expressing neuron showed no significant differences in the diabetic retinas with the exception of the bipolar cells. The numbers of nNOS bipolar cells at 12 weeks of diabetes increased threefold, showing dendritic polarity of nNOS expression. Protein levels of nNOS increased throughout the diabetic retinas reaching a peak value at 24 weeks of diabetes. Thus, diabetes up-regulates the expression of nNOS in bipolar cells, and NO from these cells may aggravate the degeneration of the outer retina in the diabetic retinas.