Teses / dissertações: "Neuronal nitric oxide synthase (nNOS)"

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Cheung, Nathan Yiutung. "Serotonin receptor and neuronal nitric oxide synthase expression in the rat brain : implications for MDMA toxicity". Thesis, King's College London (University of London), 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.368095.

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Lu, Chieh-Ju. "Neuronal nitric oxide synthase-CAPON regulation of cardiac sympathetic activity in the development of hypertension". Thesis, University of Oxford, 2015. http://ora.ox.ac.uk/objects/uuid:1204dec9-9f09-458d-b361-c8d14589fcd1.

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The studies presented in this thesis were undertaken to investigate the cellular and molecular mechanisms responsible for sympathetic hyperactivity that is observed in the Spontaneous Hypertensive Rat (SHR) and whether these abnormalities arise even before the onset of hypertension. Moreover, selected molecular candidates related to oxidative state in cardiac autonomic signalling have been explored for their potential therapeutic effects. Chapter One is an overview of (i) the relevance of autonomic dysfunction in cardiovascular disease in both human and animal models, (ii) the physiological basis of cardiac sympathetic neurotransmission, (iii) the neuromodulators of peripheral cardiac sympathetic-vagal balance discussed along with how they may be involved in cardiac adrenergic control of neurotransmission and NO-cGMP signalling. This develops the formulation of the specific aims of the thesis. Chapter Two outlines a detailed rationale for the experimental approach taken to (i) characterise protein expression in the pre-hypertensive animal model with immunohistochemistry and Western blotting, (ii) manipulate selected gene expression to amplify NO-cGMP signalling in vivo and in vitro via viral gene transfer, (iii) investigate calcium handling in cardiac sympathetic stellate neurons with calcium imaging , (iv) measure cardiac noradrenergic neurotransmission from double atria using radioactive-labelled [³H]-noradrenaline. Chapter Three demonstrated abnormal NO-cGMP signalling in pre-hypertensive SHRs. Endogenous nNOS protein residing in both cardiac parasympathetic and sympathetic neurons was significantly lower in the pre-hypertensive SHR compared to aged-matched WKYs. This was associated with lower cGMP levels. An enhanced depolarization evoked [Ca²⁺]i transient was observed in cardiac stellate neurons from pre-hypertensive SHR when compared with the WKY, an effect that was reversed by nNOS or sGC inhibition. Chapter Four investigated the role of nNOS and brain natriuretic peptide (BNP) in cGMP signalling pathways. Gene transfer of nNOS via adenoviral vector in SHR cardiac sympathetic neurons increased cGMP concentration and normalised neuronal calcium handling during depolarization. BNP significantly reduces [3H]- noradrenaline release. Overexpression of PDE2 which facilitates the breakdown of cGMP caused an increase in [³H]- noradrenaline release in response to field stimulation and also prevented the inhibitory action of BNP. Chapter Five examined the role of the nNOS adaptor protein, CAPON in NO-cGMP signalling. Endogenous CAPON protein is present in cardiac sympathetic neurons in the WKY, and is significantly reduced in pre-hypertensive SHR cardiac neurons. Artificial up-regulation of cardiac sympathetic CAPON via targeted gene transfer directly attenuated neuronal Ca²⁺ transients, resulting in decreased noradrenaline release in the SHR. Chapter Six is a concluding discussion summarising the main findings from this thesis, placing them in a physiological context and discussing avenues for further research.

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Bird, Diane Carol. "An investigation into the role of neuronal nitric oxide synthase (nNOS) in the phencyclidine mouse model of schizophrenia". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape4/PQDD_0015/MQ57249.pdf.

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4

Barua, Anupama. "The role of neuronal nitric oxide synthase (nNOS) in ischaemia/reoxygenation-induced injury and in protection of the mammalian myocardium". Thesis, University of Leicester, 2010. http://hdl.handle.net/2381/8754.

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Background: In physiological condition, NO is produced by two constitutive NOS isoform; eNOS and nNOS. Both isoforms have specific cellular locations and although the role of eNOS in myocardial ischaemic injury and in cardioprotection has been thoroughly addressed, but the role of nNOS remains unclear. Therefore, the aims of the thesis were to: (i) investigate the role of nNOS in ischaemia/reoxygenation-induced injury, (ii) determine whether its effect is species-dependent, (iii) elucidate the relationship of nNOS with mitoKATP channels and p38MAPK, two key components of IP and (iv) investigate whether modulation of the NO metabolism can overcome the unresponsiveness of the diabetic myocardium to IP. Methods and Results: Ventricular myocardial slices from rats and mice, nNOS knockout mice, and also from human right atrial slices were subjected to 90min ischaemia and 120min reoxygenation (37°C). Muscles were randomized to receive various treatments. Both the provision of exogenous NO and the inhibition of endogenous NO production significantly reduced tissue injury (creatine kinase release, cell necrosis and apoptosis), an effect that was species–independent. The protection seen with nNOS inhibition was as potent as that of IP, however, in nNOS-knocked out mice the cardioprotective effect of non-selective NOS (L-NAME) and selective nNOS inhibition (TRIM) and also that of IP was blocked while the benefit of exogenous NO remained intact. Additional studies revealed that the cardioprotection afforded by of exogenous NO and by inhibition of nNOS were unaffected by the mitoKATP channel blocker 5-HD although it was abrogated by p38MAPK blocker SB203580. Finally, in diabetic myocardium, IP did not decrease CK release neither reduced cell necrosis or apoptosis. In diabetic myocardium NO donor SNAP, inhibitor L-NAME and TRIM significantly reduced CK leakage, cell necrosis and apoptosis. Conclusions: nNOS plays a dual role in ischaemia/reoxygenation on that its presence is necessary to afford cardioprotection by IP but its inhibition reduces myocardial ischaemic injury. The role of nNOS is species-independent and exerted downstream of the mitoKATP channels and upstream of p38MAPK. Moreover, both the provision of exogenous NO and the suppression of endogenous NO production resulted in potent protection of diabetic human myocardium, overcoming the unresponsiveness of these tissues to IP.

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Balda, Mara A. "Ontogeny- and Sex-Dependent Contributions of the Neuronal Nitric Oxide Synthase (nNOS) Gene to Rewarding and Psychomotor Stimulating Effects of Cocaine". Scholarly Repository, 2009. http://scholarlyrepository.miami.edu/oa_dissertations/257.

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Multiple interactions between dopamine (DA), glutamate, and nitric oxide (NO) in mesolimbic and corticostriatal circuits suggest that NO may play a critical role in cocaine-induced behavioral and neural plasticity. Clinical and preclinical studies have revealed that females and adolescents display unique vulnerabilities to the behavioral and neurochemical effects of cocaine as a result of sex-dependent and ontogeny-dependent differences in dopaminergic systems. Thus, my research objectives were to investigate the contributions of the neuronal nitric oxide synthase (nNOS) gene, ontogeny, and gender on the rewarding and sensitizing effects of cocaine. I found that nNOS significantly influences the rewarding aspects of cocaine in adolescent mice and adult male mice (i.e., major deficits in several phases of cocaine conditioned place preference (CPP) were detected in nNOS knockout (KO) adolescent mice and nNOS KO adult male mice). However, the contribution of nNOS was sex-dependent as CPP phases were normal in KO adult females. In contrast to CPP, I found a major ontogeny-dependent contribution of nNOS to the sensitizing effects of cocaine. Namely, while nNOS is essential for the development of behavioral sensitization in adult males, this type of behavioral plasticity develops independently of nNOS during adolescence. The contribution of nNOS was once again sex-dependent as behavioral sensitization was normal in adult KO females. Together, this line of investigation has revealed that the NO-signaling pathway has a) a sex-dependent role in the neuroplasticity underlying cocaine CPP and b) a sex-dependent and ontogeny-dependent influence on cocaine-induced behavioral sensitization. Stereological and western blot analysis revealed that a sensitizing regimen of cocaine resulted in an increase in nNOS and tyrosine hydroxylase (TH) immunoreactivity in the dorsal striatum (dST) of adult, but not adolescent, wild-type (WT) male mice. In the absence of nNOS, dopaminergic neurons in the ventral tegmental area (VTA) were severely reduced and cocaine caused a downregulation of dST TH suggesting that nitrergic levels modulate TH. Thus, the finding that nNOS is essential for the development of sensitization in adulthood, but not adolescence, together with the fact that cocaine upregulated nNOS and TH in the dST in adult, but not adolescent mice, strongly suggest that the nitrergic system underlies behavioral sensitization through modulation of the dopaminergic system in adulthood. These findings suggest different approaches in the clinical treatment of drug craving and drug-seeking behavior in adolescent and adult patients.

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Schonhoff, Christopher M. "The Regulation of nNOS During Neuronal Differentiation and the Effect of Nitric Oxide on Hdm2-p53 Binding: a Dissertation". eScholarship@UMMS, 2000. https://escholarship.umassmed.edu/gsbs_diss/57.

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Nitric oxide is a ubiquitous signaling molecule with both physiological and pathological functions in biological systems. Formed by the enzymatic conversion of arginine to citrulline, NO, has known roles in circulatory, immune and nervous tissues. In the nervous system nitric oxide has been implicated in long-term potentiation, neurotransmitter release, channel function, neuronal protection and neuronal degeneration. Much of our work has focused on yet another role for nitric oxide in cells, namely, neuronal differentiation. During development, neuronal differentiation is closely coupled with cessation of proliferation. We use nerve growth factor (NGF)-induced differentiation of PC12 pheochromocytoma cells as a model and find a novel signal transduction pathway that blocks cell proliferation. Treatment of PC12 cells with NGF leads to induction of nitric oxide synthase (NOS). The resulting nitric oxide (NO) acts as a second messenger, activating the p21(WAF1) promoter and inducing expression of p21(WAF1) cyclin-dependent kinase inhibitor. NO activates the p21(WAF1) promoter by p53-dependent and p53-independent mechanisms. Blocking production of NO with an inhibitor of NOS reduces accumulation of p53, activation of the p21(WAF1) promoter, expression of neuronal markers, and neurite extension. To deternine whether p21(WAF1) is required for neurite extension, we prepared a PC12 line with an inducible p21(WAF1) expression vector. Blocking NOS with an inhibitor decreases neurite extension, but induction of p21(WAF1) with isopropyl-1-thio-beta-D-galactopyranoside restored this response. Levels of p21(WAF1) induced by isopropyl-1-thio-beta-D-galactopyranoside were similar to those induced by NGF. Therefore, we have identified a signal transduction pathway that is activated by NGF; proceeds through NOS, p53 and p21(WAF1) to block cell proliferation; and is required for neuronal differentiation by PC12 cells. In further studies of this pathway, we have examined the role of MAP kinase pathways in neuronal nitric oxide synthase (nNOS) induction during the differentiation of PC12 cells. In NGF-treated PC12 cells, we find that nNOS is induced at RNA and protein levels, resulting in increased NOS activity. We note that neither nNOS mRNA, nNOS protein nor NOS activity is induced by NGF treatment in cells that have been infected with a dominant negative Ras adenovirus. We have also used drugs that block MAP kinase pathways and assessed their ability to inhibit nNOS induction. Even though U0126 and PD98059 are both MEK inhibitors, we find that U0126, but not PD98059, blocks nNOS induction and NOS activity in NGF-treated PC12 cells. Also, the p38 kinase inhibitor, SB 203580, does not block nNOS induction in our clone of PC12 cells. Since the JNK pathway is not activated in NGF-treated PC12 cells, we determine that the Ras-ERK pathway and not the p38 or JNK pathway is required for nNOS induction in NGF-treated PC12 cells. We find that U0l26 is much more effective than PD98059 in blocking the Ras-ERK pathway, thereby explaining the discrepancy in nNOS inhibition. We conclude that the Ras-ERK pathway is required for nNOS induction. The activation of soluble guanylate cyclase and the production of cyclic GMP is one of the best characterized modes of NO action. Having shown that inhibition of NOS blocks PC12 cell differentiation we tested whether nitric oxide acts through soluble guanylate cyclase to lead to cell cycle arrest and neuronal differentiation. Unlike NOS inhibition, the inhibition of soluble guanylate cylcase does not block the induction of neuronal markers. Moreover, treatment of NGF-treated, NOS-inhibited PC12 cells with a soluble analog of cyclic GMP was unable to restore differentiation of those cells. Hence, cGMP is not a component of this pathway and we had to consider other mechanisms of NO action. It has become increasingly evident that another manner by which NO may exert its effects is by S-nitrosylation of cysteine residues. We tested, in vitro whether nitric oxide may control p53 by S-nitrosylation and inactivation of the p53 negative regulator, Hdm2. Treatment of Hdm2 with a nitric oxide donor inhibits Hdm2-p53 binding, the first step in Hdm2 regulation of p53. The presence of cysteine or DTT blocks this inhibition of binding. Moreover, nitric oxide inhibition of Hdm2-p53 binding was found to be reversible. Sulfhydryl-sensitivity and reversibility are consistent with nitrosylation. Finally, we have identified a critical cysteine residue that nitric oxide modifies in order to disrupt Hdm2-p53 binding. Mutation of this residue from a cysteine to an alanine does not interfere with binding but rather eliminates the sensitivity of Hdm2 to nitric oxide inactivation.

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7

Silva, Maria Isabel. "Distribuição de celulas imunorreativas para sintase neuronal do oxido nitrico (nNOS) no hipocampo de pombos (Columba livia) apos aprendizagem de escolha alimentar". [s.n.], 2007. http://repositorio.unicamp.br/jspui/handle/REPOSIP/314142.

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Orientadores: Elenice Aparecida de Moraes Ferrari, Claudio Antonio Barbosa de Toledo
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: O hipocampo exerce papel fundamental no processamento de aprendizagem e memória espaciais. Comparações das características funcionais, anatômicas e neuroquímicas do hipocampo são favorecidas por evidência oriunda de estudo sobre aprendizagem especial em mamíferos e aves. O objetivo do presente estudo foi analisar a marcação imunohistoquímica de células nNOS- positivas no hipocampo de pombos (C. lívia) após diferentes duração do treino em aprendizagem especial. Foram analisados grupos de animais não treinados (MAN), treinados em 1 sessão (EXP1), treinados em 5 sessões (EXP5), exposto à arena em 1 sessão (CONT1) ou em 5 sessões (CONT5). As sessões foram realizadas numa arena onde havia quatro comedouros, um dos quais com alimento. Em cada sessão, com seis tentativas, registrou-se a latência (seg) e a assertividade da escolha de um comedouro. Após os testes comportamentais, usou-se imunoistoquímica para a análise da marcação de células nNOS-positiva no hipocampo dorsal e ventral. O grupo EXP5 teve diminuição da latência de escolha ('F IND. 4,28¿= 23,74; p < 0,001) e aumento das respostas corretas ('F IND. 4,35¿= 8,66; p < 0,001) em função do treino. A marcação das células nNOS-positivas no hipocampo foi significativamente maior no hipocampo dorsal dos animais EXP5 em comparação com o hipocampo ventral ('F IND. 4,22¿= 104,79; p<0,001) e com os demais grupos ('F IND. 4,22¿= 10,17; p < 0,001). O aumento da imunorratividade de células nNOS- positivas no hipocampo dorsal de pombos após a aprendizagem da localização do comedouro correto sugere o envolvimento desta região e de processos mediados pro transmissão glutamatérgica nesse processo de aprendizagem e memória em pombos
Abstract: The hippocampus has fundamental role in spatial learning and memory processes. Functional and neurochemical analysis of the hippocampus are favored by evidence on spatial learning in mammals and birds. The present study examined the immunohistochemical expression of nNOS-positive cells in the hippocampus of pigeons (C. livia) after training in food location task. Animals were trained in one (EXP1) or five (EXP5) sessions or had one (CONT1) or five sessions (CONT5) of exposure to the experimental arena. The six trials sessions were conducted daily in one arena with 4 food bowls, one of which had food. Latency and accuracy of choise recorded. After behavioral tests, nNOS immunoractivity in hippocampal cells was analyzed. EXP 5 showed reduction imunoreactivity in hippocampal cells was analysed. EXP5 showed reduction in latency of choise ('F IND. 4,28¿= 23,74; p < 0,001) and increassis in correct choise ('F IND. 4,35¿= 8,66; p < 0,001) as function of the training. The expression of nNOS- positive cells was significantily higher in the dorsal hippocampus of EXP5 group as compared with the ventral hippocampus ('F IND. 4,22¿= 104,79; p < 0,001) and the other groups ('F IND. 4,28¿= 10,17; p < 0,001). The increases of nNOS immunoreactive neurons after learning of the food location suggest that nNOS is involved in processes of spatial learning and memory that are mediated by the dorsal hippocampus of pigeons
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8

Denadai, Magda Aline. "Efeitos do 7-nitroindazole, um inibidor da sintase neuronal do oxido nitrico (nNOS), sobre o condiciomaneto contextural em pombos". [s.n.], 2008. http://repositorio.unicamp.br/jspui/handle/REPOSIP/314745.

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Orientador: Elenice Aparecida de Moraes Ferrari
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: O óxido nítrico (NO), um neurotransmissor não convencional, tem papel importante em processos neurobiológicos de comportamento e de memória. Sua síntese é mediada por três isoformas de sintase do óxido nítrico (NOS): a neuronal (nNOS), a endotelial (eNOS) e induzível (iNOS). Este trabalho analisou o efeito do 7-nitroindazole (7-NI), um inibidor seletivo da nNOS, no condicionamento clássico aversivo em pombos. Foram usados 4 grupos: tratados com 7-NI (grupo 7-nitroindazole; G7-NI, n=5), tratados com óleo de amendoim (grupo veículo; GV, n=5), controle/sem tratamento (grupo controle; GC, n=5) e grupo não tratado/não condicionado (grupo manipulação; GM, n=5). A administração i.p. de 7-NI (25 mg/kg), ou do óleo de amendoim foi feita imediatamente após o treinamento. O G7-NI, o GV e o GC receberam três associações som-choque (5°, 10° e 15º minutos) numa sessão de 20 min. O teste a o contexto foi realizado 24 horas depois. As sessões foram gravadas para posterior transcrição e análise comportamental. A ocorrência da resposta de congelamento durante o treino não diferiu entre os grupos (p>0,05), mas durante o teste foi menor para o G7-NI em comparação ao treino (p<0.01) e aos demais grupos no teste (p<0.001). A atividade da NOS dependente de Ca++ no hipocampo foi menor no G7-NI do que nos outros grupos (p<0,01). Análise por Western blot indicou aumento na expressão de nNOS no G7-NI (p<0,05). A administração sistêmica de 7-NI teve um efeito amnésico sobre a memória contextual aversiva, indicando que a atividade da NOS dependente de Ca++ é importante para os processos de condicionamento clássico aversivo em pombos.
Abstract: Nitric oxide (NO) is an unsual neurotransmitter that plays an important role in neurobiological functions underlying behavior and memory. NO synthesis and release can be mediated by three isoforms of NO synthases (NOS): neuronal (nNOS), endothelial (eNOS) and inducible (iNOS). This study examined the effect of 7-nitroindazole (7-NI), a selective nNOS inhibitor, on contextual fear conditioning in pigeons. Four groups of pigeons were used: treated with 7-NI (7-NI; n=5), treated with peanut oil (Vehicle; n=5), non treated controls (Control; n=5) and non treated and no-trained controls (Non-trained; n=5). Treatment consisted in 7-NI (25 mg/kg; i.p.) or vehicle (peanut oil) administration, immediately after training. All the animals were trained in one 20 min session during which three tone-shock pairings (5th, 10th and 15th minutes) were presented. The test to the context was conducted 24h later. Behavioral categories were analyzed through the transcription of video-tapes of the sessions. The groups 7-NI, Vehicle and Control showed no significant differences in freezing during the conditioning session (p>0.05). During the test to the context the group 7-NI expressed significantly lower freezing as compared to Vehicle and Control (p<0.05). The 7-NI pigeons showed lower hippocampal activity of Ca++ dependent-NOS than Vehicle and Control groups (p<0.01). Western blot analysis indicated significant increase in nNOS expression (p<0.05). The systemic administration of 7-NI induced amnestic effects on contextual fear memory that evidence that Ca++-dependent NOS activity is required for fear conditioning in pigeons.
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Fisiologia
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9

Machado, Aline Vilar da Silva. "Variação circadiana da expressão da sintase neuronal de óxido nítrico (nNOS) no hipocampo e o condicionamento contextual aversivo em pombos (Columba livia)". [s.n.], 2011. http://repositorio.unicamp.br/jspui/handle/REPOSIP/314744.

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Orientador: Elenice Aparecida de Moraes Ferrari
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: A ritmicidade circadiana, expressa na alteração do comportamento e em aspectos morfofisiológicos e moleculares ao longo das 24 horas do dia, é uma das funções básicas dos organismos vivos. Os processos comportamentais e os mecanismos moleculares no hipocampo, que estão envolvidos na aprendizagem e memória, demonstram oscilação circadiana. Vários estudos sugeriram que o condicionamento clássico aversivo é afetado pelo sistema de temporização circadiana e que a oscilação circadiana de vias moleculares específicas é requerida para a consolidação da memória aversiva. O presente trabalho investigou a oscilação circadiana da expressão da nNOS e da atividade da proteína NOS no hipocampo de pombos e as suas relações com a modulação temporal do condicionamento contextual aversivo. Na Parte I, caracterizou-se o padrão temporal da expressão da nNOS, que foi detectada por Western Blotting e o padrão temporal da atividade enzimática da NOS, determinada pela quantidade de L-citrulina produzida por minuto e por micrograma de proteína na reação. Na Parte II, nos horários de mínima e máxima atividade enzimática da proteína, pombos foram treinados e testados em condicionamento aversivo ao contexto. As sessões foram gravadas para posterior análise comportamental. Após o teste foi realizada a imunoistoquímica para marcação da nNOS em neurônios do hipocampo. Foi evidenciada ritmicidade circadiana significativa (p < 0,05) na expressão protéica da nNOS e na atividade enzimática da NOS, segundo os valores fornecidos pelo método Cosinor para caracterização do padrão temporal. As médias da densitometria óptica dos grupos com horários mais próximos da acrofase - ZT04 (10hs; 0,944 ± 0,12) e a batifase - ZT16-(22hs; 0,572 ± 0,16) foram significativamente diferentes (F5,18 p < 0,0001). Os grupos condicionados, em ambos os horários, mostraram maior duração e maior ocorrência do comportamento de congelamento do que os controles (p < 0,05). Houve uma variação dia-noite para o comportamento de congelamento nos grupos controles (p < 0,05). A marcação de células nNOS-positivas foi maior no hipocampo dos grupos condicionados sendo que o total de células nNOS-positivas na área dorsal do grupo experimental testado à noite foi maior do que aquele observado nos grupos controles e no experimental da manhã (p < 0,05). Os dados mostraram que a expressão protéica da nNOS e da atividade enzimática da NOS no hipocampo de pombos mostram uma oscilação que caracteriza um padrão temporal circadiano. Tanto no horário de máxima como no de mínima atividade da nNOS, o condicionamento contextual aversivo resultou em medo condicionado ao contexto e em expressão de células nNOS-positivas no hipocampo que foi maior nos pombos condicionados do que nos controles. Contudo, no hipocampo do grupo testado à noite houve um maior número de células nNOS-positivas. Esse dado estimula questionamento sobre se ocorreria a ativação de mecanismos compensatórios para o aumento da expressão da proteína nNOS, quanto essa é requisitada em situações de baixa disponibilidade
Abstract: The circadian rhythm, expressed in changing behavior and the morphophysiologic and molecular aspects over 24 hours of the day is one of the basic functions of living organisms. The behavioral processes and molecular mechanisms in the hippocampus, which are involved in learning and memory, show circadian oscillation. Several studies have suggested that classical fear conditioning is affected by the circadian timing system and the circadian oscillation of specific molecular pathways is required for the consolidation of aversive memory. This study investigated the circadian oscillation of nNOS expression and activity of NOS protein in the hippocampus of pigeons and their relationship with the temporal modulation of aversive contextual conditioning. In Part I, we have characterized the temporal pattern of nNOS expression, which was detected by Western blotting and temporal pattern of NOS enzyme activity, determined by the amount of L-citrulline produced per minute and per microgram of protein in the reaction. In Part II, at the times of minimum and maximum activity of the protein, pigeons were trained and tested in aversive conditioning to context. The sessions were taped for later behavioral analysis. After the test was performed immunohistochemical for labeling of nNOS in neurons in the hippocampus. Circadian rhythm was evident (p <0.05) in nNOS protein expression and enzyme activity of NOS, according to figures provided by Cosinor method to characterize the temporal pattern. The mean optical density of groups with times closer to the acrophase - ZT04 (10hrs; 0.944 ± 0.12) and nadir - ZT16-(22hs; 0.572 ± 0.16) were significantly different (F5, 18 p <0.0001 ). The groups conditioned in both schedules, showed more frequent and longer duration of freezing behavior than controls (p <0.05). There was a day-night variation for freezing behavior in control groups (p <0.05). Labeling of nNOS-positive cells was higher in the hippocampus of the groups conditioned with total nNOS-positive cells in the dorsal area of the experimental group tested at night was higher than that observed in control groups and experimental group in the morning (p <0.05). The data showed that nNOS protein expression and enzymatic activity of NOS in the hippocampus of pigeons show an oscillation that characterizes a circadian temporal pattern. Both at the time of maximum as the low activity of nNOS, the aversive contextual conditioning resulted in fear conditioning to context and expression of nNOS-positive cells in the hippocampus was higher in pigeons conditioned than in controls. However, in the hippocampus of the group tested in the evening there was a greater number of nNOS-positive cells. This fact encourages questioning of whether there would be activation of compensatory mechanisms for the increased expression of nNOS protein, as this is required in situations of low availability
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Fisiologia
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10

Faria, Larissa Oliveira Melloni de 1985. "Participação da sintase neuronal de óxido nítrico (nNOS) na consolidação e reconsolidação da memória do condicionamento clássico aversivo em pombos (Columba livia)". [s.n.], 2013. http://repositorio.unicamp.br/jspui/handle/REPOSIP/314128.

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Orientador: Elenice Aparecida de Moraes Ferrari
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: O óxido nítrico (NO) é um neurotransmissor não convencional o qual tem papel importante em processos neurobiológicos de comportamento e de memória. Sua síntese é mediada por três isoformas de sintase do óxido nítrico (NOS): a neuronal (nNOS), a endotelial (eNOS) e a induzível (iNOS). Este trabalho investigou os efeitos da administração do 7- nitroindazol (7-NI), inibidor preferencial da nNOS, na consolidação e reconsolidação da memória do condicionamento clássico aversivo. Pombos adultos foram atribuídos a 5 grupos: Foram usados 5 grupos: grupo 7-nitroindazole (7-NI) (100nmol/0.5?/l; DMSO (20%), NaOH (50mM) e Tween-80 (16%) diluído em PBS; i.c.v.), grupo veículo (VEIC) (0,5?/l; DMSO (20%), NaOH (50mM) e Tween-80 (16%) diluído em PBS, i.c.v.), grupo condicionado/não tratado (COND), grupo contexto/não-tratado (CONT) e grupo não tratado/não condicionado (NÄIVE). Sete dias após implante de microcânula intracerebroventricular (i.c.v.), ocorreu o condicionamento com três associações contextochoque numa sessão de 20 min. O teste e o re-teste consistiram na re-exposição ao contexto do condicionamento por 5 min. O intervalo entre sessões foi de 24h. A administração de 7-NI ou do veículo ocorreu imediatamente após o treino (Experimento 1) ou após o re-teste (Experimento 2). A atividade enzimática da NOS dependente e independente de Ca2+ e da expressão protéica da nNOS foram realizadas no tecido hipocampal. No Experimento 1, a ocorrência de congelamento no teste do 7-NI foi menor do que no treino (p<0.01) e no teste do COND e VEIC (p < 0.001). A atividade da NOS dependente de Ca++ no 7-NI foi menor do que no COND e VEIC (p<0,01), mas não diferiu do CONT e do NÄIVE. A expressão protéica de nNOS não diferiu entre os grupos (p<0,05). No Experimento 2, houve diminuição dos comportamentos defensivos, incluindo o congelamento, no re-teste do 7-NI comparado com VEIC e COND (p<0.05), mas os grupos não diferiram quanto à atividade de NOS dependente de Ca2+ ou à expressão protéica da nNOS. Conclui-se que o 7-NI interferiu na consolidação e a reconsolidação da memória, indicando a ativação da via de sinalização do óxido nítrico no hipocampo em processos da memória de medo condicionado ao contexto em pombos
Abstract: Nitric oxide (NO) is an unconventional neurotransmitter which plays an important role in neurobiological processes of behavior and memory. Its synthesis is mediated by three isoforms of nitric oxide synthase (NOS): the neuronal (nNOS), the endothelial (eNOS) and the inducible (iNOS). This study investigated the effects of the administration of 7- nitroindazole (7-NI), a preferential nNOS inhibitor, in the consolidation and reconsolidation of aversive classical conditioning memory. Adult male pigeons were assigned to 5 groups: 7-nitroindazole, 7-NI (100nmol/0.5?/l; DMSO (20%), NaOH (50 mM) and Tween-80 (16%) diluted in PBS; i.c.v.) Vehicle group; VEH (0.5 ? / L; DMSO (20%), NaOH (50 mM) and Tween-80 (16%) diluted in PBS; i.c.v.), conditioning/non-treated group (COND), context/non-treated group (CONT) and non-conditioning/non-treated group (NÄIVE). Seven days after implantation of intracerebral ventricular (i.c.v.) microcannula the conditioning occurred with three context-shock associations in a session of 20 min. During the testing and retesting sessions pigeons were reexposed to the conditioning context for 5 min. The between sessions interval was 24h. Administration of 7-NI or vehicle occurred immediately after training (Experiment 1) or after testing (Experiment 2). The enzymatic activity of Ca2+ dependent and independent NOS and protein expression of nNOS in the hippocampus tissue were carried out following the behavioral test or retest. In Experiment 1, the occurrence of freezing in the testing session of 7-NI group was lower than in the training (p <0.01) and the testing sessions of COND and VEH groups (p <0.001). The activity of Ca2+ dependent NOS in the 7-NI group was lower than in COND and VEH groups (p <0.01) but did not differ from CONT and NÄIVE groups. The nNOS protein expression in the hippocampus did not differ among the different groups (p<0.05). In Experiment 2, there was a decrease of defensive behaviors, which include freezing, in the retest of the 7-NI compared with VEH and COND groups (p <0.05), but the groups did not differ in the activity of Ca2+ dependent NOS or the protein expression of nNOS. We conclude that 7-NI interfered on the consolidation and reconsolidation of memory, indicating activation of the nitric oxide signaling pathway in the hippocampus and in memory processes of conditioned fear context in pigeons
Mestrado
Fisiologia
Mestra em Biologia Funcional e Molecular

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Tajouri, Lotfi, e n/a. "Gene Expression Analysis and Genetic Studies in Multiple Sclerosis". Griffith University. School of Health Science, 2005. http://www4.gu.edu.au:8080/adt-root/public/adt-QGU20060111.123933.

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Multiple Sclerosis (MS) is a neurodegenerative disease of the central nervous system (CNS). As part of this disorder the myelin sheath undergoes degeneration, leading to alterations in the conductivity of axons, and impaired function. The onset of the disease occurs in young adults and clinical pathology is characterised by varying severity. These include i) Relapsing Remitting MS (RR-MS), ii) Secondary Progressive MS (SP-MS) and iii) Primary Progressive MS (PP-MS). MS is more prevalent in women and accounts for more than two thirds of all MS sufferers. MS is considered to be a multifactorial disorder with both genetic and environmental components. The prevalence of MS is dependent on geographical localisation, with lower sunlight exposure linked to higher prevalence. Also, studies show an increased risk in close relatives, or in identical twins, indicating a significant genetic component to the disorder. There are a number of genes that may plausibly be involved in MS pathophysiology. These include myelin-related genes, such as the myelin basic protein (MBP), immune-related genes, such FC receptor and osteopontin, and heat shock proteins such as xb crystallin. These candidate genes have been implicated in a variety of ways but usually through immunological and/or genetic studies. One of the most consistent findings in recent years has been the association of disease with alterations in the specific major histocompatibility complex (MHC) localised to chromosome 6p21.3, and includes MHC I, II, III. Genome wide screens have permitted the identification of loci in the genome, which are associated with MS susceptibility. The number of genes involved in MS is unknown and several case-control association studies have been undertaken to reveal the involvement of potential candidate genes. In general terms, current research is aimed at determining allelic variation of candidate genes. Such genes have been implicated in MS because they reside within susceptible regions of the chromosome associated with MS or they have a plausible potential pathophysiological role in MS. Candidate loci investigated in this study, for association with MS susceptibility, include members of the nitric oxide synthase family of metabolic proteins (inducible NOS, iNOS/NOS2A and neuronal NOS, nNOS), methylenetetrahydrofolate reductase (MTHFR), catechol-O-methyl transferase (COMT), and vitamin D receptor (VDR). The MS population used in all studies consisted of over 100 MS cases and gender, age and ethnicity matched controls. In our study of inducible and neuronal NOS genes, PCR based assays were developed to amplify a region of both promoters that contained known microsatellite variation. Supporting phyisological data suggests that the neuroinflammatory aspects of MS are associated with aberrant NO production, which may be due to aberrant regulation of NOS activity. Specific amplified products were identified by fluorescent capillary electrophoresis and allele frequencies were statistically compared using chi-squared analysis. In the nNOS and iNOS study, no association was identified with allele frequency variation and MS susceptibility (nNOS: ?2=5.63, P=0.962; iNOS: ?2=3.4; P=0.082). Similarly, no differences in allele frequencies were observed for gender or clinical course for both markers (Pvalue greater than 0.05). In short, results from this study indicate that the NOS promoter variations studied do not play a significant role in determining susceptibility to MS in the tested population. The COMT and MTHFR genes are localised at 22q12-13 and 1p36.3 respectively, regions of the genome that have been found to be positively associated with MS susceptibility. In our research, we set out to examine the G158A change in the 4th exon of the COMT gene. This functional mutation leads to an amino acid change (valine to methionine) that is directly associated with changes in the activity of COMT. The MTHFR enzyme plays a role in folate metabolism, and can be implicated in the turnover of homocysteine. Previous investigations have shown that high levels of homocysteine are encountered in MS patients, where it is also linked to demyelination in the CNS. In our study the aim was to examine the C677T variation (alanine to valine amino acid change) in the exon 4 coding region of the MTHFR gene and the G158A variation in the COMT gene. Restriction fragment length polymorphism (RFLP) analysis and gel electrophoresis was used to identify specific alleles for both COMT and MTHFR. However, as with the NOS study, no specific association was identified between MS susceptibility and variation for either of the tested COMT or MTHFR (Pvalue greater than 0.05) variants. In a final genomic investigation of the MS population, three variations in the VDR gene were analysed for association with MS susceptibility and pathology. Using RFLP analysis, three VDR variants were investigated with genotypes detected using the Taq I, Apa I and Fok I restriction enzymes. In contrast to previous genotypic analyses, this study did show a positive association, specifically between the functional variation in exon 9 of the VDR gene and MS (Taq I, 2= 7.22, P= 0.0072). Interestingly, the Apa I variant of VDR was also found to be associated with MS ( 2=4.2, P=0.04). The Taq I and Apa I variants were also found to be in very strong and significant linkage disequilibrium (D'=0.96, Pvalue less than 0.0001) and their associations were more prominent with the progressive forms of MS (SP-MS and PP-MS). In addition to genotypic analysis of a clinical population, additional research was undertaken to identify novel targets for MS susceptibility studies. Global gene expression analysis was undertaken using comparative subtractive fluorescent microarray technology to examine differences in gene activity (expression) in age and sex matched MS plaque tissue and anatomically matched normal white matter (NWM). MS plaques were obtained post mortem from MS sufferers with no drug history in the last two months before death and matched anatomically to healthy white matter from donors with no previous neurological disorders. Target arrays consisted of 5000 cDNAs and analysis was conducted using the Affymetrix 428 scanner. In this way, 139 genes were shown to be differentially regulated in MS plaque tissue compared to NWM. Of these, 69 genes showed a common pattern of expression in the chronic active and acute plaque tissues investigated (Pvalue less than 0.0001, a=0.73); while 70 transcripts were uniquely differentially expressed ( 1.5-fold) in either acute or chronic active lesions. To validate the gene expression profile results, quantitative real time reverse transcriptase (RT) PCR (Q-PCR) analysis was performed. 12 genes were selected because they were shown to be differentially expressed by array analysis in this study, or because of their involvement in MS pathology. These included transferrin (TF), superoxide dismutase 1 (SOD1), glutathione peroxidase 1 (GPX1), glutathione S-transferase pi (GSTP1), crystallin, alpha-B (CRYAB), phosphomannomutase 1 (PMM1), tubulin beta-5 (TBB5), inositol 1,4,5-trisphosphate 3-kinase B (ITPKB), calpain 1 (CAPNS1), osteopontin (SPP1 or OPN), as well as the signal transducer and activator of transcription 1 (STAT1) and protein inhibitor of activated STAT1 (PIAS1). Both absolute (copy number) and comparative differences in the relative levels of expression in MS lesions and NWM were determined for each gene. The results from this study revealed a significant correlation of real time PCR results with the microarray data, while a significant correlation was also found between comparative and absolute determinations of fold. As with the results of array analysis, a significant difference in gene expression patterning was identified between chronic active and acute plaque pathologies. For example, a up to 50-fold increase in SPP1 and ITPKB levels in acute plaques contrasted with the 5-fold or less increase in chronic active plaques (P less than 0.0.1, unpaired t-Test). Of particular note, gamma-amino butyric acid receptor ?2 (GABG2), integrin ?5 (ITGB5), complement component 4B (C4B), parathyroid hormone receptor 1 (PTHR1) were found up-regulated in MS and glial derived neurotropic factor ?2 (GDNFA2), insulin receptor (INSR), thyroid hormone receptor ZAKI4 (ZAKI4) were found down-regulated in MS. Data also revealed a decreased expression of the immune related genes STAT1 and PIAS1 in acute plaques. In conclusion, this research used both genomic analysis and technologies in gene expression to investigate both known and novel markers of MS pathology and susceptibility. The study developed tools that may be used for further investigation of clinical pathology in MS and have provided interesting initial expression data to further investigate the genes that play a role in MS development and progression.

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van, Erp Christel. "Modifying function and fibrosis of cardiac and skeletal muscle from mdx mice". University of Southern Queensland, Faculty of Sciences, 2005. http://eprints.usq.edu.au/archive/00001521/.

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Duchenne Muscular Dystrophy (DMD) is a fatal condition occurring in approximately 1 in 3500 male births and is due to the lack of a protein called dystrophin. Initially DMD was considered a skeletal myopathy, but the pathology and consequences of cardiomyopathy are being increasingly recognised. Fibrosis, resulting from continual cycles of degeneration of the muscle tissues followed by inadequate regeneration of the muscles, is progressive in both cardiac and skeletal dystrophic muscle. In the heart fibrosis interferes with contractility and rhythm whereas it affects contractile function and causes contractures in skeletal muscles. This study utilised the mdx mouse which exhibits a pathological loss of muscle fibres and fibrosis characteristic of DMD, to examine a range of mechanisms that can influence muscle function and fibrosis. Ageing and workload both appear to contribute to the development of dystrophic features in cardiac and skeletal muscle of the mdx mouse. Therefore the effect of eccentric exercise on cardiac and skeletal muscle was examined in older mdx mice. Mice ran in 30 minute sessions for five months, 5 days per week. Downhill treadmill running did not exacerbate the contractile function or fibrosis of the mdx heart or the EDL, SOL or diaphragm muscles suggesting that cytokines influence function and fibrosis to a greater extent than workload alone. The role of the cytokine TGF-beta was examined by treating mdx mice with the TGF-beta antagonist pirfenidone at 0.4, 0.8 or 1.2 per cent in drinking water for six months. Pirfenidone improved cardiac contractility (P<0.01) and coronary flow (P<0.05), to levels comparable to control mice, despite no reduction in cardiac fibrosis. Pirfenidone did not reduce fibrosis or improve function in skeletal muscle. A deficiency of neuronal nitric oxide synthase (nNOS) in DMD and mdx mice causes a lowered production of nitric oxide indicating that the substrate of nNOS, l-arginine, may be beneficial to cardiac and skeletal muscle function in mdx mice. Oral l-arginine (5 mg/g bw) improved cardiac contractility, coronary flow and reduced cardiac fibrosis (P<0.05) without improving skeletal muscle function or fibrosis. In contrast, 10 mg/g bw l-arginine improved cardiac function and coronary flow (P<0.01), despite also elevating cardiac collagen. This increment in collagen was prevented by co-administration of prednisone. The experiments described in this dissertation reveal for the first time that pharmacological treatments in mdx mice can improve cardiac structure and function. Further elucidation of the optimum time and doses of such treatments may result in future pharmacological treatments to improve cardiac function and fibrosis in DMD.

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Barreiro, Portela Esther. "Study of reactive oxygen species (ROS) and nitric oxide (NO) as molecular mediators of the sepsis-induced diaphragmatic contractile dysfunction : protective effect of heme oxygenases". Doctoral thesis, Universitat Pompeu Fabra, 2002. http://hdl.handle.net/10803/7066.

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Protein nitration is considered as a marker of reactive nitrogen species formation. Heme oxygenases (HOs) are important for the defence against oxidative stress. We evaluated the involvement of the neuronal (nNOS), the endothelial (eNOS), and the inducible (iNOS) in nitrotyrosine formation and localitzation, and both the expression and funcional significance (HO inhibition and contractility studies) of HOs in sepsis-induced muscle contractile dysfunction. Sepsis was elicited by injecting rats and transgenic mice deficient in either nNOS, eNOS, or iNOS isoforms with E.Coli lipolysaccharide (LPS). Nitrotyrosine formation and HO expressions were assessed by immunoblotting. Oxidative stress was assessed measuring protein oxidation, lipid peroxidation, and muscle glutathione. We conclude that protein tyrosine nitration occurs in normal muscles, and sepsis-mediated increase in nitrotyrosine formation is limited to the mitochondria and membrane muscle fractions. The iNOS isoform is mostly involved in nitrotyrosine formation. HOs protect normal and septic muscles from the deleterious effects of oxidants.
En un model de sepsi de disfunció diafragmàtica, s´ha avaluat el paper de les sintetases de l'òxid nítric (NOS) en la formació i localitzacio de 3-nitrotirosina, i l´expressió i significat biològic de les hemo oxigenases (HOs) (inhibidor de les HOs i estudis de contractilitat) davant l' estrès oxidatiu. La sepsi s'induí mitjançant injecció de 20 mg/kg del lipolisacàrid (LPS) d´Escherichia Coli a rates, i a ratolins deficients en les NOS induïble (iNOS), neuronal (nNOS) i endotelial (eNOS). Les proteïnes nitrificades i les HOs es van detectar amb anticossos específics. L' estrès oxidatiu s' avaluà mitjançant l' oxidació proteica, la peroxidació lipídica i el glutation muscular. Concloem que hi han proteïnes nitrificades en el múscul normal i aquestes s'incrementen durant la sepsi en les fraccions mitocondrial i membranar. L'isoforma iNOS és majorment responsable de la formació de nitrotirosina. Les HOs protegirien el múscul normal i sèptic dels efectes deleteris dels oxidants.

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Chachlaki, Konstantina. "Molecular characterization of NO-synthesizing neurons and assessment of their function in the maturation of the hypothalamic - pituitary - gonadal axis". Thesis, Lille 2, 2016. http://www.theses.fr/2016LIL2S047.

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L’apparition de la puberté et la régulation de la fertilité chez les mammifères sont contrôlées par un réseau neuronal complexe, situé principalement dans l'hypothalamus, et qui converge vers les neurones synthétisant l'hormone de libération des gonadotrophines (GnRH). Ces neurones régulent la sécrétion des gonadotrophines, la croissance et le fonctionnement des gonades. Le développement correct du système à GnRH, incluant des changements rapides dans l'expression et la signalisation de l’hormone GnRH au sein de cette population clairsemée de quelques centaines de neurones, est essentiel pour la maturation sexuelle et le fonctionnement normal de l'axe hypothalamo-hypophyso-gonadique. Lors du développement embryonnaire, ces neurones migrent de la placode olfactive vers leur emplacement définitif, l’hypothalamus, pour y recevoir les connexions afférentes qui permettront une libération pulsatile de la GnRH et la libération subséquente des gonadotrophines (l'hormone de stimulation des follicules (FSH) et l'hormone lutéinisante (LH)). Dès les années 90, l'oxyde nitrique (NO) a été identifié comme molécule clé dans la décharge pré-ovulatoire de GnRH/LH. En effet, de nombreux travaux ont suggéré que des interactions entre les neurones exprimant la forme neuronale de l’enzyme de synthèse du NO (la nNOS) et le système GnRH étaient impliquées dans le contrôle central de la fonction de reproduction à l'âge adulte. De plus, si le NO est reconnu depuis longtemps comme un acteur majeur du contrôle central de l’ovulation à l’âge adulte, la possibilité qu’il soit aussi impliqué dans la maturation sexuelle en régulant l’activité des neurones à GnRH à des stades précoces précédant la puberté n’a pas été explorée auparavant. Cependant, même si nous avons progressé dans la connaissance des interactions entre les neurones à nNOS et des différents acteurs importants de l’axe gonadotrope, l’identité moléculaire de ces neurones reste mal connue. Au cours de cette étude, nous avons recherché 1) l'identité moléculaire des neurones á nNOS dans l'hypothalamus au cours de développement 2) si le NO régule la migration et l’intégration des neurones à GnRH dans l’hypothalamus et 3) si le NO régule la maturation sexuelle. Pendant ma thèse nous avons répertorié pour la première fois les différents neurotransmetteurs et les principaux récepteurs dans les neurones à nNOS au cours du développement post-natal. De plus, les résultats de ma thèse montrent pour la première fois une implication de la signalisation du NO dans la migration des neurones à GnRH vers l'hypothalamus et font échos à l'identification d'une série de mutations de la NOS1 chez des patients atteints du syndrome de Kallmann, une maladie génétique congénitale rare qui associe une carence en GnRH, due à un défaut de migration neuronale, et une anosmie. Enfin, mes travaux montrent que le NO est un nouveau protagoniste dans la maturation post-natale du système à GnRH, la survenue de la puberté et l’acquisition de la capacité à se reproduire. Plus généralement, les résultats de ce travail de thèse permettent d’identifier de nouveaux mécanismes potentiellement responsables de troubles développementaux dans la mise en place des circuits neuronaux contrôlant l’axe gonadotrope chez les mammifères en général et l’homme en particulier. Nous espérons que ces résultats élargiront notre compréhension de la régulation de l'axe reproducteur, offrant ainsi des possibilités nouvelles de stratégies thérapeutiques contre les troubles de la fertilité
The onset of puberty and the regulation of fertility in mammals are governed by a complex neural network, primarily in the hypothalamus, that converges onto gonadotropin-releasing hormone (GnRH)-producing neurons, the master regulators of gonadotropin secretion and postnatal gonadal growth and function. The proper development of the GnRH system, including timely changes in GnRH expression and signaling by this sparse population of a few hundred neurons, is essential for sexual maturation and the normal functioning of the hypothalamic-pituitary-gonadal axis. As the brain develops during embryogenesis, these neurons should move from the olfactory placode into the correct brain location in adequate numbers, and then establish the afferent connections that will allow the pulsatile release of GnRH peptide, and the subsequent release of the gonadotropins (follicle stimulating hormone, i.e FSH and luteinizing hormone, ie. LH). As early as in the 90’s NO was presented as a key molecule in the preovulatory GnRH/LH surge, and results from different groups, have suggested the interaction of NOS-containing neurons with the GnRH system, and their involvement in the regulation of reproductive capacity. Even though nitric oxide has now been long recognized as a key player in the central hormonal regulation of ovulation during adulthood, no one has considered the possibility that it could act in an earlier stage as the master regulator of GnRH neurons before puberty, hence participating in the actual maturation of the neuroendocrine axis. The relationship of nNOS-expressing neurons with other important molecules of the hypothalamic axis has been well studied, whilst the molecular identity of this neuronal NOS-expressing population is poorly documented. . To this end, we address the hitherto unaddressed questions concerning 1) the molecular identity of nNOS-expressing neurons in the developing hypothalamus, 2) the putative involvement of the NO molecule in the migration of GnRH neurons and the proper establishment of their afferent connections in the hypothalamic region and 3) the plausible determinant role of NO signaling in the maturation of the reproductive system. During this study we identified for the first time the cohort of the principal neurotransmitters and important receptors expressed by these cells in the hypothalamic region during development. Additionally, our results reveal for the first time an involvement of NO signaling in the migration of GnRH neurons in the hypothalamus and are in line with the identification of a series of NOS1 mutations in Kallmann syndrome (KS), a rare congenital genetic condition presenting a unique combination of GnRH deficiency, arising from a faulty migration of the neuronal population, and anosmia. Lastly, our study identifies NO as a novel protagonist during postnatal development, in the regulation of the onset of puberty and the acquisition of reproductive competence. Overall, the results of my Phd thesis identify putative new targets causing alterations of developmental programming under pathophysiological gestational environment in mammals in general, and in humans in particular. Here we thus provide new insights into the mechanisms by which the alteration of GnRH neuronal function leads to hypogonadotropic hypogonadism and infertility. We are hopeful that our results will expand our understanding of how the neuroendocrine axis is regulated and will possibly provide opportunities for therapeutic strategies against debilitating conditions

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Hope, Bruce Thomas. "Neuronal NADPH-diaphorase is a nitric oxide synthase". Thesis, University of British Columbia, 1991. http://hdl.handle.net/2429/30932.

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The enzyme responsible for the neuronal NADPH-diaphorase histochemical reaction was identified in rat brain by employing a variety of histochemical and biochemical techniques. The histochemical reaction catalyzes the NADPH-dependent reduction of tetrazolium dyes to colored insoluble formazans. Although the histochemical reaction has been widely employed in neuroanatomical and neuropathological studies, the identity of the enzyme responsible for the reaction has been unknown. Previous attempts to determine the identity of the enzyme have failed due to the lack of a specific biochemical assay. Some biochemical characteristics of the histochemical reaction in rat striatum were determined in order to develop a specific biochemical assay for neuronal NADPH-diaphorase. The histochemical reaction used several different analogs of NADPH but did not use β-NADH. All tetrazolium analogs with redox potentials above that for β-NADPH were reduced, although the reduction of some tetrazoliums was oxygen-sensitive. The reaction appeared not to require metal ions, flavins, peroxides, or superoxide anions as all methods to remove these factors did not influence staining. The DT-diaphorase activator, menadione, and the inhibitor, dicumarol, did not affect neuronal NADPH-diaphorase. Electron microscopic results suggested neuronal NADPH-diaphorase was membrane-bound, particularly with the endoplasmic reticulum. These results correlate with no known enzymes, including those previously proposed as neuronal NADPH-diaphorase. Employing an antiserum which specifically detected neuronal NADPH-diaphorase, we found the enzyme to be nitric oxide (NO) synthase. NO synthase produces the membrane-permeable second messenger NO. Immunoreactive NADPH-diaphorase activity was copurified to apparent homogeneity with NO synthase activity. The antiserum specifically immunoprecipitated both NO synthase and NADPH-diaphorase activities and specifically labelled a 150 kD band on Western blots, similar to NO synthase from previous reports. The NADPH-diaphorase substrate, NBT, competed with the NO synthase substrate, arginine, for electrons from NADPH. As expected, immunoreactivity for citrulline was found only in NADPH-diaphorase neurons. Citrulline is produced along with NO from the substrate, arginine. NADPH-diaphorase activity was weak to moderate in the cerebellum even though this region contains high levels of NO synthase. The cerebellum also had no citrulline- or NADPH-diaphorase immunoreactivity. We suggest the NO synthase in the cerebellum is a different form from that in the rest of the brain. We conclude that the neuronal NADPH-diaphorase histochemical reaction is due to a form of NO synthase and therefore NADPH-diaphorase is a histochemical marker of NO synthase in the brain. The product NO is the endogenous activator of soluble gu any late cyclase in the brain. This allows a discussion of the anatomical and functional relationships between NO synthase/NADPH-diaphorase and guanylate cyclase to determine the possible functions of these enzymes and their second messenger products in the brain.
Medicine, Faculty of
Graduate

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Padayachee, Eden Rebecca. "Neuronal nitric oxide synthase : a biomarker for Alzheimers disease : interaction of neuronal nitric oxide synthase with beta-amyloid peptides in the brain". Thesis, Rhodes University, 2011. http://hdl.handle.net/10962/d1007677.

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High levels of the amino acid arginine and low levels of the product citrulline in the cerebrospinal fluid of Alzheimer's patients could mean that there is a decrease in the enzymes that metabolize this amino acid. One such enzyme is neuronal nitric oxide synthase (nNOS). In this study, neuronal nitric oxide synthase (nNOS), sourced from bovine brain was extracted and concentrated using two methods of precipitation: poly (ethylene glycol) 20 000 (PEG) and ammonium sulphate [(NH₄)₂S0₄). These two techniques gave no increase in yield nor fold purification and hence were abandoned in favour of ion exchange chromatography by DEAE-Sepharose. The enzyme was then successfully purified by anion-exchange and after dialysis produced a 38% yield and three fold purification and yielded the highest specific activity of 2.27 U/mg. Neuronal nitric oxide synthase (nNOS) was a heterodimeric protein with a total molecular mass of ± 225 kDa (95 and 130 kDa monomers). The temperature and pH optima of the enzyme were 40⁰C and 6.5, respectively. The kinetic parameters (KM and Vmax) of nNOS were 70 μM and 0.332 μmol.min⁻¹, respectively. Moreover neuronal nitric oxide synthase (nNOS) was relatively stable at 40⁰C (t½ = 3 h). It was also confirmed that β-amyloid peptides inhibited nNOS when bound to the enzyme and that nNOS behaved as a catalyst in fibril formation through association-dissociation between enzyme and β-amyloid peptide. It was further shown that Aβ₁₇₋₂₈ inhibited nNOS the most with a Ki of 1.92 μM and also had the highest Stern-Volmer value (Ksv) of 0.11 μM⁻¹ indicating tight binding affinity to nNOS and easier accessibility to fluor molecules during binding. Congo red, turbidity, thioflavin-T assays and transmission electron microscopy were successfully used to detect and visualize the presence of fibrils by studying the process of fibrillogenesis. Computerized molecular modeling successfully studied protein dynamics and conformational changes of nNOS. These results correlated with resonance energy transfer (FRET) results which revealed the distance of tryptophan residues from the arginine bound at enzyme active site. Both the aforementioned techniques revealed that in the natural state of the enzyme with arginine bound at the active site, the tryptophan residues (TRP₆₂₅ and TRP₇₂₁) were positioned at the surface of the enzyme 28 Å away from the active site. When the amyloid peptide (Aβ₁₇₋₂₈) was bound to the active site, these same two amino acids moved 14 Å closer to the active site. A five residue hydrophobic fragment Aβ₁₇₋₂₁ [Leu₁₇ - Val₁₈ - Phe₁₉ - Phe₂₀ - Ala₁] within Aβ₁₇₋₂₈ was shown by computer modeling to be critical to the binding of the peptide to the active site of nNOS.

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McNamara, Tanner. "ENOS and nNOS contribution to reflex cutaneous vasodilation during dynamic exercise in humans". Thesis, Kansas State University, 2012. http://hdl.handle.net/2097/13788.

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Master of Science
Department of Kinesiology
B.J. Wong
Recent data suggests nNOS mediates the NO-component of reflex cutaneous vasodilation with passive heat stress. Our hypothesis was nNOS, but not eNOS, inhibition would attenuate reflex cutaneous vasodilation during dynamic exercise. Protocol 1: subjects performed a VO[subscript]2 peak test on a supine cycle ergometer. Protocol 2: with experimental arm at heart level subjects cycled in supine posture at 60% VO[subscript]2 peak to raise core temperature (Tc) 0.8-1.0°C (35-45 min). In protocol 2 subjects were equipped with 4 microdialysis fibers on the forearm and each randomly assigned as: 1) lactated Ringer’s (control); 2) 5mM NPLA (nNOS inhibition); 3) 10mM L-NIO (eNOS inhibition); and 4) 20mM L-NAME (non- selective NOS inhibition). At the end of protocol 2 all sites were locally heated to 43°C and infused with SNP to elicit maximal dilation. Mean arterial pressure (MAP), skin blood flow via laser- Doppler flowmetry (LDF), and Tc via ingestible telemetric pill were measured; cutaneous vascular conductance (CVC) was calculated as LDF/MAP and normalized to maximum. In protocol 2 there was no significant difference between control (62±5 %CVCmax) and NPLA (61±6 %CVCmax). L-NIO (38±4 %CVCmax) and L-NAME (41±7 %CVCmax) significantly attenuated CVC compared to control and NPLA (p<0.001 all conditions). There was no difference between L-NIO and L- NAME. We conclude eNOS, not nNOS, contributes to reflex cutaneous vasodilation during dynamic exercise.

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18

Dai, Yue. "Study of Electron Transfer through the Reductase Domain of Neuronal Nitric Oxide Synthase and Development of Bacterial Nitric Oxide Synthase Inhibitors". Cleveland State University / OhioLINK, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=csu1472477836.

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19

Sobolewska-Stawiarz, Anna. "Probing the dynamics and conformational landscape of neuronal nitric oxide synthase". Thesis, University of Manchester, 2014. https://www.research.manchester.ac.uk/portal/en/theses/probing-the-dynamics-and-conformational-landscape-of-neuronal-nitric-oxide-synthase(82903814-5474-42e3-9339-d9a7a98ead6d).html.

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Rat neuronal nitric oxide synthase (nNOS) is a flavo-hemoprotein that catalyses the NADPH and O2-dependent conversion of L-arginine (L-arg) to L-citrulline and nitric oxide (NO) via the intermediate N-hydroxyarginine. nNOS is a homodimer, where the subunits are modular and are comprised of an N-terminal oxygenase domain (nNOSoxy) that binds iron protoporphyrin IX (heme), (6R)-5,6,7,8-tetrahydro-biopterin (H4B) and L-arg, and a C-terminal flavoprotein or reductase domain (nNOSred) that binds NADPH, FAD and FMN. Regulation of NO biosynthesis by nNOS is primarily through control of interdomain electron transfer processes in NOS catalysis. The interdomain electrons transferred from the FMN to the heme domain are essential in the delivery of electrons required for O2 activation (which occurs in the heme domain) and the subsequent NO synthesis by NOS. Both spectroscopic and kinetic approaches have been used in studying the nature and control of interdomain electron transfer, reaction mechanism and structural changes during catalysis in WT and R1400E nNOS in both full length (FL) and nNOSred. Cytochrome c reduction activity of nNOS was used to determine kinetic parameters for NADPH for FL and nNOSred, WT and R1400E nNOS in the presence and absence of calmodulin (CaM). FL nNOS, where both domains (nNOSred and nNOSoxy) were present, was proven to be more stable and more catalytically efficient than nNOSred by itself. Additionally it was observed that R1400E is still promoting electron transfer despite being thought to lower the affinity of the enzyme to the substrate (NADPH); R1400E also showed lower catalytic efficiency and lower dependence on CaM/Ca2+ compared to the WT. The structure of the functional output state has not yet been determined. In the absence of crystallographic structural data for the NOS holoenzyme, it was important to experimentally determine conformational changes and distances between domains in nNOS. A pulsed EPR spectroscopy (PELDOR) approach has been utilised to gain important and unique information about the conformational energy landscape changes in nNOS. In the presence of CaM, PELDOR results for FL WT nNOS shows a complex energy landscape with multiple conformational states, while in the absence of CaM the interflavin distance distribution matches that exhibited by nNOSred CaM- in the presence of NADP+, suggesting that CaM binding affects some major large-scale conformational changes which are involved in internal electron transfer control in nNOS. A high-pressure stopped-flow technique was also used to perturb an equilibrium distribution of conformational states, to observe the effect of the pressure on the internal electron transfer and to study the kinetics of NADPH oxidation, flavin reduction by NADPH and NO formation. It was shown that high pressure is forcing major changes in the conformational energy landscape of the protein, affecting internal electron transfer. NO formation studies under pressure show that the R1400E mutation in FL nNOS may be affecting protein/NADPH affinity and flavin reduction, but it has no effect on the heme reduction step.

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20

Traynham, Christopher J. "Effects of Neuronal Nitric Oxide Synthase Signaling On Myocyte Contractile Function". The Ohio State University, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=osu1305058816.

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21

Pierson, Shawn M. "Aspects of the transcriptional and translational regulation of nitric oxide synthase 1". Connect to this title online, 2005. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1111595828.

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Thesis (Ph. D.)--Ohio State University, 2005.
Title from first page of PDF file. Document formatted into pages; contains x, 156 p.; also includes graphics (some col.) Includes bibliographical references (p. 146-156). Available online via OhioLINK's ETD Center

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22

Garnaud, Pierre-Emmanuel F. "Calmodulin activation of the reductase domain of mammalian neuronal nitric oxide synthase". Thesis, University of Edinburgh, 2006. http://hdl.handle.net/1842/12034.

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In order to investigate the CaM activation mechanism of nNOS, the effect of the conformational changes of nNOSrd and the binding of NADP(H) on the redox potential of the flavins, cofactors were assessed for the isolated FAD and FMN sub-domains by OTTLE potentiometry. The results showed that the presence of the FAD/FMN sub-domain interface does not alter the thermodynamic properties of the redox couples involved in the catalysis. This is consistent with the fact that CaM binding has a small effect on the flavins reduction potentials. Only the FMN/FMNH^· redox couple was found to be stabilised by the presence of the FAD sub-domain (increase of 80 mV). The same redox couple was also kinetically stabilised toward oxidation. The isolated FAD sub-domain was found to have similar redox potentials to the isolated nNOSrd. In the presence of NADP⁺, both the FAD sub-domain and the nNOSrd formed the charge-transfer complex with a long-wavelength absorption band centred at 780nm. Formation of this complex was found to stabilise the FADH^·/FADH^-redox couple by approx 30 mV. It is possible that in the CaM-free enzyme, the conformation of the bound NADP⁺ may control both electron transfers between the FAD and FMN and from FMN to heme by modulating the potential of the FAD hydroquinone. The accessibility of the FMN cofactor to the heme was assessed. The results showed that if the FMN, in the isolated FMN domain, is assumed to be fully accessible, then it is 100% accessible in the CaM-bound enzyme, 45% accessible in the uncompleted enzyme and only 3% accessible in the NADPH-bound nNOSrd in the absence of CaM. This suggests that the binding of CaM is responsible for a structural reorganisation of nNOS rd that “unlocks” the conformation of the enzyme and enables the FMN sub-domain motion in order to shuttle an electron from FAD cofactor to the heme. The specificity of NADP(H) in repressing the electron transfer from the reductase domain to cytochrome c was studied by using NADP(H) analogues. Results showed that the specificity of NADPH in inducing nNOS rd conformational change relies upon the interaction of both the tightly-bound ADP substituents and the labile nicotinamide substituents and that the tightly bound ADP substituents is essential to position the nicotinamide moieties for full electron transfer repression. It appears that the “locked” conformation of the enzyme, believed to inhibit the electron transfer to the heme, is specific for the NADP(H).

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23

Hartt, Gregory Thomas. "Regulation of the human neuronal nitric oxide synthase gene via alternate promoters". Columbus, OH : Ohio State University, 2003. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1056034844.

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Thesis (Ph. D.)--Ohio State University, 2003.
Title from first page of PDF file. Document formatted into pages; contains xii, 152 p. : ill., (some col.). Includes abstract and vita. Advisor: Anthony Young, Molecular, Cellular, and Developmental Biology Program. Includes bibliographical references (p. 137-150).

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24

Ngqwala, Nosiphiwe Patience. "Interaction of metallic nanoparticles with biomedical enzyme target: neuronal nitric oxide synthase". Thesis, Rhodes University, 2013. http://hdl.handle.net/10962/d1001536.

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Alzheimer's disease (AD) is the most common type of dementia characterized by intracellular appearance of neurofibrillary tangles, synaptic and neuronal loss; and extracellular accumulation of amyloid-β (Aβ) peptide in senile plaques. The initial causes leading to AD are unknown, and the available treatments are only effective at slowing the degeneration process. The accumulation of arginine in the brain of Alzheimer patients indicates a possible disruption of enzymes responsible for its metabolism. One such enzyme is neuronal nitric oxide synthase (nNOS) and controlling its activity by interacting with nanoparticles may lead to a delay in the onset of the disease. Neuronal nitric oxide synthase was purified using DEAE-Sephacel ion exchange resulting in 10 % yield, 0.43 fold recovery and specific activity 0.09 U/mg. The enzyme was found to be a dimer with a molecular mass of 150 kDa. Characterisation of the nNOS showed an optimum temperature and pH of 50°C and 7.5 respectively, and it was relatively stable at the optimum conditions (t½ = 100 min). The purity was analysed by SDS-PAGE followed by Western blot. Purified nNOS was challenged with 3-7 nm silver and 4-15 nm gold nanoparticles of between synthesized chemical using AgNO3 and either sodium borohydride or sodium citrate. Results showed that gold nanoparticles are more effective at low concentration (5 μM) than silver nanoparticles due to their size difference. Incubation of different concentration of nanoparticles (5, 15, 25, 50 μM) with the purified nNOS showed an initial decrease of 5% in enzyme activity which over time was restored to 80%. This suggests that different nanoparticles are produced in different sizes and interaction over a given time may result in enzyme association–dissociation mechanism. Inhibition studies showed a strong binding of both nanoparticles with Ki values of 1.4 μM and 0.2 μM for silver and gold, respectively. Both nanoparticles inhibited the activity of nNOS extensively as they bound strongly to the inhibition site on the enzyme and were more in contact with fluorophores nanoparticles. This was confirmed by fluorimetry with binding constants of 0.0084 μM and 0.01092 μM for silver and gold, respectively. Results of this study suggest that silver and gold nanoparticles competitively inhibit nNOS.

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25

Sangüesa, Ferrer Juan F. "Modulation fonctionnelle et distribution du canal calcique Cav3. 2 : rôle de la nNOS". Montpellier 1, 2008. http://www.theses.fr/2008MON1T015.

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Le canal calcique Cav3. 2 joue un rôle capital dans de nombreux processus physiologiques, notamment dans la transmission de la douleur aigüe et chronique. Cependant, les mécanismes régulant la distribution et les propriétés de ces canaux restent encore mal connus. Au cours de ma thèse, j'ai participé au développement d'un nouveau modèle pour étudier ces mécanismes : la souris knock-in Cav3. 2-GFPécliptique. Cette souris permettra d'étudier la localisation et la dynamique de Cav3. 2 in vivo grâce à une étiquette fluorescente insérée dans la partie extracellulaire du canal. Des sites de recombinaison loxP ont été également introduits dans le génome de cette souris afin d'effectuer des invalidations tissu-spécifiques et/ou inductibles du canal. En parallèle, une approche protéomique m'a permis d'identifier un nouveau partenaire de Cav3. 2 : la nNOS, une enzyme responsable de la production d'oxyde nitrique (NO). En effet, nous avons montré que Cav3. 2 possède un motif très conservé dans sa région C-terminale qui lui permet d'interagir avec le domaine de PDZ de la nNOS. Cette interaction a des conséquences fonctionnelles pour les deux protéines. D'une part, la présence de la nNOS induit une diminution de la densité des courants générés par le canal Cav3. 2, grâce à un mécanisme qui nécessite l'activité de l'enzyme et la présence d'un centre coordinateur d'ions métalliques dans la partie extracellulaire du canal. D'autre part, nous avons montré que Cav3. 2 est capable de modifier la distribution subcellulaire de la nNOS. L'enzyme est emmenée par le canal à proximité de la membrane plasmique où elle est plus facilement activée par l'entrée de calcium. Cette association fonctionnelle Cav3. 2-nNOS pourrait être impliquée dans des processus comme la nociception, la mémoire et la régulation du tonus vasculaire.

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26

Roof, Steve. "Neuronal Nitric Oxide Synthase Signaling Contributes to the Beneficial Cardiac Effects of Exercise". The Ohio State University, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=osu1354048916.

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Tang, Lifei. "Effects of Neuronal Nitric Oxide Synthase Signaling on Myocyte Contraction during Beta-Adrenergic Stimulation". The Ohio State University, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=osu1385336408.

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Copp, Steven Wesley. "Enzymatic regulation of skeletal muscle oxygen transport: novel roles for neuronal nitric oxide synthase". Diss., Kansas State University, 2013. http://hdl.handle.net/2097/15512.

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Doctor of Philosophy
Department of Anatomy and Physiology
Timothy I. Musch
Nitric oxide (NO) is synthesized via distinct NO synthase (NOS) enzymes and constitutes an essential cardiovascular signaling molecule. Whereas important vasomotor contributions of endothelial NOS (eNOS) have been well-described, the specific vasomotor contributions of nNOS-derived NO in healthy subjects during exercise are unknown. The purpose of this dissertation is to test the global hypothesis that nNOS-derived NO is a critical regulator of exercising skeletal muscle vascular control. Specifically, we utilized the selective nNOS inhibitor S-methyl-L-thiocitrulline (SMTC) to investigate the effects of nNOS-derived NO on skeletal muscle vascular function within established rodent models of exercise performance. The first investigation (Chapter 2) identifies that nNOS inhibition with SMTC increases mean arterial pressure (MAP) and reduces rat hindlimb skeletal muscle blood flow at rest whereas there are no effects during low-speed (20 m/min) treadmill running. In Chapter 3 it is reported that nNOS inhibition with SMTC reduces blood flow during high-speed treadmill running (>50 m/min) with the greatest relative effects found in highly glycolytic fast-twitch muscles and muscle parts. Chapter 4 demonstrates that nNOS-derived NO modulates contracting skeletal muscle blood flow (increases), O2 consumption (VO2, increases), and force production (decreases) in the rat spinotrapezius muscle and thus impacts the microvascular O2 delivery-VO2 ratio (which sets the microvascular partial pressure of O2, PO2mv, and represents the pressure head that drives capillary-myocyte O2 diffusion). In Chapter 5 we report that systemic administration of the selective nNOS inhibitor SMTC does not impact lumbar sympathetic nerve discharge. This reveals that the SMTC-induced peripheral vascular effects described herein reflect peripheral nNOS-derived NO signaling as opposed to centrally-derived regulation. In conclusion, nNOS-derived NO exerts exercise-intensity and muscle fiber-type selective peripheral vascular effects during whole-body locomotor exercise. In addition, nNOS-derived NO modulates skeletal muscle contractile and metabolic function and, therefore, impacts the skeletal muscle PO2mv. These data identify novel integrated roles for nNOS-derived NO within healthy skeletal muscle and have important implications for populations associated with reduced NO bioavailability and/or impaired nNOS structure and/or function specifically (e.g., muscular dystrophy, chronic heart failure, advanced age, etc.).

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29

Kececioglu, Ekin. "Analysis Of Immunoreactivity Of Nos Isoforms (nnos, Enos, Inos) In Hippocampus Of Young Rats Classified As Good And Poor Learners". Master's thesis, METU, 2012. http://etd.lib.metu.edu.tr/upload/12614994/index.pdf.

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Despite very extensive studies on molecular mechanisms of learning and memory formation it is little known about individual variation in the learning skills within a random animal population and about the differences in the brain biochemistry behind this variation. In the present study, we have focused on the expression and distribution of nitric oxide synthase (NOS), one of the molecules implemented in activity-dependent neuroplasticity, in the rat hippocampus, the structure critical for episodic memory in humans and animals. The aim of the present study was to investigate the differences in expression of three different NOS isoforms: neural (n), epithelial (e), and inducible (i), in four hippocampal subregions (CA1, CA3, DG, and hilus) between Wistar rats classified on the basis of their performance in partially baited 12-arm radial maze as &ldquo
good&rdquo
and &ldquo
poor&rdquo
learners. The NOS isoforms were visualized on coronal hippocampal sections using fluorescent immunohistochemistry technique and n- and eNOS images were processed using ImageJ software, while iNOS immunoreactivity (IR) was assessed by counting immunoreactive cells. In this study, overall hippocampal levels of nNOS were significantly higher than those of eNOS and iNOS. The level of n and eNOS was higher in CA1 compared to DG/hilus areas, but lower than that in CA3 region. The expression of iNOS was the highest in CA1 and the lowest in hilus region. nNOS IR was significantly higher in &ldquo
poor&rdquo
than in &ldquo
good&rdquo
learners but only in CA1 region. No significant between-group differences were found in eNOS expression. iNOS expression was higher in &ldquo
poor&rdquo
learners but it did not reach the required significance level.

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30

Wang, Lijun. "Gene transfer strategy to study the role of neuronal nitric oxide synthase in cardiac neurotransmission". Thesis, University of Oxford, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.497136.

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31

Shabeeh, Husain. "Role of neuronal nitric oxide synthase in human vascular tone and systemic haemodynamics in vivo". Thesis, King's College London (University of London), 2014. https://kclpure.kcl.ac.uk/portal/en/theses/role-of-neuronal-nitric-oxide-synthase-in-human-vascular-tone-and-systemic-haemodynamics-in-vivo(cf1a8409-f8f6-4058-9af7-40b6c39d88ec).html.

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Endothelial and neuronal nitric oxide synthase (eNOS and nNOS respectively) are constitutively expressed in vivo. Recent data showed that selective local inhibition of nNOS reduced basal blood flow without affecting endothelial-mediated vasodilatation induced by acetylcholine or increased shear stress - suggesting that eNOS and nNOS have distinct roles in vasoregulation. This thesis aimed to investigate the role of nNOS-derived nitric oxide (NO) in the regulation of skeletal blood flow during exercise and myocardial blood flow during increased cardiac workload. At a systemic level, the role of nNOS on blood pressure and haemodynamics was investigated in a first-in-man study. We used the non-selective NOS inhibitor, NG-monomethyl-Larginine (L-NMMA), and selective nNOS inhibitor, S-methyl-L-thiocitrulline (SMTC), to determine the role of eNOS and nNOS in opposing an increase in sympathetically mediated increases in arteriolar tone in the human forearm during handgrip exercise. We found that despite reducing basal forearm blood flow (FBF), intra-brachial L-NMMA or SMTC had no significant effect on the increase in FBF or conduit artery diameter induced by local handgrip exercise, even in the face of increased sympathetic stimulation with lower body negative pressure. We investigated the relative contribution of eNOS and nNOS in the regulation of coronary vascular tone during increasing metabolic demand as achieved through incremental cardiac pacing. We found that the pacing induced increase in coronary blood flow and artery diameter was blunted by intra-coronary L-NMMA but not so by SMTC. We then undertook the first investigation in humans of the effects of systemic nNOS inhibition on haemodynamics. We found that intravenous SMTC increased systemic vascular resistance and blood pressure, whilst stroke volume, cardiac output and heart rate were reduced. Importantly, there was no effect on flow-mediated dilatation, an effect mediated by eNOS. These results suggest that nNOS has a major contribution to basal regulation of systemic vascular resistance in humans.

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32

Coelho, Camila Henriques. "Análise da inibição da óxido nítrico sintase neuronal (nNOS) na liberação de vasopressina durante sepse experimental". Universidade de São Paulo, 2009. http://www.teses.usp.br/teses/disponiveis/60/60135/tde-30102009-022744/.

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A fisiopatologia da sepse se caracteriza por hipotensão acompanhada de aumento da secreção de vasopressina (AVP) na fase inicial e diminuição numa fase mais tardia. Essa hipotensão é em parte devido ao aumento da quantidade de óxido nítrico, que juntamente com outros mediadores tem sua produção aumentada durante a sepse. A óxido nítrico sintase (NOS) é responsável pela síntese deste mediador, e sua isoforma neuronal (nNOS) está presente no músculo esquelético, pulmões, testículos, próstata, pele e também nos neurônios vasopressinérgicos do hipotálamo. O presente trabalho avaliou a participação do óxido nítrico produzido pela isoforma neuronal de NOS sobre a secreção temporal de AVP durante a sepse experimental. Ratos Wistar receberam injeção i.p. de 7-nitroindazol (50mg/kg ou 80mg/kg), inibidor específico da NOS neuronal, ou DMSO 10% + óleo de gergelim na proporção 1:9 (veículo) e após 30 minutos foram submetidos ao estímulo séptico por ligadura e perfuração cecal (CLP) ou à operação fictícia (OF). Em um grupo de animais, a sobrevida foi avaliada durante 5 dias. Em outro grupo, os animais foram decapitados 0, 4, 6, 18 e 24 horas após a cirurgia e o sangue processado para determinação do hematócrito, sódio sérico, osmolalidade, proteínas, glicose, creatinina, nitrato sérico e AVP plasmática. A neurohipófise foi removida para a determinação do conteúdo de AVP, e o hipotálamo dissecado para a determinação da atividade da NOS total. A mortalidade observada após CLP não foi modificada com o pré-tratamento com 7-NI (50mg/kg), assim como os aumentos temporais de hematócrito, glicose e nitrato sérico observados. As proteínas plasmáticas e o sódio sérico apresentaram diminuição após CLP e o pré-tratamento com 7-NI antecipou a perda proteica e postergou a diminuição do sódio sérico. Os animais após CLP não apresentaram alterações de creatinina e osmolalidade, entretanto quando prétratados com 7-NI, apresentaram aumento em 6 e 18 horas e diminuição a partir de 4hs, respectivamente. A atividade da NOS total no hipotálamo aumentou nos tempos determinados de 4 e 24 horas após CLP e este aumento foi reduzido com o prétratamento com o 7-NI nas doses de 50 e 80mg/kg, respectivamente. O conteúdo neurohipofisário de AVP diminuiu em 4, 6 e 18 horas após CLP e o pré-tratamento com 7-NI reduziu os estoques apenas em 0 e 6 horas. As concentrações plasmáticas de vasopressina apresentaram-se aumentadas sómente 6 horas após CLP e o pré-tratamento não alterou essas concentrações. Esses resultados permitem concluir que o NO produzido pela NOS neuronal não teria uma tarefa substancial na secreção de vasopressina durante sepse experimental.
The pathophysiology of sepsis is caracterized by hypotension accompanied by increase of vasopressin secretion (AVP) in early phase and decrease during late phase. This hypotension is due, in part, to the increase of nitric oxide (NO) production, that, like other mediators, shows high production during sepsis. Nitric oxide synthase (NOS) is responsible by synthesis of NO. The neuronal isoform of NOS is present in skeletic muscle, testicles, prostate, skin and vasopressinergics neurons of hypothalamus. The present work evaluated the participation of nitric oxide produced by neuronal NOS in temporal vasopressin secretion during experimental sepsis. Rats Wistar received intraperitoneal injection of 7-nitroindazole (50 or 80 mg/kg), an inhibitor of neuronal nitric oxide synthase activity, or DMSO 10% + sesame oil in the proportion 1:9 (vehicle) and after 30 minutes, they were submited to septic stimulus by cecal ligation and puncture (CLP) or to sham operation. In one of the groups, the survival rate was evaluated during 5 days. In other group, the animals were decapited 0, 4, 6, 18 and 24 hours after CLP and the blood was processed to determinate haematocrit, serum sodium, osmolality, proteins, glucose, creatinine, serum nitrate, and plasma AVP. Neurohypophysis was removed to determination of vasopressin content, and hypotalamus was dissected to determinate total NOS activity. Mortality observed after CLP was not affected by periferal injection of 7-nitroindazole (50 mg/kg) as well as haematocrit, glucose and nitrate increase. Serum sodium and plasma protein decreased after CLP and the treatment antecipated the loss protein, and delayed serum sodium decrease. CLP animals didn\'t show creatinine and osmolality alterations, but when treated with 7-nitroindazole, showed increase 6 and 18 hours, and decrease 4 hours, respectively. NOS activity in hypothalamus increased 4 and 24 hours after CLP, and was reduced with 7-NI pretreatment (50 and 80 mg/kg respectively). AVP neurohypophysis content diminished 4, 6 and 18 hours after CLP and 7-NI reduced the content just at 0 and 6 hours. Vasopressin plasma concentration increased just 6 hours after CLP and 7-NI pretreatment didn\'t alter this parameter. We concluded that NO produced by neuronal NOS doesn\'t have a substantial role in vasopressin secretion during experimental sepsis.

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33

Lemaire, Jean-François 1980. "Binding of Vac14 to neuronal nitric oxide synthase : characterisation of a new internal PDZ-recognition motif". Thesis, McGill University, 2006. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=101723.

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Protein interactions mediated by PDZ domains involve specific modes of recognition. The most common type depends on the recognition of a classical peptide motif found at the very carboxyl end of the PDZ domain binding protein. Although less common, other modes of recognition have been described involving internal sequences. The best characterized involves an internal beta-finger structure that mimics the carboxyl terminus of a protein. This work describes a new PDZ domain recognition sequence, that is found internally but which does not adopt a beta-finger conformation. The sequence mediates the previously uncharacterized interaction between the neuronal nitric oxide synthase (nNOS) and the PtdIns(3)P 5-kinase PIKfyve-activator Vac14. Mutational analyses reveal essential residues within the motif allowing for the description of a new type of PDZ domain interaction.

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34

Welland, Andrew David. "The importance of domain-domain interactions in the regulation and activity of neuronal nitric oxide synthase". Thesis, University of Edinburgh, 2008. http://hdl.handle.net/1842/3179.

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Nitric oxide synthases (NOSs) catalyse the production of the physiological messenger molecule NO from L-arginine in a unique two step oxygenation reaction. Constitutive forms of NOS are activated by the binding of calmodulin (CaM) to a 20- amino-acid inter-domain region in the presence of calcium ions, causing inter-domain electron transfer to occur from FAD to heme via FMN. This electron transfer step involves a large scale movement of the FMN-binding domain and is influenced by a number of structural features unique to NOS which include an autoinhibitory loop, a C-terminal extension, a number of phosphorylation sites, and the hinge region that connects the FAD- and FMN- binding domains. X-ray crystallographic data indicate that all of these regulatory elements lie at the interface between the two domains, restricting their motion relative to each other. The importance of this interface region in the CaM-dependent activation mechanism of neuronal NOS (nNOS) was investigated by site-directed mutagenesis of interface residues in the isolated reductase domain (nNOSrd). A range of kinetic and thermodynamic analytical techniques were employed in order to determine which catalytic steps are affected by changes in domain mobility. The rate-limiting step in the turnover of nNOSrd has been speculated to be one of three catalytic events; the hydride transfer from NADPH to FAD, the electron transfer between the bound flavin cofactors, or the electron transfer between FMN and external electron acceptors such as cytochrome c. In each case, the binding of CaM enhances the rate of reduction. In wild-type nNOSrd, NADPH reduced the FAD by hydride transfer in what should have been a simple 1-step reaction but was in fact a biphasic process. Isotope effects and the use of differing ionic strength indicated that different conformations of enzyme have different rates of reaction with NADPH. The redox state of the FMN cofactor also influenced the rate of reduction of FAD, through the interaction with the peptide backbone in the interface region. A putative proton transfer pathway exists between the bound FAD cofactor and solvent, involving Ser1176, Asp1393, His1032 and Arg1229. Mutation of the former three residues diminished the catalytic activity, specifically focused on the rate of hydride transfer, while the R1229E mutation had a much more dramatic effect. Arg1229 forms one of only two electrostatic contacts between the FAD and FMNbinding domains in the interface region and the charge reversal substitution introduced a likely inter-domain repulsion. This was expected to cause the two domains to separate, favouring a hinged-open conformation. The hydride transfer step from NADPH to FAD was activated in the CaM-free enzyme, while FAD to FMN electron transfer was inhibited. Electron transfer from reduced FMN to the artificial electron acceptor horse-heart cytochrome c was also activated in the CaMfree state. The effect on the three catalytic events meant that during steady-state turnover with cytochrome c, CaM deactivated the enzyme and caused cytochrome cdependent inhibition. Evidently, domain-domain separation was large enough in the mutant to accommodate cytochrome c, a 12 kDa protein, in the space between the cofactors at the interface. The effects of this single charge-reversal on the three distinct catalytic events illustrated how each is differently dependent on the enzyme conformation. FAD to FMN electron transfer was shown to occur exclusively in the hinged-closed form, consistent with the crystal structure of nNOSrd. The remaining two events, hydride transfer from NADPH to FAD, and electron transfer from FMN to cytochrome c, occur in the hinged-open state. In the wild-type enzyme, the hinged-open and hinged-closed states are tightly regulated by a conformational equilibrium which is affected by CaM binding. It appears that CaM activates the enzyme by shifting this equilibrium to an open form.

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Muszkiewicz, Anna. "Multi-scale modelling and simulations into the mechanisms linking neuronal nitric oxide synthase and atrial fibrillation". Thesis, University of Oxford, 2016. https://ora.ox.ac.uk/objects/uuid:244d6f3a-00da-4ae8-8d5c-31ebb847806f.

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Atrial fibrillation (AF) is the most common cardiac arrhythmia. Its incidence is projected to rise due to population ageing and increasing prevalence of associated risk factors. AF alters, or remodels, the affected atrial tissue, promoting future occurrences of itself and increasing resistance to treatment. Mechanisms underlying AF initiation and remodelling are not well understood. Recent experimental evidence indicates that decreased levels of the neuronal isoform of Nitric Oxide Synthase (nNOS) may be related to AF onset and precede remodelling. However, the potential mechanisms cannot be easily elucidated with experiments alone. Furthermore, experiments are complicated by inter-subject variability, which is particularly important in human studies due to the wide heterogeneity of the human population. In this thesis, I use multi-scale modelling and simulations in synergy with experimental information to investigate mechanistic links between nNOS and AF at the level of ionic currents/cellular action potential/whole atria in human. First, I construct populations of models spanning experimentally-observed variability in human atrial myocytes under control conditions. Second, I use those populations of control models to identify key ionic mechanisms underpinning nNOS-mediated regulation of the cellular action potential in human atrial myocytes. I show that two of those currents - I_Kur and I_K1 - play a key role in explaining the phenotypic shortening of the action potential observed under nNOS inhibition conditions and preceding AF-induced tissue remodelling. Finally, I build models of human whole atria and establish that this action potential shortening leads to the establishment of a vulnerable substrate, and hence is the main mechanism of pro-arrhythmia at this level. Overall, I provide a picture of nNOS-mediated mechanisms related to AF onset from ionic currents to the whole organ level.

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Xie, Jinling. "Transcriptional control via multiple promoters in the cns: glial filamin and neuronal nitric oxide synthase gene expression /". The Ohio State University, 1996. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487940665435278.

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Higuchi, Yoshihisa. "Increased Neurons Containing Neuronal Nitric Oxide Synthase in the Brain of a Hypoxic-Ischemic Neonatal Rat Model". Kyoto University, 1997. http://hdl.handle.net/2433/202233.

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Lee, Graham. "An investigation of nitric oxide synthase in neuronal function and in phencyclidine models of relevance to schizophrenia". Thesis, University of Strathclyde, 2014. http://oleg.lib.strath.ac.uk:80/R/?func=dbin-jump-full&object_id=23201.

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Schizophrenia is a complex and debilitating psychiatric disorder. Dysfunction of the NMDA subtype of glutamate receptors is implicated in deficits found in schizophrenia, and some of these deficits may be reproduced in rodents using the NMDA receptor antagonist, phencyclidine (PCP). Nitric oxide synthase (NOS) is the synthesising enzyme of the gaseous neuromodulator, nitric oxide. The neuronal isoform of NOS (nNOS) is functionally associated with NMDA receptors. The role of NOS in schizophrenia is not fully understood. The primary aim of this thesis is to investigate NOS signalling in cultured neurones and in rodent PCP models of relevance to schizophrenia. Using diaminofluorescein microscopy of primary neuronal cultures, it is shown that non-selective inhibition of NOS using L-NAME, and selective inhibition of nNOS using L-NPA reversed glutamate-stimulated nitric oxide generation in hippocampal and cerebellar neurones, but inhibition of endothelial NOS using L-NIO did not. Novel c ompounds that modulate the NOS cofactor, tetrahydrobiopterin, altered nitric oxide generation in cerebellar neurones. NOS activity was increased in the hippocampus, and decreased in the reticular thalamus in mice administered acute PCP (5 mg.kg-1, i.p.), as determined by NADPH-diaphorase activity. NOS activity normalised in these areas with subchronic PCP (5 mg.kg-1 twice daily for 5 days), and NOS activity was decreased in the prefrontal cortex. Decreased activity of thioredoxin reductase was found in the hippocampus and thalamus with acute PCP, but was unchanged with subchronic PCP. Pretreatment with L-NAME (40 mg.kg-1) and L-NIO (20 mg.kg-1) improved hyperlocomotion and III deficits in prepulse inhibition observed with PCP, but L-NPA (20 mg.kg-1) did not. In conclusion, the results presented in this thesis give evidence for a role of NOS in deficits observed in rodent PCP models of relevance to schizophrenia. Selective inhibition of NOS isoforms is a potential therapeutic strategy to improve deficits associated with NMDA receptor dysfunction found in schizophrenia.

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Candemir, Esin [Verfasser], e Andreas [Gutachter] Reif. "Involvement of neuronal nitric oxide synthase (NOS-I) PDZ interactions in neuropsychiatric disorders / Esin Candemir ; Gutachter: Andreas Reif". Würzburg : Universität Würzburg, 2018. http://d-nb.info/1162444371/34.

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Isin, Emre M. "Potential Prodrugs of the Neuronal Nitric Oxide Synthase and Monoamine Oxidase Inhibitor 7-Nitroindazole and Structurally Related Compounds". Thesis, Virginia Tech, 2000. http://hdl.handle.net/10919/35829.

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Parkinson's disease (PD) is a progressive neurodegenerative disorder of unknown cause that afflicts about 1.5 million Americans. The characteristic feature of PD is a deficiency of dopamine in the terminals of nigrostriatal neurons. Two enzyme systems, the neuronal form of nitric oxide synthase (nNOS) and monoamine oxidase B (MAO-B), have been linked to neurodegenerative pathways leading to PD. Several MAO-B and nNOS inhibitors have been evaluated for their neuroprotective properties in the mouse model of neurodegeneration which employs the parkinsonian inducing neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). One such compound is 7-nitroindazole (7-NI), a compound which is reported to inhibit both enzymes.

This thesis focuses on the synthesis and biological evaluation of a potential prodrug form of 7-NI and related indazolyl containing compounds which are designed to release the active drugs following a metabolic bioactivation process. These studies have led to a detailed description of the nucleophilic aromatic substitution reactions between 4-chloro-1-methylpyridinium iodide and the indazolyl reactants that were employed as the initial step in the synthesis of the target compounds. The MAO-B substrate and inhibition properties of these "prodrugs" as well as the parent indazolyl compounds were examined. The results are discussed in relation to a previously developed active site model of MAO-B.
Master of Science

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Ollerstam, Anna. "Macula Densa Derived Nitric Oxide and Kidney Function". Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Univ.-bibl. [distributör], 2002. http://publications.uu.se/theses/91-554-5293-0/.

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Isaak, Andreas [Verfasser]. "Neuronal nitric oxide synthase is involved in the induction of nerve growth factor-induced neck muscle nociception / Andreas Isaak". Aachen : Hochschulbibliothek der Rheinisch-Westfälischen Technischen Hochschule Aachen, 2011. http://d-nb.info/1018200746/34.

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Mulatz, Kirk James. "A PDZ-3 mediated physical and functional interaction between the CaV3.2 T-type calcium channel and neuronal nitric oxide synthase". Thesis, University of British Columbia, 2013. http://hdl.handle.net/2429/43790.

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T-type voltage-gated calcium channels are expressed throughout the central and peripheral nervous systems as well as in several non-neuronal tissues and contribute to variety of functions such as neuronal excitability, intracellular calcium influx, shaping action potentials, pace-making activity, hormone secretion, and neurotransmitter release. Of the three T-type channel isoforms, Cav3.2 is uniquely sensitive to redox modulation with oxidizing reagents inhibiting and reducing compounds enhancing channel activity. This modulation has been shown to alter firing patterns of reticular thalamic neurons and to affect the nociceptive threshold in vivo suggesting that redox modulation of Cav3.2 may play an important role in regulating neuronal activity. A potential source of oxidizing molecules in vivo is neuronal nitric oxide synthase (nNOS), a calcium dependent enzyme which synthesizes nitric oxide (NO) from arginine. Interestingly, the carboxyl terminus of Cav3.2 possesses a putative PDZ-3 binding ligand which is compatible with the PDZ-3 domain of nNOS. I hypothesize that Cav3.2 and nNOS physically interact via the PDZ-3 binding ligand of Cav3.2 and that this physical interaction mediates a functional interaction whereby Cav3.2 activity stimulates nNOS to produce NO which, in turn, inhibits Cav3.2 activity. Cav3.2 and nNOS were expressed in a heterologous system which allowed us to examine the putative PDZ-3 mediated interactions between the two proteins. Immunoprecipitation experiments using Cav3.2 specific antibodies demonstrate that Cav3.2 and nNOS can interact via the carboxyl PDZ-3 ligand of Cav3.2 and that this interaction is disrupted when the PDZ-3 ligand is mutated. Utilizing a NO sensitive fluorometric assay we show that Cav3.2 activity can stimulate nNOS to produce NO and that disruption the PDZ-3 interaction precludes nNOS activation. We also demonstrate that the PDZ-3 mediated physical interaction facilitates the inhibition of Cav3.2 by nNOS derived NO. Disruption of the Cav3.2/nNOS interaction in vivo using intraperitoneal injection of membrane permeable peptides designed to competitively disrupt the PDZ-3 interaction produces an exaggerated respiratory response to changes in available oxygen and a blunted response in the hyperoxic response test. These results indicate that Cav3.2 and nNOS physically and functionally interact to contribute to normal physiological processes.

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Idigo, Winifred. "Role of neuronal nitric oxide synthase in the regulation of B3- Adrenergic responses in the healthy and remodelled murine myocardium". Thesis, University of Oxford, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.526494.

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Takimoto, Yoshihito. "Augmented expression of neuronal nitric oxide synthase in the atria parasympathetically decreases heart rate during acute myoccardial infarction in rats". Kyoto University, 2003. http://hdl.handle.net/2433/149374.

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Moraes, Juliana Contin. "Efeitos do fator de necrose tumoral - alfa sobre a expressão da sintase de oxido nitrico neuronal e induzivel em hipotalamo de ratos : implicações sobre o controle da fome". [s.n.], 2006. http://repositorio.unicamp.br/jspui/handle/REPOSIP/310355.

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Orientador: Licio Augusto Velloso
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
Made available in DSpace on 2018-08-06T23:35:52Z (GMT). No. of bitstreams: 1 Moraes_JulianaContin_M.pdf: 1484102 bytes, checksum: 31482341aabc46ac3c7f655aead3fbd6 (MD5) Previous issue date: 2006
Resumo: Durante as últimas décadas tem se observado um aumento surpreendente na prevalência de obesidade e diabetes mellitus em populações de várias regiões do mundo, inclusive no Brasil. Diversos estudos epidemiológicos apontam o consumo de dietas ricas em lípides como um dos mais importantes fatores de risco para o desenvolvimento dessas patologias. Em recente trabalho em nosso laboratório, foi demonstrado que a oferta de uma dieta rica em lípides leva a uma maior expressão hipotalâmica de proteínas participantes de respostas pró-inflamatórias como TNF-a, IL-2, IL-6 e IL-ß. A citocina TNFa, agindo no hipotálamo, modula a ingestão alimentar e o gasto energético através de mecanismos incompletamente elucidados. Neste trabalho exploramos a hipótese de que, para modular a sinalização anorexigênica induzida por insulina no hipotálamo, o TNFa deve requerer a síntese de óxido nítrico. O TNFa ativa sinalização intracelular canónica no hipotálamo, com pico na concentração de 10-8 M. Esse efeito é acompanhado pela indução da expressão das formas neuronal e induzível da enzima NOS, em ambos os casos com pico em 10-12 M. Em adição, TNFa estimula a atividade catalítica de NOS. O pré-tratamento com TNFa em baixa dose (10-12 M) inibe a sinalização anorexigênica insulino-dependente. Esse efeito é abolido em camundongos iNOS mas não em camundongos nNOS knockout. Portanto, o efeito inibitório exercido pela baixa dose de TNFa sob a inibição da ingestão alimentar induzida por insulina depende, pelo menos em parte, da expressão de iNOS. Embora nNOS hipotalâmica seja induzida por TNFa, esta não participa da modulação TNFa-dependente dos sinais anorexigênicos da insulina.
Abstract: Obesity has reached epidemic proportion in several regions of the world. General changes in life-style, including consumption of fat-rich food, are amongst the most important factors leading to an unprecedented increase in the prevalence of this disease. In a recently work on our laboratory, we showed that high fat feeding (hyperlipidic diet) induced the expression of several pro-inflammatory cytokines and inflammatory responsive proteins in hypothalamus, like TNFa, IL-2, IL-6 e IL-lß. The cytokine TNFa, acting on the hypothalamus, modulates food intake and energy expenditure through mechanisms incompletely elucidated. Here, we explored the hypothesis that, to modulate insulin-induced anorexigenic signaling in hypothalamus, TNF-a would require the synthesis of NO. TNF-a activates canonical intracellular signaling in hypothalamus, peaking at 10-8 M. This is accompanied by the induction of expression of the inducible and neuronal forms of NOS, in both cases peaking at 10-12 M. In addition TNF-a stimulates NOS catalytic activity. The pre-treatment with TNF-a at low dose (10-12 M) inhibits insulin-dependent anorexigenic signaling. This effect is abolished in iNOS but not in Nnos knockout mice. Thus, the inhibitory effect exerted by low dose TNF-a upon the insulin-induced inhibition of food intake depends, at least in part, on the expression of iNOS. Although hypothalamic nNOS is induced by TNF-a it does not participate on TNF-a-dependent modulation of the insulin anorexigenic signals.
Mestrado
Ciencias Basicas
Mestre em Clinica Medica

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Minnaar, Estella Lily. "Regional neurochemical characterization of the flinders sensitive line rat with regard to glutamate-nitric oxide and cGMP signalling pathways / Estella Lily Minnaar". Thesis, North-West University, 2008. http://hdl.handle.net/10394/4214.

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The serious nature of MDD has intensified the need to identify and elucidate new neurobiological targets for antidepressant drug action. Depression presents with evidence for degenerative pathology that relates to disturbances in excitatory glutamatergic pathways, particularly the N-methyl-D-aspartate (NMDA) receptormediated release of the pleiotropic molecule, nitric oxide (NO), and cyclic guanosine monophosphate (cGMP). The contribution of the glutamate-NO/cGMP pathway may realize great importance as a fundamental substrate underlying the pathophysiology of major depression. In the next generation of antidepressant drugs, the nitric oxide pathway could playa dynamic role in addressing urgent therapeutic needs. In this study, we have used a genetic model of depression, the Flinders Sensitive Line (FSL) rat, to investigate the surrogate markers of the NO/cGMP pathway. The aim was to determine whether the depressive-like behaviour of the hypercholinergic FSL rat is accompanied by altered activation of the NO/cGMP pathway. To this end, the extent to which the FSL and Flinders Resistant Line (FRL) rats differ neurochemically with regard to basal hippocampal and frontal cortical NOS-activity, as well as nitric oxide (NO) and cGMP accumulation, were determined. Additionally, select behavioural assessments were performed to confirm the anxiogenic phenotype of the FSL strain. For neurochemical determinations a sensitive fluorometric reversed phase highperformance liquid chromatographic (HPLC) assay was developed to analyze total nitrite and nitrate in brain tissue. Nitrate was enzymatically converted to nitrite before derivatization with 2,3-diaminonaphthalene (DAN). The stable and highly fluorescent product, 2,3-naphthotriazole (NAT), was quantified. Secondly, the quantity of the amino acid L-citrulline was measured by HPLC with electrochemical detection after o-phthalaldehyde (OPA) derivatization. L-citrulline formation was used as an index for nNOS activity. Finally, a direct, competitive enzyme immunoassay kit was used to determine the downstream activity of the NO-pathway in brain tissue. FSL rats were compared to FRL rats with respect to sensitivity to serotonin 5-HT1A . receptor-mediated hypothermia under our lab-conditions. The Open Field Test (OFT) behavioural assessment was performed to compare FSL with FRL groups under baseline conditions according to their level of inherent anxiety. The parameters used to measure anxiety were number of line crosses (locomotor activity), time spent in middle blocks and social interaction time between pairs of rats. As an additional behavioural assessment, the Forced Swim Test (FST) was performed to assess behavioural restraint measured as time of immobility. Basal cGMP levels in the frontal cortex were found to be significantly less in FSL than in FRL rats, whereas the levels in the hippocampus did not differ significantly. No other significant differences with respect to NO and nNOS activity were apparent in either of the brain areas. The hypothermia test confirmed a significantly greater decrease in temperature in the FSL rat than the FRL rat. The FST did not confirm any differences in immobility time between the two rat strains. In the OFT, FSL rat groups exhibited behaviour that indicated significantly more anxiety than FRL rats. Under basal conditions, FSL rats do not present with significant changes in markers of the NO cascade in the hippocampus and frontal cortex compared to FRL controls, including NOS activity as well as NO accumUlation. However, cGMP levels were found to be significantly lower in the frontal cortex of FSL rats versus FRL rats, although not in the hippocampus. Since the FSL rat is known to be hypercholinergic, these data support an interaction between the NO/cGMP pathway and the cholinergIc system in the frontal cortex but not hippocampus of FSL animals. The mechanisms and implications of such a mutual involvement need further clarification. Further, this anatomical differentiation may have important implications for understanding the role of NO in the depressive-like behaviour of the FSL rat and, indeed, may reveal more on the neurobiology and treatment of depression. Through the performed behavioural assessments, the FSL and FRL rats were successfully separated with respect to their anxiety phenotype as well as their heightened response to serotonergic challenge, thus confirming a contribution of both the serotonergic and cholinergic systems to the depressogenic nature of these animals. As concluding remark can be said that under normal basal conditions markers of the NO/cGMP signalling cascade are not altered in FSL vs FRL rats, although cGMP levels are reduced in the frontal cortex of FSL rats, supportive of an NO-independent mechanism of cGMP regulation, possibly involving ACh.
Thesis (M.Sc. (Pharmacology)--North-West University, Potchefstroom Campus, 2009.

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Karolewicz, Beata, Katalin Szebeni, Tempestt Gilmore, Dorota MacIag, Craig A. Stockmeier e Gregory A. Ordway. "Elevated Levels of NR2A and PSD-95 in the Lateral Amygdala in Depression". Digital Commons @ East Tennessee State University, 2009. https://dc.etsu.edu/etsu-works/8607.

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Compelling evidence suggests that major depression is associated with dysfunction of the brain glutamatergic transmission, and that the glutamatergic N-methyl-d-aspartate (NMDA) receptor plays a role in antidepressant activity. Recent post-mortem studies demonstrate that depression is associated with altered concentrations of proteins associated with NMDA receptor signalling in the brain. The present study investigated glutamate signalling proteins in the amygdala from depressed subjects, given strong evidence for amygdala pathology in depression. Lateral amygdala samples were obtained from 1314 pairs of age- sex-, and post-mortem-interval-matched depressed and psychiatrically healthy control subjects. Concentrations of NR1 and NR2A subunits of the NMDA receptor, as well as NMDA receptor-associated proteins such as post-synaptic density protein-95 (PSD-95) and neuronal nitric oxide synthase (nNOS) were measured by Western immunoblotting. Additionally, levels of enzymes involved in glutamate metabolism, including glutamine synthetase and glutamic acid decarboxylase (GAD-67), were measured in the same amygdala samples. NR2A protein levels were markedly and significantly elevated (+115%, p=0.03) in depressed subjects compared to controls. Interestingly, PSD-95 levels were also highly elevated (+128%, p=0.01) in the same depressed subjects relative to controls. Amounts of NR1, nNOS, glutamine synthetase, and GAD-67 were unchanged. Increased levels of NR2A and PSD-95 suggest that glutamate signalling at the NMDA receptor in the amygdala is disrupted in depression.

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Karolewicz, Beata, Laurel Johnson, Katalin Szebeni, Craig A. Stockmeier e Gregory A. Ordway. "Glutamate Signaling Proteins and Tyrosine Hydroxylase in the Locus Coeruleus of Alcoholics". Digital Commons @ East Tennessee State University, 2008. https://dc.etsu.edu/etsu-works/8610.

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It has been postulated that alcoholism is associated with abnormalities in glutamatergic neurotransmission. This study examined the density of glutamate NMDA receptor subunits and its associated proteins in the noradrenergic locus coeruleus (LC) in deceased alcoholic subjects. Our previous research indicated that the NMDA receptor in the human LC is composed of obligatory NR1 and regulatory NR2C subunits. At synapses, NMDA receptors are stabilized through interactions with postsynaptic density protein (PSD-95). PSD-95 provides structural and functional coupling of the NMDA receptor with neuronal nitric oxide synthase (nNOS), an intracellular mediator of NMDA receptor activation. LC tissue was obtained from 10 alcohol-dependent subjects and eight psychiatrically healthy controls. Concentrations of NR1 and NR2C subunits, as well as PSD-95 and nNOS, were measured using Western blotting. In addition, we have examined tyrosine hydroxylase (TH), the rate-limiting enzyme in the synthesis of norepinephrine. The amount of NR1 was lower in the rostral (-30%) and middle (-41%) portions of the LC of alcoholics as compared to control subjects. No differences in the amounts of NR2C, PSD-95, nNOS and TH were detected comparing alcoholic to control subjects. Lower levels of NR1 subunit of the NMDA receptor in the LC implicates altered glutamate-norepinephrine interactions in alcoholism.

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Zoerner, Frank. "Novel Interventions in Cardiac Arrest : Targeted Temperature Management, Methylene Blue, S-PBN, Amiodarone, Milrinone and Esmolol, Endothelin and Nitric Oxide In Porcine Resuscitation Models". Doctoral thesis, Uppsala universitet, Anestesiologi och intensivvård, 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-236312.

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It is a major clinical problem that survival rates after out-of-hospital cardiac arrest have not markedly improved during the last decades, despite extensive research and the introduction of new interventions. However, recent studies have demonstrated promising treatments such as targeted temperature management (TTM) and methylene blue (MB). In our first study, we investigated the effect of MB administered during experi-mental cardiopulmonary resuscitation (CPR) in the setting of postponed hypother-mia in piglets. We set out to study if MB could compensate for a delay to establish targeted TTM. The study demonstrated that MB more than compensated for 30 min delay in induction of TTM. The effect of MB added to that of TTM. The second study examined the effects of TTM and S-PBN on the endothelin system and nitric oxide synthases (NOS) after prolonged CA in a porcine CPR mod-el. The study was designed to understand the cardioprotective mechanism of S-PBN and TTM by their influence on the endothelin system and NOS regulation. We veri-fied for the first time, that these two cardioprotective postresuscitative interventions activate endothelin-1 and its receptors concomitantly with eNOS and nNOS in the myocardium. We concluded that nitric oxide and endothelin pathways are implicated in the postresuscitative cardioprotective effects of TTM. The third study compared survival and hemodynamic effects of low-dose amio-darone and vasopressin to vasopressin in a porcine hypovolemic CA model. The study was designed to evaluate whether resuscitation with amiodarone and vasopressin compared to vasopressin alone would have an impact on resuscitation success, survival, and hemodynamic parameters after hemorrhagic CA. We found that combined resuscitation with amiodarone and vasopressin after hemorrhagic circulatory arrest resulted in greater 3-hour survival, better preserved hemodynamic parameters and smaller myocardial injury compared to resuscitation with vasopressin only. In our fourth study we planned to compare hemodynamic parameters between the treatment group (milrinone, esmolol and vasopressin; MEV) and control group (vasopressin only) during resuscitation from prolonged cardiac arrest in piglets. The study was designed to demonstrate if MEV treatment improved hemodynamics or cardiac damage compared to controls. We demonstrated that MEV treatment reduced cardiac injury compared with vasopressin alone.

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