Nitric oxide (NO) overproduction is correlated with numerous human diseases, such as arthritis, asthma, diabetes, inflammation and septic shock. The enzyme activities of both NO synthase (NOS) and dimethylarginine dimethylaminohydrolase-1 (DDAH-1) promote NO production. DDAH-1 mainly colocalizes in the same tissues as the neuronal isoform of NOS and catabolizes the endogenously-produced competitive inhibitors of NOS, N[omega]-monomethyl-L-arginine (NMMA) and asymmetric N[omega], N[omega]-dimethyl-L-arginine (ADMA). Inhibition of DDAH-1 leads to elevated concentrations of NMMA and ADMA, which subsequently inhibit NOS. To better understand DDAH-1, I first characterized the catalytic mechanism of human DDAH-1, where Cys274, His173, Asp79 and Asp127 form a catalytic center. Particularly, Cys274 is an active site nucleophile and His173 plays a dual role in acid/base catalysis. I also studied an unusual mechanism for covalent inhibition of DDAH-1 by S-nitroso-L-homocysteine (HcyNO), where an N-thiosulfoximide adduct is formed at Cys274. Using a combination of site directed mutagenesis and mass spectrometry, we found that many residues that participate in catalysis also participate in HcyNO mediated inactivation. Following these studies, I then screened a small set of known NOS inhibitors as potential inhibitors of DDAH-1. The most potent of these, an alkylamidine, was selected as a scaffold for homologation. Stepwise lengthening of the alkyl substituent changes an NOS-selective inhibitor into a dual-targeted NOS/DDAH-1 inhibitor then into a DDAH-1 selective inhibitor, as seen in the inhibition constants of N5-(1-iminoethyl)-, N5-(1-iminopropyl)-, N5-(1-iminopentyl)- and N5-(1-iminohexyl)-L-ornithine for neuronal NOS (1.7, 3, 20, >1,900 [mu]M, respectively) and DDAH-1 (990, 52, 7.5, 110 [mu]M, respectively). X-ray crystal structures suggest that this selectivity is likely due to active site size differences. To rank the inhibitors' in vivo potency, we constructed a click-chemistry based activity probe to detect inhibition of DDAH-1 in live mammalian cell culture. In vivo IC50 values for representative alkylamidine based inhibitors were measured in living HEK293T cells. Future application of this probe will address the regulation of DDAH-1 activity in pathophysiological states. In summary, this work identifies a versatile scaffold for developing DDAH targeted inhibitors to control NO overproduction and provides useful biochemical tools to better understand the etiology of endothelial dysfunction. / text

Nos últimos anos, nosso grupo de pesquisa vem descrevendo vários efeitos da SAA em células do sistema imune no que diz respeito à expressão e liberação de citocinas pró-inflamatórias. Neste estudo centramos nossa atenção na verificação dos efeitos da SAA sobre células mononucleares. Para isto, usamos três modelos experimentais. Em murinos, descrevemos a habilidade da SAA induzir a produção de NO por macrófagos peritoneais e, com uso de animais knockout para TLR4, sugerimos que SAA seja um ligante endógeno do TLR4. Em células mononucleares de sangue periférico humano, a SAA induz a expressão e liberação de CCL20, uma quimiocina importante na transição da resposta imune inata para adaptativa, bem como a expressão dos fatores M-CSF e VEGF. Em células THP-1, mostramos a cinética de fosforilação de proteínas tirosina quinases promovida pela SAA e comparamos com LPS, um estímulo pró-inflamatório clássico. Ainda em células THP-1 mostramos que a SAA induz a fosforilação de duas proteínas importantes no processo inflamatório por induzirem a ativação de NFκB; a p38 e a ERK1/2. Com este estudo contribuímos com o conhecimento a respeito do papel regulatório da SAA em células mononucleares. A ação da SAA sobre estas células torna-se importante, pois estas são cruciais na resposta imune inata e também atuam como células acessórias na resposta imune adaptativa. Desta forma, evidencia-se que, no processo de fase aguda, a expressão e a síntese de SAA resultam na modulação de etapas que controlam este processo e sua progressão. / In the past few years, our research group has described various effects of serum amyloid A (SAA) on cells of the immune system regarding the expression and release of pro-inflammatory cytokines. In this study we have focused on the effects of SAA on mononuclear cells. In order to do this, we have used three experimental models. In the murine experimental model, we described SAA\'s ability to induce the production of NO through peritoneal macrophages and, by using knockout animals for TLR4, we suggested that SAA is an endogenous agonist of TLR4. In mononuclear cells of peripheral human blood, SAA induced the expression and release of CCL20, an important chemokine in the transition from the innate to the adaptive immune response, as well as the expression of M-CSF and VEGF-factors. In THP-1 cells, we showed the phosporylation kinetics of tyrosine protein kinases induced by SAA, and we compared it to LPS, a classic pro-inflammatory stimulus. We also demonstrated, in THP-1 cells, that SAA induced the phosphorylation of two proteins, namely p38 and ERK1/2, that are crucial in the inflammatory process because they induce the activation of transcription factors. With this study, we contributed to the knowledge of the regulatory role of SAA in mononuclear cells. Activity of SAA on these cells is highly important, for they are crucial in the innate immune response and act as accessory cells in the adaptive immune response. Hence it is evident that, in the acute phase process, the expression and synthesis of SAA result in the modulation of the phases that control this process and its progression.

Les troubles métaboliques caractéristiques d’une alimentation de type « Western diet », sont à l’origine de pathologies cardiovasculaires, première cause de mortalité dans le monde. Il apparait nécessaire d’améliorer la compréhension des mécanismes impliqués dans l’installation des dysfonctions cardiovasculaires afin de pouvoir proposer des stratégies thérapeutiques ou préventives adaptées. Ainsi, le premier objectif de la thèse a été d’évaluer les effets d’une boisson sucrée sur la fonction vasculaire macro- et micro-circulatoire chez des sujets sains, par une approche translationnelle allant de la clinique humaine à un modèle expérimental de rongeur. Nos résultats montrent une altération de la fonction endothéliale en réponse à une prise de boisson sucrée, dans l’ensemble des lits vasculaires. L’exploration des mécanismes sous-jacents ces altérations nous a permis d’identifier l’implication du couple stress-oxydant/voie du NO. Un second objectif de thèse, a été d’étudier l’impact d’un stress métabolique chronique sur la fonction vasculaire et son incidence sur la régulation de la pression artérielle. Comme observé chez certains sujets souffrant de syndrome métabolique, notre modèle de rat ne présentait pas d’hypertension artérielle, malgré une hyperactivité du système sympathique. Ceci semble être expliqué par une compensation endothéliale eNOS-dépendant, qui permet de garantir le maintien d’une pression artérielle normale en dépit de l’effet vasopresseur adrénergique élevé. Le troisième objectif de thèse a porté sur un nouvel élément participant au maintien de l’homéostasie vasculaire et impacté par les situations pathologiques : le tissu adipeux périvasculaire (PVAT). Nos travaux démontrent dans le contexte du SMet, une altération de la voie adiponectine/eNOS dans le PVAT, en parallèle d’une augmentation de la production d’espèces oxygénées réactives.La pratique régulière d’un exercice physique est aujourd’hui reconnue comme une stratégie non-pharmacologique permettant d’impacter à la fois les désordres métaboliques et cardiovasculaires, notamment via une amélioration de la voie du NO. Nos résultats démontrent une limitation de l’apparition des dysfonctions endothéliales causée par une hyperglycémie aigue lorsqu’un protocole d’exercice physique chronique est réalisé. Enfin, l’exercice physique permet également de prévenir les modifications des propriétés vaso-actives du PVAT dans un modèle de rat SMet. Ce phénomène pourrait être expliqué par une amélioration du statut oxydant de la paroi artérielle, et à une potentialisation de la voie adiponectine/eNOS par l’exercice physique / The globalization of the western diet has mediated prevalence in cardiovascular disease related mortality, the single leading cause of death worldwide. Considering this, it is imperative that the underlying mechanisms of cardiovascular dysfunctions are continually investigated to establish a greater understanding of its pathogenesis from a healthy state to the presence of cardiometabolic diseases; and to improve upon current treatment and preventative strategies. Therefore, the first aim of this research was to identify vascular impact of acute hyperglycaemic stress induced by sweet sugar beverage consumption, with a translational approach. The results of this study demonstrated that consumption of a single commercially available sugar-sweetened beverage (SSB) induced transient micro- and macrovascular endothelial dysfunction, even in a healthy population. Further exploration into the underlying mechanisms of SSB-mediated endothelial dysfunction indicated that an increase in oxidative stress disrupts normal function of the nitric oxide pathway. Although disturbances in cardiovascular function may initially be transient, repetitive acute metabolic stress may translate to chronic cardiometabolic disease. Therefore, the second aim of this research was to assess the impact of a chronic metabolic disorder, metabolic syndrome (MetS), on vascular function in a rat model. Despite increasing sympathetic activity, the MetS rats didn’t present elevated arterial pressure. Such findings may be explained by a compensatory adaptation of endothelial function that increases production of nitric oxide in response to α-adrenergic agonist and, thus, regulates arterial pressure despite sympathetic hyperactivity. Considering this, the third aim of this research evaluated the impact of perivascular adipose tissue (PVAT) on vascular fucntion in MetS rats; demonstrating that MetS altered the adiponectin-endothelial nitric oxide synthase pathway in PVAT, in an oxidative stress-dependant manner.Exercise training is well recognized as a non-pharmacological strategy that has a beneficial impact on both metabolic and cardiovascular disorders via an improvement in function of the nitric oxide pathway. Considering this, research also assessed the efficacy of this approach to prevent vascular injury induced by acute hyperglycaemia in a healthy population and by PVAT in those with MetS. It was demonstrated that exercise attenuated acute hyperglycemia-mediated endothelial dysfunction; and restored endothelium-dependent vascular reactivity in rats with MetS, due to an improvement in the biocommunication between PVAT and arterial tissue and a notable enhancement of the adiponectine-endothelial nitric oxide synthase pathway.

Removing NO from the air with a reusable material at room temperature is challenging. In this study, a series of urea–MnOₓ/ACF and urea–x(CeO₂–(1 − x)MnO₂)/ACF materials were prepared and used for removing NO at room temperature. The results showed that 10% urea–8% (0.5CeO₂–0.5MnO₂)/ACF yielded the highest NO conversion, which showed an NO conversion ratio above 90% with 1000 ppm NO in the initial mixed gases. Moreover, the NO conversion exceeded 98% when the NO concentration was 100 ppm in the initial mixed gases. More importantly, 10% urea–8% (0.5CeO2–0.5MnO₂)/ACF was stable even after it was regenerated by reloading with urea, demonstrating that the material could be easily reused and its highperformance was maintained. Finally, the mechanism and kinetics of the NO removal was discussed.

L'ischémie/reperfusion (I/R) est un phénomène très fréquent en clinique humaine. Ce phénomène est observé lors de la désobstruction d'une artère digestive, du traitement d'un état de choc, ainsi qu'au cours d'autres pathologies. L'interruption de la perfusion tissulaire (ischémie) et le rétablissement de celle-ci (reperfusion) sont la cause de la mise en place de troubles hémodynamiques et métaboliques. L'I/R est souvent présentée comme étant la principale source de l'hyperlactatémie et le moteur de la réponse inflammatoire lors des états de choc (cardiogénique, hypovolémique, septique). Parallèlement, elle est responsable de l'induction de la production de la libération des espèces réactives de l'oxygène, des cytokines et du monoxyde d'azote. Suite à un choc hémorragique par Ischémie/reperfusion chez le rat, nous avons montré que 1) le NaHS, donneur d'H2S limite la diminution de la pression artérielle moyenne et diminue le lactate plasmatique, témoin de la souffrance tissulaire, 2) cette amélioration hémodynamique est associée à une baisse de l'expression myocardique des ARNm d'iNOS, une diminution de la concentration des dérivés NOx plasmatiques et une diminution des concentrations aortiques et myocardiques de NO et d'anion superoxyde et 3) l'inhibition d'H2S par la DL-propargylglycine aggrave le tableau hémodynamique et les conséquences tissulaires du choc. Dans un autre modèle d'ischémie/reperfusion intestinale, les résultats obtenus, montrent que l'administration de la Protéine C activée (PCa) ou de la dexaméthaosne (Dexa) : 1) améliore la PAM et la réactivité vasculaire, 2) permet d'augmenter le pH et de diminuer la lactatémie, 3) diminue la production des cytokines pro-inflammatoires et 4) inhibe les médiateurs de l'apoptose. Ces résultats sont reliés à une down régulation d'iNOS, une restauration de la voie Akt/eNOS et à une resensibilisation des adrénorécepteurs alpha. Ces résultats ouvrent de nouvelles perspectives cliniques dans les traitements de l'I/R / Ischemia/reperfusion (I/R) is a very common phenomenon, observed during intestinal artery surgery, shock treatment, as well as in several other diseases. The disruption of tissue perfusion (ischemia) and recovery (reperfusion) induce hemodynamic and metabolic dysfunction. Gut ischemia/reperfusion is often presented as the main source of lactate and the motor of the inflammatory response, such as cardiogenic, hypovolemic and septic shock. In parallel, gut reperfusion produces numerous mediators such as reactive oxygen metabolites, pro-inflammatory cytokines, and high concentrations of nitric oxide. In a model of ischemia/reperfusion induced by hemorrhagic shock, we found that 1) NaHS an injectable form of H2S, limited the decrease in arterial pressure induced by shock and decreased plasmatic lactate, a witness of tissue suffering, 2) this hemodynamic improvement was associated with a fall in myocardial iNOS mRNA expression, a reduction in the concentration of plasmatic NOx and a reduction of aortic and myocardial concentrations of NO and superoxide anion and 3) the inhibition of H2S with DL-propargylglycine worsened hemodynamics and tissue consequences of shock An experimental model of intestinal I/R has been developed, we demonstrated that the administration of APC or Dexa : 1) Improves MAP and vascular reactivity, 2) increased pH and decreased lactate, 3) decreased pro-inflammatory cytokines production and 4) inhibited apoptosis mediators expression. These results are related to a down regulation of iNOS, to a restoration of the AKT/eNOS pathway, and to alpha-adrenoreceptor resensitization. These results open new perspectives in clinical treatment of I/R

Das Bone Morphogenetic Protein 2 (BMP2) gehört zur TGF-beta Superfamilie und findet seinen Fokus in der osteogenen Aktivierung und in der Anwendung bei der Frakturheilung. Es wird angenommen, dass weitere, bisher unbekannte Verbindungen existieren, die die BMP2-Signalübertragung und die osteogene Aktivität verbessern und somit zu einer verbesserten klinischen Wirksamkeit von BMP2 führen. Für den Stickstoffoxid (NO)-Signalweg ist bereits bekannt, dass im endothelialen Kontext eine Verbindung zum BMP2-Signalweg existiert. Ziel dieser Arbeit war es daher, eine Verbindung zwischen dem NO- und BMP2-Signalweg bezüglich der Regulierung des BMP2-abhängigen Signalwegs und der Osteoinduktion aufzuzeigen. Dies erfolgte durch Anwendung von Inhibitoren (LNAME, ODQ und LY83583) und Aktivatoren (L-Arginin, Deta NONOate, SNAP und YC-1) des NO-Signalwegs, in Kombination mit BMP2. Eine mögliche Verbindung zwischen dem BMP2- und NO-Signalweg, über eine Protein Kinase A (PKA) Brücke, wurde durch die Anwendung des PKA Inhibitors H89 untersucht. Zusammenfassend zeigen diese Ergebnisse, dass der NO-Stoffwechselweg den BMP2-vermittelten Signalweg und die osteoinduktive Aktivität modulieren kann, wobei PKA beide Signalwege im Rahmen der BMP Signalübertragung verbindet, jedoch nicht zu einer BMP2-vermittelten Osteoinduktion führt. / Bone Morphogenetic Protein 2 (BMP2) is a TGF-beta superfamily member, with a major focus on osteogenic activity and application in fracture healing. In order to improve efficiency of BMP2 in the clinic, it is assumed that additional, yet unknown compounds can improve BMP2 signalling and osteogenic activity. The Nitric Oxide (NO) pathway has previously shown to be connected with the BMP2 pathway in an endothelial context. Therefore, it was the aim of this study to unravel connections between the NO and BMP2 pathway in regulating BMP2 mediated signalling and osteoinduction. This was carried out through the application of inhibitors (LNAME, ODQ and LY83583) and activators (L-Arginine, Deta NONOate, SNAP and YC-1) of the NO pathway in combination with BMP2. A proposed connection between BMP2 and NO pathways via a Protein Kinase A (PKA) bridge was investigated by application of H89 inhibitor. In summary, these results show that the NO pathway can modulate BMP2 mediated signalling and osteoinductive activity. The PKA bridge connects NO and BMP2 only for the process of BMP2 signalling, but not for BMP2 mediated osteoinduction.

Le réticulum endoplasmique et la mitochondrie sont deux organites majeurs impliqués dans la régulation du métabolisme glucido-lipidique. Ces structures interagissent au niveau de points de contact étroits appelés Mitochondria-Associated Endoplasmique Reticulum Membranes (MAMs). Les MAMs sont une zone de communication et d’échanges, de lipides et de calcium entre autre, indispensables à l’activité des deux organites et au maintien de l’homéostasie cellulaire. Des connexions physiques sont assurées par l’interaction de protéines complémentaires, comme le canal anionique voltage-dépendant (VDAC)-1, la protéine chaperonne (Grp)-75 et le récepteur de l'inositol 1,4,5-triphosphate (IP3R)-1, constituant un complexe impliqué dans le transfert de calcium. D’autres acteurs comme les mitofusines 1 et 2 (MFN1/2) assurent également un rapprochement entre les deux organites et semblent jouer un rôle dans les échanges des lipides. Récemment, les MAMs sont apparues comme un nouveau carrefour de la signalisation de l’insuline dans le foie. Le monoxyde d’azote (NO) participe également au contrôle de la réponse à l’insuline hépatique et a une action spécifique sur la mitochondrie. Mes travaux de thèse ont montré que le NO à des concentrations physiologiques module les interactions entre le RE et la mitochondrie dans le foie et que son action sur les MAMs implique la voie de signalisation sGC/cGMP/PKG. J’ai également démontré que la modulation des MAMs par le NO semble jouer un rôle clé dans la régulation de la voie de signalisation à l’insuline (projet1). Par ailleurs, j’ai exploré l’importance des MAMs dans la régulation du métabolisme lipidique. Pour cela, j’ai modulé l’expression protéique de Grp75 et Mfn2 sur un modèle d’hépatocarcinome humain (Huh7). Mes résultats ont montré qu’une surexpression des deux protéines améliore les MAMs et la β-oxydation mitochondriale mais conduit à une accumulation intracellulaire de lipides. Ceci serait dû à un défaut de sécrétion des lipides dans les lipoprotéines VLDL et pourrait impliquer l’apparition d’un stress mitochondrial et une altération des échanges de phospholipides entre les deux organites (projet 2). Par conséquence mon travail confirme le rôle physiologique des MAMs et éclaire les mécanismes d’actions de cette plateforme cellulaire dans la régulation du métabolisme glucido-lipidique hépatique. A plus long terme ces connaissances participeront peut-être à l’identification de potentielles cibles thérapeutiques afin de prévenir la stéatose et la résistance à l’insuline hépatiques et leurs complications / The endoplasmic reticulum and mitochondria are two major organelles involved in the regulation of glucose and lipid metabolism. These structures interact at close contact points called Mitochondria-Associated Endoplasmic Reticulum Membranes (MAMs). MAMs constitute an area of communication and exchange, of lipids and calcium among others, essential for the activity of both organelles and the maintenance of cellular homeostasis. Physical connections are ensured by the interaction of complementary proteins, such as the voltage-dependent anionic channel (VDAC)-1, the chaperone protein (Grp)-75 and the inositol 1,4,5-triphosphate receptor (IP3R)-1, constituting a complex involved in calcium transfer. Other actors such as mitofusins 1 and 2 (MFN1/2) also connect the two organelles and are involved in lipid exchanges. Recently, MAMs have emerged as a new carrefour for insulin signaling in the liver. Nitric oxide (NO) also helps control the response to hepatic insulin and has a specific action on mitochondria. My thesis work showed that NO at physiological concentrations modulates the interactions between RE and mitochondria in the liver and that its action on MAMs involves the sGC/cGMP/PKG signalling pathway. I also demonstrated that NO modulation of MAMs plays a key role in regulating the insulin signaling pathway (project 1). In addition, I explored the importance of MAMs in the regulation of lipid metabolism. For that purpose, protein expression of Grp75 and Mfn2 was modulated in a human hepatocarcinoma model (Huh7). Results showed that overexpression of both proteins improves MAMs and mitochondrial β-oxidation but leads to intracellular lipid accumulation. This could be due to a defect in lipid secretion in VLDL lipoproteins and could imply the appearance of mitochondrial stress and an alteration of phospholipid exchanges between the two organelles (project 2). Consequently, my work confirms the physiological role of MAMs and sheds light on the mechanisms of action of this cellular platform in the regulation of glucose and lipid metabolism in the liver. In the longer term, this knowledge may contribute to the identification of potential therapeutic targets to prevent steatosis and hepatic insulin resistance and their complications

Nitric oxide (NO) is an important biological molecule within the body, which over production of this molecule in response to different stimulations can cause various inflammatory diseases. Over production of this molecule is caused by the induction of the inducible nitric oxide synthase (iNOS) enzyme. This enzyme uses L-arginine as a substrate and therefore the presence and transport of this amino acid into the cells can be a key factor in regulating NO over production. Different signalling mechanisms have been implicated in the regulation of this pathway and one of which involves the Mitogen Activated Protein Kinases (MAPK). This family of proteins respond to inflammatory conditions and may mediate effects induced by inflammatory mediators. Of the MAPKs, the role of the c-Jun-N-terminal kinase (JNK) pathway in the induction of iNOS is still controversial. JNK and its downstream target, the transcription factor Activator Protein-1 (AP-1), have shown contradictory effects on iNOS induction leading to controversies over their role in regulating iNOS expression in different cell systems or with various stimuli. The studies described in this thesis have determined the role of JNK/AP-1 on iNOS expression, NO production, L-arginine uptake and also on the transporters responsible for L-arginine transport into the cells. The studies were carried out in two different cell types: rat aortic smooth muscle cells (RASMCs) and J774 macrophages which are both critically associated with the over production of NO in vascular inflammatory disease states. The first approach was to block the expression of the inducible L-arginine-NO pathway using SP600125 and JNK Inhibitor VIII which are both pharmacological inhibitors of JNK. The results from these studies showed that the pharmacological intervention was without effect in RASMCs, but inhibited iNOS, NO and L-arginine transport in J774 macrophages. In contrast, the molecular approach employed using two dominant negative constructs of AP-1 (TAM-67 and a-Fos) revealed a different profile of effects in RASMCs, where a-Fos caused an induction in iNOS and NO while TAM-67 had an inhibitory effect on iNOS, NO, L-arginine transport and CAT-2B mRNA expression. The latter was unaffected in RASMCs but suppressed in J774 macrophages by SP600125. Examination of JNK isoforms expression showed the presence of JNK1 and 2 in both cell systems. Moreover, stimulation with LPS/IFN- or LPS alone resulted in JNK phosphorylation which did not reveal any difference between smooth muscle cells and macrophages. In contrast, expression and activation of AP-1 subunits revealed differences between the two cell systems. Activation of cells with LPS and IFN- (RASMCs) or LPS alone (J774 macrophages) resulted in changes in the activated status of the different AP-1 subunit which was different for the two cell systems. In both cell types c-Jun, JunD and Fra-1 were increased and in macrophages, FosB activity was also enhanced. Inhibition of JNK with SP600125 caused down-regulation in c-Jun in both cell types. Interestingly this down-regulation was in parallel with increases in the subunits JunB, JunD, c-Fos and Fra-1 in RASMCs or JunB and Fra-1 in J774 macrophages. Since, SP600125 was able to exert inhibitory effects in the latter cell type but not in RASMCs, it is possible that the compensatory up-regulation of certain AP-1 subunits in the smooth muscle cells may compensate for c-Jun inhibition thereby preventing suppression of iNOS expression. This notion clearly needs to be confirmed but it is potentially likely that hetero-dimers formed between JunB, JunD, c-Fos and Fra-1 could sustain gene transcription in the absence of c-Jun. The precise dimer required has not been addressed but unlikely to exclusively involve JunB and Fra-1 as these are up-regulated in macrophages but did not sustain iNOS, NO or induced L-arginine transport in the presence of SP600125. To further support the argument above, the dominant negatives caused varied effects on the activation of the different subunits. a-Fos down-regulated c-Jun, c-Fos, FosB, Fra-1 whereas TAM-67 reduced c-Jun and c-Fos but marginally induced Fra-1 activity. Associated with these changes was an up-regulation of iNOS-NO by a-Fos and inhibition by TAM-67. Taken together, the data proposes a complex mechanism(s) that regulate the expression of the inducible L-arginine-NO pathway in different cell systems and the complexity may reflect diverse intracellular changes that may be different in each cell type and not always be apparent using one experimental approach especially where this is pharmacological. Moreover, these findings strongly suggest exercising caution when interpreting pure pharmacological findings in cell-based systems particularly where these are inconsistent or contradictory.

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.