Chronic postoperative pain impacts millions of individuals worldwide that undergo a variety of surgical procedures. Opioids remain the mainstay analgesics of acute and perioperative pain; however, prolonged opioid therapy may lead to life-threating adverse effects, tolerance, dependence, and addiction. Therefore, unraveling the cellular mechanisms that drive persistent pain states and opposing endogenous analgesia provided by opioid receptor signaling, may lead to novel analgesics. Evidence suggests that tissue injury leads to increased sensitization of the spinal cord nociceptive neurons which increases susceptibility to chronic pain via an N-methyl-D-aspartate (NMDA) receptor activation of calcium-sensitive adenylyl cyclase isoform 1 (AC1). This phenomenon, named latent pain sensitization (LS), is mediated by a compensatory response of endogenous inhibitory systems. In this dissertation, we test the hypothesis that surgical insult promotes prolonged activation of kappa opioid receptors (KOR) which mask LS via attenuation of pro-nociceptive AC1 signaling pathways in both male and female animals. We employed a murine model of chronic postoperative pain that promotes LS in the spinal cord and closely resembles the phenotypic features of postoperative pain in human subjects. When behavioral signs of hyperalgesia resolved, we targeted spinal opioid receptor systems and pronociceptive modulators with intrathecal delivery of selective pharmacological antagonists and assessed behavioral signs of hyperalgesia and spinal nociceptive sensitization. We propose that LS is kept in remission by a long-lasting compensatory response of tonic endogenous KOR signaling that hinders a pronociceptive LS pathway that includes not only AC1 but also two downstream targets: protein kinase A (PKA) and exchange protein activated by cAMP (Epac1/2) - in a sex-dependent manner. Our results propose new therapeutic targets for the management of persistent postoperative pain and underscore the importance of tailoring sex-specific pain management strategies.

Le récepteur endogène du NO, la guanylate cyclase (sGC) est l'objet de thèse. Cette enzyme synthétise le GMPc après fixation du NO. L'outil principal utilisé est la spectroscopie d'absorption résolue en temps picoseconde-nanoseconde. Nous avons montré que la fixation simultanée du CO et d'activateurs (YC-1, Bay 41-2272) induisent un hème 5c-CO, à l'instar du NO seul, expliquant l'activation synergique. Nous avons identifié toutes les étapes de l'interaction sGC-NO en mesurant la dynamique du NO de la picoseconde à la seconde. Cette dynamique dans la protéine entière est comparée à celle de la sous-unité beta (1-190) isolée et celle de senseurs de NO bactériens. Un mutant de la myoglobine (H93C) à été utilisé comme modèle pour l'étude de l'hème dans les états 4- et 5-coordonnés. Enfin, nous avons mesuré la variation d'absorption dans la bande III de la Mb et Hb pour mesurer le mouvement du Fer de l'hème aprés fixation du NO. Nous avons cherché un inhibiteur potentiel et un ligand endogène de la sGC.

L'autorenouvellement est une propriété fondatrice du concept de cellule souche. Cependant, malgré l'avancée des connaissances actuelles, les mécanismes moléculaires sous-jacents restent mal compris. Nous nous sommes donc intéressés à cette question, en étudiant l'équilibre entre autorenouvellement et différenciation dans des progéniteurs érythrocytaires primaires. D'une part, grâce à une étude combinant des approches pharmacologiques et de génétique fonctionnelle, nos résultats montrent que le contrôle de la synthèse cellulaire du cholestérol joue un rôle essentiel dans la régulation du basculement de l'autorenouvellement vers la différenciation. D'autre part, nous avons étudié la nature stochastique de l'expression génique au cours du passage de l'autorenouvellement vers la différenciation. En effet, contrairement au caractère déterministe initialement attribué à l'expression des gènes, les données accumulées au cours des dernières années démontrent que cette expression repose sur des processus stochastiques. Nous avons en particulier oeuvré à la conception et à la mise en place d'un dispositif permettant de suivre en temps réel l'expression génique dans des cellules individualisées, afin de pouvoir mesurer et évaluer cette stochasticité. Au final, l'ensemble de ces travaux participent à la compréhension des bases moléculaires de l'autorenouvellement et du contrôle des choix du devenir cellulaire.

During development, neurons must find their way to and make connections with their appropriate targets. Growth cones are dynamic, motile structures that are integral to the establishment of appropriate connectivity during this wiring process. As growth cones migrate through their environment, they encounter guidance cues that direct their migration to their appropriate synaptic targets. The gaseous messenger nitric oxide (NO), which diffuses across the plasma membrane to act on intracellular targets, is a signaling molecule that affects growth cone motility. However, most studies have examined the effects of NO on growth cone morphology when applied in large concentrations and to entire cells. In addition, the intracellular second messenger cascade activated by NO to bring about these changes in growth cone morphology is not well understood. Therefore, this dissertation addresses the effects that a spatially- and temporally-restricted application of physiological amounts of NO can have on individual growth cone morphology, on the second messenger pathway that is activated by this application of NO, and on the calcium cascades that result and ultimately affect growth cone morphology. Helisoma trivolvis, a pond snail, is an excellent model system for this type of research because it has a well-defined nervous system and cultured neurons form large growth cones. In the present study, local application of NO to Helisoma trivolvis B5 neurons results in an increase in filopodial length, a decrease in filopodial number, and an increase in the intracellular calcium concentration ([Ca2+]i). In B5 neurons, the effects of NO on growth cone behavior and [Ca2+]i are mediated via sGC, protein kinase G, cyclic adenosine diphosphate ribose, and ryanodine receptor-mediated intracellular calcium release. This study demonstrates that neuronal growth cone pathfinding in vitro is affected by a single spatially- and temporally-restricted exposure to NO. Furthermore, NO acts via a second messenger cascade, resulting in a calcium increase that leads to cytoskeletal changes. These results suggest that NO may be a signal that promotes appropriate pathfinding and/or target recognition within the developing nervous system. Taken together, these data indicate that NO may be an important messenger during the development of the nervous system in vivo.

Signal transduction is a crucial event that enables cells to sense and respond to cues from their immediate environment. Guanylyl cyclase C (GC-C) is a member of the family of receptor guanylyl cyclases. GC-C is a single transmembrane protein that responds to its ligands by the production of the second messenger cGMP. The guanylin family of peptides, (including the bacterially produced heat-stable enterotoxin ST) is the ligand for GC-C, elevates intracellular cGMP levels and activates downstream pathways. GC-C regulates the cystic fibrosis transmembrane conductance regulator (CFTR) by inducing phosphorylation by protein kinase G, resulting in chloride ion and fluid efflux. GC-C also regulates cell cycle progression through cGMP-gated Ca2+ channels. These functions are seen in the intestinal epithelium, the primary site for GC-C expression. GC-C as a molecule has been studied in detail, but its functioning in the context of other signaling pathways remains unknown. The aim of the present investigation was to understand the regulation of signal transduction by GC-C and its cross-talk with other signaling pathways operating in the cell. Molecular events that commonly connect components in a signaling pathway are protein phosphorylation and protein-protein interaction. These two aspects are explored in this thesis. The possibility of tyrosine phosphorylation of GC-C has been explored earlier in our laboratory. In vitro studies indicated that the residue Tyr820 was a site for phosphorylation by the Src family of non-receptor tyrosine kinases and those studies also suggested that phosphorylated Tyr820 could bind to the SH2 domain of Src. We generated a nonphosphorylatable mutant of GC-C, GC-CY820F, and a phosphomimetic mutant GC-CY820E to study the effect of phosphorylation of Tyr820, on the functioning of GC-C. A stable cell line of HEK293:GC-CY820F cells was generated and compared with HEK293:GC-CWT. Dose response to ST in the two cell lines showed that cGMP accumulation by GC-CY820F was greater than that of GC-CWT, although the EC50 remained unchanged. The phosphomimetic GC-CY820E mutant receptor was non-responsive to ST. Further in HEK293 cells, phosphorylation of GC-CWT by constitutively active v-Src resulted in decreased ST stimulation and this effect of v-Src was reduced with GC-CY820F. Inhibition of ST stimulation brought about by v-Src required catalytically active Src, as the kinase inactive v-SrcK295R did not inhibit ST stimulation. These results were corroborated by in vitro studies by using the recombinant catalytic domain of GC-C expressed in insect cells and by phosphorylation using a purified kinase, Hck. Observations suggested that phosphorylation of Tyr820 in the catalytic domain of GC-C compromises the guanylyl cyclase activity of GC-C. T84 and Caco-2 colon carcinoma cells endogenously express GC-C. The effect of tyrosine phosphorylation of GC-C was studied by using HgCl2, a known activator of Src kinases, and by the inhibition of protein tyrosine phosphatases using pervanadate, an irreversible inhibitor. Both these ways of achieving increased tyrosine phosphorylation resulted in decreased ST-stimulated cGMP production by GC-C, as suggested from v-Src transfection studies. This decrease was reversed by using a Src kinase specific inhibitor PP2, confirming the role of Src kinases in the inhibition of GC-C activity. Interestingly, in Caco-2 cells that differentiate in culture, the effect of pervanadate on the inhibition of ST-stimulated GC-C activation was dependent on the differentiation stage. Crypt-like cells showed higher inhibition with pervanadate. As they matured into villus-like cells, the effect of pervanadate on GC-C activation was gradually lost. This effect also correlated with a decrease in the expression of Lck, suggesting that in the context of the intestine there could be differential regulation of tyrosine phosphorylation of GC-C along the crypt-villus axis. Intestinal ligated loop assays in rats demonstrated that ST-induced fluid accumulation in the intestine was abrogated on pervanadate treatment. Reduction in this fluid accumulation by pervanadate was not observed with 8-Br-cGMP, a cell permeable analogue of cGMP. This indicated that tyrosine phosphorylation of proteins is important for ST-induced fluid accumulation, and perhaps pervanadate modulates this by phosphorylation of GC-C, thereby causing a reduction in fluid accumulation. Earlier in vitro studies on Src-SH2 binding from the laboratory had suggested the possibility of activation of Src family kinases by GC-C. The activation status of Src kinases was monitored by using phosphorylation-state specific antibody, pSFK416. ST stimulation in T84 cells increased Tyr416 phosphorylation of Src kinases in a time dependent manner, indicating that Src kinases are activated downstream of GC-C. This activation of Src kinases was also seen with the endogenous ligand of GC-C, uroguanylin. Interestingly, 8-Br-cGMP a cell permeable analogue of cGMP that is known to mimic other cellular effects of GC-C, namely Cl-secretion and cell cycle progression, did not activate Src kinases, suggesting that the mechanism of Src kinase activation by GC-C could be independent of cGMP. Binding affinities of Src, Lck, Fyn and Yes SH2 domains to Tyr820 phosphorylated GCC peptide were in the nM range, indicating a high affinity of interaction. In vitro GST-SH2 pull down experiments suggested that phosphorylation of Tyr820 in full length GC-C allows interaction of GC-C to the SH2 domain of Src. These studies suggest a dual cross-talk between Src kinases and GC-C; Src phosphorylation inhibits GC-C signaling and stimulation of GC-C by its ligands activates Src kinases. Interaction of proteins containing SH2 and SH3 domains are commonly found in signaling molecules. In accordance with the observation that there are three PXXP motifs in GCC, many SH3 domains could interact with GC-C. GC-C appears to show a preference to bind the SH3 domains of Fyn, Hck, Abl tyrosine kinases, Grb2 and Crk adaptor proteins, the α-subunit of P85 PI3 kinase, PLC-γ and cortactin to various extents. The SH3 domains of spectrin and Nck did not show any detectable interaction with GC-C. In SH3 pull-down assays, the N-terminal SH3 domain of Crk, CrkSH3 (N), bound GC-C maximally, suggesting that Crk is a good candidate for interaction with GC-C. By overlay analysis, the region of GC-C that binds CrkSH3 (N) was narrowed down to the catalytic domain of GC-C containing a ‘PGLP’ motif. Mutations were generated in GC-C at this site to generate GC-CP916Q and GC-CW918R. These mutations compromised the binding of full length receptor to CrkSH3 (N). In cells, CrkII and GC-C co-transfection inhibited the ST stimulation of GC-C. A CrkII mutant, that has compromised binding through its SH3 domain, did not inhibit the activity of GC-C. CrkII from T84 cells co-immunoprecipitated with GC-C and interestingly, the phosphorylated form of CrkII did not, indicating that GC-C - Crk interaction could be regulated by the phosphorylation of Crk. In summary, this study places GC-C, in the context of tyrosine kinase signaling pathway and interaction with the adaptor protein Crk. These studies suggest that GC-C signal transduction can be altered by cross-talk with other signaling events in the cell. Reversible phosphorylation of tyrosine residues inhibits the activity of GC-C, and this is mediated by Src family kinases. Src kinases themselves are activated on stimulation of GC-C by its ligands, possibly because of SH2 domain interaction with GC-C. Association of Crk by its SH3 domain regulates GC-C functioning primarily by inhibiting ST-stimulated cGMP production. This opens up the possibility of GC-C signaling through a multimeric complex involving other binding partners of Crk, and these cross-talks involving GC-C with the two proto-oncogenes, Src and Crk, might have far reaching consequences in the regulation of cellular functions.

Natural products represent an incredibly diverse set of chemical structures and activities. Given this fathomless, ever-evolving diversity, a reasonable approach to designing new molecules entails taking a closer look at the biochemistry that Nature has crafted over billions of years on Earth. In particular, much can be learned by unveiling the architectures of proteins, life’s molecular machines, through methods like X-ray crystallography. Acquiring the blueprints of an enzyme brings us closer to understanding the mechanism by which the enzyme transforms a simple substrate it into a complex product with biological function, and inspires us to engineer such systems to our own ends. With a focus on macromolecular structural characterization, this document elaborates on five Gram-negative bacterial biosynthetic enzymes from two categories: Cell-surface modifiers and polyketide synthases. Among the first category are the glycyl carrier protein AlmF and its ligase AlmE of Vibrio cholerae and the phosphoethanolamine transferase EptC of Campylobacter jejuni. These proteins are responsible for decorating cell-surface molecules (e.g., lipid A) of pathogenic bacteria with small functional groups to promote antibiotic resistance, motility, and host colonization. AlmE and EptC represent potential drug targets and their structures lay the groundwork for the design of therapeutics against food-borne illnesses. Included in the second category are the [4+2]-cyclase SpnF and two ketoreductase-linked dimerization elements, each from the spinosyn biosynthetic pathway in Saccharopolyspora spinosa. The former catalyzes a putative Diels-Alder reaction to form a tricyclic precursor of the insecticide spinosad, while the latter two organize ketoreductase domains within modules of a polyketide synthase. The second category also includes Ralstonia eutropha β-ketoacyl thiolase B, a substrate-permissive enzyme that can make or break carbon-carbon bonds with assistance from Coenzyme A or an analogous thiol. Each of these proteins exhibit intriguing structural features or catalyze reactions that show promise for biochemical engineering. / text

Le récepteur A des peptides natriurétiques (NPRA) fait partie de la famille des guanylates cyclases membranaires. L’activation du NPRA par ses agonistes naturels, ANP et BNP, induit une production de GMPc qui est responsable de leur rôle dans l’homéostasie cardiovasculaire, l’inhibition de l’hypertrophie et de la fibrose cardiaques et la régulation de la lipolyse. Le NPRA est un homodimère non covalent composé d’un domaine extracellulaire de liaison du ligand (ECD), d’un unique domaine transmembranaire (TM), d’un domaine d’homologie aux kinases et d’un domaine guanylate cyclase. Bien que le NPRA ait un rôle physiologique important, les mécanismes moléculaires régissant son processus d’activation restent inconnus. Nous avons donc analysé les premières étapes du processus d’activation du NPRA. Nous avons d'abord étudié le rôle de la dimérisation des ECD dans l’activation du récepteur. Nous avons utilisé les techniques de liaison de radioligand, de FRET et de modélisation moléculaire, pour caractériser la liaison à l’ECD des agonistes naturels, d’un superagoniste et d’un antagoniste. L’ANP se lie à un dimère d’ECD préformé et la dimérisation spontanée est l’étape limitante du processus de liaison. De plus, comme le démontrent nos études de FRET, tous les peptides, incluant l’antagoniste, stabilisent le récepteur sous sa forme dimérique. Cependant, l’antagoniste A71915 stabilise le dimère d’ECD dans une conformation différente de celle induite par l’ANP. La dimérisation du NPRA semble donc nécessaire, mais non suffisante à l’activation du récepteur. L’état d’activation du NPRA dépend plutôt de l’orientation des sous unités dans le dimère. Nous avons ensuite étudié le mécanisme moléculaire de transduction du signal à travers la membrane. Plusieurs études ont suggéré que l’activation du NPRA implique un changement de conformation du domaine juxtamembranaire (JM). Cependant, les études de cristallographie de l’ECD soluble de NPRA n’ont pas permis de documenter la structure du JM et le changement de conformation impliqué dans la transduction du signal reste inconnu. Pour analyser ce changement de conformation, nous avons d’abord séquentiellement substitué les neuf acides aminés du JM par une cystéine. En étudiant la capacité des mutants à former des dimères covalents de façon constitutive ou induite par l’ANP, nous avons pu évaluer la proximité relative des résidus du JM, avant et après activation du NPRA. Ces résultats ont démontré la proximité élevée de certains résidus spécifiques et sont en contradiction avec les données cristallographiques. Nous avons également démontré que le domaine intracellulaire impose une contrainte conformationnelle au JM à l’état de base, qui est levée après liaison de l’ANP. En introduisant de 1 à 5 alanines dans l’hélice-α transmembranaire, nous avons montré qu’une rotation des TM de 40° induit une activation constitutive du NPRA. Le signal d’activation pourrait donc être transmis à travers la membrane par un mécanisme de rotation des TM. En utilisant nos données expérimentales, nous avons généré le premier modèle moléculaire illustrant la conformation active du NPRA, où les domaines JM et TM sont représentés. Dans son ensemble, cette étude apporte une meilleure compréhension des mécanismes moléculaires régissant les premières étapes du processus complexe d’activation du NPRA. / Natriuretic peptide receptor-A (NPRA) is a member of the particulate guanylate cyclase family. NPRA activation by natural agonists, ANP and BNP, leads to cGMP production, which is responsible for their role in cardiovascular homeostasis, cardiac hypertrophy and fibrosis inhibition and lipolysis regulation. NPRA is a non covalent dimer composed of an extracellular domain (ECD) with a ligand binding site, a single transmembrane region (TM), a kinase homology domain, and a guanylyl cyclase domain. Although NPRA plays an important physiologic role, molecular mecanisms driving its activation process are yet unknown. We thus analysed the first steps of NPRA’s activation process. First, we studied the role of ECD dimerization in receptor activation and determined the sequential steps of this dimerization process. We used radioligand binding, FRET and molecular modeling to characterize the interaction of ECD with natural agonists, a superagonist and an antagonist. ANP binds to preformed ECD dimers and spontaneous dimerization is the rate-limiting step of the ligand binding process. Furthermore, like demonstrated with fluorescence homoquenching, all the studied peptides, including A71915 antagonist, stabilize a dimeric form of the receptor. However, A71915 stabilizes the ECD dimer in a conformation distinct from those induced by ANP. Thus, ECD dimerization is necessary but not sufficient for NPRA activation. The activation state of NPRA seems to depend on the orientation of the receptor subunits within the dimer. Then, we tried to identify the molecular mechanism of signal transduction through the plasma membrane. Previous studies have shown that activation of NPRA involves a conformational change of the juxtamembrane domain (JM). However, crystallographic study of the soluble ECD of NPRA has failed to document JM structure, and the conformational change involved in transmembrane signal transduction is still unknown. To analyse this conformational change, we first sequentially substituted nine amino acids of JM by a cysteine residue. By studying the mutant’s capacity to form ANP-induced or constitutive covalent disulfide dimers, we evaluated the relative proximity of JM residues, before and after NPRA activation. These results demonstrate a high proximity of specific JM residues and are in disagreement with crystallography data. We also demonstrated that intracellular domain imposes a conformational constraint on JM at basal state, which becomes relaxed upon ANP binding. We finally confirmed, with a full-length receptor, that A71915 stabilizes NPRA in a dimeric form where JM are in a conformation distinct from the basal state. By introducing 1 to 5 alanine residues in the transmembrane α-helix, we showed that a TM rotation of 40° leads to constitutive NPRA activation. Activation signal could thus be transmitted through the membrane by a TM rotation mechanism. We finally studied the role of the TM in NPRA dimerization. By using the ToxR system, we demonstrated that the last JM residues are required to stabilize the TM dimer. Using these experimental data, we generated the first molecular model illustrating the active conformation of NPRA, where JM and TM are depicted. In summary, this study allows a better understanding of molecular mecanisms driving the first steps of NPRA’s complex activation process.

No description available.

Human schistosomiasis popularly known as bilharzias in many regions of Africa is a freshwater snail-transmitted disease caused by parasitic flatworms known as schistosomes. The growth and development of schistosomes typically requires developmental stages in multiple hosts and transmission stages in freshwater. These life cycle environments present a plethora of stressors. Certain gene families including heat shock proteins (HSPs/Hsps) and universal stress proteins (USPs) help schistosomes to respond to unfavourable conditions. The availability of genomes sequences information for Schistosoma japonicum, Schistosoma mansoni and Schistosoma haematobium provide unique research resources to apply bioinformatics analysis of its associated USPs to predict regulatory features from sequence analysis. The objectives of the research were to (i) Infer the biochemical and environmental regulation of universal stress proteins of Schistosoma species; (ii) Identify biological function relevant protein sequence and structure features for prioritized universal stress proteins from Schistosoma species; (iii) Determine the distinctive structural features of a predicted regulator of Schistosoma adenylate cyclase activity that has possible influence on the functioning of universal stress proteins. The findings revealed that (i) schistosomes USPs are hydrophilic and very reactive in the water environment or in aqueous phase, which seems adaptive with their immediate environment and developmental stages; (ii) The functions of Smp_076400 and Sjp_0058490 (Q86DW2) are regulated by conserved binding site residues and metallic ions ligands (Ca2+, Mg2+ and Zn2+), particularly Ca2+ predicted to bind to both USPs; (iii) The S. mansoni life cycle and stress resistance pathway protein (Smp_059340.1) is regulated by Ser53, Thr188, Gly210 and Asp207 residues. The overall scope has highlighted the role of bioinformatics in predicting exploitable regulatory features of schistosome universal stress proteins and biological pathways that might lead to identification of putative functional biomarkers of common environmental diseases. The findings of this research can be applicable to other areas of environmental health and environmental genomics. / Environmental Sciences / (D. Litt et Phil. (Environmental Sciences)

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No. of bitstreams: 2 Tese - Patrícia Ferreira da Silva Castro - 2015.pdf: 4272048 bytes, checksum: bb8709720dbc4fb91db81ca23004d59d (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2015-03-26 / Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG / Nitric oxide is a potent bronchodilator and compounds capable of increasing its supply have demonstrated clinical interest in the treatment of obstructive airways diseases. This study evaluated and compared the mechanisms of relaxation of two nitric oxide donors, ruthenium complex [Ru(terpy)(bdq)NO+]3+ (TERPY) and sodium nitroprusside (SNP) in healthy isolated trachea, Sham group, or experimental model of asthma induced by ovalbumin in rats, OVA group. The isolated trachea was sectioned into rings and contracted with carbachol in an organ chamber for studying relaxation. The relaxing effect of TERPY and SNP was evaluated at increasing concentrations from 10 nM to 100 μM. Thus, we verified the contribution of the different types of K+ channels, the importance of sGC/cGMP pathway, the inhibition of PDEs (for IBMX, amrinone, rolipram and dypiridamole), the influence of the extra and intracellular Ca2+ sources (for cyclopiazonic acid) and the influence of the store and voltage operated calcium channels. Besides inhibition of COX (diclofenac), antagonism of leukotriene receptor (montelukast) and superoxide anion scavenger (TIRON). Analyses were performed under light microscopy for evidence of inflammatory infiltration and bronchoditation by TERPY in slices bronchioles of asthmatic animals. The results verified that sensitization with ovalbumin led to intense inflammatory process and hyperresponsives to carbachol in compared Sham group. TERPY and SNP led to the relaxation of tracheal smooth muscle preparations in a dependent-concentration mode in both groups. However, the maximum effect induced by TERPY was higher than the effect of SNP in Sham and OVA groups. The relaxation mechanism of TERPY in boht groups showed differences. In Sham group, TERPY relaxation by the activation of Kv, Kir, KCa and KATP channels, cGMP-independent mechanisms and by reduction of calcium influx by blocking the store and voltage operated calcium channels. In OVA group, TERPY acts through activation K+ channels, NO/GCs/GMP way and blocking the store and voltage operated calcium channels. The relaxing effect induced by SNP in OVA group was dependent of NO/GCs/GMP pathway, Kv, KCa and BKCa channels and blocking the store and voltage operated calcium channels. However, the activation of the enzyme sGC seems to be reduced in inflamed smooth muscle, as well as the role of the sarcoplasmic reticulum calcium pump. Diclofenac, montelukast and TIRON improved relaxation of the TERPY and SNP in OVA group. The TERPY is able to reverse the contraction of carbachol in asthmatic bronchioles. In conclusion, TERPY and SNP have their mechanisms of relaxation modified by the inflammatory process. However, this modification was not able to alter the pharmacological parameters potency and efficacy to TERPY. Since SNP has less efficacy effect in asthmatic tracheas. This may result from the lower participation of the enzyme sGC and reticular calcium pump, making TERPY a promising drug to reverse the narrowing of the airways. / O óxido nítrico é um potente broncodilatador e compostos capazes de aumentar a sua oferta têm demonstrado interesse clínico no tratamento das doenças obstrutivas das vias aéreas. Estee trabalho avaliou comparativamente os mecanismos de relaxamento de dois doadores de óxido nítrico, o complexo de rutênio [Ru(terpy)(bdq)NO+]3+ (TERPY) e o nitroprussiato de sódio (SNP) em traqueias isoladas de ratos saudáveis, grupo Sham, e com asma experimental induzida por ovalbumina, grupo OVA. As traqueias isoladas foram cortadas em anéis, montadas em banho para órgãos isolados e contraída com carbacol para estudo do relaxamento. O efeito relaxante do TERPY e do SNP foi avaliado em concentrações crescentes e cumulativas (10 nM a 100 μM). Foi verificada a participação dos diferentes tipos de canais de K+, a participação da via GCs/GMPc, inibição das PDEs (pelo IBMX, amrinona, rolipram e dipiridamol), a participação dos estoques internos de Ca2+ (pelo ácido ciclopiazônico), assim como a participação do influxo deste íon pelos canais de cálcio controlados por estoque e por voltagem, além da inibição da COX (pelo diclofenaco), do antagonismo dos receptores de leucotrienos (pelo montelucaste) e do sequestro dos íons superóxidos (pelo TIRON). Foram realizadas análises em microscopia de luz para comprovação da presença de infiltrado inflamatório e da broncodilatação exercida pelo TERPY em cortes de bronquíolos de animais asmáticos. Como resultados, verificou-se que a sensibilização com ovalbumina levou a um intenso processo inflamatório com migração celular e hiperreatividade ao carbacol. Evidenciou-se que o TERPY e o SNP relaxaram o músculo liso traqueal de forma concentração-dependente em ambos os grupos. Entretanto, o efeito máximo induzido pelo TERPY foi maior do que o do SNP tanto no grupo Sham quanto no OVA. O mecanismo de relaxamento do TERPY mostrou-se diferente entre os grupos. No grupo Sham, o TERPY exerce relaxamento por ativação dos canais Kv, Kir, KCa e KATP independentemente de GMPc e por redução do influxo de cálcio através do bloqueio dos canais de cálcio operados por voltagem e por estoque. No grupo OVA, o TERPY exerce seu efeito através da por ativação dos canais K+, via NO/GCs/GMP e redução do influxo de cálcio por bloqueio dos canais de cálcio operados por voltagem e operados por estoque. O efeito relaxante do SNP no grupo OVA ocorre através da ativação da via NO/GCs/GMPc, dos canais Kv, KCa e SKCa e por redução do influxo de cálcio pelos canais de cálcio operados por voltagem e operados por estoque. Entretanto, a ativação da enzima GCs parece estar reduzida em músculo liso inflamado, assim como o papel da bomba de cálcio do retículo sarcoplasmático. O diclofenaco, o montelucaste e o TIRON melhoraram o perfil de relaxamento tanto do TERPY quanto do SNP no grupo OVA. O TERPY é capaz de reverter a contração do carbacol em bronquíolos asmáticos. Em conclusão, tanto o SNP quanto o TERPY têm seus mecanismos de relaxamento modificados pelo processo inflamatório. Entretanto, esta modificação não foi capaz de alterar os parâmetros farmacológicos de potência e eficácia do TERPY. Já o SNP, tem menor eficácia em traqueias de ratos do grupo OVA e isso pode decorrer da menor participação da enzima GCs e da bomba de cálcio reticular, fazendo do TERPY uma droga promissora para reversão do estreitamento das vias aéreas.