A computational model for mitochondrial function has been developed from oxygen concentration data measured in the Oroboros Oxygraph-2k and oxygen consumption rates measured in the Seahorse XF24 Analyzer. Measurements were acquired using embryonic-cultured cortical neurons and isolated mitochondria from CD1 mice. Based on the biological mechanism of mitochondrial activity, a computational model was developed using biochemical kinetic modelling. To modulate mitochondrial activity, dysfunctions were introduced by injecting the inhibiting reagents oligomycin, rotenone, and antimycin A, and the uncoupling reagent carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) during measurements. To incorporate these changes, model equations were adapted and globally calibrated to experimental data using the genetic algorithm developed by Jason Fiege of the University of Manitoba by fitting oxygen concentration data. The model was coded in MATLAB R2014a along with the development of a graphical user interface for simulating mitochondrial bioenergetics in silico. / October 2016

eEF-2K, also known as CaM kinase-III, is an atypical protein kinase which negatively regulates the global rate of protein synthesis through the phosphorylation and inactivation of its substrate eEF-2. Recently eEF-2K has been validated as a novel target for anti-cancer therapy. However, a detailed understanding of the role of eEF-2K in cancer biology is unavailable. Mechanistic studies can often provide an understanding of enzyme function. Therefore, we determined the kinetic mechanism of eEF-2K using a peptide substrate (Acetyl-RKKYKFNEDTERRRFL-amide). We found that eEF-2K adopts a ternary-complex, steady state ordered mechanism, with ATP binding required before the peptide substrate. A good cellular inhibitor is required for elucidating the role of eEF-2K in cancer biology. To date, NH125 is the only inhibitor used to investigate the activity of eEF-2K in cells. Although it is reported as a specific inhibitor of eEF-2K, its exact mode of action has not been reported. Through in-vitro assays and cellular studies, we found that NH125 is a non-specific inhibitor of eEF-2K that blocks eEF-2 phosphorylation in cells. There is a great demand for specific inhibitors of eEF-2K. We developed a fluorescence high throughput assay system for eEF-2K. The assay utilizes the peptide substrate labeled with a Sox moiety whose phosphorylation can be monitored at 485 nm in the presence of magnesium. We also validated the assay in a screen of 30,000 compounds in 384 well plates. We found the assay to be robust and identified a relatively specific inhibitor of eEF-2K and determined its mechanism of action. We found it behaved as a slowly reversible inhibitor of eEF-2K with a two step inhibition mechanism - fast initial binding at the enzyme active site, followed by a slower inactivation step. We propose that the nitrile group on the compound binds to the active site thiol in the enzyme covalently forming a reversible thioimidate adduct to inactivate the enzyme. / text

<p>A successful flaviviral life cycle involves several coordinated events between viral proteins and host factors. The polyprotein processing at the surface of the ER membrane results in the formation of several replication proteins that bring about changes in the ER membrane making it permissive for viral genome amplification. Non-structural proteins 4A (NS4A) and non-structural protein 4B (NS4B) are two of the most important integral membrane proteins of DENV that are essential part of the viral replicase complex. The cleavage at NS4A-2K-NS4B is temporally and spatially regulated. The cleavage at the N-terminal of 2K is carried out by viral NS2B/3 protease while host signalase cleaves on the C-terminal side at the ER lumen to give rise to a mature NS4B protein. This thesis primarily focuses on demonstrating the function of 2K as an independent peptide rather than simply a signal sequence, and the role 2K plays, when present as 2K-NS4B vs NS4B. Moreover, this thesis has attempted to explore the function of transmembrane domains (TMDs) in replication separating them from their membrane anchor function. This thesis will also describe the development of a ZIKV replicon and its use in screening small molecule inhibitors in the last chapter.</p><p>In Chapter 2 of the thesis, we established 2K as an independent, information carrying peptide rather than just a signal peptide. A strategy involving chimeric virus generation and mutational analysis supported the notion that 2K is rather unique and important for viral replication and infectious particle production. Using an interserotypic 2K chimeric virus, it was established that the 2Ks of DENV are serotype specific, however, they are interchangeable with a huge fitness cost in infectious particle production. We further showed that individual amino acid residues towards then end of h-region and C-terminus of the 2K peptide affect viral replication and infectious particle production. Moreover, it was shown that the 2K peptide consists of a highly conserved ‘DNQL’ region at its N-terminal that plays an important role in viral replication.</p><p>Chapter 3 details the mechanistic aspect of the effects observed in interserotypic 2K chimeric viruses. The interserotypic chimeric viruses were comparable to wild type in replication, however, they were deficient in infectious particle production early in the life cycle. The major change to be noted in the chimeric viruses was the absence of signalase cleavage at the 2K-NS4B junction. We demonstrated that in a virus infected system, 2K-NS4B and NS4B populations are always present which led us to look for any specific functions of the cleaved vs uncleaved 2K-NS4B protein. Using a transcomplementation system where NS4B was presented in the absence of 2K, we showed that particle production can be rescued in the interserotypic 2K chimeric viruses. It was further concluded using NS4B truncations that the property of NS4B to rescue particle production was concentrated in the ER luminal loop. Further, alanine scanning mutagenesis of the conserved residues of ER loop resulted in pinpointing T198 and its involvement in the early stages of viral packaging.</p><p>Chapter 4 examined the role of TMDs of NS4A and NS4B and attempted to define their roles separately from their membrane anchoring functions. Several interserotypic TMD chimeric viruses were generated to address the function of these domains. We concluded that TMD1 and TMD3 of NS4A could be replaced with partial success across the DENV serotypes, whereas, TMD2 was serotype specific. The specificity of TMD2 of NS4A is not contributed by a single amino acid and should be a function of the secondary structure formed by TMD2 as it sits on the inner leaflet of the ER membrane. We demonstrated the variable roles different TMDs of NS4B play in viral replication using a similar strategy of reverse genetics of chimeric viruses. TMD1 of NS4B was replaceable with no to minimal effect, whereas, the remaining four showed variable effect upon substitution. More importantly, we demonstrated how the reorientation of TMD5 of NS4B post NS2B/3 cleavage might vary in different serotypes of DENV using revertant virus obtained from the TMD5 interserotypic chimera. Analysis of interserotypic cytosolic and ER luminal loop chimeras of NS4B pointed to functional conservation of the cytosolic loop between DENV-2 and DENV-3, whereas, the remaining cytosolic loops and the ER loops showed variable level of defects upon substitution, suggesting their functions in serotype-dependent manner.</p><p>Chapter 5 describes the construction and characterization of a ZIKV replicon system and use of it to screen several small molecule inhibitors of the flaviviruses MTase. Several small molecule inhibitors of flavivirus N-7-MTase were designed/synthesized in Dr. Arun K Ghosh’s lab which would target the extra pocket unique to the flavivirus SAM-binding site. We analyzed the docking of a set of these compounds into MTase domain of NS5 of ZIKV, DENV and YFV and screened them for their ability to inhibit replication of ZIKV, DENV and YFV. A huge variation in the activity profile of these compounds were observed against different flaviviruses even though these compounds were targeted against the highly conserved MTase domain of flavivirus NS5. GRL-002- and GRL-004-16-MT specifically inhibited ZIKV replication with low micromolar IC₅₀ value, while these compounds showed little to no effect on DENV and YFV.<b> </b>On the other hand, compounds GRL-007-, GRL-0012- and GRL-0015-16-MT demonstrated a dual inhibitory effect against DENV and YFV albeit the CC₅₀ values of the GRL-012 and GRL-015 were concerning. Compounds GRL-007-16-MT showed broad spectrum activity against ZIKV, DENV and YFV even though it was slightly cytotoxic to Vero cells. Moreover, GRL-002-16 was inhibitory to YFV while ineffective against DENV, whereas, GRL-016-16 had the opposite effect. Our results reveal the differential efficacies of the small molecule inhibitors targeting N-7-MTase. The experimental data suggests these compounds have different cytotoxicities in different cell lines and the compounds act in a virus-specific way. Nonetheless, we were able to shortlist some potent compounds for future modifications.</p>

O óxido nítrico (NO) é o principal agente vasodilatador endógeno que regula o tônus e a homeostase vascular, além de controlar o fluxo sanguíneo. Doadores de NO, entre eles os nitratos orgânicos, são importantes medicamentos para o tratamento de doenças cardiovasculares. O grande benefício clínico desses nitratos é atribuído ao seu efeito venodilatador. Isto se deve ao seu efeito sobre a redução do retorno venoso, da pré-carga cardíaca e da demanda de oxigênio pelo miocárdio. Porém, um dos efeitos adversos mais comuns dos nitratos orgânicos é a cefaléia causada pela vasodilatação cerebral. Os doadores de NO utilizados clinicamente, nitroglicerina (NTG) e nitroprussiato de sódio (NPS), possuem algumas limitações como indução de tolerância e toxicidade, respectivamente. Dentre os compostos amplamente estudados, que são capazes de liberar NO, estão os complexos nitrosilos de rutênio. Estes complexos têm interesse terapêutico devido à sua baixa toxicidade. Recentemente, verificamos que o complexo de rutênio [Ru(terpy)(bdq)NO]3+ (Terpy), sintetizado em nosso departamento, reduz a pressão de ratos hipertensos renais 2R-1C e promove relaxamento vascular da aorta desses animais e de ratos normotensos controles (2R). Desta forma, a hipótese deste trabalho é de que o Terpy seja capaz de induzir relaxamento vascular em anéis de artéria basilar e veia cava inferior, tanto de ratos normotensos (2R) quanto de ratos 2R-1C. O nosso estudo teve como objetivo estudar os efeitos deste composto doador de NO e do doador de NO de referência, NPS, e os seus mecanismos de relaxamento vascular na artéria basilar e veia cava inferior de ratos normotensos 2R e hipertensos renais 2R-1C. Nossos resultados demonstram que o Terpy, ao contrário do NPS, não promoveu relaxamento vascular na artéria basilar de ratos 2R e 2R-1C. Da mesma forma, o Terpy não liberou NO nas células do músculo liso vascular. O NPS liberou NO e induziu relaxamento da artéria basilar pela ativação da enzima guanililciclase solúvel (GCs), com conseqüente ativação da proteína quinase dependente de GMPc (GK) e ativação dos canais para K+(KV, KATP e KIR). Ambos doadores, assim como a NTG, promoveram relaxamento vascular em anéis de veia cava e artéria basilar de ratos 2R e 2R- 1C de forma dependente da concentração. O relaxamento das veias de ratos 2R-1C foi menor do que em veias de 2R para os doadores de NO: Terpy, NPS e NTG. A liberação do NO pelo Terpy foi menor nas veias de 2R-1C. O NPS induz relaxamento da veia cava inferior pela ativação da enzima GCs com conseqüente ativação da proteína GK e ativação de canais para K+ sensíveis ao TEA. O Terpy induziu relaxamento da veia cava inferior pela ativação da enzima GCs, com conseqüente ativação da GK, ativação da Ca2+-ATPase reticular (SERCA) e ativação dos canais para K+ (KV, SKca e BKca). Em conjunto, nossos resultados mostraram que o Terpy é menos potente que o doador de referência (NPS) na veia cava inferior de ratos 2R e 2R-1C. Sua resposta vasodilatadora se deve principalmente à ativação da GCs, de canais para K+,proteína GK e SERCA. O Terpy, ao contrário do NPS, não induz relaxamento na artéria basilar de ratos 2R e 2R-1C. A resposta vasodilatadora do NPS nesses vasos se deve principalmente à ativação da GCs, de canais para K+ e proteína GK. A SERCA parece não estar envolvida no mecanismo de relaxamento vascular induzido pelo NPS. / Nitric oxide (NO) is an endogenous vasodilator that regulates vascular tone, homeostasis and blood flow. NO donors, including organic nitrates are important drugs for the treatment of cardiovascular diseases. A major clinical benefit of NO donors is attributed to their venodilator effect, resulting in decreased venous return, cardiac preload, arterial pressure and decreased myocardial oxygen demand. But the most common side effect of these drugs is the headache, which is caused by cerebral vasodilatation. The clinically used NO donors, nitroglycerin (NTG) and sodium nitroprusside (SNP), have some limitations such as induction of tolerance and toxicity, respectively. Among the widely studied compounds, which are capable of releasing NO are the nitrosyl ruthenium complexes, which have therapeutic interest due to its low toxicity. Recently, we found that the ruthenium complex [Ru (terpy)(BDQ)NO]3+ (Terpy) reduces the blood pressure of renal hypertensive rats (2K- 1C) and promotes vascular relaxation in aorta from 2K-1C and normotensive rats (2R). Thus, the hypothesis of the present work was that Terpy is able to induce vascular relaxation in basilar artery and inferior vena cava rings in 2K and 2K-1C rats. Our study aimed to investigate the effects of Terpy and SNP (the classical NO donor) and their vascular mechanisms in basilar artery and inferior vena cava from 2K and 2K-1C rats. Our results demonstrate that Terpy, unlike the SNP, did not promote vascular relaxation in basilar artery of 2K and 2K-1C. Terpy did not release NO in vascular smooth muscle cells. SNP released NO and induced relaxation in basilar artery rings by activating the enzyme soluble guanylyl cyclase (sGC) with consequent activation of cGMP-dependent protein kinase (GK) and activation of K + channels (KV, KATP and KIR). Both NO donors and NTG promoted vascular relaxation in vena cava rings from 2K and 2K-1C rats in concentration-dependent way. We have observed an impaired relaxation to NO in cava vein from 2K-1C rats. The NO release by Terpy was lower in 2K-1C veins. NPS induces relaxation in inferior vena cava by the activation of GCs, GK and K+ channels. Terpy induced relaxation in inferior vena cava by the activation of the enzyme sGC, with consequent activation of GK, reticular Ca2 + ATPase (SERCA) and activation of K + channels (KV and BKCa SKca). Taken together, our results demonstrate that Terpy is less potent than the reference NO donor (SNP) in the inferior vena cava of 2K and 2K-1C. Its vasodilator effect is mainly due to activation of sGC, K + channels, SERCA and GK protein. In basilar artery Terpy, unlike SNP, does not induce relaxation in 2K and 2K-1C rats. Vasodilator response to SNP in basilar artery is mainly due to activation of sGC, K + channels and GK protein. SERCA appears not to be involved in the mechanism of vascular relaxation by SNP.

O óxido nítrico (NO) é o principal agente vasodilatador endógeno que regula o tônus e a homeostase vascular, além de controlar o fluxo sanguíneo. Doadores de NO, entre eles os nitratos orgânicos, são importantes medicamentos para o tratamento de doenças cardiovasculares. O grande benefício clínico desses nitratos é atribuído ao seu efeito venodilatador. Isto se deve ao seu efeito sobre a redução do retorno venoso, da pré-carga cardíaca e da demanda de oxigênio pelo miocárdio. Porém, um dos efeitos adversos mais comuns dos nitratos orgânicos é a cefaléia causada pela vasodilatação cerebral. Os doadores de NO utilizados clinicamente, nitroglicerina (NTG) e nitroprussiato de sódio (NPS), possuem algumas limitações como indução de tolerância e toxicidade, respectivamente. Dentre os compostos amplamente estudados, que são capazes de liberar NO, estão os complexos nitrosilos de rutênio. Estes complexos têm interesse terapêutico devido à sua baixa toxicidade. Recentemente, verificamos que o complexo de rutênio [Ru(terpy)(bdq)NO]3+ (Terpy), sintetizado em nosso departamento, reduz a pressão de ratos hipertensos renais 2R-1C e promove relaxamento vascular da aorta desses animais e de ratos normotensos controles (2R). Desta forma, a hipótese deste trabalho é de que o Terpy seja capaz de induzir relaxamento vascular em anéis de artéria basilar e veia cava inferior, tanto de ratos normotensos (2R) quanto de ratos 2R-1C. O nosso estudo teve como objetivo estudar os efeitos deste composto doador de NO e do doador de NO de referência, NPS, e os seus mecanismos de relaxamento vascular na artéria basilar e veia cava inferior de ratos normotensos 2R e hipertensos renais 2R-1C. Nossos resultados demonstram que o Terpy, ao contrário do NPS, não promoveu relaxamento vascular na artéria basilar de ratos 2R e 2R-1C. Da mesma forma, o Terpy não liberou NO nas células do músculo liso vascular. O NPS liberou NO e induziu relaxamento da artéria basilar pela ativação da enzima guanililciclase solúvel (GCs), com conseqüente ativação da proteína quinase dependente de GMPc (GK) e ativação dos canais para K+(KV, KATP e KIR). Ambos doadores, assim como a NTG, promoveram relaxamento vascular em anéis de veia cava e artéria basilar de ratos 2R e 2R- 1C de forma dependente da concentração. O relaxamento das veias de ratos 2R-1C foi menor do que em veias de 2R para os doadores de NO: Terpy, NPS e NTG. A liberação do NO pelo Terpy foi menor nas veias de 2R-1C. O NPS induz relaxamento da veia cava inferior pela ativação da enzima GCs com conseqüente ativação da proteína GK e ativação de canais para K+ sensíveis ao TEA. O Terpy induziu relaxamento da veia cava inferior pela ativação da enzima GCs, com conseqüente ativação da GK, ativação da Ca2+-ATPase reticular (SERCA) e ativação dos canais para K+ (KV, SKca e BKca). Em conjunto, nossos resultados mostraram que o Terpy é menos potente que o doador de referência (NPS) na veia cava inferior de ratos 2R e 2R-1C. Sua resposta vasodilatadora se deve principalmente à ativação da GCs, de canais para K+,proteína GK e SERCA. O Terpy, ao contrário do NPS, não induz relaxamento na artéria basilar de ratos 2R e 2R-1C. A resposta vasodilatadora do NPS nesses vasos se deve principalmente à ativação da GCs, de canais para K+ e proteína GK. A SERCA parece não estar envolvida no mecanismo de relaxamento vascular induzido pelo NPS. / Nitric oxide (NO) is an endogenous vasodilator that regulates vascular tone, homeostasis and blood flow. NO donors, including organic nitrates are important drugs for the treatment of cardiovascular diseases. A major clinical benefit of NO donors is attributed to their venodilator effect, resulting in decreased venous return, cardiac preload, arterial pressure and decreased myocardial oxygen demand. But the most common side effect of these drugs is the headache, which is caused by cerebral vasodilatation. The clinically used NO donors, nitroglycerin (NTG) and sodium nitroprusside (SNP), have some limitations such as induction of tolerance and toxicity, respectively. Among the widely studied compounds, which are capable of releasing NO are the nitrosyl ruthenium complexes, which have therapeutic interest due to its low toxicity. Recently, we found that the ruthenium complex [Ru (terpy)(BDQ)NO]3+ (Terpy) reduces the blood pressure of renal hypertensive rats (2K- 1C) and promotes vascular relaxation in aorta from 2K-1C and normotensive rats (2R). Thus, the hypothesis of the present work was that Terpy is able to induce vascular relaxation in basilar artery and inferior vena cava rings in 2K and 2K-1C rats. Our study aimed to investigate the effects of Terpy and SNP (the classical NO donor) and their vascular mechanisms in basilar artery and inferior vena cava from 2K and 2K-1C rats. Our results demonstrate that Terpy, unlike the SNP, did not promote vascular relaxation in basilar artery of 2K and 2K-1C. Terpy did not release NO in vascular smooth muscle cells. SNP released NO and induced relaxation in basilar artery rings by activating the enzyme soluble guanylyl cyclase (sGC) with consequent activation of cGMP-dependent protein kinase (GK) and activation of K + channels (KV, KATP and KIR). Both NO donors and NTG promoted vascular relaxation in vena cava rings from 2K and 2K-1C rats in concentration-dependent way. We have observed an impaired relaxation to NO in cava vein from 2K-1C rats. The NO release by Terpy was lower in 2K-1C veins. NPS induces relaxation in inferior vena cava by the activation of GCs, GK and K+ channels. Terpy induced relaxation in inferior vena cava by the activation of the enzyme sGC, with consequent activation of GK, reticular Ca2 + ATPase (SERCA) and activation of K + channels (KV and BKCa SKca). Taken together, our results demonstrate that Terpy is less potent than the reference NO donor (SNP) in the inferior vena cava of 2K and 2K-1C. Its vasodilator effect is mainly due to activation of sGC, K + channels, SERCA and GK protein. In basilar artery Terpy, unlike SNP, does not induce relaxation in 2K and 2K-1C rats. Vasodilator response to SNP in basilar artery is mainly due to activation of sGC, K + channels and GK protein. SERCA appears not to be involved in the mechanism of vascular relaxation by SNP.

La maladie de Charcot-Marie-Tooth (CMT) représente un large groupe hétérogène de neuropathies périphériques héréditaires. Les mutations du gène GDAP1 (ganglioside-induced differentiation-associated protein 1), codant pour une protéine de la membrane externe mitochondriale, sont associées à des formes récessives (CMT4A) et à des formes dominantes de CMT (CMT2K). GDAP1 participerait au processus de fission des mitochondries sans que son rôle soit bien défini. L'objectif de ce travail a été d'étudier le métabolisme énergétique mitochondrial de cellules de peau issues de patients porteurs de mutations du gène GDAP1. Nous avons mis en évidence un déficit énergétique mitochondrial associé au complexe I sans altération majeure du réseau mitochondrial. Ce déficit fonctionnel du complexe I est associé à une production accrue de radicaux libres et un défaut de régulation de la protéine sirtuine 1, une désacétylase NAD-dépendante impliquée dans la biogenèse mitochondriale. Nos travaux ont ainsi montré que GDAP1 a un rôle important dans le métabolisme énergétique mitochondrial. Dans le but d'établir des corrélations génotype-phénotype, nous avons créé une base de données internationale permettant de répertorier les données cliniques et les variations de séquence de GDAP1.

O relaxamento vascular induzido pelo óxido nítrico (NO) está prejudicado em aorta de ratos hipertensos renais (2R-1C). A nossa hipótese é de que o menor efeito do NO na aorta de ratos 2R-1C pode estar relacionada com a maior degradação do NO e/ou modificação das cavéolas no músculo liso vascular (MLV), considerando que o NO pode ser degradado rapidamente e que as cavéolas parecem ser importantes para a redução da concentração citosólica de Ca2+ ([Ca2+]c). O presente trabalho teve por objetivo estudar as alterações nos mecanismos vasodilatadores do NO em aorta de ratos hipertensos renais 2R-1C. Inicialmente, estudamos a influência do estresse oxidativo sobre o efeito do NO liberado dos doadores [Ru(NH.NHq)(terpy)NO+]3+ (TERPY) e nitroprussiato de sódio (NPS) em aorta de ratos normotensos (2R) e 2R-1C. Verificamos que o relaxamento foi menor na aorta dos ratos 2R-1C do que de 2R para o TERPY e NPS e que nas células do MLV da aorta de 2R-1C o efeito do TERPY em reduzir a [Ca2+]c também foi menor. Porém, o tratamento das aortas de ratos 2R-1C com antioxidante normalizou o relaxamento para ambos doadores e o efeito do TERPY sobre a [Ca2+]c. A concentração basal de superóxido (O2-) nas aortas dos ratos 2R-1C é maior do que em 2R e foi reduzida pelos antioxidantes. A concentração de NO basal e liberada do TERPY é menor em aortas de ratos 2R-1C. Estudamos a influência das cavéolas sobre o efeito do TERPY e NPS, em aorta de ratos 2R e 2R-1C. Somente em aortas de ratos 2R, a desorganização das cavéolas com ciclodextrina inibiu o relaxamento dos doadores de NO utilizados e a redução da [Ca2+]c para o TERPY. O número de cavéolas é menor tanto nas células do MLV como nas células endoteliais da aorta de ratos 2R-1C. Estudamos ainda o efeito do TERPY sobre a pressão arterial de ratos 2R e 2R-1C acordados. O TERPY possui efeito hipotensor somente nos ratos 2R-1C e este efeito foi mais prolongado do que o efeito hipotensor com NPS. O NPS teve efeito hipotensor tanto em ratos 2R como 2R-1C, porém este efeito foi maior em 2R-1C. Os resultados obtidos neste estudo indicam que a elevada concentração de O2- e o menor número de cavéolas encontrados na aorta dos ratos 2R-1C, devem contribuir de forma importante para o menor relaxamento da aorta de ratos 2R-1C. / The vascular relaxation induced by nitric oxide (NO) donors is impaired in aortas from renal hypertensive rats (2K-1C). Our hypothesis was that the lower NO effect in aortas from 2K-1C rats could be related with the higher degradation of NO and/or caveolae changes in vascular smooth muscle cells (VSMCs), considering that NO can be rapidly degraded and caveolae seems to play important role in the reduction of cytosolic Ca2+ concentration [Ca2+]c. The present study aimed to investigate the alterations on aorta relaxation induced by NO in 2K-1C rat aorta. At first, we studied the influence of oxidative stress on the effect of NO released from the NO donors [Ru(NH.NHq)(terpy)NO+]3+ (TERPY) and sodium nitroprusside (SNP) in aortas from normotensive (2K) and 2K-1C rats. The relaxation induced by both NO donors was impaired in aortas from 2K-1C rats and the reduction on [Ca2+]c induced by TERPY was also impaired in 2K-1C VSMCs. However, in aortas treated with antioxidants the relaxation to both NO donors and the reduction on [Ca2+]c to TERPY were normalized. The basal concentration of superoxide (O2-) was greater in 2K-1C than in 2K, which was reduced by the antioxidants. The basal cytosolic NO concentration ([NO]c) and the NO released from TERPY were lower in aortas from 2K-1C rats. We studied the influence of caveolae on the effects of NO released from the NO donors, in aortas from 2K and 2K-1C rats. We verified that caveolae disassemble with ciclodextrin impaired the relaxation to NO donors and the reduction on [Ca2+]c to TERPY only in aortas from 2K rats. The number of caveolae is reduced in aortic VSMCs and in the endothelial cells from 2K-1C rats. We studied the effect of TERPY on arterial pressure from 2K and 2K-1C rats. TERPY reduced the arterial pressure only in 2K-1C rats, which effect was longer than that produced by SNP. The hypotensive effect of SNP was greater in 2K-1C than in 2K rats. Taken together, our results indicate that the higher concentration of O2- and the reduced number of caveolae on aortas from 2K-1C rats could contribute to impaired aorta relaxation of 2K-1C rats.

O relaxamento vascular induzido pelo óxido nítrico (NO) está prejudicado em aorta de ratos hipertensos renais (2R-1C). A nossa hipótese é de que o menor efeito do NO na aorta de ratos 2R-1C pode estar relacionada com a maior degradação do NO e/ou modificação das cavéolas no músculo liso vascular (MLV), considerando que o NO pode ser degradado rapidamente e que as cavéolas parecem ser importantes para a redução da concentração citosólica de Ca2+ ([Ca2+]c). O presente trabalho teve por objetivo estudar as alterações nos mecanismos vasodilatadores do NO em aorta de ratos hipertensos renais 2R-1C. Inicialmente, estudamos a influência do estresse oxidativo sobre o efeito do NO liberado dos doadores [Ru(NH.NHq)(terpy)NO+]3+ (TERPY) e nitroprussiato de sódio (NPS) em aorta de ratos normotensos (2R) e 2R-1C. Verificamos que o relaxamento foi menor na aorta dos ratos 2R-1C do que de 2R para o TERPY e NPS e que nas células do MLV da aorta de 2R-1C o efeito do TERPY em reduzir a [Ca2+]c também foi menor. Porém, o tratamento das aortas de ratos 2R-1C com antioxidante normalizou o relaxamento para ambos doadores e o efeito do TERPY sobre a [Ca2+]c. A concentração basal de superóxido (O2-) nas aortas dos ratos 2R-1C é maior do que em 2R e foi reduzida pelos antioxidantes. A concentração de NO basal e liberada do TERPY é menor em aortas de ratos 2R-1C. Estudamos a influência das cavéolas sobre o efeito do TERPY e NPS, em aorta de ratos 2R e 2R-1C. Somente em aortas de ratos 2R, a desorganização das cavéolas com ciclodextrina inibiu o relaxamento dos doadores de NO utilizados e a redução da [Ca2+]c para o TERPY. O número de cavéolas é menor tanto nas células do MLV como nas células endoteliais da aorta de ratos 2R-1C. Estudamos ainda o efeito do TERPY sobre a pressão arterial de ratos 2R e 2R-1C acordados. O TERPY possui efeito hipotensor somente nos ratos 2R-1C e este efeito foi mais prolongado do que o efeito hipotensor com NPS. O NPS teve efeito hipotensor tanto em ratos 2R como 2R-1C, porém este efeito foi maior em 2R-1C. Os resultados obtidos neste estudo indicam que a elevada concentração de O2- e o menor número de cavéolas encontrados na aorta dos ratos 2R-1C, devem contribuir de forma importante para o menor relaxamento da aorta de ratos 2R-1C. / The vascular relaxation induced by nitric oxide (NO) donors is impaired in aortas from renal hypertensive rats (2K-1C). Our hypothesis was that the lower NO effect in aortas from 2K-1C rats could be related with the higher degradation of NO and/or caveolae changes in vascular smooth muscle cells (VSMCs), considering that NO can be rapidly degraded and caveolae seems to play important role in the reduction of cytosolic Ca2+ concentration [Ca2+]c. The present study aimed to investigate the alterations on aorta relaxation induced by NO in 2K-1C rat aorta. At first, we studied the influence of oxidative stress on the effect of NO released from the NO donors [Ru(NH.NHq)(terpy)NO+]3+ (TERPY) and sodium nitroprusside (SNP) in aortas from normotensive (2K) and 2K-1C rats. The relaxation induced by both NO donors was impaired in aortas from 2K-1C rats and the reduction on [Ca2+]c induced by TERPY was also impaired in 2K-1C VSMCs. However, in aortas treated with antioxidants the relaxation to both NO donors and the reduction on [Ca2+]c to TERPY were normalized. The basal concentration of superoxide (O2-) was greater in 2K-1C than in 2K, which was reduced by the antioxidants. The basal cytosolic NO concentration ([NO]c) and the NO released from TERPY were lower in aortas from 2K-1C rats. We studied the influence of caveolae on the effects of NO released from the NO donors, in aortas from 2K and 2K-1C rats. We verified that caveolae disassemble with ciclodextrin impaired the relaxation to NO donors and the reduction on [Ca2+]c to TERPY only in aortas from 2K rats. The number of caveolae is reduced in aortic VSMCs and in the endothelial cells from 2K-1C rats. We studied the effect of TERPY on arterial pressure from 2K and 2K-1C rats. TERPY reduced the arterial pressure only in 2K-1C rats, which effect was longer than that produced by SNP. The hypotensive effect of SNP was greater in 2K-1C than in 2K rats. Taken together, our results indicate that the higher concentration of O2- and the reduced number of caveolae on aortas from 2K-1C rats could contribute to impaired aorta relaxation of 2K-1C rats.