Os hormônios tireoidianos (HT) executam um papel lipolítico importante no Tecido Adiposo Branco (TAB), sendo este efeito mediado por meio do aumento da expressão de receptores beta adrenérgicos na membrana do adipócito, o que aumenta a sensibilidade deste tecido as catecolaminas. Sabe-se que os principais efetores da ação lipolítica nesse tecido são a lipase hormônio sensível (LHS) e a lipase dos triglicerídeos dos adipócitos (ATGL), as quais hidrolisam os triglicerídeos em ácidos graxos e glicerol. Além disso, outros componentes estão envolvidos na atividade lipolítica, como as perilipinas, proteínas estas que envolvem a gota de gordura, formando uma barreira contra a ação da LHS e ATGL, de modo que precisam ser fosforiladas para que a LHS e ATGL possam exercer seu efeito lipolítico. Considerando: (a) a importância do tecido adiposo na homeostase energética e como fonte de citocinas, as quais estão relacionadas com a sensibilidade tecidual à insulina; (b) que a função e o metabolismo do tecido adiposo variam com a sua distribuição regional, e (c) que as ações lipolíticas dos HT, importantes reguladores da homeostase energética, têm sido muito pouco exploradas, pretendemos investigar, em ratos, (i) se os HT interferem na expressão da LHS, ATGL, perilipina A e dos receptores beta3 adrenérgicos no tecido adiposo branco, e (ii) se essas ações diferem nos distintos depósitos de gordura, o que poderia ampliar o campo de conhecimento sobre os efeitos lipolíticos destes hormônios e a nossa compreensão sobre a contribuição deles nas complicações associadas à obesidade e suas co-morbidades. / Thyroid hormones (TH) play an important lipolytic role in white adipose tissue (WAT). This effect is mediated by increased expression of beta-adrenergic receptors on adipocytes membrane, which increases the sensitivity of that tissue to catecholamines. It is known that the main effectors of the lipolytic action in WAT enzymatic activity, especially: hormone sensitive lipase (HSL) and adipose triglyceride lipase (ATGL), which hydrolyze triglycerides into fatty acids and glycerol. In addition, other components are involved in the lipolytic activity, such as perilipin. These proteins support the fat droplet, forming a protective barrier against HSL and ATGL action. Considering: (a) the importance of adipose tissue in energy homeostasis and as a source of cytokines which are related to insulin tissue sensitivity; (b) function and metabolism of adipose tissue vary with their regional distribution; and (c) lipolytic actions of HT, important regulators of energy homeostasis, have been little explored, we investigated in rats with hypothyroidism and submitted to T3 treatment: (i) TH effects on the expression of hormone sensitive lipase (HSL), adipose triglyceride lipase (ATGL),perilipin A and beta-3 adrenergic receptors in WAT, and (ii) if this action are different on subcutaneous and visceral fat depot. This study has increased our understanding about the contribution of these hormones on WAT metabolism and metabolic disease as obesity.

by Ng Ka On. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1995. / Includes bibliographical references (leaves 100-122). / Abstract --- p.i / Acknowledgement --- p.vi / Chapter 1. --- Introduction / Chapter 1.1 --- Testicular vasculature --- p.1 / Chapter 1.1.1 --- Structural organization --- p.1 / Chapter 1.1.2 --- Peculiar structural organization pertinent to the Consideration of function --- p.3 / Chapter 1.2 --- Importance of the blood flow to testicular function --- p.6 / Chapter 1.3 --- Measurement of testicular blood flow --- p.8 / Chapter 1.4 --- Control of testicular blood flow --- p.16 / Chapter 1.5 --- Adrenergic control in the testis --- p.18 / Chapter 1.5.1 --- Adrenergic innervation and source of catecholamines --- p.18 / Chapter 1.5.2 --- Regulation of testicular function --- p.20 / Chapter 1.5.3 --- Effect on testicular blood flow --- p.22 / Chapter 1.6 --- Serotonergic control in the testis --- p.23 / Chapter 1.6.1 --- Serotonergic innervation and source of serotonin --- p.23 / Chapter 1.6.2 --- Regulation of testicular function --- p.24 / Chapter 1.6.3 --- Effect on testicular blood flow --- p.25 / Chapter 1.7 --- Cholinergic control in the testis --- p.26 / Chapter 1.7.1 --- Cholinergic innervation and source of acetylcholine --- p.26 / Chapter 1.7.2 --- Regulation of testicular function --- p.28 / Chapter 1.7.3 --- Effect on testicular blood flow --- p.29 / Chapter 1.8 --- Aims of the study --- p.30 / Chapter 2. --- Materials and methods / Chapter 2.1 --- Animals --- p.31 / Chapter 2.2 --- Drugs and chemicals --- p.32 / Chapter 2.3 --- In vivo videomicroscopy method --- p.33 / Chapter 2.4 --- Hydrogen gas clearance method --- p.37 / Chapter 2.5 --- Data and statistical analyses --- p.45 / Chapter 3. --- Results / Chapter 3.1 --- Adrenergic control --- p.46 / Chapter 3.1.1 --- Response of the testicular subcapsular artery to adrenergic agonists and antagonists --- p.46 / Chapter 3.1.2 --- Effect of adrenergic agonists on testicular capillary blood flow --- p.57 / Chapter 3.2 --- Serotonergic control --- p.60 / Chapter 3.2.1 --- Response of the testicular subcapsular artery to serotonergic agonists and antagonists --- p.60 / Chapter 3.2.2 --- Effect of serotonergic agonists on testicular capillary blood flow --- p.69 / Chapter 3.3 --- Cholinergic control --- p.76 / Chapter 3.3.1 --- Response of the testicular subcapsular artery to serotonergic agonists and antagonists --- p.76 / Chapter 3.3.2 --- Effect of serotonergic agonists on testicular capillary blood flow --- p.79 / Chapter 4. --- Discussion / Chapter 4.1 --- Adrenergic control --- p.86 / Chapter 4.2 --- Serotonergic control --- p.90 / Chapter 4.3 --- Cholinergic control --- p.96 / Chapter 4.4 --- General discussion --- p.98 / Chapter 5. --- References --- p.100

As metaloproteases da matriz (MMPs) são enzimas proteolíticas que participam da degradação da matriz extracelular no organismo de vertebrados. Estudos mostram a grande importância dessas enzimas no processo de remodelação do tecido cardíaco, além de sugerirem a participação da MMP-2 em doenças cardiovasculares. Em estudo recente foi demonstrado que as MMPs clivam o receptor ?2-adrenérgico, contribuindo para o aumento do tônus arteriolar de ratos espontaneamente hipertensos (SHR). Acredita-se que processo semelhante possa ser verificado em relação ao receptor ?-1 adrenérgico e proteínas das junções, que são fundamentais para o funcionamento do coração. As análises in sílico realizadas mostraram regiões prováveis de clivagem pela metaloprotease da matriz 2 humana recombinante (rhMMP-2) na porção extracelular, especificamente na região Nterminal deste receptor, no entanto, as análises de comparação de similaridade de substratos não apresentaram resultados significativos, embora os resultados preliminares obtidos no teste in vitro mostraram que houve hidrolise logo no início do peptídeo sintético ASPPASLLPPAS, entre os resíduos alanina e serina, entre as duas prolinas e por fim entre o resíduo de prolina e alanina, regiões com grandes chances de ocorrer a hidrólise, pois o substrato nativo desta enzima é o colágeno que é composto por uma cadeia polipeptídica com uma sequência de repetições onde geralmente temos glicina-X-Y, onde X normalmente é uma prolina e Y frequentemente uma hidroxiprolina, e raramente lisina e hidroxilisina, no entanto a replicação deste experimento não apresentou o mesmo resultado. Já os resultados obtidos no western blotting mostraram que a expressão do receptor é diminuída quando os cardiomiócitos são previamente tratados com 40mM e 120mM de rhMMP- 2 e esse efeito tem uma reversão significativa quando as células são previamente tratadas com inibidores doxiciclina ou ONO-4817, corroborando com os trabalhos apresentados na literatura em que a rhMMP-2 atua no receptor ?1adrenérgico. / Matrix metalloproteinases (MMPs) are proteolytic enzymes that participate in the degradation of the extracellular matrix in the vertebrate organism. Studies show the great importance of these enzymes in the remodeling process of cardiac tissue, besides suggesting the participation of MMP-2 in cardiovascular diseases. In a recent study, MMPs were shown to cleave the ?2-adrenergic receptor, contributing to the increase in arteriolar tone of spontaneously hypertensive rats (SHR). It is believed that a similar process can be verified also in the ?-1 adrenergic receptor and junction proteins, which are fundamental to the heart function. The in situ analyzes performed revealed sections prone to be cleaved by matrix metalloproteinase 2 recombinant human (rhMMP-2) in the extracellular portion, specifically in the Nterminal region of this receptor, however, the comparative analyzes of the similarity of substrates did not present significant results, but those obtained in the in vitro test showed that there was hydrolysis right at the beginning of the synthetic peptide ASPPASLLPPAS, between alanine and serine residues, between the two proline and finally between the proline residue and alanine. Hydrolysis among proline residues was expected, even though it was not predicted for in silica cleavage, since the native substrate of this enzyme is collagen, which is composed of a polypeptide chain with a sequence of repetitions in which it usually has glycine-XY, where X usually is a proline and Y often a hydroxyproline, and rarely lysine and hydroxylysine, however the replication of this experiment di not present the same result. The obtained results in western blotting have presented that receptor expression is decreased when cardiomyocytes are pretreated with 40mM and 120mM rhMMP-2 and this effect has a significant reversion when cells are pretreated with doxycycline or ONO-4817 inhibitors, supporting previous studies which demonstrate that rhMMP- 2 acts on the ? 1 adrenergic receptor.

by Shan Sze Wan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 145-166). / Abstracts in English and Chinese. / Title --- p.i / Abstract --- p.ii / 摘要 --- p.v / Acknowledgements --- p.vii / Table of Contents --- p.viii / List of Abbreviations --- p.xiv / List of Figures --- p.xvii / List of Tables --- p.xx / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- What are the general functions of cytokines? --- p.2 / Chapter 1.2 --- What is TNP-α? --- p.4 / Chapter 1.3 --- Actions of TNF-α --- p.5 / Chapter 1.4 --- General functions of TNF-α in astrocytes --- p.6 / Chapter 1.5 --- TNF-α receptors (TNF-Rs) --- p.8 / Chapter 1.6 --- Second messengers induced by TNP-α --- p.10 / Chapter 1.7 --- Glial Cells --- p.11 / Chapter 1.7.1 --- Oligodendroglia --- p.12 / Chapter 1.7.2 --- Brain Macrophages (Microglia) --- p.12 / Chapter 1.7.3 --- Astrocytes --- p.14 / Chapter 1.7.3.1 --- Functions of astrocytes --- p.15 / Chapter 1.8 --- "Brain injury, astrogliosis and scar formation" --- p.20 / Chapter 1.9 --- β-Adrenergic receptors (β-ARs) --- p.21 / Chapter 1.9.1 --- The active functional unit: the receptor complex --- p.22 / Chapter 1.9.2 --- General functions and distribution of β-ARs --- p.22 / Chapter 1.10 --- Functions of β-ARs in astrocytes --- p.24 / Chapter 1.10.1 --- Regulations of astrogliosis by β-ARs --- p.24 / Chapter 1.10.1.1 --- β-ARs are expressed in normal optic nerves and up-regulated after nerve crush --- p.24 / Chapter 1.10.1.2 --- Injury-induced alterations in endogenous catecholamine leads to enhanced β-AR activation --- p.25 / Chapter 1.10.1.3 --- β-AR blockade suppresses glial scar formation --- p.25 / Chapter 1.10.1.4 --- β-AR agonists affect the proliferation of astrocytes in normal brain --- p.26 / Chapter 1.11 --- Manganese Superoxide Dismutase (MnSOD) --- p.27 / Chapter 1.11.1 --- MnSOD is the target gene of NF-kB --- p.29 / Chapter 1.11.2 --- Induction of MnSOD by proinflammatory cytokines in rat primary astrocytes --- p.29 / Chapter 1.11.3 --- SMase and ceramides induce MnSOD in various cell types --- p.30 / Chapter 1.12 --- Why do we use C6 glioma cells? --- p.31 / Chapter 1.13 --- Aims and Scopes of this project --- p.32 / Chapter Chapter 2 --- MATERIALS AND METHODS / Chapter 2.1 --- Materials --- p.36 / Chapter 2.1.1 --- Cell Line --- p.36 / Chapter 2.1.2 --- Cell Culture Reagents --- p.36 / Chapter 2.1.2.1 --- Complete Dulbecco´ةs modified Eagle medium (CDMEM) --- p.36 / Chapter 2.1.2.2 --- Rosewell Park Memorial Institute (RPMI) medium --- p.37 / Chapter 2.1.2.3 --- Phosphate buffered saline (PBS) --- p.37 / Chapter 2.1.3 --- Recombinant cytokines --- p.38 / Chapter 2.1.4 --- Chemicals for signal transduction study --- p.38 / Chapter 2.1.4.1 --- Modulators of protein kinase C (PKC) --- p.38 / Chapter 2.1.4.2 --- Modulator of protein kinase A (PKA) --- p.39 / Chapter 2.1.4.3 --- β-Adrenergic agonist and antagonist --- p.39 / Chapter 2.1.5 --- Antibodies --- p.40 / Chapter 2.1.5.1 --- Anti-TNF-receptor type 1 (TNF-R1) antibody --- p.40 / Chapter 2.1.5.2 --- Anti-TNF-receptor type 2 (TNF-R2) antibody --- p.41 / Chapter 2.1.5.3 --- Anti-βi-adrenergic receptor (βl-AR) antibody --- p.42 / Chapter 2.1.5.4 --- Anti-β2-adrenergic receptor (β2-AR) antibody --- p.42 / Chapter 2.1.5.5 --- Antibody conjugates --- p.43 / Chapter 2.1.6 --- Reagents for RNA isolation --- p.43 / Chapter 2.1.7 --- Reagents for reverse transcription-polymerase chain reaction (RT-PCR) --- p.43 / Chapter 2.1.8 --- Reagents for electrophoresis --- p.45 / Chapter 2.1.9 --- Reagents and buffers for Western blot --- p.45 / Chapter 2.1.10 --- Other chemicals and reagents --- p.47 / Chapter 2.2 --- Maintenance of rat C6 glioma cell line --- p.47 / Chapter 2.3 --- RNA isolation --- p.48 / Chapter 2.3.1 --- Measurement of RNA yield --- p.49 / Chapter 2.4 --- Reverse transcription-polymerase chain reaction (RT-PCR) --- p.50 / Chapter 2.5 --- Western blot analysis --- p.52 / Chapter Chapter 3 --- RESULTS / Chapter 3.1 --- Effect of TNF-α on the expression of TNF-receptors (TNFRs) in C6 glioma cells --- p.55 / Chapter 3.1.1 --- Effect of TNF-α on TNF-R1 and -R2 mRNA expression in C6 cells --- p.56 / Chapter 3.1.2 --- The signaling systems mediating TNP-α-induced TNF-R2 expression in C6 cells --- p.57 / Chapter 3.1.2.1 --- The involvement of PKC in TNF-α-induced TNF-R2 expression in C6 cells --- p.57 / Chapter 3.1.2.2 --- Effect of PMA on the TNF-R protein levels in C6 cells --- p.63 / Chapter 3.1.2.3 --- Effect of Ro31 on the TNF-α-induced TNF-R protein level in C6 cells --- p.65 / Chapter 3.1.2.4 --- Effect of PKA activator on the level of TNF-R2 mRNA in C6 cells --- p.67 / Chapter 3.2 --- Effect of TNP-α on the expression of β1- and β2-adrenergic receptors (β1- and β2-ARs) in C6 glioma cells --- p.69 / Chapter 3.2.1 --- Effect of TNF-α on β1- and β2-ARs mRNA expression in C6 cells --- p.70 / Chapter 3.2.2 --- The signaling systems mediating TNF-α-induced β1- and β2-AR expression in C6 cells --- p.70 / Chapter 3.2.2.1 --- The involvement of PKC mechanism between TNF-α and β-ARs in C6 cells --- p.71 / Chapter 3.2.2.2 --- Effect of PMA on the β1- and β2-ARs protein level in C6 cells --- p.76 / Chapter 3.2.2.3 --- Effect of Ro31 on the TNF-α-induced β1- and β2-AR protein levels in C6 cells --- p.78 / Chapter 3.2.2.4 --- Effect of dbcAMP on the levels of βl- and β2-ARs mRNA in C6 cells --- p.80 / Chapter 3.3 --- Relationship between TN1F-R2 and β-adrenergic mechanism in C6 cells --- p.82 / Chapter 3.3.1 --- Effects of isproterenol and propranolol on endogenous TNF-α mRNA levels in C6 cells --- p.82 / Chapter 3.3.2 --- Effects of isoproterenol and propranolol on TNF-R2 mRNA levels in C6 cells --- p.83 / Chapter 3.3.3 --- Effects of β1-agonist and antagonist on endogenous TNF-α mRNA expression in C6 cells --- p.87 / Chapter 3.3.4 --- Effects of β1-agonist and antagonist on TNF-R2 mRNA expression in C6 cells --- p.91 / Chapter 3.3.5 --- Effects of β2-agonist and antagonist on endogenous TNF-α mRNA in C6 cells --- p.93 / Chapter 3.3.6 --- Effects of β2-agonist and antagonist on TNF-R2 mRNA in C6 cells --- p.100 / Chapter 3.4 --- Effect ofTNF-α on the expression of a transcriptional factor nuclear factor kappa B (NF-kB) in C6 glioma cells --- p.102 / Chapter 3.4.1 --- Effect ofTNF-α on NF-kB (p50) mRNA expression in C6 cells --- p.106 / Chapter 3.4.2 --- Effect of β-agonist and antagonist on NF-kB (p50) mRNA expression in C6 cells --- p.108 / Chapter 3.4.3 --- Effect of PMA and Ro31 on the levels of NF-kB mRNA in C6 cells --- p.109 / Chapter 3.5 --- Effects of TNF-α on the expression of manganese superoxide dismutase (MnSOD) in C6 glioma cells --- p.111 / Chapter 3.5.1 --- Effects of TNF-α on MnSOD and Cu-ZnSOD mRNAs expression in C6 cells --- p.114 / Chapter 3.5.2 --- Effects of β-agonist and β-antagonist on MnSOD mRNA expression in C6 cells --- p.115 / Chapter 3.5.3 --- Effects of PKC activator and inhibitor on the levels of MnSOD mRNA in C6 cells --- p.117 / Chapter Chapter 4 --- DISCUSSION AND CONCLUSION / Chapter 4.1 --- Effects of TNF-α on the expression of TNF-receptors (TNFRs) in C6 glioma cells --- p.122 / Chapter 4.2 --- Effects of TNF-a on the expression of β1- and β2-adrenergic receptors (β1 and β2-ARs) in C6 glioma cells --- p.126 / Chapter 4.3 --- Relationship between TNF-α and β-adrenergic mechanism in C6 cells --- p.128 / Chapter 4.4 --- Effects of TNF-α on the expression of a transcriptional factor nuclear factor kappa B (NF-kB) in C6 glioma cells --- p.131 / Chapter 4.5 --- Effects of TNF-α on the expression of manganese superoxide dismutase (MnSOD) in C6 glioma cells --- p.133 / Chapter 4.6 --- Possible sources of β-agonists --- p.136 / Chapter 4.7 --- Conclusions --- p.137 / Appendix A --- p.143 / References --- p.145

La voie β-adrénergique (β-AR)/AMPc est cruciale pour l’adaptation de la fonction cardiaque. Dans l’insuffisance cardiaque (IC), cette signalisation est perturbée et une part importante des patients meurt de troubles du rythme. Classiquement, les effets inotrope et lusitrope positifs de l’AMPc sont attribués à la phosphorylation par la protéine kinase AMPc dépendante (PKA) des protéines clés du couplage excitation–contraction (CEC). L’AMPc active aussi le facteur d’échange Epac, impliqué dans l’hypertrophie cardiaque et le contrôle de l’homéostasie calcique. Une cible d’Epac est la CaMKII, une kinase modulée par le Ca2+ et la calmoduline qui phosphoryle aussi les protéines clés du CEC, et dont l’activation est pro-arythmique.Les phosphodiestérases (PDEs) de type 3 et 4 sont majeures pour dégrader l’AMPc et contrôler l’homéostasie calcique et le CEC. Les inhibiteurs de PDE3 sont de puissants cardiotoniques mais leur utilisation est limitée par leurs effets pro-arythmiques. De plus, l’invalidation de gènes codant pour PDE4 conduit à des arythmies ventriculaires. Mon travail a permis d’identifier les perturbations de l’homéostasie calcique responsables de la survenue d’arythmies lorsque l’activité des PDE3 et des PDE4 est diminuée. Mes résultats montrent que les inhibiteurs de PDEs exercent des effets inotropes via PKA, mais suscitent des vagues de Ca2+ pro-arythmiques impliquant la PKA et la CaMKII activée en partie via Epac. Ceci suggère l'utilisation potentielle d'inhibiteurs de CaMKII comme compléments aux inhibiteurs de PDEs pour limiter leurs effets délétères, une hypothèse que j’ai pu vérifier dans un modèle porcin plus proche du patient. / The β-adrenergic pathway (β-AR)/cAMP is crucial for the adaptation of the cardiac function upon stress. In heart failure (HF), this signaling pathway is disrupted and a significant proportion of patients dies of cardiac arrhythmias. Classically, the inotropic and lusitropic effects of cAMP are attributed to the phosphorylation by the cAMP-dependent protein kinase (PKA) of the key proteins of the excitation-contraction coupling (ECC). cAMP also activates the exchange factor Epac, which is involved in cardiac hypertrophy and controls intracellular Ca2+ homeostasis. Epac activates CaMKII, another kinase modulated by Ca2+ and calmodulin which phosphorylates the same key proteins of the ECC, and is involved in arrhythmogenesis.Phosphodiesterases (PDEs) type 3 and 4 are crucial enzyme to degrade cAMP and to control Ca2+ homeostasis, thus ECC. PDE3 inhibitors are potent cardiotonic drugs but their use is limited by their pro-arrhythmic effects. Furthermore, the invalidation of genes encoding PDE4 results in ventricular arrhythmias. My work allowed characterizing the perturbations of Ca2+ homeostasis which lead to arrhythmias when PDE3 and PDE4 activities are decreased. My results show that PDE inhibitors exert inotropic effects via PKA, but evoke pro-arrhythmic Ca2+ waves via both PKA and CaMKII, the latter being activated in part via Epac. Altogether, these results suggest the potential use of CaMKII inhibitors as adjuncts to PDEs inhibitors to limit their deleterious effects, a hypothesis I also tested in a porcine model closer to the patient.

As metaloproteases da matriz (MMPs) são enzimas proteolíticas que participam da degradação da matriz extracelular no organismo de vertebrados. Estudos mostram a grande importância dessas enzimas no processo de remodelação do tecido cardíaco, além de sugerirem a participação da MMP-2 em doenças cardiovasculares. Em estudo recente foi demonstrado que as MMPs clivam o receptor ?2-adrenérgico, contribuindo para o aumento do tônus arteriolar de ratos espontaneamente hipertensos (SHR). Acredita-se que processo semelhante possa ser verificado em relação ao receptor ?-1 adrenérgico e proteínas das junções, que são fundamentais para o funcionamento do coração. As análises in sílico realizadas mostraram regiões prováveis de clivagem pela metaloprotease da matriz 2 humana recombinante (rhMMP-2) na porção extracelular, especificamente na região Nterminal deste receptor, no entanto, as análises de comparação de similaridade de substratos não apresentaram resultados significativos, embora os resultados preliminares obtidos no teste in vitro mostraram que houve hidrolise logo no início do peptídeo sintético ASPPASLLPPAS, entre os resíduos alanina e serina, entre as duas prolinas e por fim entre o resíduo de prolina e alanina, regiões com grandes chances de ocorrer a hidrólise, pois o substrato nativo desta enzima é o colágeno que é composto por uma cadeia polipeptídica com uma sequência de repetições onde geralmente temos glicina-X-Y, onde X normalmente é uma prolina e Y frequentemente uma hidroxiprolina, e raramente lisina e hidroxilisina, no entanto a replicação deste experimento não apresentou o mesmo resultado. Já os resultados obtidos no western blotting mostraram que a expressão do receptor é diminuída quando os cardiomiócitos são previamente tratados com 40mM e 120mM de rhMMP- 2 e esse efeito tem uma reversão significativa quando as células são previamente tratadas com inibidores doxiciclina ou ONO-4817, corroborando com os trabalhos apresentados na literatura em que a rhMMP-2 atua no receptor ?1adrenérgico. / Matrix metalloproteinases (MMPs) are proteolytic enzymes that participate in the degradation of the extracellular matrix in the vertebrate organism. Studies show the great importance of these enzymes in the remodeling process of cardiac tissue, besides suggesting the participation of MMP-2 in cardiovascular diseases. In a recent study, MMPs were shown to cleave the ?2-adrenergic receptor, contributing to the increase in arteriolar tone of spontaneously hypertensive rats (SHR). It is believed that a similar process can be verified also in the ?-1 adrenergic receptor and junction proteins, which are fundamental to the heart function. The in situ analyzes performed revealed sections prone to be cleaved by matrix metalloproteinase 2 recombinant human (rhMMP-2) in the extracellular portion, specifically in the Nterminal region of this receptor, however, the comparative analyzes of the similarity of substrates did not present significant results, but those obtained in the in vitro test showed that there was hydrolysis right at the beginning of the synthetic peptide ASPPASLLPPAS, between alanine and serine residues, between the two proline and finally between the proline residue and alanine. Hydrolysis among proline residues was expected, even though it was not predicted for in silica cleavage, since the native substrate of this enzyme is collagen, which is composed of a polypeptide chain with a sequence of repetitions in which it usually has glycine-XY, where X usually is a proline and Y often a hydroxyproline, and rarely lysine and hydroxylysine, however the replication of this experiment di not present the same result. The obtained results in western blotting have presented that receptor expression is decreased when cardiomyocytes are pretreated with 40mM and 120mM rhMMP-2 and this effect has a significant reversion when cells are pretreated with doxycycline or ONO-4817 inhibitors, supporting previous studies which demonstrate that rhMMP- 2 acts on the ? 1 adrenergic receptor.

O peso corporal dos animais tende a ser relativamente estável durante longos períodos de tempo. Situações de restrição calórica ou aumento na ingestão de calorias levam a alterações fisiológicas compensatórias que resistem aos efeitos destas perturbações. De fato, o gasto energético aumenta em animais submetidos à dieta hipercalórica, a chamada termogênese facultativa, de modo a manter os estoques energéticos constantes. É possível que defeitos na termogênese facultativa estejam envolvidos no desenvolvimento da obesidade. O BAT, o principal sítio de termogênese facultativa, é ativado pela liberação de NE pelo Sistema Nervoso Simpático, que se liga aos receptores adrenérgicos b1, b2 e b3 expressos nos adipócitos marrons. Diversos estudos demonstram que os receptores b são importantes na proteção contra a obesidade, mas ainda não é claro qual o papel de cada isoforma neste processo. Assim sendo, o objetivo do nosso trabalho foi avaliar o papel das isoformas b1 e b2 na mediação da termogênese facultativa induzida pela dieta. Para tanto, nós tratamos camundongos com nocaute para o receptor adrenérgico b1 (KOb1) e camundongos com nocaute para o receptor b2 (KOb2) com dieta hipercalórica por 22 semanas. O peso corporal foi medido diariamente e o consumo de oxigênio foi determinado usando-se um sistema de respirometria aberto ao final do experimento. A composição corporal foi determinada pela análise da carcaça. Animais foram expostos ao frio de 4ºC por 4h e sua temperatura corporal foi medida em vários tempos e a resposta térmica do iBAT foi determinada pela infusão de NE ou agonista b adrenérgico. Além disso, foram determinados os níveis de RNAm das isoformas de receptores adrenérgicos b nos animais nocaute. Os resultados obtidos em nosso estudo mostram que os animais KOb1 e KOb2 tratados com dieta hipercalórica não desenvolvem obesidade mais severa do que os animais selvagens mas não são capazes de aumentar o consumo de oxigênio induzido pela dieta, sugerindo que estes receptores não são relevantes na termogênese induzida pela dieta. Por outro lado, nossos dados indicam que a presença do receptor b1 é exigida para termogênese induzida pelo frio, uma vez que os camundongos KOb1 são sensíveis ao frio e a capacidade termogênica do BAT destes animais em reposta à NE é bastante reduzida quando comparados com animais selvagens. A ausência do receptor b2 não piora a resposta dos animais ao frio sugerindo que esta isoforma não esteja envolvida na termogênese induzida pela dieta ou pelo frio. Os nossos achados indicam que a isoforma do receptor adrenérgico b1 é fundamental na termogênese induzida pelo frio, mas não pela dieta. Além disso, é provável que a termogênese induzida pela dieta seja regulada por mecanismos distintos da termogênese induzida pelo frio. / The body weight of animals tends to be relatively stable over long periods of time. Situations of caloric restriction or increase in intake of calories lead to compensatory physiological changes that resist the effects of these disorders. In fact, the energy expenditure increases in animals treated with diet hypercaloric called facultative thermogenesis, in order to keep to energy stock constant. Defects in this facultative thermogenesis may be related to the development of obesity. Brown adipose tissue is the main site of facultative thermogenesis and is activated by signaling of b1, b2 e b3 adrenergic receptors by Norepinephrine released by Sympathetic Nervous System. Several studies showed that the isoforms b of adrenergic receptors are important in mechanisms involved in obesity and also in promoting cold tolerance. Nonetheless, it is unclear the role of each isoform in these process. Therefore, the purpose of our study was to evaluate the role of isoforms b1 and b2 in mediate the facultative thermogenesis. For that, we fed nocaute mice for the adrenergic receptor b1 (KOb1) and nocaute mice for the adrenergic receptor b2 (KOb2) with high fat diet for 22 weeks. During treatment body weight was determined daily. By the end of the experiment oxygen consumption was measured using a system of open respirometry and body composition was determined by analysis of the carcass. We also exposed KOb1 and KOb2 animals to cold (4C). The thermogenic response of iBAT was evaluated through i.v NE infusion. The results obtained in our study showed that the animals KOb1 and KOb2 treated with high fat diet did not gain more fat when compared to wild type animals, but are unable to increase the oxygen consumption, suggesting that these receptors are not relevant in development of obesity. Furthermore, our data indicate that the presence of the b1 receptor is required for cold-induced thermogenesis, since the KOb1 mice are sensitive to cold and BAT thermogenic response is significantly impaired when compared with animals wild type. The absence of b2 receptor does not worsen the response of animals to cold suggesting that this isoform is not involved in the diet- or cold- induced thermogenesis. In conclusion, our findings indicate that the b1 isoform of the adrenergic receptor is critical in the cold-induced thermogenesis, but not in diet induced thermogenesis. Moreover, it is likely that the diet-induced thermogenesis and cold-induced thermogenesis are regulated by different mechanisms.

Einleitung: / Introduction:

Há evidências que o treinamento induz alterações plásticas e funcionais na alça suprabulbar do controle autonômico cardiovascular, mediados pelos barorreceptores arteriais. O presente estudo objetivou verificar os efeitos da desnervação sino-aórtica associada ou não ao treinamento (T) na expressão de neurônios noradrenérgicos, ocitocinérgicos e vasopressinérgicos, assim como o efeito temporal induzido pelo T. Foi possível constatar que o T aumenta a expressão de dopamina beta-hidroxilase e ocitocina, sem alteração a expressão de vasopressina, e que essas alterações neuropeptídicas precedem ao surgimento das alterações benéficas induzidas pelo T como o aumento da sensibilidade barorreflexa e bradicardia de repouso. Foi possível observar que a desnervação sino-aórtica bloqueia todos esses efeitos benéficos induzidos pelo T, diminuindo a expressão de dopamina beta hidroxilase, vasopressina e ocitocina no PVN. Contudo, esses dados sugerem que a via aferente Noradrenérgica, via barorreceptor, é essencial para ajustes benéficos cardiovasculares. / There is evidence aerobic training (T) determines plastic and functional changes in the suprabulbar modulatory loop of the cardiovascular autonomic control, many of them mediated by arterial baroreceptors. The present study aim to investigate the effects of sinoaortic denervation associated or not to T on noradrenergics, oxitocinergics, vasopressinergics neurons expression, as temporal effects induced by T. It was possible find augment on dopamine beta-hydroxilase and oxytocin expression, without changes on vasopressin expression, and that neuropeptidergic changes preceding appearance of beneficial training-induced, as well the increases baroreflex sensitivity and resting bradycardia. Data also indicate that sinoaortic denervation blocks all of these effects training-induced, decreases of dopamine beta-hidroxylase, vasopressin and oxytocin on the PVN. Therefore, those data suggests that noradrenergic afferents driven by barorreceptors are essencial to trigger beneficial training-induced cardiovascular adjustments.

O presente trabalho teve por objetivo investigar a variação na secreção de LH em resposta ao tratamento com neurotransmissores, na presença ou não de esteróides gonadais e desta forma gerar informações sobre os mecanismos neuroendócrinos envolvidos na maturação sexual de novilhas da raça Nelore. A concentração de LH foi quantificada por radioimunoensaio e as novilhas ovariectomizadas (OVX) apresentaram maior concentração basal de LH (P≤0,05), que as novilhas inteiras (INT). A administração de um antagonista gabaérgico (picrotoxina, 0,18 mg/kg, iv, amostras 15 min por 10 h) aos 8, 10, 14 e 17 meses de idade, aumentou (P≤0,05) a concentração média e área total de secreção de LH nas novilhas INT tratadas aos 14 meses, a área total dos picos e área do maior pico de secreção de LH foi maior (P≤0,05) nas novilhas OVX aos 14 e 17 meses de idade . A administração de um antagonista dopaminérgico (sulpiride, 0,59 mg/kg, sc, amostras 15 min por 10 h) aos 8, 12 e 16 meses de idade diminuiu (P≤0,05) a secreção de LH (concentração média, área total, área total dos picos e área do maior pico secreção de LH) nas novilhas OVX aos 8 meses de idade. A administração de um estimulador alfa-adrenérgico (clonidina, 10 µg/kg, iv, amostras 15 min por 4 h) aos 8, 12 e 15 meses de idade, diminuiu (P≤0,05) o número de picos nas novilhas OVX com 8 meses de idade. A administração do 17β-estradiol (2 µg/kg, iv, amostra 15 min por 3 h, 1 h por 7 h e 3 h por 22 h) aos 10, 13 e 17 meses de idade diminuiu a diferença (P≥0,05) entre os grupos OVX e INT em relação ao número de picos, área total de picos, área do maior pico e tempo necessário para acontecer o maior pico. Foi avaliada a secreção de LH da desmama à primeira ovulação em novilhas INT e OVX. A concentração de LH aumentou durante a maturação sexual, tanto nas novilhas INT quanto nas OVX. O número de picos de secreção de LH e amplitude máxima de secreção de LH foi maior (P≤0,05) nas novilhas OVX com o aproximar da primeira ovulação. Os resultados indicam uma diminuição da sensibilidade do hipotálamo aos esteróides gonadais durante o processo de maturação sexual nas novilhas da raça Nelore e a participação alternada de neurotransmissores, inibindo e estimulando a secreção de LH. Concluímos que, em novilhas pré-púberes da raça Nelore o desenvolvimento da retroalimentação positiva aos esteróides gonadais no hipotálamo foi importante para aumentar a secreção de LH antes da primeira ovulação, com a participação de neurotransmissores estimulando ou inibindo a secreção de LH. / The variation on LH secretion after neurotransmitter administration, on the presence or absence of gonadal steroid, was investigated, generating information about the mechanisms evolved on sexual maturation in Nellore heifers. LH concentration was quantified by RIA. As expected ovariectomized heifers higher basal LH concentration (P≤0,05) than intact heifers. The picrotoxin administration (GABA antagonist, 0,18 mg/kg, iv, samples 15 min for 10 h) at 8, 10, 14 and 17 months of age increased (P≤0,05) average concentration and total secretion area on intact treated heifers at 14 months and peak total area and area of highest peal on ovariectomized heifers at 14 and 17 months of age. The dopaminergic antagonist (sulpiride, 0.59 mg/kg, sc, sample 15 min for 10 h) administrated at 8, 12 and 16 months of age decreased (P≤0,05) LH secretion (average levels, total peak area and area of the highest secretion peak) on ovariectomized heifers at 8 months of age. The administration of an alfa-adrenérgic stimulatory (clonidine 10 µg/kg, iv, samples 15 min for 4 h) at 8, 12 and 15 months of age decreased (P≤0,05) decreased the number of peaks at 8 months of age. 17β-estradiol administration (2 µg/kg, iv, samples every 15 min for 3 h, every 1 h for 7 h and every 3 h for 22 h) decreased the differences between ovariectomized and intact heifers on number of peaks, total peak area, highest peak area and time to highest peak occurrence. The LH secretion from weaning to first ovulation in non treated intact and ovariectomized heifers was also evaluated. LH concentration increase during sexual maturation in both ovariectomized and intact heifers. The number of peaks, and maximum LH secretion amplitude was higher in ovariectomized heifer closest to first ovulation, when compared to intact heifers. The results suggested a decrease on hipothalamus sensitivity to gonadal steroid during the sexual maturation in Nelore heifer associated with neurotransmitter participation either stimulating or inhibiting LH secretion. It was possible to conclude that the decrease of negative feedback associated with the increase on positive feedback of gonadal steroids over hipothalamus was necessary to increase LH secretion before first ovulation, that was associated with neurotransmitter participation on LH secretion.