Les travaux présentés dans ce mémoire ont pour objectif d’approfondir la compréhension de l’altération de la réponse à la stimulation des récepteurs β-adrénergiques dans plusieurs contextes physiopathologiques. La première étude confirme l’existence d’une dysfonction β-adrénergique à l’échelle du cardiomyocyte au cours de la sénescence. Elle met en lumière le rôle de la protéine MRP4 (multidrug resistance associated protein 4) dans cette diminution de réponse inotrope positive à l’isoprotérénol. La deuxième étude évalue la réponse à la stimulation des récepteurs β-adrénergiques dans le syndrome métabolique et montre que la dysfonction est modérée dans ce contexte même en cas de diabète associé à l’obésité. Ces résultats fonctionnels sont expliqués par la diminution d’expression des récepteurs β1- et β2-adrénergiques mais l’absence de surexpression du récepteur β3-adrénergique comme observée dans le diabète de type 1. La troisième étude analyse le rôle de l’atorvastatine sur la réponse β-adrénergique chez les diabétiques et les mécanismes de modulation de cette réponse par une étude du transcriptome cardiaque. Elle montre également que l’inhibition de la production d’oxyde nitrite améliore la réponse β-adrénergique. La quatrième étude a expliqué une part de la dysfonction β-adrénergique chez les diabétiques par la surexpression de MRP4. L’inhibition de MRP4 a permis de restaurer la réponse à l’isoprotérénol au cours de la cardiopathie diabétique. Au total, l’ensemble de nos travaux poursuit la description des mécanismes de la dysfonction β-adrénergique dans la sénescence et le diabète et souligne le rôle de MRP4. / The studies presented in this report looked for a better understanding of the altered response to stimulation of the β-adrenergic receptors in several physiopathological contexts. The first study confirms the alteration of the β-adrenergic response at the cardiomyocyte level in the senescent cardiomyopathy. The role of MRP4 (multidrug resistance associated protein 4) in the reduced inotropic response to isoproterenol is emphasized. The second study evaluates the response to β-adrenoceptors stimulation in the metabolic syndrome and shows mild dysfunction in this context even in obesity associate with diabetes. These functional results are explained by a reduced expression of β1- and β2-adrenergic receptors but no overexpression of β3-adrenoceptor as observed in type 1 diabetes. The third study analyzes the role of atorvastatin on the β-adrenergic response in the diabetic cardiomyopathy and the mechanisms involved by study of the cardiac transcriptome. The inhibition of nitrite oxide production improves the response to β-adrenoceptors stimulation in diabetic heart. The fourth study explained part of the β-adrenergic dysfunction in the diabetic cardiomyopathy by the overexpression of MRP4. The inhibition of this protein restored the response to isoproterenol during diabetic cardiomyopathy. All together the present results carry on with description of the mechanisms involved in the β-adrenergic dysfunction in aging and diabetes and underline the role of MRP4.

Indivíduos portadores do glutamato na posição 27 do gene que codifica para o receptor beta2-adrenérgico têm resposta vasodilatadora muscular aumentada durante manobras fisiológicas. No entanto, o impacto do consumo agudo de gordura nessa resposta não é conhecido. Neste estudo, testou-se a hipótese de que o consumo gordura afetaria a resposta vasodilatadora aumentada destes indivíduos durante manobras fisiológicas. Vinte e cinco indivíduos saudáveis foram subdivididos em dois grupos: 11 homozigotos para o glutamato (Glu27Glu, 40+-3 anos; 65+-3kg) e 14 homozigotos para a glutamina (Gln27Gln, 40+-2 anos; 64+-2kg). O fluxo sangüíneo muscular foi medido por pletismografia de oclusão venosa. A resposta vasodilatadora muscular foi avaliada durante 3 minutos de exercício e estresse mental em jejum e 3 horas após consumo de 62 g de gordura. A condutância basal foi semelhante entre grupos (Glu27Glu=2,3+-0,1; Gln27Gln=2,2+-0,1; P=0,21). O aumento da condutância vascular durante exercício e durante o estresse mental foi maior no grupo Glu27Glu (0,73+-0,2 vs 0,22+-0,1; P=0,008 e 1,8?0,3 vs 1,2+-0,2; P=0,04, respectivamente). O consumo agudo de uma preparação rica em gordura eliminou esta diferença. A resposta de pressão arterial e freqüência cardíaca foi semelhante antes e após a ingestão de gordura. Os níveis de triglicérides, glicose e insulina foram semelhantes ao longo de todo período de estudo. O consumo agudo de gordura elimina a resposta aumentada do fluxo sangüíneo muscular durante manobras fisiológicas dos indivíduos portadores do genótipo Glu27Glu no receptorbeta2- adrenérgico. / Subjects who have glutamic acid at position 27 in gene encoding to beta2-adrenoceptor have increased muscle vasodilatory response during physiological maneuvers. However, the impact of a high-fat meal in this response is unknown. We tested the hypothesis that a high-fat meal would modify the increased muscle vascular reactivity during handgrip and mental stress in these subjects. Twenty-five healthy subjects were subdivided in two groups: 11 were homozygous to glutamic acid (Glu27Glu, 40?3 years; 65+-3kg) and 14 were homozygous to glutamine (Gln27Gln, 40+-2 years; 64+-2kg). Forearm blood flow was measured by venous occlusion pletysmography. Forearm blood flow was recorded for 3 minutes of handgrip and mental stress during fasting and three hours after 62g of fat consumption. Baseline forearm vascular conductance was similar between groups (Glu27Glu=2.3+-0.1; Gln27Gln=2.2+-0.1; P=0.21). Forearm vascular conductance during handgrip and mental stress was greater in the genotype Glu27Glu (0.73+-0.2 vs 0.22+-0.1; P=0.008 and 1.8+-0.3 vs 1.2+-0.2; P=0.04, respectively). Acute fat consumption eliminated the difference of vasodilatory response previously achieved. Blood pressure and heart rate response were similar before and after fat intake. Triglycerides, glucose and insulin levels were also similar between groups. We concluded that high-fat ingestion abolishes the augmented muscle blood flow responses during physiological maneuvers in individuals who are homozygous for the Glu27 allele of the beta2-adrenoceptor gene.

Adicionalmente às alterações estruturais e funcionais observadas na insuficiência cardíaca (IC), as alterações na dinâmica do Ca2+ intracelular apresentam uma relação negativa com a contratilidade e função cardíaca. Para evitar a progressão da IC, o tratamento desta síndrome, conta atualmente com intervenções multifatoriais, incluindo a terapia farmacológica, com destaque aos -bloqueadores, e a terapia não medicamentosa incluindo o treinamento físico aeróbico, que quando realizado em intensidade adequada melhora a tolerância ao esforço e a qualidade de vida do portador de insuficiência cardíaca. É bem conhecido que os -bloqueadores melhoram a função cardíaca e dinâmica do Ca2+ intracelular na IC, além de minimizar a remodelação cardíaca, no entanto, ainda existem controvérsias sobre qual o melhor -bloqueador a ser utilizado e seus efeitos específicos na regulação de Ca2+ intracelular. Quanto aos efeitos do treinamento físico aeróbico na IC, seu efeito é reconhecido na melhora da tolerância aos esforços e qualidade de vida do paciente, o que é atribuído principalmente, a ganhos vasculares e músculos-esqueléticos. Em relação à função cardíaca, em trabalho anterior do nosso laboratório, o treinamento físico aeróbico melhorou a função cardíaca associado à melhora nos transientes de cálcio do miócito cardíaco. Como tanto o treinamento físico como os -bloqueadores apresentaram seus respectivos efeitos positivos, mas diferenciados, na presente tese estudou-se o efeito associado do treinamento físico ao tratamento com -bloqueadores sobre a função cardíaca e o fluxo de cálcio no cardiomiócito. Além disso, comparou-se qual -bloqueador (metoprolol ou carvedilol) seria mais efetivo na associação com o treinamento físico. Dividimos nossa amostra em 6 grupos, todos com IC. Dentre eles, 3 foram mantidos sedentários; salina (S), metoprolol (M) e carvedilol (C) e 3 foram treinados; treinados (T), treinados e tratados com metoprolol (MT) e treinados e tratados com carvedilol (CT). Verificamos que apenas os camundongos com IC treinados melhoraram a tolerância ao esforço, medida por teste máximo em esteira rolante, o que não foi observado no tratamento farmacológico isoladamente. Quanto à função cardíaca, observamos uma melhora da função nos grupos que realizaram treinamento físico ou foram tratados com metoprolol ou carvedilol isoladamente. Nos grupos que associaram as terapias farmacológicas e nãofarmacológica, observamos uma melhora da função apenas no grupo treinado carvedilol associada a um aumento da expressão da SERCA 2, proteína esta, envolvida na recaptação do Ca2+ para o reticulo sarcoplasmático, e, portanto, relacionada ao relaxamento da bomba cardíaca, apesar de não se observar aumento no pico do transiente de cálcio em cardiomiócitos isolados. Em conjunto, os resultados sugerem uma associação preferencial entre o treinamento físico e o carvedilol o que pode trazer implicações clínicas futuras importantes para o tratamento da IC. / In heart failure (HF), structural and functional alterations in myocardium are often paralleled by, defects in intracellular Ca2+ transients.. Within therapeutical strategies for heart failure, it is worth mentioning -blockers and aerobic exercise training. It is known that -blockers improve cardiac function and Ca2+ handling in heart failure. Indeed, -blockers present cardiac antiremodeling effects, but there are many controversies concerning which - blocker is more efficient in HF treatment and which would be its specific effects in Ca2+ regulation. In relation to aerobic exercise training effects in HF, it is important to highlight its positive effects in exercise tolerance and life quality improvement associated with vascular and skeletal muscle gains. We also observed previously that exercise training improves cardiac function associated with improved calcium transients in cardiac myocytes. Taking into consideration the positive impact of both exercise training and -blockers, presently we studied the associative effect of exercise training and -blockers on cardiac function and Ca2+ handling in cardiomyocytes in sympathetic hyperactivity induced HF in mice. We further compared which -blocker (metoprolol or carvedilol) would be more effective to be associated with exercise training. HF mice were assigned into 6 different groups, being 3 of them sedentary: saline (S), metoprolol (M) and carvedilol (C) and 3 exercisetrained: trained (T), trained and treated with metoprolol (MT), and trained and treated with carvedilol (CT). We observed that only HF mice exercise-trained improved exercise tolerance evaluated by maximum exercise test on a treadmill, which was not observed in -blocker treatment alone. Both exercise training and -blockers improved cardiac function evaluated by echocardiography, respectively. When exercise training was associated with -blockers, only HF mice exercise-trained and carvedilol-treated improved cardiac function associated with increased expression of SERCA 2 protein levels, which is involved in sarcoplasmic Ca2+ reuptake and cardiac relaxation. This response was not paralleled by changes in peak Ca2+ transients in isolated cardiac myocytes. Our results provide evidence for a superior effect of exercise training associated with carvedilol for improving cardiac function in HF mice.

Fibroblast growth factor 21 (FGF21) is a unique peptide hormone involved in the energy homeosta- sis, as well as in the regulation of glucose and lipid metabolism. Numerous animal studies suggest that FGF21 may be used as a potential treatment for obesity and type 2 diabetes mellitus. It was found out, that FGF21 counteracts the development of obesity presumably by increasing energy expenditure through activation of thermogenesis in brown and white adipose tissue. FGF21 apparently also inhibits lipolysis. However, the specific mechanism of action of FGF21 is not clear. In our experiments we studied the antiobesogenic effects of FGF21 on mice model of diet-induced obesity at thermoneutrality. It is assumed that this model approach (in contrast to housing mice at standard laboratory temperature) mimics closely the metabolic status of humans. During the 4- to 8-day FGF21 treatment we observed a gradual reduction of lipid content in the brown and white adipose tissue and liver, especially in combination with β3-adrenergic stimulation. We have confirmed that FGF21 inhib- its lipolysis and also stimulates browning in certain adipose tissue depots. Furthermore, we have found that the effect of FGF21 on fatty acid secretion by adipose tissue is not mediated by changes in the fatty acid re-esterification...

Endogenous cardiac protection against acute ischemia/reperfusion injury can by increased by cardiac adaptation to various forms of chronic hypoxia. Chronic hypoxia induces a large variety of adaptive changes in the myocardium that could be considered as protective, but the exact mechanism of increased ischemic tolerance is unknown. Different studies suggest that catecholamine release and their effect on -adrenergic signaling after adaptation to chronic hypoxia contributes to cardioprotection. In this study we focused on characterization of -adrenergic receptors ( -ARs) in the myocardium of rats after adaptation to three different hypoxic conditions: 1. intermittent normobaric hypoxia - INH/R (23 h hypoxia, 1 h reoxygenation), 2. intermittent normobaric hypoxia - INH (8 h hypoxia, 16 h normoxia), 3. continuous normobaric hypoxia - CNH (24 h hypoxia). We compared how each hypoxic model affects the total number of -adrenergic receptors and proportion of individual subtypes ( 1-and 2-ARs) in the left and right ventricles compared control normoxic rats. The INH model had apparently no effect on -ARs in either ventricles. On the other hand, adaptation to INH/R and CNH was accompanied by a significant decrease (by about 25%) in the total number of -adrenergic receptors in the right ventricles. Our present...

Flies are a widely distributed pest insect that poses a significant threat to food security. Flight is essential for the dispersal of the adult flies to find new food sources and ideal breeding spots. The supply of metabolic fuel to power the flight muscles of insects is regulated by adipokinetic hormones (AKHs). The fruit fly, Drosophila melanogaster, the flesh fly, Sarcophaga crassipalpis, and the oriental fruit fly, Bactrocera dorsalis all have the same AKH that is present in the blowfly, Phormia terraenovae; this AKH has the code-name Phote-HrTH. Binding of the AKH to the extracellular binding site of a G protein-coupled receptor causes its activation. In this thesis, the structure of Phote-HrTH in SDS micelle solution was determined using NMR restrained molecular dynamics. The peptide was found to bind to the micelle and be reasonably rigid, with an S 2 order parameter of 0.96. The translated protein sequence of the AKH receptor from the fruit fly, Drosophila melanogaster, the flesh fly, Sarcophaga crassipalpis, and the oriental fruit fly, Bactrocera dorsalis were used to construct two models for each receptor: Drome-AKHR, Sarcr-AKHR, and Bacdo-AKHR. It is proposed that these two models represent the active and inactive state of the receptor. The models based on the crystal structure of the β-2 adrenergic receptor were found to bind Phote-HrTH with a predicted binding free energy of –107 kJ mol–1 for Drome-AKHR, –102 kJ mol–1 for Sarcr-AKHR and –102 kJ mol–1 for Bacdo-AKHR. Under molecular dynamics simulation, in a POPC membrane, the β-2AR receptor-like complexes transformed to rhodopsin-like. The identification and characterisation of the ligand-binding site of each receptor provide novel information on ligand-receptor interactions, which could lead to the development of species-specific control substances to use discriminately against these pest flies.

Katecholamine beeinflussen durch direkte Stimulation über adrenerge Rezeptoren die Funktion von Immunzellen. Ziel der Untersuchungen an Patienten mit Rheumatoider Arthritis war es, das Expressionsprofil unterschiedlicher adrenerger Rezeptorsubtypen in CD4(+)T-Lymphozyten dieser Patienten zu bestimmen. Zur Quantifizierung der Expression wurden semiquantitative RT-PCR-Analysen durchgeführt. Die Untersuchung zeigte, dass alpha1-adrenerge Rezeptoren in CD4(+)-T-Lymphozyten von RA-Patienten exprimiert werden. Es scheint eine Korrelation zwischen bestimmten extraartikulären Organmanifestationen (z.B. Sicca-Sydrom und Tenosynovitis) und der Expression alpha1-adrenerger Rezeptoren zu bestehen. Die gefundene differenzielle Expression der Rezeptoren in CD4(+)-T-Lymphozyten von RA-Patienten legen vertiefende Untersuchungen zur Relevanz des adrenergen Systems bei der Lymphozytenfunktionsmodulation nahe.

α2-Adrenergic receptor (AR) subtype mRNA (α2a, α2b, α2c) neuronal localization in human spinal cord has not been described. We therefore performed in situ hybridization to identify cell bodies at four levels of human spinal cord (cervical, thoracic, lumbar, sacral) containing α2AR subtype specific mRNA. α2AR mRNA is present in gray matter only (ventral > dorsal; sacral > cervical > thoracic = lumbar). In addition to α2AR mRNA in cell bodies in thoracic and lumbar intermediolateral (sympathetic) and sacral intermediate (parasympathetic) cell columns (lamina VII), all levels in dorsal horn laminae I, II, V, and ventral horn lamina IX, we demonstrate α2AR mRNA in dorsal horn laminae III and IV, and dorsal nucleus of Clarke, where α2ARs have not been described. Previously unreported heterogeneity in α2AR subtype distribution (α2a and α2bAR mRNA present, α2cAR mRNA virtually absent) is found at all sites of α2AR mRNA expression in human spinal cord, including locations known to mediate effects of α2AR agonist drugs on nociception, autonomic function and motor tone. Cervical spinal cord demonstrates a predominance of α2a mRNA signal, while thoracic, lumbar, and sacral spinal cord demonstrate an increasing predominance of α2bAR mRNA. If confirmed at a protein level, these findings have profound implications for therapeutic strategies in managing human pain.

Stress hormones, adrenaline and noradrenaline, have been shown to be critical for heart development. Mice lacking dopamine greek lower case letter beta]-hydroxylase (Dbh), an enzyme responsible for synthesis of these adrenergic hormones, die during mid-gestation due to cardiac failure. Prior research showed that adrenergic cells are found within the electrical conduction system of the heart, and adrenergic deficiency leads to slowed cardiac conduction during embryogenesis. Microarray analysis of wild-type (Dbh+/+) and knockout (Dbh-/-) mouse hearts revealed significant differences in expression of retinoic acid (RA) signaling genes. RA signaling has also been shown to be critical for heart development. These data suggest that heart failure due to adrenergic deficiency may be dependent upon RA signaling. This led to the hypothesis that adrenergic hormones promote the development of the electrical conduction system through modulation of RA signaling. To test this, embryonic mouse hearts were cultured with LE 135, a RA receptor blocker. Heart rate, arrhythmic index (AI) and conduction time were measured. Under these conditions there was a marked increase in arrhythmias. Hearts treated with LE 135 showed a mean AI of 0.232±0.057 after 24 hours of treatment while when untreated had an AI of 0.083±0.028 (p<0.05;n=15). In contrast, there was no significant change in heart rate or conduction speed after 24 hours with or without the retinoic acid receptor blocker. To determine if adrenergic stimulus influences retinoic acid response, an established RA-sensitive reporter cell line was employed. These F9-RARE-LacZ cells were treated with forskolin (cAMP regulator) and isoproterenol (greek lower case letter beta]-agonist) to measure changes in RA signaling. Evaluation of RA signaling showed an increase in retinoic acid responsiveness when treated with an adrenergic signaling agonist.; These results suggest that proper retinoic acid signaling is essential for maintaining cardiac rhythmicity during embryonic development and adrenergic stimulation can influence this response.

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