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Le système nerveux sympathique (SNS) a été identifié à être progressivement activé dans de nombreuses maladies cardio-vasculaires, du processus chroniques différents, y compris l'hypertension et de cardiomyopathie, à l'insuffisance cardiaque congestive[82,156,412-414]. L'hyperactivité du système nerveux sympathique peut modifier le nombre, la fonction et la voie de signalisation en aval des récepteurs β-adrénergiques. Bien qu'il n'yait pas de mécanisme unifié pour interpréter ces découvertes divergentes, des anomalies de calcium ont été reconnu comme une cause fondamental de la fonction systolique défectueuse du ventricule gauche [82]. Le niveau de calcium intracellulaire est directement impliqué dans l'interaction entre l'actine et la myosine, qui controle la contractilité du muscle. Les stimulations des récepteurs β-adrénergiques peuvent induire des réponses inotropes / lusitropes positifs par la production d'adénosine monophosphate cyclique (AMPc) et l'activation de la protéine kinase A (PKA). Le calcium de type transitoire est augmenté suite à la phosphorylation des protéines ciblées (cannaux calciques, récepteurs de la ryanodine, SERCA2a et troponine). Dans les conditions physiologique, la dysfonctionnement des récepteurs β-adrénergiques peut être un mécanisme commun de la réduction de la fonction cardiaque. Dans ce contexte, nous avons effectué des expériences au sujet de la voie de stimulation β-récepteurs adrénergiques en deux états physiopathologiques différents, cardiomyopathie du syndrome métabolique et de personnes âgées.La première expérience est menée au sein de rats sénescents. Au coeur sénescent, un dysfonctionnement diastolique et une réponse réduite à la stimulation β-adrénergiques ont été identifiés, qui sont associés à une sous-expression des récepteurs β1- et β2-adrénergiques, ainsi qu’à une sur-expression des récepteurs β3-adrénergiques.[73,415,416] Ces changements permettent de réduire la production d'AMPc ou facilitent l'hydrolyse de l'AMPc. Ainsi, un mécanisme complémentaire de la régulation de l'AMPc a été impliqué dans le cœur. La protéine de multirésistance 4 (MRP4) joue un rôle important dans la régulation de l'AMPc intracellulaire et les réponses cardiaques stimulées par les récepteurs β-adrénergiques. Mais le rôle de MRP4 au coeur sénescent n'a jamais été étudié. Dans ce fait, nous avons mené de l'expérience pour étudier l'expression de MRP4 et son influence sur le dysfonctionnement β-adrénergique dans le cœur sénescent. L’expression de MRP4 a été quantifiée dans ventriculaires gauches par Western Blotting. Les réponses des récepteurs β-adrénergiques à l'isoprotérénol ont été étudiés in vivo (échocardiographie de stress) et in vitro (raccourcissement des sarcomères et de calcium de type transitoire dans les cardiomyocytes isolés par Ionoptix®) chez les rats jeunes (âgés de 3 mois) et les rats sénescents (âgés de 24 mois) prétraités ou non avec MK571, un inhibiteur spécifique de MRP4.... / The sympathetic nervous system (SNS) has been identified to be progressively activated in many cardiovascular diseases, from different chronic process including hypertension and cardiomyopathy, to congestive heart failure [82,156,412-414]. The hyperactivity of sympathetic nervous system may change the number, function and downstream mechanisms of β-adrenoceptors. Although there is no unified mechanism to interpret those divergent findings, calcium abnormalities has been recognized to be fundamental in the defective systolic function in left ventricle [82]. The intracellular calcium level is directly involved in the interaction of actin and myosin, thus reflects the contractility of muscle. β-adrenergic stimulation can induce the positive inotropic / lusitropic responses via the production of cyclic adenosine monophosphate (cAMP) and the activation of protein kinase A (PKA). Thus calcium transient is increased after phosphorylation of serials targeted proteins (calcium channel, ryanodine receptor, SERCA2a, and troponin). Under the pathophysiological condition, β-adrenergic dysfunction may be a common mechanism of decreased cardiac function. So, we performed experiments about β-adrenoceptor stimulation pathway in two different pathophysiological status, cardiomyopathy of the elderly and metabolic syndrome. The first experiment is conducted within senescent rat. In the senescent heart, diastolic dysfunction and reduced response to β-adrenergic stimulation have been identified, which are associated with the down-regulation of 1- and 2-adrenoceptors, along with the up-regulation of 3-adrenoceptor. [73,415,416] These changes either reduce the cAMP production or facilitate the hydrolysis of cAMP. Meanwhile, a complementary mechanism of the regulation of cAMP has been involved in heart. The multidrug resistance protein 4 (MRP4) plays an important role in the regulation of intracellular cAMP and β-adrenergic stimulated cardiac responses. But the role of MRP4 in the senescent heart has never been studied. Thus, we conducted the experiment to study the MRP4 expression and its influence on β-adrenergic dysfunction in the senescent rat heart. MRP4 was quantified in left ventricular homogenates by Western blotting. The β-adrenergic responses to isoproterenol were investigated in vivo (stress echocardiography) and in vitro (sarcomere shortening and calcium transient of isolated cardiomyocyte by Ionoptix®) in young (3-month age) and senescent (24-month age) rats pretreated or not with MK571, a specific MRP4 inhibitor. As a result, we confirmed that the MRP4 overexpression contributes to the decrease of positive inotropic response to β-adrenoceptor stimulation in the senescent heart...

2017-02-09全文追加 / 京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18453号 / 医博第3908号 / 新制||医||1004(附属図書館) / 31331 / 京都大学大学院医学研究科医学専攻 / (主査)教授 坂田 隆造, 教授 木村 剛, 教授 小池 薫 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Some forms of neuropathic pain are maintained by sympathetic fibers, but the underlying mechanisms are poorly understood. Thus, the aim of this study was to investigate the possible involvement of the TRPA1 receptor as well as the role of the sympathetic nervous system (involved in sympathetically maintained neuropathic pain) in a model of neuropathic pain induced by chronic sciatic nerve constriction injury (CCI) in mice. The systemic injection of the selective TRPA1 antagonist HC-030031 reversed both mechanical and cold allodynia induced by chronic sciatic nerve constriction injury. Nerve injury also sensitizes mice to the nociception induced by the intraplantar injection of a low dose of the TRPA1 agonist allyl isothiocyanate without changing TRPA1 immunoreactivity in the injected paw. Furthermore, the chemical sympathectomy produced by guanethidine largely prevented CCI-induced mechanical and cold allodynia. CCI also induced a norepinephrine-trigged nociception that was inhibited by α-adrenoceptor antagonism and norepinephrine transporter and monoamine oxidase inhibition. Finally, the peripheral injection of HC-030031 also largely reduced CCI-induced nociception by norepinephrine and mechanical or cold allodynia. Taken together, the present findings reveal the critical role of TRPA1 in mechanical and cold hypersensitivity as well as in hypersensitivity to norepinephrine following nerve injury. This article presents the role of TRPA1 receptor on the sympathetically-maintained nociception induced by nerve injury in mice. Our results suggest that TRPA1 antagonists may be useful to treat neuropathic patients that present sympathetically maintained pain. / Algumas formas de dor neuropática são mantidos por fibras simpáticas, contudo os mecanismos subjacentes são pouco compreendidos. Assim, o objetivo deste estudo foi investigar o possível envolvimento do receptor TRPA1, bem como o papel do sistema nervoso simpático num modelo de dor neuropática induzido pela constrição crônica do nervo ciático (CCI) em camundongos. A administração sistêmica do antagonista seletivo do receptor TRPA1, HC-030031, reverteu a alodinia mecânica e ao frio induzida pela CCI. A injeção de uma baixa dose do agonista do receptor TRPA1, isotiocianato de alila, induziu uma resposta nociceptiva nos animais que sofreram a lesão no nervo, sem alterar a imunorreatividade do TRPA1 na pata injetada. Além disso, a simpatectomia química produzida pela guanetidina amplamente previniu a alodínia mecânica e ao frio induzida pela CCI. Ainda, a injeção intraplantar de norepinefrina induziu nocicepção espontânea, a qual foi reduzida pelo antagonista α-adrenérgico, inibidor do transportador de norepinefrina e inibidor da enzima monoamina oxidase. Finalmente, a administração periférica do HC-030031 reduziu a nocicepção espontânea induzida pela norepinefrina e também a alodínia mecânica e térmica de animais neuropáticos. Assim, nossos resultados revelam o papel crítico do receptor TRPA1 na alodínia mecânica e ao frio, bem como na hipersensibilidade à norepinefrina após lesão do nervo em um modelo de dor neuropática mantida pelo simpático. Dessa forma, o receptor TRPA1 poderá ser um alvo para o desenvolvimento de novos tratamentos para esse tipo de dor.