Thesis (M.S.)--Duquesne University, 2009. / Abstract included in electronic submission form. Title from document title page. Includes bibliographical references (p. 65-71) and index.

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 157-188).

京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第21047号 / 薬科博第90号 / 新制||薬科||10(附属図書館) / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 竹島 浩, 教授 中山 和久, 教授 金子 周司 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

Kationenkanäle der Canonical Transient Receptor (TRPC)-Familie spielen eine wichtige Rolle in der pathologischen Herzhypertrophie. Neben anderen Isoformen besitzt TRPC4 die Potenz, den strukturellen und funktionellen Umbau des Herzens im Rahmen der pathologischen Hypertrophie über Ca2+-Transienten zu bestärken. TRPC4-Kanäle sind nicht-selektive Kationenkanäle, die für Na+ und Ca2+ durchlässig sind. Sie setzen sich in der Plasmamembran zu Homo- oder Heterotetrameren zusammen. Die TRPC4-Kanalaktivität wird durch die Stimulation von Gq-Protein-gekoppelten Rezeptoren (GPCR) reguliert und führt zu einem Ca2+-Einstrom, der für die Aktivierung von Calcineurin und des nuclear factor of activated T-cells (NFAT) notwendig ist. Eine weitere Aktivierungsform lässt sich über die Entleerung von intrazellulären Ca2+-Speichern (SOCE) aus dem Sarkoplasmatischen Retikulum (SR) nachweisen. Die funktionelle Wirkung des TRPC4 ist von der Expression der beiden Splice-Varianten TRPC4α und TRPC4β abhängig. Um diese funktionelle Abhängigkeit der Splice-Variante C4β genauer zu charakterisieren, wurden in der vorliegenden Studie zytosolische Ca2+-Signale und deren Aktivierungsmechanismen analysiert. Für die Untersuchungen wurden neonatale Rattenkardiomyozyten (NRC) verwendet, die mit adenoviralen Vektoren infiziert wurden und TRPC4beta (Ad-TRPC4β), TRPC4alpha (Ad-TRPC4α) und beta-Galaktosidase (Ad-ßgal) als Kontrolle exprimierten. Es erfolgte eine Auswertung der Ca2+-Transienten, in der gezeigt werden konnte, dass TRPC4β den Ca2+-Einstrom in schlagenden Kardiomyozyten beeinflusst. Dies machte sich in einer erhöhten Ca2+-Amplitude unter basalen Bedingungen bemerkbar. Ebenfalls konnte deutlich gemacht werden, dass eine Ca2+-Entleerung des SR TRPC4β als sogenannten SOC (speicher-regulierten Kanal, store-operated channel) aktiviert. Außerdem reagierten TRPC4β-infizierte NRCs mit einem gesteigerten Ca2+-Maximalspitzenwert (peak) unter Stimulation mit dem GPCR-Agonisten Angiotensin II. Die Amplitude der Ca2+-Transienten bei Überexpression von Ad-TRPC4β war im Vergleich zur Ad-ßgal-Kontrollgruppe deutlich gesteigert. Darüber hinaus war der Abfall der Ca2+-Transienten der TRPC4β-exprimierenden Zellen beschleunigt. Dies lässt einen kompensatorischen Mechanismus vermuten, mit dem Ziel, einer Ca2+-Überladung der Zelle durch den TRPC4β-induzierten Ca2+-Einstrom entgegenzuwirken. In zusätzlichen Experimenten zeigte sich TRPC4β ebenfalls deutlich sensitiver gegenüber der Angiotensin II-Stimulation als TRPC4α. Weiterführende Untersuchungen ließen erkennen, dass TRPC4β, im Gegensatz zu anderen TRPC-Isoformen, keinen pro-hypertrophen, sondern vielmehr einen pro-apoptotischen Einfluss auf Kardiomyozyten ausübt. Zusammenfassend zeigt die vorliegende Studie, dass eine erhöhte Aktivität der Splice-Variante TRPC4β mit kritischen Veränderungen zytosolischer Ca2+-Signale verbunden ist und somit ein entscheidender Faktor für die Entstehung und Progression kardialer Pathologien sein könnte. / The Transient Receptor Potential Channel Subunit 4 (TRPC4) has been considered as a crucial Ca2+ component in cardiomyocytes promoting structural and functional remodeling in the course of pathological cardiac hypertrophy. Functional properties of TRPC4 are also based on the expression of the TRPC4 splice variants TRPC4α and TRPC4β. Aim of the present study was to analyze cytosolic Ca2+ signals, signaling, hypertrophy and vitality of cardiomyocytes in dependence on the expression level of either TRPC4α or TRPC4β.

Kationenkanäle der Canonical Transient Receptor (TRPC)-Familie spielen eine wichtige Rolle in der pathologischen Herzhypertrophie. Neben anderen Isoformen besitzt TRPC4 die Potenz, den strukturellen und funktionellen Umbau des Herzens im Rahmen der pathologischen Hypertrophie über Ca2+-Transienten zu bestärken. TRPC4-Kanäle sind nicht-selektive Kationenkanäle, die für Na+ und Ca2+ durchlässig sind. Sie setzen sich in der Plasmamembran zu Homo- oder Heterotetrameren zusammen. Die TRPC4-Kanalaktivität wird durch die Stimulation von Gq-Protein-gekoppelten Rezeptoren (GPCR) reguliert und führt zu einem Ca2+-Einstrom, der für die Aktivierung von Calcineurin und des nuclear factor of activated T-cells (NFAT) notwendig ist. Eine weitere Aktivierungsform lässt sich über die Entleerung von intrazellulären Ca2+-Speichern (SOCE) aus dem Sarkoplasmatischen Retikulum (SR) nachweisen. Die funktionelle Wirkung des TRPC4 ist von der Expression der beiden Splice-Varianten TRPC4α und TRPC4β abhängig. Um diese funktionelle Abhängigkeit der Splice-Variante C4β genauer zu charakterisieren, wurden in der vorliegenden Studie zytosolische Ca2+-Signale und deren Aktivierungsmechanismen analysiert. Für die Untersuchungen wurden neonatale Rattenkardiomyozyten (NRC) verwendet, die mit adenoviralen Vektoren infiziert wurden und TRPC4beta (Ad-TRPC4β), TRPC4alpha (Ad-TRPC4α) und beta-Galaktosidase (Ad-ßgal) als Kontrolle exprimierten. Es erfolgte eine Auswertung der Ca2+-Transienten, in der gezeigt werden konnte, dass TRPC4β den Ca2+-Einstrom in schlagenden Kardiomyozyten beeinflusst. Dies machte sich in einer erhöhten Ca2+-Amplitude unter basalen Bedingungen bemerkbar. Ebenfalls konnte deutlich gemacht werden, dass eine Ca2+-Entleerung des SR TRPC4β als sogenannten SOC (speicher-regulierten Kanal, store-operated channel) aktiviert. Außerdem reagierten TRPC4β-infizierte NRCs mit einem gesteigerten Ca2+-Maximalspitzenwert (peak) unter Stimulation mit dem GPCR-Agonisten Angiotensin II. Die Amplitude der Ca2+-Transienten bei Überexpression von Ad-TRPC4β war im Vergleich zur Ad-ßgal-Kontrollgruppe deutlich gesteigert. Darüber hinaus war der Abfall der Ca2+-Transienten der TRPC4β-exprimierenden Zellen beschleunigt. Dies lässt einen kompensatorischen Mechanismus vermuten, mit dem Ziel, einer Ca2+-Überladung der Zelle durch den TRPC4β-induzierten Ca2+-Einstrom entgegenzuwirken. In zusätzlichen Experimenten zeigte sich TRPC4β ebenfalls deutlich sensitiver gegenüber der Angiotensin II-Stimulation als TRPC4α. Weiterführende Untersuchungen ließen erkennen, dass TRPC4β, im Gegensatz zu anderen TRPC-Isoformen, keinen pro-hypertrophen, sondern vielmehr einen pro-apoptotischen Einfluss auf Kardiomyozyten ausübt. Zusammenfassend zeigt die vorliegende Studie, dass eine erhöhte Aktivität der Splice-Variante TRPC4β mit kritischen Veränderungen zytosolischer Ca2+-Signale verbunden ist und somit ein entscheidender Faktor für die Entstehung und Progression kardialer Pathologien sein könnte. / The Transient Receptor Potential Channel Subunit 4 (TRPC4) has been considered as a crucial Ca2+ component in cardiomyocytes promoting structural and functional remodeling in the course of pathological cardiac hypertrophy. Functional properties of TRPC4 are also based on the expression of the TRPC4 splice variants TRPC4α and TRPC4β. Aim of the present study was to analyze cytosolic Ca2+ signals, signaling, hypertrophy and vitality of cardiomyocytes in dependence on the expression level of either TRPC4α or TRPC4β.

No description available.

No description available.

Triptofano (TRP), um amino ácido essencial, é metabolizado por duas vias principais, a via das quinureninas e a via serotonérgica. Em ambas as vias há a possibilidade de formação de compostos ativos no sistema imune que se caracterizam pelas ações imunossupressoras e indutoras de tolerância. Na via serotonérgica há a formação de serotonina (5-HT) e em alguns tecidos de melatonina (MEL). Este composto pode ainda ser oxidado por ação de peroxidases aos seus produtos de abertura de anel indólico o AFMK (N1-acetil-n2-formil-5-metoxiquinuramina) e AMK (N1-acetil-5-metoxiquinuramina). Já na via das quinureninas, o TRP é diretamente metabolizado à N-formilquinurenina (NFK) e este é rapidamente deformilado a quinurenina (QUIN). Neste projeto avaliamos qual o efeito do choque endotóxico induzido por injeção endovenosa de LPS (1 mg/kg) sobre a biodisponibilidade de TRP e formação de seu metabólito QUIN. Este estudo foi realizado em condições controle e na vigência de sobrecarga de TRP (administração subcutânea de 0,8 mg/kg). Utilizamos ratos machos Wistar com 30 dias separados em quatro grupos: GI (controle), GII (LPS), GIII (TRP) e GIV (TRP+LPS). TRP (0,8 mg/Kg) foi injetado por via subcutânea nos tempos 0 e 2 horas. Quando injetado, LPS (1 mg/kg) foi administrado por via intravenosa no tempo 2 horas. Após 1 hora da última administração, sangue e cérebro foram coletados. O cérebro foi seccionado em três regiões: cerebelo, córtex e mesencéfalo, os quais foram processados para obtenção de homogenatos. Tanto os homogenatos quanto o soro foram tratados com acetona para extração de TRP e seus metabólitos. A análise destes compostos foi realizada por cromatografia líquida de alta eficiência (HPLC). A administração de TRP elevou significativamente a sua concentração no soro e no SNC. Quando da administração de LPS no grupo que já havia recebido sobrecarga de TRP (GIV) houve uma marcada elevação de TRP e de QUIN séricos e das regiões do SNC, especialmente na região do córtex. Concluímos que na vigência de choque endotóxico há um aumento da biodisponibilidade de TRP, tanto no soro como no SNC e que há um aumento da metabolização deste pela rota das quinureninas, possivelmente via IDO. Estes resultados contribuem para a compreensão da toxicidade de TRP, especialmente relevante no caso em que haja um choque endotóxico concomitante e evidencia o córtex como uma região mais susceptível para os efeitos tóxicos do TRP. / Tryptophan (TRP) is an essential amino acid, metabolized by two main paths; the kynurenine and the serotonergic pathways. In both, there is the possibility of generation of biologic active compounds, especially on the immune system leading to immunosuppression and tolerance. In the serotonergic path there is the formation of serotonine (5-HT) and in some tissues of melatonine (MEL). The latter can be oxidized by the action of peroxidases to its indole ring opening product AFMK (N1-acetil-n2-formil-5-methoxikynuramine) and AMK (N1-acethyl-5-methoxykynuramine). In the kynurenine path, TRP is metabolized to N-formylkynurenine (NFK) that is deformilated to kynurenine (KYN). In this study we evaluated the effect of a endotoxic skock induced by an intravenous injection of LPS (1 mg/kg) on the bioavailability of TRP and formation of KYN. This study was carried out in control conditions and on TRP overload (subcutaneous administration of 0,8 mg/Kg). One month old male Wistar rats were divide in four groups: GI(control), GII(LPS), GIII(TRP) and GIV (TRP+LPS). TRP (0,8 mg/kg) was subcutaneously injected at zero and 2h times. When injected, LPS (1mg/kg) was intravenously administered at 2 h. After one hour from the last administration, blood and brain were collected. Brain is separated in cerebellum, midbrain and cortex and was lysed for the preparation of homogenates. Both, serum and homogenates were extracted in acetone; TRP and KYN were analyzed by HPLC. TRP overload caused a significant increase in its concentration in serum and brain. When LPS was administered in conjunction with TRP overload (GIV) there was a remarkable increase in TRP and KYN in serum and brain, especially in cortex. Our conclusion is that in the bioavailability of TRP, in serum and in brain, and its metabolization to kynurenine is increased by inflammation. IDO is probably involved in this condition. Our results contribute to the knowledge of TRP toxicity, particularly with a concomitant inflammation and demonstrate the cortex as a region of more susceptibility to TRP toxicity.

En este trabajo de tesis doctoral se caracteriza funcional y molecularmente el canal TRPV4 en varios modelos de células epiteliales respiratorias mostrando por primera vez la participación de este canal en la función osmoreguladora a nivel celular así como la identificación de nuevas variantes del canal. Se demuestra que la entrada de Ca2+ en respuesta a un hinchamiento hipotónico se produce a través del canal TRPV4 y es necesaria para una eficiente recuperación del volumen o RVD. Por su parte, las células epiteliales respiratorias con fenotipo de fibrosis quística no son capaces de reducir su volumen en un medio hipotónico a causa de una regulación defectuosa del canal, indicando, al mismo tiempo, que la regulación del TRPV4 por el estímulo hipotónico es dependiente de la CFTR.La caracterización de las variantes del canal TRPV4 demuestra que los dominios de ANK son determinantes moleculares claves en el proceso de oligomerización del canal. Al mismo tiempo este trabajo describe nuevos aspectos relacionados con la biogénesis del TRPV4 hasta ahora desconocidos: la oligomerización del canal tiene lugar en el RE, orgánulo donde es N-glicosilado de forma simple antes de ser transportado hacia el Golgi donde sus N-glicanos son madurados. / This thesis characterizes molecularly and funcionally the TRPV4 channel in various models of airway epithelial cells showing, for the first time, the involvement of this channel in an osmoregulatory cellular function as well as the isolation of new splice variants of this channel. It is demonstrated that the TRPV4 channel is the molecular Ca2+ pathway activated by hypotonic estimulus needed to trigger the RVD response. Furthermore, the cystic fibrosis airway epithelial cells showed an impaired RVD due to the misregulation of the TRPV4 channel, indicating that the regulation by the hypotonic stimulus is CFTR-dependent.The characterization of the new variants demonstrated that the ANK domains are key structural determinants in the oligomerization process of the TRPV4. This work also describes new aspects related to the biogenesis of this channel: oligomerization is achieved in the ER, where the TRPV4 is N-glycosilated and then transported to the Golgi where the glycans are matured.

Submitted by Maike Costa (maiksebas@gmail.com) on 2017-09-12T11:16:41Z No. of bitstreams: 1 arquivototal.pdf: 71034772 bytes, checksum: 9c21531ff7d7e2de79d846d056ac6485 (MD5) / Made available in DSpace on 2017-09-12T11:16:41Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 71034772 bytes, checksum: 9c21531ff7d7e2de79d846d056ac6485 (MD5) Previous issue date: 2015-02-24 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The monoterpenes found in essential oils from plants act on transient receptor potential channels (TRP). Some TRP channels with altered expression in hypertensive rats may be new therapeutic targets for the control of hypertension. Aim: Compare the responses induced by Essential Oil of Mentha x villosa Hudson (OEMV), rotundifolone and menthol in Spontaneously Hypertensive rats (SHR) and normotensive Wistar Kyoto (WKY), evaluating the role of TRP channels. Methods and Results: In vivo (blood pressure measurement and heart rate), in vitro (measure of the frequency and force of contraction in the atria and the isometric tension in superior mesenteric arteries) and biochemical (PCR and Western blot) studies were used. The OEMV (3, 5, 10 and 20 mg/kg), the rotundifolone (10, 20 and 30 mg/kg) and the menthol (3, 5, 10 and 20 mg/kg) induced significant hypotensive and bradycardic response in non-anesthetized SHR and WKY rats. The reduction in the diastolic blood pressure was significantly greater than the decrease in the systolic blood pressure, suggesting a greater action on the vascular component of blood pressure. However, the significant bradycardic effect and reduction in the systolic blood pressure also suggest an action on the cardiac component. Furthermore, the decrease in the blood pressure and heart rate induced by rotundifolone and by menthol were significantly more potent in SHR. The action of OEMV, the rotundifolone and menthol in the right atrium (with spontaneous activity) and left (electrically stimulated) showed negative inotropic and chronotropic effects and culminating in complete inhibition of cardiac activity. Moreover, the negative inotropic effect was more potent in SHR and protein TRPM8 channel showed increased expression in the ventricles (left > right) and atria (left > right) of SHR rats. Also, OEMV, rotundifolone and menthol induced vasorelaxant response in superior mesenteric arteries of SHR and WKY rats, precontracted with PHE. The major mechanism involves the endothelium-independent route, which was more potent in SHR. The mechanism of the endothelium-independent vasorelaxant response induced by rotundifolone and menthol probably involves TRPM8 channels, which showed increased expression in SHR, and TRPC1, TRPC3 and TRPC6 channels. However, the response induced by menthol in WKY rats involves other TRP channels (probably TRPM6 and TRPM7). In addition, the flow cytometry showed an increase in [Ca2+]i induced by rotundifolone in SHR vascular myocytes, probably by activating of the TRPM8 channel. Conclusions: The hypotensive, bradycardia, negative inotropic and vasorelaxant responses induced by OEMV, rotundifolone and menthol were significantly more potent in SHR than in WKY rats. The mechanism of the endothelium-independent vasorelaxant response induced by rotundifolone and menthol involves TRPM8, TRPC (probably TRC1, TRPC3 and TRPC6), BKCa and CaV channels, but menthol may be acting in other TRP channels (probably TRPM6 and TRPM7) in WKY rats. The TRPM8 channel showed increased expression in SHR rats. Thus, the action of OEMV, rotundifolone and menthol on these channels can be related with the higher potency observed in SHR rats. / Os monoterpenos presentes em óleos essenciais de plantas atuam sobre canais Potencial Receptor Transiente (TRP). Alguns canais TRP com expressão alterada em ratos hipertensos podem ser novos alvos terapêuticos para o controle da hipertensão arterial. Objetivo: Comparar as respostas induzidas pelo Óleo Essencial de Mentha x-villosa Hudson (OEMV), pela rotundifolona e pelo mentol em Ratos Espontaneamente Hipertensos (SHR) e normotensos Wistar Kyoto (WKY), avaliando o papel de canais TRP. Métodos e Resultados: Estudos in vivo (medida de pressão arterial e freqüência cardíaca), in vitro (medida da freqüência e força de contração em átrios e da tensão isométrica em artérias mesentéricas superiores) e bioquímicos (PCR e Western blot) foram usados. O OEMV (3, 5, 10, 20 mg/kg), a rotundifolona (10, 20 e 30 mg/kg), e o mentol (3, 5, 10 e 20 mg/kg) induziram significativa resposta hipotensora e bradicárdica em ratos SHR e WKY não-anestesiados. A redução na pressão arterial diastólica foi significativamente maior do que a redução na pressão arterial sistólica, sugerindo uma maior ação sobre o componente vascular da pressão arterial. Entretanto, o significativo efeito bradicárdico e a redução na pressão arterial sistólica sugerem também uma ação sobre o componente cardíaco. Além disso, a diminuição na pressão arterial e freqüência cardíaca induzida por rotundifolona e por mentol foram significativamente mais potentes em ratos SHR. A ação do OEMV, da rotundifolona e do mentol em átrios direito (com atividade espontânea) e esquerdo (estimulado eletricamente) mostrou efeitos cronotrópico e inotrópico negativos e culminando na completa inibição da atividade cardíaca. Além disso, o efeito inotrópico negativo foi mais potente em ratos SHR e a proteína do canal TRPM8 mostrou expressão aumentada nos ventrículos (esquerdo > direito) e nos átrios (esquerdo > direito) de ratos SHR. O OEMV, a rotundifolona e o mentol também induziram resposta vasorrelaxante em artérias mesentéricas superiores de ratos SHR e WKY, pré-contraídos com FEN. O mecanismo majoritário envolve a via independente do endotélio, que foi mais potente em ratos SHR. O mecanismo da resposta vasorrelaxante independente do endotélio induzida por rotundifolona e mentol envolve provavelmente canais TRPM8, que apresentaram expressão aumentada em ratos SHR, e canais TRPC1, TRPC3 e TRPC6. Entretanto, a resposta induzida por mentol em ratos WKY envolve outros canais TRP (provavelmente TRPM6 e TRPM7). Além disso, a citometria de fluxo mostrou um aumento na [Ca2+]i induzido por rotundifolona em miócitos vasculares de ratos SHR, provavelmente por ativação de canais TRPM8. Conclusões: As respostas hipotensora, bradicárdica, inotrópica negativa e vasorrelaxante induzidas por OEMV, rotundifolona e mentol foram significativamente mais potentes em ratos SHR do que em ratos WKY. O mecanismo da resposta vasorrelaxante independente de endotélio induzida por rotundifolona e mentol envolve canais TRPM8, TRPC (provavelmente TRPC1, TRPC3 e TRPC6), BKCa e CaV, porém o mentol pode estar atuando em outros canais TRP (provavelmente TRPM6 e TRPM7) em ratos WKY. Os canais TRPM8 mostraram expressão aumentada em ratos SHR. Dessa forma, a ação do OEMV, da rotundifolona e do mentol sobre esses canais pode estar relacionada com a maior potência observada em ratos SHR.