Melanome zählen zu den zehn häufigsten Tumorentitäten weltweit. Bei frühzeitiger Diagnose ist eine Exzision im Gesunden kurativ. Sobald eine Resektion im Gesunden jedoch nicht mehr möglich ist, sinken die Heilungschancen drastisch. Maligne Melanome sprechen wenig auf konventionelle Tumortherapien wie Radiatio und zytostatische Chemotherapie an. Daher werden neue Therapieoptionen in der Melanomtherapie getestet. Neueste Ansätze beziehen sich auf die Modulation von Immunzellen mittels monoklonaler Antikörper sowie die Modifikation der Signaltransduktion über die Mitogen-aktivierte Protein Kinase Kinase (MAPKK = MEK), BRAF und c-KIT. Auch Ionenkanäle stellen eine vielversprechende, zukünftige Option in der Behandlung maligner Melanome dar. Ich konnte zeigen, dass neben der bereits beschriebenen funktionellen Expression des „transient receptor potential“ Kanals TRPM8 in Melanomzelllinien auch TRPA1 in verschiedenen Melanomzelllinien exprimiert und funktionell ist. Die Phytopharmaka Senföl (Allylisothiozyanat; AITC) und Zimtaldehyd zeigen in Melanom-Modellen antitumoröse Effekte. Zudem sind beide Substanzen potente Stimulatoren von TRPA1. In dieser Arbeit wurde untersucht, ob AITC und Zimtaldehyd TRPA1-vermittelt die Proliferation, Apoptose und Migration von Melanomzellen beeinflussen. Das Vorkommen von TRPA1 in verschiedenen Melanomzelllinien wurde auf molekularbiologischer Ebene, mit fluorometrischen Bestimmungen des TRPA1-vermittelten Ca2+-Einstroms sowie in elektrophysiologischen Messungen nachgewiesen. Anschließend wurde die funktionelle Relevanz von TRPA1 bezüglich tumorhemmender Eigenschaften geprüft. Durch die Anwendung von TRPA1-Blockern konnte die AITC- und Zimtaldehyd-induzierte Verminderung der Proliferation nicht aufgehoben werden. Auch bezüglich der Migration und Apoptose konnte keine Korrelation zu einer TRPA1-Modulation festgestellt werden. Daher scheinen die durch AITC und Zimtaldehyd induzierten Effekte höchstwahrscheinlich nicht durch TRPA1 vermittelt zu werden.

Diabetic cystopathy is a chronic and common complication of diabetes with a classical triad of symptoms; decreased bladder sensation, increased bladder capacity and impaired detrusor muscle contractility (Hunter and Moore, 2003). In animal models of diabetes such as streptozotocin-induced diabetes in the rat, abnormalities of bladder function have been reported (Longhurst and Belis, 1986). The prototypic TRPV channel, TRPV1, is activated by capsaicin, which has been shown to cause contraction of the rat bladder (Saitoh et al., 2007), and this is reduced in STZ-diabetic rat bladder (Pinna et al., 1994). Therefore we hypothesize that TRPV1 function will be reduced in the diabetic bladder. The aim of this study are the following: Firstly, to investigate the effect of the streptozotocin (STZ) model of diabetes on a range of TRP channel functions in the urinary bladder smooth muscle preparation using TRP channel agonists and antagonists and to study the neurotransmitters involved in the contractile or relaxant responses. Some studies were also performed on colon tissues. Secondly, to explore the involvement of cholesterol modudation in TRP channel signalling. Thirdly, to study the change in TRP channel response with time following the treatment with streptozotocin. The results showed that the contractile responses to the TRPV1 agonist capsaicin, TRPV4 agonist 4-α-PDD, and TRPA1 agonist allyl isothiocyanate were significantly reduced in diabetic bladder. The selective TRPV1 antagonist, SB-366791, inhibited the contractile responses to capsaicin confirming the involvement of TRPV1 channels. The effect of diabetes is unlikely to be at the level of contractile machinery since the contractile responses to muscarinic receptor agonist carbachol were not significantly reduced in diabetic tissues. It is reported for the first time that the combination of neurokinin 1 and 2 antagonists GR-205171 and SB-207164 inhibited the contractile responses to capsaicin suggesting that a neurokinin may be the neurotransmitter involved in the capsaicin responses. In addition, the reduction of the responses to capsaicin in STZ-induced diabetic tissues occurred not only in urinary bladder but also in colon. Cholesterol-PEG significantly lowered the maximal contractile responses to capsaicin of rat bladder strips. Methyl-β-cyclodextrin, α-cyclodextrin and β-cyclodextrin at the same concentrations enhanced the contractile responses to capsaicin in the control and diabetic rat bladder strips. These effects of cyclodextrin are specific to capsaicin activated contractions and not seen with TRPA1 activation, suggesting that the effects are not mediated downstream of channel activation. Since α-cyclodextrin does not sequester cholesterol, the enhanced responses to cyclodextrins may not be due to the cholesterol modulations. Instead, theses novel findings may possibly occur by changing the local membrane lipid environment of the TRPV1 channel. As early as 36 hours after induction of diabetes by STZ, the contractile responses to capsaicin were significantly reduced in comparison to those of the controls and this reduction persisted until the eight weeks time point. In contrast, responses to the TRPA1 agonist allyl isothiocyanate were not affected at early time points but were reduced one week after STZ treatment. This detailed time course analysis suggests that there are novel mechanisms of modulation of the TRPV1 channels in this STZ model. In conclusion, in the rat urinary bladder or colon preparations, diabetes mellitus using STZ animal model caused 1) the impairment of a number of TRP channel subfamily functions, TRPV1, TRPV4 and TRPA1 but not TRPM8. The combination of NK1 and NK2 antagonists significantly inhibited the responses to capsaicin. This may suggest the involvement of neurokinin in postsynaptic transmission in rat bladder following the activation of TRPV1 channel, 2) the impairment caused by STZ-induced diabetes occurred very early (within 36 hours after diabetes induction) in TRPV1 channel but not TRPA1 channel. There are specific early effects of STZ treatment on TRPV1 channel function at a time when other afferent nerve terminal channels (TRPA1) are functioning normally, suggesting that early onset of dysfunction in TRPV1 signalling may not merely be the consequence of nerve damage, 3) the mechanism of this impairment may not be the effect of neuropathy on neurotransmitter release or nerve damage. Improving the responsiveness of nerves of bladder in diabetic patients might be of therapeutic benefit. The present studies suggest that it is possible to enhance function using indirect modulators such as bradykinin which potentiated the TRPV1 channel function in diabetic rat bladders.

Thesis (Ph. D.)--Univeristy of Nevada, Reno, 2002. / Includes bibliographical references. Online version available on the World Wide Web.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2010. / Title from PDF title page (viewed on June 25, 2010). Includes bibliographical references.

Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(工学) / 甲第15408号 / 工博第3287号 / 新制||工||1495(附属図書館) / 27886 / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 森 泰生, 教授 濵地 格, 教授 跡見 晴幸 / 学位規則第4条第1項該当

Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(工学) / 甲第15728号 / 工博第3342号 / 新制||工||1505(附属図書館) / 28273 / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 森 泰生, 教授 濵地 格, 教授 跡見 晴幸 / 学位規則第4条第1項該当

The transient receptor potential (TRP) are cation channels mostly permeable to both monovalent and divalent cations. ThermoTRP is a specific group of directly thermally activated TRP channels. The vanilloid transient receptor potential 3 (TRPV3) is an ion channel widely expressed in keratinocytes, that is implicated in the regulation of skin homeostasis, thermo- sensing, nociception and development of itch sensation. Our results show the importance of the cytoplasmic inter-subunit interface in the heat sensitivity of TRPV3. As there is a structural analogy within the vanilloid receptors, our hypothesis of the identified important region is supposed to be valid also for other thermally activated TRPV receptors (TRPV1, TRPV2 and TRPV4). We have proved that TRPV3 is a substrate for ERK1/2 protein kinase (kinase regulated by extracellular signal 1 and 2) and we have identified TRPV3 phosphorylation sites that may be direct targets for ERK1/2. Of these residues, threonine 264 has been shown to be the main phosphorylation site responsible for TRPV3 sensitization mediated by ERK kinase. In human keratinocytes, the phosphorylation might be physiologically and pathophysiologically important in processes of TRPV3 sensitization mediated by MAPK signaling pathway. The transient receptor potential ankyrin 1...

L’hypertension pulmonaire (HTP) est la principale pathologie de la circulation pulmonaire et a un très mauvais pronostic. Elle se caractérise par une hyperréactivité et un remodelage des petites artères pulmonaires (AP) entraînant une augmentation progressive des résistances vasculaires pulmonaires, qui, ultimement, aboutit à une insuffisance cardiaque droite et au décès du patient. Il est admit que le calcium joue un rôle très important aussi bien dans les mécanismes de remodelage que dans l’hyperréactivité des AP observés dans l’HTP. Dans le présent travail, nous avons étudié l’expression et le rôle d’une famille particulière de canaux calciques, les TRPV, dans les AP de rats contrôles (normoxiques) et souffrant d’hypertension pulmonaire (rats hypoxiques chroniques et traités à la monocrotaline). Nous montrons que (1) les canaux TRPV1, V2 et V4 sont exprimés dans les AP et que cette expression est augmentée au cours de l’HTP ; (2) la stimulation de ces canaux par des agonistes spécifiques induit une augmentation de la concentration calcique intracellulaire dans les cellules musculaires lisses (CML) ; (3) le récepteur à la ryanodine de type 2 (RRy 2) du réticulum sarcoplasmique est impliqué dans la voie de signalisation dépendante de TRPV4 et que son expression est également augmentée au cours de l’HTP ; (4) les canaux TRPV1 et TRPV4 sont impliqués dans la migration des CML, processus fondamental du remodelage ; (5) les contractions induites par l’activation de TRPV2 et TRPV4 dans les AP de rats hypertendus sont significativement diminuées par la streptomycine, un inhibiteur des canaux SAC (stretch activated channels). Ce travail démontre donc l’implication des canaux TRPV à la fois dans l’hyperréactivté et le remodelage des AP. De nouveaux traitements ciblant les canaux TRPV pourraient constituer une approche thérapeutique innovante de l’hypertension pulmonaire. / Pulmonary hypertension (PH)) is the primary pathology of the pulmonary circulation and has a very bad prognostic. This disease is characterized by a hyperreactivity and remodelling of small pulmonary arteries (PA) leading to a progressive increase in pulmonary vascular resistance which ultimately leads to right heart failure and death of the patient. It is admitted that calcium plays an important role both in the mechanisms of remodelling and in the hyperresponsiveness of PA observed in PH. In the present work, we studied the expression and the role of a particular family of calcium channels, TRPV channels, in PA from control rats (normoxic) and pulmonary hypertensive rats (chronically hypoxic and monocrotaline-treated rats). We show that (1) TRPV1, V2 and V4 channels are expressed in the PA and that their expression are increased in PH; (2) stimulation of these channels by specific agonists induces an increase in the intracellular calcium concentration in smooth muscle cells (SMC), (3) the ryanodine receptor type 2 (RRy2) of the sarcoplasmic reticulum is involved in the TRPV4-dependent signaling pathway and its expression is also increased in PH, (4) TRPV1 and TRPV4 channels are involved in the migration of SMC, the fundamental process of remodelling, (5) contractions induced by activation of TRPV2 and TRPV4 in the PA from hypertensive rats are significantly decreased by streptomycine, an inhibitor of stretch activated channels (SAC). This work thus demonstrates the involvement of TRPV channels in both the hyperreactivity and remodelling of PA. New treatments targeting TRPV channels could be an innovative therapeutic approach for pulmonary hypertension.

Melanome zählen zu den zehn häufigsten Tumorentitäten weltweit. Bei frühzeitiger Diagnose ist eine Exzision im Gesunden kurativ. Sobald eine Resektion im Gesunden jedoch nicht mehr möglich ist, sinken die Heilungschancen drastisch. Maligne Melanome sprechen wenig auf konventionelle Tumortherapien wie Radiatio und zytostatische Chemotherapie an. Daher werden neue Therapieoptionen in der Melanomtherapie getestet. Neueste Ansätze beziehen sich auf die Modulation von Immunzellen mittels monoklonaler Antikörper sowie die Modifikation der Signaltransduktion über die Mitogen-aktivierte Protein Kinase Kinase (MAPKK = MEK), BRAF und c-KIT. Auch Ionenkanäle stellen eine vielversprechende, zukünftige Option in der Behandlung maligner Melanome dar. Ich konnte zeigen, dass neben der bereits beschriebenen funktionellen Expression des „transient receptor potential“ Kanals TRPM8 in Melanomzelllinien auch TRPA1 in verschiedenen Melanomzelllinien exprimiert und funktionell ist. Die Phytopharmaka Senföl (Allylisothiozyanat; AITC) und Zimtaldehyd zeigen in Melanom-Modellen antitumoröse Effekte. Zudem sind beide Substanzen potente Stimulatoren von TRPA1. In dieser Arbeit wurde untersucht, ob AITC und Zimtaldehyd TRPA1-vermittelt die Proliferation, Apoptose und Migration von Melanomzellen beeinflussen. Das Vorkommen von TRPA1 in verschiedenen Melanomzelllinien wurde auf molekularbiologischer Ebene, mit fluorometrischen Bestimmungen des TRPA1-vermittelten Ca2+-Einstroms sowie in elektrophysiologischen Messungen nachgewiesen. Anschließend wurde die funktionelle Relevanz von TRPA1 bezüglich tumorhemmender Eigenschaften geprüft. Durch die Anwendung von TRPA1-Blockern konnte die AITC- und Zimtaldehyd-induzierte Verminderung der Proliferation nicht aufgehoben werden. Auch bezüglich der Migration und Apoptose konnte keine Korrelation zu einer TRPA1-Modulation festgestellt werden. Daher scheinen die durch AITC und Zimtaldehyd induzierten Effekte höchstwahrscheinlich nicht durch TRPA1 vermittelt zu werden.:1 Bibliographische Beschreibung 4 2 Abkürzungsverzeichnis 6 3 Einführung 8 3.1 Die Superfamilie der TRP-Kanäle und ihre Expression in malignen Tumoren 8 3.2 Weitere Ionenkanalentitäten im malignen Melanom 10 3.3 Klassifikation und Therapie maligner Melanome 11 3.4 Naturstoffe mit TRP-Kanal-aktivierenden Eigenschaften in der Therapie maligner Tumore 13 3.5 TRPA1 - ein Ionenkanal mit chemosensorischen Eigenschaften 14 4 Ableitung der Fragestellung 17 5 Publikation 18 6 Zusammenfassung der Arbeit 28 7 Literaturverzeichnis 31 8 Anlagen 36 8.1 Erklärung über die eigenständige Abfassung der Arbeit 36 8.2 Curriculum Vitae 37 8.3 Publikationen 39 8.4 Danksagung 40

Doctor of Philosophy / Department of Entomology / David C. Margolies and Yoonseong Park / Organisms are capable of sensing environmental conditions through diverse mechanisms. Transient receptor potential channels (TRPs) are a cation channel family that has been found to function in diverse sensing mechanisms. In this dissertation, I identified the function of several TRPs in thermosensing and mechanosensing in the red flour beetle, Tribolium castaneum. Candidate TRPs were chosen based on homology to TRPs found and studied in Drosophila melanogaster. To identify the function of candidate TRPs in T. castaneum, I suppressed the expression of target genes by RNA interference technique and investigated the phenotype of each treated beetle. Temperature is a major limiting environmental factor for organisms. I tested the function of candidate TRPs in thermotaxis (behavior) and thermal acclimation (physiology). Using bioinformatics approaches, I identified three candidate TRPs – painless, pyrexia, and trpA1 – involved in high temperature sensing. To test thermotactic behavior, I investigated beetle movement on a temperature arena with two separate temperature zones. Thermal acclimation was tested by pre-exposing beetles to either 42 °C for 10 min. When treated with double stranded RNA of TRPA1 (dstrpA1), the thermotactic response of beetles at 39 and 42 °C was reduced when compared to control groups. With pre-exposure at 42 °C, survivorship of dstrpA1-treated beetles significantly increased after one minute exposure at 52 °C compared to beetles that were not pre-exposed. With dspainless treatment, beetles showed lower response to thermal acclimation and lower long-term survivorship. Beetles treated with dspyrexia showed lower recovery after heat treatment without pre-exposure at 42 °C. To identify the function of candidate TRPs in mechanosensing, I evaluated dsRNA treated beetles for survival, walking behavior, and tonic immobility. Treatment with dsnompC and dstrpA5 resulted in failure in eclosion, causing 93 % mortality in both treatments. Survivors in dsnompC showed defects in elytra sclerotization. In dsnanchung and dsinactive treatments, adults showed abnormal walking behavior and reduced walking speed that were likely caused by defects of mechanosensing in folding of the joint between the femur and tibia. For tonic immobility, beetles with dsnanchung, dsinactive, dswaterwitch and dsick2 (insect cytokine 2) treatments showed increased sensitivity to mechanical stimulation leading to tonic immobility.