The effect of capsazepine and nonylphenol on cytosolic free Ca2+ concentrations ([Ca2+]i) in MDCK renal tubular cells is unclear. This study explored whether capsazepine and nonylphenol changed basal [Ca2+]i levels in suspended MDCK cells by using fura-2 as a Ca2+-selective fluorescent dye. Capsazepine at concentrations between 10 and 200 microM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced partially by 40% after removing extracellular Ca2+. Capsazepine induced Mn2+ quench of fura-2 fluorescence, indirectly implicating Ca2+ entry. Capsazepine-induced Ca2+ influx was not changed by L-type Ca2+ entry inhibitors and protein kinase C modulators [phorbol 12-myristate 13-acetate (PMA) and GF109203X]. In Ca2+-free medium, 100microM capsazepine-induced Ca2+ release was substantially suppressed by pretreatment with thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor). Pretreatment with capsazepine nearly abolished thapsigargin-induced Ca2+ release. Nonylphenol also increased [Ca2+]i in a concentration- dependent manner like capsazepine does. Similar response in [Ca2+]i rise can be found by inhibition of phospholipase C and using thapsigargin. Different from capasazpine, the [Ca2+]i rise was inhibited by PMA. At concentrations between 5 and 100microM, nonylphenol killed cells in a concentration-dependent manner. Collectively, in MDCK cells, capsazepine induced [Ca2+]i rises by causing phospholipase C-independent Ca2+ release from the endoplasmic reticulum and Ca2+ influx via non-L-type Ca2+ channels. Nonylphenol induced [Ca2+]i increase in MDCK cells via evoking Ca2+ entry through protein kinase C-regulated Ca2+ channels, and releasing Ca2+ from endoplasmic reticulum and other cellular storage in a phospholipase C-independent manner.

>Magister Scientiae - MSc / Cancer is defined as the abnormal growth of genetically mutated or perturbant cells. Nicotine is a known cancer promoter and an apoptotic suppressor. This alkaloid acts on the nicotinic acetylcholine receptors which affects the ubiquitin-proteasome protein degradation pathway and ultimately hinders apoptosis. The endoplasmic reticulum (ER) is an interconnecting organelle which synthesises proteins and its quality control processes ensures the proper protein folding, post-translational modifications and conformation of secretory and trans-membrane proteins. Studies demonstrated that the antibiotic, Tunicamycin (Tm) and the sesquiterpene lactone, Thapsigargin (Tg) causes ER stress and consequently cellular arrest. Tm interferes with N-glycosylation of newly synthesised proteins triggering the unfolded protein response, while Tg inhibits intracellular Ca2+ ATPases resulting in increased cytosolic Ca2+. Studies showed that these compounds have potential pro-apoptotic effects. The combinatorial effects of nicotine, Tm and Tg may produce antagonistic or synergistic effects and provide a therapeutic tool against breast cancer. The aim of the study was to determine the apoptotic effects of nicotine, Tm, and Tg on human breast carcinoma (MCF-7) at various time intervals and further to elucidate whether selected ratios of their combinations resulted in synergistic or antagonistic effects.

Abstract The effect of mercury (Hg2+), a known nephrotoxicant, on intracellular free Ca2+ levels ([Ca2+]i) in Madin Darby canine kidney (MDCK) cells was explored. [Ca2+]i was measured by using the Ca2+-sensitive dye fura-2. Hg2+ increased [Ca2+]i in a concentration-dependent manner with an EC50 of 6 mM. The Ca2+ signal comprised a gradual increase. Removal of extracellular Ca2+ decreased the Hg2+-induced [Ca2+]i increase by 67%, suggesting that the Ca2+ signal was due to both extracellular Ca2+ influx and store Ca2+ release. In Ca2+-free medium, the Hg2+-induced [Ca2+]i increase was nearly abolished by pretreatment with 1 mM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), and conversely, pretreatment with Hg2+ abolished thapsigargin-induced Ca2+ increase. Hg2+-induced Ca2+ release was not altered by inhibition of phospholiase C but was potentiated by activation of protein kinase C. Overnight treatment with 1 mM Hg2+ did not alter cell proliferation rate, but 10 mM Hg2+ killed all cells. Collectively, this study shows that Hg2+ induced protein kinase C-regulated [Ca2+]i increases in renal tubular cells via releasing store Ca2+ from the endoplasmic reticulum in a manner independent of phospholipase C activity. Hg2+ also caused cytotoxicity at higher concentrations.

The present study was undertaken to investigate the effects of the T-type calcium channel blocker, Mibefradil, on white adipocytes. Unexpected for a T-type channel blocker, Mibefradil was found to increase intracellular calcium levels, cause lipid droplet fusion, and result in cell death. Calcium imaging of white adipocytes showed an increase of calcium concentration by Mibefradil at concentrations ranging from10-50 µM. The elevation in calcium by Mibefradil was significantly reduced by pretreatment of cells with Thapsigargin, an endoplasmic reticulum (ER) specific Ca ATPase inhibitor. Additionally, lipid droplet fusion and cell death were also attenuated by Thapsigargin pretreatment in white adipocytes. We conclude that Mibefradil elevated intracellular calcium levels, induced lipid droplet fusion and cell death in white adipocytes via mobilizing intracellular calcium stores from the ER. These results describe novel effects of Mibefradil on white adipocytes and may provide new insight into how this drug might be repurposed in obesity research.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 103-131).

La thapsigargine, lactone sesquiterpénique de type guaïanolide d’origine naturelle, est un puissant inhibiteur des enzymes ubiquitaires SERCAs (Sarco-Endoplasmic Reticulum Ca2+-ATPases) et est actuellement en phase clinique pour le traitement du cancer de la prostate non-hormono dépendant. Au cours de cette thèse, nous avons développé une nouvelle approche pour la synthèse de cette molécule, suffisamment flexible pour permettre de préparer des analogues ayant un bon degré de diversité structurale. Cette voie met en jeu une métathèse cyclisante d’ényne pour former le squelette bicyclique [5-7] de cette molécule. La préparation du précurseur de métathèse a nécessité le développement de deux voies différentes. La première utilise le ditertbutylacétylène dicarboxylate comme matière de départ et met en jeu un procédé original Michael-Wittig en un pot. Malgré une étude approfondie de cette étape ayant permis d’optimiser les conditions opérationnelles avec un rendement de 65%, cette voie s’est finalement avérée non applicable pour la synthèse envisagée ceci en raison d’un manque de chimiosélectivité au niveau d’une étape décisive. Une deuxième voie a donc été développée. Elle utilise la 2-méthylcyclopentane-1,3-dione comme produit de départ et offre le précurseur de métathèse en série racémique en 10 étapes et un rendement de 8%. Les quatre centres stéréogènes C1, C3, C10 et C6 sont mis en place grâce à des réactions diastéréosélectives (d.r ≥ 80%). La dernière étape de cette voie est une réaction d’alkynylation d’un aldéhyde conjugué avec piégeage in-situ de l’alcool secondaire obtenu. La configuration relative du centre C-6 de ce précurseur de métathèse s’est avéré par la suite épimère de celui de la thapsigargine (nOe mesurés après RCEYM). Des essais de métathèse cyclisante ont ensuite été réalisés sur l’ényne obtenue dans différentes conditions incluant l’utilisation de catalyseurs originaux fournis par le Pr. Marc Mauduit (université de Rennes). Ces réactions se sont montrées concluantes, fournissant le squelette bicyclique [5,7] de la thapsigargine avec de bons rendements (de 61 à 89%).A la suite de cette étape, un ensemble de réactions a été réalisé afin de mettre en place les groupements fonctionnels nécessaires à la transformation de la plateforme bicyclique obtenue en thapsigargine. De nombreuses difficultés ont été rencontrées : les tentatives d’épimérisation du centre C-6, d'époxydation de l'adduit de cyclisation ou encore de formation de -lactone se sont révélées infructueuses ou ont conduit à des résultats inattendus. L’idée d’exploiter la présence du centre stéréogène C-6 de configuration non naturelle pour installer le centre C-8 crucial pour l’activité biologique de la thapsigargine, via un réarrangement sigmatropique [1,3] de type suprafacial catalysé à l’or (catalyseur de Nolan), a été également testée. De manière inattendue, le produit de réarrangement est obtenu avec inversion faciale, indiquant le passage probable par un cation allylique.Ainsi, au cours de ce travail, deux composés bicycliques [5,7] oxygénées en C8, précurseurs raisonnables de la thapsigargine et analogues, ont été synthétisés par deux voies qui diffèrent au niveau du réarrangement final des produits de cyclisation hydroxylés en C6obtenu après métathèse cyclisante. A partir de de la 2-méthylcyclopentan-1,3-dione commerciale l’un est obtenu en 13 étapes avec 3% de rendement et l’autre en 12 étapes avec 5% de rendement. L’ensemble des essais de réactions effectuées sur les adduits de métathèse permettent de mieux cerner leur réactivité. / Thapsigargin, a guaianolide sesquiterpene lactone of natural origin is a potent inhibitor of ubiquitous SERCA enzymes (Sarco-Endoplasmic Reticulum Ca2+-ATPase) and is currently in clinical trial phase 1 for the treatment of non-hormono dependent prostate cancer. In this work a new approach for the synthesis of thapsigargin has been developed, flexible enough to allow the elaboration of analogues with a high degree of structural diversity. The strategy developed involves an enyne metathesis cyclization reaction (RCEYM) to form the bicyclic [5,7] scaffold of these molecules. For the synthesis of the required precursor of RCEYM, two successive routes have been explored. The first one uses tert-butylacetylene dicarboxylate as starting material and involves a one-pot Michael-intramolecular Wittig reaction. Conditions have been found to allow an efficient synthesis of trisubstituted cyclopentenones. Unfortunately this route has proved difficult to use for the programmed synthesis because of the lack of chemoselectivity at a decisive strategic level. A second approach was developed starting from commercial 2-methyl-cyclopentane-1,3-dione, providing the required racemic precursor of the metathesis reaction in 10 steps and 8% yield. The four C1, C3, C6 and C10 stereogenic centers are set up through diastereoselective reactions (dr ≥ 80%). The final step of the synthesis of this precursor involves an alkynylation reaction of an aldehyde combined with in-situ trapping of the secondary alcohol obtained. The relative configuration of center C6 was shown to be the inverse of that of thapsigargin. Metathesis cyclization assays were then carried out on this intermediate enyne under different conditions, including the use of original catalysts provided by Prof. Marc Mauduit (University of Rennes). These reactions provided the expected [5,7] bicyclic core of thapsigargin with good yields (61 to 89%). In the third part of the thesis, a set of reactions have been attempted to put in place the necessary functional groups for the achievement of the synthesis of thapsigargin. Many difficulties were encountered: attempted epimerization of the C6 center, epoxidation of the RCEYM cyclization adduct or attempts to form a  -lactone either were unsuccessful or led to unexpected results. The idea of using the presence of the stereogenic center of unnatural configuration at C6 to install the hydroxyl group at C8, crucial for the biological activity of thapsigargin, via a gold-catalyzed (Nolan catalyst) suprafacial sigmatropic [1,3] rearrangement was experimented. Unexpectedly, the rearranged acetate adduct was obtained with facial inversion, indicating a probable allyl cation intermediate. Thus, in this work, two [5,7] bicyclic oxygenated compounds at C8 have been synthesized from commercial 2-methylcyclopentane-1,3-dione through two pathways that differ in the conditions leading to the final rearrangement of the cyclic C6-hydroxylated RCEYM adducts. One of them is obtained in 13 steps with 3% yield and the second in 12 steps with 5% yield. Both compounds appear to be suitable intermediates for the syntesis of thapsigargin and its analogues. All tests performed on the adducts, obtained after RCEYM, helps us to identify their reactivity.

Nicotinic acid-adenine dinucleotide phosphate (NAADP) is a recently described potent intracellular Ca2+-mobilizing messenger active in a wide range of diverse cell types. In the present study, we have investigated the interaction of NAADP with other Ca2+-mobilizing messengers in the release of transmitter at the frog neuromuscular junction. We show, for the first time, that NAADP enhances neurosecretion in response to inositol 1,4,5-trisphosphate (IP3), cADP-ribose (cADPR) and sphingosine 1-phosphate (S1P), but not sphingosylphosphorylcholine. Thapsigargin was without effect on transmitter release in response to NAADP, but blocked the responses to subsequent application of IP3, cADPR and S1P and their potentiation by NAADP. Asynchronous neurotransmitter release may therefore involve functional coupling of endoplasmic reticulum Ca2+ stores with distinct Ca2+ stores targeted by NAADP.

La cycloaddition [2+2] du dichlorocétène sur le 7-méthylcycloheptatriène conduit, après expansion de cycle au diazométhane et déshydrohalogénation, à une -chlorotriénone, ceci de manière très sélective. Cet intermédiaire s'est montré très efficace par le passé dans la synthèse de sesquiterpènes naturels possédant le squelette bicyclo[5.3.0]décane (guaianes, guaianolides, azulènes…). Nous avons utilisé cette méthodologie dans une approche synthétique des thapsigargines, des guaianolides naturels à la structure complexe dont un dérivé est à l'étude contre le cancer de la prostate. Nos recherches et notre savoir-faire nous ont permis d'accéder à un intermédiaire avancé pouvant mener à cette famille de composés, il possède les groupements, insaturations et configurations clé nécessaires. Il peut par ailleurs servir de plateforme idéale pour accéder à des azulènes naturels. Nous avons alors en paralèle synthétisé des azulènes bleu, violet et rouge (dont il s'agit de la première synthèse pour ces derniers) / The [2+2] cycloaddition of dichloroketene and 7-methylcycloheptatriene, followed by ring expansion with diazomethane and dehydrohalogenation, affords -chlorotrienone with high selectivity. In the past, this compound proved to be very efficient in the synthesis of natural sesquiterpenes containing the bicyclo[5.3.0]decane squeletton (guaianes, guaianolides, azulenes…). This methodology was used in an efficient synthetic approach of thapsigargins, which are complex guaianolides currently studied to treat prostate cancer via a derivative. Our work led us to get an advanced intermediate for this family, with key moities and configurations. It can also be used to access to natural azulenes. Then we synthetized blue, purple and red azulenes (the first ones for the latters).

1. The effect of urotensin II (U-II) on spontaneous transmitter release was examined in the frog to see if the biological activity of this vasoactive peptide extended to neural tissues. 2. In normal Ringer solution, frog and human U-II (fU-II and hU-II, respectively) caused concentration-dependent, reversible increases in miniature endplate potential (MEPP) frequency, with hU-II about 22 times more potent than fU-II. hU-II caused a dose-dependent increase in MEPP amplitude, whereas fU-II caused an increase, followed by a decrease with higher concentrations. 3. Increasing extracellular Ca 2+ three-fold had no effect on the MEPP frequency increase to 25 μM hU-II. Pretreatment with thapsigargin to deplete endoplasmic reticulum Ca 2+ caused a 61% reduction in the MEPP frequency increase to 25 μM hU-II. 4. Pretreatment with the phospholipase C inhibitor U-73122 caused a 93% reduction in the MEPP frequency increase to 25 μM hU-II and a 15% reduction in the increase in MEPP amplitude. Pretreating with antibodies against the inositol 1,4,5-trisphosphate (IP 3) type 1 receptor using liposomal techniques reduced the MEPP frequency increase by 83% but had no effect on MEPP amplitude. 5. Pretreating with protein kinase C inhibitors (bisindolylmaleimide I and III) had no effect on the response to 25 μM hU-II, but pretreating with protein kinase A inhibitors (H-89 and KT5720) reduced the MEPP frequency increase by 88% and completely abolished the increase in MEPP amplitude. 6. Our results show that hU-II is a potent stimulator of spontaneous transmitter release in the frog and that the effect is mediated by IP 3 and cyclic AMP/protein kinase A.

No description available.