The vacuolar H+-ATPase (V-ATPase) in the apical membrane of blowfly (Calliphora vicina) salivary gland cells energizes the secretion of a KCl-rich saliva in response to the neurohormone serotonin (5-HT). We have shown previously that exposure to 5-HT induces a cAMP-mediated reversible assembly of V-0 and V-1 subcomplexes to V-ATPase holoenzymes and increases V-ATPase-driven proton transport. Here, we analyze whether the effect of cAMP on V-ATPase is mediated by protein kinase A (PKA) or exchange protein directly activated by cAMP (Epac), the cAMP target proteins that are present within the salivary glands. Immunofluorescence microscopy shows that PKA activators, but not Epac activators, induce the translocation of V1 components from the cytoplasm to the apical membrane, indicative of an assembly of V-ATPase holoenzymes. Measurements of transepithelial voltage changes and microfluorometric pH measurements at the luminal surface of cells in isolated glands demonstrate further that PKA-activating cAMP analogs increase cation transport to the gland lumen and induce a V-ATPase-dependent luminal acidification, whereas activators of Epac do not. Inhibitors of PKA block the 5-HT-induced V-1 translocation to the apical membrane and the increase in proton transport. We conclude that cAMP exerts its effects on V-ATPase via PKA.

Biogenic amines and their receptors regulate and modulate many physiological and behavioural processes in animals. In vertebrates, octopamine is only found in trace amounts and its function as a true neurotransmitter is unclear. In protostomes, however, octopamine can act as neurotransmitter, neuromodulator and neurohormone. In the honeybee, octopamine acts as a neuromodulator and is involved in learning and memory formation. The identification of potential octopamine receptors is decisive for an understanding of the cellular pathways involved in mediating the effects of octopamine. Here we report the cloning and functional characterization of the first octopamine receptor from the honeybee, Apis mellifera . The gene was isolated from a brain-specific cDNA library. It encodes a protein most closely related to octopamine receptors from Drosophila melanogaster and Lymnea stagnalis . Signalling properties of the cloned receptor were studied in transiently transfected human embryonic kidney (HEK) 293 cells. Nanomolar to micromolar concentrations of octopamine induced oscillatory increases in the intracellular Ca2+ concentration. In contrast to octopamine, tyramine only elicited Ca2+ responses at micromolar concentrations. The gene is abundantly expressed in many somata of the honeybee brain, suggesting that this octopamine receptor is involved in the processing of sensory inputs, antennal motor outputs and higher-order brain functions.

The biogenic amine serotonin (5-HT) plays a key role in the regulation and modulation of many physiological and behavioural processes in both vertebrates and invertebrates. These functions are mediated through the binding of serotonin to its receptors, of which 13 subtypes have been characterized in vertebrates. We have isolated a cDNA from the honeybee Apis mellifera (Am5-ht7) sharing high similarity to members of the 5-HT7 receptor family. Expression of the Am5-HT7 receptor in HEK293 cells results in an increase in basal cAMP levels, suggesting that Am5-HT7 is expressed as a constitutively active receptor. Serotonin application to Am5-ht7-transfected cells elevates cyclic adenosine 3',5'-monophosphate (cAMP) levels in a dose-dependent manner (EC50 = 1.1-1.8 nM). The Am5-HT7 receptor is also activated by 5-carboxamidotryptamine, whereas methiothepin acts as an inverse agonist. Receptor expression has been investigated by RT-PCR, in situ hybridization, and western blotting experiments. Receptor mRNA is expressed in the perikarya of various brain neuropils, including intrinsic mushroom body neurons, and in peripheral organs. This study marks the first comprehensive characterization of a serotonin receptor in the honeybee and should facilitate further analysis of the role(s) of the receptor in mediating the various central and peripheral effects of 5-HT.

Epidemiologic data suggest that soy consumption may protect against cancer induction in several tissues in humans, including urothelial carcinoma. Genistein have been reported to regulate genes that are involved in several cellular events. However, the molecular mechanism of genistein -induced upregulation of epithelial membrane protein 2 (EMP2), candidate urothelial tumor suppressor, is not entirely understood. At first, we found that the mRNA and protein expression levels of EMP2 were significantly greater in the normal urothelial tissues and human urothelial cells than those in urothelial bladder carcinoma tissues and urothelial cell carcinoma-derived cell lines. Second, EMP2 knockdown via RNA interference markedly enhanced cell proliferation, colony formation, migration and invasiveness. By contrast, EMP2 overexpression suppressed these malignant behaviors. Third, we showed that genistein-induced inhibition in cell proliferation is associated with an increase in EMP2 expression. Using various deleted EMP2 promoter constructs, we defined that the EMP2 core promoter is enough to observe the genistein-induced upregulation of EMP2 transcriptional activity. Using site direct mutagenesis and chromatin immunoprecipitation assays demonstrated that cyclic-AMP response element binding protein 1 (CREB1) acts as a positive regulator of EMP2 transcription by directly binding to its promoter. These results showed EMP2 suppressed urothelial cell carcinoma-derived cell growth, motility and invasion and for the first time that genistein promoted EMP2 expression in urothelial cell carcinoma-derived cells by inducing EMP2 transcriptional activity via CREB1 binding.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 68-82).

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 67-89).

Das Rett Syndrom (RTT) gehört zu den tiefgreifenden Entwicklungsstörungen des Gehirns von dem fast ausschließlich Mädchen betroffen sind (ICD-10, F84.10). Ursächlich für die Pathogenese sind Mutationen im X-chromsomalen MECP2-Gen, welches für den Transkriptionsfaktor Methyl-CpG binding protein 2 (MeCP2) kodiert. Unterschiedliche neurologische Symptome treten zwischen 6 und 18 Monaten nach der Geburt auf, wobei schwere Rhythmussstörungen der Atmung für ein Viertel plötzlicher Todesfälle bei Rett-Patientinnen verantwortlich gemacht werden. Der neuronale Atmungsrhythmus bei Säugern wird in verschieden Regionen des ponto-medullären Hirnstammes generiert, wobei der Prä-Bötzinger Komplex als essentiell für die Rhythmogenese der Atmung angesehen wird. Mittels Genexpressionsstudien in der Ventralen Respiratorischen Gruppe (VRG), die den Prä-Bötzinger-Komplex einschließt, zeigte sich eine massiv erhöhte, pathologische Expression des Serotoninrezeptor 5B sowohl auf mRNA-, als auch auf Proteinebene bei MeCP2-defizienten Mäusen zum postnatalen Entwicklungstag P40. Der Serotoninrezeptor 5B (5-HTR5B) gehört zur Klasse der G-Protein-gekoppelten Rezeptoren. Durch detaillierte Analysen des 5-HTR5B-Proteins konnte eine natürliche Trunkierung des Rezeptors nachgewiesen werden. Des Weiteren wurde eine ungewöhnliche intrazelluläre Lokalisierung in Membranen von vesikulären und tubulären Kompartimenten beobachtet. Trotz dieser ungewöhnlichen Eigenschaften besitzt der Rezeptor weiterhin die Fähigkeit, das inhibitorische G-Protein Gαi3 konstitutiv zu aktivieren und somit den Anstieg von cAMP zu verhindern. Durch genetisches Ausschalten des 5-HTR5B Proteins (knockout) konnte gezeigt werden, dass die durch 5-HTR5B-verminderte cAMP-Konzentration in der VRG ursächlich für den gestörten Atmungsrhythmus MeCP2-defizienter Mäuse ist. Die sich aus diesen Ergebnissen ableitende pharmakologische Strategie, die cAMP Konzentration zu erhöhen, führte zu einem deutlich verbesserten Atmungsrhythmus. Die Ergebnisse dieser Arbeit implizieren neue Therapieansätze zur Behandlung der Atmungs-störungen von Rett-Patienten.

Diss. (sammanfattning) Uppsala : Univ., 2002. / Härtill 5 uppsatser.

Thesis (Ph. D.)--University of Washington, 1997. / Vita. Includes bibliographical references (leaves [87]-116).

Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves [115]-133).