TY - GEN A1 - Troppmann, Britta A1 - Balfanz, Sabine A1 - Baumann, Arnd A1 - Blenau, Wolfgang T1 - Inverse agonist and neutral antagonist actions of synthetic compounds at an insect 5-HT1 receptor N2 - Background and purpose: 5-Hydroxytryptamine (5-HT) has been shown to control and modulate many physiological and behavioural functions in insects. In this study, we report the cloning and pharmacological properties of a 5-HT1 receptor of an insect model for neurobiology, physiology and pharmacology. Experimental approach: A cDNA encoding for the Periplaneta americana 5-HT1 receptor was amplified from brain cDNA. The receptor was stably expressed in HEK 293 cells, and the functional and pharmacological properties were determined in cAMP assays. Receptor distribution was investigated by RT-PCR and by immunocytochemistry using an affinity-purified polyclonal antiserum. Key results: The P. americana 5-HT1 receptor (Pea5-HT1) shares pronounced sequence and functional similarity with mammalian 5-HT1 receptors. Activation with 5-HT reduced adenylyl cyclase activity in a dose-dependent manner. Pea5-HT1 was expressed as a constitutively active receptor with methiothepin acting as a neutral antagonist, and WAY 100635 as an inverse agonist. Receptor mRNA was present in various tissues including brain, salivary glands and midgut. Receptor-specific antibodies showed that the native protein was expressed in a glycosylated form in membrane samples of brain and salivary glands. Conclusions and implications: This study marks the first pharmacological identification of an inverse agonist and a neutral antagonist at an insect 5-HT1 receptor. The results presented here should facilitate further analyses of 5-HT1 receptors in mediating central and peripheral effects of 5-HT in insects. KW - Biogenic amine KW - constitutive activity KW - cellular signalling KW - G-protein-coupled receptor KW - insect Y1 - 2010 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-44346 ER - TY - JOUR A1 - Godec, Aljaž A1 - Metzler, Ralf T1 - First passage time statistics for two-channel diffusion JF - Journal of physics : A, Mathematical and theoretical N2 - We present rigorous results for the mean first passage time and first passage time statistics for two-channel Markov additive diffusion in a 3-dimensional spherical domain. Inspired by biophysical examples we assume that the particle can only recognise the target in one of the modes, which is shown to effect a non-trivial first passage behaviour. We also address the scenario of intermittent immobilisation. In both cases we prove that despite the perfectly non-recurrent motion of two-channel Markov additive diffusion in 3 dimensions the first passage statistics at long times do not display Poisson-like behaviour if none of the phases has a vanishing diffusion coefficient. This stands in stark contrast to the standard (one-channel) Markov diffusion counterpart. We also discuss the relevance of our results in the context of cellular signalling. KW - first passage time KW - Markov additive processes KW - Fokker-Planck equation KW - random search processes KW - coupled initial boundary value problem KW - cellular signalling KW - asymptotic analysis Y1 - 2017 U6 - https://doi.org/10.1088/1751-8121/aa5204 SN - 1751-8113 SN - 1751-8121 VL - 50 IS - 8 PB - IOP Publ. Ltd. CY - Bristol ER - TY - THES A1 - Röser, Claudia T1 - Charakterisierung der Serotonin-Rezeptoren in den Speicheldrüsen von Calliphora vicina T1 - Characterization of serotonin receptors in the salivary gland of Calliphora vicina N2 - Die Fähigkeit, mit anderen Zellen zu kommunizieren, ist eine grundlegende Eigenschaft aller lebenden Zellen und essentiell für die normale Funktionsweise vielzelliger Organismen. Die Speicheldrüsen der Schmeißfliege Calliphora vicina bilden ein ausgezeichnetes physiologisches Modellsystem um zelluläre Signaltransduktionsprozesse an einem intakten Organ zu untersuchen. Die Speichelsekretion wird dabei hormonell durch das biogene Amin Serotonin (5-Hydroxytryptamin; 5-HT) reguliert. 5-HT aktiviert in den sekretorischen Zellen der Drüsen über die Bindung an mindestens zwei membranständige G-Protein gekoppelte Rezeptoren (GPCR) zwei separate Signalwege, den IP3/Ca2+- und den cAMP-Signalweg. Zur Identifizierung und Charakterisierung der 5-HT-Rezeptoren in den Speicheldrüsen von Calliphora wurden unter Anwendung verschiedener Klonierungsstrategien zwei cDNAs (Cv5-ht2α und Cv5-ht7) isoliert, die große Ähnlichkeit zu 5-HT2- und 5-HT7-Rezeptoren aus Säugetieren aufweisen. Die Hydropathieprofile der abgeleiteten Aminosäuresequenzen postulieren die für GPCRs charakteristische heptahelikale Architektur. Alle Aminosäuremotive, die für die Ligandenbindung, die Rezeptoraktivierung und die Kopplung an G-Proteine essentiell sind, liegen konserviert vor. Interessanterweise wurde für den Cv5-HT7-Rezeptor eine zusätzliche hydrophobe Domäne im N Terminus vorhergesagt. Die Cv5-HT2α-mRNA liegt in zwei alternativ gespleißten Varianten vor. Mittels RT-PCR-Experimenten konnte die Expression beider Rezeptoren in Gehirn und Speicheldrüsen adulter Fliegen nachgewiesen werden. Ein Antiserum gegen den Cv5-HT7 Rezeptor markiert in den Speicheldrüsen die basolaterale Plasmamembran. Die Expression der Rezeptoren in einem heterologen System (HEK 293-Zellen) bestätigte diese als funktionelle 5-HT Rezeptoren. So führte die Stimulation mit Serotonin für den Cv5-HT2α zu einer dosis-abhängigen Erhöhung der intrazellulären Ca2+ Konzentration ([Ca2+]i, EC50 = 24 nM). In Cv5-HT7-exprimierenden Zellen löste 5-HT dosisabhängig (EC50 = 4,1 nM) einen Anstieg der intrazellulären cAMP Konzentration ([cAMP]i) aus. Für beide heterolog exprimierten Rezeptoren wurden pharmakologische Profile erstellt. Liganden, die eine Rezeptorsubtyp-spezifische Wirkung vermuten ließen, wurden daraufhin auf ihre Wirkung auf das transepitheliale Potential (TEP) intakter Speicheldrüsenpräparate getestet. Drei 5-HT-Rezeptoragonisten: AS 19, R-(+)-Lisurid und 5-Carboxamidotryptamin führten zu einer cAMP-abhängigen Positivierung des TEP durch eine selektive Aktivierung der 5 HT7-Rezeptoren. Eine selektive Aktivierung des Ca2+-Signalweges durch den Cv5-HT2 Rezeptor ist mit Hilfe von 5-Methoxytryptamin möglich. Dagegen konnte Clozapin im TEP als selektiver Cv5-HT7-Rezeptorantagonist bestätigt werden. Die Kombination eines molekularen Ansatzes mit physiologischen Messungen ermöglichte somit die Identifikation selektiver Liganden für 5-HT2- bzw. 5-HT7-Rezeptoren aus Calliphora vicina. Dies ermöglicht zukünftig eine separate Aktivierung der 5-HT-gesteuerten Signalwege und erleichtert dadurch die weitere Erforschung der intrazellulären Signalwege und ihrer Wechselwirkungen. N2 - Cellular communication is a fundamental property of living cells and essential for normal functioning of multicellular organisms. The salivary glands of the blowfly Calliphora vicina are a well established physiological model system to study cellular signaling in an intact organ. Fluid secretion in this gland is hormonally regulated by the biogenic amine serotonin (5-hydroxytryptamine, 5-HT). In the secretory cells, 5-HT causes a parallel activation of the InsP3/Ca2+- and the cAMP-signaling pathways through binding and stimulation of at least two G protein coupled receptors (GPCR). In order to characterize the respective 5-HT receptors on the secretory cells, we have cloned two cDNAs (Cv5-ht2α, Cv5-ht7) that share high similarity with mammalian 5-HT2 and 5-HT7 receptor classes. Analysis of the deduced amino acid sequences postulates the typical heptahelical architecture of GPCRs for both receptors. Sequence motifs that are essential for ligand binding, receptor activation and coupling to G-proteins are well conserved. Interestingly, a computer-based structural analysis of Cv5-HT7 predicts an additional eighth hydrophobic region in the N-terminus of the receptor. We also found an alternative splice variant of the Cv5-HT2α mRNA. Using RT-PCR experiments, transcripts of both receptor mRNAs could be detected in brain and salivary gland tissue. An antiserum raised against the Cv5 HT7 receptor stained the basolateral region of the salivary glands. Heterologous receptor expression in HEK 293 cells leads to a dose-dependent increase in the intracellular Ca2+-concentration ([Ca2+]i) for Cv5-HT2α (EC50 = 24 nM) and cAMP-concentration for Cv5-HT7 (EC50 = 4,1 nM) upon application of 5-HT. A pharmacological profile was established for both receptors. Ligands that appeared to act as specific ligands of either Cv5-HT2α or Cv5-HT7 in this approach, were then tested for their effect on the transepithelial potential (TEP) of intact blowfly salivary gland preparations. Three 5-HT receptor agonists: AS 19, R-(+)-lisuride and 5-carboxamidotryptamine showed a cAMP dependent positivation of the TEP, caused by a selective activation of the Cv5-HT7 receptor. 5-methoxytryptamine exclusively activates the Ca2+ pathway via Cv5-HT2α. Clozapine antagonizes the effects of 5-HT in blowfly salivary glands and was confirmed as a Cv5-HT7 antagonist. The combination of a molecular approach with physiological measurements enabled us to identify selective ligands for 5-HT2 and 5-HT7 receptors of Calliphora vicina. These results facilitate a selective activation of the intracellular signaling pathways activated by 5-HT and will facilitate future research on different aspects of intracellular signaling and crosstalk mechanisms. KW - Calliphora KW - GPCR KW - Serotonin KW - Rezeptor KW - zelluläre Signalübertragung KW - Calliphora KW - G-protein-coupled receptors KW - serotonin KW - cellular signalling Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-61486 ER - TY - GEN A1 - Schlenstedt, Jana A1 - Balfanz, Sabine A1 - Baumann, Arnd A1 - Blenau, Wolfgang T1 - Am5-HT7 : molecular and pharmacological characterization of the first serotonin receptor of the honeybee (Apis mellifera) N2 - 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. KW - Behaviour KW - biogenic amine KW - cellular signalling KW - constitutive activity KW - cyclic AMP Y1 - 2006 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-44423 ER -