TY - JOUR A1 - Richter, Katharina Natalia A1 - Rolke, Daniel A1 - Blenau, Wolfgang A1 - Baumann, Otto T1 - Secretory cells in honeybee hypopharyngeal gland: polarized organization and age-dependent dynamics of plasma membrane JF - Cell & tissue research N2 - The honeybee hypopharyngeal gland consists in numerous units, each comprising a secretory cell and a canal cell. The secretory cell discharges its products into a convoluted tubular membrane system, the canaliculus, which is surrounded at regular intervals by rings of actin filaments. Using probes for various membrane components, we analyze the organization of the secretory cells relative to the apicobasal configuration of epithelial cells. The canaliculus was defined by labeling with an antibody against phosphorylated ezrin/radixin/moesin (pERM), a marker protein for the apical membrane domain of epithelial cells. Anti-phosphotyrosine visualizes the canalicular system, possibly by staining the microvillar tips. The open end of the canaliculus leads to a region in which the secretory cell is attached to the canal cell by adherens and septate junctions. The remaining plasma membrane stains for Na,K-ATPase and spectrin and represents the basolateral domain. We also used fluorophore-tagged phalloidin, anti-phosphotyrosine and anti-pERM as probes for the canaliculus in order to describe fine-structural changes in the organization of the canalicular system during the adult life cycle. These probes in conjunction with fluorescence microscopy allow the fast and detailed three-dimensional analysis of the canalicular membrane system and its structural changes in a developmental mode or in response to environmental factors. KW - Hypopharyngeal gland KW - Cell polarity KW - Moesin KW - Actin cytoskeleton KW - Honeybee Y1 - 2016 U6 - https://doi.org/10.1007/s00441-016-2423-9 SN - 0302-766X SN - 1432-0878 VL - 366 SP - 163 EP - 174 PB - Springer CY - New York ER - TY - JOUR A1 - Bogen, Oliver A1 - Bender, Olaf A1 - Loewe, Jana A1 - Blenau, Wolfgang A1 - Thevis, Beatrice A1 - Schroeder, Wolfgang A1 - Margolis, Richard U. A1 - Levine, Jon D. A1 - Hucho, Ferdinand T1 - Neuronally produced versican V2 renders C-fiber nociceptors IB4-positive JF - Journal of neurochemistry N2 - A subpopulation of nociceptors, the glial cell line-derived neurotrophic factor (GDNF)-dependent, non-peptidergic C-fibers, expresses a cell-surface glycoconjugate that can be selectively labeled with isolectin B4 (IB4), a homotetrameric plant lectin from Griffonia simplicifolia. We show that versican is an IB4-binding molecule in rat dorsal root ganglion neurons. Using reverse transcriptase polymerase chain reaction (RT-PCR), insitu hybridization and immunofluorescence experiments on rat lumbar dorsal root ganglion, we provide the first demonstration that versican is produced by neurons. In addition, by probing Western blots with splice variant-specific antibodies we show that the IB4-binding versican contains only the glycosaminoglycan alpha domain. Our data support V2 as the versican isoform that renders this subpopulation of nociceptors IB4-positive (+). A subset of nociceptors, the GDNF-dependent non-peptidergic C-fibers can be characterized by its reactivity for isolectin B4 (IB4), a plant lectin from Griffonia simplicifolia. We have previously demonstrated that versican V2 binds IB4 in a Ca2+-dependent manner. However, given that versican is thought to be the product of glial cells, it was questionable whether versican V2 can be accountable for the IB4-reactivity of this subset of nociceptors. The results presented here prove - for the first time - a neuronal origin of versican and suggest that versican V2 is the molecule that renders GDNF-dependent non-peptidergic C-fibers IB4-positive. KW - IB4 KW - nociceptors KW - pain KW - sensory neurons KW - V2 KW - versican Y1 - 2015 U6 - https://doi.org/10.1111/jnc.13113 SN - 0022-3042 SN - 1471-4159 VL - 134 IS - 1 SP - 147 EP - 155 PB - Wiley-Blackwell CY - Hoboken ER - TY - JOUR A1 - Blankenburg, Stefanie A1 - Balfanz, Sabine A1 - Hayashi, Y. A1 - Shigenobu, S. A1 - Miura, T. A1 - Baumann, Otto A1 - Baumann, Arnd A1 - Blenau, Wolfgang T1 - Cockroach GABA(B) receptor subtypes: Molecular characterization, pharmacological properties and tissue distribution JF - Neuropharmacology N2 - gamma-aminobutyric acid (GABA) is the predominant inhibitory neurotransmitter in the central nervous system (CNS). Its effects are mediated by either ionotropic GABA(A) receptors or metabotropic GABA(B) receptors. GABA(B) receptors regulate, via Gi/o, G-proteins, ion channels, and adenylyl cyclases. In humans, GABA(B) receptor subtypes are involved in the etiology of neurologic and psychiatric disorders. In arthropods, however, these members of the G-protein-coupled receptor family are only inadequately characterized. Interestingly, physiological data have revealed important functions of GABA(B) receptors in the American cockroach, Periplaneta americana. We have cloned cDNAs coding for putative GABA(B) receptor subtypes 1 and 2 of P. americana (PeaGB1 and PeaGB2). When both receptor proteins are co-expressed in mammalian cells, activation of the receptor heteromer with GABA leads to a dose-dependent decrease in cAMP production. The pharmacological profile differs from that of mammalian and Drosophila GABA(B) receptors. Western blot analyses with polyclonal antibodies have revealed the expression of PeaGB1 and PeaGB2 in the CNS of the American cockroach. In addition to the widespread distribution in the brain, PeaGB1 is expressed in salivary glands and male accessory glands. Notably, PeaGB1-like immunoreactivity has been detected in the GABAergic salivary neuron 2, suggesting that GABA(B) receptors act as autoreceptors in this neuron. KW - GABA(B) receptor KW - G-protein-coupled receptor KW - Periplaneta americana KW - Central nervous system KW - Adenylyl cyclase KW - Salivary gland Y1 - 2015 U6 - https://doi.org/10.1016/j.neuropharm.2014.08.022 SN - 0028-3908 SN - 1873-7064 VL - 88 SP - 134 EP - 144 PB - Elsevier CY - Oxford ER - TY - JOUR A1 - Troppmann, Britta A1 - Balfanz, Sabine A1 - Krach, Christian A1 - Baumann, Arnd A1 - Blenau, Wolfgang T1 - Characterization of an Invertebrate-Type Dopamine Receptor of the American Cockroach, Periplaneta americana JF - International journal of molecular sciences N2 - We have isolated a cDNA coding for a putative invertebrate-type dopamine receptor (Peadop2) from P. americana brain by using a PCR-based strategy. The mRNA is present in samples from brain and salivary glands. We analyzed the distribution of the PeaDOP2 receptor protein with specific affinity-purified polyclonal antibodies. On Western blots, PeaDOP2 was detected in protein samples from brain, subesophageal ganglion, thoracic ganglia, and salivary glands. In immunocytochemical experiments, we detected PeaDOP2 in neurons with their somata being located at the anterior edge of the medulla bilaterally innervating the optic lobes and projecting to the ventro-lateral protocerebrum. In order to determine the functional and pharmacological properties of the cloned receptor, we generated a cell line constitutively expressing PeaDOP2. Activation of PeaDOP2-expressing cells with dopamine induced an increase in intracellular cAMP. In contrast, a C-terminally truncated splice variant of this receptor did not exhibit any functional property by itself. The molecular and pharmacological characterization of the first dopamine receptor from P. americana provides the basis for forthcoming studies focusing on the significance of the dopaminergic system in cockroach behavior and physiology. KW - G-protein-coupled receptor KW - dopamine KW - insect KW - cellular signaling KW - salivary gland KW - biogenic amine Y1 - 2014 U6 - https://doi.org/10.3390/ijms15010629 SN - 1422-0067 VL - 15 IS - 1 SP - 629 EP - 653 PB - MDPI CY - Basel ER - TY - JOUR A1 - French, Alice S. A1 - Simcock, Kerry L. A1 - Rolke, Daniel A1 - Gartside, Sarah E. A1 - Blenau, Wolfgang A1 - Wright, Geraldine A. T1 - The role of serotonin in feeding and gut contractions in the honeybee JF - Journal of insect physiology KW - Honeybee KW - Apis mellifera KW - Serotonin KW - 5-HT KW - 5-HT receptor KW - Gut contractions Y1 - 2014 U6 - https://doi.org/10.1016/j.jinsphys.2013.12.005 SN - 0022-1910 SN - 1879-1611 VL - 61 SP - 8 EP - 15 PB - Elsevier CY - Oxford ER - TY - JOUR A1 - Thamm, Markus A1 - Rolke, Daniel A1 - Jordan, Nadine A1 - Balfanz, Sabine A1 - Schiffer, Christian A1 - Baumann, Arnd A1 - Blenau, Wolfgang T1 - Function and distribution of 5-HT2 receptors in the honeybee (apis mellifera) JF - PLoS one N2 - Background: Serotonin plays a pivotal role in regulating and modulating physiological and behavioral processes in both vertebrates and invertebrates. In the honeybee (Apis mellifera), serotonin has been implicated in division of labor, visual processing, and learning processes. Here, we present the cloning, heterologous expression, and detailed functional and pharmacological characterization of two honeybee 5-HT2 receptors. Methods: Honeybee 5-HT2 receptor cDNAs were amplified from brain cDNA. Recombinant cell lines were established constitutively expressing receptor variants. Pharmacological properties of the receptors were investigated by Ca2+ imaging experiments. Quantitative PCR was applied to explore the expression patterns of receptor mRNAs. Results: The honeybee 5-HT2 receptor class consists of two subtypes, Am5-HT2 alpha and Am5-HT2 beta. Each receptor gene also gives rise to alternatively spliced mRNAs that possibly code for truncated receptors. Only activation of the full-length receptors with serotonin caused an increase in the intracellular Ca2+ concentration. The effect was mimicked by the agonists 5-methoxytryptamine and 8-OH-DPAT at low micromolar concentrations. Receptor activities were blocked by established 5-HT receptor antagonists such as clozapine, methiothepin, or mianserin. High transcript numbers were detected in exocrine glands suggesting that 5-HT2 receptors participate in secretory processes in the honeybee. Conclusions: This study marks the first molecular and pharmacological characterization of two 5-HT2 receptor subtypes in the same insect species. The results presented should facilitate further attempts to unravel central and peripheral effects of serotonin mediated by these receptors. Y1 - 2013 U6 - https://doi.org/10.1371/journal.pone.0082407 SN - 1932-6203 VL - 8 IS - 12 PB - PLoS CY - San Fransisco ER - TY - JOUR A1 - Reim, Tina A1 - Thamm, Markus A1 - Rolke, Daniel A1 - Blenau, Wolfgang A1 - Scheiner, Ricarda T1 - Suitability of three common reference genes for quantitative real-time PCR in honey bees JF - Apidologie : a quality journal in bee science N2 - Honey bees are important model organisms for neurobiology, because they display a large array of behaviors. To link behavior with individual gene function, quantitative polymerase chain reaction is frequently used. Comparing gene expression of different individuals requires data normalization using adequate reference genes. These should ideally be expressed stably throughout lifetime. Unfortunately, this is frequently not the case. We studied how well three commonly used reference genes are suited for this purpose and measured gene expression in the brains of honey bees differing in age and social role. Although rpl32 is used most frequently, it only remains stable in expression between newly emerged bees, nurse-aged bees, and pollen foragers but shows a peak at the age of 12 days. The genes gapdh and ef1 alpha-f1, in contrast, are expressed stably in the brain throughout all age groups except newly emerged bees. According to stability software, gapdh was expressed most stably, followed by rpl32 and ef1 alpha-f1. KW - gene expression KW - quantitative PCR KW - reference gene KW - stability program KW - Apis mellifera Y1 - 2013 U6 - https://doi.org/10.1007/s13592-012-0184-3 SN - 0044-8435 VL - 44 IS - 3 SP - 342 EP - 350 PB - Springer CY - Paris ER - TY - JOUR A1 - Schewe, Bettina A1 - Blenau, Wolfgang A1 - Walz, Bernd T1 - Intracellular pH regulation in unstimulated Calliphora salivary glands is Na+ dependent and requires V-ATPase activity JF - The journal of experimental biology N2 - Salivary gland cells of the blowfly Calliphora vicina have a vacuolar-type H+-ATPase (V-ATPase) that lies in their apical membrane and energizes the secretion of a KCl-rich primary saliva upon stimulation with serotonin (5-hydroxytryptamine). Whether and to what extent V-ATPase contributes to intracellular pH (pH(i)) regulation in unstimulated gland cells is unknown. We used the fluorescent dye BCECF to study intracellular pH(i) regulation microfluorometrically and show that: (1) under resting conditions, the application of Na+-free physiological saline induces an intracellular alkalinization attributable to the inhibition of the activity of a Na+-dependent glutamate transporter; (2) the maintenance of resting pHi is Na+, Cl-, concanamycin A and DIDS sensitive; (3) recovery from an intracellular acid load is Na+ sensitive and requires V-ATPase activity; (4) the Na+/H+ antiporter is not involved in pHi recovery after a NH4Cl prepulse; and (5) at least one Na+-dependent transporter and the V-ATPase maintain recovery from an intracellular acid load. Thus, under resting conditions, the V-ATPase and at least one Na+-dependent transporter maintain normal pH(i) values of pH.7.5. We have also detected the presence of a Na+-dependent glutamate transporter, which seems to act as an acid loader. Despite this not being a common pH(i)-regulating transporter, its activity affects steady-state pH(i) in C. vicina salivary gland cells. KW - Calliphora vicina KW - salivary gland KW - intracellular pH regulation KW - Na+/H+ antiporter KW - NHE KW - vacuolar H+-ATPase KW - V-ATPase KW - intracellular pH KW - insect KW - blowfly KW - BCECF KW - NH4Cl prepulse Y1 - 2012 U6 - https://doi.org/10.1242/jeb.063172 SN - 0022-0949 VL - 215 IS - 8 SP - 1337 EP - 1345 PB - Company of Biologists Limited CY - Cambridge ER - TY - JOUR A1 - Heindorff, Kristoffer A1 - Blenau, Wolfgang A1 - Walz, Bernd A1 - Baumann, Otto T1 - Characterization of a Ca2+/calmodulin-dependent AC1 adenylyl cyclase in a non-neuronal tissue, the blowfly salivary gland JF - Cell calcium N2 - Crosstalk between intracellular signalling pathways is a functionally important and widespread phenomenon in cell physiology across phyla. In the salivary gland of the blowfly, serotonin induces fluid secretion via parallel activation of both the InsP(3)/Ca2+ and the cAMP/PKA signalling pathways, which interact on multiple levels. We have determined the molecular identity of a link between both pathways that mediates a Ca2+-dependent rise of intracellular cAMP. Whereas hydrolysis of cAMP via phosphodiesterases is largely independent of Ca2+, cAMP synthesis by adenylyl cyclases (AC) is potentiated in a Ca2+/calmodulin (Ca2+/CaM)-dependent manner. The existence of a Ca2+/CaM-dependent AC is supported by physiological data and a molecular approach. We have cloned Cv rutabaga cDNA, encoding the first blowfly AC, and confirmed its expression in the salivary gland via reverse transcription followed by polymerase chain reaction. The putative gene product of Cv rutabaga is a Ca2+/CaM-dependent type I AC and shows highest homology to Rutabaga from Drosophila. Thus, a Ca2+/CaM-dependent AC serves as a link between the InsP(3)/Ca2+ and the cAMP/PKA signalling pathways in the salivary gland of the blowfly and might be important for the amplification and optimization of the secretory response. KW - Adenylyl cyclase KW - Phosphodiesterase KW - Crosstalk KW - Ca2+ KW - cAMP KW - Intracellular signalling KW - Salivary gland KW - Calliphora vicina KW - Rutabaga Y1 - 2012 U6 - https://doi.org/10.1016/j.ceca.2012.04.016 SN - 0143-4160 VL - 52 IS - 2 SP - 103 EP - 112 PB - Churchill Livingstone CY - Edinburgh ER - TY - JOUR A1 - Röser, Claudia A1 - Jordan, Nadine A1 - Balfanz, Sabine A1 - Baumann, Arnd A1 - Walz, Bernd A1 - Baumann, Otto A1 - Blenau, Wolfgang T1 - Molecular and pharmacological characterization of serotonin 5-HT2 alpha and 5-HT7 receptors in the salivary glands of the blowfly calliphora vicina JF - PLoS one N2 - Secretion in blowfly (Calliphora vicina) salivary glands is stimulated by the biogenic amine serotonin (5-hydroxytryptamine, 5-HT), which activates both inositol 1,4,5-trisphosphate (InsP(3))/Ca2+ and cyclic adenosine 3',5'-monophosphate (cAMP) signalling pathways in the secretory cells. In order to characterize the signal-inducing 5-HT receptors, we cloned two cDNAs (Cv5-ht2 alpha, Cv5-ht7) that share high similarity with mammalian 5-HT2 and 5-HT7 receptor genes, respectively. RT-PCR demonstrated that both receptors are expressed in the salivary glands and brain. Stimulation of Cv5-ht2 alpha-transfected mammalian cells with 5-HT elevates cytosolic [Ca2+] in a dose-dependent manner (EC50 = 24 nM). In Cv5-ht7-transfected cells, 5-HT produces a dose-dependent increase in [cAMP](i) (EC50 = 4 nM). We studied the pharmacological profile for both receptors. Substances that appear to act as specific ligands of either Cv5-HT2 alpha or Cv5-HT7 in the heterologous expression system were also tested in intact blowfly salivary gland preparations. We observed that 5-methoxytryptamine (100 nM) activates only the Cv(5)-HT2 alpha receptor, 5-carboxamidotryptamine (300 nM) activates only the Cv5-HT7 receptor, and clozapine (1 mu M) antagonizes the effects of 5-HT via Cv5-HT7 in blowfly salivary glands, providing means for the selective activation of each of the two 5-HT receptor subtypes. This study represents the first comprehensive molecular and pharmacological characterization of two 5-HT receptors in the blowfly and permits the analysis of the physiological role of these receptors, even when co-expressed in cells, and of the modes of interaction between the Ca2+- and cAMP-signalling cascades. Citation: Roser C, Jordan N, Balfanz S, Baumann A, Walz B, et al. (2012) Molecular and Pharmacological Characterization of Serotonin 5-HT2a and 5-HT7 Receptors in the Salivary Glands of the Blowfly Calliphora vicina. Y1 - 2012 U6 - https://doi.org/10.1371/journal.pone.0049459 SN - 1932-6203 VL - 7 IS - 11 PB - PLoS CY - San Fransisco ER -