Abstracts - Association for Chemoreception Sciences

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P O S T E R S OSNs of the OMP-KO mouse. Here we address the molecular interactions between NCX and olfactory marker protein (OMP) or calmodulin (CaM) and their functional consequences in the regulation of intracellular Ca. We assayed reverse-mode NCX activity to determine Na dependent Ca influx in CHO cells stably expressing NCX1 protein. Pretreatment with CaM antagonists e.g. Ophiobolin-A or W7 significantly inhibits NCX activity, suggesting a modulatory role for CaM. The NCX -CHO cells were transiently transfected with pCMV-OMP-IRES-GFP or vector, and NCX1 activity was determined. In cells expressing OMP, NCX activity was significantly inhibited compared to control, indicating OMP modulates NCX1 activity. Addition of OMP to the membrane lysates obtained from porcine heart or from NCX1-CHO cells inhibited NCX1activity, as determined by solid support membrane based electrophysiology (Iongate) confirming our cell-based findings. By contrast, in SGLT1- CHO cells (sodium-dependent glucose transporter1) OMP had no effect, demonstrating the selectivity of OMP for NCX1. Interaction of OMP and/or CaM and NCX activity will be tested in freshly isolated rat cardiac myocytes (a rich source of endogenous NCX1). Although our prior analyses of the OMP- KO mice indicated that NCX activity was reduced in the absence of OMP, these findings suggest that OMP inhibits NCX activity. This apparent discrepancy may relate to the differential interaction of NCX with CaM and OMP to modulate NCX activity. Acknowledgements: NIH Grant RO1 DC003112 #P170 POSTER SESSION IV: CHEMOSENSORY TRANSDUCTION AND SIGNALING Inhibition or Loss of Plasma Membrane Calcium ATPases Prolongs Desensitization In Mouse Olfactory Sensory Neurons Judith Van Houten 1 , Samsudeen Ponissery Saidu 2 1 University of Vermont Burlington, VT, USA, 2 Monell Chemical Senses Center Philadelphia, PA, USA Olfactory sensory neurons (OSNs) respond to odorant stimuli with increases in intracellular Ca 2+ . Termination of the Ca 2+ signal is achieved through removal from the cell by Na + / Ca 2+ exchanger (NCX) and plasma membrane calcium ATPases (PMCAs) or through sequestration by the SERCA pump or binding proteins. Since Ca 2+ is involved in feedback regulation of the olfactory signal transduction pathway, its clearance also contributes to recovery from desensitization. We have previously demonstrated that the inhibition of PMCAs significantly slows down Ca 2+ clearance from mouse OSN knobs after odor stimulation. In order to understand the effects of PMCA inhibition and the subsequent slower Ca 2+ clearance on OSN functionality, we examined the adaptation properties of wild type (WT) and PMCA2 knockout (PMCA2KO) OSNs in response to two successive 8s IBMX/Fsk stimulations. While WT OSN knobs showed significantly attenuated second Ca 2+ responses when stimulated at 150 and 200s after first stimulation, in the KO, the attenuation was larger and significant even at 250s interval. Comparison of the reduction in Ca 2+ amplitudes between the WT and PMCA2KO showed that the attenuation was stronger in the KO at 150 and 200s. When WT OSNs were treated with the PMCA blocker carboxyeosin, there was a further reduction in the second Ca 2+ responses. These results indicate that slower Ca 2+ clearance prolongs desensitization in PMCA-inhibited OSNs. We also show that the desensitization observed with our choice of stimulus (8s application of IBMX/Fsk) is mediated through CaMKII. Inhibition of CaMKII with an inhibitory peptide (AIP) caused the Ca 2+ responses to two successive IBMX/Fsk stimuli to be similar in amplitude in both WT and PMCA2KO at 150 and 200s intervals. Supported by R21 DC006643. Acknowledgements: R21 DC006643 #P171 POSTER SESSION IV: CHEMOSENSORY TRANSDUCTION AND SIGNALING Exogenous Odorant Receptor Suppresses Endogenous Receptor Expression in Cultured Olfactory Sensory Neurons Huaiyang Chen, Qizhi Gong University of California Davis, CA, USA Discrimination of odorants is achieved by a large number of odorant receptors (OR) expressed by olfactory sensory neurons (OSNs). Each OSN expresses only one OR gene among the repertoire of about 1000 genes in the mouse genome. ORs are believed to play an important role in the maintenance of the selected OR expression. The regulatory mechanisms of OR selection and maintenance in OSN are still unclear. We established a dissociated OSN culture system, which allows efficient genetic manipulation of gene expression in vitro. In the OSN culture, endogenous OR expression was assessed by real-time RT-PCR. Among 30 selected OR genes, we detected the expression of 18 ORs in cultured OSNs. Lentiviral vectors expressing a selected OR can be infected into cultured OSNs efficiently and functional exogenous OR expressions are detected. Exogenous I7 was expressed in OSNs for 5 days in vitro (DIV). Levels of endogenous OR transcripts in exogenous I7 expressing culture were compared with that of the control GFP expressing culture. We observed reduced expression levels for all tested endogenous OR genes in exogenous I7 expressing OSN culture. We further rested this phenomenon by introducing either exogenous P2 or MOR118 to the culture. Consistently, exogenous OR expression suppress the transcript levels of endogenous ORs at 5 DIV. Furthermore, we observed that exogenous MOR118 expression suppresses the expression of endogenous MOR118 as well. Whether other exogenous OR expressions also suppress their endogenous OR transcripts is being investigated. Acknowledgements: NIH/NICDC DC010237 #P172 POSTER SESSION IV: CHEMOSENSORY TRANSDUCTION AND SIGNALING Heterologous Expression of Mouse Pheromone Receptors Identifies Cognate Ligands Sandeepa Dey, Hiroaki Matsunami Molecular Genetics and Microbiology, Duke University Medical Center Durham, NC, USA Pheromones are chemicals from conspecifics that affect innate behavior or hormonal changes. In mammals, the vomeronasal organ (VNO) is thought to play a prominent role in detecting pheromones; the vomeronasal sensory neurons (VSNs) express three families of seven-transmembrane G-protein coupled receptors (GPCRs): the V1Rs, V2Rs, and FPRs, in two molecularly and spatially-distinct regions. In mice, VSNs that express the V2Rs are thought to detect peptide cues, including MHC-presenting peptides, major urinary proteins (MUPs), and exocrine gland-secreting peptides (ESPs). They are thought to be involved in various pheromone-mediated behaviors and physiological changes, such as mating, aggression, and selective 84 | AChemS Abstracts 2010 Abstracts are printed as submitted by the author(s)
pregnancy block. In order to understand how pheromones are detected by the vomeronasal receptors, it is essential to know which receptors are activated by a given chemical. However, identifying cognate ligands for the V2Rs has been challenging, partly because they are poorly localized to the surface of heterologous cells. Here, we show the establishment of heterologous cell system to functionally identify the V2Rs and demonstrate that the ESP ligands can differentially activate the V2Rs. We also show the large extracellular domain of the V2Rs plays a critical role in ligand selectivity. Our results provide a platform to characterize ligand selectivity of the V2Rs and suggest that a unique mechanism that regulates the functional expression of the V2Rs. #P173 POSTER SESSION IV: CHEMOSENSORY TRANSDUCTION AND SIGNALING Muscarinic Receptor M3 Potentiates the Function of a Broad Range of Mammalian Odorant Receptors Yun R. Li 1 , Hiroaki Matsunami 1, 2 1 Department of Molecular Genetics and Microbiology, DurhamDepartment of Molecular Genetics and Microbiology, Duke University Medical Center Durham, NC, USA, 2 Department of Neurobiology, Duke University Medical Center Durham, NC, USA Mammalian olfaction begins with the binding of odorant molecules to specific odorant receptors (ORs), which are expressed on the surface of olfactory sensory neurons (OSNs) and make up the largest family of G-protein coupled receptors (GPCRs). Here we show that type 3 muscarinic acetylcholine receptor M3, a non-OR Class A GPCR expressed in OSNs, enhances odorant-specific response of a large set of ORs. Coexpression of M3 in heterologous cells produces an ~10-fold leftward shift in EC50 values and increases the maximum response by 25-1000% for a broad range of ORs in the cAMP-mediated luciferase reporter gene assays for OR activity. Coexpression of M3 does not enhance the cell-surface expression of the ORs. Thus, the action of M3 is distinct from that of known accessory factors, such as the RTPs. The M3-dependent potentiation of OR activity is significantly enhanced in the presence of RTP1, indicating that the effect of M3 is synergistic and increases the response of ORs already at the cell surface. M3 potentiation of OR activation is further enhanced by muscarinic agonist carbachol, but inhibited by the muscarinic inverse agonist atropine. Conversely, OR activation by cognate odors causes the activation of M3 downstream signaling by eliciting a Ca 2+ response, an effect that is inhibited when cells are simultaneously exposed to odor and atropine. The interaction and crosstalk observed between ORs and M3 in heterologous cells is likely dependent on the ability of these GPCRs to form stable heterometric complexes, as ORs and M3 coprecipitate each other in HEK293T cells. Our study suggests that OR and M3 heteromers functionally couple with one another, and will serve as an effective screening tool to identify active ligands for the ORs. This work was supported by NIH-NIDCD. Acknowledgements: NIH-NIDCD Duke Undergraduate Research Support Grants #P174 POSTER SESSION IV: CHEMOSENSORY TRANSDUCTION AND SIGNALING The OR37 subfamily: establishment of the clustered expression pattern Jörg Strotmann, Andrea Bader, Verena Bautze, Desirée Haid, Heinz Breer Institute of Physiology, University of Hohenheim Stuttgart, Germany In contrast to most other olfactory sensory neuron (OSN) populations, cells expressing a member from OR37 subfamily of odorant receptor (OR) genes are restricted to a small central patch of the mouse olfactory epithelium (OE). To obtain insight into the regulatory mechanisms which determine this unique spatial organization, a lineage tracing approach (mOR37C-IRES-Cre) was performed to visualize all cells that transcribed OR37C at any time during differentiation. As expected, labelled OSNs were found in the typical central patch; surprisingly, however, numerous additional OSNs were found which were broadly dispersed throughout the OE. Using in situ hybridization, the mRNA for OR37C could only be detected in those cells located in the typical patch, suggesting that all ectopic OSNs had ceased OR37C expression. The question whether these cells may undergo premature apoptosis was addressed by analysis for active caspase-3; none of them, however, expressed this pro-apoptotic marker. A close examination of the ectopically positioned OSNs revealed that they all extended an axon towards the olfactory bulb (OB), and indeed many glomeruli could be detected which contained a few labelled fibers. The location of these glomeruli in the medial and lateral domains of the bulb indicated that these represented glomeruli that receive input from OSN populations expressing other ORs than OR37C. Altogether, these data indicate that OSNs which initially express OR37C outside the typical patch do not continue, but switch to the expression of a different OR gene, suggesting the involvement of a mechanism downstream of gene choice that restricts OR37C expression to the central patch. Acknowledgements: Supported by the Deutsche Forschungsgemeinschaft #P175 POSTER SESSION IV: CHEMOSENSORY TRANSDUCTION AND SIGNALING Expression of odorant receptor genes on the olfactory epithelium following olfactory nerve transection Yongxiang Wei 1 , Yuehong Liu 2 , Ling Yang 2 , Xutao Miao 3 , Yayan Lu 2 , Xiaochao Liu 2 1 Beijing Chaoyang Hospital,Capital Medical University Beijing , China, 2 Beijing Tongren Hospital,Capital Medical University Beijing, China, 3 Beijing Jishuitan Hospital Beijing, China Objective To construct unilateral olfactory nerve transection model of rats and observe the change of expression quantity and position of odorant receptors in regenerated olfactory epthelium. Methods Experimental group and control group consisted of 20 and 12 rats respectively. The left olfactory bulb of the rats in the former group was exposed under microscope and the olfactory nerve was transected along cribriform plate; the rats in control group didn’t accept any treatment. The change of cell morphology, and quantity and thickness of epithelium were observed by HE staining. The expression pattern of olfactory receptor genes Olr287, Olr226, Olr1493 and Olr1654 in olfactory epithelium and the distribution and quantity changes of each gene P O S T E R S Abstracts are printed as submitted by the author(s) Abstracts | 85
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AChemS Association for Chemorecepti
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AChemS Association for Chemorecepti
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We are pleased to announce that sev
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#1 GIVAUDAN LECTURE Normal and Canc
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and against delta ENaC is being pur
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#13 SYMPOSIUM - GENETICS OF HUMAN O
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#19 SYMPOSIUM - CILIA, SENSORY DYSF
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#26 PLATFORM PRESENTATIONS - POLAK
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esponse selectivity for both OSNs a
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elicit glucagon-like peptide-1 (GLP
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contrast, changing the concentratio
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of OBPs, we have systematically sup
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whereas mouse and joystick reaction
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POSTER PRESENTATIONS #P1 POSTER SES
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PROP strips and PROP solutions. PAV
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#P11 POSTER SESSION I: TASTE IMAGIN
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#P325 POSTER SESSION VII: OLFACTORY
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of 7/10, and causing eyes to water
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showed that N1/P2 latencies were lo
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differentiated patients with AD (M=
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pleasantness 3)of mixtures (A,B), A
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Index Aarts, H - P328 Abe, K - P88,
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Haase, L - P3, P4, P29, P355 Haddad
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Murata, Y - P272 Murphy, C - P3, P4
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Veldhuizen, M - P7, P24, P25, P242,
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Registration 7:30 am to 1:00 pm, 6:
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See you next year! AChemS 33rd Annu
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Abstracts - Association for Chemoreception Sciences

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