1 1 Symposium Chemosensory Receptors Satellite DEVELOPMENT ...

69 Poster Peripheral Olfaction and Peripheral TasteINHIBITION OF THE IP3 PATHWAY PERMITS FLY SUGARRECEPTOR CELL RESPONSES TO NA-SACCHARINMiller S.E. 1 , Kennedy L.M. 1 1 Neuroscience Laboratory, BiologyDepartment, Clark University, Worcester, MASince flies give no behavioral, and little “sugar cell” responses to“artificial” sweeteners (Higgins & Kennedy, 2001), it has been thoughtthat they lack excitatory receptor mechanisms for these compounds. Yetthe fly “deterrent cell” responds to Na-saccharin (Na-S), as well asbitter stimuli, and there is reciprocal inhibition for Na-S and sugarstimuli (Liscia et al., 2004). We studied sugar cell responses to Na-Sduring inhibition of the IP 3 pathway with 1-(5-isoquinolinesulfonyl)-2-methylpiperazine 200 µM and Na-deoxycholate 0.03 % w/v (H7).Receptor cell action potentials were tip-recorded from single sensilla inisolated Phormia regina proboscises. Addition of H7 to Na-S 4.87 mMincreased sugar cell firing rates from medians of 1.5 to 5 actionpotentials/ first 100 msec (p < 0.002, Mann-Whitney). There was atypical concentration-dependent response curve for firing to Na-S (p =0.05, Kramer), while the adaptation rate increased with Na-Sconcentration, in the presence of H7. These data show that the fly sugarcell has an excitatory receptor mechanism for Na-S. An IP 3 bittertransduction pathway in the deterrent cell, leading to inhibition of sugarcell firing, could be responsible for the lack of fly responses to Na-S.But an IP 3 pathway is known to mediate adaptation of sugar cellresponses to sugars (Amakawa et al., 1992). Given the positiverelationship for adaptation rate and Na-S concentration, and the lack ofsynapses between sensillum receptor cells, activation of the sugar cellIP 3 -mediated adaptation process so as to cut off an excitatory responseseems a more likely mechanism.70 Poster Peripheral Olfaction and Peripheral TasteINVESTIGATING CYCLIC AMP IN TASTE TRANSDUCTIONUSING REAL TIME IMAGINGRoberts C.D. 1 , Chaudhari N. 2 , Roper S.D. 2 1 Program in Neurosciences,Miller School of Medicine, University of Miami, Miami, FL;2 Physiology & Biophysics and Program in Neurosciences, MillerSchool of Medicine, University of Miami, Miami, FLThere is unequivocal biochemical and physiological evidence that thediffusible second messenger 3'-5' cyclic adenosine monophosphate(cAMP) is involved in taste transduction. Yet, recent reports haveemphasized the role of Ca 2+ signaling in taste transduction (i.e. tastereceptor → G protein → PLCβ2 → IP 3 → ∆[Ca 2+ ] i ) and attention hasbeen shifted from cAMP signaling in taste. The significance of theoriginal cAMP data is presently unresolved. The goal of our research isto re-investigate cAMP in taste transduction in the light of currentunderstanding. We have developed techniques to image real-timechanges in intracellular cAMP in taste cells using genetically-encodedcAMP reporters. These FRET-based reporters are modifications of PKAsubunits and permit one to measure single-cell cAMP levels withexcellent spatial and temporal resolution (Zaccolo & Pozzan 2002,Science 295:1711). Using a biolistic approach we have transfected ratfungiform taste buds with cAMP reporter plasmids. Focal application ofthe bitter tastant cycloheximide (100 µM) to living fungiform tastebudsin situ produced a decrease in [cAMP] i within individual taste receptorcells. These results are qualitatively similar to previous biochemicalmeasurements from homogenized taste tissue (Yan et al 2001, Am JPhysiol Cell Physiol 280:C742) but are now allowing us to examine thecAMP response in individual, identified cells. (Supported by DC006021[NC])71 Poster Peripheral Olfaction and Peripheral TasteIDENTIFICATION OF TWO PUTATIVE TASTE SIGNALTRANSDUCTION COMPONENTSLopezjimenez N.D. 1 , Cavenagh M.M. 1 , Sainz E. 1 , Battey J.F. 1 , SullivanS.L. 1 1 National Institute on Deafness and Other CommunicationDisorders, National Institutes of Health (NIH), Rockville, MDTo identify genes important for taste receptor cell function, weanalyzed the sequences and expression patterns of clones isolated froma mouse taste receptor cell-enriched cDNA library. Here, we report theanalyses of two of these genes. One, Gpr113, encodes a G-proteincoupledreceptor belonging to family 2B, members of which arecharacterized by having long N-terminal, extracellular domains. Thepredicted N-terminal extracellular domain of GPR113 contains 696amino acids with two functional domains, a peptide hormone-bindingdomain and a G-protein-coupled receptor proteolytic site. The secondencodes a novel member of the TRP family of ion channels, manymembers of which have been implicated in sensory signal transduction.Expression analyses with both of these genes indicate that theirexpression is highly restricted to subsets of taste receptor cells.Furthermore, co-localization studies with various taste receptor cellmarkers suggest that GPR113 plays a role in sweet taste, whereas theTRP channel plays a role in either salty or sour taste transduction.Knock out mouse models are currently being developed to test thesehypotheses. This work was sponsored by the Divisions of IntramuralResearch of the NIDCD and NINDS, NIH.72 Poster Peripheral Olfaction and Peripheral TasteDROSOPHILA NORPA EXPRESSION IN TASTE NEURONS:ROLE IN TREHALOSE DETECTIONChyb S. 1 , Sadiq F. 2 , Robert P. 2 , Chyb M. 2 1 CSIRO Entomology,Canberra, Australian Capital Territory, Australia; 2 Molecular CellBiology, Imperial College London, Wye, Kent, United KingdomDrosophila norpA (no receptor potential A) gene encodesphosphatidylinositol (PI)-specific phospholipase C (PLC-ß) and yieldstwo products: subtype I & II. PLC hydrolyses phosphatidylinositol 4,5-biphosphate (PIP 2 ) into second messengers diacylglycerol (DAG) andinositol trisphosphate (InsP 3 ), which ultimately leads to Ca 2+ releasefrom the intracellular stores. The best studied example of a transductionpathway involving norpA product is the Drosophila photoreception;flies with strong alleles of norpA are blind due to a dramatic decrease inthe photoreceptor PLC levels. Subsequently, the norpA-encoded PLChas been shown to be required for Drosophila olfaction (Riesgo-Escovaret al., 1995). Here, we report that norpA may also be involved inDrosophila gustation. Firstly, RT-PCR results indicate that major tasteorgans, labella and tarsi, contain detectable levels of subtype II norpAtranscript; in contrast compound eyes show high levels of subtype I.Secondly, using a GAL4/UAS approach with the minimal norpApromoter (Doh et al., 1997) we show that norpA is expressed is arelatively large subset of gustatory neurons. Finally, genetic ablation ofnorpA-expressing taste neurons leads to a marked decrease in Gr5atranscript levels and to significantly reduced feeding responses totrehalose. Our findings suggest that both norpA-encoded PLC mayfunction in trehalose detection pathway.18