1 1 Symposium Chemosensory Receptors Satellite DEVELOPMENT ...

37 Poster Peripheral Olfaction and Peripheral TasteEFFECT OF EXTERNAL NA + ON NA-CA EXCHANGE-MEDIATED CURRENT RECOVERY IN FROG ORNsAntolin S. 1 , Matthews H.R. 1 1 Department of Physiology, University ofCambridge, Cambridge, United KingdomDuring the olfactory response, Ca 2+ enters through CNG channels,opening Ca 2+ activated Cl - channels which augment the depolarisingcurrent. The dependence of Ca 2+ extrusion upon external [Na + ] wasstudied using the decay of Ca Cl current to monitor the recovery of [Ca 2+ ]following a brief exposure to the PDE inhibitor IBMX, which elevatesciliary [cAMP]. The solution bathing the cilia was rapidly exchanged bytranslating the suction pipette, which recorded receptor current, betweensolution streams. Cilia were exposed for 1 s to 100 µM IBMX, andreturned to IBMX-free solution, allowing the receptor current to decaywith monoexponential kinetics. The decay time constant (τ c ) wasgreatly prolonged by a factor of 34.6 ± 8.2 if [Na + ]o was reduced to10% (11mM) of its value in Ringer by substitution with guanidinium,an ion which permeates the CNG channel but does not support Na-Caexchange. When the cilia were returned to Ringer after 3 s in low-Na +solution, the τ c was similar to that when returning immediately toIBMX-free Ringer, suggesting that Ca 2+ extrusion via Na-Ca exchangedominates current decay, since [cAMP] falls rapidly after IBMXremoval. The τ c was remarkably insensitive to [Na + ]o, beingsubstantially retarded only after reduction to third or less of that inRinger. Rate constants at different [Na + ]o were fitted by a Hill equationwith Hill coeff. of 3.1, suggesting a stoichiometry of 3Na + -1Ca 2+ for theexchanger. A K d of 58.7mM indicates that Na-Ca exchange in frogORN´s is surprisingly insensitive to [Na + ], in contrast to thephotoreceptor and cardiac exchangers. The high affinity of the olfactoryexchanger for external Na + allows normal response termination evenfollowing mucus dilution.38 Poster Peripheral Olfaction and Peripheral TasteINVESTIGATING THE ROLE OF SODIUM CALCIUMEXCHANGERS IN CULTURED HUMAN OLFACTORY CELLSCostanzo J. 1 , Gomez G. 1 1 Biology Dept., University of Scranton,Scranton, PAOlfactory receptor neurons (ORNs) respond to odorants with changesin intracellular calcium concentrations ([Ca 2+ ] i ). In human ORNs, aboutone fourth of these responses are decreases in [Ca 2+ ] i ; thus this type ofresponse represents a large portion of the input into the olfactory bulb,yet the mechanism behind this type of response in unknown. Wetherefore investigated the involvement of Na + /Ca exchangers (NCX) inthis system, and used human olfactory cultured cells due to theiravailability, ease of use, and because they demonstrate structural andfunctional characteristics that are similar to those found in acutelyisolated human ORNs. Cells were grown in vitro on 6-well plates andtested with odorants in the presence or absence of extracellular Na + ;responses were measured using calcium imaging techniques.Immediately following imaging, cells were localized on the cultureplate, fixed, and tested with immunocytochemistry using antibodiesagainst NCX. Our results have shown that NCX is present andfunctional in these cells, suggesting that this may be the primarymechanism for mediating [Ca 2+ ] i decreases. An understanding of themechanism responsible for generating [Ca 2+ ] i changes may haveimportant implications for the study of the physiology of olfaction.This work is part of the undergraduate Honors research of JC and waspartially supported by NIH 5 RO3DC4954-2.39 Poster Peripheral Olfaction and Peripheral TasteMECHANISMS OF CHLORIDE ACCUMULATION IN INTACTMOUSE OLFACTORY EPITHELIUMNickell W.T. 1 , Kleene N.K. 1 , Kleene S.J. 1 1 University of Cincinnati,Cincinnati, OHA depolarizing chloride current is a substantial part of the olfactoryresponse to odors; this requires a mechanism for accumulation ofchloride against an electrochemical gradient. The sodium-potassiumchlorideexchanger NKCC1 has been shown to play an important role inthis process. However, two other classes of transporter that might play arole in chloride accumulation are common. We used theelectroolfactogram (EOG) to investigate the mechanisms of chlorideaccumulation in intact mouse olfactory epithelium. In mice lacking theNKCC1 gene, an EOG was present that was 60% of the amplitude ofthe EOG in wild-type mice. Niflumic acid, a chloride channel blocker,reduced this response by 80% in both wild-type and knockout mice.Bumetanide, a blocker of NKCC, reduced the amplitude of the EOG inwild-type epithelium by 53%. There was no effect of bumetanide on theEOG in mice lacking NKCC1. These results strongly suggest thatNKCC1 is a major part of a more complex system of chlorideaccumulation. Hence we are testing other blockers of chloride transport.DIDS (1 mM), a blocker of chloride-bicarbonate exchangers, reducedthe EOG amplitude by about 60% in both wild-type and NKCC1 knockoutmice. In frog, DIDS also blocks the ciliary chloride channels by23%, but this is not sufficient to account for the reduction of the EOGby DIDS. Thus it is probable that both NKCC and chloride-bicarbonatetransporters contribute to maintenance of the chloride gradient inolfactory neurons, but further experiments are necessary to fullyunderstand the system. This work was supported by NIDCD grant R01DC00926.40 Poster Peripheral Olfaction and Peripheral TasteDUAL EFFECT OF ATP IN THE OLFACTORY EPITHELIUMOF XENOPUS LAEVIS TADPOLES: ACTIVATION OF BOTHRECEPTOR AND SUSTENTACULAR SUPPORTING CELLSManzini I. 1 , Czesnik D. 1 , Kuduz J. 1 , Schild D. 1 1 University ofGoettingen, Goettingen, Lower Saxony, GermanyNucleotides and amino acids are acknowledged categories of waterborneolfactory stimuli. In previous studies it has been shown thatlarvae of Xenopus laevis are able to sense amino acids. Here we reporton the effect of ATP in the olfactory epithelium (OE) of Xenopus laevistadpoles. First, ATP activates a subpopulation of cells in the OE. TheATP-sensitive subset of cells is almost perfectly disjoint from the subsetof amino acid-activated cells. Both responses are not mediated by thewell-described cAMP transduction pathway as the two subpopulationsof cells do not overlap with a third, forskolin-activated subpopulation.We further show that in contrast to amino acids, which act exclusivelyas olfactory stimuli, ATP appears to feature a second role. Surprisinglyit activated a large number of sustentacular supporting cells (SCs), andto a much lower extent olfactory receptor neurons. The cells of theamino acid- and ATP-responding subsets featured differences in shape,size and position in the OE. The latencies to activation upon stimulusapplication differed markedly in these subsets. To obtain these resultstwo technical points were important. We used a novel dextrantetramethylrhodaminebackfilled slice preparation of the OE and wefound out that an antibody to calnexin, a known molecular chaperone,also labels SCs. Our findings thus show a strong effect of ATP in theOE and we discuss some of the possible physiological functions ofnucleotides in the OE. [Supported by DFG:SFB 406 (B5) and by DFGResearch Center for Molecular Physiology of the Brain (CMPB, ProjectB4)]10