Abstracts - Association for Chemoreception Sciences

any potential role of ryanodine receptors (RyRs) in this signaling
pathway has not been identified in mouse taste cells. Using RT-
PCR and immunocytochemistry, we detected the RyR isoform 1
in multiple taste papillae types, while calcium imaging studies
revealed that RyRs are physiologically functional in mouse taste
cells. We found that RyRs significantly contribute to taste-evoked
Ca 2+ responses in approximately 30% of type II cells but did not
significantly contribute to the taste-evoked responses in dualresponsive
taste cells. The discovery of RyRs in mouse taste cells
indicates that multiple Ca 2+ release mechanisms contribute to the
formation of evoked taste responses and our findings reveal an
important role for RyRs in the transduction of these taste-evoked
responses. Acknowledgements: This work was supported by
NIDCD DC00635801 and NSF 0917893 to KM.
#5 PLATFORM PRESENTATIONS -
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Acetylcholine, released from taste buds during gustatory
stimulation, enhances taste responses
Robin Dando 1 , Yijen A. Huang 1 , Stephen D. Roper 1,2
1
Department of Physiology and Biophysics, Miller School of
Medicine, University of Miami Miami, FL, USA, 2 Program in
Neuroscience, Miller School of Medicine, University of Miami
Miami, FL, USA
Mammalian taste buds possess apical taste receptors as well as
basolateral neurotransmitter receptors for synaptic transmission
and neuromodulation. One class of basolateral receptors
expressed in taste buds are the muscarinic acetylcholine (ACh)
receptors (Eguchi et al, 2008), reported to be activated by bathapplied
ACh (Ogura, 2002). Acetylcholinesterase has long been
known to surround taste buds, suggesting a physiological role for
ACh in taste reception. Collectively, these findings implicate a
role for ACh in taste bud function. We tested this hypothesis by
imaging taste cell responses in lingual slices, and by measuring
neurotransmitter secretion from isolated taste buds using cellular
biosensors. When we applied ACh or carbachol (1 mM) to a
lingual slice, we observed that taste-evoked calcium responses
were potentiated. The effects of ACh were blocked by atropine
(5 mM), suggesting muscarinic receptor activation. Using ATP
biosensors, we measured the effect of bath-applied ACh on ATP
secretion from isolated taste buds. ACh augmented taste-evoked
ATP release, complementing the ACh potentiation observed in
the lingual slice preparation. Moreover, bath-applied atropine
itself, in the absence of any added ACh, reduced taste-evoked
ATP release from taste buds, suggesting endogenous cholinergic
mechanisms are activated during taste stimulation. Finally, we
used ACh biosensor cells to test whether taste buds secrete ACh.
We observed that ACh is indeed released from isolated taste buds
upon tastant stimulation. The source of taste-evoked ACh
secretion may be from cholinergic nerve fibers that innervate taste
buds or directly from taste cells themselves, as implied by Ogura
et al (2007). Our findings strongly implicate ACh as a
neurotransmitter that enhances taste responses.
Acknowledgements: Supported by NIH/NIDCD grants
5R01DC000374 and 5R01DC007630 to SDR.
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Epithelial Sodium Channel (ENaC) is Involved in Reception of
Sodium Taste: Evidence from Mice with a Tissue-Specific
Conditional Targeted Mutation of the ENaCa Gene
Natalia P. Bosak 1 , Masashi Inoue 2 , Theodore M. Nelson 1 , Edith
Hummler 3 , Yutaka Ishiwatari 1,4 , Alexander A. Bachmanov 1
1
Monell Chemical Senses Center Philadelphia, PA, USA, 2 Tokyo
University of Pharmacy and Life Science Tokyo, Japan,
3
University of Lausanne Lausanne, Switzerland, 4 Institute of Life
Sciences, Ajinomoto Co., Inc. Kawasaki, Japan
Observations that amiloride and other ENaC blockers alter taste
responses to sodium salts generated the hypothesis that ENaC is
involved in salt taste reception. To directly test this hypothesis, we
have generated mice with the ENaCa subunit selectively
eliminated in the lingual epithelium using the Cre-loxP mediated
conditional gene deletion technique. Electrophysiological
experiments have shown that mice with the tissue-specific
conditional ENaCa deletion lacked the amiloride-sensitive
component of chorda tympani nerve responses to lingual
application of sodium salts. However, the amiloride-insensitive
component of the response to sodium salts, responses to nonsodium
salts, sweet, bitter and sour taste stimuli, or responses to
irritants were not affected in these mice. In brief-access tests,
ENaCa knockout mice had an attenuated aversion to higher
concentrations of NaCl compared with wild-type mice. These
data provide direct evidence that ENaC is involved in detecting
sodium taste. Our results further demonstrate that there is a
significant ENaC-independent component of taste responses to
salts.
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Novel proteolyzed ENaC isoforms and corresponding
salt taste enhancing compounds
Kambiz Shekdar, Jessica Langer, Purvi Shah, Joseph Gunnet,
Dennis Sawchuk
Chromocell Corporation North Brunswick, NJ, USA
Modulators of novel proteolyzed human ENaC isoforms were
identified and confirmed in sensory testing to either potentiate or
block human salt taste perception. First, a stable ENaC cell line
was produced using Chromovert®, a method that enables testing
of millions of individual cells to rapidly identify and isolate
individual clones stably expressing all desired native subunits.
Next, a functional 384-well assay specific for the activity of ENaC
comprising alpha, beta and gamma subunits was produced. The
cell line was treated with limiting proteolysis in conjunction with
the functional assay and a series of novel proteolytic ENaC
isoforms was defined based on the differing pharmacology of
reference compounds. Interestingly, ENaC isoforms that were less
sensitive to inhibition by amiloride were identified, providing a
cell based platform consistent with both amiloride-sensitive and
amiloride-insensitive components of human salt taste perception.
High throughput screening of proteolyzed and non-proteolyzed
isoforms resulted in at least 12 distinct chemical series with varied
activity against the ENaC isoforms. The creation of and access to
multiple distinct ENaC isoforms allowed discovery of
corresponding compounds for use as research tools to determine
which isoforms correlate with in vivo ENaC activity. Medicinal
chemistry and testing of compounds in Ussing chamber models
8 | AChemS Abstracts 2010 Abstracts are printed as submitted by the author(s)