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development. After we injected 5 day old mice with BrdU, both
CBCs and TEC were labeled 5 days post-BrdU. Over the following
60 days, the percent of labeled CBCs increased while the percent of
labeled TECs decreased. These data suggest that CBCs are
generated from non-CBC progenitor cell during development,
particularly postnatally while the trachea is growing. We then asked
if new CBCs could be generated from the adult epithelium or if the
capacity to generate new CBCs was lost after the perinatal period.
TECs were recovered from adult A/J mice and grown in an air-
liquid interface (ALI) culture. After 14 days at ALI, the number of
CBCs increased to 1% of the total population. BrdU added to ALI
cultures labeled CBCs, indicating that the increase in CBC density
was due to new cell divisions. We conclude that: 1) CBCs are
normally a static population in adult mice; 2) CBC progenitors
proliferate during neonatal development while the trachea is
growing; and 3) CBCs can be regenerated from a proliferative
population resident in the adult epithelium. Acknowledgements:
Supported by NIDCD & NHLBI
#P51
POSTER SESSION II:
TRIGEMINAL SYSTEM; TASTE CNS;
NEUROIMAGING; OLFACTION CNS
Biophysical Characterization of the Human Nociceptor
Channel, hTRPA1
Yuriy V. Bobkov
1
, Elizabeth A. Corey
1
, Kirill Y. Ukhanov
1
,
Barry W. Ache
1,2
1
Whitney Laboratory, Center for Smell and Taste, and McKnight
Brain Institute, University of Florida Gainesville, FL, USA,
2
Depts. of Biology and Neuroscience, University of Florida
Gainesville, FL, USA
There is striking similarity in the functional role of TRPA channel
orthologs among evolutionary diverse species. Abundantly
expressed in the somatosensory system, including in trigeminal
neurons, mammalian TRPA channels detect and integrate noxious
stimuli of different sensory modalities including mechanical and
thermal. They also function as broad-spectrum “alarm”
chemoreceptors, signaling potentially harmful exposure to irritants
and pungent compounds. Despite a growing understanding of
potential modulators, agonists, and antagonists for these channels,
the exact mechanisms of channel regulation and activation remain
mostly unknown or are controversial, including even the basic
biophysical parameters of both native and heterologously expressed
TRPA channels. Here we transiently express the human TRPA1
(hTRPA1) channel in HEK293T cells and use conventional
single channel inside-out and outside-out patch recording to
characterize the basic properties and the ion selectivity of the
channel. The relative permeability of the channel to inorganic
cations is: Ca
2+
(5.1) > Ba
2+
(3.5) > Mg
2+
(2.8) > NH
4 +
(1.5) >
Li
+
(1.62) > Na
+
(1.0) ≥ K
+
(0.98) ≥ Rb
+
(0.98) > Cs
+
(0.95). To
organic cations it is: Na
+
(1.0) ≥ DiMA(0.99) > TriMA(0.7) >
TetraMA(0.4) > NMDG(0.1). Activation of the channel appears to
recruit the channel to a conformational state with increased
permeability to large organic cations. The pore of the channel in
this state can be characterized as being dilated approximately
1-2.5Å (from ~6.5/8.2 to ~7.4/10.6 Å) based on estimates of the
minimum cation diameter/mean cation diameter). Overall our
findings further characterize and refine the basic parameters of the
hTRPA1 channel, in particular to help predict water soluble (low
logD) ligands and pharmacological compounds that can permeate
the channel. Acknowledgements: This work was supported by
National Institute on Deafness and Other Communication Disorders
(DC005995 and DC001655). We thank Drs. Gunter Gisselmann
and Hanns Hatt for generously providing hTRPA1.
#P52
POSTER SESSION II:
TRIGEMINAL SYSTEM; TASTE CNS;
NEUROIMAGING; OLFACTION CNS
The involvement of TRP channels in trigeminal odour sensation
J. Kyereme
1
, M. Luebbert
1,2
, N. Schoebel
3
, H. Hatt
1
1
1Faculty of Biology and Biotechnology, Ruhr Universität Bochum,
Universitätsstr. 150, 44780 Bochum, Germany,
2
Ruhr University
Research School Bochum, Germany,
3
Leibniz Reseach Center for
Working Environment and Human Factors, Ardeystr. 67, 44139
Dortmund, Germany
To date, the detection of different odorants by the olfactory system
is well investigated and understood. However, much less is known
about the activation of the trigeminal system (TS) caused by the
majority of odorants in higher concentrations. Some members of
the transient receptor potential (TRP) superfamily of ion channels
are highly expressed in trigeminal sensory neurons. In order to
investigate if different TRP channels are involved in the detection
of several odorants by the TS, we used primary cultures of rat
trigeminal sensory neurons (TSN’s) to perform an overlapping
study with classical TRP channel agonists and different odorants
using calcium imaging. The findings of this study gave first hints
to an involvement of TRPV1, TRPM8, and TRPA1 in the detection
of the odorants we used. To obtain a better understanding of the
odorant-effect on the specific TRP channels, we performed
current clamp measurements on TSN’s during which we applied
different odorants in the presence and absence of respective
antagonists. Although the blockers failed to completely inhibit
odorant induced depolarisations, the effects on the membrane
potentials were significantly decreased in the presence of these
substances. Finally, we used CHO cells heterologously expressing
either rTRPA1, rTRPV1, or rTRPM8 performing whole-cell
measurements, in order to test the direct effect of the odorants used
on the respective channel proteins. Our experiments indicate that
TRPV1, TRPM8, and TRPA1 seem to play crucial roles in the
detection of different odorants via the TS. A better understanding of
the odorant interaction with different TRP channels might lead to a
more detailed understanding of the properties of the TS.
#P53
POSTER SESSION II:
TRIGEMINAL SYSTEM; TASTE CNS;
NEUROIMAGING; OLFACTION CNS
Microglial Responses After Injury of the Chorda
Tympani Nerve
Dianna L. Bartel, Thomas E. Finger
Rocky Mtn Taste & Smell Center, Univ Colorado School of
Medicine Aurora, CO, USA
The chorda tympani (CT) nerve, which innervates taste buds on the
anterior tongue, is vulnerable to injury during dental and inner ear
procedures. The CT is primarily a sensory nerve restricted to fine
caliber fibers that convey special visceral, or gustatory, sensation.
When the CT is damaged, robust microglial responses occur in the
first central gustatory relay, the nucleus of the solitary tract (nTS).
Microglial cells rapidly become morphologically reactive and their
density increases significantly on the injured side as a result of
proliferation and migration. Although the CT nerve does not carry
pain fibers, the general profile of microglial responses in the nTS is
similar to that described in nerve injuries that model chronic pain.
In pain models, microglial responses actively contribute to the
initiation of chronic pain signaling that persists long after the initial
nerve damage. Recent studies have demonstrated a crucial role for
the microglial Toll-like receptor 4 (TLR4) in this microglial
Abstracts | 45
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