Identifying TRPA1 Agonists by Monitoring Intracellular Calcium Levels in HEK Cells
Paige Roe, Erik Johnson, and Wayne Silver
Department of Biology, Wake Forest University
roepm7@wfu.edu
Introduction
Figure 1. Dose response curves for 13 tested stimuli.
Conclusions
Nasal irritation by ammonia, wasabi, onions
and other irritants is due to stimulation of
trigeminal nerves. These nerves appear to be
stimulated by virtually all volatile compounds
if presented in high concentrations (Bryant
and Silver, 2000).
Relative fluorescence (RFU) for thirteen stimuli at various concentrations. The RFU calculation is shown in the bottom right corner of this section. n=9 for all concentrations. A twotailed, independent T-test was conducted to determine statistical significance relative to the vehicle (p<0.05). Stars indicate statistical differences from vehicle addition. Error bars =
standard error. Examples of where the stimulus occurs are denoted in parentheses.
Based on both the dose response curves and
inhibitor assays, the following conclusions
were made about the tested stimuli:
• Stimulates TRPA1 (previously known
agonists)
Trigeminal nerve endings contain several
types of receptors; however, the specific
receptors stimulated by many trigeminal
stimuli are unknown.
*
• Stimulates TRPA1 (Newly identified
Transient receptor potential (TRPs) proteins
are non-specific cation channels with an
affinity for calcium, and are associated with
the trigeminal nerve.
agonists)
▫Benzaldehyde
▫Toluene
TRPA1, a TRP channel found in a subset of
neurons in the trigeminal ganglion, is currently
known to be activated by at least ninety
different compounds.
• Could stimulate TRPA1 (needs to be
confirmed by additional assays)
▫alpha-Terpineol
▫Acetic Acid
▫Amyl Acetate
▫D-Limonene
This study tested thirteen trigeminal stimuli to
determine if they activate TRPA1.Three
stimuli were previously shown to activate
TRPA1 (AITC, CIN, EUG). Two do not
activate TRPA1 (CAP, NIC). It was not known
whether eight of the stimuli activated TRPA1
(ACA, AMA, ATER, BEN, LIM, DEN, CYC,
TOL).
• Does not stimulate TRPA1
▫Capsaicin
▫Cyclohexanone
▫Denatonium benzoate
▫Nicotine (Andre et al., 2008)
More experiments, both in this heterologous
expression system and in whole animals, are
currently underway to further characterize these
stimuli. Hopefully, these additional assays will
add insight and help classification of the four
compounds where current results are not clear.
Methods
To maintain cell health, hTRPA1-HEK cells
were cultured in a tetracycline-inducible
system. In normal culture conditions, this
system results in the repression of TRPA1
expression in the cell. Tetracycline binds a
repressor allowing for TRPA1 expression to
occur more abundantly.
Final fluorescence value of well containing hTRPA1-HEK cells
Initial fluorescence value of well containing hTRPA1-HEK cells
RFU =
Final fluorescence value of well containing naive HEK cells
Initial fluorescence value of well containing naive HEK cells
Both naive HEK and induced hTRPA1-HEK
cells (tetracycline added) were allowed to
grow in a black-walled, 96-well plate for a
minimum of 24 hours.
Intracellular calcium levels were monitored
using the Ca+2-sensitive fluorescent dye
FLUO-3AM. Baseline fluorescence of each
well was measured. A potential stimulus was
then added to the well and fluorescence was
measured again.
The response elicited in hTRPA1-HEK cells
by particular stimuli were normalized to the
responses elicited in naïve HEK cells.
The experiment was repeated using the TRPA1
inhibitor HC-030031 to confirm increases in
intracellular calcium were due to TRPA1
activation. In the inhibitor assays, hTRPA1HEK cell responses to stimulus addition were
normalized to vehicle addition instead of naive
HEK cells.
▫Allyl-Isothiocyanate (Jordt et al., 2004)
▫Cinnamaldehyde (Bautista et al., 2004)
▫Eugenol (Bandell et al., 2005)
Andre E., Campi B, Materazzi S, Trevisani M, Amadesi S, Massi
D, Creminon C, Vaksman N, Nassini R, Civelli M, Baraldi PG,
Poole DP, Bunnett NW, Geppetti P, and Patacchini R. (2008).
Cigarette smoke-induced neurogenic inflammation is mediated by
α,β-unsaturated aldehydes and the TRPA1 receptor in rodents. J
Clin Invest 118: 2574-2582.
Bandell M, Story GM, Hwang SW, Wiswanath V, Eid SR, Petrus
MJ, Early TJ, and Patapoutian A. (2004). Noxious cold ion
channel TRPA1 is activated by pungent compounds and
bradykinin." Neuron 41: 849-857.
Figure 2. Responses to five stimuli are inhibited
when using the TRPA1 inhibitor, HC-030031.
Mean RFU for four stimuli when hTRPA1-HEK cells are
either incubated with or without the inhibitor HC-030031.
The RFU calculation is shown on the bottom side of this
section. Note that the normalization calculations are
different than those used for dose response curves above.
Stimuli shown are 1 mM allyl isothiocyanate, 33 mM
benzaldehyde, 33 mM cinnamaldehyde, 33 mM eugenol
and 100 mM toluene. Error bars = standard error. A twotailed, independent T-test was conducted to determine
statistical significance between wells incubated with or
without inhibitor (p<0.05). Other tested stimuli either
showed no increase in fluorescence in hTRPA1-HEK cells
without inhibitor or inconclusive results in this assay.
Literature Cited
Bautista DM, Movahed P, Hinman A, Axelsson HE, Sterner O,
Hogestatt ED, Julius D, Jordt SE, and Zygmunt PM. (2005).
Pungent products from garlic activate the sensory ion channel
TRPA1. PNAS 102:12248-12252.
RFU =
Final fluorescence value of well receiving stimulus
Initial fluorescence value of well receiving stimulus
Final fluorescence value of well receiving vehicle
Initial fluorescence value of well receiving vehicle
Bryant, B. and Silver, W.L. (2000) Chemesthesis: The common
chemical sense. In T.E. Finger, W.L. Silver, and D. Restrepo.
(editors) Neurobiology of Taste and Smell 2nd Edition . WileyLiss, Inc. 479 pp. 73-100.
Jordt, S.E., Bautista, D.M., Chuang, H.H., McKemy, D.D.,
Zygmunt, P.M., Hogestatt, E.D., Meng, I.D. and Julius, D. (2004)
Mustard oils and cannabinoids excite sensory nerve fibres through
the TRP channel ANKTM1. Nature. 427:260-265.
Acknowledgements
Dr. David Julius (UCSF) for the hTRPA1-HEK cells.
Dr. Gloria Muday for aid in data analysis