Mol Cell Biochem (2009) 331:239–245

DOI 10.1007/s11010-009-0165-7

QGP-1 cells release 5-HT via TRPA1 activation; a model

of human enterochromaffin cells
Hitoshi Doihara Æ Katsura Nozawa Æ Ryosuke Kojima Æ
Eri Kawabata-Shoda Æ Toshihide Yokoyama Æ
Hiroyuki Ito

Received: 28 January 2009 / Accepted: 21 May 2009 / Published online: 9 June 2009
Ó Springer Science+Business Media, LLC. 2009

Abstract Recently, we discovered that transient receptor Serotonin (5-hydroxytryptamine: 5-HT) is a biological
potential ankyrin1 channel (TRPA1) is highly expressed in amine that has a various physiological functions [1]. In the
human and rat enterochromaffin (EC) cells, and those gastrointestinal tract, 5-HT is biosynthesized and stored in
TRPA1 agonists such as allyl isothiocyanates (AITC) and enterochromaffin (EC) cells and released by various lumi-
cinnamaldehyde (CA) enhance the release of serotonin nal stimuli, including glucose, lipid, or distension pressure
(5-hydroxytryptamine; 5-HT) from EC cells in vitro. In this [2, 3]. The dysfunction of EC cells has been implicated in a
study, QGP-1 cells, a human pancreatic endocrine cell line, number of diseases, including carcinoid disease and irrita-
were found to highly express TRPA1 and EC cell marker ble bowel syndrome [1, 4–7]. Identifying the signaling
genes, such as tryptophan hydroxylase 1 (TPH1), chro- mechanisms which activate EC cells is necessary in
mogranin A (CgA), synaptophysin, ATP-dependent vesic- understanding the symptoms of several diseases as well as
ular monoamine transporter 1 (VMAT1), metabotropic its normal physiology. Human EC cells are difficult to study
glutamate receptor 4 (mGluR4), b1-adrenergic receptor as established under the last 100 years [8, 9], however,
(ADB1), muscarinic 4 acetylcholine receptor (ACM4), recent studies revealed that some of endocrine cell lines
substance P, serotonin transporter (SERT), and guanylin. such as BON, KRJ-I, CNDT2.5, HC45, and HC49 are close
Furthermore, the TRPA1 agonists AITC, CA, and acrolein to the normal EC cells to mimic the EC features [10–13].
concentration dependently evoked an increase in intracel- The transient receptor potential ankyrin1 channel
lular Ca2? influx and the release of 5-HT in QGP-1 cells. (TRPA1) was shown to be activated by cold stimuli or
The effects of these TRPA1 agonists were inhibited by pungent irritants such as allyl isothiocyanates (AITC),
ruthenium red, a TRPA1 antagonist, and TRPA1-specific cinnamaldehyde (CA), and acrolein [14–19] and inhibited
siRNA. These results indicate that the Ca2? influx increase by ruthenium red, a non-competitive TRPA1/TRPV1
and 5-HT release induced by AITC, CA and acrolein in antagonist [20]. Although the physiological function of
QGP-1 cells were mediated by TRPA1, and that the QGP-1 TRPA1 in somatic sensory neurons has been well studied,
cell line could be a new model for the investigation of little is known about the physiological function of TRPA1
TRPA1 function in the human EC cell. in the gastrointestinal tract. Recently, we found that TRPA1
is highly expressed in the EC cells of humans and rats [21].
Keywords QGP-1 cells
EC cells
TRPA1
5-HT Moreover, we showed that TRPA1 agonists concentration-
dependently evoked an increase in intracellular Ca2? influx
as well as the release of 5-HT from purified rat EC cells and
Hitoshi Doihara and Katsura Nozawa have contributed equally to this
RIN14B cells, a rat pancreatic endocrine cell line [21].
work. However, there have been no reports on investigating
TRPA1 function in human EC cells.
H. Doihara (&)
K. Nozawa
R. Kojima
The aim of this study is to obtain human EC cell-like
E. Kawabata-Shoda
T. Yokoyama
H. Ito
lines that have a function of TRPA1. Thirteen human cell
Pharmacology Laboratories, Drug Discovery Research, Astellas
Pharma Inc, 21 Miyukigaoka, Tsukuba, Ibaraki 305-8585, Japan lines derived from tissues that highly express TRPA1 [22]
e-mail: hitoshi.doihara@jp.astellas.com and carcinoid cell lines [10, 23, 24] were screened.

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240 Mol Cell Biochem (2009) 331:239–245

Materials and methods NM_003053), 50 -CACCACCAAGGTCTTCTTAGTTGG-30

and 50 -GTAGAGGAAGGCAAACACGGC-30 for human
Cell culture synaptophysin (GenBank accession No. NM_003179.2),
50 -CCTGGGACTAGGGATGAGCA-30 and 50 -TTGGTC
A375 cells (ATCC; CRL-1619) were cultured in Dul- TTGGTGAGCAGGG-30 for human mGluR4 (GenBank
becco’s Modified Eagle’s Medium (Invitrogen Japan, accession No. NM_000841), 50 -AGTACGGCTCCTTC
Tokyo). AGS and PC-3 cells (ATCC; CRL-1739 and CRL- TTCTGCG-30 and 50 -AGGGTCTCGATGCTGGC-30 for
1435, respectively) were cultured in F-12K Medium human ADB1R (GenBank accession No. NM_000684),
(Invitrogen Japan). Caco-2 cells and Calu 6 cells (ATCC; 50 -A AGACAGGCAATGAGTGTGTGACA-30 and 50 -CG
HTB-37 and HTB-56, respectively) were cultured in CAGGGCGCATGC-30 for human ACM4 (GenBank
Eagle’s Minimum Essential Medium (Invitrogen Japan). accession No. NM_000741), 50 -TGCCGGAGCCCTTTG
COR-L23 (ECACC; 92031919), NCI-H358, NCI-N87, SK- AG-30 and 50 -AATCCAAAGAACTGCTGAGGCTT-30 for
MEL-6 (NCI), COLO320DM, QGP-1, and MKN28 cells human substance P (GenBank accession No. NM_001058),
(HSRRB; JCRB0225, JCRB0183, and JCRB0253, respec- 50 -ATGGCCTGGGCGCTATACTAC-30 and 50 -GGAATG
tively) were cultured in RPMI 1640 (Invitrogen Japan). GAGGGTCCAGGTG-30 for human SERT (GenBank
SK-BR-3 cells (ATCC; HTB-30) were cultured in accession No. NM_001045), and 50 -CCCATCCCTGGTGA
McCoy’s 5a Medium (Invitrogen Japan). All culture ACCTG-30 and 50 -GAGAGGCTTGAGTTCTTCTGGAA
medium was supplemented with 10% fetal bovine serum AG-30 for human guanylin (GenBank accession No.
(FBS) and antibiotics (100 U/ml penicillin and 100 lg/ml NM_033553.2).
streptomycin) except for that used to culture Caco-2 cells,
which contained 20% FBS and antibiotics. All cell lines Ca2? influx assay
were cultured at 37°C in 5% CO2 atmosphere.
To analyze the function of TRPA1 in QGP-1 cells, the
RT-PCR analysis effects of TRPA1 agonists (AITC, CA, and acrolein) on
Ca2? influx were studied. QGP-1 cells were plated at
The total RNA from each cell line mentioned above was 2 9 104 cells per well in black 96-well clear-bottom plates
isolated using an RNeasy Kit (Qiagen-Japan, Tokyo) (Biocoat, Becton Dickinson, CA, USA) and incubated for
according to the manufacturer’s instructions. The total RNA 48 h at 37°C and 5% CO2 to achieve 80–100% confluence.
was reverse-transcribed into cDNA using the Superscript-II The cell culture medium was removed, and the cells of
enzyme (Invitrogen Japan) according to the manufacturer’s each well were washed once with 100 ll of assay buffer
instructions. The GenBank coding sequences for chro- (140 mM NaCl, 0.15 mM CaCl2, 3.3 mM KH2PO4,
mogranin A (CgA), synaptophysin, ATP-dependent vesic- 0.8 mM K2HPO4, 1.2 mM MgCl2, 10 mM glucose, and
ular monoamine transporter 1 (VMAT1), metabotropic 20 mM HEPES [pH 7.4]). The cells were incubated in
glutamate receptor 4 (mGluR4), b1-adrenergic receptor assay buffer containing 2 lM Fluo-4AM for 120 min at
(ADB1), muscarinic 4 acetylcholine receptor (ACM4), 37°C in 5% CO2 before each experiment to allow for the
substance P, serotonin transporter (SERT), and guanylin equilibration of Fluo-4AM across the cell membranes.
were used to generate specific oligonucleotide primers for Fluorescence was recorded using the fluorometric imaging
the selected gene sequences shown below. Quantitive PCR plate reader (FLIPR) instrument at an excitation wave-
was performed using an ABI PRISM 7900HT sequence length of 488 nm and an emission wavelength of 540–
detection system (Applied Biosystems, CA, USA) with 590 nm. Assays were carried out at room temperature. The
Power SYBR Green PCR master mix. The primer sequences EC50 values were calculated as maximum responses
were 50 -GAGAGTCCTTCCTAGAACCATATCTGA-30 and induced by each TRPA1 agonist at highest concentration
50 -CATGAGGACAATTGGGACAAATATT-30 for human (300 lM, respectively).
TRPA1 (GenBank accession No. NM_007332), 50 -CCC
TTTGATCCCAAGATTAC-30 and 50 -CATTCATGGCAC 5-HT release experiments
TGGTTATG-30 for human TPH1 (GenBank accession No.
NM_004179), 50 -GGAAGAGCCATCATCCAAG-30 and To investigate the TRPA1-mediated 5-HT release, we
50 -TTCACCACTTTTCTCTGCCT-30 for human CgA examined the effects of TRPA1 agonists (AITC, CA, and
(GenBank accession No. NM_001275), 50 -TGGTGCCAT acrolein) and TRPA1 antagonist (ruthenium red) in QGP-1
TGTAAAGGCC-30 and 50 -GCAGGTAGTAGCAGAGTG cells. QGP-1 cells were seeded at 2 9 105 cells/0.5 ml/
GAGCAT-30 for human VMAT1 (GenBank accession No. well in 24-well plates and cultured for 72 h. The medium

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Mol Cell Biochem (2009) 331:239–245 241

was removed before washing the cells with HBSS con- Results
taining 0.1% BSA and 2 lM fluoxetine (Tocris, MO,
USA). The HBSS was again removed and replaced with TRPA1 and TPH1 expression in various human cell
0.25 ml HBSS containing different stimulants at the indi- lines
cated concentrations for 20 min at 37°C. The assay buffer
was collected and centrifuged for 5 min to remove any Analyses performed to determine the expression of TRPA1
detached cells. The supernatants were collected and stored mRNA in the 13 cell lines using RT-PCR showed that
at -80°C until 5-HT measurement using an enzyme expression was most abundant in QGP-1 cells (Fig. 1a).
immunoassay (EIA) kit (Beckman Coulter, CA, USA). The Expression of TRPA1 mRNA was moderate in A375 cells,
EC50 values were calculated as maximum responses Caco-2 cells, NCI-H358 cells, and SK-MEL-6 cells. High
induced by each TRPA1 agonist at highest concentration expression of TPH1 mRNA was found only in QGP-1 cells
(300 lM, respectively). among the 13 cell lines tested (Fig. 1b). Other EC cell
marker genes, CgA, VMAT1, synaptophysin, mGluR4,
siRNA experiments ADB1, ACM4, substance P, SERT, and guanylin, were
also expressed in QGP-1 cells (Table 1). As like to previ-
A siRNA from the human TRPA1 sequence was designed ous studies [25, 26], colonic epithelial Caco-2 cells
TM
using the siDirect designing program (RNAi Co., Ltd., expressed only SERT and guanylin, but not other EC
Tokyo, Japan). The sequence was 50 -rGUrArArCUr- marker genes.
AUUrArArGrCUrArArArAUrGrC-30 for the sense primer
and 50 -rAUUUUrArGrCUUrArAUrArGUUrArCrAU-30 Ca2? influx assay
for the antisense primer. These 21-nucleotide siRNA
sequences were obtained from Sigma Genosys (Tokyo, To analyze the function of TRPA1 in QGP-1 cells, we
Japan). Negative control siRNA (Ambion, Applied Bio- examined the effects of TRPA1 agonists using the FLIPR
systems, Tokyo, Japan) was used as a control. The siRNA assay. AITC, CA, and acrolein concentration-dependently
was transfected using Lipofectamine RNAimax (Invitrogen increased intracellular Ca2? influx (Fig. 2a). The increased
Japan) according to the manufacturer’s instructions. The intracellular Ca2? influx induced by AITC and acrolein
cells were used for 5-HT release and gene expression reached the maximum responses at highest doses
experiments 2 days after transfection. (300 lM), but did not reach the maximum response in CA.
The EC50 values ± SE of AITC, CA, and acrolein were
Drugs 36.1 ± 9.4, 72.5 ± 17.1, and 17.7 ± 6.5 lM, respectively.
To investigate whether the effects were mediated through
AITC and CA were purchased from Wako Pure Chemical TRPA1, we examined the effects of TRPA1 agonists on
Co. (Osaka, Japan), and acrolein was purchased from ruthenium red treated cells. In this study, taking into
Sigma-Aldrich Japan. All drugs were dissolved in dimethyl account of signal/noise ratio, we used 2–6 folds higher
sulphoxide (DMSO). concentrations of TRPA1 agonists than that of EC50 values.

300
Ratio (% of G3PDH))

0.400
a b
Ratio (% of G3PDH )

0.350 250
0.300
TRPA1 TPH1
200
0.250
0.200 150

0.150 100
0.100
50
0.050
0 000
0.000 0
COLO320DM

N87
NCI-N87
7

SK-MEL-6
H358
8
Caco2
2

NCI-H358
6
Calu6

COR-L23
L23
MKN28
8
AGS
S
5
A375

PC -3
QGP-1
1
3
BR-3
MKN28
COLO320DM
M

NCI-H358
Calu6
Caco2

NCI-N87
N87
AGS
A375

COR L23
COR-L23

SK-MEL-6
PC 3
PC-3
QGP 1
QGP-1
SK BR 3
SK-BR-3

QG
P

SK- B
NC
CO

NCI
NC
N

Fig. 1 Expression of TRPA1 (a) and TPH1 (b) mRNA in cell lines. melanoma, AGS cells: stomach, Caco-2 cells: large intestine, Calu 6:
mRNA expression is presented as the ratio of TRPA1 or TPH1 to lung, COLO320DM: large intestine, COR-L23: lung, MKN28:
glyceraldehyde-3-phosphate dehydrogenase (G3PDH) mRNA stomach, NCI-H358: lung, NCI-N87: stomach, PC-3: prostate gland,
obtained using RT-PCR, and is expressed as the mean ± SD. The QGP-1: pancreas, SK-BR-3: mammary gland, and SK-MEL-6:
cell lines used were obtained from the following sources: A375 cells: melanoma

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242 Mol Cell Biochem (2009) 331:239–245

Table 1 Expression of EC cells marker genes in QGP-1 and Caco-2 5-HT release studies were 122 ± 6.8, 144 ± 20.5, and
cells 81.8 ± 20 lM, respectively. In 5-HT release experiments,
QGP-1 Caco-2 the effects of TRPA1 agonists were not reached to the
maximum responses compared to Ca2? influx studies. We
CgA 279 ± 35.6 N.D.
could not examine further higher concentration of TRPA1
VMAT1 9.45 ± 2.1 N.D. agonists because of their low solubility. The release of
Synaptophysin 92.9 ± 34 N.D. 5-HT induced by 300 lM of each agonist was completely
mGluR4 6.98 ± 0.59 N.D. inhibited by 30 lM of ruthenium red (Fig. 4a–c). We also
ADB1 1.88 ± 0.20 N.D. found that the 5-HT secretion induced by 300 lM TRPA1
ACM4 10.9 ± 2.3 N.D. agonists in QGP-1 cells was inhibited by siRNA transfec-
Substance P 2.10 ± 0.38 N.D. tion (Fig. 4d). We also studied LDH release induced by
SERT 0.777 ± 0.15 0.678 ± 0.14 TRPA1 agonists, but no difference was observed compared
Guanylin 0.273 ± 0.022 0.464 ± 0.25 with DMSO treated cells (data not shown).
mRNA expressions were presented as the % ratio to G3PDH on three
experiments as the mean ± SD
N.D. Not detected Discussion

Ruththenium red (30 lM) strongly blocked the increases in In the previous study, we showed that TRPA1 is highly
the Ca2? influx induced by AITC (100 lM), CA (300 lM), expressed in the EC cells of human and rat intestine, and
and acrolein (100 lM) (Fig. 2b). The effect of TRPA1- that the stimulation of TRPA1 releases 5-HT in purified EC
specific siRNA was also evaluated for further confirmation cells from rat small intestine [21]. Here, we show that
that the increases in the Ca2? influx were mediated by TRPA1 and EC cell marker genes are expressed, and
TRPA1. RT-PCR showed that the transfection of siRNA TRPA1 can function in QGP-1 cells, a human-derived cell
reduced TRPA1 mRNA expression by approximately 50% line.
in QGP-1 cells (Fig. 3a). TRPA1 agonist-induced Ca2? To obtain a cell line with an EC cell-like nature, we
influx was also inhibited by siRNA transfection (Fig. 3b). screened various cell lines derived from gastrointestinal
and endocrine tissues as well as those highly expressing
5-HT release experiments TRPA1 [22]. TPH1 is regarded as a good EC cell marker
gene because TPH1 is specifically expressed in these cells
To examine the functional significance of TRPA1 in QGP- [3]. Among the 13 cell lines, only QGP-1 highly expressed
1 cells, we attempted to determine whether TRPA1 ago- both TPH1 and TRPA1 mRNAs. QGP-1 cells were also
nists induced the release of 5-HT in QGP-1 cells. AITC, found to express CgA, VMAT1, synaptophysin, mGluR4,
CA, and acrolein were found to concentration-dependently ADB1, ACM4, substance P, SERT, and guanylin, which
enhance the release of 5-HT in QGP-1 cells (Fig. 4a–c). were known to be expressed in EC cells [23]. These results
The EC50 values ± SD of AITC, CA, and acrolein on the indicate that QGP-1 cells can have an EC cell-like nature.

a b
units)

units)

2000 none
2000
ve fluorescent u

AITC
ve fluorescent u

Ruthenium Red
1500 CA
Acrolein 1500

1000
1000
[Ca2+]i (relativ

[Ca2+]i (relativ

500
500

0
-6 -5 -4 -3 0
AITC CA Acrolein
Concentration
C i (l(log M)

Fig. 2 TRPA1 agonist increases Ca2? influx in QGP-1 cells. a AITC, change in fluorescence represents the inhibitory ratio of ruthenium red
CA, and acrolein evoked concentration dependent increases in Ca2? to AITC-, CA-, or acrolein-induced Ca2? influx. Each point
influx. b 30 lM Ruthenium red inhibited Ca2? influx induced by represents the mean value of duplicate FLIPR assays. Data are
AITC (100 lM), CA (300 lM), and acrolein (100 lM) (filled expressed as the mean ± SE of five experiments
columns) compared to that induced by DMSO (open columns). The

123
Mol Cell Biochem (2009) 331:239–245 243

0.12

unitts)
a 900
b

H)
0.1 800 nega .cont.
cont.

ratio (% of G3PDH

a2+]i (relative fluorescent

700 siRNA
0.08
600

0.06 500

f
400
0.04 300
200
0.02
100

[Ca
0 0
nega.. cont. siRNA DMSO AITC CA Acrolein

Fig. 3 a Inhibitory effect of mRNA expression by TRPA1-specific acrolein (100 lM) in QGP-1 cells. The change in fluorescence
siRNA. mRNA expression is presented as the ratio of TRPA1 mRNA represents the inhibitory ratio of siRNA to DMSO-treated cells. Each
to G3PDH mRNA in triplicate assays using real time RT-PCR. point represents the mean values of duplicate FLIPR assays. Data are
b siRNA (filled columns), but not control siRNA (open columns) expressed as the mean ± SE of three experiments
inhibited Ca2? influx induced by AITC (100 lM), CA (300 lM), and

Fig. 4 TRPA1 agonists 60

5- HT (nM/10 cells/well)
b
(nM/10cccells/well)

stimulate 5-HT release in QGP- 70 a 50

60
1 cells. a AITC, b CA, and c
50 40
acrolein enhanced the release of

5
5

5-HT in QGP-1 cells 40

H (nM/10

30

concentration-dependently. 30
20
Ruthenium red (RR, 30 lM) 20
10
HT

inhibited the release of 5-HT 10

5-H

induced by 300 lM AITC, CA, 0 0

5

and acrolein in QGP-1 cells. DMSO 30 100 300 300+RR DMSO 30 100 300 300+RR
d siRNA inhibited the release of AITC (µ M) CA ( µM)
5-HT induced by 300 lM 180
0cells/well)
0 cells/well)

AITC, CA, and acrolein in

160
c 0cells/well)
d
140 160 Nega. cont.
QGP-1 cells (filled columns) 140
120 siRNA
compared to negative control 120
100
5

siRNA (open columns). The

5
5 - HT (nM/10

5-- HT (nM/10

100
80
change in 5-HT secretion is 80
60
presented as the ratio of the 60
40
mean ± SE to the basal level 40
20
(DMSO) of three experiments 20
0 0
DMSO 30 100 300 300+RR DMSO AITC CA Acrolein
Acrolein ( µM)

The EC cells are known to function as a luminal sensing similar to previous studies [21, 32]. The rank order potency
receptor in the gut [27], and release the 5-HT induced by of TRPA1 agonists in 5-HT release experiments was sim-
various stimulus [28, 29]. For example, amine, tastants, or ilar to that in Ca2? influx studies and the ratio of the EC50
olfactant induce the increase of cAMP, intracellular Ca2? values on 5-HT release experiments to Ca2? influx studies
influx, or PKC activation through GPCRs and stimulates were 3.4, 2.0, and 4.6 (AITC, CA, and acrolein, respec-
the 5-HT release in EC cells [30]. Increased intracellular tively). These results indicated that the agonists-induced
Ca2? influx is known as a one of the major pathway for the Ca2? influx and 5-HT release are correlated.
release of 5-HT from EC cells [31]. In this study, we found The gene expression profiling and proteomic analyses of
that QGP-1 cells concentration-dependently increase the QGP-1 cells revealed that QGP-1cells have a similar
intracellular Ca2? influx and 5-HT release in response to nature to that of BON cells [33, 34]. The BON cells,
TRPA1 agonists, and that this increase was inhibited by derived from human pancreatic cells, are known to have a
TRPA1 antagonist and TRPA1-specific siRNA. These nature of EC cells [35]. The QGP-1 cells could be also
results indicated that QGP-1 cells release the 5-HT through derived from human pancreatic islet delta cells, but not EC
the TRPA1 channels. cells. Therefore, it is not surprising that QGP-1 cells, a
The rank order potency of TRPA1 agonists in Ca2? pancreatic islet delta cell line, have an EC cell-like nature.
influx studies was acrolein [ AITC [ CA and the effects We did not test the BON, KRJ-I, CNDT2.5, HC45, and
of AITC and acrolein were reached to the maximum HC49 cells, which are known to secrete 5-HT in response
responses, whereas it was partial in CA. These results were to mechanical or chemical stimuli [10, 12, 13, 23], because

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244 Mol Cell Biochem (2009) 331:239–245

we could not obtain them. Further studies are needed to 13. Stilling GA et al (2007) Characterization of the functional and
elucidate the function of TRPA1 in EC cell using various growth properties of cell lines established from ileal and rectal
carcinoid tumors. Endocr Pathol 18:223–232. doi:10.1007/
EC-like cell lines. s12022-007-9001-3
To summarize, we demonstrated for the first time that 14. Obata K et al (2005) TRPA1 induced in sensory neurons con-
QGP-1, a human cell line, expresses TRPA1 and EC cell tributes to cold hyperalgesia after inflammation and nerve injury.
marker genes and releases 5-HT via TRPA1 activation. Our J Clin Invest 115:2393–2401. doi:10.1172/JCI25437
15. Bandell M, Story GM, Hwang SW, Viswanath V, Eid SR, Petrus
results indicate that the QGP-1 cell line could be a useful MJ, Earley TJ, Patapoutian A (2004) Noxious cold ion channel
model for the study of the physiological and pharmaco- TRPA1 is activated by pungent compounds and bradykinin.
logical nature of TRPA1 in EC cells. Neuron 41:849–857. doi:10.1016/S0896-6273(04)00150-3
16. Tominaga M, Caterina MJ (2004) Thermosensation and pain.
Acknowledgments We would like to thank T. Koizumi, J Neurobiol 61:3–12. doi:10.1002/neu.20079
M. Yamano, Y. Takinami, R. Takezawa, Y. Takemoto, T. Goto, 17. Zhang XF, Chen J, Faltynek CR, Moreland RB, Neelands TR
H. Tanaka, H. Okada, H. Kamada, and M. Okada for their excellent (2008) Transient receptor potential A1 mediates an osmotically
technical assistance and advice. activated ion channel. Eur J Neurosci 27:605–611. doi:10.1111/
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