Group Title: Molecular Pain 2008, 4:22
Title: TRPM8 mechanism of autonomic nerve response to cold in respiratory airway
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Title: TRPM8 mechanism of autonomic nerve response to cold in respiratory airway
Series Title: Molecular Pain 2008, 4:22
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Creator: Xing H
Ling JX
Chen M
Johnson RD
Tominaga M
Wang CY
Gu J
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Molecular Pain


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BioMed Central


Research

TRPM8 mechanism of autonomic nerve response to cold in
respiratory airway
Hong Xing', Jennifer X Ling', Meng Chen', Richard D Johnson2,
Makoto Tominaga3, Cong-Yi Wang4 and Jianguo Gu*l


Address: 'Department of Oral & Maxillofacial Surgery and Diagnostic Sciences, McKnight Brain Institute and College of Dentistry, University of
Florida, Gainesville, Florida 32610, USA, 2Department of Physiological Sciences, McKnight Brain Institute and College of Veterinary Medicine,
University of Florida, Gainesville, Florida 32610, USA, 3Section of Cell Signaling, Okazaki Institute for Integrative Bioscience, National Institutes
of Natural Sciences, Higashiyama 5-1, Myodaiji, Okazaki, Aichi 444-8787, Japan and 4Center for Biotechnology and Genomic Medicine, Medical
College of Georgia, 1120 15th Street, CA4098, Augusta, GA 30912, USA
Email: Hong Xing hongxing@ufl.edu; Jennifer X Ling jlingl964@gmail.com; Meng Chen meng.chen@duke.edu;
Richard D Johnson johnson@mbi.ufl.edu; Makoto Tominaga tominaga@nips.ac.jp; Cong-Yi Wang cwang@mcg.edu;
Jianguo Gu* jgu@dental.ufl.edu
* Corresponding author


Published: 5 June 2008
Molecular Pain 2008, 4:22 doi: 10.1 186/1744-8069-4-22


Received: 12 May 2008
Accepted: 5 June 2008


This article is available from: http://www.molecularpain.com/content/4/1/22
2008 Xing et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.



Abstract
Breathing cold air without proper temperature exchange can induce strong respiratory autonomic
responses including cough, airway constriction and mucosal secretion, and can exacerbate existing
asthma conditions and even directly trigger an asthma attack. Vagal afferent fiber is thought to be
involved in the cold-induced respiratory responses through autonomic nerve reflex. However,
molecular mechanisms by which vagal afferent fibers are excited by cold remain unknown. Using
retrograde labeling, immunostaining, calcium imaging, and electrophysiological recordings, here we
show that a subpopulation of airway vagal afferent nerves express TRPM8 receptors and that
activation of TRPM8 receptors by cold excites these airway autonomic nerves. Thus activation of
TRPM8 receptors may provoke autonomic nerve reflex to increase airway resistance. This putative
autonomic response may be associated with cold-induced exacerbation of asthma and other
pulmonary disorders, making TRPM8 receptors a possible target for prevention of cold-associated
respiratory disorders.


Background
Normally, a breath of cold air is warmed up to near body
temperature through heat exchange in the upper airway,
mainly the nose, before the air enters the bronchopulmo-
nary system. Temperature exchange, however, is compro-
mised under conditions including flu, allergy, and other
respiratory diseases. Exercise in cold weather can also
result in the rapid inhalation of cold air into the trachea
and bronchi, and the air temperature there can drop as
low as about 200C due to an insufficient temperature


exchange [1,2]. Respiratory responses to cold air are
reflexive, including cough, airway constriction and
mucosal secretion. These responses may have some pro-
tective roles for bronchopulmonary tissues when exposed
to potentially hazardous cold environment. However, the
responses can be harmful in people having certain respi-
ratory diseases. For example, cold is a major environmen-
tal factor that exacerbates existing asthma conditions and
directly triggers asthma [3]. Inhalation of cold air is a
direct cause of airway constriction to trigger exercise


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asthma in athletes performing winter sports [4,5]. Clini-
cally, the "cold air challenge test", a test of bronchopul-
monary reactivity and airway resistance, has been used for
asthma diagnosis for over 20 years [6,7] because many
asthma patients show bronchopulmonary hyper-reactiv-
ity and increased airway resistance to cold air challenge.

Respiratory responses to cold may be through a neural
reflex mechanism [8,9]. The main afferent nerves that
innervate the bronchopulmonary system are derived from
the vagus nerve. Factors that stimulate these nerves trigger
an autonomic reflex to cause airway constriction and
mucosal secretion [ 10,11 If respiratory responses to cold
are indeed mediated by bronchopulmonary vagal affer-
ents, what is the molecular mechanism by which cold ini-
tiates the autonomic responses?

Recently, studies have identified a molecular mechanism
for sensing cold by the somatic sensory nerve endings of
the skin [12-19]. It has been demonstrated that cool tem-
perature opens a new type of ion channels (receptors) on
the membranes of a subpopulation of somatic sensory
nerves, which causes sensory nerve excitation [20,21]. The
ion channels were cloned from somatic sensory neurons
of rats [12], mice, and humans [13], and were named tran-
sient receptor potential channel M8 (TRPM8) [12,13];
since it belongs to the transient receptor potential (TRP)
super-family. When expressed on heterologous cell sys-
tems, cooling temperatures below 24-28 C start to evoke
depolarizing currents. TRPM8-mediated currents increase
with decreasing temperatures and reach maximum cur-
rents near 10 C. TRPM8 can also be activated by menthol,
the active ingredient of peppermint, and by other cooling
compounds [12]. Electrophysiological studies have indi-
cated that TRPM8 is highly permeable to Ca2+ [12,13,21],
and activation of TRPM8 results in a large increase of
intracellular Ca2+ levels [12,13,20-22] through both Ca2+
entry from extracellular sites and Ca2+ release from intrac-
ellular Ca2+ stores [22].

Vagal afferent nerves and somatic sensory nerves are two
different nervous systems. Functionally, somatic sensory
afferent fibers sense stimuli to produce conscious sensa-
tions. On the other hand, vagal afferent nerves belong to
autonomic nervous system and are not involved in any
conscious sensation. Stimulation of vagal afferent nerves
only produces autonomic reflex. However, several sensory
molecules that are found in somatic sensory neurons are
also found in vagal afferent nerves. For example, VR1
receptor (vallinoid receptor-1) is found in nociceptive
somatic sensory fibers and serves as a sensor for noxious
heat [23]. This receptor is also expressed on some vagal
afferent nerves and activation of this receptor by capsai-
cin, an active ingredient of hot chili pepper can produce
cough reflex and neurogenic inflammation in the bron-


chopulmonary system [24]. In the present study, we have
tested the hypothesis that cold excites bronchopulmonary
vagal afferent nerves through the activation of TRPM8
receptors.

Methods
Retrograde labeling and preparation of vagal ganglion
neurons
Adult Sprague-Dawley rats (200 to 300 g, n = 48) were
used according to the Institutional Animal Care and Use
Committee guideline of the University of Florida. Retro-
grade labeling of the vagal ganglion (VG) neurons that
innervate low airway tissues was performed based on a
method described previously [25]. In brief, rats were con-
tinuously anesthetized with isoflurane using an anaesthe-
tizing machine. A small amount of 1,1'-dioctadecyl-
3,3,3',3'- tetramethylindocarbocyanine perchlorate (Dil,
20 gl, 0.25% in DMSO) was gradually instilled into the
caudal region of rat trachea using a 50 gl Hamilton
syringe. The animals were positioned supine during dye
instillation and kept the same position for 30 min before
recovery from anesthesia. Seven days after dye instillation,
both left and right vagal ganglions nodosee ganglions)
were harvested from the animals.

The acutely dissociated neurons were prepared in a man-
ner described in our previous studies. In brief, the gangli-
ons were incubated for 45 min at 370C in S-MEM
medium (Gibco, Grand Island, NY) with 0.2% colla-
genase and 1% dispase (Sigma) and then triturated to dis-
sociate neurons. The VG neurons were then plated on
glass coverslips previously coated with poly-D-lysine
(PDL), maintained in normal bath solution (see below) at
an ambient temperature of 26 C. Cells were used within
4 hours after plating. In a different set of experiments,
acutely dissociated dorsal root ganglion (DRG) neurons
were used and DRG cell preparation was performed in the
same was as VG neurons [26]. Dil-labeled VG neurons
were identified under the fluorescence microscope (exci-
tation, 550 nm; emission, 650 nm).

Ca2+ Imaging
VG neurons were incubated with 2 gM Fluo3-AM for 30
min at 370C to load Ca2+ indicator. Fluo3-AM was pre-
pared in 20% pluronic acid (Molecular Probes, Eugene,
OR). The cells were then perfused with normal bath solu-
tion flowing at 1 ml/min in a 0.5 ml chamber. The normal
bath solution contained (in mM): 150 NaC1, 5 KC1, 2
MgCl2, 2 CaCl2, 10 glucose, 10 HEPES, pH 7.2, osmolarity
adjusted to 320 mOsm with sucrose. Fluo-3 fluorescence
in the cells was detected using a peltier-cooled charge-cou-
pled device (CCD) camera (PentaMAX-III System, Roper
Scientific, Trenton, NJ) under a fluorescence microscope
(1OX objective). Excitation was at 450 nm and emission at
550 nm, achieved by a fluorescence filter sets. Images were


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taken at one frame per second, and digitized using
MetaFluor software (Universal Imaging Corporation).
Unless otherwise indicated, all experiments were carried
out at an ambient temperature of 26 C. Effects of cold on
intracellular Ca2+ levels were tested by application of a
cold bath solution, which yield temperature drop from
26 C to 19 C within 1 min in the recording chamber.
Effects of menthol were tested by application of 100 iM
menthol for 20 s. Cold and menthol solutions were deliv-
ered through a glass tube; the tube had internal diameter
of 0.5 mm and was positioned 0.5 mm away from the
recorded cells.

Patch-clamp recordings
Whole-cell recordings were performed on VG neurons
and DRG neurons. For most voltage-clamp recordings,
electrode internal solution contained (in mM): 110
Cs2SO4, 2 MgCl2, 0.5 CaCl2, 5 TEA-C1, 5 EGTA, 5 HEPES,
pH 7.3. When both voltage-clamp and current-clamp
recordings were applied to the same cells, electrode intra-
cellular solution contained (in mM) 135 K-gluconate, 2
MgCl2, 0.5 CaCl2, 5 EGTA, 10 HEPES, 2 Na2ATP, 0.5
NaGTP, pH 7.3. Recording electrode resistance was ~5
MO. Unless otherwise indicated, voltage-clamp record-
ings were performed on cells held at -70 mV. Signals were
amplified with MultiClamp 700A (Axon Instruments,
Union City, CA), filtered at 2 kHz and sampled at 5 kHz.
Cold bath solution and menthol were applied to neurons
in the same manner as that in Ca2+ -imaging experiments.
A voltage ramp from -90 mV to 70 mV was used to obtain
I-V relationship of menthol-evoked currents. The voltage
ramp was applied during the steady states of menthol-
evoked currents. I-V relationship was constructed after a
subtraction of a control ramp test. In the voltage-ramp
test, lidocaine (1 mM) was used to block both TIX-sensi-
tive and TIX-resistant Na+ channels and Ca2+ channels
[27].

Immunostaining
Adult Sprague-Dawley rats (200-250 g, n = 6) were per-
fusion fixed and the inferior parts of vagal ganglions
nodosee ganglions) were dissected out. The ganglions
were cut with a cryostat into transverse sections at thick-
ness of 16 gim. Sections were incubated in a rabbit anti-
TRPM8 antibody (1:300) overnight at 40C and then 3 hrs
with a secondary antibody (goat-anti-rabbit, conjugated
with Alexa 594). The TRPM8 antibody was generated in
rabbits using a sequence of N-terminus, EGARLSMRSR-
RNG, of rat TRPM8 receptors. Double immunostaining of
TRPM8 and P2X3 receptors were performed by first incu-
bating ganglion sections with a guinea pig anti-P2X3 anti-
body (1:3000) overnight at 40C, followed by 3 hrs of
incubation with a secondary antibody (goat-anti-guinea,
conjugated with Alexa 488). Immunostaining for TRPM8
receptors were subsequently performed as described


above. Images were acquired using a fluorescence micro-
scope. To determine immunoreactive positive neurons in
each sample, a threshold is set at 2.5 times of averaged
cytoplasmic density level. All neurons sectioned through
their nucleus for which mean optical density exceeded the
threshold were counted as positive.

Data analysis
For Ca2+-imaging experiments, relative fluorescence inten-
sity AF/F0 was used and a AF/F0 value of > 0.1 was consid-
ered to have response. All the data were represented as
mean + SEM. Paired-t tests were used for statistical com-
parison, and significance was considered at the p < 0.05.

Results
Seven days after the instillation of Dil into the respiratory
airway (Figure la), the inferior parts of vagal ganglions
(VG) were harvested from these animals and then dissoci-
ated for functional studies. Of 1638 acutely dissociated
VG neurons, 170 of them showed a high intensity of Dil
labeling (Figure Ib). We studied effects of the TRPM8
receptor ligand menthol [12,13] on the airway VG neu-
rons using a Ca2+ imaging technique. As shown in Figure
Ic-e, application of menthol (100 iM, 20 s) resulted in
increases of intracellular Ca2+ in 7% of VG neurons (116/
1638 cells), including both Dil-labeled and non-labeled
neurons. Of the 170 Dil-labeled cells, 28 of them (17%)
showed response to menthol (Figure Ic).

We determined whether cold stimulation might produce
a response similar to menthol on VG neurons. Since there
was little activation of TRPM8 receptors at temperatures
above 240C when cells were near resting membrane
potential [28], a basal temperature of 26 C was used in
our experiments. As shown in Figure Ic, f &lg, a 7C of
temperature drop, from 260C to 19 C, resulted in
increases of intracellular Ca2+ in 6.5% of VG neurons
(106/1638 cells), including both Dil-labeled and non-
labeled neurons. All these cold-responsive cells were men-
thol-responsive neurons. Among the 170 Dil-labeled
cells, 27 of them (16%) were both menthol- and cold-
responsive neurons. These results indicate that a subpop-
ulation of VG neurons innervating respiratory airways
responds to both cold and the TRPM8 receptor agonist
menthol.

We next determined whether TRPM8 proteins were
expressed on VG neurons by immunostaining sections of
ganglions using a TRPM8 antibody. TRPM8 immunoreac-
tivity was found to be present in 7.2% ofVG neurons (75/
1041 cells, Figure 2a). The cell sizes were 24 + 0.7 inm,
ranged from 12 to 36 gim. Many vagal afferent nerves
innervating respiratory airways express P2X3 receptors
and these nerves were suggested to be involved in vagal
nerve reflex [29]. We determined whether TRPM8 recep-


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a b


:xr.
Dii


5

S -, *,- -
./ J


Control


Menthol


Cold Recovery


4095


Menthol


0 40 80 120


40 80
Time (s)


0.6
S0.4
0.2
0
0 40 80 120 160 200
Time (s)
o
0 26
C 22 --
E 18
W 0 40 80 120 160 200
Time (s)


g 0.6

40.4
0 0.2
0
0 40 80 120160200
Time (s)
C)
o 26
C. 22
E 18
W 0 40 80 120 160 200
Time (s)


Figure I
Responses to menthol and cold in vagal ganglion neurons that innervate respiratory airways. a, Diagram illus-
trates the instillation of Dil into the lower segment of rat trachea to retrograde label airway VG neurons. b, The micrographs
show a field of acutely dissociated VG neurons under bright light (left) and fluorescence light (right). Dil-labeled VG neurons
show strong fluorescence intensity. c, An example shows menthol- and cold-induced increases of [Ca2],in in Dil-labeled VG
neurons. The image was selected from the boxed region in (b) where two Dil-labeled VG neurons were included. Images were
taken before (control), following a 20-s application of 100 piM menthol, following a 7oC temperature drop, and recovery in
normal bath solution. d&e, Time course of menthol induced-responses in a Dil-labeled neuron (d) and pooled results (n = 28)
(e). f&g, Time course of cold induced-responses in a Dil-labeled neuron (f) and pooled results (n = 27) (g). The fluorescence
intensity changes (AF/F0) were shown on the top panels and temperature ramps on the bottom panels.





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0.6
~0.4


0.5
S0.4
0.3
< 0.2
0.1
0


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Figure 2
TRPM8-immunoreactivity in VG neurons, a, Image shows TRPM8 immunoreactivity (TRPM8-ir) in a portion of an infe-
rior part of a VG section. TRPM8-ir neurons were not evenly distributed throughout the sections. B, Image shows P2X3
immunoreactivity (P2X3-ir) in the same VG section as in (a). c, An overlay image made with (a) and (b) shows co-localization
of TRPM8-ir in many P2X3-ir positive neurons.


tors may be located on P2X3-expressing vagal neurons
using double immunostaining with both the TRPM8 anti-
body and a P2X3 receptor antibody. About 40% of VG
neurons (415/1041 cells, Figure 2b) were found to be
P2X3-ir positive. The TRPM8-ir and P2X3-ir double posi-
tive neurons accounted for majority (68%) of TRPM8-ir
positive neurons (Figure 2c). Furthermore, when men-
thol, cold, and ATP responses were tested using Ca2+ imag-
ing technique, we found that most menthol-responsive
VG neurons were ATP-sensitive (Figure 3). Of 368 cells
tested for cold, menthol and ATP, 30 of them were men-
thol- and cold-responsive cells. Of these 30 menthol- and
cold-responsive cells, 27 of them were sensitive to ATP.
These results indicate that TRMP8 receptors are expressed
predominantly on a subpopulation of VG neurons that
are ATP-sensitive.

To confirm that the responses ofVG neurons to cold and
menthol were indeed mediated by TRPM8 receptors, we
characterized electrophysiological properties of cold- and
menthol-induced responses. Figure 4a shows a Dil
labeled VG neuron that was included in patch-clamp
recording. All neurons used for the electrophysiological
study were pre-identified to be menthol-responsive VG
neurons using the Ca2+ imaging technique. Under voltage-
clamp configuration with cells held at -70 mV, a tempera-
ture drop from 26 C to 210C within 20 sec evoked
inward currents (Fig. 4b &4d, 57 9 pA, n = 11). Menthol
(100 gM, 20 s) was tested subsequently in 7 cells and all
of them showed inward currents (Figure 4c &4d, 191 29
pA, n = 7). The relationship between menthol-evoked cur-
rents and holding potentials showed strong outward rec-
tification with a reversal potential near 0 mV (-2 0.3 mV,
n = 4, Figure 4e). These properties are consistent with the


electrophysiological characteristics of the cloned TRPM8
that were expressed in heterologous expression system
[12,13] as well as the characteristics of cold-sensing
somatic sensory neurons acutely dissociated from rat dor-
sal root ganglions (DRGs) (Figure 4f). These results indi-
cate that cold and menthol responses in respiratory airway
VG neurons were mediated by TRPM8 receptors.

TRPM8 currents in VG neurons were found to be substan-
tially smaller than those in DRG neurons (Figure 4g). Can
TRPM8 activation be sufficient to excite VG neurons? To
address this issue we made recordings on VG neurons
under current-clamp configuration and tested the effect of
menthol and cold. Menthol (100 iM, 20 s) depolarized
VG neurons from the resting membrane potential of -60
mV (-60 0.6 mV) to -38 mV (-38 1.5 mV) and caused
action potential firing (n = 4, Figure 4h). Similar to the
response induced by menthol, a temperature drop from
26 to 21 C within 20 s depolarized VG neurons from the
resting membrane potential of-61 mV (-61 0.4 mV) to -
36 mV (-36 2.1 mV), which was followed by action
potential firing (n = 4, Figure 4i). These results indicate
that a 5 C of temperature drop to 21 C is sufficient to
excite respiratory airway VG neurons.

Discussion
In the present study, we have shown that a subpopulation
ofvagal afferent neurons innervating bronchopulmonary
tissues expresses TRPM8 receptors and that cold can excite
these autonomic afferent fibers through the activation of
TRPM8 receptors. These findings provide a putative
molecular mechanism by which cold induces autonomic
responses in respiratory system. Thus, TRPM8 receptors



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20Lm








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g Cold bath solution


(30/368)


S0.2

0


0.6
LIL
L 0.4
0.2
0


0 50 100 150 200
Time (s)

menthol


(30/368)




120


(27/368)


0 40 80
Time (s)


LL
10.2

0


0 40 80 120
Time (s)


Figure 3
ATP-sensitivity of cold- and menthol-responsive VG neurons. a-e, Fluorescence intensity in a retrograde labeled VG
neurons (arrow indicated) in control (a), following a temperature drop to 19C (b), the application of 100 UiM menthol (c), the
application of 100 UiM ATP (d), and recovery (e). f, The micrograph shows the Dil retrograde-labeled VG neurons. g-i, Fluores-
cence intensity changes over time in the cell shown in b, c, and d. j, Bright filed.







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cold


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d


Cold


300-


Menthol


< 200-
(--

E 100-


0-
00


i-mV
50 100


DRG nA

2.0

1.0

-100 -50
1.0


1200-


mV
50 100


o 800


z 400
0


Cold Menthol


VGN


Cold


Menthol


50 mV

10s


DRG


Figure 4
TRPM8 receptor-mediated excitation of airway VG neurons. a. Micrographs show a Dil-labeled VG neuron (top) and
subsequent patched-clamp recording (bottom). b. A sample trace shows a whole-cell inward current evoked from the neuron
by a 5C of temperature drop within 20 s. c. Inward current evoked by 100 IM menthol in the same neuron as in (b). d.
Pooled results from experiments shown in b&c. Holding potential was -70 mV in b-d. e. I-V relationship of menthol-evoked
currents from a VG neuron. f. I-V relationship of menthol-evoked currents from an acutely dissociated DRG neuron. g. a com-
parison of menthol-evoked currents in VG neurons (n = 7) and DRG neurons (n = 21). Cells were held at -70 mV. h&i. Action
potentials elicited by 100 IM menthol (h) and cold (i) in the same VG neuron. All recordings were made from menthol-sensi-
tive neurons pre-identified using Ca2+-imaging technique. Menthol was applied at 100 IM for 20 s. Cold stimulation was
achieved by a 20-s application of cold bath solution which yielded a 5C temperature drop from 26 to 21 C.


have functions beyond encoding for consciousness of
cold sensation in somatic sensory system.

Retragrade labeling with Dil used in this study allowed us
to identify the vagal afferent neurons whose peripheral


never endings innervated bronchopulmonary tissues. The
cell bodies of these afferent fibers were used in the present
work to study TRPM8 receptor expression and functions.
Similar approaches have been used for studying expres-
sion and functions of other sensory receptors, e.g. P2X


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VG n,
1.2
0.8
0.4
-100 -50

-0.4


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receptors, on vagal afferent neurons that innervate bron-
chopulmonary tissues [25]. Usually, a receptor that is
used for detecting environmental stimulants is expressed
on peripheral nerve endings as well as on their cell bodies.
Sensory neuron cell bodies have been used as model sys-
tems to study sensory receptors because they have many
advantages including the feasibility of using functional
approaches such as calcium imaging technique and elec-
trophysiological method. Nerve endings, on the other
hand, are difficult to be studied directly using these func-
tional approaches. Using calcium imaging approach, we
have shown that 7% of neurons in the total neuron pop-
ulation responded to both menthol and cold. This is con-
sistent with our immunostaining results. However, in the
retrograde-labeled neurons, the cold- and menthol-
responsive neurons were over 16%. This result suggests
that bronchopulmonary system is a visceral tissue that is
preferentially innervated by TRPM8-expression vagal
afferent fibers. We have shown that most cold- and men-
thol-responsive neurons are also ATP-sensitive, suggesting
that TRPM8-expressing neurons belong to polymodal sen-
sory afferent neurons. The basal temperature for which
cells were maintained was 26 C in the present study. Use
of lower basal temperature is to minimize metabolic stress
under in vitro experimental condition. In addition,
TRPM8 receptors are not significantly activated at temper-
ature above 24 C when cells were at resting membrane
potentials [28], and thereby higher basal temperature is
not necessary for the present study. We have shown that a
few degree of temperature drop from 26 C results in sig-
nificant increase of intracellular Ca2+ and that all cold-
responsive neurons are also sensitive to the TRPM8 ago-
nist menthol. Patch-clamp recordings showed that men-
thol-evoked currents were outward rectified with a
reversal potential near 0 mV. Thus, cold-induced response
in these cells is mainly, if not absolutely, mediated by
TRPM8 receptors.

The presence of TRPM8 receptors on neurons whose
peripheral nerve endings innervate bronchopulmonary
tissues predicts the expression of TRPM8 receptors on
these nerve endings. Sensing cold temperature by these
autonomic nerves and subsequent autonomic reflex may
play a role in respiratory regulation responding to envi-
ronmental temperature changes. In the earlier studies,
TRPM8 receptors were reported to be only expressed on a
subpopulation of dorsal root ganglion and trigeminal
ganglion neurons in normal animals [12,13]. The expres-
sion of TRPM8 receptors on autonomic afferent nerves
innervating bronchopulmonary system has not been
reported previously. However, our finding is not com-
pletely unexpected because respiratory system opens to
environments and sensing cooling temperatures can be
physiologically important. In addition to bronchopulmo-
nary vagal afferent fibers, recent studies have provided evi-


dence suggesting that TRPM8 receptors are involved in
bladder cooling reflex and micturition [30,31]. Thus,
TRPM8 receptors may be widely expressed on afferent fib-
ers innervating visceral organs and to be involved in
reflexive responses.

Since only a small population of neurons expresses
TRPM8 receptors, it raises a question as to whether
TRPM8-expressing neurons randomly innervate airway
trees, or whether there is a regional innervation ofTRPM8-
expressing neurons. Under physiological conditions, tem-
peratures in the airway tree of the lung lobes are unlikely
to drop below 35 C. However, temperatures in the upper
airway such as laryngeal tracheal region may drop below
25 C. It is possible that TRPM8 receptors are mainly
expressed on upper airway trees where these receptors
serve as a sensor of cold temperatures to mediate reflexive
responses.

An intriguing issue is whether TRPM8 receptors may be
involved in cold-induced asthma and asthma exacerba-
tion. This potential pathological function, if proven to be
true, can be very significant clinically. Cold-induced
asthma and asthma exacerbation is a well known phe-
nomenon such that people susceptible to asthma are
often advised to stay away from cold air and keep warm in
winter season. In addition to cold, the factors responsible
for the development of asthma also include genetic pre-
disposition and other factors such as smoking and inflam-
mation [32]. However, these factors often interact with
each other to contribute to asthma etiology [32]. A
number of previous studies in both animals and human
have shown that cold increases airway resistance and the
cold-induced responses were mediated by vagal afferent
nerves [33-35]. In human, inhalation of cold air is known
to be a direct cause of airway constriction in athletes per-
forming winter sports, i.e. exercise asthma [4,36]. If
TRPM8 is involved in cold-induced asthma and asthma
exacerbation, it would be interesting to know whether
TRPM8 receptor expression is up-regulated in those peo-
ple who are susceptible to asthma such that they are more
sensitive to cold than normal subjects. In addition to
direct cold stimulation, inhalation of the vapors of men-
thol or peppermint oil can rapidly trigger airway constric-
tion and asthma in some people [37,38]. However, low
concentrations of menthol are often used for suppressing
respiratory reactions [39]. Thus, TRPM8 receptors may
have complicated respiratory functions under physiologi-
cal and pathological conditions.

Competing interests
The authors declare that they have no competing interests.






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http://www.molecularpain.com/content/4/1/22


Authors' contributions
HX carried out the calcium imaging and electrophysiolog-
ical recordings. JXL performed immunochemical experi-
ments. MC performed Dil injection and assisted
electrophysiological recordings. RDJ guided the dissection
of nodose ganglia. MT provide TRPM8 antibody and
instructed immunochemical experiments. CYW partici-
pated in data analysis. JG designed the experiments, inter-
preted the data and wrote the paper.


Acknowledgements
We thank Victoria Dugan for technical assistance. The study was supported
by a research funding to JG from the University of Florida.

References
I. McFadden ER Jr, Pichurko BM, Bowman HF, Ingenito E, Burns S,
Dowling N, Solway J: Thermal mapping of the airways in
humans. J Appl Physiol 1985, 58:564-570.
2. Ingenito EP, Solway J, McFadden ER Jr, Pichurko B, Bowman HF,
Michaels D, Drazen JM: Indirect assessment of mucosal surface
temperatures in the airways: theory and tests. J Appl Physiol
1987, 63:2075-2083.
3. Ellis EF: Asthma in childhood. J Allergy Clin Immunol 1983,
72:526-539.
4. Weiler JM, Ryan EJ 3rd: Asthma in United States olympic ath-
letes who participated in the 1998 olympic winter games. J
Allergy Clin Immunol2000, 106:267-271.
5. Carlsen KH, Carlsen KC: Exercise-induced asthma. Paediatr
Respir Rev 2002, 3:154-160.
6. Galdes-Sebaldt M, McLaughlin FJ, Levison H: Comparison of cold
air, ultrasonic mist, and methacholine inhalations as tests of
bronchial reactivity in normal and asthmatic children. J Pedi-
atr 1985, 107:526-530.
7. Koskela HO, Hyvarinen L, Brannan JD, Chan HK, Anderson SD:
Responsiveness to three bronchial provocation tests in
patients with asthma. Chest 2003, 124:2171-2177.
8. Sant'Ambrogio FB, Sant'Ambrogio G, Mathew OP: Effects of airway
cooling on tracheal stretch receptors. Respir Physiol 1986,
66:205-214.
9. Pisarri TE, Giesbrecht GG: Reflex tracheal smooth muscle con-
traction and bronchial vasodilation evoked by airway cooling
in dogs. J Appl Physiol 1997, 82:1566-1572.
10. de Jongste JC, Jongejan RC, Kerrebijn KF: Control of airway cal-
iber by autonomic nerves in asthma and in chronic obstruc-
tive pulmonary disease. Am Rev Respir Dis 1991, 143:1421-1426.
I I. Mazzone SB, Canning BJ: Central nervous system control of the
airways: pharmacological implications. Curr Opin Pharmacol
2002, 2:220-228.
12. McKemy DD: How cold is it? TRPM8 and TRPAI in the molec-
ular logic of cold sensation. Mol Pain 2005, 1:16.
13. Peier AM, Moqrich A, Hergarden AC, Reeve AJ, Andersson DA, Story
GM, Earley TJ, Dragoni I, Mclntyre P, Bevan S, Patapoutian A: A TRP
channel that senses cold stimuli and menthol. Cell 2002,
108:705-715.
14. McKemy DD, Neuhausser WM, Julius D: Identification of a cold
receptor reveals a general role for TRP channels in thermo-
sensation. Nature 2002, 416:52-58.
15. Dhaka A, Murray AN, Mathur Earley TJ, Petrus MJ, Patapoutian A:
TRPM8 is required for cold sensation in mice. Neuron 2007,
54:371-378.
16. Colburn RW, Lubin ML, Stone DJ Jr, Wang Y, Lawrence D, D'Andrea
MR, Brandt MR, Liu Y, Flores CM, Qin N: Attenuated cold sensi-
tivity in TRPM8 null mice. Neuron 2007, 54:379-386.
17. Bautista DM, Siemens J, Glazer JM, Tsuruda PR, Basbaum Al, Stucky
CL,Jordt SE, Julius D: The menthol receptor TRPM8 is the prin-
cipal detector of environmental cold. Nature 2007,
448:204-208.
18. Takashima Y, Daniels RL, Knowlton W, Teng Liman ER, McKemy
DD: Diversity in the neural circuitry of cold sensing revealed
by genetic axonal labeling of transient receptor potential
melastatin 8 neurons. J Neurosci 2007, 27:14147-14157.


19. Dhaka A, Earley TJ, Watson J, Patapoutian A: Visualizing cold
spots: TRPM8-expressing sensory neurons and their projec-
tions. J Neurosci 2008, 28:566-575.
20. Okazawa M, Takao K, Hori A, Shiraki T, Matsumura K, Kobayashi S:
Ionic basis of cold receptors acting as thermostats. J Neurosci
2002, 22:3994-4001.
21. Reid G, Babes A, Pluteanu F: A cold- and menthol-activated cur-
rent in rat dorsal root ganglion neurones: properties and
role in cold transduction. J Physiol2002, 545:595-614.
22. Tsuzuki K, Xing H, Ling J, Gu JG: Menthol-induced Ca2+ release
from presynaptic Ca2+ stores potentiates sensory synaptic
transmission. J Neurosci 2004, 24:762-771.
23. Caterina MJ, Schumacher MA, Tominaga M, Rosen TA, Levine JD,
Julius D: The capsaicin receptor: a heat-activated ion channel
in the pain pathway. Nature 1997, 389:816-824.
24. Kollarik M, Undem BJ: Activation of bronchopulmonary vagal
afferent nerves with bradykinin, acid and vanilloid receptor
agonists in wild-type and TRPVI-/- mice. J Physiol 2004,
555:115-123.
25. Undem BJ, Chuaychoo B, Lee MG, Weinreich D, Myers AC, Kollarik
M: Subtypes of vagal afferent C-fibres in guinea-pig lungs. J
Physiol 2004, 556:905-917.
26. Xing H, Chen M, LingJ, Tan W, Gu JG: TRPM8 mechanism of cold
allodynia after chronic nerve injury. J Neurosci 2007,
27:13680-13690.
27. Gu JG, MacDermott AB: ATP P2x receptor activation elicits
glutamate release from sensory neuron synapses. Nature
1997, 389:749-753.
28. Voets T, Droogmans G, Wissenbach U,Janssens A, Flockerzi V, Nilius
B: The principle of temperature-dependent gating in cold-
and heat-sensitive TRP channels. Nature 2004, 430:748-754.
29. Brouns I, Van Genechten J, Hayashi H, Gajda M, Gomi T, Burnstock
G, Timmermans JP, Adriaensen D: Dual sensory innervation of
pulmonary neuroepithelial bodies. Am Respir Cell Mol Biol 2003,
28:275-285.
30. Stein RJ, Santos S, Nagatomi J, Hayashi Y, Minnery BS, Xavier M, Patel
AS, Nelson JB, Futrell WJ, Yoshimura N, Chancellor MB, De Miguel
F: Cool (TRPM8) and hot (TRPVI) receptors in the bladder
and male genital tract. J Urol 2004, 172:1175-1178.
31. Tsukimi Y, Mizuyachi K, Yamasaki T, Niki T, Hayashi F: Cold
response of the bladder in guinea pig: involvement of tran-
sient receptor potential channel, TRPM8. Urology 2005,
65:406-410.
32. Kleeberger SR, Peden D: Gene-environment interactions in
asthma and other respiratory diseases. Annu Rev Med 2005,
56:383-400.
33. Suzuki S, Ishii M, Sasaki J, Takishima T: Bronchial responsiveness
to methacholine during airway cooling in normal subjects.
Clin Allergy 1986, 16:33-40.
34. Giesbrecht GG, Pisarri TE, Coleridge JC, Coleridge HM: Cooling
the pulmonary blood in dogs alters activity of pulmonary
vagal afferents. J Appl Physiol 1993, 74:24-30.
35. Giesbrecht GG, Younes M: Exercise- and cold-induced asthma.
Con J Appl Physiol 1995, 20:300-314.
36. Gotshall RW: Exercise-induced bronchoconstriction. Drugs
2002, 62:1725-1739.
37. Kawane H: Menthol and aspirin-induced asthma. Respir Med
1996, 90:247.
38. dos Santos MA, Santos Galvao CE, Morato Castro F: Menthol-
induced asthma: a case report. J Investig Allergol Clin Immunol
2001, 11:56-58.
39. Morice AH, Marshall AE, Higgins KS, Grattan TJ: Effect of inhaled
menthol on citric acid induced cough in normal subjects. Tho-
rax 1994, 49:1024-1026.















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