Am J Physiol Regul Integr Comp Physiol 291: R541–R550, 2006.
First published March 2, 2006; doi:10.1152/ajpregu.00016.2006.
CALL FOR PAPERS
Physiology and Pharmacology of Temperature Regulation
Thermal sensitivity of isolated vagal pulmonary sensory neurons: role of
transient receptor potential vanilloid receptors
Dan Ni,1 Qihai Gu,1 Hong-Zhen Hu,2 Na Gao,2 Michael X. Zhu,3 and Lu-Yuan Lee1
1
Department of Physiology, University of Kentucky Medical Center, Lexington, Kentucky; and 2Department of Physiology and
Cell Biology, 3Neuroscience and Center for Molecular Neurobiology, The Ohio State University, Columbus, Ohio
Submitted 6 January 2006; accepted in final form 23 February 2006
Ni, Dan, Qihai Gu, Hong-Zhen Hu, Na Gao, Michael X. Zhu, and
Lu-Yuan Lee. Thermal sensitivity of isolated vagal pulmonary sensory
neurons: role of transient receptor potential vanilloid receptors. Am J
Physiol Regul Integr Comp Physiol 291: R541–R550, 2006. First published March 2, 2006; doi:10.1152/ajpregu.00016.2006.—A recent study
has demonstrated that increasing the intrathoracic temperature from 36°C
to 41°C induced a distinct stimulatory and sensitizing effect on vagal
pulmonary C-fiber afferents in anesthetized rats (J Physiol 565: 295–308,
2005). We postulated that these responses are mediated through a direct
activation of the temperature-sensitive transient receptor potential vanilloid (TRPV) receptors by hyperthermia. To test this hypothesis, we
studied the effect of increasing temperature on pulmonary sensory neurons that were isolated from adult rat nodose/jugular ganglion and
identified by retrograde labeling, using the whole cell perforated patchclamping technique. Our results showed that increasing temperature from
23°C (or 35°C) to 41°C in a ramp pattern evoked an inward current,
which began to emerge after exceeding a threshold of ⬃34.4°C and then
increased sharply in amplitude as the temperature was further increased,
reaching a peak current of 173 ⫾ 27 pA (n ⫽ 75) at 41°C. The
temperature coefficient, Q10, was 29.5 ⫾ 6.4 over the range of 35⫺41°C.
The peak inward current was only partially blocked by pretreatment with
capsazepine (⌬I ⫽ 48.1 ⫾ 4.7%, n ⫽ 11) or AMG 9810 (⌬I ⫽ 59.2 ⫾
7.8%, n ⫽ 8), selective antagonists of the TRPV1 channel, but almost
completely abolished (⌬I ⫽ 96.3 ⫾ 2.3%) by ruthenium red, an effective
blocker of TRPV1⫺4 channels. Furthermore, positive expressions of
TRPV1– 4 transcripts and proteins in these neurons were demonstrated
by RT-PCR and immunohistochemistry experiments, respectively. On
the basis of these results, we conclude that increasing temperature within
the normal physiological range can exert a direct stimulatory effect on
pulmonary sensory neurons, and this effect is mediated through the
activation of TRPV1, as well as other subtypes of TRPV channels.
HYPERTHERMIA CAN OCCUR under both physiological and pathophysiological conditions. For example, the body core temperature can increase to ⬎41.5°C in rats during strenuous exercise
(5) and in humans during marathon running (28). Body temperature also frequently exceeds 41°C in the case of severe
fever. However, our understanding of the effect of hyperthermia on the regulation of airway functions is extremely limited.
The afferent activities that arise from sensory terminals
located in the lungs and airways are conducted primarily by
branches of vagus nerves (25, 40). These vagal afferent fibers
play an important role in the regulation of respiratory functions
and airway defense reflexes, and their cell bodies reside in the
nodose and intracranial jugular ganglia (25). Among these
bronchopulmonary sensory nerves, a majority (⬃75%) are
unmyelinated (C-fiber) afferents that function as a primary
sensor for detecting inhaled irritants and become even more
sensitive under various pathophysiological conditions in the
airways and lungs (12, 24). One of the characteristic features of
these C-fiber afferents is their exquisite sensitivity to capsaicin
and the expression of the transient receptor potential vanilloid
type 1 (TRPV1) channel (18). A recent study in anesthetized
rats surgically prepared with isolated perfused thoracic chamber showed that hyperthermia activates and sensitizes vagal
pulmonary C fibers (35). However, whether these effects are
caused by a direct action of hyperthermia on sensory nerves or
by indirect effects via local release of inflammatory mediators
(35) and/or cytokines (21) is not known, because some of these
endogenous mediators (e.g., PGE2, hydrogen ion, etc.) are
known to activate C-fiber endings (12, 24, 25).
The subtypes of TRPV channels, TRPV 1⫺4, are generally
recognized as the primary thermal sensors in mammalian
species (31, 38) . Despite the fact that these TRPVs can also be
activated by many nonthermal stimuli (10, 30, 32), these
channels respond commonly to an increase in temperature, and
each type of TRPV is activated in a different temperature range
(⬎43°C for TRPV1, ⬎52°C for TRPV2, ⬎34⫺38°C for
TRPV3, and ⬎27⫺35°C for TRPV4) (3, 38). A recent report
from our laboratory has demonstrated that 2-aminoethoxydiphenyl borate (2APB), a common activator of TRPV1⫺3
receptors (9, 19), exerted a direct stimulatory effect on isolated
vagal bronchopulmonary sensory neurons. The 2APB-induced
stimulation was only partially blocked by a TRPV1 antagonist,
suggesting the possible presence of TRPV1⫺3 channels in
these neurons (16). However, whether and to what extent these
channels are involved in the expression of thermal sensitivity
of these neurons under normal physiological conditions is yet
unknown.
In view of the background information described above and
the existing unanswered questions, this study was carried out to
investigate whether isolated vagal pulmonary sensory neurons
could be activated by an increase in temperature within the
physiological range, and if so, whether the response was
mediated through TRPV channels. RT-PCR and immunohistochemistry were used to determine whether these thermal-
Address for reprint requests and other correspondence: L.-Y. Lee, Dept. of
Physiology, Univ. of Kentucky Medical Center, Lexington, KY (e-mail:
lylee@uky.edu).
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
capsaicin; C fibers; airway reflexes; temperature; exercise; fever
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0363-6119/06 $8.00 Copyright © 2006 the American Physiological Society
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TRPV CHANNELS AND THERMAL SENSITIVITY OF LUNG SENSORY NEURONS
Table 1. Primers for RT-PCR
Size of the 1st
PCR Product
Channels
Primers for the 1st PCR
TRPV1
Rat/NM031982
TRPV2
Rat/NM017207
TRPV3
Mouse/AF510316 & human/AF514998
TRPV4
Rat/NM023970
5⬘-AGCGAGTTCAAAGACCCAGA-3⬘
5⬘-GTTCAAGGGTTCCACGAGAA-3⬘
5⬘-CAGAAGGCTCCACTGGAAAG-3⬘
5⬘-CTGCAGAATGTGCCTGAAAA-3⬘
5⬘-GCAAGGCKGAGATCCTGAAG-3⬘
5⬘-AGGCCTATGGTGAGCTTGAA-3⬘
5⬘-CCCCATCCTCAAAGTCTTCA-3⬘
5⬘-TTGAACTTGCGAGACAGGTG-3⬘
822
614
862
792
Primers for the Nested PCR
5⬘-TGGAGGTGGCAGATAACACA-3⬘
5⬘-TTAGGGGTCTCACTGCTGCT-3⬘
5⬘-TGATGAAGGCTGTGCTGAAC-3⬘
5⬘-CAGAGCATGCAGGACTGTGT-3⬘
5⬘-ATTGCCATYTTCCTGCTGAG-3⬘
5⬘-GCTGAAGCTGCCRTAGGARC-3⬘
5⬘-ATCAACTCGCCCTTCAGAGA-3⬘
5⬘-GGGAGCACTTGAGAAGCAAC-3⬘
Size of the Nested
PCR Product
349
398
372
385
Channels include the species and GenBank numbers. Primers are forward (top) and backward (bottom). The nested PCR was performed using the product of
the first PCR as the template. The PCR product is given in nucleotides. TRPV, transient receptor potential vanilloid subfamily.
sensitive TRPV channels were, indeed, expressed in these
neurons.
MATERIALS AND METHODS
This study was performed in accordance with the Guide for the
Care and Use of Laboratory Animals published by the National
Institutes of Health and was also approved by the University of
Kentucky Institutional Animal Care and Use Committee.
Labeling vagal pulmonary sensory neurons. Sensory neurons innervating the lungs and airways were identified by retrograde labeling
from the lungs by using the fluorescent tracer, 1,1’-dioctadecyl3,3,3’,3’-tetramethylindocarbocyanine perchlorate (DiI), as described
previously (23). Briefly, young adult Sprague-Dawley rats (⬃160 g)
were anesthetized with an intraperitoneal injection of pentobarbital
sodium (50 mg/kg) and intubated with a polyethylene (PE) catheter
(PE-150) with its tip positioned in the trachea above the thoracic inlet.
DiI was initially sonicated and dissolved in ethanol, diluted in saline
(1% ethanol vol/vol), and then instilled into the lungs (0.2 mg/ml; 0.2
ml ⫻ 2) with the animal’s head tilted up at ⬃30°.
Isolation of nodose and jugular ganglion neurons. After 7⫺10
days, an interval previously determined to be sufficient for DiI to
diffuse to the cell body, the rats were anesthetized with halothane
inhalation and decapitated. The head was immediately immersed in
ice-cold Hank’s balanced salt solution. Nodose and jugular ganglia
were extracted under a dissecting microscope and placed in ice-cold
DMEM/F12 solution. Each ganglion was desheathed, cut into ⬃10
pieces, placed in 0.125% type IV collagenase, and incubated for 1 h
in 5% CO2 in air at 37°C. The ganglion suspension was centrifuged
(150 g, 5 min), and the supernatant was aspirated. The cell pellet was
resuspended in 0.05% trypsin in Hanks’ balanced salt solution for 5
min and centrifuged (150 g, 5 min); the pellet was then resuspended
in a modified DMEM/F12 solution (DMEM/F12 supplemented with
10% (vol/vol) heat-inactivated fetal bovine serum, 100 units/ml penicillin, 100 ␮g/ml streptomycin, and 100 ␮M MEM nonessential
amino acids) and gently triturated with a small bore fire-polished
Pasteur pipette. The dispersed cell suspension was centrifuged (500 g,
8 min) through a layer of 15% bovine serum albumin to separate the
cells from the myelin debris. The pellets were resuspended in the
modified DMEM/F12 solution supplemented with 50 ng/ml 2.5S
nerve growth factor, plated onto poly-L-lysine-coated glass coverslips,
and then incubated overnight (5% CO2 in air at 37°C).
Electrophysiology. Patch-clamp recordings were performed in a
small-volume (0.2 ml) perfusion chamber that was continuously
perfused by gravity-feed (VC-6, Warner Instruments, Hamden, CT)
with extracellular solution (ECS) at 1 ml/min. Recordings were made
in the whole cell perforated patch configuration (50 ␮g/ml gramicidin)
using Axopatch 200B/pCLAMP 8.2 (Axon Instruments, Union City,
CA). Borosilicate glass electrodes had tip resistance of 2⫺4 M⍀. The
series resistance was usually in the range of 4⫺8 M⍀ and was not
compensated. The ECS contained (in mM): 136 NaCl, 5.4 KCl, 1.8
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CaCl2, 1 MgCl2, 0.33 NaH2PO4, 10 glucose, 10 HEPES, with a pH of
7.4. The intracellular solution contained (in mM): 92 potassium
gluconate, 40 KCl, 8 NaCl, 1 CaCl2, 0.5 MgCl2, 10 EGTA, 10
HEPES, with a pH of 7.2. Chemical and temperature stimulations
were applied by using a three-channel fast-stepping perfusion system
(SF-77B, Warner), with its tip positioned to ensure that the cell was
fully within the stream of the perfusate. The temperature of the
solution perfusing the neurons was raised progressively (TC-344B
and SHM-6, Warner) from the baseline, either 23°C (room temperature) or 35°C (body temperature), to 41°C in a “ramp” pattern in
⬃20 s. The actual temperature was measured by a microthermal probe
(time constant ⫽ 0.005 s) (Harvard Apparatus, Holliston, MA) placed
within 100 ␮m downstream from the cell being recorded. The data
were filtered at 5 kHz and digitized at 5 kHz. The resting membrane
potential was held at ⫺70 mV.
Fig. 1. Representative experimental records illustrating responses of isolated
rat vagal pulmonary sensory neurons to increase in temperature. A: in currentclamp mode, increasing the temperature in a ramp pattern evoked depolarization and action potentials in a jugular neuron (21.0 pF). V, membrane potential;
notice that the membrane began to depolarize as the temperature reached
⬃35°C, but action potentials were not evoked until ⬃40°C. Insets display the
action potential signals at an expanded time scale. B: in voltage-clamp mode
(holding potential ⫽ ⫺70 mV), capsaicin (0.5 ␮M, 2-s duration), and hyperthermia were applied to a jugular neuron (28.6 pF). In both A and B, ⬃2 min
elapsed between the two hyperthermia challenges.
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TRPV CHANNELS AND THERMAL SENSITIVITY OF LUNG SENSORY NEURONS
The temperature coefficient over a 10°C temperature range (Q10)
and the activation energy Ea were used to characterize the temperature
dependency of the membrane current (39). An Arrhenius plot was
obtained by plotting the common logarithm of the current against the
reciprocal of the absolute temperature. In the temperature range in
which the Arrhenius plot was linear (correlation coefficient r ⬎ 0.95),
Ea was expressed using the slope of the regression line: ⫺Ea ⫽
2.303Rlog10(I2/I1)/[(1/T2) ⫺ (1/T1)], where I1 and I2 are the values of
normalized currents at the lower and higher absolute temperatures T1
and T2, respectively, and R is the gas constant (8.314 J 䡠 K⫺1 䡠 mol⫺1).
Q10 was determined using the formula: Q10 ⫽ exp[10Ea/(RT1T2)].
RT-PCR. Cytoplasm of ⬃20 individual DiI-labeled nodose/jugular
ganglion neurons was collected, and RT-PCR was performed by using
Titan One Tube RT-PCR Kit (Roche Applied Science; Indianapolis,
IN). Because of the low level of mRNA messages from limited
pulmonary sensory neurons, a second PCR was performed using the
product of the first PCR as the template. The primers used for the first
and the nested PCR amplification are summarized in Table 1. Reverse
transcription was performed at 50°C for 30 min. Cycling conditions
for the first PCR were the following: 1) initial denaturation at 94°C for
3 min; 2) 10 cycles of denaturation at 94°C for 30 s, annealing at 55°C
for 30 s, elongation at 68°C for 1 min; 3) 25 cycles of denaturation at
94°C for 30 s, annealing at 55°C for 30 s, elongation at 68°C (45 s-4
min) cycle elongation of 5 s for each cycle; and 4) a prolonged
elongation at 68°C for 7 min. Reaction products were subsequently
maintained at 4°C until they were used as templates for nested
reactions.
The nested PCR was performed as follows: an initial denaturation
at 94°C for 4 min was followed by 30 cycles of denaturation at 94°C
for 1 min, annealing at 55°C for 2 min, elongation at 72°C for 2 min.
Reaction products were separated on 1% agarose gel in Tris-acetate
EDTA buffer, stained with 0.5% ethidium bromide, and analyzed by
AlphaEaseFC software (Alpha Innotech, San Leandro, CA).
Immunohistochemistry. Seven to ten days after receiving a high
dose of DiI labeling (1 mg/ml; 0.2 ml ⫻ 2), rats (200 –250 g; n ⫽ 3)
were anesthetized with halothane inhalation and decapitated. Nodose
and jugular ganglia were immediately dissected and placed in 4%
paraformaldehyde overnight at 4°C. The ganglia were then incubated
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in 15% sucrose in PBS (0.15 M NaCl in 0.01 M sodium phosphate
buffer pH 7.2) overnight at 4°C. The tissue was embedded in optimal
cutting temperature compound (Richard-Allan Scientific, Kalamazoo,
MI) and sectioned at 8 ␮m with a cryostat (model HM550; MICROM
International, Walldorf, Germany). The sections were incubated in
10% normal donkey serum in 0.02 M PBS for 1 h at room temperature
before exposure to the primary antisera diluted in PBS containing
10% normal donkey serum and 0.3% Triton X-100. The primary
antibodies used were rabbit anti-TRPV1 (1:50) (cat. no.: PC420;
EMD Bioscience, La Jolla, CA), rabbit anti-TRPV2 (1:2,000) (cat.
no.: AB5398; Chemicon International, Temecula, CA), rabbit antiTRPV3 (1:1,000) (gift from Dr. M. Caterina, John Hopkins University), and rabbit anti-TRPV4 (1:100) (cat. no.: ACC034; Alomone
Labs, Jerusalem, Israel). The preparations were incubated for 24 h
with the primary antibody at 4°C followed by 3 ⫻ 10-min washes
with PBS and then incubated with FITC-labeled donkey anti-rabbit
secondary IgG (product code: 711–165-152; Jackson ImmunoResearch Laboratories, West Grove, PA) for another 2 h at room
temperature followed by 3 ⫻ 10-min washes with PBS. The preparations were mounted with coverslips in Vectorshield (Vector Laboratories, Burlingame, CA). Fluorescent labeling was examined and
photographed by using a Nikon Eclipse TE2000-U fluorescent microscope.
Chemicals. All chemicals were obtained from Sigma (St. Louis,
MO), except for AMG 9810 [(E)-3-(4-t-butylphenyl)-N-(2,3dihydrobenzo[b][1,4]dioxin-6-yl)acrylamide], which was obtained
from Tocris (Ellisville, MO). A stock solution of capsaicin (1 mM)
was prepared in 1% Tween 80, 1% ethanol, and 98% saline. Stock
solutions of capsazepine, AMG 9810 and ruthenium red were prepared in DMSO at the concentration of 15, 50, and 10 mM, respectively. Solutions of these chemical agents at desired concentrations
were then prepared daily by dilution with ECS. No detectable effect
of the vehicles of these chemicals was found in our preliminary
experiments.
Statistical analysis. Data were analyzed with a one-way ANOVA,
unless mentioned otherwise. When the ANOVA showed a significant
interaction, pair-wise comparisons were made with a post hoc analysis
Fig. 2. Thermal-sensitive properties of isolated rat vagal pulmonary sensory neurons. A:
the temperature-current relationships of the
two hyperthermia challenges in the same neuron (data are identical to that in Fig. 1B). TI20%
was determined by locating the temperature
point at which the amplitude of the current
reached 20% of the peak current generated at
41°C. B: the relationship between the peak
current induced by hyperthermia (I41°C) and
that by capsaicin (Icap) of either a low dose (F,
n ⫽ 60) or a high dose (E, n ⫽ 15). C: the
relationship between cell diameter and TI20%
(n ⫽ 34). D: correlation between cell diameter
and current density; the latter was calculated
by dividing the peak current to hyperthermia
by the capacitance of each individual neuron
(n ⫽ 34). In both C and D: closed circle,
capsaicin sensitive (Cap ⫹); open circle, capsaicin insensitive (Cap ⫺).
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TRPV CHANNELS AND THERMAL SENSITIVITY OF LUNG SENSORY NEURONS
(Fisher’s least significant difference). A P value ⬍0.05 was considered significant. Data are expressed as means ⫾ SE.
RESULTS
Responses of isolated rat vagal pulmonary sensory neurons
to increase in temperature. Sensory neurons innervating the
lungs and airways were identified by the fluorescence intensity
of DiI. When the temperature of the extracellular solution
surrounding the neuron was elevated from room temperature
(⬃23°C) to 41°C in ⬃20 s in a ramp pattern, a whole cell
inward current was consistently evoked in 78% of the neurons
(87 out of 112) tested in voltage-clamp mode at the holding
potential of ⫺70 mV (e.g., Fig. 1B). The increase in temperature also induced membrane depolarization in six of the nine
neurons tested, and action potentials in three of them in
current-clamp mode; an example is shown in Fig. 1A.
The inward current induced by increasing temperature in
vagal pulmonary sensory neurons became apparent when temperature exceeded a certain threshold (e.g., Fig. 1B) and increased sharply in amplitude as the temperature was further
increased; this is clearly illustrated when the temperaturecurrent relationship of the response shown in Fig. 1B is plotted
in Fig. 2A. The temperature threshold for activation was
defined as TI20%, which is calculated as the temperature when
the current reached 20% of the peak current amplitude generated at 41°C (e.g., Fig. 2A). The average TI20% was 34.4 ⫾
0.4°C (n ⫽ 87). In the majority of the neurons, the currenttemperature relationship obtained from the ascending and the
subsequent descending phases of the temperature ramp (be-
tween 23°C and 41°C, as in Fig. 3A) exhibited a counterclockwise hysteresis (Fig. 3B). In order to quantitatively express the
neuron sensitivity to temperature increase, the temperature
coefficient, Q10, was measured; Q10 was derived from the
linear fits of the semilog Arrhenius plot, in which the evoked
current was plotted against 1/T (T, absolute temperature) (e.g.
Fig. 3C). In general, a Q10 value of ⬎5 indicates the presence
of temperature-sensitive channels (17). In this study, the Q10
over the higher temperature range (35⫺41°C) was 29.5 ⫾ 6.4
(n ⫽ 22), which was distinctly higher than that over the lower
temperature range (23⫺30°C, Q10 ⫽ 2.84 ⫾ 0.56; P ⬍ 0.01,
n ⫽ 22) (Fig. 3D).
Correlation between sensitivities to capsaicin and increasing temperature. Because capsaicin is known to be a selective
activator of TRPV1 channel (7), we examined the correlation
between the responses of whole cell inward currents evoked by
capsaicin and increasing temperature in the same neurons.
Indeed, inward currents were evoked by both capsaicin (0.3,
0.5, or 1.0 ␮M; 2⫺6 s) and hyperthermia (41°C) in the same
neuron in 58% (65 of 112) of the cells tested (e.g., Fig. 1B).
However, no significant correlation was found between the
peak amplitude of the currents evoked by capsaicin and increasing temperature (r2 ⫽ 0.01, n ⫽ 75; Fig. 2B).
Correlation between the neuron size and the response to
increasing temperature. We further investigated whether the
neuron sensitivity to hyperthermia is related to the cell size.
However, no correlation was found either between TI20%
(33.4 ⫾ 0.7°C, n ⫽ 34) and cell diameter (32.5 ⫾ 1.0 ␮m)
(r2 ⫽ 0.01; Fig. 2C) or between the peak current density at
Fig. 3. Whole cell inward current induced by increasing
temperature and its temperature dependency in isolated rat
vagal pulmonary sensory neurons. A: representative experimental record illustrating that an inward current was evoked
in a jugular neuron (37.4 pF) when a temperature ramp of
23– 41°C was applied. B: the temperature-current relationship of the same response in A exhibits a counterclockwise
hysteresis; arrows indicate the direction of temperature
change. C: Arrhenius plot of the data in A illustrating two
distinct phases of the response to increase in temperature.
Q10 values were derived from linear fits of the data in low
and high temperature ranges. D: group data for Q10 values
over the two temperature ranges. Data are means ⫾ SE (n ⫽
22). *P ⬍ 0.01, significant difference between the Q10 values
in these two ranges with paired t-test.
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41°C (6.37 ⫾ 1.54 pA/pF, n ⫽ 34) and cell diameter (r2 ⫽
0.0004; Fig. 2D). Furthermore, there was no significant difference in the peak inward current evoked by increasing temperature (41°C) between the neurons isolated from nodose and
jugular ganglia (nodose: 3.22 ⫾ 0.90 pA/pF, n ⫽ 23; jugular:
5.07 ⫾ 0.84 pA/pF, n ⫽ 11; P ⬎ 0.1; unpaired t-test). In the 34
neurons tested, 24 responded to capsaicin, and 10 did not; in
comparison, both the peak current density and TI20% were
significantly higher in the capsaicin-sensitive neurons (7.47 ⫾
2.13 pA/pF and 34.7 ⫾ 0.7°C, respectively) than that in the
capsaicin-insensitive neurons (3.70 ⫾ 0.54 pA/pF, P ⬍ 0.01;
30.2 ⫾ 0.8°C, P ⬍ 0.01; unpaired t-test).
Pulmonary neuron response to increasing temperature
within the physiological range. Normal body temperature of
the rat during sleep is around 35⫺36°C (33). To determine
whether the neuron response to increasing temperature in the
range tested in this study (23⫺41°C; room temperature:
⬃23°C) is different from that in the normal physiological
temperature range (35⫺41°C), we compared the current response to increase in temperature over these two different
temperature ranges in the same neurons; the temperaturecurrent relationship obtained from the two temperature ranges
overlapped almost completely in individual cells (e.g., Fig.
4A), clearly indicating that the neuron response to hyperthermia was mainly generated in the temperature range of
35⫺41°C (Fig. 4B). A similar pattern of responses was also
found in seven other pulmonary sensory neurons tested in this
study series following the same experimental protocols as that
described above.
Role of TRPV channels in the response of pulmonary sensory neurons to increasing temperature. To investigate the
relative contributions of different subtypes of TRPV channels
to the effect of hyperthermia on pulmonary sensory neurons,
currently available channel blockers were used in the subsequent experiments; both capsazepine (4) and AMG 9810 (14)
have been shown to be selective antagonists of TRPV1 channel, whereas ruthenium red is used frequently as a nonselective
antagonist for TRPV1⫺4 channels (3). The whole cell inward
current evoked by increasing temperature (41°C) was only
partially blocked by pretreatment with capsazepine (10 ␮M, 5
min) or AMG 9810 (0.3 ␮M, 4 min): before and after capsazepine, 389.3 ⫾ 117.4 pA and 186.4 ⫾ 38.5 pA (P ⬍ 0.05, n ⫽
11), respectively; before and after AMG 9810, 367.1 ⫾ 177.8
pA and 84.6 ⫾ 17.4 PA (P ⬍ 0.05, n ⫽ 8), respectively (Fig.
5). Furthermore, TI20% was significantly reduced from 34.9 ⫾
0.7°C at control to 31.0 ⫾ 0.7°C after capsazepine (P ⬍ 0.05,
n ⫽ 11; paired t-test) and from 33.7 ⫾ 1.5°C at control to
30.9 ⫾ 1.0°C after AMG 9810 (P ⬍ 0.05, n ⫽ 8; paired t-test).
In sharp contrast, the response was almost completely abolished by pretreatment with ruthenium red (3 ␮M, 5 min) alone
(15.0 ⫾ 6.8 pA; P ⬍ 0.05, n ⫽ 9) (Fig. 5). The inward current
evoked by increasing temperature returned to the control level
after ⬃60 min washout of ruthenium red (235.0 ⫾ 84.3 pA;
P ⬎ 0.1, n ⫽ 9) (Fig. 5C). In contrast, the vehicle (DMSO) at
the same concentration (10⫺15 ␮M) as that of capsazepine and
ruthenium red applied in the same manner did not produce any
detectable effect (n ⫽ 3). The blocking effect of capsazepine
was completely reversed after 30-min washout, whereas that of
AMG 9810 lasted for ⬎60 min (data not shown).
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Detection and identification of TRPV1⫺4 mRNAs and proteins in pulmonary sensory neurons. To determine whether
mRNAs coding for the four thermal-sensitive TRPV channels,
namely TRPV1⫺4, are present in the vagal pulmonary sensory
neurons, RT-PCR was performed using total RNA isolated
from ⬃20 DiI-labeled pulmonary nodose or jugular ganglion
neurons, with primers specifically against each of these TRPV
channels (Table 1). As shown in Fig. 6, electrophoresis of the
nested PCR products clearly revealed the presence of mRNAs
of all four thermal-sensitive TRPV channels. The expression of
these channels were confirmed in four distinct trials using
neurons collected from four different cultures; in contrast,
controls that did not contain RNA template or the RT enzyme
did not yield any product (n ⫽ 4).
To confirm the RT-PCR results, immunohistochemistry was
performed by using antisera raised against TRPV1⫺4. Indeed,
the immunoreactivities for all these four thermal-sensitive
TRPV channels were separately detected in DiI-labeled vagal
pulmonary sensory neurons (e.g., Fig. 7). Because of the
relatively limited number of these neurons that expressed each
of the four TRPV channels, we were unable to correlate the
expression of the TRPV channels with the cell size.
DISCUSSION
Results of this study clearly demonstrated that isolated vagal
pulmonary sensory neurons can be directly activated by an
increase in temperature within the normal physiological range,
Fig. 4. Representative experimental records illustrating that the responses of
rat vagal pulmonary sensory neurons to increase in temperature are generated
mainly within the physiological temperature range. A: inward currents evoked
by increasing temperature from a baseline of room (25°C; left trace) or body
temperature (35°C; right trace) to hyperthermia (41°C) in a jugular neuron
(21.0 pF). B: temperature-current relationships plotted from the two current
traces in A; F, from the baseline of 25°C; E, from 35°C.
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TRPV CHANNELS AND THERMAL SENSITIVITY OF LUNG SENSORY NEURONS
Fig. 5. Blocking effects of capsazepine,
AMG 9810, and ruthenium red on the hyperthermia-induced current in rat vagal pulmonary sensory neurons. A: representative traces
illustrating the inward currents evoked by
increasing temperature at control, after pretreatments with capsazepine (CPZ, 10 ␮M)
and ruthenium red (RR, 3 ␮M), and after
washout in a jugular neuron (28.6 pF). At
least 10 min was allowed for the cell to
recover between tests. B: the temperaturecurrent relationships of the four current traces
shown in A. C: group data showing the effects
of different treatments on cell response to
increasing temperature. Data are means ⫾ SE
(n ⫽ 11; RR was studied in only nine of these
cells). D: representative traces illustrating the
inward currents evoked by capsaicin (Cap, 1
␮M, 2-s duration) and increasing temperature,
before (control) and after pretreatment with
AMG 9810 (AMG, 0.3 ␮M) in a jugular
neuron (22.3 pF). E: group data showing the
effect of AMG treatment on cell response to
capsaicin and increasing temperature (n ⫽ 8)
because of slow recovery after the AMG
treatment, data after washout were not collected. *Significantly different (P ⬍ 0.05)
from the control response. †Significant difference (P ⬍ 0.05) between the responses after
CPZ and RR treatments.
as demonstrated by the evoked inward currents (voltage-clamp
mode) and membrane depolarization/action potentials (currentclamp mode) (Fig. 1). The inward current induced by hyperthermia was temperature dependent; the distinctly higher Q10
value (29.5 ⫾ 6.4) over the range of 35⫺41°C suggests that the
increase in temperature probably leads to the opening of
temperature-sensitive ion channels. The inward current induced by increasing temperature was partially blocked by
capsazepine and AMG 9810, selective TRPV1 antagonists, but
almost completely abolished by ruthenium red, an effective
blocker of TRPV1⫺4 channels, indicating the involvement of
TRPV1, as well as other subtypes of thermal-sensitive TRPV
channels. In addition, the RT-PCR and immunohistochemistry
studies have further demonstrated the expressions of
TRPV1⫺4 channel transcripts and proteins, respectively, in
these neurons. Taken together, these observations led us to
conclude that TRPV1⫺4 channels play a primary role in
regulating the responses of these pulmonary sensory neurons to
hyperthermia.
TRPV channels are a subfamily of the TRP superfamily of
ion channel proteins containing six transmembrane domains
that form either nonselective or Ca2⫹-selective (e.g., TRPV5
and TRPV6) cationic channels (11, 29). In addition to their
sensitivity to increase in temperature, the TRPV channels can
also be activated by a variety of physiological and pharmacoAJP-Regul Integr Comp Physiol • VOL
logical stimuli, including acid, capsaicin, anandamide, and
certain lipooxygenase products for TRPV1, hypotonicity/low
osmolarity, and phorbol esters for TRPV4 (2), and 2APB for
TRPV1⫺3 (9, 19). The function of TRPV1 as a polymodal
transducer for nociceptive stimuli in primary sensory neurons
has been well recognized (6). Increasing evidence from recent
studies has collectively suggested that TRPV1 may also play
an important role in the manifestation of various symptoms of
airway hypersensitivity (cough, reflex bronchoconstriction,
etc.) associated with airway inflammatory reactions (15, 20,
25). However, the potential roles of other TRPV channels
(TRPV2⫺4) in the neural regulation of airway functions are
yet unknown.
The lack of any correlation between peak current amplitude
evoked by increasing temperature (41°C) and capsaicin, a
selective agonist of TRPV1 channel, in these neurons (Fig. 2B)
suggests a possibility that the hyperthermia-induced response
is not solely mediated through TRPV1 channel. Considering
the possible limitation of capsazepine to block the heat-induced
current mediated through the TRPV1 channel (3), we tested in
a subsequent experiment the effect of a newly developed,
selective TRPV1 antagonist, AMG 9810, which has been
shown to effectively block capsaicin and heat-induced activation of TRPV1 channel in both in vivo and in vitro preparations
(14). The current induced by the increase in temperature was
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TRPV CHANNELS AND THERMAL SENSITIVITY OF LUNG SENSORY NEURONS
Fig. 6. Detection of the presence of TRPV1– 4 mRNAs in rat vagal pulmonary
sensory neurons using RT-PCR. Cytoplasm of 20 individual DiI-labeled
nodose neurons was collected into single PCR tubes. One-step RT-PCR and
the nested PCR were carried out to detect the presence of these four thermalsensitive TRPV channels.
not totally blocked by either capsazepine or AMG 9810 (Fig.
5). Although the experiments for obtaining more definitive
evidence were limited by the unavailability of specific agonists/antagonists for other subtypes of TRPV channels, the
combination of the partial blocking effect of either capsazepine
or AMG 9810 and the almost complete abolition by ruthenium
red (Fig. 5) indicated the possible involvement of other thermal-sensitive TRPV channels, in addition to TRPV1, in the
response of these neurons to increase in temperature. Future
studies in the knockout mice of specific TRPV channels should
allow us to further delineate the relative contributions of
different subtypes of TRPV channels to the thermal sensitivity
of pulmonary sensory neurons.
Another interesting observation in this study is that the peak
current density evoked by the same heat stimulation (41°C)
was significantly smaller in the capsaicin-insensitive pulmonary neurons than the capsaicin-sensitive neurons (3.70 vs.
7.47 pA/pF). Presumably, the capsaicin-insensitive neurons
express either no or a low density of TRPV1 channel, and the
whole cell current recorded during increasing temperature
represents a summation of all of the currents conducted
through open channels of that neuron. Furthermore, the temperature threshold for activation, TI20%, was also markedly
higher (mean ⌬ ⫽ 4.5°C) in the capsaicin-sensitive neurons.
These differences may be related to the fact that the temperature threshold for activating TRPV1 channel is much higher
(⬃43°C) than that of TRPV3 and V4 (38).
The average temperature threshold for activation of the
pulmonary sensory neurons measured by the evoked current in
the present study was ⬃34°C (Figs. 2A and 3A), which is
considerably lower than that previously reported in the primary
sensory neurons innervating other organ systems (e.g., dorsal
root ganglion neurons) (⬃43°C) (3, 31). This difference is
probably related to the different native environments and
temperature ranges to which these sensory endings are normally exposed. We also believe that this threshold may reflect
the relative contributions of different subtypes of thermalsensitive TRPV channels to the response of these neurons to
AJP-Regul Integr Comp Physiol • VOL
R547
hyperthermia for the following reasons. First, the activation
threshold varied between cells in a wide range of 30⫺39°C.
Second, the proportion of the high-temperature-induced peak
current that was blocked by capsazepine also varied substantially between cells. Lastly, the threshold temperature observed
in our study does not match that of any existing subtypes of the
thermal-sensitive TRPV channels, namely, TRPV1⫺4. The
functional diversity of TRPV channels, especially in primary
sensory neurons, has not been fully explored. However, it has
been suggested that TRPV channels can coassemble into heteromeric pore complexes in native cells and can possibly
exhibit distinct channel gating functions from the individual
homomeric channels. Indeed, a recent study by Liapi and
Wood (26) has demonstrated the heteromultimer formation
between TRPV1 and TRPV2 in adult rat cerebral cortex. In a
TRPV1 and TRPV3 heterologously coexpressed system, a
positive coimmunoprecipitation and an increased sensitivity to
capsaicin and proton (compared with the transfection with
TRPV1 alone) has been reported (36). It seems possible that
one of the functional alterations of these heteromultimers could
be a change in the activation threshold in response to hyperthermic stimulus. In the present study, the temperature threshold for activation is lower than that of TRPV1 or TRPV2 but
higher than that of TRPV3 or TRPV4 (Fig. 2, A and C).
Whether this difference is related to the possible heteromultimer formation between different TRPV subunits, as well as a
difference in the subunit composition remains to be determined.
Previous studies of other types of primary sensory neurons
have reported that different subtypes of TRPV channels are
expressed in sensory neuron populations of different cell sizes.
For example, TRPV1 is expressed exclusively in small- and
medium-diameter neurons (6, 7), whereas TRPV2 is preferentially expressed in medium- and large-diameter dorsal root
ganglion neurons (1, 8). In contrast, our results do not indicate
any correlation between the cell response to hyperthermia,
expressed as the peak current density at 41°C, and the cell
diameter (Fig. 2D). Furthermore, there is no clear correlation
between the temperature threshold for activation and the cell
size, regardless of the sensitivity to capsaicin (Fig. 2C). These
results do not seem to indicate a clear pattern of size-dependent
distribution of specific subtypes of TRPV channels in these
pulmonary sensory neurons. Sensory neurons derived from
different ganglionic origin are known to express distinct physiological and pharmacological properties (34). However, we
did not find any significant difference in the response to
increase in temperature between nodose and jugular pulmonary
neurons in this study.
It is well documented that vagal bronchopulmonary afferents play an important role in regulating various cardiopulmonary functions (13, 25). However, the existing knowledge about the thermal sensitivity of these afferent nerves is
extremely limited. It is, therefore, of particular significance
to know that increasing temperature within the physiological range has a direct effect on isolated pulmonary sensory
neurons as shown in the present study (Fig. 1). The fact that
a high percentage of these neurons (78%) displayed pronounced temperature sensitivity suggests that this is a general property of the pulmonary sensory neurons. It also lends
support to our previous observation of a stimulatory effect
of hyperthermia on pulmonary C fibers in anesthetized rats
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TRPV CHANNELS AND THERMAL SENSITIVITY OF LUNG SENSORY NEURONS
Fig. 7. Identification by immunohistochemistry of TRPV1– 4 expression in rat vagal pulmonary sensory neurons. The sections of nodose ganglion (8 ␮m) were
fixed and subject to immunohistochemistry for the four thermal-sensitive TRPV channels. Left: immunoreactivities for TRPV1– 4; middle: pulmonary sensory
neurons as identified by DiI labeling; right: merge of the stainings for TRPVs and DiI. Scale bars ⫽ 30 ␮m.
(35). However, we should also point out that there are
noticeable differences in the results between the present
study and our previous in vivo study. The average temperature thresholds of activation for bronchopulmonary C-fiber
endings and the isolated sensory neurons are 39.2°C and
34.4°C, respectively. Several factors may account for this
difference. In the in vivo study, the temperature threshold
was determined from the action potential signal generated
from the sensory terminal and recorded from the axon (35).
In contrast, the temperature threshold measured in this study
was determined from the inward current signal, which
reflected the increase in channel conductance. However,
subthreshold depolarization does not necessarily lead to the
generation of action potentials unless it reaches the firing
threshold. This is illustrated in the example shown in Fig.
1A, in which membrane depolarization and action potential
occurred at the temperatures of ⬃35°C and ⬃40°C, respectively. In addition, the differences in membrane properties,
AJP-Regul Integr Comp Physiol • VOL
the expression of channels/receptors, and the signal transduction mechanism between the neuronal soma and the
sensory terminal may have also contributed to this difference in the temperature threshold. Moreover, the cellular
environment and milieu that surround and interact with the
sensory terminals in living tissue are very different from that
used in the cultured neurons, which may also influence the
thermal sensitivity of these afferents.
In our previous in vivo study, the stimulatory effect of
hyperthermia was only observed in the capsaicin-sensitive
afferents in the lungs (35), whereas the thermal sensitivity was
found in both capsaicin-sensitive and capsaicin-insensitive
pulmonary neurons in the present study. Although this discrepancy cannot be adequately explained in this study, the various
factors discussed above may have also contributed to the
difference. More importantly, it is well documented that when
capsaicin-sensitive bronchopulmonary afferents are stimulated
(e.g., by hyperthermia), they can elicit centrally mediated
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TRPV CHANNELS AND THERMAL SENSITIVITY OF LUNG SENSORY NEURONS
reflex responses such as cough, bronchoconstriction, and hypersecretion of mucus (12, 24), and evoke the local “axon
reflex” via the release of tachykinins (TKs) and CGRPs (27,
37). In fact, it has been recently reported that tissue hyperthermia triggers the local release of TKs and CGRPs (22, 41). In
the airways and lungs, these neuropeptides are known to act on
a number of effector cells (e.g., airway smooth muscles,
cholinergic ganglia, inflammatory cells, and mucous glands)
and generate potent local effects such as bronchoconstriction,
extravasation of macromolecules, and chemotactic responses
(27, 37).
In summary, this study has established the first evidence that
isolated vagal pulmonary sensory neurons can be activated by
an increase in temperature within the physiological range. Our
results also suggest that the thermal sensitivity of these neurons
is mediated through the activation of TRPV1 and other TRPV
channels. This conclusion is further supported by the evidence
of expression of thermal-sensitive TRPV1⫺4 channels in these
sensory neurons. However, the relative contributions of these
different subtypes of TRPV channels to the thermal sensitivity
of these neurons remain to be determined. Although this
finding has provided the definitive evidence of a direct stimulatory effect of hyperthermia on vagal bronchopulmonary sensory nerves, the influence of the activation of these neurons by
hyperthermia on the overall regulation of airway function
under normal (e.g., strenuous exercise) or pathophysiological
conditions (e.g., fever, airway inflammation, heat stroke) requires further investigations.
ACKNOWLEDGMENTS
We thank Michelle E. Wiggers, Robert Morton, Shaoxin Feng, and Yiqin
Xiong for their technical assistance. We thank Dr. Michael J. Caterina (Johns
Hopkins University) for the gift of TRPV3 antibody. We also thank Dr.
Timothy S. McClintock (University of Kentucky) for his support and valuable
suggestions in the RT-PCR experiment.
GRANTS
This study was supported by grants from National Institutes of Health
(HL-67379) and the Kentucky Lung Cancer Research Program (to L. Y. Lee).
Q. Gu is a Parker B. Francis Fellow in Pulmonary Research.
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