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Am J Physiol Regul Integr Comp Physiol 291: R541-R550, 2006. First published March 2, 2006; doi:10.1152/ajpregu.00016.2006
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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

1Department 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


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
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 patch-clamping 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 ({Delta}I = 48.1 ± 4.7%, n = 11) or AMG 9810 ({Delta}I = 59.2 ± 7.8%, n = 8), selective antagonists of the TRPV1 channel, but almost completely abolished ({Delta}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.

capsaicin; C fibers; airway reflexes; temperature; exercise; fever

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-sensitive TRPV channels were, indeed, expressed in these neurons.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
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’-dioctadecyl-3,3,3’,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 x 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 µg/ml streptomycin, and 100 µM 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 µg/ml gramicidin) using Axopatch 200B/pCLAMP 8.2 (Axon Instruments, Union City, CA). Borosilicate glass electrodes had tip resistance of 2–4 M{Omega}. The series resistance was usually in the range of 4–8 M{Omega} and was not compensated. The ECS contained (in mM): 136 NaCl, 5.4 KCl, 1.8 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 µm 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.

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·K–1·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.


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  		Table 1. Primers for RT-PCR

 
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 x 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 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 µm 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 anti-TRPV3 (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 x 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 x 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-(2,3-dihydrobenzo[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 (Fisher's least significant difference). A P value <0.05 was considered significant. Data are expressed as means ± SE.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
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.


Figure 1
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  		Fig. 1. Representative experimental records illustrating responses of isolated rat vagal pulmonary sensory neurons to increase in temperature. A: in current-clamp 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 µM, 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.

 
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 temperature-current 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 current-temperature relationship obtained from the ascending and the subsequent descending phases of the temperature ramp (between 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).


Figure 2
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  		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 (bullet, n = 60) or a high dose ({circ}, 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 –).

 

Figure 3
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  		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.

 
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 µM; 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 µm) (r2 = 0.01; Fig. 2C) or between the peak current density at 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 temperature-current 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.


Figure 4
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  		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; bullet, from the baseline of 25°C; {circ}, from 35°C.

 
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 µM, 5 min) or AMG 9810 (0.3 µM, 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 µM, 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 µM) 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).


Figure 5
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  		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 µM) and ruthenium red (RR, 3 µM), 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 temperature-current 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 µM, 2-s duration) and increasing temperature, before (control) and after pretreatment with AMG 9810 (AMG, 0.3 µM) 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. {dagger}Significant difference (P < 0.05) between the responses after CPZ and RR treatments.

 
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).


Figure 6
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  		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 thermal-sensitive TRPV channels.

 
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.


Figure 7
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  		Fig. 7. Identification by immunohistochemistry of TRPV1–4 expression in rat vagal pulmonary sensory neurons. The sections of nodose ganglion (8 µm) 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 µm.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
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, as demonstrated by the evoked inward currents (voltage-clamp mode) and membrane depolarization/action potentials (current-clamp 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 pharmacological 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 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 {Delta} = 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 thermal-sensitive TRPV channels to the response of these neurons to 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 (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, 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 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.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
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.


   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.


   FOOTNOTES
 

Address for reprint requests and other correspondence: L.-Y. Lee, Dept. of Physiology, Univ. of Kentucky Medical Center, Lexington, KY (e-mail: lylee{at}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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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