Effect of increasing temperature on TRPV1-mediated responses in isolated rat pulmonary sensory neurons

Dan Ni, Lu-Yuan Lee

Abstract

Hyperthermia has been shown to sensitize vagal pulmonary C-fibers in anesthetized rats. However, it was not clear whether the effect was due to a direct action of hyperthermia on these sensory neurons. To answer this question, we carried out this study to determine the effect of increasing temperature on the responses to various chemical stimuli in isolated nodose and jugular ganglion neurons innervating the rat lungs. In the whole cell perforated patch-clamp study, when the temperature was increased from normal (∼36°C) to hyperthermic (∼40.6°C) level of the rat body temperature, the inward currents evoked by capsaicin, a selective activator of the transient receptor potential vanilloid type 1 (TRPV1), and 2-aminoethoxydiphenyl borate (2-APB), a nonselective activator of TRPV1–3 receptors, were both significantly increased. This potentiating effect was clearly present even at a moderate level of hyperthermia (∼39°C). However, only the slow, sustained component of acid-evoked current mediated through the TRPV1 receptor was potentiated by hyperthermia, whereas the rapid, transient component was inhibited. In contrast, the currents evoked by adenosine 5′-triphosphate and acetylcholine, neither of which is known to activate the TRPV1 channel, did not increase when the same temperature elevation was applied. Furthermore, the hyperthermia-induced potentiation of the cell response to 2-APB was significantly attenuated by either capsazepine or AMG 9810, selective TRPV1 antagonists. In conclusion, increasing temperature within the physiological range exerts a potentiating effect on the response to TRPV1 activators in these neurons, which is probably mediated through a positive interaction between hyperthermia and these chemical activators at the TRPV1 channel.

  • C-fibers
  • transient receptor potential vanilloid channel
  • acid-sensing ion channel
  • exercise
  • inflammation

hyperthermia can occur during strenuous exercise, severe fever, or other pathophysiological conditions. For example, during exhaustive exercise, the body core temperature can increase to 41.9°C in humans (33) and 43.4°C in rats (5). In addition, tissue inflammation and infection can lead to an increase in the local temperature. Indeed, a significant increase in the exhaled air temperature (Δ = 2.7°C) in patients with airway inflammation has been recently reported (41). However, the effect of hyperthermia on the neural regulation of airway function is poorly understood.

Reflex responses elicited by activation of vagal bronchopulmonary afferents are known to play an important role in the regulation of various airway functions under both normal and disease conditions (14, 32). Among these sensory nerves, the majority (∼75%) are unmyelinated (C-fiber) afferents that function as an important sensor for detecting inhaled chemical irritants, and they become even more sensitive under various pathophysiological conditions in the airways and lungs (15, 31). One of the most prominent functional characteristics of these C-fiber afferents is their distinct sensitivity to capsaicin (24), suggesting the presence of the transient receptor potential vanilloid type 1 (TRPV1) channel in the sensory terminals (12). TRPV1 and other TRPV channels, namely TRPV2-4, have been shown to be involved in temperature sensing in various sensory systems (9, 40), and each type of TRPV is activated in a different temperature range (9). These TRPVs not only respond to increase in temperature, but also can be activated by certain non-thermal stimuli, including several endogenous chemical mediators (13, 26). More importantly, they are known to be expressed in pulmonary sensory neurons (36).

Previous work in our laboratory has shown that the responses of bronchopulmonary C-fiber afferents to both lung inflation and capsaicin injection were markedly potentiated when the intrathoracic temperature was increased in anesthetized rats (44). However, whether the stimulation was generated by a direct action of hyperthermia on these sensory nerves could not be determined in that study because these TRPV channels are also expressed on other cell types in the lung (e.g., epithelial cells, endothelial cells, airway smooth muscles, etc.) (26), which upon activation can generate a secondary effect on these afferent endings. The present study was, therefore, designed to investigate the direct effect of increasing temperature within the physiological range on the excitability of isolated pulmonary sensory neurons and to determine the role of TRPV receptors in these responses.

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 with DiI.

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 (30). Briefly, young adult male Sprague-Dawley rats (∼160 g; Harlan, Indianapolis, IN) were anesthetized with an intraperitoneal injection of pentobarbital (40 mg/kg) and intubated with a polyethylene 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 upwards at ∼30°. Animals were used after 7–10 days to allow time for the dye to reach the cell body located in the nodose and jugular ganglia.

Isolation of nodose and jugular ganglion neurons.

The DiI-labeled rats were anesthetized with halothane inhalation and decapitated. The head was immediately immersed in ice-cold Hanks’ balanced salt solution. Nodose and jugular ganglia were extracted under a dissecting microscope and placed in ice-cold DMEM/F-12 solution. Each ganglion was desheathed, cut into approximately eight pieces, placed in 0.125% type IV collagenase, and incubated for 1.5 h in 5% CO2 in air at 37°C. The ganglion suspension was centrifuged (150 g, 5 min) and the supernatant aspirated. The cell pellet was resuspended in 0.05% trypsin in Hanks’ balanced salt solution for 1 min and centrifuged (150 g, 5 min); the pellet was then resuspended in a modified DMEM/F-12 solution (DMEM/F-12 supplemented with 10% (vol/vol), heat-inactivated FBS, 100 U/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% BSA to separate the cells from the myelin debris. The pellets were resuspended in the modified DMEM/F-12 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 (50 μg/ml gramicidin) configuration, using Axopatch 200B/pCLAMP 8.2 (Axon Instruments, Union City, CA). Borosilicate glass electrodes had tip resistance of 2–4 MΩ. The series resistance was usually in the range of 6–10 MΩ and was not compensated. The intracellular solution contained (in mM): 92 potassium gluconate, 40 KCl, 8 NaCl, 1 CaCl2, 0.5 MgCl2, 10 EGTA, 10 HEPES, pH 7.2. ECS contained (in mM): 136 NaCl, 5.4 KCl, 1.8 CaCl2, 1 MgCl2, 0.33 NaH2PO4, 10 glucose, 10 HEPES, pH 7.4. For solutions with pH ≤6, MES was used instead of HEPES for pH buffering.

Chemical solutions were applied to the recorded neuron by using a three-channel fast-stepping perfusion system (SF-77B, Warner), with its tip positioned within 500 μm from the cell recorded to ensure that the cell was fully within the stream of the perfusate. The temperature of the ECS perfusing the neurons was maintained (TC-344B and SHM-6, Warner) at a constant level of either resting body temperature (BT; ∼36°C) or hyperthermic temperature (HT; ∼40.6°C) in rats for >60 s before applying the chemical solution at the same temperature. The actual temperature was measured by a microthermal probe (time constant = 5 ms) (Harvard Apparatus, Holliston, MA) placed within 100 μm downstream from the cell being perfused and recorded on computer. Data were filtered at 5 kHz and digitized at 50 kHz. For all the experiments performed under voltage-clamp mode, the resting membrane potential was held at −70 mV.

Chemicals.

Capsaicin, 2-aminoethoxydiphenyl borate (2-APB), acid (pH 5.5, 6.0, and 6.5), acetylcholine (ACh), and adenosine 5′-triphosphate (ATP) are activators of pulmonary sensory neurons, and they were chosen in this study for the following reasons: capsaicin is a known selective TRPV1 agonist (12); 2-APB is a common activator of TRPV1, V2, and V3 (25); acid activates both TRPV1 and acid-sensing ion channels (ASICs) (12, 48); in contrast, ACh and ATP are not known to directly activate the TRPV1 channel.

All chemicals were obtained from Sigma Chemical (St. Louis, MO) except for 2-APB (Tocris, Ellisville, MO). A stock solution of capsaicin (1 mM) was prepared in 1% Tween 80, 1% ethanol, and 98% saline. Stock solutions of AMG 9810, capsazepine (CPZ), 2-APB, and amiloride were prepared in DMSO at the concentrations of 0.001, 0.015, 0.1, and 1 M, respectively. Solutions of these chemical agents at desired concentrations were then prepared daily by dilution with ECS. The responses of neurons to the vehicles of these chemicals were tested in our preliminary experiments, and no detectable effect was found.

Statistical analysis.

Data were analyzed with a one-way ANOVA analysis, followed by a post hoc Newman-Keuls test, unless mentioned otherwise. A P value <0.05 was considered significant. Data are means ± SE.

RESULTS

Sensory neurons innervating the lungs and airways were identified by the fluorescent intensity of DiI; those with spherical shape and smooth membrane were chosen for the study. Experimental protocols were completed in a total of 90 pulmonary sensory neurons isolated from nodose and jugular ganglia. The whole cell capacitances of these neurons were in the range of 12.3–40.8 pF (25.5 ± 0.8 pF; n = 90); the majority of them (77 out of 90) were small in size (capacitance ≤30 pF); 41.1% (n = 37) of the cells were nodose neurons, and 58.9% (n = 53) were jugular neurons. Although neurons were not selected based on their sensitivity to capsaicin (except in the study of cell response to capsaicin), 81.1% (73 out of 90) of the cells were activated by a low concentration of capsaicin (0.3 or 1.0 μM, 1–6 s).

Effect of increasing temperature on responses of vagal pulmonary sensory neurons to capsaicin and 2-APB.

In current-clamp mode, when the temperature of the ECS surrounding the neuron was elevated from normal BT (35.9 ± 0.06°C) to HT (40.6 ± 0.07°C) in rats, baseline membrane potential (Vm) was significantly elevated (Vm = −69.5 ± 3.08 mV at BT, Vm = −64.8 ± 4.3 mV at HT; n = 7, P < 0.05) (Fig. 1, A and B), and membrane depolarization evoked by capsaicin challenge (0.3 or 1.0 μM, 1–8 s) was also potentiated (ΔVm (Cap) = 29.3 ± 7.0 mV at BT, ΔVm (Cap) = 42.1 ± 8.3 mV at HT; n = 7, P < 0.01); in four of the seven cells, capsaicin evoked firing of action potentials, and the number of action potentials in response to the same capsaicin challenge was also clearly increased during HT (3 ± 3 at BT, 15 ± 7 at HT; n = 4, P < 0.05) (Fig. 1, A and C). To minimize a possible involvement of the voltage-sensitive currents generated by changes in membrane potentials, the rest of our experiments were conducted in voltage-clamp mode. Similarly, capsaicin (0.3 μM, 2–4 s)-evoked current (Δ I) was greatly potentiated by increasing the temperature from ∼36°C to ∼40.6°C (Δ I = 775 ± 140 pA at BT, Δ I = 2,242 ± 597 pA at HT; n = 7, P < 0.05) (Fig. 1, D and E).

Fig. 1.

Effect of increasing temperature on the response of vagal pulmonary sensory neurons to capsaicin (Cap). A: experimental records illustrating that both membrane depolarization and number of action potentials evoked by Cap (1 μM, 4 s) were increased in current-clamp mode when the temperature was increased from 36.0 to 40.6°C in a jugular neuron (22.9 pF); the response recovered when the temperature was returned. Vm, membrane potential; T, temperature. B: group data for the baseline membrane potential at the 2 different temperatures: BT, body temperature (35.7 ± 0.09°C); HT, hyperthermic temperature (40.5 ± 0.11°C). C: group data for capsaicin (0.3–1 μM, 1–8 s)-evoked membrane depolarization, ΔVm (Cap), at the 2 temperatures. D: experimental records illustrating that the Cap (0.3 μM, 2 s)-evoked current was increased when the temperature was increased from 35.8 to 40.6°C in a nodose neuron (23.8 pF) in voltage-clamp mode. E: group data for the Cap (0.3 μM, 2–4 s)-evoked current response (Δ I) at the 2 different temperatures. *P < 0.05 and **P < 0.01 compared with the corresponding response at BT.

2-APB (0.3 mM, 2–8 s)-evoked current was also potentiated at the hyperthermic temperature (Δ I = 484 ± 99 pA at BT, Δ I = 1,019 ± 209 pA at HT; n = 19, P < 0.01) (Fig. 2, A and B). To further investigate the contribution of the TRPV1 channel, we studied the effect of CPZ, a selective TRPV1 channel antagonist (4). Increasing the temperature from 36.0 ± 0.10°C to 40.6 ± 0.08°C markedly enhanced the 2-APB (0.3 mM; 2–8 s)-evoked inward current both before (Δ I = 435 ± 123 pA at BT; Δ I = 1,061 ± 282 pA at HT; n = 8, P < 0.01) and after (Δ I = 116 ± 25 pA at BT; Δ I = 305 ± 85 pA at HT; n = 8, P < 0.05) the pretreatment with CPZ (10 μM, 2 min); CPZ at this concentration has been shown to completely block the effect of capsaicin on the TRPV1 channel in this model system (22). However, this increase in the current response to 2-APB resulting from the same temperature elevation (between BT and HT) was significantly attenuated by the CPZ pretreatment (Fig. 2, C and D; before CPZ, Δ I = 625 ± 177 pA; after CPZ, Δ I = 189 ± 65 pA; n = 8, P < 0.05; analyzed by the linear mixed model 2-factor ANOVA). In addition, another selective and more potent TRPV1 antagonist, AMG 9810 (AMG), which has been shown to effectively block both capsaicin and heat-induced activation of the TRPV1 channel (18), was also tested. Similarly, pretreatment of AMG (1 μM, 5 min) significantly attenuated the increase in the current response to 2-APB resulting from the temperature elevation (before AMG, Δ I = 689 ± 192 pA; after AMG, Δ I = 218 ± 47 pA; n = 5, P < 0.05; the linear mixed model 2-factor ANOVA) (Fig. 2, E and F). Pretreatment with the vehicle of CPZ (0.7 mM DMSO, 2 min) and AMG (10 μM DMSO, 5 min) had no effect. These results indicate that the stimulatory effect of 2-APB on these neurons consisted of two components: one mediated through TRPV1, and the other through non-TRPV1; both of these components were potentiated by increasing the temperature from BT to HT.

Fig. 2.

Effect of increasing temperature on the response of vagal pulmonary sensory neurons to 2-aminoethoxydiphenyl borate (2-APB). A: experimental records illustrating that the 2-APB (0.3 mM, 3 s)-evoked current was clearly augmented when the temperature was increased from 36.4 to 40.4°C in a jugular neuron (17.0 pF), and the response recovered when the temperature was returned. B: group data for the 2-APB (0.3 mM, 2–8 s)-evoked current response at the 2 different temperatures: BT (36.0 ± 0.10°C) and HT (40.6 ± 0.08°C). C: experimental records illustrating the effect of capsazepine (CPZ; 10 μM, 2 min) pretreatment on the 2-APB (0.3 mM, 3 s)-evoked current at 36.2°C and 40.9°C in a nodose neuron (25.2 pF). D: group data illustrating the effect of CPZ pretreatment on 2-APB (0.3 mM, 2–8 s)-evoked currents at BT (36.1 ± 0.06°C) and HT (40.9 ± 0.07°C). E: experimental records illustrating the effect of AMG 9810 (AMG; 1 μM, 5 min) pretreatment on the 2-APB (0.3 mM, 2 s)-evoked current at 35.7°C and 40.4°C in a jugular neuron (21.4 pF). F: group data illustrating the effect of AMG pretreatment on 2-APB (0.3 mM, 2–6 s)-evoked currents at BT (35.8 ± 0.05°C) and HT (40.4 ± 0.08°C). *P < 0.05 and **P < 0.01 compared with the corresponding current response at BT.

In our preliminary studies, we found no difference between nodose and jugular pulmonary sensory neurons in the potentiating effect of increasing temperature on the cell responses to capsaicin or 2-APB; for example, the hyperthermia-induced increases in response to the same challenge of 2-APB (0.3 mM, 2–8 s) were 102 ± 17% (n = 10) and 138 ± 24% (n = 9) in nodose and jugular neurons, respectively (P > 0.05). Therefore, the data obtained from the neurons of these two different ganglion origins were pooled for group analysis in this study.

Effect of increasing temperature on the response of pulmonary sensory neurons to acid.

Acid-induced currents in pulmonary sensory neurons have been shown to be mediated through the activation of both ASICs and the TRPV1 channel (22). Figure 3A illustrates the typical response of these neurons evoked by acid (pH 5.5, 2 s) at room temperature, consisting of a rapidly activated and inactivated current and a slow, sustained current, defined as the transient and sustained components, respectively. When the temperature was raised to ∼36°C, the sustained current was potentiated compared with that at room temperature. Interestingly, however, the transient current induced by acid was inhibited by the increase in temperature. When the temperature was further increased to 40.2°C, the sustained current response induced by the same degree of acidity became larger, and the transient current disappeared completely. Both the transient and sustained responses almost completely returned when the temperature was returned to body and room temperatures (Fig. 3A). Similar responses were also seen in five other cells. To further identify the types of channels involved in the effect of hyperthermia, amiloride, a known blocker of ASICs (48), and CPZ were applied separately in this experiment. Figure 3A showed that the transient but not the sustained component was completely blocked by amiloride (1 mM, 1 min). In contrast, CPZ (10 μM, 3 min) significantly attenuated the sustained but not the transient component. These results indicate that the transient and sustained components of the acid-evoked current seen at room temperature are mediated mainly through ASICs and the TRPV1 channel, respectively, which is consistent with those reported previously in pulmonary sensory neurons (22).

Fig. 3.

Effect of increasing temperature on the response of vagal pulmonary sensory neurons to low pH. A: experimental records illustrating the typical acid-induced current exhibiting both rapid, transient and slow, sustained components in a jugular neuron (22.5 pF). Transient component of the acid (pH 5.5, 2 s)-induced current was almost completely inhibited, whereas the sustained component was potentiated when the temperature was increased from 24.4 to 36.4°C, and then to 40.2°C; the responses recovered when the temperature was returned. Amiloride (1 mM, 1 min) pretreatment completely blocked the transient component, and the response was recovered after 2-min wash out. Pretreatment with CPZ (10 μM, 3 min) almost completely blocked the sustained component. B: experimental records illustrating that the acid (pH 6.5, 6 s)-evoked rapid transient current was completely inhibited when the temperature was increased from 24.1 to 36.0°C in a nodose neuron (27.9 pF). C: group data for the acid (pH 6 or 6.5, 1–6 s)-evoked transient current tested at the 3 temperatures in the order shown in A: RT, room temperature (22.8 ± 0.58°C); BT (35.8 ± 0.10°C); HT (40.6 ± 0.12°C). **P < 0.01, *P < 0.05 compared with the current response at RT. D: experimental records illustrating that the acid (pH 6.0, 4 s)-evoked slow, sustained current was increased when the temperature was increased from 35.7 to 40.3°C in a nodose neuron (24.6 pF). E: group data for the acid (pH 5.5 or 6.0, 4–6 s)-evoked sustained current response at the 2 temperatures: BT, 36.0 ± 0.07°C; HT, 40.5 ± 0.08°C. *P < 0.05 compared with the current response at BT.

As shown in the previous study (22), the acid-evoked responses in different pulmonary sensory neurons exhibited varying degrees of expression of these two different phenotypes of inward currents. In the following experiments, we selectively chose the neurons exhibiting a specific type of current response to acid to further investigate the effect of increasing temperature on these two different types of acid-induced current components. In the neurons that exhibited only the transient current component in response to acid stimulation (e.g., Fig. 3B), the inward current evoked by low pH (6.0 or 6.5, 2–6 s) at room temperature (Δ I = 593 ± 116 pA) was almost completely inhibited at 36.0 ± 0.07°C (Δ I = 28 ± 18 pA), and completely disappeared at 40.7 ± 0.05°C (Δ I = 0 pA) (Fig. 3, B and C) (n = 7, P < 0.01). The response was fully recovered upon returning to room temperature (Fig. 3C). In a sharp contrast, in the neurons that only exhibited sustained current in response to acid stimulation (e.g., Fig. 3D), the sustained inward current evoked by low pH (5.5 or 6.0, 2–6 s) was markedly potentiated by an increase in temperature (Fig. 3, D and E; Δ I = 558 ± 208 pA at BT, Δ I = 1,074 ± 353 pA at HT; n = 9, P < 0.05).

Effect of increasing temperature on pulmonary neuron response to non-TRPV1 activators.

The results described above clearly indicate the potentiating effects of hyperthermia on the responses of pulmonary sensory neurons to TRPV1 channel activators. To further determine whether these potentiating effects are specific to TRPV1 channel activators, cell responses to other chemical stimulants that do not directly activate TRPV1, such as ACh and ATP, were studied.

ACh is known to activate both nicotinic and muscarinic ACh receptors, but its possible effects on pulmonary sensory neurons have not been well characterized. In our experiments, ACh in the range of 50–100 μM evoked an inward current in 22 of the 68 pulmonary sensory neurons tested, with a current magnitude comparable to that evoked by capsaicin (0.3 μM) (e.g., Fig. 4B). Furthermore, our results showed that hexamethonium (0.1 mM, 5 min), a specific antagonist of nicotinic ACh receptor, almost completely abrogated the ACh (100 μM, 2–6 s)-evoked current (Δ = 90.1 ± 5.0%; n = 6) (e.g., Fig. 4, A and B), indicating that the response was mostly mediated through nicotinic ACh receptors.

Fig. 4.

Effect of increasing temperature on the response of vagal pulmonary sensory neurons to ACh. A: experimental records illustrating that pretreatment with hexamethonium (Hex; 100 μM, 5 min) almost completely abolished the ACh (100 μM, 3 s)-evoked current in a nodose neuron (19.8 pF). B: group data illustrating the effect of Hex (100 μM, 5 min) on the ACh (50 or 100 μM, 2–6 s)-evoked current. Con, control; Rec, recovery. *P < 0.05 compared with control response. C: experimental records illustrating that the ACh (50 μM, 4 s)-evoked current was not changed when the temperature was increased from 36.0 to 40.3°C in a nodose neuron (27.9 pF), and the response remained unchanged when the temperature was returned. D: group data for the ACh (50 or 100 μM, 1–6 s)-evoked current response at the 2 different temperatures: BT (35.9 ± 0.08°C); HT (40.7 ± 0.08°C). No significant difference (P = 0.27) of the current response was found between the 2 temperatures.

ATP is a known activator of both P2X and P2Y receptors (6). ATP (0.3 μM, 1–6 s) reproducibly evoked an inward current in 34 of the 39 pulmonary sensory neurons (460 ± 135 pA), but the pattern of the current response varied between different cells (e.g., Fig. 5, A and C). Pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonate (30 or 50 μM, 2 min), a nonselective P2X receptor antagonist (28), completely abolished the ATP-evoked current (Δ = 99.1 ± 0.6%; n = 7) (e.g., Fig. 5, A and B), indicating that the response was mostly mediated through P2X receptors.

Fig. 5.

Effect of increasing temperature on the response of vagal pulmonary sensory neurons to adenosine 5′-triphosphate (ATP). A: experimental records illustrating that pretreatment with pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonate (PPADS; 50 μM, 2 min) completely abolished the ATP (0.3 μM, 2 s)-evoked current in a nodose neuron (21.9 pF). B: group data illustrating the effect of PPADS (30 or 50 μM, 2 min) on ATP (0.3 μM, 2–6 s)-evoked current. *P < 0.01 compared with control response. C: experimental records illustrating that the ATP (0.3 μM, 6 s)-evoked current was not changed when the temperature was increased from 36.2 to 40.6°C in a jugular neuron (12.5 pF), and the response remained unchanged when the temperature was returned. D: group data for the ATP (0.3 μM, 1–6 s)-evoked current response at the 2 different temperatures: BT (35.9 ± 0.08°C), HT (40.7 ± 0.07°C). No significant difference (P = 0.36) of the current response was found between the 2 temperatures.

Our results clearly showed that neither ACh (50 or 100 μM, 2–6 s)- nor ATP (0.3 μM, 1–6 s)-evoked current was potentiated by hyperthermia (Fig. 4, C and D, Fig. 5, C and D). ACh-induced current responses were 602 ± 397 pA and 640 ± 415 pA at 35.9 ± 0.08°C and 40.7 ± 0.08°C, respectively (n = 8, P = 0.27). Similarly, ATP-induced current responses were 460 ± 136 pA and 415 ± 118 pA at 35.9 ± 0.08°C and 40.7 ± 0.07°C, respectively (n = 13, P = 0.19).

To determine the temperature threshold of the potentiating effect of hyperthermia on capsaicin-evoked responses, in a subsequent series of experiments, we successively increased the temperature in 1.5°C increments between 36.0 and 40.5°C, each step held in a steady state for 3 min with 15 min recovery between two steps, and tested the response to capsaicin (0.1 or 0.3 μM, 1–3 s) in each neuron. Although capsaicin-evoked current increased progressively when the temperature was raised (Δ I = 416 ± 68 pA at BT, Δ I = 601 ± 83 pA at 37.5°C, Δ I = 1,092 ± 157 pA at 39.0°C, Δ I = 1,336 ± 248 pA at 40.5°C), significant potentiation was found only after the temperature reached 39.0°C (n = 10, P < 0.05) (Fig. 6).

Fig. 6.

A: experimental records illustrating that Cap (0.1 μM, 1 s)-evoked current was increased when temperature was elevated from 35.9 to 37.4, 39.0, and 40.4°C in a jugular neuron (29.9 pF). B: group data for the Cap (0.1 or 0.3 μM, 1–3 s)-evoked current response at the 5 different temperatures as indicated. *P < 0.05 compared with the current response at 36°C.

DISCUSSION

The present study demonstrated that increasing temperature within the normal physiological range (36–40.6°C) resulted in sensitization of isolated pulmonary sensory neurons to chemical activators of the TRPV1 channel; this was clearly indicated by the observation that both membrane depolarization and the number of action potentials evoked by capsaicin were markedly potentiated by hyperthermia. When the membrane potential was held constant in voltage-clamp mode, both capsaicin- and 2-APB-induced currents were significantly augmented when temperature was increased. This potentiating effect was temperature dependent and became clearly evident when the temperature reached ∼39°C. The potentiating effect of hyperthermia on 2-APB-evoked current was attenuated by either CPZ or AMG 9810, selective antagonists of the TRPV1 receptor. In contrast, the responses to ATP and ACh (neither is known to activate the TRPV1 receptor) remained unchanged with increasing temperature in these neurons. More interestingly, increasing temperature exerted a paradoxical effect on the acid-evoked current: the rapid, transient component mediated by ASICs was consistently inhibited, and the slow, sustained component mediated by the TRPV1 receptor was markedly potentiated.

Several possible physiological implications of these findings should be considered. The temperature change applied in this study was well within the normal physiological range. The BT in rats was measured at 35.5–36.0°C during sleep (43), whereas the HT (∼40.5°C) employed in this study can occur under both normal and pathophysiological conditions. Most importantly, the potentiating effect observed in our study was clearly present even at the moderate level of hyperthermia (39.0°C; Fig. 6). In healthy subjects, the most common cause of hyperthermia is an increase in metabolic rate (e.g., during vigorous exercise). Body core temperature exceeding 41°C has been reported during exertional exercise in healthy men (33) and in animals (5). Hyperthermia also occurs frequently under pathophysiological conditions caused by endogenous pyrogens or infection, such as in patients suffering from severe fever. Moreover, tissue inflammation is known to lead to local hyperemia and an increase in temperature in the inflamed area (20). In fact, a recent study has reported a higher temperature (Δ = 2.7°C) in the expired air of asthmatic patients (41). The present study has provided the first evidence that hyperthermia exerts a direct potentiating effect on the sensitivity of pulmonary sensory neurons to chemical activators of the TRPV1 receptor. We believe that this finding is physiologically relevant because some of the endogenous TRPV1 activators, such as hydrogen ion and certain lipoxygenase metabolites of arachidonic acid (26), may be released in the airway tissue concurrently with an increase in tissue temperature during inflammatory reaction. In addition, recent studies have presented compelling evidence of an important role of TRPV1 in various symptoms of airway hypersensitivity associated with airway inflammation (19, 26). For example, an elevated cough sensitivity to TRPV1 activators, such as capsaicin or citric acid aerosol, has been reported in patients with asthma or airway inflammation (16, 39).

Depolarization of baseline membrane potential at hyperthermic temperature shown in the current study (Fig. 1, A and B) is consistent with the results reported recently by Ni et al. (36). The results of that study indicate that TRPV1 and possibly other thermal sensitive TRPV channels (TRPV2-4) are involved in the expression of thermal sensitivity of these cells. However, we cannot rule out the possible involvement of other ion channels exhibiting thermal sensitivity. Recent studies have demonstrated that the activities (e.g., single channel conductance, frequency of opening, etc.) of certain potassium channels, such as TREK-1, TREK-2, and TRAAK, were elevated with increasing temperature (1, 27), which is expected to induce membrane hyperpolarization. Hence, it is unlikely that these potassium channels play a part in inducing the membrane depolarization found in pulmonary sensory neurons during hyperthermia in this study.

In addition, the small and sustained elevation of temperature by ∼4.6°C (from 36 to 40.6°C) generated only a small inward current (average ∼40 pA) in these neurons, which is considerably smaller than the average current response (∼135 pA) evoked by a larger and transient temperature elevation (from 23 to 41°C in a ramp pattern) found in our previous study (36). However, despite the relatively mild increase in baseline current during hyperthermia, the inward currents evoked by various chemical activators of the TRPV1 receptor (e.g., capsaicin, proton, 2-APB) in these neurons were drastically and consistently augmented not only in voltage-clamp mode (Figs. 1, 2, and 3D) but also in current-clamp mode (capsaicin; Fig. 1, AC), clearly indicating a synergistic, not additive, effect of hyperthermia on the responses to these chemical activators. Although the specific site(s) and mechanism of the sensitizing effect cannot be determined in this study, cytoplasmic COOH-terminal tail of the TRPV1 receptor may play an important part in mediating the positive interaction between hyperthermia and chemical activators of the channel since this region has been shown to be involved in the conformational changes leading to channel activation (46). Furthermore, the evidence that CPZ or AMG pretreatment markedly attenuated the potentiating effect of hyperthermia on the neuron response to 2-APB suggests a primary role of the TRPV1 receptor (Fig. 2, CF). However, because the potentiated response to 2-APB was not completely abolished by CPZ or AMG (Fig. 2, CF), the involvements of TRPV2 and V3 receptors, although to a lesser extent, cannot be dismissed.

Our results clearly demonstrated that incremental increases in temperature induced a progressive increase in the capsaicin-evoked response in pulmonary sensory neurons (Fig. 6). Because of the long recovery time required after each capsaicin challenge and the limited time for maintaining a stable recording of these neurons, we were not able to identify more precisely the threshold temperature of the potentiating effect of hyperthermia. Nevertheless, our data indicated that the response to capsaicin was pronouncedly potentiated after the temperature exceeded 39°C, which is at a level of moderate hyperthermia. This temperature is substantially lower than the threshold temperature (43°C) for activating the heterologously expressed TRPV1 channel reported in the literature (12, 13, 40), but is comparable to that found in pulmonary C-fibers recorded in anesthetized rats (39.7°C; Ref. 44). Whether this discrepancy is due to a different activation threshold of the TRPV1 expressed in pulmonary sensory neurons (22) or a different transduction mechanism involved in the sensitizing effect of hyperthermia on the response to TRPV1 activators remains to be determined.

Lactic acid is produced by anaerobic metabolism such as during strenuous exercise (17), and local tissue acidosis frequently occurs in airway inflammatory and ischemic conditions (29). It is known that protons are capable of modulating the activity of a number of ion channels expressed on primary afferent sensory nerves, including ASIC channels (48) and the TRPV1 receptor (12). Analyses of the stimulatory effect of acid on native and cloned TRPV1 receptors suggest that the TRPV1 channel is involved in the sustained response to acid challenge in vivo (3, 10). This is further supported by the profoundly reduced responses to acid in cultured dorsal root ganglion (DRG) neurons after a targeted disruption of the TRPV1 gene (11). Similarly, our recent study (36) showed that the acid-evoked current in the majority of pulmonary sensory neurons consists of two components: the transient component is mainly mediated through activation of ASICs, whereas the slow, sustained component is mostly mediated through the TRPV1 channel. Our results in this study further demonstrated that hyperthermia exerted opposite effects on these two components of the acid-induced current in pulmonary sensory neurons (Fig. 3A). The inhibitory effect of hyperthermia on the ASICs in pulmonary sensory neurons is in general agreement with observations reported previously (2, 45), which showed that the proton-gated channel activation was inhibited by increasing temperature in both transfected cells and DRG neurons. Hyperthermia appears to exert an effect on the same site as proton activation on the ASIC channel, because mutation of a conserved residue that determines the channel gating abolished the desensitizing effect of increasing temperature on proton-induced current (2). On the other hand, the potentiating effect of hyperthermia on the TRPV1-mediated response to low pH is consistent with the finding of a positive interaction at the TRPV1 channel between proton and heat (47). In addition, more rapid activation and inactivation of the TRPV1-mediated current were noticeable during hyperthermia compared with that at room temperature (e.g., Fig. 3A), which was probably due to the faster opening and closing of the TRPV1 channel at a higher temperature; indeed, a similar increase in rate of gating caused by increasing temperature is known to occur in voltage-gated channels (23).

These results clearly indicated the distinct functional roles of these two types of channels in sensing the acidity under different temperatures. TRPV1 played a dominant role, whereas ASICs exhibited very little or no response to acid in the physiological range of body temperature in pulmonary sensory neurons. Whether this finding is applicable to sensory neurons (e.g., DRG neurons) innervating somatic tissue, where the temperature is generally lower than that of the viscera, remains to be determined. Nevertheless, the complete inhibition of ASICs after exceeding 40°C shown in this study may explain, at least in part, the lack of a significant increase in pulmonary C-fiber sensitivity to lactic acid with increasing temperature reported in anesthetized rats (44).

Our conclusion on the primary role of TRPV1 in the synergistic effect of hyperthermia on the response to TRPV1 chemical activators is further supported by the observation that the same temperature elevation failed to potentiate the response to either ACh or ATP in pulmonary sensory neurons (Fig. 4, C and D; Fig. 5, C and D). ACh, a primary neurotransmitter of the autonomic nervous system, evoked an inward current in pulmonary sensory neurons via activation of nicotinic ACh receptors (Fig. 4, A and B). ATP, in addition to its key role in cellular metabolism, also functions as an active extracellular messenger, producing its effects via the activation of both P2X receptor, a ligand-gated cation channel, and P2Y receptor, a G protein-coupled receptor (7). Our results showed that ATP stimulated pulmonary sensory neurons mainly via activation of P2X receptors (Fig. 5, A and B). Neither ACh nor ATP is known to activate TRPV1 directly, and their stimulatory effects on pulmonary sensory neurons were not affected by pretreatment with CPZ (Ni and Lee, unpublished data).

Potential of the TRPV1 channel as a signal integrator has attracted much attention in recent years. For example, a potentiating effect of chemical and physical stimuli on the gating of the TRPV1 channel has been suggested to play an important role in certain diseases (37). Furthermore, a number of endogenous inflammatory mediators [e.g., prostaglandin E2 (PGE2), bradykinin, acid, etc.] can sensitize TRPV1 during tissue inflammation, which leads to nociceptor hypersensitivity and hyperalgesia (8). In pulmonary sensory neurons, the hyperthermic temperature activates TRPV1 and/or other subtypes of TRPV channels (36), which in turn can induce the influx of cations, mainly Ca2+ and Na+ (34). These cations could possibly trigger several cascading events. For example, the modulatory effects of cytosolic Ca2+, such as increasing fusion of receptor-containing vesicles to the plasma membrane (13), may contribute to the hyperthermia-induced sensitizing effects on the response of pulmonary sensory neurons to capsaicin, 2-APB, and acid (sustained component) by increasing the density of receptor expression on the cell membrane. In addition, the protein kinase C- and protein kinase A-mediated phosphorylation of the TRPV1 channel is believed to induce TRPV1 sensitization caused by endogenous inflammatory mediators such as PGE2 and bradykinin (21, 35, 42). Furthermore, a recent study has shown that high temperature (42°C) shifted the TRPV1 channel activation curve (open probability vs. voltage) from a non-physiological positive voltage range towards the negative potential (38). This large shift of voltage-dependent activation curve to a physiologically relevant voltage range with a relatively small gating charge may contribute to not only the hyperthermia-induced hypersensitivity demonstrated in this study, but also the diverse functional properties of TRPV1 (38).

In conclusion, this study showed that increasing temperature within the physiological range potentiated the responses of isolated pulmonary sensory neuron to TRPV1 activators. This potentiating effect was probably mediated through a positive interaction between hyperthermia and chemical activators primarily at the TRPV1 channel. Because tissue hyperthermia and endogenous release of certain chemical activators of the TRPV1 are known to occur concurrently during airway inflammatory reaction, selecting the TRPV1 channel as a potential therapeutic target for treating airway inflammatory diseases should merit further investigations.

GRANTS

This study was supported by National Heart, Lung, and Blood Institute Grants HL-58686 and HL-67379.

Acknowledgments

We thank Michelle E. Wiggers and Robert F. Morton for technical assistance and Chunxu Liu for statistical analysis.

Footnotes

  • 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

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