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Hyperthermia increases sensitivity of pulmonary C-fibre afferents in rats
http://www.100md.com 《生理学报》 2005年第10期
     1 Department of Physiology, University of Kentucky Medical Center, Lexington, KY, USA

    2 Institutes of Physiology, National Yang-Ming University, Taipei, Taiwan, ROC

    Abstract

    This study was carried out to investigate whether an increase in tissue temperature alters the excitability of vagal pulmonary C-fibres. Single-unit afferent activities of 88 C-fibres were recorded in anaesthetized and artificially ventilated rats when the intrathoracic temperature (Tit) was maintained at three different levels by isolated perfusion of the thoracic chamber with saline: control (C: 36°C), medium (M: 38.5°C) and high (H: 41°C), each for 3 min with 30 min recovery. Our results showed: (1) The baseline fibre activity (FA) of pulmonary C-fibres did not change significantly at M, but increased drastically (>5-fold) at H. (2) The C-fibre response to right-atrial injection of capsaicin (0.5 μg kg–1) was markedly elevated at H (FA = 5.94 ± 1.65 impulses s–1 at C and 13.13 ± 2.98 impulses s–1 at H; P < 0.05), but not at M. Similar increases in the C-fibre responses to other chemical stimulants (e.g. adenosine, etc.) were found at H; all the enhanced responses returned to control in 30 min. (3) The C-fibre response to lung inflation was also significantly potentiated at H. In sharp contrast, there was no detectable change in either the baseline activity or the responses to lung inflation and deflation in 10 rapidly adapting pulmonary receptors and 10 slowly adapting pulmonary receptors at either M or H. (4) The enhanced C-fibre sensitivity was not altered by pretreatment with indomethacin or capsazepine, a selective antagonist of the transient receptor potential vanilloid type 1 (TRPV1) receptor, but was significantly attenuated by ruthenium red that is known to be an effective blocker of all TRPV channels. (5) The response of pulmonary C-fibres to a progressive increase in Tit in a ramp pattern further showed that baseline FA started to increase when Tit exceeded 39.2°C. In conclusion, a pronounced increase in the baseline activity and excitability of pulmonary C-fibres is induced by intrathoracic hyperthermia, and this enhanced sensitivity probably involves activation of temperature-sensitive ion channel(s), presumably one or more of the TRPV receptors, expressed on the C-fibre endings.
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    Introduction

    External body surface, such as skin, can be exposed to high temperature and even noxious heat under certain unusual circumstances. In contrast, the lung structures are enclosed within the thoracic cavity and constantly exposed to the core temperature of the body that is usually maintained within a relatively narrow physiological range. However, body temperature can increase when the metabolic rate increases, such as during exercise. Tissue inflammation can also lead to an increase in local tissue temperature (Planas et al. 1995). Thus, the lung tissue and the sensory endings residing within the lung structures are subjected to hyperthermia under various conditions; for example, core temperature of the body can exceed 41°C during strenuous exercise or acute heatstroke in humans and in animals (Pugh et al. 1967; Brooks et al. 1971; Greenleaf, 1979; Bouchama et al. 1991).
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    Non-myelinated (C-fibre) vagal afferents arising from the lungs and airways are known to play an important role in regulating the airway functions under both normal and pathophysiological conditions of the lungs (Coleridge & Coleridge, 1984; Lee & Pisarri, 2001). Capsaicin, the major pungent active ingredient of hot peppers, is a potent and selective stimulant of C-fibre endings in the lungs. Hence, it has been used extensively as a tool for identifying the presence of these endings in a number of species including humans (Coleridge & Coleridge, 1984; LaMotte et al. 1992; Ho et al. 2001; Lee & Pisarri, 2001). The existence of ‘capsaicin receptor’ was first suggested upon the discovery of capsazepine (CPZ) that selectively blocked the various pharmacological effects of capsaicin (Bevan et al. 1992). Indeed, the transient receptor potential vanilloid type 1 (TRPV1) receptor was cloned in 1997 by Caterina et al. (1997) using an expression cloning technique to isolate a functional cDNA that encoded the capsaicin receptor from rodent dorsal root ganglion (DRG) neurones. More importantly, extensive evidence further demonstrated that the TRPV1 receptor also functions as the transducer for nociceptive thermal stimulation in the somatic afferents (Julius & Basbaum, 2001; Vlachova et al. 2001). Furthermore, expression of the TRPV1 receptor and other members of the TRPV family in the vagal sensory (nodose and jugular) ganglia has recently been demonstrated (Helliwell et al. 1998; Zhang et al. 2004). However, whether pulmonary C-fibre endings are sensitive to temperature change is not yet known. This study was therefore carried out to answer the following specific questions: (1) Are vagal pulmonary C-fibres sensitive to an increase in the temperature within the normal physiological range If so, does an increase in the temperature modulate the excitability of vagal pulmonary C-fibres to chemical and mechanical stimuli (2) What is the role of TRPV1 and other TRPV channels in the expression of thermal sensitivity in pulmonary C-fibres (3) Are cyclooxygenase metabolites of arachidonic acid involved in the hypersensitivity of pulmonary C-fibres induced by hyperthermia (4) Do other types of vagal lung afferents that are insensitive to capsaicin also exhibit thermal sensitivity in a manner similar to that in pulmonary C-fibres
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    The preliminary data of this study have been presented in an abstract form at the 2004 American Thoracic Society International Conference.

    Methods

    Animal preparation

    The procedures described below were performed in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health, USA, and were also approved by the University of Kentucky Institutional Animal Care and Use Committee.
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    Sprague-Dawley rats (393 ± 4 g, n= 96) were initially anaesthetized with an intraperitoneal injection of -chloralose (100 mg kg–1) and urethane (500 mg kg–1) dissolved in a 2% borax solution; supplemental doses of -chloralose (10 mg kg–1 h–1) and urethane (50 mg kg–1 h–1) were injected intravenously (I.V.) to maintain abolition of pain reflexes elicited by paw-pinch. One femoral artery was cannulated for recording the arterial blood pressure (ABP) with a pressure transducer (Statham P23AA). For I.V. administration of pharmacological agents, the left jugular vein was cannulated and a catheter was advanced until its tip was positioned just above the right atrium. A short tracheal cannula was inserted just below the larynx via a tracheotomy. Tracheal pressure (Pt) was measured (Validyne MP45-28) via a side-port of the trachea cannula. Body temperature was maintained at 36°C by means of a heating pad placed under the animal which was lying in a supine position. At the end of the experiment, the animal was killed by an I.V. injection of KCl.
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    Isolated perfused thoracic chamber

    To elevate and maintain the intrathoracic temperature (Tit) at a constant level, a thoracic chamber was prepared and perfused by isotonic saline that was kept at a constant temperature from an external reservoir. After a midline thoracotomy, the inlet of the perfusion circuit, the tip of a PE-190 catheter, was sutured to the interior dorsal wall (bottom) of the thoracic cage that was then partially closed by sutures to form a chamber; the tip of the outlet catheter was positioned at the opening (top) of the thoracic chamber and connected to a suction pump. The perfusion was driven by gravity feed and maintained at a rate of 150 ml min–1. A miniature temperature probe (Harvard NP52-1757) was sutured to the interior wall of the thoracic cage to measure the Tit; probes (Harvard NP52-1583) were also inserted in the external reservoir and rectus to monitor the perfusate and animal's core temperatures, respectively. Tit reached and remained at a steady state of higher temperature in <30 s after the onset of perfusion, and returned rapidly (<30 s) to control upon perfusion with saline at body temperature.
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    Measurement of single fibre activity

    The open-chest rat was artificially ventilated with a respirator (UGO Basile 7025); the expiratory outlet of the respirator was placed under 3 cmH2O pressure to maintain a near-normal functional residual capacity. Tidal volume and respiratory frequency were set at 8–10 ml kg–1 and 50 breaths min–1, respectively, to mimic those of vagotomized rats. Both cervical vagus nerves were separated from carotid arteries and then sectioned to eliminate any possible influence of vagus-mediated reflex bronchoconstriction on afferent responses. The caudal end of the cut right vagus nerve was placed on a small dissecting platform and immersed in a pool of mineral oil. A thin filament was teased away from the desheathed nerve trunk and placed on a platinum–iridium hook electrode. Action potentials were amplified (Grass P511K), monitored by an audio monitor (Grass AM8RS) and displayed on an oscilloscope (Tektronix 2211). The thin filament was further split until the afferent activity arising from a single unit was electrically isolated. The nerve trunk was ligated just above the diaphragm to eliminate afferent signals arising from lower visceral organs.
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    The procedures for identifying pulmonary C-fibres have been described in detail previously (Ho et al. 2001). Briefly, our criteria were as follows: (1) an abrupt and intense response to right-atrial injection of capsaicin (1 μg kg–1) within 2 s after the injection; (2) a weak response to lung inflation (Pt= 30 cmH2O) (e.g. Fig. 1); and (3) the general location of the receptors identified by their responses to gentle probing and pressing of the lungs with a blunt-ended glass rod. Slowly adapting pulmonary receptors (SARs) and rapidly adapting pulmonary receptors (RARs) were identified initially by their distinct phasic discharge synchronous with the respirator cycles, and single units of these receptors were further classified by their adaptation indexes (AIs) in response to lung inflation; AI was calculated in each fibre by dividing the difference in fibre activity (FA) between the first two seconds of a constant-pressure (Pt= 30 cmH2O) lung inflation, by the FA of the first second, and expressed as a percentage (Knowlton & Larrabee, 1946; Widdicombe, 1954). Fibres with AIs of <80% and >80% were classified as SARs and RARs, respectively. The signals of the afferent activity, Pt and ABP were recorded on a thermal writer (Gould TW11) and on a VCR format data recorder (Vetter 4000 A). FA was analysed by a computer and a data acquisition system (Biocybernetics TS-100) in 0.1 s (for RARs and SARs) or 0.5 s intervals (for C-fibres), unless indicated otherwise.
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    Left panels: responses to lung inflation (Pt= 30 cmH2O for 10 s); right panels: responses to capsaicin (0.5 μg kg–1 in 0.1 ml volume) that was first slowly injected into the catheter and then flushed (at arrow) into the right atrium as a bolus with saline (0.3 ml). Receptor location, right lower lobe; rat body weight, 380 g. Lung inflation and capsaicin injection were tested at the 2nd minute and the 3rd minute, respectively, during the 3 min constant perfusion of thoracic chamber with perfusate at a given temperature; the measured Tit is shown at the top left corner of each panel. At least 30 min were allowed to elapse between tests to avoid possible tachyphylaxis. AP, action potential; Pt, tracheal pressure; ABP, arterial blood pressure.
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    Experimental design and protocol

    Five series of experiments were carried out. Study series 1 was designed to determine the effect of increasing Tit on the excitability of pulmonary C-fibres. The baseline activity and the response to chemical stimulation or lung inflation were determined when Tit was altered between three different levels: control (C: 36°C), medium (M: 38.5°C) and high (H: 41°C) in each fibre; the control temperature was chosen because the normal body (core) temperature of the rats during sleep is 36°C (Refinetti & Menaker, 1992; Briese, 1998). The sequence of applying three levels of Tit was alternated between fibres to achieve a balanced design. Each level of Tit was maintained for 3 min, and at least 30 min was allowed to elapse between tests for a complete recovery. The response to each of the five chemical agents known to stimulate pulmonary C-fibres was studied by a right-atrial bolus injection: capsaicin (0.5 μg kg–1), phenylbiguanide (PBG, 4 μg kg–1), adenosine 5'-triphosphate (ATP, 0.6 mg kg–1), adenosine (170 μg kg–1) and lactic acid (8 mg kg–1). The volume of each bolus injection was 0.1 ml, which was first slowly injected into the catheter (dead space 0.2 ml) and then flushed into the right atrium by an injection of 0.3 ml saline. Only one chemical agent was studied in a given animal. Constant-pressure lung inflation was applied by inflating the lung with a constant air flow (12 ml s–1) until Pt reached 30 cmH2O, and maintained at that pressure for 10 s after turning off the respirator. In Study series 2, the possible involvement of TRPV channels in the temperature sensitivity of pulmonary C-fibres was investigated. At the H level of Tit, the baseline activity and the response to lung inflation were determined and compared before, during and after recovery from administration of CPZ (0.3 mg kg –1min–1 for 20 min, I.V.), a selective antagonist of the TRPV1 receptor; Tit was elevated to H during the last 3 min of CPZ infusion. The same protocols were followed in a separate group of rats to determine the blocking effect of ruthenium red (ruthenium oxychloride ammoniated, RR), an inorganic dye and a non-specific blocker of Ca2+ channels (Hamilton & Lundy, 1995); RR is also known for its non-selective but effective blocking effect on all the TRPV channels (Clapham et al. 2001; Gunthorpe et al. 2002). The C-fibre response to hyperthermia was tested 10 min after the RR injection (0.3 mg kg–1, I.V.). In Study series 3, a possible involvement of cyclooxygenase metabolites of arachidonic acid in the C-fibre sensitivity to hyperthermia was determined. At the H level of Tit, the baseline activity and the responses to lung inflation and capsaicin injection were determined and compared before and 30 min after administration of indomethacin (10 mg kg–1, I.V.), a cyclooxygenase inhibitor. In Study series 4, the effects of increasing Tit on the excitability of two other major types of vagal pulmonary receptors were investigated; the baseline activity and the responses to lung inflation (Pt= 30 cmH2O) and deflation (Pt= 0 cmH2O) of SARs and RARs were determined and compared between three different levels of Tit (C, M and H) in each receptor. In Study series 5, the relationship between the C-fibre baseline activity and Tit was determined by progressively increasing the temperature in the perfusate reservoir, which led Tit to rise continuously in a ramp fashion from C (36°C) to H (41°C) in 60 s. In this study series, the baseline FA was measured as the reciprocal of the interspike interval or analysed in 20 s intervals (when the fibre was quiescent for >20 s).
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    Materials

    Stock solution of capsaicin (250 μg ml–1) was prepared in 1% Tween 80, 1% ethanol and 98% saline. Stock solutions of PBG (1 mg ml–1), ATP (20 mg ml–1) and adenosine (10 mg ml–1) were prepared in saline and stored at –20°C. Injected solutions of these chemical agents at desired concentrations were then prepared daily by dilution with isotonic saline on the basis of the animal's body weight. Lactic acid (30% solution) was stored at 4°C and diluted in distilled water. CPZ was first dissolved in dimethyl sulphoxide at a concentration of 40 mg ml–1, and further diluted with saline containing 10% Tween 80 and 10% ethanol, to a final concentration of 3 mg ml–1. RR (3 mg ml–1) solution was prepared daily in isotonic saline. Indomethacin (5 mg ml–1) was dissolved in a mixture of polyethylene glycol and saline (1: 1 ratio). All chemical agents were purchased from Sigma Chemical (St Louis, MO, USA).
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    Statistical analysis

    The change in FA (FA) in response to a stimulus was calculated as the difference between the peak FA and the baseline FA (60 s average) in each fibre. The peak response of FA was averaged over 2 s intervals after injections of capsaicin, PBG and lactic acid, and 10 s intervals after the injections of ATP and adenosine, because the fibre discharge evoked by the latter two chemicals lasted substantially longer. In the response to lung inflation or deflation, FA was averaged over the 10 s duration of inflation or deflation. Data were then analysed with a one-way repeated-measures 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 reported as means ±S.E.M.
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    Results

    When Tit was raised from C (35.9 ± 0.1°C) to M (38.5 ± 0.1°C) and H (41.1 ± 0.1°C), the rectal temperature increased only very slightly at the end of the 3 min perfusion (n= 65, P < 0.05; Table 1); heart rate also increased slightly but significantly (P < 0.05) during hyperthermia (Table 1). However, there was no detectable change in mean arterial blood pressure (P > 0.05; Table 1). The increases in both rectal temperature and heart rate returned to control shortly (<5 min) after the Tit was returned to C by switching to the perfusate at 36°C.
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    A total of 88 pulmonary C-fibres, 10 RARs and 10 SARs were studied in 96 rats. The distribution of locations of these receptors was as follows: 18 in the upper lobe (15 C-fibres, 2 RARs, 1 SAR); 28 in the middle lobe (22 C-fibres, 3 RARs, 3 SARs); 36 in the lower lobe (26 C-fibres, 5 RARs, 5 SARs), and 7 in the accessory lobe (6 C-fibres, 1 SAR); all were located in the right lung. The locations of the remaining 19 C-fibres were not identified, but all of them were activated by lung inflation and responded to bolus injection of capsaicin with a latency of <1 s; these receptors were therefore considered to be pulmonary C-fibres (Ho et al. 2001).
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    Study series 1

    Pulmonary C-fibres had either no or very low and irregular baseline activity when Tit was maintained at the C level (Figs 1 and 2). When Tit was raised from C to M, the baseline activity of pulmonary C-fibres did not change significantly (Fig. 2). Although the responses to lung inflation and capsaicin injection increased (FA > 50% baseline FA) in 31.6% and 27.3% of the C-fibres, respectively, when Tit reached the M level (e.g. Fig. 1), the group data showed no significant increase in either of these responses (Fig. 2). In sharp contrast, when Tit was elevated to the H level, pulmonary C-fibre activity increased drastically (e.g. Fig. 1), and the average baseline FA increased almost five-fold: 0.04 ± 0.02 impulses s–1 (imp s–1) at C and 0.22 ± 0.06 imp s–1 at H (P < 0.05; Fig. 2). Furthermore, the C-fibre responses to lung inflation and capsaicin injection were also markedly increased; for example, FA induced by capsaicin injection was 5.94 ± 1.65 imp s–1 at C and 13.13 ± 2.98 imp s–1 at H (n= 11, P < 0.05; Fig. 2). These increased C-fibre responses completely returned to control 30 min after Tit was returned to C level. The responses to other chemical stimulants of pulmonary C-fibres (e.g. PBG, ATP and adenosine) were also markedly increased when Tit was elevated to H (Fig. 3), with only one exception: the C-fibre response to lactic acid was only slightly increased at H, and the group data were not significantly different from those at control (Fig. 3). None of the responses to these chemical stimulants were significantly elevated when Tit was raised to M (Fig. 3).
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    Left panels, fibre activity (FA) was analysed by computer in 0.5 s intervals. Upper, average baseline FA (n= 53; data of only 30 s are shown to avoid clustering); middle, average response to lung inflation (Pt= 30 cmH2O for 10 s; n= 19), depicted by the horizontal bar; lower, average response to right-atrial injection of capsaicin (arrow, 0.5 μg kg–1 in 0.1 ml; n= 11). Responses were tested at three different levels of Tit in each fibre: control (36°C), medium (38.5°C) and high (41°C). Each level of Tit was maintained for 3 min, and at least 30 min elapsed between tests in each fibre; the average Tit is shown in each panel. Right panels, peak responses at three different levels of Tit. FA represents the difference between the peak FA (average over 10 s and 2 s intervals for lung inflation and capsaicin, respectively) and the baseline FA (average over 60 s intervals) in each fibre. Responses during recovery were not tested in every fibre due to time limitation; recovery data shown in upper, middle and lower panels were collected from 36, 13 and 10 fibres, respectively. *Significantly different (P < 0.05) from the corresponding data at control and medium Tit, respectively. Data are means ±S.E.M.
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    Average peak responses of pulmonary C-fibres to right-atrial injection of PBG (4 μg kg–1, n= 10), ATP (0.6 mg kg–1, n= 9), adenosine (170 μg kg–1, n= 10), and lactic acid (8 mg kg–1, n= 13) were measured when Tit was maintained at three different levels: control (36°C), medium (38.5°C) and high (41°C). Chemicals (in 0.1 ml) were first slowly injected into the catheter and then flushed into the right atrium as a bolus with saline (0.3 ml). FA represents the difference between the peak FA (average over 2 s intervals for PBG and lactic acid injections, and 10 s intervals for ATP and adenosine injections), and the baseline FA (average over 60 s intervals). *Significantly different (P < 0.05) from the corresponding data at control and medium Tit, respectively. Data are means ±S.E.M.
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    Although both rectal temperature and heart rate returned to their prehyperthermia control levels shortly (<5 min) after Tit was returned to 36°C, the increases in the baseline activity and the sensitivity to chemical stimulation and lung inflation of pulmonary C-fibres persisted for a substantially longer duration (>15 min) in five of the fibres tested.

    Study series 2

    As shown in our previous report (Lee & Lundberg, 1994), I.V. infusion of CPZ at a constant rate of 0.25–0.35 mg kg –1min–1 for 20 min did not change the baseline heart rate or mean ABP, but significantly increased the arterial pulse pressure (P < 0.05). This dose of CPZ completely abolished the stimulatory effect of capsaicin (0.5 μg kg–1) regardless whether Tit was at C or H (data not shown), indicating that the dose of CPZ administered was sufficient to block the TRPV1 receptor. However, CPZ treatment did not significantly alter the increase in either the baseline FA or the response to lung inflation induced by increasing Tit to H (Figs. 4A and 5). In contrast, RR (0.3 mg kg–1) significantly reduced both the baseline FA and the response to lung inflation (30 cmH2O) of the pulmonary C-fibres at H (baseline FA at H: before RR, 0.20 ± 0.05 imp s–1; after RR, 0.07 ± 0.03 imp s–1; n= 10, P < 0.05) (FA to lung inflation at H: before RR, 2.41 ± 0.35 imp s–1; after RR, 1.49 ± 0.53 imp s–1; n= 10, P < 0.05) (Figs. 4B and 5). This dose of RR was sufficient to block the response to capsaicin injection (0.5 μg kg–1) completely in the same group of pulmonary C-fibres. The effect of RR was reversible after 30 min.
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    A, receptor location, right lower lobe; rat body weight, 400 g. B, receptor location, right middle lobe; rat body weight, 390 g. During hyperthermia, Tit was elevated from control (36°C) to high (41°C) for 3 min, and lung inflation (Pt= 30 cmH2O) was tested during the last 30 s of hyperthermia. CPZ was infused I.V. at 0.3 mg kg –1min–1 for 20 min, and RR was injected I.V. slowly at 3 mg kg–1; hyperthermia was then applied during the last 3 min of CPZ infusion or at 10 min after the RR injection, and again after 30 min recovery. The measured Tit is shown at the top left corner of each panel. At least 30 min were allowed to elapse between tests to avoid possible tachyphylaxis. Notice that the activity of another receptor (smaller height of the action potential) in B also increased when Tit was raised to 41°C. See legend of Fig. 1 for further explanation.
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    Upper panels: baseline FA (average over 60 s intervals); lower panels: FA, the difference between the peak FA (average over the 10 s duration of lung inflation) and the average baseline FA (60 s) in each fibre. During hyperthermia, Tit was elevated from control (36°C) to high (41°C) for 3 min, and lung inflation (Pt= 30 cmH2O) was tested during the last 30 s of hyperthermia. CPZ was infused I.V. at 0.3 mg kg –1min–1 for 20 min (n= 10), and RR was injected I.V. slowly at 3 mg kg–1 (n= 10); hyperthermia was then applied during the last 3 min of CPZ infusion or 10 min after the RR injection, and again after 30 min recovery. *Significantly different (P < 0.05) from the corresponding data at control and high Tit, respectively. Data are means ±S.E.M.
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    Study series 3

    Indomethacin (10 mg kg–1) pretreatment did not significantly alter the increase in either the baseline FA or the responses to lung inflation (30 cmH2O) and capsaicin injection (0.5 μg kg–1) induced by increasing Tit to H (Fig. 6). For example, the baseline FA at H was 0.21 ± 0.06 imp s–1 and 0.21 ± 0.05 imp s–1 (P > 0.05, n= 7) before and after indomethacin, respectively. Similarly, pretreatment with indomethacin failed to prevent the hyperthermia-induced potentiation of the responses to lung inflation and capsaicin (Fig. 6).
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    Left panel: baseline FA (average over 60 s intervals); right and middle panels: FA, the difference between the peak FA (average over 2 s after capsaicin injection and average over 10 s duration of lung inflation) and the average baseline FA (60 s) in each fibre. During hyperthermia, Tit was elevated from control (36°C) to high (41°C) for 3 min; capsaicin injection (0.5 μg kg–1) and lung inflation (Pt= 30 cmH2O) were tested during the last 30 s of hyperthermia. *Significantly different (P < 0.05) from the corresponding data at control. Data are means ±S.E.M. (n= 7).
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    Study series 4

    At control Tit, all the SARs exhibited distinct phasic baseline activity that peaked during the inspiratory phase of the respiratory cycles. The phasic discharge was present during the expiratory phase in 8 out of 10 RARs, and there was no phasic baseline activity in the remaining two. When constant-pressure lung inflation (Pt= 30 cmH2O) was applied for 10 s, the discharge in RARs increased abruptly but ceased rapidly (<2 s). When prolonged lung deflation was produced by exposing the tracheal catheter to atmospheric pressure (Pt= 0 cmH2O) in the open-chest preparation, the RAR discharge declined to 40% and was then maintained at that level (Fig. 7). In contrast, the discharge in SARs was sustained during the entire duration of lung inflation, but ceased completely during deflation (Fig. 7). Increasing the Tit to the levels of M and H in an identical manner as that in Study series 1 did not induce any detectable change in the intensity and pattern of the baseline activity (averaged over 60 s duration) from those at control Tit in either RARs (19.0 ± 4.6 imp s–1 at C; 18.2 ± 5.1 imp s–1 at M; 20.0 ± 5.0 imp s–1 at H; n= 10, P > 0.05) or SARs (35.5 ± 8.4 imp s–1 at C; 35.1 ± 8.8 imp s–1 at M; 36.7 ± 8.7 imp s–1 at H; n= 10, P > 0.05) (Fig. 7). Furthermore, the responses of RARs and SARs to constant-pressure lung inflation and deflation were not altered by increasing Tit to M or H (P > 0.05; Fig. 7). In addition, the effect of increasing Tit to H on the FA response to capsaicin was tested in five RARs, including the two receptors without phasic activity. Even at H, none of these RARs were activated by the bolus injection of capsaicin at a much higher dose (2 μg kg–1) than that applied to C-fibres.
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    Afferent responses to lung inflation (Pt= 30 cmH2O for 10 s; upper panels) and lung deflation (Pt= 0 cmH2O for 10 s; lower panels) when Tit was maintained at C (36°C, ) and H (41°C, ) were measured in 10 RARs (left panels) in 10 rats and 10 SARs (right panels) in 10 other rats. Fibre activity (FA) was measured in 0.1 s intervals by computer. Data are means ±S.E.M.

    Study series 5

    This study series was carried out to examine more closely the relationship between fibre activity and Tit, and to determine more precisely the temperature threshold for pulmonary C-fibre activation during hyperthermia. When Tit was increased progressively from 36°C to 41°C in a ramp pattern over 60 s, it was apparent that the baseline FA of pulmonary C-fibres did not start to increase until Tit exceeded 39–40°C in six of the seven fibres studied (e.g. Fig. 8); the remaining fibre was not activated even after Tit exceeded 41°C. The relationship between baseline FA and Tit was analysed by piecewise linear regression method in all six fibres, and the inflection point was identified by the lowest value of the residual sum of squares of FA expressed as a function of temperature (Vieth, 1989). The temperature coefficient (the slope of the regression line) was 0.011 ± 0.005 imp s–1°C–1 below the inflection point, and increased drastically when Tit exceeded the inflection point (0.407 ± 0.125 imp s–1°C–1; n= 6, P < 0.05). The average threshold temperature for activation was 39.2 ± 0.4°C (n= 6), which was determined by the point of intersection of the two regression lines (e.g. bottom of Fig. 8).
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    Baseline fibre activity (FA) was measured when Tit was increased progressively in a ramp fashion from 36°C to 41°C in 60 s by gradually raising the temperature in the perfusate reservoir. The relationship between the corresponding data of FA and Tit is then plotted in the lower panel, and analysed by the piecewise linear regression method (for details, see Results); FA was measured as the reciprocal of the interspike interval or analysed in 20 s intervals when the fibre was quiescent for >20 s. The temperature threshold for activating this fibre is 39.7°C, determined by the intersection point (arrow of the broken line) of lines A and B. Notice the drastic increase in FA as Tit exceeded 39.7°C, which is also illustrated by the distinct increase in the slope of the FA–Tit relationship: 0.018 imp s–1°C–1 in line A (r= 0.889) and 0.685 imp s–1°C–1 in line B (r= 0.730). Receptor location, right upper lobe; rat body weight, 375 g.
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    Discussion

    Our results show that increasing the intrathoracic temperature to 41°C induced a significant and consistent increase in the baseline activity of vagal pulmonary C-fibres. Furthermore, intrathoracic hyperthermia also produced a distinct elevation in the sensitivities of these afferents to chemical stimulants and to lung inflation. These effects were reversible after Tit was returned to normal body temperature, and were reproducible in the same fibre after 30 min of recovery. No significant change was found at the intermediate level of hyperthermia (38.5°C) in the same fibres. In addition, the same levels of hyperthermia did not induce any change in either the baseline activity or the sensitivity to lung inflation in the other two major types of lung afferents, RARs and SARs. These results suggest that the sensitivity to hyperthermia represents a characteristic feature of the capsaicin-sensitive pulmonary C-fibres.
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    Tissue hyperthermia occurs when the rate of heat production is elevated, or heat dissipation is diminished, either locally or systemically. For example, hyperthermia is generated during vigorous exercise or under pathophysiological conditions caused by infection or endogenous pyrogens (e.g. cytokines, etc.). Body temperature exceeding 41°C has been reported during exertional exercise in healthy individuals (Pugh et al. 1967; Greenleaf, 1979) as well as in patients suffering from acute heatstroke (Bouchama et al. 1991). In addition, body temperature rising above 40°C is frequently found in patients with severe fever. Furthermore, an increase in local tissue temperature is generally considered as one of the common signs of tissue inflammation. For example, the tissue temperature in the inflamed areas was elevated by 3–4°C above the normal temperature when an inflammatory reaction was induced in the rat paw (Planas et al. 1995). A recent report has further revealed a higher tissue temperature in the airways of asthmatic patients (Paredi et al. 2002). Taken together, the intrathoracic temperature applied in this study is certainly within the physiological range.
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    Bronchopulmonary C-fibres represent >75% of the vagal afferents innervating the airways and lung structures (Jammes et al. 1982). Several studies have clearly illustrated the presence of C-fibre sensory endings in the airway mucosa in a number of species including humans (Komatsu et al. 1991; Adriaensen et al. 1998). The exquisite sensitivity of these sensory endings to inhaled chemical irritants (e.g. cigarette smoke, etc.) and certain endogenous chemical substances (e.g. lactic acid, etc.) is one of the most important properties and functions of these afferents (Coleridge & Coleridge, 1984; Lee & Pisarri, 2001). One of the anatomical characteristics of these sensory endings is the prominent presence of numerous varicosities containing tachykinins (TKs) and calcitonin gene-related peptide (CGRP) along the length of single axons. Because of the superficial location of the sensory terminals in the airway lumen, and the potent biological activities of TKs, these afferents play a very important role in the regulation of airway protective functions under both normal and pathophysiological conditions (Coleridge & Coleridge, 1984; Lee & Pisarri, 2001). Activation of these afferents is known to evoke airway irritation and cough, and to elicit reflex responses such as bronchoconstriction and hypersecretion of mucus, which are mediated through the central nervous system and cholinergic pathway (Coleridge & Coleridge, 1984; Lee & Pisarri, 2001). In addition, TKs and CGRP released locally from these nerve endings upon activation can produce airway constriction, protein extravasation, mucosal oedema and chemotactic effects on various inflammatory cells (Lundberg & Saria, 1987; Solway & Leff, 1991). Intense and/or sustained stimulation of these endings can lead to the development of ‘neurogenic inflammatory reaction’ in the airways (Lundberg & Saria, 1987; Solway & Leff, 1991). Based upon the results obtained in this study, it seems reasonable to suggest that an increase in the local tissue temperature in the lung (e.g. during airway inflammation) (Paredi et al. 2002) may activate and sensitize C-fibre endings. Thus, these afferent endings will be more sensitive to inhaled irritants and certain endogenous chemical mediators. Consequently, both cholinergic and tachykininergic effects resulting from stimulation of these afferents will be augmented.
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    The mechanisms underlying the hyperthermia-induced C-fibre hypersensitivity observed in this study are not fully understood. An increase in temperature is known to increase the rate of gating of all voltage-gated ion channels in neurones, because the rates of conformational changes of channel proteins are temperature sensitive (Schwarz, 1986). However, this non-specific action seems unlikely to account for the distinct sensitizing effect of hyperthermia on pulmonary C-fibres in this study for the following reasons. The same increase in intrathoracic temperature did not elevate either the baseline activity or the sensitivity to lung inflation/deflation in RARs and SARs (Fig. 7) that are innervated by larger diameter, faster conducting myelinated axons. Furthermore, a distinctly higher temperature coefficient during hyperthermia calculated from the FA–Tit relationship in six of the seven fibres tested clearly indicates the presence of temperature-sensitive channel(s) in these afferents (Pehl et al. 1997). The threshold temperature for activating these channels is indicated by the abrupt change in the slope of the FA–Tit curve during the progressive increase in Tit (e.g. Fig. 8). Taken together, the observed effect seems to result from the action of hyperthermia on a temperature-sensitive ion channel(s) that is expressed selectively on the non-myelinated C-fibre endings.
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    We cannot identify the specific ion channel(s) that mediates the responses in this study, but certain potential candidates merit careful consideration. Transient receptor potential (TRP) channels are a family of non-voltage-gated cation channels, and TRPVs are a subfamily known as temperature-gated ion channels (Clapham et al. 2001; Gunthorpe et al. 2002; Benham et al. 2003). The temperature threshold for activation varies among different subtypes of mammalian heat-sensitive TRPV receptors: 43°C, 52°C, 31–39°C and 24–33°C for TRPV1, TRPV2, TRPV3 and TRPV4, respectively (Benham et al. 2003); the thresholds for the last two, TRPV3 and TRPV4, vary within a range between different reports (Benham et al. 2003). TRPV1 and TRPV2 are believed to be the primary transducers for detecting thermal stimulation in the DRG nociceptive neurones (Caterina et al. 1997, 1999; Benham et al. 2003), whereas TRPV3 and TRPV4 are recognized as the temperature sensor for the warm-sensitive receptor (Smith et al. 2002; Benham et al. 2003). Functional expression of the TRPV1 receptor at the vagal bronchopulmonary C-fibre terminals has been clearly documented (Lee & Lundberg, 1994; Ho et al. 2001; Carr et al. 2003). However, the intrathoracic temperature applied in this study never reached 43°C, the threshold for activating the TRPV1 receptor (Caterina et al. 1997; Vlachova et al. 2001). Moreover, capsazepine, a selective antagonist of the TRPV1 (Bevan et al. 1992), applied at a dose sufficient to block the stimulatory effect of capsaicin on pulmonary C-fibres did not significantly alter the sensitizing effect of hyperthermia (Figs 4 and 5). On the basis of these results, we believe that activation of TRPV1 is not primarily responsible for generating the responses observed in this study. On the other hand, we cannot rule out the possible involvement of the TRPV3 and/or TRPV4 receptors, because their reported threshold temperatures for activation (Benham et al. 2003) are either below or within the temperature range reached during the hyperthermia condition in our experiment. In addition, ruthenium red, known to be a non-selective but effective blocker of the TRPV channels (Clapham et al. 2001; Gunthorpe et al. 2002), significantly attenuated the hyperthermia-induced potentiation of C-fibre responses (Figs 4 and 5). A recent study demonstrating the functional expression of TRPV receptors in pulmonary C neurones (Gu et al. 2005) has lent further support to this hypothesis. Activation of these TRPV receptors by hyperthermia may therefore evoke inward current that leads to membrane depolarization from the resting potential of the nerve terminal and shortening of the intervals between action potentials, thus increasing its excitability. However, despite the fact that the weight of the evidence seems to support such a possibility, the lack of specific antagonists of the TRPV3 and TRPV4 receptors precludes our ability to reach a more definitive conclusion.
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    Since the hyperthermia-induced increases in baseline activity and sensitivity were not completely abolished by ruthenium red (Fig. 5), other factors in addition to TRPV channels should also be considered. After the hyperthermia challenge was terminated by returning Tit to 36°C, the rectal temperature of the rats as well as the heart rate returned rather quickly (<5 min) to control, but the increases in both the baseline activity and sensitivity of pulmonary C-fibres declined slowly and lingered for a substantially longer duration (>15 min). The cause of this slow recovery of the fibre sensitivity cannot be determined in this study. It is known that the threshold temperature for activating TRPV1 can be modulated by the presence of certain chemical ligands or the phosphorylation states of the channel; for example, the threshold temperature was reduced to 30°C in cloned TRPV1 when pH in the external solution was lowered to 6.3 (Tominaga et al. 1998). Presumably, an increase in the intrathoracic temperature will increase the tissue metabolic rate and therefore the production of CO2 and hydrogen ion locally in the lung tissue. Acute hyperthermia is also known to elevate the levels of several proinflammatory cytokines (e.g. tumour necrosis factor (TNF), interleukin 6 (IL-6), etc.) in the circulating blood (Bouchama et al. 1991; Jiang et al. 1999). Cytokines such as TNF and IL-6 have been shown to sensitize DRG nociceptive neurones and play a part in the inflammatory hyperalgesia (Cunha et al. 1992; Sommer & Kress, 2004). Certain cytokines are known to cause an increased release of prostaglandins (Ericsson et al. 1997), such as PGE2, and PGE2 can enhance the sensitivity of C-fibres in the lung (Lee & Pisarri, 2001). However, the involvement of cyclooxygenase metabolites can be ruled out because the hyperthermia-induced C-fibre hypersensitivity persisted even after applying indomethacin. An increase in tissue temperature has been shown to promote the mitochondrial production of reactive oxygen species (Zuo et al. 2000). Evidence of a stimulatory effect of hydroxyl radical and other reactive oxygen species on pulmonary C-fibres has been previously reported (Lee, 1990; Ruan et al. 2003). Whether and to what extent the enhanced sensitivity of these C-fibre endings is caused by cytokines and other endogenous substances released locally in the lung tissue during hyperthermia remain to be determined.
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    When the intrathoracic temperature was elevated in this study, it was maintained at the new level for 3 min by continuous perfusion of the thoracic chamber to ensure that the tissue temperature in the deeper regions of the lung would rise and reach equilibrium with that of the perfusate. To determine whether there is a difference in the temperature, we turned off the respirator momentarily after Tit had been maintained at 41°C for 0.5–2 min, and inserted a needle thermocouple microprobe (Harvard BS4 52-1732) into the lung parenchyma at various depth (2–6 mm) at the end of the experiment in three rats. Surprisingly, the difference in temperature between lung tissue and perfusate was consistently smaller than 0.3°C, regardless of the depth of the needle probe insertion. However, when the fibre activity and temperature relationship was obtained by progressively increasing the intrathoracic temperature in a ramp fashion in Study series 5, the measured Tit may be slightly higher than the actual tissue temperature in the deeper regions of the lung. Therefore, the threshold temperature (39.2°C) determined from the FA-Tit relationship may reflect a temperature range, instead of the precise temperature, at which these nerve endings are activated. Nevertheless, a consistent increase in the baseline activity of pulmonary C-fibres was detected when Tit exceeded 39–40°C in all but one fibre tested in the ramp-hyperthermia study, which is in general agreement with that found in our steady-state (3 min) hyperthermia experiments. On the other hand, it would be interesting to know whether the thermal sensitivity of these sensory nerves changes during the course of a more sustained (>3 min) hyperthermia.
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    The selection of 36°C as the control temperature in this experiment was based upon the normal body temperature in the intact rat during sleep (Refinetti & Menaker, 1992; Briese, 1998). During wakefulness, the rat core temperature increases to 37–37.5°C at rest (Refinetti & Menaker, 1992; Briese, 1998). However, based on our results (Fig. 2), we do not expect any significant change in either the baseline activity or the sensitivity of pulmonary C-fibres caused by this small increase in body temperature (1.0–1.5°C).
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    In conclusion, intrathoracic hyperthermia induces a distinct stimulatory and sensitizing effect on vagal pulmonary C-fibre afferents, whereas the same level of hyperthermia does not alter the sensitivity of myelinated pulmonary afferents. Hence, the sensitivity to hyperthermia represents a characteristic feature of the afferent properties of pulmonary C-fibres. Furthermore, the relationship between the C-fibre activity and temperature reveals that the threshold for activation is near 39–40°C. Based upon these results, we further suggest that the sensitizing effect of hyperthermia may be mediated through temperature-sensitive ion channel(s), probably TRPV3 and/or TRPV4, though more definitive evidence is required to determine the extent of their involvement. In view of the important role of the pulmonary C-fibre afferents in the regulation of airway functions both in health and disease, the physiological implications and consequence of this potentiating effect of hyperthermia on these sensory nerves certainly need to be further explored.
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