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J Appl Physiol 101: 644-654, 2006. First published April 20, 2006; doi:10.1152/japplphysiol.00192.2006
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HIGHLIGHTED TOPIC
Reflexes From the Lung and Airways

Mediator mechanisms involved in TRPV1 and P2X receptor-mediated, ROS-evoked bradypneic reflex in anesthetized rats

Ting Ruan,1,2 You Shuei Lin,3 Kae-Shin Lin,4 and Yu Ru Kou1

Departments of 1Physiology and 4Pharmacology, School of Medicine, National Yang-Ming University, Taipei; 2Department of Physiology, College of Medicine, Chung-Shan Medical University, Taichung; and 3Department of Physiology, Taipei Medical University, Taipei, Taiwan

Submitted 15 February 2006 ; accepted in final form 11 April 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Inhalation of H2O2 is known to evoke bradypnea followed by tachypnea, which are reflexes resulting from stimulation by reactive oxygen species of vagal lung capsaicin-sensitive and myelinated afferents, respectively. This study investigated the pharmacological receptors and chemical mediators involved in triggering these responses. The ventilatory responses to 0.2% aerosolized H2O2 were studied before and after various pharmacological pretreatments in anesthetized rats. The initial bradypneic response was reduced by a transient receptor potential vanilloid 1 (TRPV1) receptor antagonist [capsazepine; change ({Delta}) = –53%] or a P2X purinoceptor antagonist [iso-pyridoxalphosphate-6-azophenyl-2',5'-disulphonate (PPADS); {Delta} = –47%] and was further reduced by capsazepine and iso-PPADS in combination ({Delta} = –78%). The initial bradypneic response was reduced by a cyclooxygenase inhibitor (indomethacin; {Delta} = –48%), ATP scavengers (apyrase and adenosine deaminase in combination; {Delta} = –50%), or capsazepine and indomethacin in combination ({Delta} = –47%), was further reduced by iso-PPADS and indomethacin in combination ({Delta} = –75%) or capsazepine and ATP scavengers in combination ({Delta} = –83%), but was not affected by a lipoxygenase inhibitor (nordihydroguaiaretic acid) or by any of the various vehicles. No pretreatment influenced delayed tachypnea. We concluded that 1) the initial bradypneic response to H2O2 results from activation of both TRPV1 and P2X receptors, possibly located at terminals of vagal lung capsaicin-sensitive afferent fibers; 2) the functioning of the TRPV1 and P2X receptors in triggering the initial bradypnea is, in part, mediated through the actions of cyclooxygenase metabolites and ATP, respectively; and 3) these mechanisms do not contribute to the H2O2-evoked delayed tachypnea.

lung; vagal sensory receptors; sensory transduction; cyclooxygenase metabolites; adenosine 5'-triphosphate

VARIOUS LUNG DISEASES ARE manifested by increased production of pulmonary reactive oxygen species (ROS) (10, 13, 14, 44, 46, 48, 62). Excess pulmonary ROS have been suggested to stimulate vagal lung afferent fibers and trigger respiratory reflexes under pathological conditions, such as pulmonary air embolism or sepsis (8, 9, 30), or when the airways are insulted by inhaled irritants, such as cigarette or wood smoke (19, 26, 28, 29, 33, 36, 51, 53). These observations promote the concept that vagal lung afferent fibers may function as a sensor system that detects excess pulmonary ROS. However, the underlying mechanisms are still incompletely understood.

Our laboratory recently reported (51) that inhalation of aerosolized H2O2 acutely evokes initial reflex bradypnea followed by delayed reflex tachypnea and that these responses may result from stimulation of vagal lung capsaicin-sensitive and myelinated afferents, respectively. A subsequent electrophysiological study (53) revealed that delivery of aerosolized H2O2 indeed stimulates capsaicin-sensitive vagal lung afferent fibers, and this sensory transduction is mediated through transient receptor potential vanilloid 1 (TRPV1) receptors and P2X purinoceptors. Both the reflex (51) and afferent responses (53) to H2O2 are largely or totally suppressed by antioxidants targeted at H2O2 or hydroxyl radicals, suggesting the involvement of ROS. Capsaicin-sensitive vagal lung afferent fibers are composed of mainly C fibers and some A{delta} fibers, which are important to the regulation of respiratory functions under pathophysiological conditions (6, 35, 59). The TRPV1 and P2X receptors are two ligand-gated nonselective cation channels (41, 56) that are located at terminals of these vagal lung afferent fibers (5, 15, 25, 34, 47, 58, 60). While the importance of TRPV1 and P2X receptors in the sensory transduction of pulmonary ROS is recognized, their relative contributions to H2O2-evoked initial bradypnea and delayed tachypnea remain to be delineated.

TRPV1 receptors are activated by multiple stimuli, such as capsaicin, noxious heat, acid, and several products of arachidonate metabolism (5, 15, 16, 25, 37, 41, 56, 58). They can be viewed as an integrator of painful chemical and physical stimuli of the somatosensory system (56). On the other hand, P2X receptors can be activated only by ATP and its congeners (49). ROS are known to increase the release of arachidonate metabolites in lung tissue (4, 7, 40). To synthesize these metabolites, arachidonic acid is converted to prostaglandins and thromboxane via cyclooxygenase or to leukotrienes via lipoxygenase (2). Additionally, ROS may cause rapid release of cytosolic ATP, which activates the P2X receptors of pain nociceptors in the vicinity (11, 41). After its formation, ATP is sequentially degraded by enzymes to ADP, AMP, and adenosine (45). Collectively, it is possible that arachidonate metabolites and ATP are part of a signaling cascade for the sensory transduction of pulmonary ROS, leading to elicitation of respiratory reflexes. Nevertheless, this possibility remains to be investigated.

The present study was undertaken in anesthetized rats 1) to investigate the role of TRPV1 and P2X receptors in triggering initial bradypnea and delayed tachypnea evoked by inhalation of aerosolized H2O2, 2) to assess the involvement of arachidonate metabolites and ATP in triggering these ventilatory responses, and 3) to delineate the chemical mediator mediating the functioning of the TRPV1 or P2X receptors in triggering these ventilatory responses. To accomplish these objectives, the acute ventilatory responses to aerosolized H2O2 were compared before and after pretreatment with antagonists of the TRPV1 and P2X receptors, inhibitors of cyclooxygenase and lipoxygenase, and scavengers of ATP.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Animal preparation.   Male Sprague-Dawley rats were anesthetized with an intraperitoneal injection of chloralose (100 mg/kg; Sigma Chemical, St. Louis, MO) and urethane (500 mg/kg; Sigma) dissolved in a borax solution (2%; Sigma). A polyethylene catheter was inserted into the jugular vein and advanced until the tip was close to the right atrium to allow intravenous administration of pharmacological agents. The right femoral artery was cannulated to allow measurement of the arterial blood pressure (ABP). During the course of the experiments, supplemental doses of chloralose (20 mg·kg–1·h–1) and urethane (100 mg·kg–1·h–1) were administered to maintain abolition of the corneal reflex and pain reflexes induced by pinching the animal’s tail. Body temperature was maintained at ~37°C throughout the experiment by means of a servo-controlled heating blanket.

The animal’s neck was opened along the midline. A short tracheal cannula was inserted just below the larynx via a tracheostomy and connected to a pneumotachograph (Fleisch 4/0, Richmond, VA), through which the animals breathed spontaneously. Respiratory flow was measured using a pneumotachograph coupled to a differential pressure transducer (Validyne MP45–12, Northridge, CA). The flow signal was integrated to give tidal volume (VT). Tracheal pressure was monitored using a pressure transducer (Validyne MP45–28) via a side tap to the tracheal cannula. All physiological signals were recorded on a chart recorder (Gould TA11, Cleveland, OH) and a tape recorder (Neurocorder DR-890, New York, NY) for later analysis. All protocols were in accordance with the recommendations found in the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health, USA, and were approved by the Institutional Animal Care and Use Committee of the National Yang-Ming University, Taiwan.

Generation and delivery of aerosolized H2O2.   Generation and delivery of aerosolized H2O2 were achieved by methods that have been described previously (51). In brief, H2O2 aerosol was generated by delivering an airstream (15 ml/s) through the cup of an active ultrasonic nebulizer (ULTRA-NEB 99, DeVilbiss, Sunrise Medical, Carlsbad, CA), that contained H2O2 solution (0.2%). Airway exposure to aerosolized H2O2 was achieved by turning a three-way stopcock for a 30-s period; this three-way stopcock communicates with the distal end of the pneumotachograph and a side arm of the tubing (8 mm inner diameter) attached to the outlet of the nebulizer. Through this connection, H2O2 aerosol was spontaneously inhaled into the lower airways and lungs. Before each H2O2 challenge, the animal’s lungs were hyperinflated (tracheal pressure >20 cmH2O) to establish a constant volume history (43). Using a dye tracer, the time lag between the onset of challenge and the arrival of the aerosolized tracer in the airways was estimated to be <1 s.

Pharmacological agents.   The stock solutions of capsazepine (CPZ; a TRPV1 receptor antagonist; 10 mg/ml), indomethacin (Indo; a nonselective cyclooxygenase inhibitor; 20 mg/ml), and nordihydroguaiaretic acid (NDGA; a nonselective lipoxygenase inhibitor; 20 mg/ml) (55) were prepared by first dissolving the chemicals in dimethyl sulfoxide at a concentration of 40, 100, and 100 mg/ml, respectively, and then were further diluted with saline containing 10% Tween 80 and 10% ethanol. The stock solution of capsaicin (a TRPV1 receptor agonist; 250 µg/ml) was prepared by dissolving the chemical in a solution containing 10% Tween 80, 10% ethanol, and 80% saline. The stock solutions of iso-pyridoxalphosphate-6-azophenyl-2',5'-disulphonate (iso-PPADS; a P2X receptor antagonist; 50 mg/ml) (22, 49), {alpha},beta-methylene-ATP ({alpha}beta-meATP; a P2X receptor agonist; 2 mg/ml) (24, 47), and phenylbiguanide (a serotonin 5-HT3 receptor agonist; 2 mg/ml) (18, 34) were prepared by dissolving the chemical in saline. The stock solution of apyrase (an enzyme that catalyses breakdown of ATP to AMP; 400 U/ml) was prepared by dissolving the chemical in PBS. The stock solution of adenosine deaminase (ADA; an enzyme that catalyses breakdown of adenosine; 417 U/ml) was prepared by dissolving the chemical in 50% glycerol and 50 mM potassium phosphate and was further diluted to a concentration of 200 U/ml by PBS. A combination of apyrase and ADA has previously been used as ATP scavengers (1, 54). The stock solutions of CPZ, Indo, NDGA, and ADA, which were stored at 4°C; the others were stored at –20°C. CPZ (3 mg/kg; 1 min before H2O2 challenge), iso-PPADS (15 mg/kg; 15 min before H2O2 challenge), Indo (5 mg/kg; 20 min before H2O2 challenge), NDGA (5 mg/kg; 20 min before H2O2 challenge), and their vehicles (20 min before H2O2 challenge), in a volume of 0.2 ml, were slowly injected into the vein over 30 s. Capsaicin, {alpha}beta-meATP, and phenylbiguanide, in a volume of 0.2 ml, were separately injected into the vein as a bolus at doses of 0.75, 15, and 6 µg/kg, respectively. Each of these injections was then flushed into the right atrium by an injection of 0.4 ml saline. Injected solutions of these chemical agents at the desired concentrations were prepared daily by diluting with saline on the basis of the animal’s body weight. ADA (10 U/rat), a combination of apyrase (20 U/rat) and ADA (10 U/rat), or their vehicles, in a volume of 0.1 ml, was directly instilled into the airways via the tracheal cannula 10 min before H2O2 challenge, and the animal was rotated from head up to head down and lying on its right and then its left side several times to generalize the distribution of the drug. Before each airway instillation, the animal’s lungs were hyperinflated (tracheal pressure >20 cmH2O) to establish a constant volume history (43). The doses and effective time of application of these agents were adopted or derived from studies reported previously (18, 24, 34, 47, 53, 55) or were indicated by our preliminary study. The doses of CPZ and iso-PPADS were chosen also because they could block the reflex apneic responses to capsaicin and {alpha}beta-meATP, respectively, in our preliminary study. The effects, doses, and vehicles of these pharmacological agents are summarized in Table 1. Except for iso-PPADS (Tocris Cookson, Ellisville, MO) and NDGA (Cayman Chemical, Ann Arbor, MI), the others drugs were purchased from Sigma.


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  		Table 1. Pharmacological agents used in this study

 
Experimental design and protocols.   In this study, 150 rats (weight 350–450 g) were evenly divided into 15 groups to conduct four series of experiments. In the first study series, an investigation into the role of the TRPV1 and P2X receptors was carried out, and the ventilatory response to H2O2 challenge was studied before and after pretreatment with CPZ (group 1), iso-PPADS (group 2), a combination of CPZ and iso-PPADS (CPZ+iso-PPADS; group 3), or their vehicles (vehicle 1; group 4). In the second study series, an investigation into the involvement of arachidonate metabolites and ATP was carried out, and the ventilatory response to H2O2 challenge was studied before and after pretreatment with Indo (group 5), NDGA (group 6), the vehicle for Indo or NDGA (vehicle 2; group 7), a combination of apyrase and ADA (apyrase+ADA; group 8), ADA alone (group 9), or the vehicles for apyrase+ADA (vehicle 3; group 10). In the third study series, an investigation into the chemical mediators responsible for the function of the TRPV1 or P2X receptors was carried out, and the ventilatory response to H2O2 challenges was studied before and after pretreatment with a combination of CPZ and Indo (CPZ+Indo; group 11), a combination of iso-PPADS and Indo (iso-PPADS+Indo; group 12), a combination of CPZ, apyrase, and ADA (CPZ+apyrase+ADA; group 13), or their vehicles (vehicle 4; group 14). In the fourth study series, an investigation into the reflex nature of the observed responses was carried out, and the ventilatory response to H2O2 challenge was studied before and after bilateral cervical vagotomy (group 15). Based on the results of our previous study (51), at least 60 min were allowed to elapse between the two H2O2 challenges to avoid possible tachyphylaxis. To determine whether pretreatment with CPZ or iso-PPADS was effective, the reflex apneic responses induced by an intravenous injection of capsaicin or {alpha}beta-meATP were compared before and after pretreatment. To determine whether the functioning of the vagal lung afferents was affected, reflex apneic responses were induced by an intravenous injection of capsaicin or phenylbiguanide, and these were also compared before and after other pharmacological pretreatments.

Data analysis and statistics.   Respiratory frequency (f), VT, mean ABP, and heart rate were measured at 3-s intervals. An interval of at least 2 min before and 3 min after the H2O2 challenge was measured. Baseline data were calculated as the mean over the reading 2 min immediately before the H2O2 challenge. Because changes in f induced by the H2O2 challenge exhibited a biphasic pattern (51), responses occurring from 0 s to 21 s and from 21 s to 90 s after the onset of the challenge were defined as the initial and delayed responses, respectively. The peak response was defined as the value averaged over 3 s after the H2O2 challenge. These physiological parameters were analyzed using a computer equipped with an analog-to-digital converter (DASA 4600, Gould) and software (BioCybernatics, 1.0; Taipei, Taiwan). Results obtained from the computer analysis were routinely checked with those obtained by manual calculations for accuracy. Data on the cardiopulmonary parameters were compared using a paired t-test and one-way or two-way repeated-measures ANOVA tests followed by Fisher’s least significant difference procedure when appropriate. A value of P < 0.05 was considered significant. All data are presented as the means ± SE.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Control ventilatory responses to H2O2.   Inhalation of 0.2% aerosolized H2O2 consistently evoked an initial bradypnea followed by delayed tachypnea (Fig. 1, left) in all 150 rats studied. As a group (n = 150), the initial decrease in f began at 6.8 ± 0.2 s (range, 3–12 s) after onset of the challenge and lasted for 13.7 ± 0.4 s (range, 5–25 s). Subsequently, f quickly increased and reached its peak at 36.2 ± 0.5 s (range, 25–50 s) after onset of the challenge. This delayed tachypnea lasted for 74.6 ± 3.6 s (range, 20–180 s). On average, both the lowest (68.4 ± 0.9 breaths/min) and peak f (98.3 ± 1.4 breaths/min) values during the initial and delayed periods, respectively, significantly differed from the baseline f (82.9 ± 0.6 breaths/min). Additionally, average VT values during these two periods were significantly smaller than the baseline VT (Table 2). Furthermore, at the onset of or during the delayed tachypneic period, augmented inspiration (3) was also evoked in 88 of the 150 rats studied (Fig. 1, left).


Figure 1
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  		Fig. 1. Experimental records illustrating acute responses to inhalation of 0.2% aerosolized H2O2 in two anesthetized and spontaneously breathing rats. A: responses before and after pretreatment with capsazepine and iso-pyridoxalphosphate-6-azophenyl-2',5'-disulphonate in combination (CPZ+iso-PPADS). B: responses before and after pretreatment with vehicles of CPZ+iso-PPADS (vehicle 1). VR, respiratory flow; VT, tidal volume; ABP, arterial blood pressure. Horizontal bars indicate the duration (30 s) of H2O2 challenge. Between the two H2O2 challenges, 60 min were allowed to elapse. Note that the H2O2 challenge evoked bradypnea followed by tachypnea and an augmented breathing in the control and that the former response was reduced after CPZ+iso-PPADS.

 

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  		Table 2. H2O2-evoked reduction in tidal volume and augmented breaths before and after various experimental interventions in the 15 study groups

 
Role of TRPV1 and P2X receptors in the H2O2-evoked f responses.   Pretreatment with CPZ alone (Fig. 2A), iso-PPADS alone (Fig. 2B), or CPZ+iso-PPADS (Figs. 1A and 2C) significantly suppressed the H2O2-evoked initial bradypnea, but had no effect on the H2O2-evoked delayed tachypnea. In contrast, pretreatment with vehicles (vehicle 1) failed to significantly affect these f responses (Figs. 1B and 2D). After pretreatment with CPZ, iso-PPADS, CPZ+iso-PPADS, and vehicle 1, the initial bradypneic responses were 46.7 ± 14.3, 53.1 ± 11.3, 21.5 ± 12.8, and 97.1 ± 16.5% of the control, respectively, and the delayed tachypneic responses were 99.3 ± 13.6, 109.8 ± 9.7, 96.9 ± 19.2, and 105.4 ± 19.3% of the control, respectively. At the doses tested, pretreatment with CPZ alone and iso-PPADS alone effectively blocked the apneic response to intravenous injections of capsaicin and {alpha}beta-meATP, whereas pretreatments with vehicle 1 failed to do so (Table 3). In contrast, pretreatment with CPZ+iso-PPADS did not significantly affect the apneic responses to intravenous injection of phenylbiguanide (Table 3).


Figure 2
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  		Fig. 2. Mean responses of respiratory frequency to inhalation of 0.2% aerosolized H2O2 in four groups of rats. A–D: responses before and after pretreatment with CPZ alone, iso-PPADS alone, CPZ+iso-PPADS, and vehicles of CPZ+iso-PPADS (vehicle 1). Top: periods between the two vertical dashed lines indicate the duration of aerosol challenge. Bottom: data of respiratory frequency in the initial and delayed responses were measured as the lowest values during 0–21 s and peak values during 21–90 s, respectively, postchallenge. Data are values averaged over a 3-s period. Data in each group are the means ± SE from 10 rats. *Significantly different from the corresponding baseline in the same group; asignificantly different from the corresponding control response: P < 0.05.

 

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  		Table 3. Average apneic response to intravenous injections of receptor agonists before and after various experimental interventions in the 15 study groups

 
Involvement of arachidonate metabolites and ATP in the H2O2-evoked f responses.   Pretreatment with Indo (Figs. 3A and 4A) significantly suppressed the H2O2-evoked initial bradypnea but had no effect on the H2O2-evoked delayed tachypnea. In contrast, pretreatment with NDGA (Fig. 4B) or the vehicle (vehicle 2) for Indo or NDGA (Figs. 3B and 4C) failed to significantly affect these f responses. After pretreatment with Indo, NDGA, and vehicle 2, the initial bradypneic responses were 51.6 ± 19.9, 98.4 ± 17.4, and 99.1 ± 19.1% of the control, respectively, and the delayed tachypneic responses were 97.1 ± 15.4, 95.5 ± 19.7, and 94.9 ± 14.4% of the control, respectively. On the other hand, pretreatment with apyrase+ADA (ATP scavengers) (Fig. 5A) significantly suppressed the H2O2-evoked initial bradypnea, but had no effect on the H2O2-evoked delayed tachypnea. In contrast, pretreatment with ADA alone (Fig. 5B) or the vehicles (vehicle 3) for apyrase+ADA (Fig. 5C) failed to significantly affect these f responses. After pretreatment with apyrase+ADA, ADA, and vehicle 3, the initial bradypneic responses were 50.4 ± 14.8, 108.3 ± 15.5, and 98.9 ± 12.8% of the control, respectively, and the delayed tachypneic responses were 94.9 ± 17.1, 105.8 ± 14.9, and 102.6 ± 19.5% of the control, respectively. At the doses tested, all pretreatments did not significantly affect the apneic responses to intravenous injection of capsaicin (Table 3).


Figure 3
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  		Fig. 3. Experimental records illustrating acute responses to inhalation of 0.2% aerosolized H2O2 in two anesthetized and spontaneously breathing rats. A: responses before and after pretreatment with indomethacin (Indo). B: responses before and after pretreatment with vehicle of Indo (vehicle 2). Horizontal bars indicate the duration (30 s) of H2O2 challenge. Note that the H2O2-evoked initial bradypnea was reduced after Indo. See legend to Fig. 1 for further explanations.

 

Figure 4
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  		Fig. 4. Mean responses of respiratory frequency to inhalation of 0.2% aerosolized H2O2 in three groups of rats. A–C: responses before and after pretreatment with Indo, nordihydroguaiaretic acid (NDGA), and vehicle of Indo and NDGA (vehicle 2). Data in each group are the means ± SE from 10 rats. *Significantly different from the corresponding baseline in the same group; asignificantly different from the corresponding control response: P < 0.05. See legend to Fig. 2 for further explanation.

 

Figure 5
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  		Fig. 5. Mean responses of respiratory frequency to inhalation of 0.2% aerosolized H2O2 in three groups of rats. A–C: responses before and after pretreatment, with a combination of apyrase and adenosine deaminase (apyrase+ADA), ADA alone, and vehicles of apyrase+ADA (vehicle 3). Data in each group are the means ± SE from 10 rats. *Significantly different from the corresponding baseline in the same group; asignificantly different from the corresponding control response: P < 0.05. See legend to Fig. 2 for further explanation.

 
Effects of chemical mediators of TRPV1 and P2X receptors.   Pretreatment with CPZ+apyrase+ADA (Fig. 6C), CPZ+Indo (Fig. 6A), or iso-PPADS+Indo (Fig. 6B) significantly suppressed the H2O2-evoked initial bradypnea but had no effect on the H2O2-evoked delayed tachypnea. In contrast, pretreatment with their vehicles (vehicle 4) failed to significantly affect these f responses (Fig. 6D). After pretreatment with CPZ+Indo, iso-PPADS+Indo, CPZ+apyrase+ADA, and vehicle 4, the initial bradypneic responses were 53.3 ± 12.5, 25.3 ± 6.9, 17.0 ± 11.9, and 94.3 ± 9.4% of the control, respectively, and the delayed tachypneic responses were 93.1 ± 17.7, 99.7 ± 15.9, 98.8 ± 14.1, and 97.8 ± 18.5% of the control, respectively. At their doses tested, all pretreatments did not significantly affect the apneic responses to intravenous injection of phenylbiguanide (Table 3).


Figure 6
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  		Fig. 6. Mean responses of respiratory frequency to inhalation of 0.2% aerosolized H2O2 in four groups of rats. A–D: responses before and after pretreatment with CPZ+Indo, iso-PPADS+Indo, CPZ+apyrase+ADA, and vehicles of CPZ+apyrase+ADA (vehicle 4). Data in each group are the means ± SE from 10 rats. *Significantly different from the corresponding baseline in the same group; asignificantly different from the corresponding control response: P < 0.05. See legend to Fig. 2 for further explanation.

 
Effect of various pharmacological pretreatments on the H2O2-evoked VT responses.   The H2O2-induced reduction in VT during both the initial and delayed periods was not significantly altered by any pharmacological intervention used in this study (Table 2; Figs. 1 and 3). Additionally, the number of animals displaying H2O2-evoked augmented breaths was not significantly changed by any pharmacological intervention used in this study (Table 2; Figs. 1 and 3).

Reflex nature of the H2O2-evoked ventilatory responses.   After vagotomy, both the initial bradypneic and delayed tachypneic responses to H2O2 did not occur. On average, the initial lowest (44.3 ± 3.0 breaths/min) and delayed peak f values (43.2 ± 3.1 breaths/min) did not significantly differ from the baseline f (43.0 ± 3.1 breaths/min). Furthermore, the H2O2-evoked reductions in VT and augmented breaths observed during the initial or delayed periods were also abolished by vagotomy (Table 2). Additionally, the apneic responses to intravenous injection of capsaicin were eliminated by vagotomy (Table 3).

Response of ABP to H2O2 before and after experimental interventions.   Inhalation of 0.2% aerosolized H2O2 generally produced an increase in ABP during the initial period and a decrease in ABP during the delayed period (Figs. 1 and 3). As a group, the ABP value changed from a baseline of 100.4 ± 0.7 mmHg (n = 150) to an initial peak value of 110.8 ± 0.9 mmHg and to a delayed lowest value of 87.6 ± 1.1 mmHg under control conditions. Additionally, the heart rate value changed from a baseline of 384.5 ± 3.3 beats/min (n = 150) to an initial lowest value of 342.9 ± 4.1 beats/min and to a delayed lowest value of 322.6 ± 4.0 beats/min under control conditions. The initial increase in ABP evoked by H2O2 challenge was not significantly altered by any of the pharmacological pretreatments but was abolished by vagotomy (Table 4). The delayed reduction in ABP evoked by H2O2 challenge was not significantly influenced by pretreatment with vehicles 1–4, NDGA, or ADA but was prevented by the rest of pharmacological pretreatments and vagotomy (Table 4).


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  		Table 4. Average response of mean arterial blood pressure to H2O2 challenge before and after various experimental interventions in the 15 study groups

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Results of this study demonstrate that inhalation of 0.2% aerosolized H2O2 consistently evoked initial bradypnea followed by delayed tachypnea. Furthermore, during the tachypneic period, augmented breaths were also evoked in the majority of rats (59%) studied. The characteristics of these H2O2-evoked ventilatory responses were similar to those described previously (51). Bilateral cervical vagotomy totally prevented these ventilatory responses, confirming the previous finding (51) that they are airway reflexes mediated through lung vagal afferents. In our previous study (51), we suggested that the initial response and the delayed responses to H2O2 are airway reflexes mediated through vagal lung capsaicin-sensitive and myelinated afferents, respectively, because they can be prevented by perivagal capsaicin treatment and vagal cooling to 7°C. We also suggested that the reflex effects of H2O2 are due to the action of ROS, because the ventilatory responses to H2O2 are largely or totally suppressed by an antioxidant acting against H2O2 or hydroxyl radicals (51).

We further demonstrate that pretreatment with CPZ [change ({Delta}) = –53%] or iso-PPADS ({Delta} = –47%) reduced the initial bradypneic response to H2O2 by about one-half, while pretreatment with vehicles failed to do so. CPZ and iso-PPADS are antagonists of the TRPV1 and P2X receptors, respectively (22, 34), and the doses used in this study effectively prevented the reflex apneic response to intravenous injections of capsaicin and {alpha}beta-meATP, respectively. These observations suggest that both the TRPV1 and the P2X receptors are important in eliciting the initial reflex bradypnea. The fact that pretreatment with CPZ and iso-PPADS in combination provided a more complete blockade of the initial bradypneic response ({Delta} = –78%) indicated that the functioning of the TRPV1 receptors in triggering the H2O2-evoked initial bradypneic response is, at least in part, independent from that of the P2X receptors. Furthermore, recent studies (31, 57) have demonstrated a positive interaction between TRPV1 and P2Y receptors in response to ATP in isolated cell models. The functional interaction of TRPV1 and P2X receptors, however, has not been reported and remains to be elucidated. The suppressive effects of CPZ and iso-PPADS were unlikely to be due to the possible deleterious effects of these drugs on vagal lung capsaicin-sensitive afferents, because these drugs did not affect the reflex apneic response to intravenous injections of phenylbiguanide, a serotonin 5-HT3 receptor agonist, which serves as a stimulus for this type of vagal lung afferents (18, 34). Thus it appears that the initial bradypneic response to H2O2 may result from the activation of both TRPV1 and P2X receptors, possibly those located at terminals of the vagal lung capsaicin-sensitive afferent fibers. This notion is strongly supported by our electrophysiological findings (53), which showed that delivery of aerosolized H2O2 stimulates these afferent fibers and the sensory transduction of ROS is mediated through both TRPV1 and P2X receptors. In this context, a significant number of studies of the afferent responses to receptor agonists have suggested that TRPV1 and P2X receptors may be located at the nerve terminals of vagal lung capsaicin-sensitive afferent fibers (5, 15, 25, 34, 47, 53, 58, 60). Both in vitro electrophysiological and pharmacological studies have characterized TRPV1 and P2X receptors as present on the membrane of jugular or nodose vagal neurons (12, 17, 21, 23, 56, 60). Intravenous injection of an agonist of TRPV1 or P2X receptors has also been shown to cause inhibitory airway reflexes (34, 42).

The initial reflex bradypnea that we observed required an average latency of 6.8 s to be evoked following H2O2 challenges. This long latency caused us to doubt that the TRPV1 and P2X receptors located at the nerve terminals are directly stimulated by ROS themselves. As an alternative, ROS may indirectly activate the TRPV1 and P2X receptors through released chemical mediators. In this context, we have demonstrated that pretreatment with Indo ({Delta} = –48%) or apyrase+ADA ({Delta} = –50%) reduced the initial bradypneic response to H2O2 by about one-half, whereas pretreatment with NDGA, the vehicle of Indo, or the vehicles of apyrase+ADA failed to do so. Indo is a nonselective cyclooxygenase inhibitor, whereas NDGA is a nonselective lipoxygenase inhibitor (55). Apyrase is an enzyme that catalyses breakdown of ATP to AMP, whereas ADA is an enzyme that catalyses breakdown of adenosine. A combination of apyrase and ADA has been used to act as ATP scavengers (1, 54). The suppressive effects of apyrase+ADA could not be explained by its promotion of breakdown of adenosine only, since pretreatment with ADA alone failed to alter the initial bradypneic response to H2O2. Additionally, the suppressive effects of Indo and apyrase+ADA were unlikely to be due to the possible deleterious effects of these drugs on vagal lung capsaicin-sensitive afferents, because these drugs did not affect the reflex apneic response to intravenous injections of capsaicin. Accordingly, our observations suggest that both cyclooxygenase metabolites and ATP are involved in eliciting the initial reflex bradypnea. Although lipoxygenase metabolites have been demonstrated to activate TRPV1 receptors (5, 6, 20), they do not seem to play a role in the observed response.

We subsequently delineated the chemical mediator involved in the functioning of the TRPV1 and P2X receptors during the triggering of the reflex responses to H2O2. We found that, when TRPV1 receptors were blocked by CPZ, the additional removal of ATP by scavengers could further reduce the initial bradypneic response to H2O2 ({Delta} = –83%), whereas an additional inhibition of cyclooxygenase by Indo failed to do so ({Delta} = –47%). Similarly, when P2X receptors were blocked by iso-PPADS, an additional inhibition of cyclooxygenase was able to further reduce the initial bradypneic response to H2O2 ({Delta} = –75%). These results suggest that, in the event of the triggering of the initial reflex bradypnea, cyclooxygenase metabolites are linked to the functioning of the TRPV1 receptors, whereas ATP is associated with the functioning of the P2X receptors.

The exact mechanisms by which ROS activates the pathways of the cyclooxygenase metabolites/TRPV1 receptors and ATP/P2X receptors, leading to elicitation of the initial reflex bradypnea, remain to be elucidated. One possibility is that ROS causes the release of cyclooxygenase metabolites and ATP from lung cells, which subsequently activate the TRPV1 and P2X receptors, respectively, located at the terminals of vagal lung capsaicin-sensitive afferent fibers. In fact, ROS are known to increase the release of cyclooxygenase metabolites, such as prostaglandins in the lung tissue (7, 40). Certain prostaglandins have been shown to possess capsaicin-like actions on cardiac capsaicin-sensitive afferent fibers (16). Interestingly, cyclooxygenase also catalyzes the metabolism of anandamide (27, 64), an endocannabinoid, which is postulated to be an endogenous ligand of TRPV1 receptors (37, 61). Prostaglandin ethanolamides are a novel class of the cyclooxygenase metabolites generated from this reaction (27, 64) and can bind or interact with TRPV1 receptors (39, 50). Furthermore, ROS have been shown to damage cells and cause a rapid release of cytosolic ATP, which activates P2X receptors of pain nociceptors in the vicinity (11, 41). Furthermore, ROS may have nondamaging effects, and, upon stimulation, nondamaged epithelial or endothelial cells may release ATP, exerting a paracrine effect on P2X receptors located on other cells (32). A second possibility is that baseline levels of cyclooxygenase metabolites and ATP are required to maintain the sensitivity of the TRPV1 and P2X receptors. Consequently, administration of Indo or ATP scavengers might make vagal lung capsaicin-sensitive afferents less responsive to ROS-related stimulus. However, this possibility is not likely, because pretreatment with Indo or ATP scavengers did not reduce the reflex apneic response to capsaicin or phenylbiguanide.

In contrast to their effects on initial bradypnea, all pharmacological pretreatments made in this study did not affect the H2O2-evoked delayed tachypnea and augmented breaths. These observations indicate that the pathways of the cyclooxygenase metabolite/TRPV1 receptors and ATP/P2X receptors do not contribute to these H2O2-evoked delayed responses. Thus it is conceivable that the vagal lung afferents responsible for triggering these delayed responses are myelinated afferents with a large diameter, whose activity arises from pulmonary rapidly adapting (irritant) receptors, as our laboratory has suggested previously (51). In fact, preliminary results from our electrophysiological study in anesthetized rats have revealed that delivery of aerosolized H2O2 by a respirator stimulated pulmonary rapidly adapting receptors (52). Likewise, the H2O2-evoked reduction in VT occurring during both the initial and delayed periods following H2O2 challenges was not significantly affected by any of the pharmacological pretreatments, suggesting that it is related to mechanisms other than the pathways of the cyclooxygenase metabolite/TRPV1 receptors and ATP/P2X receptors. This reduction in VT is not seen after vagotomy, indicating that it is mediated through vagal afferent and/or efferent pathways.

In accordance with our previous findings, inhalation of 0.2% aerosolized H2O2 initially produced an increase in ABP and subsequently caused a decrease in ABP. Accompanying these two periods of ABP changes, heart rate persistently dropped. Our laboratory previously (51) showed that the initial increase in ABP persisted after perivagal capsaicin treatment, but was prevented during vagal cooling to 7°C, which suggests that it may be a reflex resulting from stimulating pulmonary rapidly adapting receptors or myelinated vagal afferents that arise from other intrathoracic structures, such as the heart or large pulmonary vessels. Conversely, the delayed decrease in ABP persisted during vagal cooling to 7°C but was prevented after perivagal capsaicin treatment, indicating that it may be a reflex resulting from activation of vagal lung capsaicin-sensitive afferent fibers. These notions gain support from the observations made in this study that, when the initial bradypneic response to H2O2 was suppressed by pharmacological pretreatments relevant to the pathways of the cyclooxygenase metabolite/TRPV1 receptors and ATP/P2X receptors, the delayed decrease in ABP vanished. Conversely, when the delayed ventilatory responses to H2O2 were not altered by pharmacological pretreatments, the initial increase in ABP persisted. The cardiovascular reflexes originating from activation of vagal lung capsaicin-sensitive afferent fibers are quite clear (35), whereas that from activation of pulmonary rapidly adapting receptors has not been well defined (63). It is interesting to note that the reflex effect of pulmonary rapidly adapting receptors preceded the reflex effect of lung capsaicin-sensitive afferent fibers for ABP responses, whereas the reverse sequence was true for airway responses. It is possible that the depressor response mediated by lung capsaicin-sensitive afferent fibers was initially masked by an overriding increase in blood pressure mediated by a different set of afferent pathways. Furthermore, the depressor response seems to last for a much longer duration than bradypnea. It is possible that H2O2-induced releases of vasodilative mediators, such as histamine (38), are involved.

In summary, the initial bradypneic response to H2O2 results from the activation of both TRPV1 and P2X receptors, possibly located at terminals of vagal lung capsaicin-sensitive afferent fibers, and the functioning of the TRPV1 and P2X receptors in triggering the initial bradypnea is, in part, mediated through the actions of cyclooxygenase metabolites and ATP, respectively. These mechanisms, however, do not contribute to the H2O2-evoked delayed tachypnea and augmented breaths. While the functions of the TRPV1 and P2X receptors are relatively well established for somatosensory nociceptors and pain sensation (41, 56), their roles in the regulation of breathing, especially under pathological conditions, remain to be further explored. The results of this and our laboratory’s previous studies (51, 53) thus provide evidence to support the hypothesis that vagal lung capsaicin-sensitive afferent fibers can detect excess pulmonary ROS, thereby eliciting an inhibitory airway reflex, and that this sensory transduction is mediated through the pathways of the cyclooxygenase metabolite/TRPV1 receptors and ATP/P2X receptors. Vagal lung capsaicin-sensitive and myelinated afferents have been largely, up to the present, implicated in various airway diseases, such as airway hyperreactivity, cough, and bronchoconstriction (35, 59), all of which may be related to excess production of ROS. The above implies that interfering with the above-mentioned pathways are possible target choices for potential therapeutic regimes to treat these ROS-related airway diseases.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
ACKNOWLEDGMENTS
 REFERENCES
 
This study was supported by National Science Council of the Republic of China Grants NSC94-2320-B-010-002 and NSC94-2320-B-010-031, and Grants VGHUST95-P7-28 and 95A-C-D01-PPG-01 from the Aim for the Top University Plan supported by the Ministry of Education of the Republic of China.


   ACKNOWLEDGMENTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
REFERENCES
 
The authors are grateful to Dr. Tien Huan Hsu for statistical analysis of data and Dr. Ralph Kirby for help in language editing.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Y. R. Kou, Dept. of Physiology, School of Medicine, National Yang-Ming Univ., Shih-Pai, Taipei 112, Taiwan (e-mail: yrkou{at}ym.edu.tw)

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
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 ACKNOWLEDGMENTS
 REFERENCES
 

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