ToxSci Advance Access originally published online on July 13, 2006
Toxicological Sciences 2006 93(2):411-421; doi:10.1093/toxsci/kfl061
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Adenosine Sensory Transduction Pathways Contribute to Activation of the Sensory Irritation Response to Inspired Irritant Vapors
Toxicology Program, Department of Pharmaceutical Sciences, School of Pharmacy, University of Connecticut, Storrs, Connecticut 06269-3092
1 To whom correspondence should be addressed at Toxicology Program, Department of Pharmaceutical Sciences, 69 N. Eagleville Rd, U-3092, University of Connecticut, Storrs, CT 06269-3092. Fax: (860) 486-5792. E-mail: john.morris{at}uconn.edu.
Received May 10, 2006; accepted July 10, 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The molecular mechanisms through which sensory irritants stimulate nasal trigeminal nerves are poorly understood. The current study was aimed at evaluating the potential contribution of purinergic sensory transduction pathways in this process. Aerosols of 4–36mM adenosine 5'-triphosphate (ATP) and adenosine both acted as sensory irritants. Large dose capsaicin pretreatment to induce degeneration of transient receptor potential vanilloid type-1 (TRPV1)-expressing C fibers greatly reduced, but did not abolish, the sensory irritation response to ATP aerosol and was without effect on the response to adenosine aerosol, indicating that ATP acts largely on capsaicin-sensitive (primarily C fibers) and adenosine acts on capsaicin-insensitive (primarily A
fibers) nerves. The response to adenosine was diminished by pretreatment with the broad-based adenosine receptor antagonist theophylline (20 mg/kg) and A1-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (0.1 mg/kg), providing evidence that adenosine stimulates capsaicin-insensitive nerves via the A1 receptor. The sensory irritation responses to 275 ppm styrene and 110 ppm acetic acid vapors were significantly reduced by theophylline pretreatment suggesting a role for adenosine signaling pathways in activation of the sensory irritant response by these vapors. If sensory nerves are activated by mediators that are released from injured airway mucosal cells, then nasal sensory nerve activation may be a reflection of irritant-induced alterations in airway cell integrity. Key Words: sensory irritation; adenosine; acetic acid; styrene.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Detection and initiation of appropriate protective responses to noxious airborne chemicals are necessary for maintenance of airway integrity in polluted atmospheres (Baraniuk, 1994
; Barnes, 1996). The respiratory tract sensory nerve system plays an integral role in this process. Airborne pollutants that stimulate nasal trigeminal sensory nerves are termed sensory irritants and represent a toxicologically important class of compounds. For example, sensory irritation forms the basis for roughly one half of the occupational exposure guidelines in the United States (Schapper, 1993), and the primary complaint about poor indoor air quality is irritation (Hodgson, 2002). In healthy individuals, stimulation of sensory nerves may represent a nuisance; however, in individuals with allergic airway disease, sensory irritants exacerbate disease and have a deleterious health impact. For example, subjects with allergic rhinitis exhibit enhanced responsiveness to chlorine and acetic acid vapors (Shusterman et al., 2003, 2005) and experience more symptoms due to poor indoor air quality than healthy individuals (Hall et al., 1993; Mendell, 1993; Shusterman et al., 2003). Similarly, individuals with allergic asthma are more sensitive to pollutants than healthy individuals (Leikauff, 2002; Thurston and Bates, 2005). Neither the mechanisms through which irritants stimulate sensory nerves nor the mechanisms responsible for the heightened irritant responsiveness in allergic airway disease are known.
Activation of sensory nerves may occur via direct interaction of irritant molecules with airway nerve endings or may occur indirectly via sensory transduction pathways that involve release of paracrine mediators from nonneuronal cell types that then interact with sensory nerves. The latter pathway has not been described for nasal trigeminal nerves and would represent a novel pathway for stimulation of these nerves by irritant pollutants. Purinergic sensory transduction pathways have been described in several organ systems. For example, release of the purine adenosine 5'-triphosphate (ATP) from epithelial cells is thought to represent an important pathway for sensory nerve stimulation in the bladder and gut (Bertrand, 2003; Burnstock, 2001; Schiebert and Zsembery, 2003). It has recently been shown that airway epithelial cells release ATP in response to toxicologically relevant concentrations of ozone (Ahmad et al., 2005) raising the possibility that purinergic sensory transduction pathways may exist in the respiratory tract as well.
ATP can stimulate sensory nerves through P2X or P2Y receptors, or can be catabolized by ubiquitous extracellular ATPases and 5-nucleotidases to form adenosine, which can act through the A1, A2a, A2b, or A3 receptors. Pulmonary sensory nerves of the rodent are activated by the purines ATP and/or adenosine (Hong et al., 1998; Kollarik et al., 2003), and a common side effect of adenosine agonist therapy in humans is dyspnea and chest tightness, suggesting that respiratory tract sensory nerves of the human are responsive to this purinergic mediator as well (Burki et al., 2005). The current study was aimed at examining the potential role for purinergic sensory transduction pathways in initiation of the nasal sensory irritant response. Of particular interest were adenosine receptor–dependent pathways because humans are responsive to adenosine as evidenced by the induction of dyspnea and chest tightness by this agent.
In animal models, the sensory irritation response is characterized by "braking" at the onset of the expiratory phase of each breath due to glottis closure and increased laryngeal resistance, followed by rapid exhalation (Alarie, 1973; Bos et al., 1992; Nielsen, 1991; Vijayaraghavan et al., 1993). In the current study, the sensory irritant response was quantified by measuring the duration of the braking period by plethysmography. Mice were exposed to ATP or adenosine aerosols to examine the potential for either mediator to initiate the sensory irritation response. The broad-acting adenosine receptor antagonist, theophylline, and the A1-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) were used as tools to confirm the adenosine receptor basis of any response. The role of capsaicin-sensitive (primarily C fibers) and capsaicin-insensitive (primarily A fibers) nerves in mediating the responses to ATP and adenosine was assessed by pretreating mice with capsaicin following the protocol used previously in this laboratory (Morris et al., 2003; Symanowicz et al., 2004). The responsiveness to adenosine was examined in both healthy and ovalbumin-induced allergic airway diseased (OVA-AAD) mice using the protocols previously shown to induce enhanced sensory irritant responsiveness (Morris et al., 2003). Finally, the effects of the broad-acting adenosine antagonist theophylline on the sensory irritant response to two irritants, acetic acid and styrene, were examined to provide information on the potential participation of adenosine sensory transduction pathways in activation of the sensory irritant response by these vapors. The receptor basis for stimulation of nasal trigeminal nerves by these two vapors has been the subject of previous investigations in this laboratory (Symanowicz et al., 2004). Results indicated that nasal sensory nerves of the mouse are activated by both ATP and adenosine, with the latter likely being mediated by adenosine A1 receptor pathways in A nerves. Sensory nerve responsiveness to adenosine is enhanced in allergic airway disease and adenosine sensory transduction pathways likely contribute to elicitation of the sensory irritation response to acetic acid and styrene. If sensory nerves are activated by mediators that are released from injured airway mucosal cells, then the sensory irritation response may be a reflection of the presence of not only airborne irritants but also irritant-induced alterations in airway cell integrity.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animals and exposure methodology.
C57Bl/6J female mice, obtained from Jackson Laboratories (Bar Harbor, ME), were used in all studies. Animals were 5–6 weeks of age at purchase and were housed over hardwood shavings (Sani-Chip Dry, P.J. Murphy Forest Products, Montville, NJ) in animal rooms maintained at 22–25°C with a 12-h light-dark cycle (lights on at 6:30 A.M.). Food (Lab Diet, PMI Nutrition International, Brentwood, MO) and tap water were provided ad libitum. Animals were acclimated for at least 1 week prior to use and were used within 8 weeks of arrival. Body weights averaged
20 g at the time of use. All protocols were approved by the University of Connecticut Institutional Animal Care and Use Committee.
Experimental designs.
The initial experiments were aimed at characterizing the concentration response relationship for the sensory irritation response to ATP aerosol and the role of capsaicin-sensitive and/or capsaicin-insensitive nerves in that response. The effect of the broad-acting adenosine receptor antagonist theophylline on the response to equimolar ATP or adenosine aerosols was then examined to characterize the role of adenosine receptor pathways. In addition to acting via adenosine receptors, ATP might act via stimulation of P2X receptors (Kollarik et al., 2003); therefore, for the sake of completeness, the sensory irritant response to the P2X-selective agonist 
,ß-methylene-ATP (Chizh and Illes, 2000) was also examined. The next experiment was designed to examine the role of capsaicin-sensitive and -insensitive nerves in activation of sensory nerves by adenosine aerosol and to determine if adenosine acted through the adenosine A1 receptor subtype using the A1 antagonist DPCPX (Fredholm et al., 2001; Gu et al., 2003) as a tool. To determine if sensory nerve responsiveness to adenosine was altered in allergic airway disease, the response to adenosine aerosol was examined in mice with OVA-AAD using the protocols previously established in this laboratory (Morris et al., 2003). Finally, the effect of the broad-acting adenosine antagonist theophylline on the sensory irritation responses to styrene and acetic acid was examined to assess the potential contribution of adenosine sensory transduction pathways in the responses to these irritants.
Exposure protocols.
As in our previous studies (Morris et al., 2003, 2005; Symanowicz et al., 2004), spontaneously breathing mice were exposed and respiratory parameters monitored in a Buxco double plethysmograph (Buxco, Inc., Sharon, CT) using the Buxco noninvasive mechanics software. Animals were restrained in the plethysmograph by a latex collar, but were not anesthetized. Multiple breathing patterns were analyzed including breathing frequency, tidal volume, time of inspiration and expiration, peak flows during inspiration and expiration, the duration of braking in early expiration, and the duration of any pause at the end of expiration. Because sensory irritation is characterized by braking at the onset of expiration (Alarie, 1973; Vijayaraghavan et al., 1993) the current study focused on this parameter. Clean or irritant laden air was drawn into the head space of the double plethysmograph at a flow rate of 0.6 l/min. Air temperature ranged between 22 and 25°C and relative humidity averaged 50%. After a 10-min acclimatization period, a 10-min baseline exposure to clean air commenced followed by a 15-min exposure to irritant. Breathing parameters were collected during the baseline and exposure periods. One-minute average values were recorded and used for statistical analysis as is typical for sensory irritation protocols (Alarie, 1981). Plethysmograph headspace air samples were drawn during exposure and analyzed for irritant concentration as described below.
Drug protocols.
The adenosine receptor antagonist theophylline (Fredholm et al., 2001) was administered ip at a dose of 20 mg/kg (5 mg/ml in saline) 20–30 min prior to aerosol exposure. This was the minimally effective dose; pilot experiments revealed 5 mg/kg theophylline to be without effect on the response to adenosine. The adenosine A1 receptor antagonist DPCPX (Fredholm et al., 2001) was administered at a dose of 0.1 mg/kg (0.01 mg/ml in 2% dimethylsulfoxide in saline, ip) 20–30 min prior to aerosol. This is the dose used by Gu et al. (2003). Capsaicin pretreatments were performed by the protocol previously described (Morris et al., 2003; Symanowicz et al., 2004). Briefly, animals received two injections of capsaicin (sc): 25 mg/kg followed by 75 mg/kg 1 day later. Prior to each injection, animals were anesthetized with avertin (250 mg/kg, ip) and then treated with 10 mg/kg theophylline (sc, 5 mg/ml in distilled water) and 0.1 mg/kg terbutaline (ip, 0.05 mg/ml in saline) to minimize respiratory side effects. The capsaicin was dissolved in 1:1:8 ethanol:Tween80:saline at a concentration of 5 mg/ml. Control mice received the drugs and capsaicin vehicle injection. Animals were used 1–2 weeks after treatment. Responsiveness to capsaicin aerosol challenge is markedly reduced for at least 5 weeks by this protocol (Morris et al., 2003; Symanowicz et al., 2004 and unpublished observations).
OVA-AAD was induced by the protocol used previously in this laboratory (Morris et al., 2003). The pathophysiologic changes in this model have been thoroughly characterized (Cloutier et al., 2004; Morris et al., 2003; Schramm et al., 2000; Wu et al., 2001; Yiamouyiannis et al., 1999). Animals received three weekly ip injections of 25 µg OVA (Grade V, Sigma Chemical Company, St Louis MO) adsorbed to 2 mg aluminum hydroxide. One week after the last injection, animals were exposed for 1 h/day to aerosolized OVA in a directed airflow nose-only exposure chamber (CH Technologies, Westwood, NJ). Atmospheres were generated by nebulization (Lovelace Nebulizer, In-Tox Products, Albuquerque, NM) of 1% OVA in saline. Airborne OVA concentration averaged 20 mg/m3 (1.8-µm MMAD, 
g = 2.5, Mercer impactor, CH Technologies, Westwood, NJ). Control animals received ip OVA injections but no OVA aerosol exposures and are designated OVA-d0. OVA-AAD animals received the ip injections followed by eight daily OVA exposures and are designated OVA-d8. Responsiveness to adenosine was measured 1 day after the eighth daily exposure. The response to sensory irritants has been previously been shown to be enhanced at this time point in this model (Morris et al., 2003).
Irritant generation and analysis.
Aerosols of ATP, adenosine, and ,ß-methylene-ATP were generated with a Lovelace nebulizer. Particle size averaged 1.1-µm MMAD (g = 2.1, Mercer impactor). Nebulization solutions contained 10 µg/ml fluoroscein as a tag. During exposure headspace air was drawn through an 0.2-µm filter, the filter was eluted with 10mM NaOH, and fluorescence determined as described previously (Morris et al., 2003; Symanowicz et al., 2004). Airborne concentrations were calculated stoichiometrically from the fluoroscein tag data. Styrene and acetic acid atmospheres were generated by flash evaporation and analyzed in breathing space air during exposure as described previously (Morris et al., 2003; Symanowicz et al., 2004). Styrene concentrations were determined by drawing samples through a gas chromatograph equipped with a gas sampling valve (Varian model 3800, Varian, Sugar Land TX, DB-WAX column, J&W Scientific, Folsom, CA). Acetic acid concentrations were determined by drawing plethysmograph headspace air through two midget impingers in series, each containing 10 ml of distilled water. The concentration of acetate in the impinger fluid was determined via high-performance liquid chromatography with ultraviolet detection at 210 nm (Varian Model 2510) using a mobile phase of 5:95 vol/vol acetonitrile:0.1% H2PO4 at a flow rate of 1 ml/min.
Data analysis.
Data are presented as mean ± standard error of the mean (SEM) unless otherwise indicated. Expiratory braking data were collected as 1-min averages in each animal during the 10-min baseline and 15-min exposure period. Data were log transformed as appropriate to correct for heteroscedasticity. A repeated measures analysis of variance (ANOVA) followed by Newman-Keuls test was performed on each animal group (with the repeated measure being time) to determine if expiratory braking duration during aerosol exposure differed significantly from baseline levels as revealed by a significant effect of time. To determine if the pharmacological manipulations altered the responses, expiratory braking data obtained during the exposure period only were compared by two-factor repeated measures ANOVA with one factor being drug pretreatment and the other factor being the repeated measure, time. (Each animal was exposed only once, thus these represent between animal comparisons.) These comparisons often revealed a statistical interaction between drug pretreatment and time, if so, individual comparisons among drug groups were made at each exposure time by ANOVA (followed by Newman-Keuls if appropriate). All statistical calculations were performed with Statistica Software (StatSoft, Tulsa, OK). A p < 0.05 was required for significance.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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ATP Sensory Irritation: Concentration Response
Shown in Figure 1 are the expiratory braking responses to 7 and 36mM ATP aerosols. (Detailed information on the breathing pattern changes is provided below.) Exposure groups contained four to seven mice. Airborne ATP levels averaged approximately 50 and 240 nmol/l in these two exposure groups, respectively. Expiratory braking duration was significantly elevated over baseline by ATP. During exposure to 7mM ATP, expiratory braking was elevated over baseline during exposure minutes 1 and 2. Expiratory braking was significantly higher than baseline throughout the first 5 min of exposure to 36mM ATP. The expiratory braking durations during minutes 1 and 2 in the 7 and 36mM ATP groups differed significantly from each other. It was not possible to determine if the response to 36mM ATP represented a maximal response because solubility constraints precluded nebulization of solutions of higher concentration. Baseline breathing frequency and minute ventilation were similar in both groups and averaged 284 ± 21 breaths/min and 61 ± 9 ml/min (mean ± standard deviation [SD]), respectively.
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ATP Sensory Irritation: Role of Capsaicin-Sensitive Nerves
The effect of capsaicin pretreatment on the response to ATP is shown in Figure 2. Exposure groups contained four to seven mice. To produce a large response, animals were exposed to 28mM ATP aerosols; airborne ATP concentration averaged 150 nmol/l. Expiratory braking was significantly elevated over baseline throughout the entire 15-min exposure in the control (vehicle pretreated) group, with a peak response of 101 ms occurring at the onset of exposure. In capsaicin-pretreated animals, expiratory braking duration was significantly elevated over baseline only in the first minute of exposure at which time it averaged 25 ms; thus the response to ATP was diminished but not abolished by the capsaicin pretreatment. Two-factor ANOVA revealed a significant interaction between pretreatment group and time (p < 0.001); analysis at each time point revealed that the response in capsaicin-pretreated animals was lower than in vehicle-pretreated animals at all exposure times except 10 min (for which time p = 0.065). Baseline breathing frequency and minute ventilation were similar in both groups and averaged 295 ± 34 breaths/min and 72 ± 9 ml/min (mean ± SD), respectively.
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ATP and Adenosine Sensory Irritation: Role of Adenosine Receptor Pathways
To examine the potential role of adenosine A receptor pathways in ATP-induced sensory irritation, control mice and mice pretreated with the adenosine A receptor antagonist theophylline were exposed to 14mM adenosine or ATP aerosol. (This was the highest concentration of adenosine that would remain in solution during the nebulization.) Each exposure group contained six mice. Airborne concentrations for adenosine and ATP averaged 80 and 70 nmol/l, respectively.
Shown in Figure 3 are digitized flow signals for a typical mouse exposed to ATP. During baseline no braking was observed at early expiration (Fig. 3A). During the first minute of exposure (Fig. 3B) to the ATP, braking was observed at the onset of each expiration, a pattern characteristic of sensory irritation (see Fig. 4D in Vijayaraghavan et al., 1993). There was no apparent pause at the end of expiration suggesting that pulmonary irritation did not occur. Breathing patterns appeared similar to baseline by minute 5 of exposure (Fig. 3C) and remained similar to baseline throughout the exposure. The response to adenosine during the first minute of exposure (in a separate mouse) is shown for comparative purposes in Figure 3D. As can be seen there was an induction of braking at the onset of expiration without apparent alteration in other aspects of breathing pattern, a response pattern similar to that observed for ATP (Fig. 3B). Breathing parameter data are summarized in Table 1. Both ATP and adenosine induced a transient increase in braking duration (67 and 47 ms, respectively). Peak inspiratory and expiratory flow were not decreased (and in fact, appeared to increase) suggesting that marked airway obstruction did not occur. Pause duration between end expiration and inspiration did not increase significantly, suggesting that pulmonary irritation did not occur.
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To explore the receptor basis for the response to ATP or adenosine, groups of mice were pretreated with theophylline. Theophylline was chosen because it is a broad-based antagonist acting, albeit with differing affinities, at the A1, A2a, A2b, and A3 receptor (Fredholm et al., 2001
). In both control and theophylline-pretreated ATP exposed animals (Fig. 4A) expiratory braking was significantly elevated over baseline during the first 3 min of exposure, with the peak response occurring in the first minute. The initial response to ATP was reduced by theophylline. Two-factor ANOVA revealed a significant interaction between pretreatment group and time (p < 0.01); analysis at each time point revealed that the response in theophylline-pretreated mice was significantly lower during the first minute of exposure, but not at other times. In both control and theophylline-pretreated adenosine exposed mice (Fig. 4B) expiratory braking was significantly elevated over baseline during the first 4 min of exposure (Fig. 4B), with the peak response occurring in the first minute. The response to adenosine was reduced by theophylline. Two-factor ANOVA revealed a significant interaction between pretreatment group and time (p < 0.001); analysis at each time point revealed that the response in theophylline-pretreated animals was significantly lower than in control animals at minutes 1 and 4 of exposure, but not at other times. ATP appeared to be more potent than adenosine as evidenced by the greater peak response to 14mM ATP compared with 14mM adenosine (Fig. 4A vs. 4B). It was not possible to determine if 14mM adenosine induced a maximal response because the limited solubility of adenosine precluded nebulization of high-concentration solutions.
Baseline breathing parameters were altered by theophylline pretreatment. Specifically, in the control (nonpretreated) and theophylline-pretreated animals baseline breathing frequency averaged 300 ± 9 and 343 ± 9 breaths/min, respectively (mean ± SEM, p < 0.01) and minute ventilation averaged 60 ± 3 and 73 ± 3 ml/min, respectively (mean ± SEM, p < 0.01). Theophylline did not shorten the time of inspiration (which averaged 0.086 ± 0.002 and 0.81 ± 0.003 s, respectively, in control and theophylline-pretreated mice, p > 0.05), but did cause a shortening of expiration (131 ± 5 vs. 106 ± 3 ms, respectively, in control vs. theophylline-pretreated mice, p < 0.05). There was a concomitant increase in peak expiratory flow in the theophylline-pretreated mice. The duration of braking during the baseline period was unaltered by theophylline, averaging 7 ms in both the control and theophylline groups. Due to the increased minute ventilation rates, the theophylline-pretreated animals received a higher inspired dosage than the control animals. It is difficult to attribute the diminution of response in the theophylline groups (Fig. 4A and 4B) to the elevated inspired dosage.
Potential for P2X Receptor Pathways
Vagal nerves of the mouse can be stimulated via P2X receptor agonists (Kollarik et al., 2003). To determine if similar pathways existed on the trigeminal nerve, three mice were exposed to the P2X receptor agonist ,ß-methylene-ATP (Chizh and Illes, 2000). For exposure 2-mM solutions in saline were nebulized, airborne ,ß-methylene-ATP concentrations averaged 30 nmol/l. Expiratory braking duration was significantly elevated over baseline levels throughout the entire exposure to the ,ß-methylene-ATP (Fig. 5), with the peak average response occurring in the first minute. Baseline breathing frequency and minute ventilation averaged 287 ± 17 breaths/min and 64 ± 3 ml/min (mean ± SD), respectively.
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Adenosine Sensory Irritation: Role of Capsaicin-Sensitive Nerves and A1 Receptor Pathways
The next experiment was aimed at better characterizing the response to adenosine, specifically to assess the role of capsaicin-sensitive versus capsaicin-insensitive nerves and to explore the potential role of the A1 receptor in mediating adenosine responsiveness via use of the A1-selective antagonist DPCPX (see Fig. 6). All mice were exposed to 14mM adenosine, airborne exposure concentrations averaged 95 nmol/l. The exposure groups contained 5–12 mice. The control mice received either capsaicin vehicle injection 1–2 weeks prior to exposure or no pretreatment, and the data were pooled to form a single control group. As observed previously (Fig. 4B), adenosine at a concentration of 14mM induced a mild transient expiratory braking response in the control mice, with the peak response being 32 ms. In DPCPX-pretreated mice, the peak expiratory braking duration response to adenosine was 13 ms. In capsaicin-pretreated mice, the peak response was 31 ms. Comparison of the response data by two-factor ANOVA revealed a significant difference among groups (p < 0.05), a significant effect of time (p < 0.001). A statistically significant interaction between these factors was not detected (p > 0.05). Newman-Keuls test revealed that the response in the control and capsaicin-pretreated mice were statistically similar (p > 0.05) and the response in the DPCPX-pretreated mice was significantly (p < 0.05) lower than that in both other groups. Baseline breathing frequency and minute ventilation were similar in all groups and averaged 303 ± 26 breaths/min and 85 ± 10 ml/min, respectively.
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Adenosine Sensory Irritation: Enhancement in Allergic Airway Disease
Shown in Figure 7 is the response to 14mM adenosine aerosol in OVA-d0 (control) and OVA-d8 mice. Airborne adenosine concentrations averaged 115 nmol/l. Exposure groups contained 8–10 mice. In OVA-d0 mice, expiratory braking duration was significantly elevated over baseline levels in minutes 1–4 of exposure. In OVA-d8 mice, expiratory braking duration was significantly elevated throughout the entire exposure with the overall response being approximately two-fold greater than in healthy mice. Two-factor ANOVA revealed a significant effect of time (p < 0.001), and a difference between OVA-d0 and OVA-d8 (p < 0.05). A statistical interaction between time and ovalbumin was not detected (p > 0.05). Baseline breathing frequency was similar in control and OVA-d8 animals and averaged 286 ± 25 (mean ± SD). Minute ventilation averaged 73 ± 9 ml/min in control versus 63 ± 6 ml/min (mean ± SD) in OVA-d8. These values were significantly different (p < 0.02), indicating that the OVA-d8 mice received a lower inspired dosage than the control mice due to the reduced minute ventilation.
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Adenosine Signaling: Contribution to Sensory Irritant Response to Irritant Vapors
The effects of theophylline pretreatment on the sensory irritation response to two irritants, styrene and acetic acid, are shown in Figure 8A and 8B. Exposure groups contained three to five mice. These irritants and exposure concentrations were selected because they have been previously studied in this laboratory (Symanowicz et al., 2004
). Styrene concentration averaged 275 ppm, and acetic acid exposure concentration averaged 110 ppm. Expiratory braking was increased over baseline by styrene throughout the entire 15-min exposure in both control and theophylline-pretreated mice (Fig. 8A). The was considerable interanimal variability in the response to styrene. The reasons for this are unclear. The peak response in control animals was approximately 250 ms and occurred at the end of the exposure, at this time breathing frequency averaged 57% of the baseline frequency. This response is greater than those observed for ATP or adenosine. Theophylline significantly reduced the response to styrene; two-factor ANOVA revealed a significant effect of pretreatment (p < 0.05), an effect of time (p < 0.001) but did not detect a significant interaction between these factors (p > 0.05). As can be seen in Figure 8A, the response throughout the entire exposure was lower in theophylline-pretreated than in control mice. Expiratory braking duration was also increased throughout the entire exposure to acetic acid in both control and theophylline-pretreated mice (Fig. 8B). The peak response of 200 ms occurred at the onset of exposure, at this time breathing frequency averaged 61% of the baseline frequency. This response was also greater than that observed for ATP or adenosine. Theophylline reduced the response to acetic acid; specifically, two-factor ANOVA revealed a significant effect of pretreatment (p < 0.05) and an effect of exposure time (p < 0.001) but did not detect a significant interaction between these factors (p > 0.05). Baseline breathing frequency was significantly higher in theophylline-pretreated animals averaging 311 ± 21 in this group compared with 267 ± 12 (mean ± SD) in the control mice. Baseline minute ventilation was higher in the theophylline group than in the control group averaging 76 ± 17 (mean ± SD) and 67 ± 10, respectively, but these values did not differ significantly from each other (p > 0.05).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The sensory irritation response, a decreased breathing frequency due to a prolonged braking at the onset of the expiratory phase of each breath, is a specific response that has long been known to result from trigeminal sensory nerve activation (Alarie, 1973
). Trigeminal nerves consist of both neuropeptide-rich C fibers and neuropeptide-poor A fibers. The potential role of each nerve subtype in mediating the sensory irritation response is not known with certainty; however, previous results in our laboratory using high-dose capsaicin pretreatment as a tool to destroy transient receptor potential vanilloid type-1 (TRPV1) receptor–expressing nerves (primarily C fibers) suggests that both nerve subtypes may be involved (Morris et al., 2003; Symanowicz et al., 2004). The molecular mechanisms through which sensory irritants stimulate trigeminal nerves are also not known and may well differ for C fibers versus A fibers. In the current study, ATP aerosols were found to produce concentration-dependent sensory irritation responses in the mouse (Fig. 1), indicating that purinergic sensory transduction pathways exist for nasal trigeminal sensory nerve activation. ATP may act via P2 receptors and/or be catabolized to adenosine and act via adenosine A receptors with the balance of its effects being determined by its interaction with the P2 receptors relative to its rate of degradation via ectoenzymes to adenosine. At the concentrations used in the current study, it appears that both receptor pathways contribute to ATP-induced sensory irritation as evidenced by the facts that (1) ATP elicited a greater response than equimolar adenosine; and (2) the A receptor antagonist theophylline, at a dose which virtually abolished the sensory irritation response to adenosine, only partially diminished the response to ATP (Fig. 4).
Trigeminal nerves express P2X receptors (Xioang and Burnstock, 1998), and the current results indicate that the P2X-selective agonist ,ß-methylene-ATP (Chizh and Illes, 2000) acts as a sensory irritant (Fig. 5). This result is perhaps not surprising: ,ß-methylene-ATP has been shown to stimulate lower respiratory tract vagal C fibers in multiple species, including the dog (Pelleg and Hurt, 1996), rat (McQueen et al., 1998), guinea pig (Undem et al., 2004), and mouse (Kollarik et al., 2003). The current study revealed that the sensory irritation response to ATP aerosol was largely mediated by C fibers, as indicated by the greatly diminished response in capsaicin-pretreated mice (Fig. 2). Based on analogy to the vagus, perhaps C-fiber stimulation by ATP is mediated via P2X receptor activation.
Adenosine aerosols also induced the sensory irritation response (Figs. 4, 6, and 7) suggesting the potential involvement of A receptor activation in ATP-induced sensory irritation. Unlike ,ß-methylene-ATP, adenosine does not activate vagal C fibers in the mouse (Kollarik et al., 2003). Pretreatment with capsaicin was without effect on the sensory irritation response to adenosine (Fig. 6), a result consistent with the failure of adenosine to directly stimulate C fibers. The lack of effect of capsaicin pretreatment on adenosine cannot be easily attributed to the failure to use a pharmacologically effective dosage because the protocol produced dramatic effects on the response to ATP (Fig. 2) and virtually blocks the response to acrolein (Morris et al., 2003) and capsaicin aerosol challenge (Symanowicz et al., 2004). Capsaicin-insensitive nerves include A fibers and a subset of C fibers that do not express the TRPV1 (capsaicin) receptor (Kollarik et al., 2004). As noted above, adenosine does not activate vagal C fibers; thus, with the caveat that lower respiratory tract (vagal) sensory nerves and upper respiratory tract (trigeminal nerves) may differ phenotypically, these results suggest that adenosine is acting solely on A nerves. This is a particularly intriguing finding because there are few, if any, known A fiber–selective agonists.
Species differences may exist in the nerve subtype specificity of adenosine. Using an isolated C-fiber electrophysiological recording methodology it has been shown that vagal C fibers of the rat were activated by intravenous administration of adenosine, a response prevented by the A1-selective antagonist DPCPX (Hong et al., 1998; Kwong et al., 1998). In both the rat study and the current mouse study it is possible that adenosine acts indirectly, for example by interacting with mast cells to cause the release of mediators, which then stimulate sensory nerves (Adriaensen and Timmermans, 2004; Meade et al., 2001; Pelleg and Schulman, 2002). It is possible that these pathways are stimulated more effectively in the rat than in the mouse. Alternatively, the observed species differences may reflect differences in delivered doses in the two studies. This latter possibility is difficult to evaluate because it is difficult to compare an iv dosage in the rat study to an aerosolized dosage in the mouse. Future studies would be needed to resolve this issue.
The sensory irritation response to adenosine was diminished by pretreatment with two adenosine receptor antagonists, theophylline and DPCPX (Figs. 4 and 6), providing strong evidence that it was receptor mediated. Adenosine may act through one or more of the four known receptor subtypes: A1, A2a, A2b, and A3. The A1 receptor subtype has also been implicated in trigeminal nerves stimulation in the human (Giffin et al., 2003). DPCPX is an A1-selective antagonist (Fredholm et al., 2001); its effectiveness strongly implicates the involvement of A1 receptor pathways. In this aspect the effects of adenosine in the mouse respiratory tract appear similar to those observed in the rat, in which the A1 receptor has also been implicated (Gu et al., 2003) for C-fiber stimulation. It should be noted that in both studies the DPCPX was administered systemically; thus the possibility exists that the antagonist is not acting locally to block afferent sensory nerve responses but centrally to diminish neural integration and/or efferent respiratory reflex responses. Future studies might rely upon delivery of aerosolized antagonists to address this issue.
The sensory irritation response to adenosine aerosol challenge was enhanced in the OVA-allergic airway disease model (Fig. 6). The mechanisms of the enhanced responsiveness are not known, but may relate to the phenotypic switch that occurs in allergic airway disease in which the normally neuropeptide-poor A neurons express neuropeptides (Figueroa et al., 1998; Hunter et al., 2000; Undem et al., 2000). Dosimetric mechanisms, where there is enhanced aerosol deposition in the nose of the diseased mice, are possible, but seem unlikely because there is no change in isolated upper respiratory tract air-flow resistance or whole airway resistance in this model (Morris et al., 2003) and because minute ventilation rates were actually lower in the diseased than in healthy mice. Diseased mice in this model demonstrate enhanced responsiveness to acrolein and acetic acid (Morris et al., 2003) and, importantly, human subjects with allergic rhinitis demonstrate heightened responsiveness to acetic acid vapor (Shusterman et al., 2005). Although highly speculative, if sensory nerve activation by pollutants is mediated in part by adenosine signal transduction pathways, then enhanced responsiveness to adenosine in allergic airway disease might provide a mechanism for the enhanced irritant sensitivity in disease.
Documentation that adenosine sensory transduction pathways exist does not indicate that they are toxicologically relevant with respect to sensory nerve stimulation by airborne toxicants. Moreover, the administration of exogenous aerosolized ATP may not mimic the same balance of receptor stimulation as endogenous release of ATP because the pharmacokinetics of degradation versus receptor binding may differ in the two scenarios. The inhibition of the sensory irritation response to styrene and acetic acid by the broad-acting adenosine receptor antagonist theophylline provides evidence that adenosine sensory transduction pathways do indeed contribute to sensory nerve activation by these irritants, although it should be noted that it is not possible to exclude the possibility that theophylline is acting nonspecifically. Theophylline diminished the responses to acetic acid and styrene throughout the entire exposure (Fig. 8). In contrast, the sensory irritation response to adenosine itself (Figs. 4 and 6) was quite transient. The reasons for this discrepancy are unclear. In the rat adenosine can act as a neuromodulator, sensitizing C fibers to capsaicin (Gu et al., 2003), an effect that occurs at lower doses than those needed to directly activate the nerves (Hong et al., 1998; Kwong et al., 1998). Perhaps adenosine might contribute to sensory nerve activation in two ways: it may directly stimulate A fibers and it may also sensitize C fibers such that they are more responsive to these irritants. Further studies would be needed to resolve these possibilities.
That responses to both acetic acid and styrene, two widely differing irritants, were diminished by theophylline suggests that adenosine sensory transduction pathways may contribute to sensory nerve activation by a variety of chemicals. Much circumstantial evidence suggests that epithelial-neuronal sensory transduction pathways may well be involved in sensory nerve activation in the nose. First, these pathways are well characterized in other organ systems (Bertrand, 2003; Burnstock, 2001; Schiebert and Zsembery, 2003). Second, nasal inhalation dosimetric considerations suggest that epithelial cells might play a key role in mediating nasal responses. Specifically, highly reactive compounds, such as acrolein and acetic acid, are likely scrubbed/detoxified/neutralized efficiently by epithelial cells and do not effectively reach the depth of even superficial sensory nerves. Third, we have provided data that the sensory nerve–mediated responses to at least one irritant (acetaldehyde) are due to the epithelial intracellular formation of a metabolite (acetic acid, Stanek et al., 2001). Fourth, it has recently been shown that airway epithelial cells release ATP in response to toxicologically relevant concentrations of ozone (Ahmad et al., 2005).
In summary, purinergic sensory transduction pathways for activation of the nasal sensory irritation response are present in the mouse nose. Adenosine sensory transduction pathways appear to selectively activate A sensory nerves in this species and these pathways appear to contribute to the sensory nerve activation by two irritants, acetic acid and styrene. Responsiveness to adenosine is enhanced in OVA-AAD, providing a potential mechanism for the heightened irritant responsiveness that is observed in this model. If trigeminal sensory nerves are activated by mediators released from epithelial cells, it becomes likely that these nerves may serve as sensors of airway cell damage. In this regard, sensory nerve stimulation might best be viewed not only as an index of the presence of irritant pollutants, but also as a sentinel for pollutant-induced airway cell injury.
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ACKNOWLEDGMENTS |
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We thank the members of the University of Connecticut Pulmonary Research Consortium, in particular Roger S. Thrall and Michelle M. Cloutier for their helpful advice and suggestions. The expert technical assistance of Barbro Simmons is gratefully acknowledged.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ahmad, S., Ahmad, A., McConville, G., Schneider, B. K., Allen, C. G., Manzer, R., Mason, R. J., and White, C. W. (2005). Lung epithelial cells release ATP during ozone exposure: Signaling for cell survival. Free Radic. Biol. Med. 39, 213–226.[CrossRef][ISI][Medline]
Alarie, Y. (1973). Sensory irritation by airborne chemicals. CRC Crit. Rev. Toxicol. 3, 299–363.
Alarie, Y. (1981). Bioassay for evaluating the potency of airborne sensory irritants and predicting acceptable levels of exposure in man. Food Cosmet. Toxicol. 19, 623–626.[CrossRef][ISI][Medline]
Baraniuk, J. N. (1994). Neural control of the upper respiratory tract. In Neuropeptides in Respiratory Medicine (M. A. Kaliner, P. J. Barnes, G. H. H. Kinkel, and J. N. Baraniuk, Eds.), pp. 79–123. Marcel Dekker, New York, NY.
Barnes, P. J. (1996). Role of neural mechanisms in airway defense. In Environmental Impact on the Airways (J. Cretien and D. Dusser, Eds.), pp. 93–121. Marcel Dekker, New York, NY.
Bertrand, P. P. (2003). ATP and sensory transduction in the enteric nervous system. Neuroscientist 9, 243–260.[Abstract]
Bos, P. M., Zwart, A., Reuzel, P. B., and Bragt, P. C. (1992). Evaluation of the sensory irritation test for the assessment of occupational health risk. Crit. Rev. Toxicol. 21, 423–450.[CrossRef][ISI]
Burki, N. K., Dale, W. J., and Lee, L.-Y. (2005). Intravenous adenosine and dyspnea in humans. J. Appl. Physiol. 98, 180–185.
Burnstock, G. (2001). Purine-mediated signalling in pain and visceral perception. Trends Pharmcol. Sci. 22, 182–188.[CrossRef][Medline]
Chizh, B. A., and Illes, P. (2000). P2X receptors and nociception. Pharmacol. Rev. 55, 553–568.
Cloutier, M. M., Guernsey, L., Wu, C. A., and Thrall, R. S. (2004). Electrophysiological properties of the airway epithelium in the murine ovalbumin model of allergic airway disease. Am. J. Pathol. 164, 1849–1856.
Figueroa, J. M., Mansilla, E., and Suburo, A. M. (1998). Innervation of nasal turbinate blood vessels in rhinitic and children. Am. J. Respir. Crit. Care Med. 157(6 Pt 1), 1959–1966.
Fredholm, B. B., Ijzerman, A. P., Jacobson, K. A., Klotz, K.-N., and Linden, J. (2001). International union of pharmacology. XXV. Nomenclature and classification of adenosine receptors. Pharmacol. Rev. 53, 527–552.
Giffin, N. J., Kowacs, F., Libri, V., Williams, P., Goadsby, P. J., and Kaube, H. (2003). Effect of the adenosine A1 receptor agonist GR79236 on trigeminal nociception with blink reflex recordings in healthy human subjects. Cephalalgia 23, 287–292.[CrossRef][ISI][Medline]
Gu, Q., Ruan, T., Hong, J.-L., Burki, N., and Lee, L.-Y. (2003). Hypersensitivity of pulmonary C fibers induced by adenosine in anesthetized rats. J. Appl. Physiol. 95, 1315–1324.
Hall, H. I., Leaderer, B. P., Cain, W. S., and Fidler, A. T. (1993). Personal risk factors associated with mucosal symptom prevalence in office workers. Indoor Air 3, 206–209.[CrossRef]
Hodgson, M. (2002). Indoor environmental exposures and symptoms. Environ. Health Perspect. 110(Suppl. 4), 663–667.
Hong, J.-L., Ho, C.-Y., Kwong, K., and Lee, L.-Y. (1998). Activation of pulmonary C fibres by adenosine in anaesthetized rats: Role of adenosine A1 receptors. J. Physiol. 508, 109–118.
Hunter, D. D., Myers, A. C., and Undem, B. J. (2000). Nerve growth factor-induced phenotypic switch in guinea pig airway sensory neurons. Am. J. Respir. Crit. Care Med. 161(6), 1985–1990.
Kollarik, M., Dinh, Q. T., Fischer, A., and Undem, B. J. (2003). Capsaicin-sensitive and -insensitive vagal bronchopulmonary C-fibres in the mouse. J. Physiol. 551(pt 3), 869–879.
Kwong, K., Hong, J.-L., Morton, R. F., and Lee, L.-Y. (1998). Role of pulmonary C fibers in adenosine-induced respiratory inhibition in anesthetized rats. J. Appl. Physiol. 84, 417–424.
Leikauff, G. D. (2002). Hazardous air pollutants and asthma. Environ. Health Perspect. 110(Suppl. 4), 505–526.
Mendell, M. J. (1993). Non-specific symptoms in office workers: A review and summary of the epidemiologic literature. Indoor Air 3, 227–236.
Morris, J. B., Symanowicz, P. T., Olsen, J. E., Thrall, R. S., Cloutier, M. M., and Hubbard, A. K. (2003). Immediate sensory-nerve mediated respiratory responses to irritants in healthy and allergic airway diseased mice. J. Appl. Physiol. 94, 1563–1571.
Nielsen, G. D. (1991). Mechanisms of activation of the sensory irritant receptor by airborne chemicals. Crit. Rev. Toxicol. 21, 183–208.[ISI][Medline]
Schapper, M. (1993). Development of a database for sensory irritants and its use in establishing occupational exposure limits. J. Am. Ind. Hyg. Assoc. 54, 488–544.
Schiebert, E. M., and Zsembery, A. (2003). Extracellular ATP as a signaling molecule for epithelial cells. Biochim. Biophys. Acta 1615, 7–32.[Medline]
Schramm, C. M., Puddington, L., Yiamouyiannis, C. A., Lingenheld, E. G., Whiteley, H. E., Wolyniec, W. W., Noonan, T. C., and Thrall, R. S. (2000). Proinflammatory roles of T-cell receptor (TCR)
and TCRß lymphocytes in a murine model of asthma. Am. J. Respir. Cell Mol. Biol. 22, 218–225.
Shusterman, D., Murpy, M.-A., and Balmes, J. (2003). Influence of age, gender and allergy status on nasal reactivity to inhaled chlorine. Inhal. Toxicol. 15, 101–111.[CrossRef][ISI][Medline]
Shusterman, D. J., Tarun, A., Murphy, M. A., and Morris, J. B. (2005). Seasonal allergic rhinitic and normal subjects respond differentially to nasal provocation with acetic acid vapor. Inhal. Toxicol. 17, 147–152.[CrossRef][ISI][Medline]
Stanek, J., Symanowicz, P. T., Olsen, J. E., Gianutsos, G., and Morris, J. B. (2001). Sensory nerve mediated nasal vasodilatory response to inspired acetaldehyde and acetic acid. Inhal. Toxicol. 13, 807–822.[ISI][Medline]
Symanowicz, P. T., Gianutsos, G., and Morris, J. B. (2004). Lack of role for the vanilloid receptor in response to several inspired irritant air pollutants in the C57Bl/6J mouse. Neurosci. Lett. 362, 150–153.[CrossRef][ISI][Medline]
Thurston, G. D., and Bates, D. V. (2003). Air pollution as an underappreciated cause of asthma symptoms. JAMA 290, 1915–1918.
Undem, B. J., Kajekar, R., Hunter, D. D., and Myers, A. C. (2000). Neural integration and allergic disease. J. Allergy Clin. Immunol. 106(5 Suppl.), S213–S220.[Medline]
Vijayaraghavan, R., Schapper, M., Thompson, R., Stock, M. F., and Alarie, Y. (1993). Characteristic modification of the breathing pattern of mice to evaluate the effects of airborne chemicals on the respiratory tract. Arch. Toxicol. 67, 478–490.[CrossRef][ISI][Medline]
Wu, C. A., Puddington, L., Whiteley, H. E., Yiamouyiannis, C. A., Schramm, C. M., Mohammadu, F., and Thrall, R. S. (2001). Murine cytomegalovirus infection alters Th1/Th2 cytokine expression, decreases airway eosinophilia, and enhances mucus production in allergic airway disease. J. Immunol. 167, 2798–2807.
Xioang, A., Bo, X., and Burnstock, G. (1998). Localization of ATP-gated P2X receptor immunoreactivity in rat sensory and sympathetic ganglia. Neurosci. Lett. 256, 105–108.[CrossRef][ISI][Medline]
Yiamouyiannis, C. A., Schramm, C. M., Puddington, L., Stengel, P., Baradaran-Hosseini, E., Wolyniec, W. W., Whiteley, H. E., and Thrall, R. S. (1999). Shifts in lung lymphocyte profiles correlate with the sequential development of acute allergic and chronic tolerant stages in a murine asthma model. Am. J. Pathol. 154, 11–21.
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