1 Adenosine sensory transduction pathways ...

ToxSci Advance Access published July 13, 2006

Adenosine sensory transduction pathways contribute to activation of the sensory irritation response to inspired irritant vapors

Ryan P. Vaughan, Michael T. Szewczyk, Jr., Michael J. Lanosa, Christopher R. DeSesa, Gerald Gianutsos, and John B. Morris

Connecticut, Storrs, CT

Corresponding author:

John B. Morris Toxicology Program Department of Pharmaceutical Sciences 69 N. Eagleville Rd, U-3092 University of Connecticut Storrs, CT 06269-3092

Telephone:

860.486.3590

Fax:

860.486.5792

Email:

[email protected]

© The Author 2006. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please email: [email protected]

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Toxicology Program, Dept. of Pharmaceutical Sciences, School of Pharmacy, University of

ABSTRACT

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 4-36 mM ATP and adenosine both acted as sensory irritants. Large dose capsaicin pretreatment to induce degeneration of TRPV1-expressing C fibers greatly reduced, but did not abolish, the sensory

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 response 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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irritation response to ATP aerosol and was without effect on the response to adenosine aerosol,

INTRODUCTION

Detection and initiation of appropriate protective responses to noxious airborne chemicals is necessary for maintenance of airway integrity in polluted atmospheres (Barnes 1996; Baraniuk, 1994). 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

(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 (Mendel, 1993; Shusterman, 2003; Hall et al., 1993). Similarly, individuals with allergic asthma are more sensitive to pollutants than healthy individuals (Thurston and Bates, 2005; Leikauff, 2002). 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 non-neuronal cell types that then interact with sensory nerves.

The

latter pathway has not been described for nasal trigeminal nerves and would represent a novel

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forms the basis for roughly one-half of the occupational exposure guidelines in the US

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 ATP from epithelial cells is thought to represent an important pathway for sensory nerve stimulation in the bladder and gut (Burnstock 2001;Scheibert and Zsembery 2003; Bertrand, 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.

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 (Kollarik et al, 2003; Hong et al., 1998), and a common side effect of adenosine agonist therapy in humans is dyspnea and chest tightness, suggesting 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, 1972, Bos, 1992; Nielsen, 1991; Vijayaraghavan et al., 1993). In the current study the sensory irritant response was quantified by measuring the

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ATP can stimulate sensory nerves through P2X or P2Y receptors, or can be catabolized by

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 8cyclopentyl-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 capsaicininsensitive (primarily A fibers) nerves in mediating the responses to ATP and adenosine was assessed by pretreating mice with capsaicin by the protocol used previously in this laboratory

in both healthy and ovalbumin-induced allergic airway diseased 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 not only the presence of airborne irritants but also the presence of irritant-induced alterations in airway cell integrity.

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(Morris et al., 2003, Symanowicz et al., 2004). The responsiveness to adenosine was examined

MATERIALS AND METHODS

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 hour light-dark cycle

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 capsaicinsensitive 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 ,

methylene-adenosine 5’-triphosphate ( ,

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

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(lights on at 6:30 AM). Food (Lab Diet, PMI Nutrition International, Brentwood, MO) and tap

and to determine if adenosine acted through the adenosine A1 receptor subtype using the A1 antagonist 8-cyclopentyl-1,3-dipropylxanthine (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 ovalbumin-induced allergic airway disease 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 were examined to assess the potential contribution

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 non-invasive 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 22-25 oC and relative humidity averaged ~50%. After a 10 minute acclimatization period, a 10 minute baseline exposure to clean air commenced followed by a 15 minute 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

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of adenosine sensory transduction pathways in the responses to these irritants.

is typical for sensory irritation protocols (Alarie 1981; ASTM, 1984). Plethsymograph 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

(Fredholm et al., 2001) was administered at a dose of 0.1 mg/kg (0.01 mg/ml in 2% DMSO 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 one 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 one to two 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).

Ovalbumin-induced allergic airway disease (OVA-AAD) was induced by the protocol used previously in this laboratory (Morris et al., 2003). The pathophysiologic changes in this model

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without effect on the response to adenosine. The adenosine A1 receptor antagonist DPCPX

have been thoroughly characterized (Yiamouyiannis et al., 1999; Schramm et al., 2000, Morris et al., 2003; Cloutier et al., 2004). 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 hr/d 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,

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 one 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 , -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 10 mM 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

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Mercer impactor, CH Technologies, Westwood, NJ). Control animals received ip OVA

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 distilled water. The concentration of acetate in the impinger fluid was determined via HPLC 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.

braking data were collected as one minute averages in each animal during the 10 minute baseline and 15 min exposure period. Data were log transformed as appropriate to correct for heteroscedasticity. A repeated measures 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