Ethanol Potentiates The TRPV1-Mediated Cough In The Guinea Pig Raffaele Gatti, Eunice Andre, Campi Barbara, Thai Q. Dinh, Giovanni Fontana, Axel Fischer, Pierangelo Geppetti, Marcello Trevisani

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Raffaele Gatti, Eunice Andre, Campi Barbara, Thai Q. Dinh, Giovanni Fontana, et al.. Ethanol Poten- tiates The TRPV1-Mediated Cough In The Guinea Pig. Pulmonary Pharmacology & Therapeutics, 2009, 22 (1), pp.33. ￿10.1016/j.pupt.2008.11.001￿. ￿hal-00504932￿

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Title: Ethanol Potentiates The TRPV1-Mediated Cough In The Guinea Pig Authors: Raffaele Gatti, Eunice Andre, Campi Barbara, Thai Q. Dinh, Giovanni Fontana, Axel Fischer, Pierangelo Geppetti, Marcello Trevisani

PII: S1094-5539(08)00108-9 DOI: 10.1016/j.pupt.2008.11.001 Reference: YPUPT 876

To appear in: Pulmonary Pharmacology & Therapeutics

Received Date: 6 August 2008 Revised Date: 29 October 2008 Accepted Date: 4 November 2008

Please cite this article as: Gatti R, Andre E, Barbara C, Dinh TQ, Fontana G, Fischer A, Geppetti P, Trevisani M. Ethanol Potentiates The TRPV1-Mediated Cough In The Guinea Pig, Pulmonary Pharmacology & Therapeutics (2008), doi: 10.1016/j.pupt.2008.11.001

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. ARTICLE IN PRESS

TYPE OF CONTRIBUTION: Regular paper

DATE OF PREPARATION: 2nd August 2008

NUMBER OF TEXT PAGES: 12

NUMBER OF TABLES: 0 T IP NUMBER FIGURES: 3 R TITLE: Ethanol Potentiates The TRPV1-Mediated Cough In The Guinea PigC

AUTHORS: Raffaele Gatti1, Eunice Andre1, Campi Barbara1, Thai Q. DinhS3, Giovanni Fontana2, Axel Fischer3, Pierangelo Geppetti2 and Marcello TrevisaniU4 N 1Department of Clinical & Experimental Medicine, Pharmacology Section, University of Ferrara, Italy; 2Department of Critical Care Medicine and Surgery,A University of Florence, Florence, Italy; 3 Allergie-Zentrum Charité, Charité-UMniversitätsmedizin Berlin, Berlin, Germany, 4PharmEste srl, Ferrara, Italy. D

CORRESPONDING AUTHOR: RaffaeleE Gatti Ph.D. Department of Clinical & ExperimentalT Medicine, Pharmacology Section, University of Ferrara, 44100 Ferrara, Italy. P E

C C A *Author to who proofs should be sent to: Raffaele Gatti, Ph.D, Department of Clinical & Experimental Medicine, Pharmacology Section, University of Ferrara, 44100 Ferrara, Italy. Phone: +39 (0)532 455224; Fax: +39 (0)532 455205; e-mail: [email protected]

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ETHANOL POTENTIATES THE TRPV1-MEDIATED COUGH IN THE GUINEA PIG

Raffaele Gatti1*, Eunice Andre1, Campi Barbara1, Thai Q. Dinh3, Giovanni Fontana2, Axel Fischer3, Pierangelo Geppetti2 and Marcello Trevisani4

1Department of Clinical & Experimental Medicine, Pharmacology Section, University of Ferrara, Italy; 2Department of Critical Care Medicine and Surgery, University of Florence, T Florence, Italy; 3Allergie-Zentrum Charité, Charité-Universitätsmedizin Berlin, Berlin,I P Germany; 4PharmEste srl, Ferrara, Italy. R C

Abbreviated title: Ethanol exacerbates cough in guinea pig S U N *, Author for Correspondence: Raffaele Gatti Ph.D., Department of Clinical & Experimental Medicine, Pharmacology Section, University of Ferrara, 44100A Ferrara, Italy. Phone: +39 (0)532 455224; Fax: +39 (0)532 455205; Me-mail: [email protected]

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Abstract: Ethanol is a chemical irritant able to induce a large variety of effects in the airways. It has been reported that ethanol sensitises the transient receptor potential vanilloid 1 (TRPV1) to various stimuli and inhalation of ethanol enhances the cough mediated by TRPV1 activation () in patients suffering of airway sensory hyperreactivity. Here, we set out to investigate whether ethanol sensitizes the cough induced by TRPV1 activation in a guinea pig model and the possible mechanism of such exacerbating effect. T Aerosolized (RTX, 0.5 µM) and hypertonic saline (7%) produced a coughIP response dependent and independent of TRPV1 activation, respectively. Ethanol (3%, 10 min) inhalation, that per se did not cause any tussive response, significantly increased theR number of coughs evoked by RTX inhalation without affecting hypertonic saline (7%) inducedC cough. Potentiation by ethanol of the tussive response to RTX was prevented by the PKC inhibitor, GF109203X (GFX). S In conclusion, ethanol selectively exaggerates, via a PKC-dependentU pathway, the cough response evoked by TRPV1 stimulation. The present results mayN contribute to explain respiratory distresses sometimes associated to alcohol consumption, including cough and asthma. A M Keywords: cough, guinea pig, vanilloid receptor-1 (TRPV1), resiniferatoxin, protein kinase C (PKC). D E T P E C C A

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Introduction

Ethyl alcohol, (ethanol) is known to produce a variety of neuronal actions, including an inhibitory effect, possibly by facilitating the opening of gamma aminobutyric acid-A (GABA- A) receptor-chloride channels [1]. An opposite excitatory action of ethanol has recently been described in primary sensory neurons [2] expressing the transient receptor potential vanilloid- 1 (TRPV1) [3]. TRPV1 is a cation channel rather selectively expressed in a subset of T nociceptive primary sensory neurons with C and A-δ fibers, which are activated by noxiousIP temperature, low extracellular pH [3,4], and a variety of lipids, including , N- arachidonoyl dopamine, and certain eicosanoids [5-7]. The excitatory action of ethanolR on TRPV1 is most likely due to its ability to lower the threshold temperature forC the channel activation [2]. Ethanol has been shown also to dramatically potentiate TRPV1 activation by protons and anandamide [2]. S Different intracellular pathways have been reported to contribute to UTRPV1 sensitization. Protein kinase A (PKA) has been shown to sensitize TRPV1 [8]N and other mechanisms independent of PK [9] seem to contribute to TRPV1 exaggerated responses. Type C protein kinase (PKC) contributes to the exaggeration of TRPV1-mediatedA responses by activation of G protein coupled receptors, including the proteinase Mactivated receptor-2 (PAR2) [10], the B2 receptor [11] or by agonists of tyrosine kinase receptors [12,13]. In damaged or irritated skin or mucosal surface,D exposure to ethanol is often associated to a burning pain sensation, that has proposed to be due to the sensitizing effect of ethanol on TRPV1 [2]. A typical response mediated byE TRPV1 activation in experimental animals and man is cough [14], and alcoholic drinksT are capable of triggering a wide range of irritative and defensive responses in alcoholic drink-sensitiveP individuals, including rhinitis, itching, facial swelling, headache, cough andE asthma [15 ,16-18]. It has been reported that ethanol- containing corticosteroid medicines cause bronchoconstriction in susceptible asthmatic patients supposedly becauseC of the alcohol included in the formulation [19]. Moreover, inhalation of ethanolC 5 and 25% enhanced the cough reaction to capsaicin [20] in patients with airway sensory hyperreactivity.A Thus, the purpose of the present study was to investigate whether ethanol could exaggerate the cough response produced by TRPV1 stimulation in a guinea pig model and whether PKC could contribute to the modulation of the evoked tussive response.

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Experimental procedures

Animals. Male Dunkin-Hartley guinea pigs (250-350 g, Pampaloni, Italy) were acclimatized in cages, (24 ± 0.5 ºC) for 1 week after delivery, and were allowed free access to water and standard rodent diet (Morini, Italy). All experiments complied with the national guidelines and were approved by the regional ethics committee. T Cough study IP Experimental set-up. After the period of acclimatisation to laboratory conditions, animals were individually placed in a transparent perspex box (20 x 10 x 10 cm, Vetrotecnica, Italy)R ventilated with a constant airflow of 400 ml/min. Pro-tussive stimuli were nebulisedC via a mini- ultrasonic nebuliser (Model 2511; PulmoSonic, DeVilbiss). The particle size produced had an aerodynamic mass median diameter of 0.9 µm and the output of the nebuliserS was 0.4 ml per min. Identification of a cough response was achieved by three differentU approaches: 1) by observing/counting the typical cough posture of the guinea pig N(by a trained and blind observer) during the challenge; 2) by the presence of the investigator who confirmed the cough sounds during the challenge (transmitted from the microphoneA in the cage to the recorder and to the outside speakers); 3) by the subsequent analysisM of the sound waves recorded into a personal computer. The cough sounds were recorded, digitally stored and counted by a blind observer. D Study Protocols. All experiments were carried out at the same time of day starting at 9.00 a.m. To elicit cough, guinea pigs were exposed Efor 10 min to aerosols of RTX (0.5 µM) and hypertonic saline (7% sodium chloride,T 1.2 M). To evaluate the potential modulatory role of ethanol against the TRPV1-mediatedP cough, aerosolised ethanol (1-3%) was administered to guinea pigs for 10 min prior toE the cough challenge. In experiments aimed at evaluating the role of PKC on cough exacerbation, the PKC blocker, GF109203X (GFX, 1 µM) was aerosolised for 10 min priorC to the aerosol with ethanol 3%. The effect of aerosolisedC ethanol was also investigated against hypertonic saline (7% sodium chloride), a stimulusA that induces cough in a TRPV1-indipendent manner. Hypertonic saline was administered for 10 min after the administrations of 3% ethanol. To prevent the possible contribution of bronchoconstriction in the tussive response to the diverse stimuli, all guinea pigs were intraperitoneally administered with the β-adrenoceptor agonist, terbutaline (0.5 mg/kg), five minutes prior to the beginning of the experiment.

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Drugs. Agents were obtained from the following companies: resiniferatoxin, (Tocris, UK); GF109203X (GFX), (Alexis Corporation, Switzerland). The stock concentration of RTX (1 mM) and GFX (1 mM) were prepared in 100% DMSO. All the other drugs were dissolved in isotonic solution.

Data analysis. Values were presented as mean ± SE. Data are compared using Student’s t-test or standard one way analysis of variance (ANOVA) following by post hoc Bonferroni’s test.T A p value < 0.05 was considered significant. A minimum of 6 guinea pigs were used to testIP the effect of vehicle or of each single dose of the drugs. R Results C Aerosolised ethanol (1-3%, per 10 min) did not induce a significant increase in the number of coughs (1.2 ± 1.0, n = 8) if compared to that produced by isotonic saline S(0.9% sodium chloride, 1.0 ± 0.2, 10 min, n = 10, data not shown). U In contrast, aerosolised RTX (0.5 µM), administered for 10 min,N produced a number of coughs (10.9 ± 0.8, n = 13, fig. 1) significantly higher than those induced by aerosolised isotonic saline (0.9% sodium chloride, 1.1 ± 1.3, n = 12). Pre-treatment withA aerosolised ethanol (1% and 2%) did not modify the number of coughs induced by RTXM (fig. 1). In contrast, aerosolised 3% ethanol (per 10 min) consistently and significantly enhanced RTX-induced cough (45 ± 8% of increase, n = 12, fig. 1). D Furthermore, an aerosol pre-treatment with GFX practically abolished the cough exacerbation produced by ethanol 3% (fig. 2) suggestingE a crucial role of PKC in the observed cough exacerbation. T In the experimental set aimed to evaluateP the selectivity of the exacerbating effect of ethanol, 3% ethanol was aerosolised perE 10 min prior to the cough challenge with hypertonic saline (7%, sodium chloride) and no modification of the tussive response were observed between ethanol-treated animals (6.3C ± 1.5, n = 7) and animals that received aerosolised hypertonic saline (7.3 ± 0.3, n =C 6) (fig. 3). Finally, in an independentA experimental set, after 10 min of aerosolisation with 3% ethanol, guinea pig blood samples were collected and alcoholemia was quantified. No measurable plasmatic levels of ethanol were found in any of the blood samples (data not shown).

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Discussion

The present results show that ethanol potentiates TRPV1-mediated cough by a PKC-dependent mechanism in a guinea pig model. Specifically, ethanol at doses that per se did not produce any measurable cough response, exaggerated RTX- evoked cough. The ability of ethanol to affect TRPV1 seems to derive from its property to sensitize the channel to other stimuli. For instance, in electrophysiological experiments ethanol reduced the threshold temperature for TRPV1 T activation by 8°C [2]. Thus, ethanol, reducing the threshold temperature for TRPV1 activationIP from 43°C [3] to 35°C [2] allowed the physiological temperature to activate the channel. TRPV1 activation thresholds to anandamide and protons are also lowered by ethanol pre-treatmentR [2]. Interestingly, ethanol activity on the cough reflex appeared to be selective, sinceC it did not affect the tussive response evoked by hypertonic saline, a tussigenic agent [21,22] which is thought to induce cough through a mechanism totally independent from the TRPV1S activation [22,23]. The fact that no detectable ethanol plasma levels were attained afterU the exposure to the higher tested aerosolised concentration (3%), indicates that ethanol enhancedN TRPV1-dependent cough through a peripherally restricted action. TRPV1 channel activity is remarkably up-regulated by differentA inflammatory mediators which promote channel phosphorilation by either activation Mof PKC or cAMP-dependent PKA mechanism. Phosphorilation of TRPV1 increases th e probability of the ion channel to open in response to heat, protons and endogenous lipidsD [11,24]. Thus, TRPV1 phosphorilation seems to play an important role in the regulation of channel activity that finally results in channel sensitization. PKC is known to phosphorylateE several cellular components, including enzymes and membrane-bound receptors and ionT channels that are key regulators in the processe of nociceptor excitation and sensitizationP (for review see: [9]). Interestingly, PKC has been shown to arbitrate the effects of ethanolE on receptors [25] and membrane-bound transporters [26], but the mechanism underlying this modulation has not been elucidated. Ethanol causes also translocation of PKC in aC similar manner to that induced by esters [27] and similarly to ethanol, phorbol estersC act on PKC activating TRPV1 channels and decreasing its threshold of activation to heatA [28,29]. Because ethanol has been associated to PKC translocation [30] and PKC appears to play an integral role in TRPV1 sensitisation, we hypothesize that PKC could mediate ethanol-induced sensitization of TRPV1 in the airways in vivo. In this respect, we have previously shown that the PKC activator, 12-O-tetradecanoylphorbol- 13-acetate (TPA), that per se does not cause cough, significantly potentiated citric acid- and RTX-induced cough in guinea pig, an effect that was completely reversed by GFX [31]. The

ARTICLE IN PRESS 7 aforementioned results reinforce the hypothesis that PKC promotes activation of TRPV1 indirectly via phosphorilation of some residues of the protein [32]. Involvement of PKC in ethanol-mediated potentiation of TRPV1-dependent cough was indicated by the observation that PKC inhibition by GFX, abated the exaggerated response induced by pre-exposure to ethanol. The selectivity of this effect is supported by the fact that ethanol did not show any modulatory effect against hypertonic saline-induced cough. Furthermore, as previously shown, treatment with aerosolised TPA did not exacerbate the cough induced by hypertonic saline[31].T We shown here that aerosolized ethanol enhances the cough response to TRPV1 agonistsIP via a PKC-dependent mechanism without interfering with a cough reflex induced by a TRPV1- independent mechanism. A recent finding showed that a positive correlation betweenR the tussive response to capsaicin and the number of TRPV1-positive nerves in patients withC chronic cough exists [33]. Moreover patients with airways sensory hyperresponsiveness exhibited an exacerbated cough reflex to capsaicin after inhalation of ethanol at concentrationsS significantly higher (5 and 25%) than those used in the present work (3%) [20]. HereU we described that the mechanism of action of such exacerbating effect involves the activationN of PKC. This observations may suggest that an increased network of TRPV1 sensory fibres may predispose susceptible individuals to cough exacerbation byA a large variety of conditions, including accidental, iatrogenic or alimentary exposureM to ethanol.

Grants: This work was in part supported by ARCA,D Padua and MUIR, Rome.

CONFLICT OF INTERESTS: Authors haveE no, real or perceived competing of interest that relate to this manuscript. T

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[14] Morice AH, Geppetti P. Cough. 5: The type 1 vanilloid receptor: a sensory receptor for cough. Thorax 2004;59:257-8. [15] Vally H, de Klerk N, Thompson PJ. Alcoholic drinks: important triggers for asthma. J Allergy Clin Immunol 2000;105:462-7. [16] Myou S, Fujimura M, Nishi K, Watanabe K, Matsuda M, Ohka T, et al. Effect of ethanol on airway caliber and nonspecific bronchial responsiveness in patients with alcohol-induced asthma. Allergy 1996;51:52-5. T [17] Saito Y, Sasaki F, Tanaka I, Sato M, Okazawa M, Sakakibara H, et al. Acute severeIP alcohol- induced bronchial asthma. Intern Med 2001;40:643-5. [18] Zellweger JP. Alcohol-induced asthma: not only in Asians. J Allergy Clin RImmunol 1997;99:860. C [19] Antonicelli L, Micucci C, Bonifazi F. Bronchospasm induced by inhalant corticosteroids: the role of ethanol. Allergy 2006;61:146-7. S [20] Millqvist E, Ternesten-Hasseus E, Bende M. Inhaled ethanolU potentiates the cough response to capsaicin in patients with airway sensory hyperreactivity.N Pulm Pharmacol Ther 2008;22:[Epub ahead of print]. [21] Fahy JV, Wong HH, Geppetti P, Reis JM, Harris SC,A Maclean DB, et al. Effect of an NK1 receptor antagonist (CP-99,994) on hypertonicM saline-induced bronchoconstriction and cough in male asthmatic subjects. Am J Respir Crit Care Med 1995;152:879-84. [22] Lalloo UG, Fox AJ, Belvisi MG, ChungD KF, Barnes PJ. inhibits cough induced by capsaicin and citric acid but not by hypertonic saline in guinea pigs. J Appl Physiol 1995;79:1082-7. E [23] Trevisani M, Milan A, Gatti R,T Zanasi A, Harrison S, Fontana G, et al. Antitussive activity of iodo-resiniferatoxin in guineaP pigs. Thorax 2004;59:769-72. [24] Vellani V, MapplebeckE S, Moriondo A, Davis JB, McNaughton PA. Protein kinase C activation potentiates gating of the vanilloid receptor VR1 by capsaicin, protons, heat and anandamide. J PhysiolC 2001;534:813-25. [25] Wafford KA,C Whiting PJ. Ethanol potentiation of GABAA receptors requires phosphorylationA of the alternatively spliced variant of the gamma 2 subunit. FEBS Lett 1992;313:113-7. [26] Nagy LE, Diamond I, Casso DJ, Franklin C, Gordon AS. Ethanol increases extracellular adenosine by inhibiting adenosine uptake via the nucleoside transporter. J Biol Chem 1990;265:1946-51.

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[27] Gordon AS, Yao L, Wu ZL, Coe IR, Diamond I. Ethanol alters the subcellular localization of delta- and epsilon protein kinase C in NG108-15 cells. Mol Pharmacol 1997;52:554-9. [28] Numazaki M, Tominaga T, Toyooka H, Tominaga M. Direct phosphorylation of capsaicin receptor VR1 by protein kinase Cepsilon and identification of two target serine residues. J Biol Chem 2002;277:13375-8. [29] Sugiura T, Tominaga M, Katsuya H, Mizumura K. Bradykinin lowers the threshold temperature for heat activation of vanilloid receptor 1. J Neurophysiol 2002;88:544-8.T [30] Stubbs CD, Slater SJ. Ethanol and protein kinase C. Alcohol Clin Exp Res 1999;23:1552-IP 60. [31] Gatti R, Andre E, Amadesi S, Dinh TQ, Fischer A, Bunnett NW, et al. ProteaseR activated receptor 2 activation exaggerates TRPV1-mediated cough in guinea pigs.C J Appl Physiol 2006;101:506-11. [32] Bhave G, Hu HJ, Glauner KS, Zhu W, Wang H, Brasier DJ, et al.S Protein kinase C phosphorylation sensitizes but does not activate the capsaicinU receptor transient receptor potential vanilloid 1 (TRPV1). Proc Natl Acad Sci U S AN 2003;100:12480-5. [33] Groneberg DA, Niimi A, Dinh QT, Cosio B, Hew M, Fischer A, et al. Increased expression of transient receptor potential vanilloid-1 in airway Anerves of chronic cough. Am J Respir Crit Care Med 2004;170:1276-80. M

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Legends to Figures :

Fig. 1: Dose dependent effect of aerosolised ethanol (EtOH, 1-3%) on resiniferatoxin (RTX, 0.5 µM)-induced cough in the guinea pig. Aerosolised ethanol was administered for 10 min prior to the cough challenge with RTX. *, P < 0.05, ANOVA and Bonferroni’s test vs. vehicle (VEH). Each column is presented as mean (S.E.M.) of at least 6 experiments. T Fig. 2: Effect of aerosolized GF109203X (GFX, 1 µM) on ethanol (EtOH, 3%)-inducedI P resiniferatoxin (RTX, 0.5 µM) dependent cough exaggeration in the guinea pig. #, P < 0.05,

ANOVA and Bonferroni’s test vs. vehicle (VEH1); *, P < 0.05, ANOVA and Bonferroni’sR test vs. vehicle (VEH2). Each column is presented as mean (S.E.M.) of at least 6 experiments.C

Fig. 3: Effect of aerosolized ethanol (EtOH, 3%) on cough induced by inhalationS of hypertonic saline (HS, 7% sodium chloride) in the guinea pig. Each column is presentedU as mean (S.E.M.) of at least 6 experiments. Student’s t-Test test was used and no Nstatistically significance was observed between the two groups. A

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(10 min) A n of coughs of n M 0 VEHD EtOH E (3%) T P E C C A