Involvement of P2X receptor subtypes in ATP-induced enhancement of the cough reflex sensitivity

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Abstract

We examined the effect of inhaled ATP on the chemical irritant-induced coughs to clarify the roles of ionotropic purinergic receptors in these modulations. Although inhalation of 0.1 M citric acid by itself produced only a few coughs in guinea pigs, exposure to ATP, at concentrations of 3–10 μM, for 2 min concentration-dependently increased the number of 0.1 M citric acid-induced coughs. ATP-induced enhancement of the number of citric acid-induced coughs was abolished when animals were pretreated with 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5-triphosphate (TNP-ATP), an antagonist of P2X receptor subtypes P2X14, at a concentration of 50 μM, for 2 min. However, exposure to pyridoxal-phosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS), an antagonist of P2X receptor subtypes P2X1,2,3,5,7, but not of P2X4 receptors, at a concentration of 50 μM, for 2 min, had no effect on the ATP-induced enhancement of the number of citric acid-induced coughs. Furthermore, exposure to reactive blue 2 (RB2, 30 μM, 2 min), an antagonist of P2Y receptors, had no effect on the ATP-induced enhancement of the number of citric acid-induced coughs. Exposure to ATP, at a concentration of 10 μM, for 2 min significantly increased the number of citric acid-induced coughs in capsaicin-pretreated guinea pigs. Furthermore, ATP had no effect on the number of capsaicin-induced coughs in naive animals. These results suggest that although ATP, by itself, does not elicit spontaneous coughs, it likely enhances the cough reflex sensitivity. Furthermore, stimulation of P2X receptors, especially P2X4 receptors, on rapidly adapting receptors may be required for the ATP-induced enhancement of the cough reflex sensitivity.

Introduction

Cough is an important respiratory defense mechanism and one of the most important symptoms of pulmonary disorders. Chronic cough is generally defined as cough persisting for 8 weeks or longer. The morbidity associated with chronic cough in humans is likely to be a product of the enhanced frequency and intensity of coughs that occurs as a result of increased excitability in this behavior. Patients with a variety of pulmonary disorders show an enhanced cough sensitivity in response to inhaled irritants, but the frequency and intensity of cough can be elevated. The mechanisms by which the sensitivity, spontaneous frequency and magnitude of cough are increased in airway disease are poorly understood. Therefore, a better understanding of the pathogenic mechanisms of chronic cough is extremely important for the development of new therapeutic strategies to alleviate this stress.

ATP has long been recognized to be an activator of sensory nerves (Brouns et al., 2000). ATP can act on metabotropic and ionotropic purinergic receptors (Burnstock, 2001). The metabotropic receptors are designated P2Y receptors, whereas the ionotropic receptors are referred to as P2X receptors. In the somatosensory system, P2X receptors are most often involved in ATP-induced action potential discharge. In the canine lung, ATP was found to cause a burst of action potentials in vagal C-fibers by a mechanism that was inhibited by nonselective P2X receptor antagonism (Pelleg and Hurt, 1996). In guinea pigs, Undem and his coworkers have found that ATP effectively activates a subset of C-fibers and rapidly adapting receptors. In each case, the response is blocked by P2X receptor antagonists (Canning et al., 2004, Undem et al., 2004). Although these results strongly suggest the possibility that ATP may modulate the cough reflex through an excitatory mechanism, the role of ATP in the regulation of cough reflex is not yet clearly defined.

Therefore, the present study was designed to investigate the effect of inhaled ATP on the chemical irritant-induced coughs, and to clarify the roles of ionotropic purinergic receptors in these modulations.

Section snippets

Animals

Male Hartley guinea pigs (Tokyo Animal Laboratory Inc., Tokyo, Japan) weighing about 300–350 g were used. The animals were housed in groups of four per cage under a 12-h light–dark cycle with food and water available continuously. These studies were carried out in accordance with the Guidelines for the Use of Laboratory Animals as adopted by the Committee on the Care and Use of Laboratory Animals of Hoshi University which is accredited by the Ministry of Education, Science, Sports and Culture.

Antitussive assay

Effect of ATP on the number of citric acid-induced coughs

A concentration of 0.1 M citric acid was selected for inducing coughs, since our preliminary study showed that this concentration by itself produced only few coughs in guinea pigs (1.90 ± .6 coughs / 10 min, n = 6). As shown in Fig. 1A, exposure to ATP, at concentrations of 3–10 μM, for 2 min concentration-dependently increased the number of citric acid-induced coughs. A significant increase in the number of coughs was observed when animals were exposed to 6 and 10 μM of ATP (6 μM: pre, 1.5 ± 0.4 coughs

Discussion

In the present study, we demonstrated that ATP concentration-dependently and significantly enhanced the number of citric acid-induced coughs in guinea pigs and this effect was completely blocked by pretreatment with inhaled TNP-ATP, but not PPADS aerosol. Furthermore, exposure to RB2, an antagonist of P2Y receptors, had no effect on the ATP-induced enhancement of the number of citric acid-induced coughs. P2X receptors are a family of cation channels gated by extracellular ATP (Jahr and Jessell,

Acknowledgements

We thank Mr. S. Hayashi, Mr. D. Takemura and Miss K. Machida for their excellent technical assistance.

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