Acetylcholine-induced ex vivo ATP release from the human nasal mucosa

Abstract

Objective

The present study aimed at investigating ATP release in response to acetylcholine (Ach) and pharmacologically elucidating the intracellular signal transduction pathway of this reaction in an ex vivo experiment.

Methods

The inferior turbinate mucosa was collected from 21 patients with chronic hypertrophic rhinitis who underwent endoscopic turbinectomy. The mucosa was shaped into a filmy round piece, and incubated with chemical(s) in Hank's balanced salt solution for 10 min. After incubation, the ATP concentration was measured by a luciferin-luciferase assay.

Results

The baseline release of ATP without stimulus was 57.2 ± 10.3 fM. The ATP release was significantly increased by stimulation with 100 μM Ach. The Ach-induced ATP release was completely inhibited by removing extracellular Ca²⁺. Significant inhibition of the Ach-induced ATP release was also observed by the addition of 1 μM atropine, 40 μM 2-APB, 10 μM CBX, and 100 μM PPADS, whereas 30 nM bafilomycin A1 did not affect the ATP release.

Conclusion

These results indicate that the Ach-induced ATP release from the human nasal mucosa is dependent on the pannexin-1 channel and purinergic P2X7 receptor, suggesting that these two molecules constitute a local autocrine/paracrine signaling system in the human nasal epithelium.

Introduction

Airway mucociliary transport function is essential for the clearance of inhaled foreign particulate matter along with secreted mucus, and thereby plays an important role in the host defense system. Various inflammatory upper and lower airway diseases are associated with mucociliary dysfunction. It is known that the mucociliary transport function of the airway epithelium is regulated by extracellular adenosine triphosphate (ATP) and intracellular Ca²⁺ through the activation of purinergic receptors on the surface of the epithelial cells [1], [2], [3]. It has also been shown that mucociliary transport is promoted by acetylcholine (Ach) in the upper and lower airway epithelium [4], [5]. However, the intracellular transduction pathway that connects the Ach receptor and ATP release is scarcely understood in humans, and only partially elucidated even in experimental animals.

ATP release is thought to be mediated by two different mechanisms: vesicle- and channel-mediated pathways. Among the many channel-mediated pathways, the pannexin-1 channel is the most convincing candidate for a mediator of ATP release of ATP in a diverse range of cell types, including airway epithelial cells [6]. Pannexins constitute a family of transmembrane channel proteins in vertebrates. They are homologous to the invertebrate gap-junction proteins known as innexins [7], and consist of three subtypes, pannexin-1, pannexin-2, and pannexin-3. Pannexins have no sequence similarity to connexins, the prototypical vertebrate gap-junction proteins [8]. The pannexin genes were originally cloned as gap-junction-related proteins, but their significance in vivo has not been fully understood to date.

Several authors have reported ATP release through the pannexin-1 channel [9], [10], [11]. Bao et al. [9] observed that oocytes exogenously expressing pannexin-1 release ATP when depolarized by a high concentration of extracellular K⁺. Ransford et al. [11] found that cultured airway epithelial cells isolated from the human lung secrete ATP via pannexin-1 upon hypotonic stress. Seminario-Vidal et al. [10] also demonstrated hypotonicity-evoked pannexin-1-mediated ATP release from cultured human bronchial epithelial cells and from the excised mouse trachea. With regard to the upper airway, we have recently shown in an ex vivo experiment that the human nasal mucosa expresses pannexin-1 and releases ATP through this channel in response to a hypotonic stimulus [12]. Using the same ex vivo experimental system, the present study aimed at investigating ATP release in response to Ach and pharmacologically elucidating the intracellular signal transduction pathway of this reaction.