Acetylcholine-induced ex vivo ATP release from the human nasal mucosa
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.
Section snippets
Patients and sample collection
The inferior turbinate mucosa was collected from 21 patients with chronic hypertrophic rhinitis who underwent endoscopic turbinectomy. The study included 17 male and 4 female patients, aged 17–79 years, with an average age of 49.0 years. Total and/or specific serum IgE levels were positive in 15 patients. Bilateral inferior turbinate bones were resected together with the lateral mucosa of the turbinate under general anesthesia. Informed consent was obtained from all patients, and the study was
Results
The baseline release of ATP without stimulus was 57.2 ± 10.3 fM (n = 16), ranging from 12 to 146 fM. The ATP release was significantly increased to 168.9 ± 20.9 fM (n = 16) by stimulation with 100 μM Ach (P = 0.0004; Fig. 1). In the following experiments, 100 μM Ach was used as a stimulus. The Ach-induced ATP release was completely inhibited when Ca2+-free HBSS (HBSS(−); 8000 (in mg/L) NaCl, 400 KCl, 350 NaHCO3, 60 KH2PO4, 47.9 Na2HPO4, 1000 glucose) containing 2 mM EGTA was used as an incubation buffer (Fig. 2
Discussion
The present study showed that Ach induces ATP release from the human nasal mucosa through the activation of a muscarinic Ach receptor and that this release is probably mediated by the pannexin-1 channel but not by vesicular release. There are 5 subtypes of muscarinic Ach receptors, M1–M5. Previous studies have demonstrated that the Ach receptor dominantly expressed in the nasal mucosa is M3 [16], [17]. M3 is thought to be coupled with the Gq protein [18], which upregulates phospholipase C, and
Conflict of interest
The authors have no other funding, financial relationships or conflicts of interest to disclose.
Acknowledgement
This study was supported by a Grant-in-Aid for Scientific Research (C) (No. 16K11203; 2016–2018) to H. S. from Japan Society for the Promotion of Science.
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These three authors equally contributed to this work.