Pannexin 1 Contributes to ATP Release in Airway Epithelia

Abstract

ATP is a paracrine regulator of critical airway epithelial cell functions, but the mechanism of its release is poorly understood. Pannexin (Panx) proteins, related to invertebrate innexins, form channels (called pannexons) that are able to release ATP from several cell types. Thus, ATP release via pannexons was examined in airway epithelial cells. Quantitative RT-PCR showed Panx1 expression in normal human airway epithelial cells during redifferentiation at the air-liquid interface (ALI), at a level comparable to that of alveolar macrophages; Panx3 was not expressed. Immunohistochemistry showed Panx1 expression at the apical pole of airway epithelia. ALI cultures exposed to hypotonic stress released ATP to an estimated maximum of 255 (+/-64) nM within 1 minute after challenge (n = 6 cultures from three different lungs) or to approximately 1.5 (+/-0.4) microM, recalculated to a normal airway surface liquid volume. Using date- and culture-matched cells (each n > or = 16 from 4 different lungs), the pannexon inhibitors carbenoxolone (10 microM) and probenecid (1 mM), but not the connexon inhibitor flufenamic acid (100 microM), inhibited ATP release by approximately 60%. The drugs affected Panx1 currents in Xenopus oocytes expressing exogenous Panx1 correspondingly. In addition, suppression of Panx1 expression using lentivirus-mediated production of shRNA in differentiated airway epithelial cells inhibited ATP release upon hypotonic stress by approximately 60% as well. These data not only show that Panx1 is expressed apically in differentiated airway epithelial cells but also that it contributes to ATP release in these cells.

Alanine substitution scanning of pannexin1 reveals amino acid residues mediating ATP sensitivity

“…Furthermore, ATP release or surrogate measures are correlated with Panx1 expression levels. In knockdown experiments with Panx1 siRNA/shRNA, the ATP release is largely attenuated [3,14]. Also, the pharmacology of Panx1 channels matches that of ATP release.…”

“…However, instead of forming the intercellular channels of gap junctions as connexins do, pannexins operate as unpaired pannexons, allowing the flux of molecules from the cytoplasm to the extracellular space and vice versa [1]. It has been recognized that Pannexin1 (Panx1) exerts a key role in the release of ATP [2][3][4][5]. In this function Panx1 contributes to the propagation of intercellular calcium waves.…”

“…The evidence for the ATP release function of Panx1 channels includes that Panx1 is expressed in cells that are documented to have channel-mediated ATP release, including erythrocytes, endothelial cells, astrocytes and airway epithelial cells [3][4][5]14]. Notable in airway epithelial cells, the expression of Panx1 is restricted to the apical membrane of these cells, where the ATP release takes place [3].…”

“…Notable in airway epithelial cells, the expression of Panx1 is restricted to the apical membrane of these cells, where the ATP release takes place [3]. Furthermore, ATP release or surrogate measures are correlated with Panx1 expression levels.…”

Evidence for sustained ATP release from liver cells that is not mediated by vesicular exocytosis

“…There is evidence for ATP release through ATPbinding cassette (ABC) transporters, connexin hemichannels and multiple Cl − channels [12][13][14]. More recent studies have found that pannexin1 hemichannels play a key role in ATP release from many different cells [15][16][17][18]. Vesicular exocytosis may also contribute to ATP release [19][20][21].…”