Cell deformation at the air-liquid interface induces Ca²⁺-dependent ATP release from lung epithelial cells

Abstract

Extracellular nucleotides regulate mucociliary clearance in the airways and surfactant secretion in alveoli. Their release is exquisitely mechanosensitive and may be induced by stretch as well as airflow shear stress acting on lung epithelia. We hypothesized that, in addition, tension forces at the air-liquid interface (ALI) may contribute to mechanosensitive ATP release in the lungs. Local depletion of airway surface liquid, mucins, and surfactants, which normally protect epithelial surfaces, facilitate such release and trigger compensatory mucin and fluid secretion processes. In this study, human bronchial epithelial 16HBE14o(-) and alveolar A549 cells were subjected to tension forces at the ALI by passing an air bubble over the cell monolayer in a flow-through chamber, or by air exposure while tilting the cell culture dish. Such stimulation induced significant ATP release not involving cell lysis, as verified by ethidium bromide staining. Confocal fluorescence microscopy disclosed reversible cell deformation in the monolayer part in contact with the ALI. Fura 2 fluorescence imaging revealed transient intracellular Ca(2+) elevation evoked by the ALI, which did not entail nonspecific Ca(2+) influx from the extracellular space. ATP release was reduced by ∼40 to ∼90% from cells loaded with the Ca(2+) chelator BAPTA-AM and was completely abolished by N-ethylmalemide (1 mM). These experiments demonstrate that in close proximity to the ALI, surface tension forces are transmitted directly on cells, causing their mechanical deformation and Ca(2+)-dependent exocytotic ATP release. Such a signaling mechanism may contribute to the detection of local deficiency of airway surface liquid and surfactants on the lung surface.

Interfacial stress affects rat alveolar type II cell signaling and gene expression

“…This demonstrates that the surface tension of the I AL (in our experiments) is in a range to overwhelm the elastic modulus of AT II cells (3), and that neither cellular surface tension nor any internal scaffolding is able to sustain the loads induced by a curved I AL (50). Thus these data directly show that the I AL is able to exert a mechanical deforming stress on the cells, which is in accordance to what has been postulated and shown by different methods in recent studies before (55,56). Additionally, these data show that cell response is due neither to simple fluid removal nor to dry out phenomena, but due to a critical compression of the cells preceding an actual loss of cell water.…”

“…Furthermore, since cell flattening occurred considerably earlier than cell damage, we conclude that, at least at the beginning, it is not a harmful event (56). Importantly, these data directly show that the I AL exerts a mechanical deforming stress on the cells, which is in accordance with what has been postulated and shown by different methods in recent studies before (55,56).…”

“…Within the alveolar lining fluid, ATP might reach millimolar concentrations (58), but the source of it is essentially unknown. Beside a supposed release from scattered purinergic nerve terminals, and release due to cell membrane stress failures, a channel-gated release from AT II cells, as well as a mechanosensitive ATP-exocytosis, has been proposed (16,55). Here, a release of ATP, in concentrations similar to those reported by Patel et al (49) for cyclic stretch, was detected after a brief I AL exposure (ϳ10 s).…”

“…Interestingly, even small amounts of surfactant (0.1 mg/ml) significantly improved the viability of epithelial cells exposed to an advancing I AL , as it is probably also the case for the degree of interfacial compression of the surface films or the amount of surface-associated material (29). Consequently, one could speculate that pulmonary surfactant, in addition to its well-known functions, also serves as a protective surface coat, and that the ability of cells to produce such a coat might have been an important prerequisite in the evolution of air-exposed epithelia to further allow the development of entire gas-exchanging organs, a hypothesis also similarly raised elsewhere (55).…”

“…Previously, it has been shown that cells respond in conventional and in inverted interface experiments with intracellular Ca 2ϩ signals (55,56,63). Here we extended those studies using agonists and inhibitors for various channels and pathways (Fig.…”