Faculty Opinions recommendation of Ca2+-dependent ATP release from A549 cells involves synergistic autocrine stimulation by coreleased uridine nucleotides.

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 Ca2+ elevation evoked by the ALI, which did not entail nonspecific Ca2+ influx from the extracellular space. ATP release was reduced by ∼40 to ∼90% from cells loaded with the Ca2+ 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 Ca2+-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.

Download Full-text

Cell swelling-induced ATP release and gadolinium-sensitive channels

AJP Cell Physiology ◽

10.1152/ajpcell.00317.2001 ◽

2002 ◽

Vol 282 (1) ◽

pp. C219-C226 ◽

Cited By ~ 71

Author(s):

Francis Boudreault ◽

Ryszard Grygorczyk

Keyword(s):

Membrane Lipids ◽

Atp Release ◽

A549 Cells ◽

Cell Swelling ◽

Specific Effects ◽

Underlying Mechanisms ◽

Dependent Processes ◽

Atp Efflux ◽

Stretch Activated Channels ◽

Hypotonic Swelling

ATP release induced by hypotonic swelling is an ubiquitous phenomenon in eukaryotic cells, but its underlying mechanisms are poorly defined. A mechanosensitive (MS) ATP channel has been implicated because gadolinium (Gd3+), an inhibitor of stretch-activated channels, suppressed ATP efflux monitored by luciferase bioluminescence. We examined the effect of Gd3+on luciferase bioluminescence and on ATP efflux from hypotonically swollen cells. We found that luciferase was inhibited by ≤10 μM Gd3+, and this may have contributed to the previously reported inhibition of ATP release. In ATP efflux experiments, luciferase inhibition could be prevented by chelating Gd3+with EGTA before luminometric ATP determinations. Using this approach, we found that 10–100 μM Gd3+, i.e., concentrations typically used to block MS channels, actually stimulated hypotonically induced ATP release from fibroblasts. Inhibition of ATP release required at least 500, 200, or 100 μM Gd3+ for fibroblasts, A549 cells, and 16HBE14o− cells, respectively. Such biphasic and cell-specific effects of Gd3+ are most consistent with its action on membrane lipids and membrane-dependent processes such as exocytosis.

Download Full-text

Rhinovirus-16 increases ATP release in A549 cells without concomitant increase in production

ERJ Open Research ◽

10.1183/23120541.00159-2020 ◽

2020 ◽

Vol 6 (4) ◽

pp. 00159-2020

Author(s):

Samantha K. Atkinson ◽

Alyn H. Morice ◽

Laura R. Sadofsky

Keyword(s):

Epithelial Cells ◽

Atp Release ◽

A549 Cells ◽

Airway Epithelial Cells ◽

Alveolar Epithelial Cells ◽

Intercellular Adhesion Molecule ◽

Upper Respiratory Tract ◽

Intracellular Atp ◽

Airway Hypersensitivity ◽

Secondary Stimulation

Human rhinovirus (RV) is the most common cause of upper respiratory tract infection (URTI) and chronic airway disease exacerbation. Cough is present in 50–80% of URTI cases, accompanied by heightened airway hypersensitivity, yet no effective treatment currently exists for this infectious cough. The mechanism by which RV causes cough and airway hypersensitivity in URTI is still unknown despite recent advances in potential therapies for chronic cough.The effect of RV-16 infection (MOI 1) on intracellular ATP stores and ATP release in A549 alveolar epithelial cells was measured.RV-16 infection was found to significantly increase (by 50% from basal at 24 h) followed by decrease (by 50% from basal at 48 and 72 h) intracellular ATP concentrations, while increasing ATP release (from 72 h) independently of secondary stimulation. This effect was mimicked by intercellular adhesion molecule 1 receptor binding alone through ultraviolet-inactivated sham control. In addition, RV-16-infected cells became more sensitive to secondary stimulation with both hypotonic and isotonic solutions, suggestive of a hypersensitive response. These responses were not mediated via increased TRPV4 or pannexin-1 whole-cell expression as determined by Western blotting. Interestingly, the increased ATP release seen was not a result of increased mitochondrial ATP production.Thus, this is the first report demonstrating that RV-16 infection of airway epithelial cells causes hypersensitivity by increasing ATP release. These finding provide a novel insight into the process by which viruses may cause cough and identify a potential target for treatment of viral and post-viral cough.

Download Full-text

Lytic Release of Cellular ATP: Physiological Relevance and Therapeutic Applications

Life ◽

10.3390/life11070700 ◽

2021 ◽

Vol 11 (7) ◽

pp. 700

Author(s):

Ryszard Grygorczyk ◽

Francis Boudreault ◽

Olga Ponomarchuk ◽

Ju Jing Tan ◽

Kishio Furuya ◽

...

Keyword(s):

Tissue Injury ◽

Physiological Mechanism ◽

Atp Release ◽

A549 Cells ◽

Extracellular Atp ◽

Lung Damage ◽

Extracellular Nucleotides ◽

Purinergic Signalling ◽

Protective Effects ◽

Therapeutic Applications

The lytic release of ATP due to cell and tissue injury constitutes an important source of extracellular nucleotides and may have physiological and pathophysiological roles by triggering purinergic signalling pathways. In the lungs, extracellular ATP can have protective effects by stimulating surfactant and mucus secretion. However, excessive extracellular ATP levels, such as observed in ventilator-induced lung injury, act as a danger-associated signal that activates NLRP3 inflammasome contributing to lung damage. Here, we discuss examples of lytic release that we have identified in our studies using real-time luciferin-luciferase luminescence imaging of extracellular ATP. In alveolar A549 cells, hypotonic shock-induced ATP release shows rapid lytic and slow-rising non-lytic components. Lytic release originates from the lysis of single fragile cells that could be seen as distinct spikes of ATP-dependent luminescence, but under physiological conditions, its contribution is minimal <1% of total release. By contrast, ATP release from red blood cells results primarily from hemolysis, a physiological mechanism contributing to the regulation of local blood flow in response to tissue hypoxia, mechanical stimulation and temperature changes. Lytic release of cellular ATP may have therapeutic applications, as exemplified by the use of ultrasound and microbubble-stimulated release for enhancing cancer immunotherapy in vivo.

Download Full-text