Mechanosensitive ATP Release Through Transient Membrane Rupture in Alveolar Epithelial Cells

Abstract

Mechanical ventilation (MV) is critical for the survival of patients with respiratory distress, but it can also exacerbate their symptoms: a condition called ventilator‐induced lung injury (VILI). Innate protection to attenuate VILI symptoms includes adenosine (Ado), an anti‐inflammatory mediator known to promote resolution of pulmonary edema. Airways‐localized Ado is a breakdown product of ATP by ectoenzymes, and high level of ATP has been found in the airways at the onset of MV treatment. Since stretch is a potent ATP secretion stimulus, this elevated ATP level has been explained by the intense deformation experienced by lung cells during MV. While ATP‐permeable channels or exocytosis have been frequently proposed as probable mechanisms for mechanosensitive ATP release, the pathway for cell stretch‐induced ATP secretion remains to be firmly identified. Here, we present an investigation on alveolar cells with a quantitative ATP imaging technique.Rat‐derived primary cultures of alveolar epithelial type 2 cells grown in a compliant silicone chamber for 7 days were uniaxially stretched to 30% linear deformation at varying strain rate and hold time. Level of extracellular ATP in medium was quantified with an ATP‐dependent luciferase‐luciferin light‐emitting reaction and wide field of view imaging of the whole stretch chamber (2×18 mm). Pre‐ and post‐stretch uptake of the plasma membrane impermeant DNA intercalator‐type propidium iodide (PI) dye was evaluated by epifluorescence microscopy.We first observed a strong spatial and temporal correlation between density distribution of extracellular ATP and PI uptake in response to stretch, suggesting a conductive pathway for ATP secretion. High rate of ATP release per cell ruled out involvement of ATP‐permeable channels however, and was instead consistent with a membrane rupture process. Quantification of PI fluorescence uptake suggested that almost all of the detected extracellular ATP leaked through those transient cracks. In addition, the fast decline of ATP response as soon as cell deformation ceased (half‐life ~5 seconds), excluded the involvement of slow acting membrane repair processes for the closure of the ATP‐permeable pathways but rather suggested rapid recoil of elastic membrane components. Further proof implicating the viscoelastic plasma membrane came from two prominent characteristics of the stretch‐induced ATP response: relaxation and sensitivity to strain rate, both hallmarks of viscoelasticity.This study gathered evidence to support an alternate mechanism of ATP release by mechanical stretch; one occurring through transient membrane rupture. This lytic mechanism is most likely non‐lethal however, due to the fast resealing of these membrane breaks resulting in minimal trauma on cells. We expect such cellular ATP release mechanism to contribute for the early detection of ATP inside mechanically‐ventilated lungs. This knowledge may help to shape a MV load history optimized to stimulate ATP release, and subsequent Ado formation, in order to minimize VILI‐associated symptoms.Support or Funding InformationSupported by the Canadian Institutes of Health Research (PJT166157 to R.G.)