Paracrine stimulation of surfactant secretion by extracellular ATP in response to mechanical deformation

AS Patel, D Reigada, CH Mitchell… - … of Physiology-Lung …, 2005 - journals.physiology.org
AS Patel, D Reigada, CH Mitchell, SR Bates, SS Margulies, M Koval
American Journal of Physiology-Lung Cellular and Molecular …, 2005journals.physiology.org
We developed a heterologous system to study the effect of mechanical deformation on
alveolar epithelial cells. First, isolated primary rat alveolar type II (ATII) cells were plated onto
silastic substrata coated with fibronectin and maintained in culture under conditions where
they become alveolar type I-like (ATI) cells. This was followed by a second set of ATII cells
labeled with the nontransferable, vital fluorescent stain 5-chloromethylfluorescein diacetate
to distinguish them from ATI cells. By morphometric analysis, equibiaxial deformation …
We developed a heterologous system to study the effect of mechanical deformation on alveolar epithelial cells. First, isolated primary rat alveolar type II (ATII) cells were plated onto silastic substrata coated with fibronectin and maintained in culture under conditions where they become alveolar type I-like (ATI) cells. This was followed by a second set of ATII cells labeled with the nontransferable, vital fluorescent stain 5-chloromethylfluorescein diacetate to distinguish them from ATI cells. By morphometric analysis, equibiaxial deformation (stretch) of the silastic substratum induced comparable changes in cell surface area for both ATII and ATI cells. Surfactant lipid secretion was measured using cells metabolically labeled with [3H]choline. In response to 21% tonic stretch for 15 min, ATII cells seeded with ATI cells secreted nearly threefold more surfactant lipid compared with ATII cells seeded alone. ATI cells did not secrete lipid in response to stretch. The enhanced lipid secretion by ATII plus ATI cocultures was inhibited by treatment with apyrase and adenosine deaminase, suggesting that ATP release by ATI cells enhanced surfactant lipid secretion at 21% stretch. This was confirmed using a luciferase assay where, in response to 21% stretch, ATI cells released fourfold more ATP than ATII cells. Because ATI cells release significantly more ATP at a lower level of stretch than ATII cells, this supports the hypothesis that ATI cells are mechanosensors in the lung and that paracrine stimulation of ATII cells by extracellular ATP released from ATI cells plays a role in regulating surfactant secretion.
American Physiological Society