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1 Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, PA, USA; Department of Bioengineering, University of Pennsylvania, Philadelphia, PA, USA
2 Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, PA, USA
3 Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, PA, USA; Institute for Environmental Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA, USA
4 Department of Bioengineering, University of Pennsylvania, Philadelphia, PA, USA
* To whom correspondence should be addressed. E-mail: mkoval{at}mail.med.upenn.edu.
We developed a heterologous system to study the effect of mechanical deformation on alveolar epithelial cells. First, isolated primary rat alveolar type II (AT2) cells were plated onto silastic substrata coated with fibronectin and maintained in culture under conditions where they become alveolar type I-like (AT1) cells. This was followed by a second set of AT2 cells which were labeled with a non-transferable, vital fluorescent stain, 5-chloromethylfluorescein diacetate (CMFDA) to distinguish them from AT1 cells. By morphometric analysis, equibiaxial deformation (stretch) of the silastic substratum induced comparable changes in cell surface area for both AT2 and AT1 cells. Surfactant lipid secretion was measured using cells that were metabolically labeled with 3H-choline. In response to 21 % tonic stretch for 15 min, AT2 cells that were seeded with AT1 cells secreted nearly 3-fold more surfactant lipid as compared to AT2 cells seeded alone. AT1 cells did not secrete lipid in response to stretch. The enhanced lipid secretion by AT2 + AT1 co-cultures was inhibited by treatment with apyrase and adenosine deaminase (ADA), suggesting that ATP release by AT1 cells enhanced surfactant lipid secretion at 21% stretch. This was confirmed using a Luciferase assay, where, in response to 21% stretch, AT1 cells released 4-fold more ATP than AT2 cells. Since AT1 cells release significantly more ATP at a lower levels of stretch than AT2 cells, this supports the hypotheses that type I cells are mechanosensors in the lung and that paracrine stimulation of type II cells by extracellular ATP released from type I cells may play a role in regulating surfactant secretion.
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