Thomas Brown (Committee Member), James Olson (Advisor), Robert Putnam (Committee Member)
Master of Science (MS)
Regulation of normal volume is an important aspect of cell homeostasis. Possible mechanisms which signal volume regulation are increasing the rate of reactive oxygen species (ROS) production and release of ATP for interaction with purinergic receptors. We examined whether an increase in ROS production during cell swelling also led to cell injury of C6 glioma cells. Cells were loaded with 5,6-carboxy-2,7-dihydrofluorscein diacetate (DCFDA) to fluoroscopically measure the rate of cellular ROS production and were perfused with phosphate-buffered saline solutions (PBS) containing 100 μM carbenoxolone to inhibit dye efflux. Cell death was determined cytometrically and by measuring the release of lactate dehydrogenase (LDH) into the culture medium 24 hr after exposing cells for 60 min to isoosmotic or hypoosmotic PBS. Immediately after changing the perfusion solution from isoosmotic to hypoosmotic PBS, the production of ROS increased by 60.2% + 19.6, but returned to baseline after 5 min. Increased efflux of ATP was not observed in hypoosmotic conditions. ROS production was not directly activated by endogenously applied extracellular ATP, but ATP increased ROS production in swollen cells. Cells swollen by hypoosmotic solutions had a slight increase in the probability of necrotic cell death. Our data suggest that increased ROS produced by cells swollen during hypoosmotic stress does not lead to significant cell injury in cultured C6 cells.
Department or Program
Department of Neuroscience, Cell Biology & Physiology
Year Degree Awarded
Copyright 2008, all rights reserved. This open access ETD is published by Wright State University and OhioLINK.