Investigating the Role of Osmotically Inactive Water during Stress-Induced Dehydration in Mammalian Cells

Intracellular water content heavily contributes to the functionality of important cellular functions, including intracellular signaling and metabolism. To maintain optimal volume and functionality, cells typically tightly regulate intracellular water content. However, this regulation has been found to become impaired when cells undergo prolonged or severe stress. While ion exchange disruptions and acute cell damage have been noted to induce intracellular water accumulation, stress-induced dehydration from loss of intracellular water, particularly in response to apoptosis, has also been reported. The objective of this study is to investigate whether non-apoptotic, pre-apoptotic, or sub-apoptotic stress in mammalian cells leads to intracellular dehydration. We have hypothesized that stress-induced dehydration is the result of protein damage, in which stress-induced protein aggregation results in the release of osmotically inactive (OI) water and its subsequent expelling from the cell. Two mammalian cell lines, 3T3 and HeLa, were utilized, with intracellular protein concentrations measured through combining transmission-through-dye microscopy (TTD) and the transport-of-intensity equation (TIE) to measure cell volume and mass, respectively. Both cell types were treated overnight with 5-50 μM of ouabain or 25 μM of menadione to measure stress-induced changes in protein concentration. Ouabain-treated 3T3 cells were also probed for apoptosis using a mitochondrial potential probe. The protein aggregation detection dye PROTEOSTAT (Enzo Life) was used to probe for protein aggregation in ouabain-treated 3T3 cells. Furthermore, 3T3 cells were treated overnight with 10 nM of bortezomib as a positive control for protein aggregation. To measure OI water within cells, a modified method based on the Boyle-van 't Hoff equation was used, in which cells were exposed to increasingly hypertonic solutions before measuring cell volume and mass through TTD/TIE. Plotting the calculated protein concentration against the measured osmolarity of the hypertonic solutions gave the bound water/protein mass ratio for the measured cells, thus quantifying OI water content. Our preliminary results have shown an overall increase in protein concentration in both HeLa and 3T3 cells treated overnight with ouabain or menadione, which correlates to a decrease in intracellular water content. 3T3 cells treated with ouabain and a mitochondrial potential probe also revealed that 10% of the treated cells were undergoing detectable apoptosis, meaning that the majority of cells underwent non-apoptotic or pre-apoptotic stress and dehydration. Furthermore, we have detected protein aggregation in 3T3 cells treated overnight with ouabain through the use of the PROTEOSTAT probe, indicating stress-induced protein aggregation. We have also measured OI water in 3T3 cells treated with bortezomib overnight using the modified Boyle-van 't Hoff method described previously, which resulted in a smaller bound water/protein mass ratio compared to control cells, which supports our theory of intracellular proteins releasing OI water following stress-induced aggregation. While our research for this study is still ongoing at the time of writing, our current results support the theory that protein aggregation occurs as a response to prolonged stress in mammalian cells, resulting in the release of previously OI water and subsequent intracellular dehydration due to the removal of the newly osmotically activated water. Funding provided by the Kent State University Research Council This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.