Poster P20 - Cell Dry Mass Density Regulation Mechanism under Hypertonic Stress

Introduction: Cells suffer hypertonic stress when exposed to an environment with higher osmolarity than the regular condition. Upon exposure to a hypertonic environment, intracellular water rapidly exits the cells, causing a reduction in cell volume. Subsequently, cells activate specific ion channels to increase intracellular ion content, facilitating the restoration of cell volume. Eventually, cells synthesize organic osmolytes to replace the excess intracellular ions. Cell dry mass density, defined as the ratio of the mass of intracellular non-water molecules to cell volume. For cells, dry mass density is a critical determinant of the large molecules diffusion rates, the biochemical reaction rates, and the protein stability. Normally, cells tightly regulate their dry mass density. However, the behavior and regulatory mechanisms of cell dry mass density under hypertonic stress remain poorly understood. In this study, first, we demonstrate that a 50% hypertonic shock induces an immediate and acute increase in cell dry mass density. Second, we observed that after exposure to the hypertonic environment, it takes 48 hours for cell dry mass density to decrease back to within a 10% deviation from the isotonic control. Finally, We discovered that the regulatory dry mass density decrease is achieved through an increased volume growth rate mediated by Na+/H+ exchanger hyperactivation, and a decreased dry mass growth rate mediated by protein synthesis inhibition. Our findings reveal a novel mechanism for cell dry mass density regulation, and suggest that cells may regulate dry mass density more stringently than volume and dry mass.

Materials and

Methods: We conducted our experiments primarily using NIH3T3 cells. To create a hypertonic medium, we added 28 g/L sorbitol to the standard cell culture medium, which increased the medium's osmolarity from 330 mM to 450 mM (+50%). We used pH-rodo to measure intracellular pH, surface sensing of translation to measure protein synthesis rate, and stained ubiquitin to measure protein degradation rate.

Cell volume, was measured using the Fluorescence Exclusive method. In this approach, cells were cultured with membrane-impermeable fluorescent dextran in microfluidic channels approximately 10 μm tall. Since fluorescent dextran does not enter the cells, the areas occupied by cells exhibited lower fluorescence intensity when viewed from the bottom. For each cell, its precise volume was determined by dividing the decrease in integrated fluorescence intensity by the medium's fluorescence intensity density.

Cell dry mass, was measured using quantitative phase microscopy. A bulb directs a laser beam through both the cell and the surrounding culture medium. When operational, the laser bulb directs a laser beam through both the cell and the surrounding culture medium. The light slows down as it passes through the cell, which has a higher refractive index than the medium, resulting in detectable phase shift. The camera adeptly captures this phase shift, and we summarizes the total phase alteration caused by the entire cell. This total phase alteration, along with the medium's refractive index, the laser's wavelength, and the cell's volume, allows for an accurate calculation of the cell's refractive index, dry mass density, and dry mass.

Results, Conclusions, and Discussions: First, we show that within the first five minutes after a 50% hypertonic shock, cell volume decreases by 20%, while cell dry mass remains constant, resulting in a 20% increase in cell dry mass density. This outcome is expected, small molecules and water efflux through plasma membrane immediately, whereas large molecules remain inside the cells after exposure to hypertonic environment.
Second, we tracked cell volume, dry mass, and dry mass density for up to 96 hours post-hypertonic shock. We found that although cell volume recovery is achieved within seven hours, dry mass density recovery is not complete until 48 hours. Also, while the trajectories of cell volume and dry mass recovery exhibit a peak at 24 hours, the recovery trajectory of cell dry mass density is unimodal. A new steady state is achieved after 72 hours, where cell volume and dry mass are 20% larger, and dry mass density is 10% higher than isotonic control. This evidence suggests that dry mass density, rather than volume or dry mass, is the primary parameter restored after hypertonic shock.
Third, by measuring cell volume and dry mass growth rates, we found that the regulatory dry mass density decrease is achieved through the increased cell volume growth rate and decreased dry mass growth rate. We further observed that, following hypertonic shock, the Na+/H+ exchanger is hyperactivated, and the protein production rate decreases. This explains the volume growth rate increase and the dry mass growth rate decrease. Finally, inhibiting NHE channels with EIPA significantly impeded the recovery of volume and dry mass density.
Overall, we have elucidated the process and mechanism of cell dry mass density recovery following exposure to a hypertonic environment.

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