Figure 1.
COS-7 cells suspended in the MicroCell chamber at 22°C.
A) First image from a series of images initiated 21 s after cells had been exposed to hypotonic (157 mOsm/kg) TBS. The numbers indicate individually identified cells selected for analysis. The changes in their diameters were recorded until well after the cells had reached their maximal volume. Arrows point to two cells that show membrane damage in the form of a bleb of leaking cytosol. B) Fluorescence image of the same group of cells taken immediately after the last image of the bright light image series, about 5 min after A) was taken. Viable cells display green calcein AM fluorescence. Although the two damaged cells still display fluorescence, such cells were excluded from the analysis. Scale bar = 50 µm.
Table 1.
Initial values and constants used to calculate Lp with Equation (2).
Figure 2.
Boyle-van't Hoff plot of COS-7 cells.
The BvH plot displays the absolute volume as a function of the reciprocal of the osmolality of the TBS in which the cells were suspended for 5 to 7 min at 22°C. Each point is the mean of 250 to 828 individually analyzed cells. The volumes were calculated from the measured diameters as described in the text. Data were fitted by the method of least squares.
Figure 3.
Normalized shrink curves of COS-7 cells in hypertonic (602 mOsm/kg) TBS.
The cell volumes were normalized to the mean isotonic volume at each temperature; namely, 3324, 3920, and 3853 µm3. The data represent averages and S.E. values of 153 individually measured cells. Cells reached their equilibrium volume faster the higher the temperatures.
Table 2.
Lp values and their ratios, determined under hyper- and hypotonic conditions at 0, 10 and 22°C.
Figure 4.
Normalized swell curves of COS-7 cells in hypotonic (157 mOsm/kg) TBS.
The data represent averages and S.E. values of 205 individually measured cells. Cells reached their equilibrium volume faster the higher the temperatures. However, these times were significantly longer than in the hyperosomotic situation for all three temperatures tested.
Figure 5.
Lp values (water permeability) obtained under hyper- and hypotonic conditions.
The three bars to the left show the Lp's under hypertonic conditions and the three bars to the right under hypotonic conditions. The differences between the two Lp's determined at the same temperature are highly significant (p<0.0001). That is true for all three temperatures. These differences strongly indicate rectification of the water flow; that is, the flow rate is different out of the cell than into the cell.
Figure 6.
Arrhenius plots for COS-7 cells.
The upper line represents the fit for hypotonic conditions (157 mOsm/kg), indicated by circles, and the lower line is the fit for hypertonic conditions (602 mOsm/kg), indicated by triangles, at temperatures of 0 and 10°C. The activation energy, Ea, of the hypotonic Lp values was 12.0 kcal/mol. The value for Ea of the hypertonic Lp values was 10.7 kcal/mol. The Lp values gained at 22°C (open symbols) were not used for the calculation of Ea, for reasons given in the discussion.
Figure 7.
Comparison of the experimental data with computer simulations.
Plots of the volumes of COS-7 cells as function of time under hypotonic conditions (left column) and under hypertonic conditions (right column). The diamonds are the experimental values. The light solid line is the simulated curve obtained by calculating the cell volumes as a function of time using the classical differential equation [Eq 3]. The heavy solid line was obtained by sliding the light line in the X and Y directions to obtain the best fit by eye to the experimental points.