Hyperosmotic injury in mammalian cells. 3. Volume and alkali cation alterations of CHO cells in unprotected and DMSO-treated cultures

Abstract

Correlated studies on volume distributions and cation (Na + and K +) content of CHO cells in suspension were carried out after various exposures to hypertonic NaCl or sucrose (500–7550 m Osm in both the presence and absence of DMSO (5–20%; w v ). The effects superimposed by ouabain (10 −2–10 −4 m), amphotericin B (6–18 μg/ml), and glutaraldehyde (1.25%) on the above-mentioned parameters were also investigated. Volumetric analysis of CHO cells with the Coulter Channelyzer indicated a biphasic dose-dependent response to hypertonic media, the duration of the TABLE 2 Correlation between Volume, Survival, and Cation Content of CHO Cells Exposed to Hypertonic Media in Suspension Osmolality m Osm Exposure (min) Hypertonic agent NaCl Sucrose V a Na + K + S b V Na + K + S 1000 60 or less Small High High High Normal Low High High 1500–2000 60 or less Small High Low Low Normal Low High High 2000 or over 60 or more Small or large c High Very low Very low Small Very low Very low Very low a V, modal volume. b S, surviving fraction [data from Ref. (18)]. c Time dependency visible during a 2-hr observation period. shrinkage- re-expansion cycle being inversely proportional to medium osmolality. In hypertonic sucrose, CHO cells apparently fail to shrink up to approximately 1000 m Osm. In both hypertonic sucrose and NaCl there is a constant loss of intracellular K + at velocities which depend primarily on medium tonicity and also on exposure length. K + loss is accompanied by increased levels of intracellular Na + only in cells exposed to hypertonic salt; in hypertonic (1–2 m) sucrose intracellular Na + shows a progressive diminution. In NaCl-DMSO experiments the amount of K + lost from the cells is less in comparison to that observed in “unprotected” cell suspensions. DMSO also mitigates excessive intracellular accumulations of Na +. In contrast to ouabain and amphotericin B, DMSO was found to slow down considerably the early volume re-expansion phase. Glutaraldehyde on the other hand totally suppressed the ability of cells to readjust their volume in hypertonic solutions. By correlating the present data with several earlier published results on CHO cells' posthypertonic survival, it appears that intracellular K + plays a key role in the chain of reactions that enable nucleated mammalian cells to sustain hypertonic stresses. Whether intracellular K + is equally important for the survival of cells at subzero temperatures remains to be determined.