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Time-dependent sensitivity of bovine erythrocytes to changes in glycerol concentration at subzero temperatures: Simulation of freezing damage
320
ANNUAL
MEETING
induced volume decrease. In dramatic contrast sanonin ghosts have no semipermeability in NaCl solutions and a decreased semipermeability in sucrose solutions. The ghosts have a very high ATPase activity. Though quite permeable to ATP, they do not undergo shape or volume changes in the presence of ATP. Freeze-thaw ghosts and salt ghosts are intermediate in membrane damage. They have a decrease in semipermeability in NaCl solutions but have expected semipermeability in sucrose solutions, Their ATPase activity is intermediate between that of osmotic and saponin ghosts. Nonreconstituted freeze-thaw ghosts and salt ghosts undergo ATP-induced sphere-disc transformation but do not undergo the volume decrease. ATPase is located on the inner membrane surface (Marchesi, V. T. and Palade, G. E., J. Cell Biol. 35, 385404, 1967) as is the site of ATP-induced shape change (Weed et al., J. Clin. Invest. 48, 795-809, 1969). The data suggest that slow freezing produces an alteration rather than a major disruption of the membrane. The configuration of inner membrane protein and/or protein-lipid interaction may be altered. (Supported in part by N.I.H. grant HE12114). 48. Time-Dependent Sensitivity of Bovine Erythrocytes to Changes in Glycerol Concentration at Subzero Temperatures: Simulation of Freezing
Damage. S. P. LEIBO AND P. MAZUR (Biology Division, Oak Ridge National Laboratory, Oak Ridge, TN 37830). We have shown previously that exposing bovine erythrocytes to glycerol at 20°C for 1 and 30 min yielded similar survivals after freezing to -196°C at SO”C/min. But exposing the cells to glycerol at 20” for intermediate times prior to freezing produced dramatic but transient decreases in survival. This decrease is concentration dependent, the time at which it occurs increasing with increasing glycerol concentration. Seeking an explanation of this transient, increased sensitivity to freezing, we believe that we have uncovered a phenomenon that may play a major role in freezing damage of erythrocytes. Armed with a detailed mathematical and experimental analysis of the rate of glycerol permeation in the bovine erythrocyte, we are able to describe the state of the cell with respect to intracellular glycerol concentration and cell water volume as a function of time in glycerol at 20°C. Based on this information, we have sought to test the applicability of the following argument to freezing damage in the bovine erythrocyte. (1) When a SO~Ution freezes, the solute concentration increases as water vapor is removed in the form of ice. (2) When a cell is frozen in such a solution, it will be
ABSTRACTS
exposed to increased solute concentration, requiring the loss of cell water in response to the osmotic pressure gradient. The response must occur at subzero temperatures. The reverse, of course, will occur during thawing. The test was to ask the following question: Can bovine erythrocytes suspended in concentrated glycerol solutions at 20” withstand a second hyperosmotic exposure at subzero temperatures? Experimentally, the test consisted of suspending washed erythrocytes in 1 and 2 M glycerol at 20” for various periods of time. The suspensions were cooled to subzero temperatures, e.g., -5°C mixed with prechilled 6 M glycerol in isotonic saline, held briefly, warmed to 20°C and the amount of hemolysis then measured. Conceptually, this treatment may be considered a simulation of the events during freezing. The events during thawing were simulated by first performing the above treatment and then diluted the suspensions, still at subzero temperatures, with prechilled glycerol solutions at the same concentrations that were present initially. Controls consisted of suspensions exposed to glycerol concentration changes with no temperature change, i.e., at 20°C and suspensions exposed to temperature changes, i.e., $20” to -5”C, with no concentration changes. Briefly, we have found that the response of bovine erythrocytes suspended in concentrated glycerol solutions and then exposed to changes in glycerol concentration at subzero temperatures, in the absence of any freezing, parallels rather accurately the response of these cells when frozen to -196°C and then thawed. One interpretation of these results is that the erythrocyte membrane undergoes a transition below 0°C rendering the cell incapable of tolerating volume changes required by large changes in the osmotic pressure of the suspending solution. (Research sponsored by the U. S. Atomic Energy Commission under contract with the Union Carbide Corporation.) 49. Alteration
of Semipermeability Properties of J. PICARELLI* AND D. B. PRIBOR (Biology Department, The University of Toledo. Toledo, OH 43606).
Red Cells
by Slow Freezing.
Aliquots of human red cells were hemolyzed b> four methods and the resulting ghosts washed with buffered. isotonic saline and then resuspended in 0.05, 0.34, or 1.0 OSMphosphate-buffered NaCl or sucrose solutions (pH 7.4). Hematocrits were measured to determine volume changes in these solutions. Ghosts produced by osmotic stress. osmotic ghosts. swell or shrink, respectively, in hypotonic or hypertonic NaCl or sucrose solutions. Ghosts produced by saponin shrink in the 1.0 OSM
ANNUAL
MEETING
induced volume decrease. In dramatic contrast sanonin ghosts have no semipermeability in NaCl solutions and a decreased semipermeability in sucrose solutions. The ghosts have a very high ATPase activity. Though quite permeable to ATP, they do not undergo shape or volume changes in the presence of ATP. Freeze-thaw ghosts and salt ghosts are intermediate in membrane damage. They have a decrease in semipermeability in NaCl solutions but have expected semipermeability in sucrose solutions, Their ATPase activity is intermediate between that of osmotic and saponin ghosts. Nonreconstituted freeze-thaw ghosts and salt ghosts undergo ATP-induced sphere-disc transformation but do not undergo the volume decrease. ATPase is located on the inner membrane surface (Marchesi, V. T. and Palade, G. E., J. Cell Biol. 35, 385404, 1967) as is the site of ATP-induced shape change (Weed et al., J. Clin. Invest. 48, 795-809, 1969). The data suggest that slow freezing produces an alteration rather than a major disruption of the membrane. The configuration of inner membrane protein and/or protein-lipid interaction may be altered. (Supported in part by N.I.H. grant HE12114). 48. Time-Dependent Sensitivity of Bovine Erythrocytes to Changes in Glycerol Concentration at Subzero Temperatures: Simulation of Freezing
Damage. S. P. LEIBO AND P. MAZUR (Biology Division, Oak Ridge National Laboratory, Oak Ridge, TN 37830). We have shown previously that exposing bovine erythrocytes to glycerol at 20°C for 1 and 30 min yielded similar survivals after freezing to -196°C at SO”C/min. But exposing the cells to glycerol at 20” for intermediate times prior to freezing produced dramatic but transient decreases in survival. This decrease is concentration dependent, the time at which it occurs increasing with increasing glycerol concentration. Seeking an explanation of this transient, increased sensitivity to freezing, we believe that we have uncovered a phenomenon that may play a major role in freezing damage of erythrocytes. Armed with a detailed mathematical and experimental analysis of the rate of glycerol permeation in the bovine erythrocyte, we are able to describe the state of the cell with respect to intracellular glycerol concentration and cell water volume as a function of time in glycerol at 20°C. Based on this information, we have sought to test the applicability of the following argument to freezing damage in the bovine erythrocyte. (1) When a SO~Ution freezes, the solute concentration increases as water vapor is removed in the form of ice. (2) When a cell is frozen in such a solution, it will be
ABSTRACTS
exposed to increased solute concentration, requiring the loss of cell water in response to the osmotic pressure gradient. The response must occur at subzero temperatures. The reverse, of course, will occur during thawing. The test was to ask the following question: Can bovine erythrocytes suspended in concentrated glycerol solutions at 20” withstand a second hyperosmotic exposure at subzero temperatures? Experimentally, the test consisted of suspending washed erythrocytes in 1 and 2 M glycerol at 20” for various periods of time. The suspensions were cooled to subzero temperatures, e.g., -5°C mixed with prechilled 6 M glycerol in isotonic saline, held briefly, warmed to 20°C and the amount of hemolysis then measured. Conceptually, this treatment may be considered a simulation of the events during freezing. The events during thawing were simulated by first performing the above treatment and then diluted the suspensions, still at subzero temperatures, with prechilled glycerol solutions at the same concentrations that were present initially. Controls consisted of suspensions exposed to glycerol concentration changes with no temperature change, i.e., at 20°C and suspensions exposed to temperature changes, i.e., $20” to -5”C, with no concentration changes. Briefly, we have found that the response of bovine erythrocytes suspended in concentrated glycerol solutions and then exposed to changes in glycerol concentration at subzero temperatures, in the absence of any freezing, parallels rather accurately the response of these cells when frozen to -196°C and then thawed. One interpretation of these results is that the erythrocyte membrane undergoes a transition below 0°C rendering the cell incapable of tolerating volume changes required by large changes in the osmotic pressure of the suspending solution. (Research sponsored by the U. S. Atomic Energy Commission under contract with the Union Carbide Corporation.) 49. Alteration
of Semipermeability Properties of J. PICARELLI* AND D. B. PRIBOR (Biology Department, The University of Toledo. Toledo, OH 43606).
Red Cells
by Slow Freezing.
Aliquots of human red cells were hemolyzed b> four methods and the resulting ghosts washed with buffered. isotonic saline and then resuspended in 0.05, 0.34, or 1.0 OSMphosphate-buffered NaCl or sucrose solutions (pH 7.4). Hematocrits were measured to determine volume changes in these solutions. Ghosts produced by osmotic stress. osmotic ghosts. swell or shrink, respectively, in hypotonic or hypertonic NaCl or sucrose solutions. Ghosts produced by saponin shrink in the 1.0 OSM