The effects of incubation conditions on enzyme release from anoxic heart cell cultures

Abstract

Cultured myocytes from neonate rats were subjected to varying concentrations of glucose and serum under normoxic and anoxic conditions. Cellular metabolism was measured in terms of glucose utilization and lactate production. Leakage of cytoplasmic lactate dehydrogenase (LDH) into the medium was measured to give an indication of cell injury. The effect of variations in temperature on these parameters was evaluated. Myocytes cultured under anoxic conditions for 16 h utilized more glucose than normoxic cells with a concomitant increase in lactate production. When the initial glucose concentration in the medium was below 3 m m, anoxic myocytes released large amounts of LDH. Also, at low initial glucose concentrations there was a surge of anoxic LDH release between 12 and 15 h of incubation. The addition of exogeneous lactate to anoxic cultures had no influence on enzyme leakage. However, reduced enzyme leakage was observed when medium lactate was increased under normoxic conditions. An increased concentration of serum in the medium stimulated glucose metabolism in both normoxic and anoxic myocytes. Despite this enhancement of glycolysis, anoxic cells released significant amounts of LDH into the medium. LDH release by anoxic myocytes is minimal in medium containing 1 to 2% v/v serum. Maximum glucose metabolism and lactate production at 5 m m glucose occurred between 37 to 40°C. The relation between LDH release and temperature was not marked. Significant release of LDH did occur in anoxic cultures incubated between 37°C and 40°C when compared with normoxic cultures at the same temperature. At 4°C, in the absence of glucose metabolism, LDH release occurred from both normoxic and anoxic cells. These data demonstrate that significant cellular enzyme release occurs during anoxia: (a) below a threshold glucose concentration and (b) in proportion to the serum content of the medium.