Glutathione efflux from cultured astrocytes.
The characteristics and kinetics of GSH efflux from the monolayer culture of rat astrocytes were investigated. GSH efflux was dependent on temperature, with a Q10 value of 2.0 between 37 and 25 degrees C. The GSH efflux rate showed a hyperbolic dependency on the intracellular GSH concentration. The data were fitted well to the Michaelis-Menten model, giving the following kinetic parameter values: Km = 127 nmol/mg of protein; Vmax = 0.39 nmol/min/mg of protein. p-Chloromercuribenzenesulfonic acid, a thiol-reactive agent impermeable to the cell membrane, lowered the GSH efflux rate by 25% without affecting the intracellular GSH content. These results suggest that a carrier is involved in the efflux of GSH. The GSH content of cultured astrocytes showed a marked increase when the cells were exposed to insults, such as sodium arsenite, cadmium chloride, and glucose/glucose oxidase that lead to the generation of hydrogen peroxide. The increase in GSH content was attributed to the induction of the cystine transport activity by the agents. Although the intracellular GSH concentration and GSH efflux were increased, the kinetics of GSH efflux were not affected by those agents that imposed the oxidative stress. Because the Km value is very large, it is suggested that astrocytes release GSH depending on their GSH concentration in a wide range.
Sagara
J
J
Department of Biochemistry, University of Tsukuba, Ibaraki, Japan.
Makino
N
N
Bannai
S
S
D002731:4-Chloromercuribenzenesulfonate / Q000494:pharmacology; D000818:Animals; D001253:Astrocytes / Q000378:metabolism*; D001692:Biological Transport; D002478:Cells, Cultured; D003553:Cystine / Q000378:metabolism; D005949:Glucose Oxidase / Q000494:pharmacology; D005978:Glutathione / Q000378:metabolism*; D007700:Kinetics; D009994:Osmolar Concentration; D018384:Oxidative Stress; D051381:Rats; D017208:Rats, Wistar; D013696:Temperature
D003553:Cystine; D002731:4-Chloromercuribenzenesulfonate; D005949:Glucose Oxidase; D005978:Glutathione