Activation by reduced glutathione of methotrexate transport into isolated rat liver cells

The uptake of methotrexate (MTX) by isolated rat hepatocytes and its changes under the influence of exogenous GSH have been studied under various conditions: GSH concentration, pH of incubation medium, preincubation of cells prior to MTX and GSH addition, ionic composition of the incubation medium (standard saline, Na+-free, Na+ and K+-free, or ion-deficient), after prior treatment of cells by membrane -SH blockers (p-CMBS, 4-CMB and DIP2+) and ATP. It was found that GSH strongly accelerated MTX uptake. This effect depended on GSH concentration and on preincubation of cells. The GSH effect was not dependent on medium pH in spite of an observed close relationship between pH of incubate and MTX transport itself. Activation by GSH of MTX transport was connected to an increase in intracellular K+. It was also noted that while blockers of membrane -SH groups like p-CMBS and 4-CMB inhibited MTX uptake and increased the intracellular Na+/K+ ratio, both effects were partially overcome by GSH. After treatment by DIP2+, Na+/K+ ratio was unaffected, but MTX uptake inhibited. Still GSH abolished inhibition. Added ATP also inhibited MTX uptake and caused loss of cellular K+ and accumulation of Na+. Here neither effect could be reversed by GSH; consequently, high cellular amounts of K+ and MTX accumulated by previous action of GSH were depleted on subsequent ATP addition. MTX uptake was low in sucrose medium. But in this ion-deficient medium, GSH had the greatest stimulatory effect on MTX uptake. It is concluded that binding GSH can affect the redox state of the -S-S-/-SH groups of the cellular plasma membrane and that this effect of GSH might demonstrate involvement of the redox state in the control of MTX permeability.