Transport of 5-methyltetrahydrofolic acid and folic acid in freshly isolated hepatocytes.

Transport of 5-methyltetrahydropteroylglutamic acid (5CH,-H,PteGlu) was studied in hepatocytes isolated from rat liver by enzymatic dispersion. The hepatocytes maintained gradients for sodium and potassium ions (I Na+l = 28.1 meql liter and I K+I = 133 meqlliter of cell water) during 90 min of incubation. Uptake of 5-CH,,-H,PteGlu was found to be a complex process. At low concentrations of substrate, uptake was saturable. At higher concentrations of substrate (up to 20 PM), the uptake was not saturable. Both components were less active in the absence of extracellular sodium ions. The saturable component, both in the presence and absence of extracellular sodium, was energy-dependent; in both cases inhibition by sodium arsenate was observed and concentration of 5-CH,,-H,PteGlu proceeded against an electrochemical gradient. Ion exchange chromatography indicated that PteGlu, but not 5-CH,-H,PteGlu, was metabolized significantly after 60 to 90 min of incubation. The accumulated 5CH,-H,PteGlu was bound to macromolecules present in sonicated cell extracts to the extent of about 20%. Uptake of 5-CH,,-H,PteGlu by the primary saturable component was inhibited by the structural analogs, methotrexate, 5-CHOH,PteGlu, and to a lesser degree, by PteGlu, whereas the secondary, nonsaturable component was unaffected by high concentrations of these compounds. Uptake of 5-CH,H,PteGlu was stimulated, and efflux was inhibited by the metabolic inhibitor, sodium azide, suggesting that an energy-dependent efflux is operative. In addition, evidence is presented which suggests the conclusion that 5-CH3H,PteGlu transport is dependent on the presence of intact disulfide bonds. These studies show that 5-CH,,-H,PteGlu transport in freshly isolated hepatocytes is a saturable, active process(es) composed of sodium-dependent and sodium-independent components and that efflux may also be an energy-dependent phenomenon.