Rapid and steady-state amino acid transport in perfused human fibroblasts and colon adenocarcinoma cells: effects of methotrexate

Abstract

Initial and steady-state uptakes of serine and phenylalanine by human fibroblasts and human colon tumour cells were studied applying a double isotope dilution technique to perfused populations of cultivated cells retained on microcarrier beads. This new method permits the differentiation of the unidirectional transport parameters and can also distinguish between membrane-associated processes and independently intracellular events in isolated cells. High initial l-serine uptake values in colon adenocarcinoma cells became negative under steady-state conditions. To determine if the observed negative l-serine uptake was produced by the rapid efflux of intracellular l-[ 3H]serine, the cells were treated with methotrexate (MTX) (an inhibitor of cytosolic dihydrofolate reductase). The modified curve of l-[ 3H]serine uptake after MTX treatment suggests that, under these experimental conditions, net serine transport is non concentrative in colon tumour cells and could be modulated by the rate of intracellular serine metabolism; it also suggests that MTX does not directly affect serine transport in perfused human colon adenocarcinoma cells. Initial and steady-state uptakes of phenylalanine were high in both fibroblasts and tumour cells and were unaffected by MTX treatment.