Transport of large neutral amino acids into BeWo cells

BeWo choriocarcinoma cells were cultured onto solid microcarrier beads, packed into columns and superfused. Unidirectional influx of L-phenylalanine (L-phe) and L-leucine (L-leu) across the microvillous border of the cells was studied using a rapid paired-tracer dilution technique. Influx of L-phe and L-leu comprised both saturable and non-saturable components. K-m values for L-phe and L-leu were 0.57 +/- 0.01 mM and 0.05 +/- 0.01 mM, respectively, with V-max values of 120.4 +/- 0.5 nmol/mg/min and 41.7 +/- 0.2 nmol/mg/min. Non-saturable uptake components were 29.0 +/- 0.1 nmol/mg/mM and 37.9 +/- 0.1 nmol/mg/min/mM respectively. L-leu uptake was found to be sodium-independent. The uptake of L-[H-3]phe was strongly inhibited (90-100 per cent) by unlabelled L-phe, D-phe, L-leu or 2-aminoendobicyclo-[2,2,1]-heptane-2-carboxylic acid (BCH) but not by L-arginine (L-arg) or methyl alpha-aminoisobutric acid (Me-AIB). Pre-incubation of Bewo cultures for 24 h in the presence of an additional 1.2 mtr L-phe (simulating maternal phenylketonuria) significantly reduced both the K-m and V-max components of L-phe influx. L-arg (2 mM) had no effect on L-leu influx whereas 2 mM L-phe completely inhibited saturable L-leu influx. These data suggest that the microvillous border of differentiated BeWo cells transport large neutral amino acids predominantly via system L rather than by B(0) or y(+)L transporters. (C) 2000 Harcourt Publishers Ltd.