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 m m and 0.05+/-0.01 m m, 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/m m and 37.9+/-0.1 nmol/mg/min/m m respectively. l -leu uptake was found to be sodium-independent. The uptake of l -[(3)H]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 m ml -phe (simulating maternal phenylketonuria) significantly reduced both the K(m)and V(max)components of l -phe influx. l -arg (2 m m) had no effect on l -leu influx whereas 2 m ml -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.