Unidirectional flux of phenylalanine into Vero cells. Measurement using paired tracers in perfused cultures.
The uptake of phenylalanine by Vero cells in perfused culture was measured using a double-label technique. Cells were anchored in microcarrier beads and maintained in a column perfused at a constant rate. The extracellular tracer [14C]mannitol and the test tracer [3H]phenylalanine were injected as a bolus, and the column effluent was sampled at 10-s intervals. The proportion of the test tracer retained by the cells was calculated by analysis of time-dilution curves of test and reference tracers. Uptake measurements were specific and highly reproducible. Uptake of [3H]phenylalanine was inhibited by unlabelled phenylalanine and by other amino acids that utilize transport system L. This new approach proved useful for rapid measurement of unidirectional uptake, and for determination of kinetics parameters of uptake under steady state conditions. This rapid technique obviates some of the limitations associated with uptake measurements in whole organs and with measurements in conventional cell cultures.
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D000818:Animals; D001692:Biological Transport; D002250:Carbon Radioisotopes; D002460:Cell Line; D002522:Chlorocebus aethiops; D007668:Kidney; D007700:Kinetics; D008353:Mannitol / Q000378:metabolism*; D010477:Perfusion; D010649:Phenylalanine / Q000378:metabolism*; D014316:Tritium
D002250:Carbon Radioisotopes; D014316:Tritium; D008353:Mannitol; D010649:Phenylalanine