Effects of varying extracellular amino acid concentrations on bidirectional amino acid transport and intracellular fluxes in mammary epithelial cells

Abstract

Understanding the regulation of cellular AA uptake as protein supply changes is critical for predicting milk component yields because intracellular supplies partly regulate protein synthesis. Our objective was to evaluate cellular uptake and kinetic behavior of individual AA when cells are presented with varying extracellular AA supplies. Bovine primary mammary epithelial cells were grown to confluency and transferred to medium with an AA profile and concentration similar to that of plasma from dairy cows for 24 h. Treatments were 4 AA concentrations, 0.36, 2.30, 4.28, and 6.24 mM, which represented 16, 100, 186, and 271% of typical plasma AA concentrations, respectively, in lactating dairy cows. Twenty-four plates of cells (89.4 × 19.2 mm) were assigned to each treatment. Cells were first subjected to treatment medium enriched with 15N-labeled AA for 24 h and then incubated with treatment medium enriched with 13C-labeled AA for 0, 15, 60, 300, 900, 1,800, and 3,600 s. Intracellular free AA, intracellular protein-bound AA, and extracellular medium free AA were analyzed for concentrations and isotopic enrichment using gas chromatography-mass spectrometry. A dynamic, 12-pool model was fitted to the data for 14 AA to derive unidirectional uptake and efflux, protein turnover, transamination, oxidation, and synthesis. The derived concentration for half the maximal uptake (km) indicated no saturation of AA uptake at typical in vivo concentrations for 11 of the 14 AA. Arginine, Pro, and Val appeared to exhibit saturation kinetics. Net uptake of all essential AA except Phe was positive across treatments. Most nonessential AA exhibited negative net uptake values. Efflux of AA was quite high, with several AA exhibiting greater than 100% efflux of the respective influx. Intracellular pool turnover was rapid for most AA (e.g., 2 min for Arg), demonstrating plasticity in matching needs for protein translation to supplies. Intracellular AA concentrations increased linearly in response to treatment for most AA, whereas 9 AA exhibited quadratic responses. Amino acid uptake is responsive to varying extracellular supplies to maintain homeostasis. No saturation of uptake was evident for most AA, indicating that transporter capacity is likely not a limitation for most AA except possibly Arg, Val, and Pro in mammary epithelial cells.