Uptake of L-proline by Cultured Primary Rat Astrocytes

Abstract

The proteinogenic amino acid L-proline can be efficiently metabolized by cultured astrocytes as exogenous substrate to fuel mitochondrial ATP regeneration. To investigate the properties of the transport processes involved in astrocytic L-proline uptake, we used primary rat astrocyte cultures as model system. After application of L-proline, cultured astrocytes efficiently accumulated the amino acid in a time- and concentration-dependent saturable manner. Omission of sodium ions drastically lowered cellular L-proline accumulation to low values that increased proportional to the concentration of L-proline applied. The saturable sodium-dependent L-proline accumulation followed apparent Michaelis-Menten kinetics with a calculated K_M value of around 1.0 mM and a V_max value of around 20 nmol/(5 min x mg). In contrast to L-proline, D-proline was hardly taken up by the cells. L-Proline uptake was strongly affected by lowering the incubation temperature, while alterations of the extracellular pH value did not affect the accumulation of L-proline. An excess of the amino acids L-alanine, L-asparagine, L-methionine, L-serine, L-threonine or L-valine severely lowered the uptake of L-proline. Inhibition of uptake was also found in the presence of L-4-hydroxyproline and L-homocysteine as well as in the presence of the ASCT1 inhibitor L-4-chlorophenylgycine, but not after exposure to inhibitors of other potential astrocytic L-proline transporters. These data suggest that L-proline uptake by cultured rat astrocytes is mainly mediated by the sodium-dependent neutral amino acid transporter ASCT1.