Proline uptake by monolayers of human intestinal absorptive (Caco-2) cells in vitro.
Monolayers of the Caco-2 human intestinal cell line exhibit active and passive uptake systems for the imino acid L-proline. The active transport component is saturable and it is responsible for about two thirds of the observed flux over the nanomolar concentration range, at 37 degrees C and pH 7.4. In contrast to L-phenylalanine, specific L-proline uptake has a high degree of sodium dependency and the efficiency of the carrier system is significantly reduced when protein synthesis (cycloheximide), Na+/K(+)-ATPase (ouabain) or cellular metabolism (sodium azide) are inhibited. The expression of the L-proline carrier by Caco-2 cells was under some degree of nutritional control. Glucose deficiency, over the time scale of the experiment, had no effect. The temperature-dependence of the specific uptake process followed the Arrhenius model with an apparent activation energy of 93.5 kJ nmol-1. This pathway also displayed Michaelis-Menten concentration-dependence with a Ksdm of 5.28 mM and a maximal transport flux (Jsdmax) of 835 pmol min-1 (10(6) cells)-1. Although the passive component was unchanged, the pH of the donor phase exerted a profound effect on the active carrier component. Within the physiological pH range a local maximum efficiency was found at pH 7.4 but dramatic increases were noted as pH 5.0 was approached. In competition studies, with 100-fold excess of a second amino acid, strong inhibition of uptake was found with alpha-aminoisobutyric acid, L-alanine and L-serine whereas moderate inhibition was observed with glycine, D-proline and gamma-aminoisobutyric acid. Aromatic and branched amino acids showed weak (L-valine) or no interaction (L-phenylalanine, L-leucine) with the carrier system. These data indicate that the carrier system for the uptake of L-proline has many features in common with the A system for amino acid transport.
Nicklin
P L
PL
Pharmaceutical Sciences Institute, Aston University, Birmingham, UK.
Irwin
W J
WJ
Hassan
I F
IF
Mackay
M
M
D001386:Azides / Q000494:pharmacology; D001692:Biological Transport; D002478:Cells, Cultured / Q000378:metabolism; D003513:Cycloheximide / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007413:Intestinal Mucosa / Q000378:metabolism*; D010042:Ouabain / Q000494:pharmacology; D010649:Phenylalanine / Q000378:metabolism; D011392:Proline / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism; D019810:Sodium Azide; D013696:Temperature
D001386:Azides; D010649:Phenylalanine; D010042:Ouabain; D019810:Sodium Azide; D003513:Cycloheximide; D011392:Proline; D012964:Sodium