Uptake and intracellular fate of L-DOPA in a human intestinal epithelial cell line: Caco-2.
The aim of the present study was to examine the kinetic characteristics of the L-3,4-dihydroxyphenylalanine (L-DOPA) transporter and the fate of newly formed dopamine in Caco-2 cells. In the presence of 50 microM benserazide (an inhibitor of aromatic L-amino acid decarboxylase), L-DOPA was rapidly accumulated in Caco-2 cells. At equilibrium (30 min of incubation) the intracellular L-DOPA concentration was 10.2 +/- 0.1 microM at a medium concentration of 0.5 microM. In saturation experiments the accumulation of L-DOPA was saturable with a Michaelis-Menten constant (Km) of 60 +/- 10 microM and a maximal reaction velocity (Vmax) of 6.6 +/- 0.3 nmol . mg protein-1 . 6 min-1; at 4 degrees C the amount of L-DOPA accumulated in the cells was nonsaturable. When cells were incubated with increasing concentrations of L-DOPA (10-100 microM) in the absence of benserazide, a substantial amount of the L-DOPA that was taken up was decarboxylated to dopamine, with an apparent Km of 27.2 microM. In experiments performed in cells cultured in polycarbonate filters, the accumulation of L-DOPA in the presence of benserazide was greater when the substrate was applied from the basolateral cell border than when it was applied from the apical cell border. In the absence of benserazide, L-DOPA applied from the basolateral cell border resulted in a nonlinear formation of dopamine (Km = 43 +/- 7 microM, Vmax = 23.7 +/- 1.2 nmol . mg protein-1 . 6 min-1). The amount of dopamine leaving the cell through the apical cell border was lower than the amount that escaped through the basolateral cell border, and the process was saturable (Km = 623 +/- 238 microM, Vmax = 0.19 +/- 0.02 nmol . mg protein-1 . 6 min-1). In conclusion, the data presented here show that Caco-2 cells are endowed with an efficient L-DOPA uptake system, and intracellular L-DOPA was found to be rapidly converted to dopamine, some of which diffuses out of the cell. The utilization of Caco-2 cells cultured on polycarbonate filters probably provides a better way to look at processes such as the outward transfer of intracellular molecules, namely, the outward transfer of newly formed dopamine.
Vieira-Coelho
M A
MA
Institute of Pharmacology and Therapeutics, Faculty of Medicine, 4200 Porto, Portugal.
Soares-Da-Silva
P
P
D001142:Aromatic-L-Amino-Acid Decarboxylases / Q000378:metabolism; D001545:Benserazide / Q000494:pharmacology*; D001692:Biological Transport / Q000187:drug effects; D018938:Caco-2 Cells; D002851:Chromatography, High Pressure Liquid; D003110:Colonic Neoplasms; D004295:Dihydroxyphenylalanine / Q000378:metabolism; D004298:Dopamine / Q000378:metabolism; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism*; D007700:Kinetics; D007980:Levodopa / Q000378:metabolism*
D007980:Levodopa; D004295:Dihydroxyphenylalanine; D001545:Benserazide; D001142:Aromatic-L-Amino-Acid Decarboxylases; D004298:Dopamine