Uptake and intracellular fate of l-DOPA in a human intestinal epithelial cell line: Caco-2

Abstract

The aim of the present study was to examine the kinetic characteristics of thel-3,4-dihydroxyphenylalanine (l-DOPA) transporter and the fate of newly formed dopamine in Caco-2 cells. In the presence of 50 μM benserazide (an inhibitor of aromaticl-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 μM at a medium concentration of 0.5 μM. In saturation experiments the accumulation of l-DOPA was saturable with a Michaelis-Menten constant ( K m) of 60 ± 10 μM and a maximal reaction velocity ( V max) of 6.6 ± 0.3 nmol ⋅ mg protein−1 ⋅ 6 min−1; at 4°C the amount of l-DOPA accumulated in the cells was nonsaturable. When cells were incubated with increasing concentrations of l-DOPA (10–100 μM) in the absence of benserazide, a substantial amount of thel-DOPA that was taken up was decarboxylated to dopamine, with an apparent K m of 27.2 μM. 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 ( K m = 43 ± 7 μM, V max = 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 ( K m = 623 ± 238 μM, V max = 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 intracellularl-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.