Uptake and intracellular fate of L-DOPA in a human intestinal epithelial cell line: Caco-2
Vieira-Coelho, M.A.; Soares-Da-Silva, P.
American Journal of Physiology 275(1): C104-C112
1998
ISSN/ISBN: 0002-9513 PMID: 9688840 Document Number: 487628
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 mM 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 mM at a medium concentration of 0.5 mM. In saturation experiments the accumulation of L-DOPA was saturable with a Michaelis-Menten constant (Km) of 60 ± 10 mM and a maximal reaction velocity (Vmax) 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 mM) 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 mM. 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 mM, 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 mM, 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. Reprinted by permission of the publisher.