Uptake of l-3,4-dihydroxyphenylalanine and dopamine formation in cultured renal epithelial cells

Abstract

In the presence of benserazide (50 μM), l-3,4-dihydroxyphenylalanine ( l-DOPA) was rapidly accumulated in both LLC-PK 1 and OK cells; equilibrium was attained at 30 min of incubation. For these LLC-PK 1 and OK cells, the analysis revealed a rate constant of inward transport (k in in pmol/mg protein/min) of 3.6 ± 0.4 and 18.1 ± 0.3 and a rate constant of outward transport (k out in pmol/mg protein/min) of 1.0 ± 0.1 and 5.2 ± 0.1, respectively. Nonlinear analysis of the saturation curves for LLC-PK 1 and OK cells revealed a K m (in μM) of 86 ± 12 and 14 ± 4, respectively. The cellular accumulation of the substrate was temperature-dependent and stereoselective. Aromatic l-amino acid decarboxylase (AAAD) activity was determined in cell homogenates; nonlinear analysis of the saturation curves revealed, for LLC-PK 1 and OK cells, a K m (in μM) of 1866 ± 107 and 845 ± 153 and a V max (in nmol/mg protein/15 min) of 4.4 ± 0.1 and 0.9 ± 0.1, respectively. In the absence of benserazide, only a limited amount of the l-DOPA taken up was decarboxylated to dopamine in cell monolayers; the K m value (in μM) for decarboxylation of intracellular l-DOPA in LLC-PK 1 and OK cells was 61 ± 14 and 108 ± 36, respectively. A low amount of newly formed dopamine was found to escape to the apical bathing fluid. This outward transfer of newly formed dopamine was a nonsaturable process up to 300 μM intracellular dopamine. In conclusion, the data presented here show that OK cells are endowed with a more efficient l-DOPA uptake system than LLC-PK 1 cells, but the latter are endowed with a significantly higher AAAD activity than OK cells. In both cell lines, intracellular l-DOPA is rapidly converted to dopamine, some of which diffuses out of the cell.