UPTAKE OF L-3,4-DIHYDROXYPHENYLALANINE AND DOPAMINE FORMATION IN CULTURED RENAL EPITHELIAL-CELLS

In the presence of benserazide (50 mu M), L-3,4-dihydroxyphenylalanine (L-DOPA) was rapidly accumulated in both LLC-PK1 and OK cells; equili brium was attained at 30 min of incubation. For these LLC-PK, and OK c ells, 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 co nstant 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 saturati on curves for LLC-PK, and OK cells revealed a K-m (in mu M) of 86 +/- 12 and 14 +/- 4, respectively. The cellular accumulation of the substr ate was temperature-dependent and stereoselective. Aromatic L-amino ac id decarboxylase (AAAD) activity was determined in cell homogenates; n onlinear analysis of the saturation curves revealed, for LLC-PK1 and O K cells, a K-m (in mu M) 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, respectivel y. 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 va lue (in mu M) for decarboxylation of intracellular L-DOPA in LLC-PK1 a nd OK cells was 61 +/- 14 and 108 +/- 36, respectively. A low amount o f newly formed dopamine was found to escape to the apical bathing flui d. This outward transfer of newly formed dopamine was a nonsaturable p rocess up to 300 mu M intracellular dopamine. In conclusion, the data presented here show that OK cells are endowed with a more efficient L- DOPA uptake system than LLC-PK1 cells, but the latter are endowed with a significantly higher AAAD activity than OK cells. In both cell line s, intracellular L-DOPA is rapidly converted to dopamine, some of whic h diffuses out of the cell. (C) 1997 Elsevier Science Inc.