Uptake of L-3,4-dihydroxyphenylalanine and dopamine formation in cultured renal epithelial cells.
In the presence of benserazide (50 microM), L-3,4-dihydroxyphenylalanine (L-DOPA) was rapidly accumulated in both LLC-PK1 and OK cells; equilibrium was attained at 30 min of incubation. For these LLC-PK1 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-PK1 and OK cells revealed a Km (in microM) 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-PK1 and OK cells, a Km (in microM) of 1866 +/- 107 and 845 +/- 153 and a Vmax (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 Km value (in microM) for decarboxylation of intracellular L-DOPA in LLC-PK1 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 microM 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 lines, intracellular L-DOPA is rapidly converted to dopamine, some of which diffuses out of the cell.
Soares-da-Silva
P
P
Institute of Pharmacology & Therapeutics, Faculty of Medicine, Porto, Portugal. Patricio.Soares@mail.telepac.pt
Vieira-Coelho
M A
MA
Serrão
M P
MP
D000818:Animals; D001142:Aromatic-L-Amino-Acid Decarboxylases / Q000502:physiology; D002478:Cells, Cultured; D004298:Dopamine / Q000096:biosynthesis*; D004847:Epithelial Cells / Q000378:metabolism; D005260:Female; D007668:Kidney / Q000378:metabolism*; D007980:Levodopa / Q000378:metabolism*; D009893:Opossums; D013552:Swine
D007980:Levodopa; D001142:Aromatic-L-Amino-Acid Decarboxylases; D004298:Dopamine