Apical and basolateral uptake and intracellular fate of dopamine precursor L-dopa in LLC-PK1 cells.
The present study was aimed at the uptake of L-3,4-dihydroxyphenylalanine (L-dopa) and its intracellular decarboxylation to dopamine. The accumulation of L-dopa from the apical side in cells cultured in collagen-treated plastic was found to be a saturable process with a Michaelis constant (Km) of 123 +/- 17 microM and a maximal velocity (Vmax) of 6.0 +/- 0.2 nmol.mg protein-1.6 min-1. The uptake of L-dopa applied from either the apical or basal cell borders in cells cultured in polycarbonate filters was also found to be saturable; nonlinear analysis of saturation curves for apical and basal application revealed Km values of 63.8 +/- 17.0 and 42.5 +/- 9.6 microM and Vmax values of 32.0 +/- 5.8 and 26.2 +/- 3.4 nmol.mg protein-1.6 min-1, respectively. Cell monolayers incubated with L-dopa, applied from either the apical or the basal side, in the absence of benserazide, led to the accumulation of newly formed dopamine. The intracellular accumulation of newly formed dopamine was a saturable process with apparent Km values of 20.5 +/- 8.2 and 247.3 +/- 76.8 microM when the substrate was applied from the apical and basal side, respectively. Some of the newly formed dopamine escaped to the extracellular milieu. The basal outward transfer of dopamine was five- to sevenfold of that occurring at the apical side and was uniform over a wide range of concentrations of intracellular dopamine; the apical outward transfer of the amine depended on the intracellular concentration of dopamine and was a nonsaturable process. The apical and basal outward transfers of dopamine were insensitive to cocaine (10 and 30 microM) and GBR-12909 (1 and 3 microM). The accumulation of exogenous dopamine in LLC-PK1 cells was found to be saturable; nonlinear analysis of the saturation curves revealed for the apical and basal application of dopamine a Km of 17.7 +/- 4.3 and 96.0 +/- 28.1 microM and a Vmax of 2.0 +/- 0.1 and 2.2 +/- 0.3 nmol.mg protein-1.6 min-1, respectively. However, both cocaine (10, 30, or 100 microM) and GBR-12909 (1 or 3 microM) were found not to affect the uptake of 100 microM dopamine applied from either the apical or the basal cell border. In conclusion, the data presented here show that LLC-PK1 cells are endowed with considerable aromatic L-amino acid decarboxylase (AADC) activity and transport L-dopa quite efficiently through both the apical and basal cell borders. On the other hand, our observations support the possibility of a basal-to-apical gradient of AADC activity and the possibility that LLC-PK1 cells might constitute an interesting in vitro model for the study of the renal dopaminergic physiology.
Soares-Da-Silva
P
P
Faculty of Medicine, Institute of Pharmacology and Therapeutics, Porto, Portugal.
Serrão
M P
MP
Vieira-Coelho
M A
MA
D000818:Animals; D065105:Aromatic Amino Acid Decarboxylase Inhibitors; D001142:Aromatic-L-Amino-Acid Decarboxylases / Q000378:metabolism; D001545:Benserazide / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects; D003042:Cocaine / Q000494:pharmacology; D004298:Dopamine / Q000378:metabolism*; D018765:Dopamine Uptake Inhibitors / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000378:metabolism; D007687:Kidney Tubules, Proximal / Q000378:metabolism*; D007700:Kinetics; D018374:LLC-PK1 Cells; D007980:Levodopa / Q000378:metabolism*; D010879:Piperazines / Q000494:pharmacology; D013552:Swine
D065105:Aromatic Amino Acid Decarboxylase Inhibitors; D018765:Dopamine Uptake Inhibitors; D004791:Enzyme Inhibitors; D010879:Piperazines; D007980:Levodopa; D001545:Benserazide; C043425:vanoxerine; D001142:Aromatic-L-Amino-Acid Decarboxylases; D003042:Cocaine; D004298:Dopamine