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Faculty of Medicine, Institute of Pharmacology and Therapeutics, 4200 Porto, Portugal
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 µM and a maximal velocity
(Vmax) of 6.0 ± 0.2 nmol · mg
protein
1 · 6 min1. 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 µM and
Vmax
values of 32.0 ± 5.8 and 26.2 ± 3.4 nmol · mg
protein1 · 6 min1, 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 µM 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 µM) and GBR-12909 (1 and 3 µM). 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 µM and a
Vmax of 2.0 ± 0.1 and 2.2 ± 0.3 nmol · mg
protein1 · 6 min1, respectively.
However, both cocaine (10, 30, or 100 µM) and GBR-12909 (1 or 3 µM)
were found not to affect the uptake of 100 µM 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.
L-3,4-dihydroxyphenylalanine; decarboxylase
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