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-dihydroxyphenylalan ine (L-dopa) and its intracellular decarboxylation to dopamine. The ac cumulation of L-dopa from the epical side in cells cultured in collage n-treated plastic was found to be a saturable process with a Michaelis constant (K-m) of 123 +/- 17 mu M and a maximal velocity (V-max) 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 pol ycarbonate filters was also found to be saturable; nonlinear analysis of saturation curves for apical and basal application revealed K-m val ues of 63.8 +/- 17.0 and 42.5 +/- 9.6 mu M and V-max 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 t he basal side, in the absence of benserazide, led to the accumulation of newly formed dopamine. The intracellular accumulation of newly form ed dopamine was a saturable process with apparent K-m values of 20.5 /- 8.2 and 247.3 +/- 76.8 mu M when the substrate was applied from the apical and basal side, respectively. Some of the newly formed dopamin e escaped to the extracellular milieu. The basal outward transfer of d opamine was five-to sevenfold of that occurring at the apical side and was uniform over a wide range of concentrations of intracellular dopa mine; the apical outward transfer of the amine depended on the intrace llular concentration of dopamine and was a nonsaturable process. The a pical and basal outward transfers of dopamine were insensitive to coca ine (10 and 30 mu M) and GBR-12909 (1 and 3 mu M). The accumulation of exogenous dopamine in LLC-PK1 cells was found to be saturable; nonlin ear analysis of the saturation curves revealed for tile apical and bas al application of dopamine a K-m of 17.7 +/- 4.3 and 96.0 +/- 28.1 mu M and a V-max 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 mu M) and GB R-12909 (1 or 3 mu M) were found not to affect the uptake of 100 mu M dopamine applied from either the apical or the basal cell border. In c onclusion, 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 ba sal cell borders. On the other hand, our observations support the poss ibility of a basal-to-apical gradient of AADC activity and the possibi lity that LLC-PK1 cells might constitute an interesting in vitro model for the study of the renal dopaminergic physiology.