Apical and basolateral uptake and intracellular fate of dopamine precursor L-dopa in LLC-PK(1) cells

• Type: Article in International Scientific Journal
• Date: 1998

Authors

soares-da-silva, p (Author) FMUP; serrao, mp (Author) Other; vieira-coelho, ma (Author) Other

Journal

Title: American Journal of Physiology - Renal Physiology

Vol. 274

Pages: F243-F251

ISSN: 1931-857X

Publisher: American Physiological Society

Indexing

ISI Web of Knowledge - 48 Citations

Scopus - 55 Citations

Scientific classification

FOS: Medical and Health sciences > Basic medicine

Other information

Authenticus ID: P-001-8H4

Abstract (EN): 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 epical side in cells cultured in collagen-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 polycarbonate filters was also found to be saturable; nonlinear analysis of saturation curves for apical and basal application revealed K(m) values 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 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 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 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 mu M) and GBR-12909 (1 and 3 mu M). The accumulation of exogenous dopamine in LLC-PK(1) cells was found to be saturable; nonlinear analysis of the saturation curves revealed for the apical and basal 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 GBR-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 conclusion, the data presented here show that LLC-PK(1) 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-PK(1) cells might constitute an interesting in vitro model for the study of the renal dopaminergic physiology.

Language: English

Type (Professor's evaluation): Scientific

No. of pages: 9