title: Regulation of apical transporter of L-DOPA in human intestinal Caco-2 cells.
abstract: The present study examined the nature of the apical inward L-3,4-dihydroxyphenylalanine (L-DOPA) transporter in human intestinal epithelial Caco-2 cells, and whether protein kinases modulate the activity of this transporter. The apical inward transfer of L-DOPA was promoted through an energy-dependent and sodium-insensitive transporter (Km=33 microM; Vmax=2932 pmol/mg protein/6 min). This transporter was insensitive to N-(methylamino)-isobutyric acid, but competitively inhibited by 2-aminobicyclo(2,2,1)-heptane-2-carboxylic acid (BCH; IC50=83 microM). The organic cation inhibitor decynium 24 failed to affect the accumulation of L-DOPA, whereas the organic anion inhibitor 4,4'-diisothiocynatostilbene-2,2'-disulphonic acid (DIDS) competitively inhibited L-DOPA uptake (IC50=83 microM). However, the apical-to-basal and basal-to-apical transepithelial transport and the cell accumulation of [3H]-PAH was close to that of [14C]-sorbitol and insensitive to DIDS (300 microM). Modulators of protein kinase A (PKA) [cyclic adenosine monophosphate (cAMP), forskolin, H-89 and cholera toxin], protein kinase G (PKG) [cyclic guanosine monophosphate (GMP), zaprinast, LY 83583 and sodium nitroprusside] and protein kinase C (PKC) (phorbol 12,13-dibutirate and chelerythrine) failed to affect the accumulation of L-DOPA. The Ca2+/calmodulin inhibitors calmidazolium and trifluoperazine inhibited L-DOPA uptake (IC50s of 53 and 252 microM, respectively), but the rise of intracellular Ca2+ by A23187 (1 microM) and thapsigargin (1 microM) played no role on L-DOPA uptake. It is concluded that Caco-2 cells take up L-DOPA over the apical cell border through the sodium-independent and pH-sensitive L-type amino acid transporter.
authors.affiliation: Institute of Pharmacology and Therapeutics, Faculty of Medicine, Porto, Portugal.
authors.forename: S
authors.initials: S
authors.lastname: Fraga
authors.forename: B
authors.initials: B
authors.lastname: Sampaio-Maia
authors.forename: M P
authors.initials: MP
authors.lastname: Serrão
authors.forename: P
authors.initials: P
authors.lastname: Soares-da-Silva
mesh_terms: D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects; D018938:Caco-2 Cells; D002147:Calmodulin / Q000037:antagonists & inhibitors / Q000378:metabolism; D016764:Cell Polarity; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D020128:Inhibitory Concentration 50; D007980:Levodopa / Q000378:metabolism*; D016015:Logistic Models; D026901:Membrane Transport Proteins / Q000378:metabolism*; D013997:Time Factors; D014268:Trifluoperazine / Q000494:pharmacology
chemical_list: D002147:Calmodulin; D007093:Imidazoles; D026901:Membrane Transport Proteins; D014268:Trifluoperazine; D007980:Levodopa; C031938:calmidazolium; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid