Regulation of apical transporter of L‐DOPA in human intestinal Caco‐2 cells

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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 μM; 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 μM). 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 μM). 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 μM). 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 μM, respectively), but the rise of intracellular Ca2+ by A23187 (1 μM) and thapsigargin (1 μM) 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.

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