Molecular modulation of inward and outward apical transporters of L-dopa in LLC-PK1 cells

The present study examined the nature of the apical inward and outward L-3, 4-dihydroxyphenylalanine (L-dopa) transporters in LLC-PK1 cells and whether protein kinases differentially modulate the activities of these transporte rs. The apical inward transfer of L-dopa was promoted through an energy-dep endent and sodium-insensitive transporter (Michaelis constant = 38 mu M; ma ximum velocity = 2608 pmol.mg protein(-1).6 min(-1)). This transporter was insensitive to N-(methylamino)-isobutyric acid but competitively inhibited by 2-aminobicyclo(2,2,1)-heptane-2-carboxylic acid (BHC; IC50 = 251 mu M). Modulators of protein kinase A (cAMP, forskolin, IBMX, and cholera toxin), protein kinase G (cGMP, zaprinast, LY-83583 and sodium nitroprusside), and protein kinase C (phorbol 12,13-dibutirate and chelerythrine) failed to aff ect the accumulation of L-dopa. The Ca2+/calmodulin inhibitors calmidazoliu m and trifluoperazine inhibited L-dopa uptake (IC50 of 72 and 55 mu M, resp ectively). The inhibitory effect of calmidazolium on the accumulation of L- dopa was of the noncompetitive type. The organic anion inhibitor DIDS, but not p-aminohippurate, and the protein tyrosine kinase (PTK) inhibitor genis tein significantly increased L-dopa accumulation, which was mainly due to i nhibition of apical outward transfer of L-dopa. It is concluded that LLC-PK 1 cells take up L-dopa over the apical cell border through the L-type amino acid transporter, which appears to be under the control of Ca2+-calmodulin -mediated pathways. The apical outward transfer of L-dopa may be promoted t hrough a DIDS-sensitive transport mechanism and appears to be under the ton ic control of PTK.