Molecular modulation of inward and outward apical transporters of L-dopa in LLC-PK(1) cells.
The present study examined the nature of the apical inward and outward L-3,4-dihydroxyphenylalanine (L-dopa) transporters in LLC-PK(1) cells and whether protein kinases differentially modulate the activities of these transporters. The apical inward transfer of L-dopa was promoted through an energy-dependent and sodium-insensitive transporter (Michaelis constant = 38 microM; maximum 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; IC(50) = 251 microM). 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 affect the accumulation of L-dopa. The Ca(2+)/calmodulin inhibitors calmidazolium and trifluoperazine inhibited L-dopa uptake (IC(50) of 72 and 55 microM, respectively). 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 genistein significantly increased L-dopa accumulation, which was mainly due to inhibition 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 Ca(2+)-calmodulin-mediated pathways. The apical outward transfer of L-dopa may be promoted through a DIDS-sensitive transport mechanism and appears to be under the tonic control of PTK.
Soares-Da-Silva
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Faculty of Medicine, Institute of Pharmacology and Therapeutics, 4200 Porto, Portugal. Patricio.Soares@mail.telepac.pt
Serrão
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D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D001692:Biological Transport / Q000187:drug effects; D002118:Calcium / Q000502:physiology; D002147:Calmodulin / Q000502:physiology; D002352:Carrier Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology; D017869:Cyclic GMP-Dependent Protein Kinases / Q000502:physiology; D003513:Cycloheximide / Q000494:pharmacology; D003609:Dactinomycin / Q000494:pharmacology; D027261:Fusion Regulatory Protein-1; D018374:LLC-PK1 Cells; D007980:Levodopa / Q000378:metabolism*; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D011493:Protein Kinase C / Q000502:physiology; D011494:Protein Kinases / Q000502:physiology*; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011505:Protein-Tyrosine Kinases / Q000502:physiology; D013552:Swine
D015703:Antigens, CD; D002147:Calmodulin; D002352:Carrier Proteins; D027261:Fusion Regulatory Protein-1; D019384:Nucleic Acid Synthesis Inhibitors; D011500:Protein Synthesis Inhibitors; D003609:Dactinomycin; D007980:Levodopa; D003513:Cycloheximide; D011494:Protein Kinases; D011505:Protein-Tyrosine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D017869:Cyclic GMP-Dependent Protein Kinases; D011493:Protein Kinase C; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D002118:Calcium