title: P-glycoprotein phosphorylation/dephosphorylation and cellular accumulation of L-DOPA in LLC-GA5 Col300 cells.
abstract: 1. The present work was aimed to study the effect of PKC activation and protein-serine/threonine phosphatase (PP1/PP2 A) inhibition on P-glycoprotein (P-gp) mediated transport of L-DOPA in LLC-GA5 Col300 cells, a renal cell line expressing the human P-glycoprotein in the apical membrane. 2. L-DOPA accumulation was a time-and concentration-dependent process with the following kinetic characteristics: kin, 57.3 +/- 1.2 pmol mg protein(-1) min(-1); k(out), 3.3 +/- 0.1 pmol mg(-1) protein min(-1); Amax, 10.6 +/- 0.8; Kn, 198 +/- 64 microM; Vmax, 5.2 +/- 0.7 nmol mg protein(-1). 3. Verapamil (25 microM), a P-glycoprotein inhibitor, markedly increased (approximately 40% increase) the accumulation of a non-saturating concentration of L-DOPA (2.5 microM) at both initial rate of uptake (IRU, 6 min incubation) and at steady-state (SS, 30 min incubation). 4. PKC activation with phorbol 12,13-dibutyrate (PDBu, 1, 3 and 10 nM) produced a concentration-dependent decrease in L-DOPA accumulation at SS, but not at IRU. The inactive phorbol ester, 4alpha-phorbol 12,13-didecanoate (100 nM), produced no change in L-DOPA accumulation. The effect of PDBu was completely reverted by staurosporine (100 nM). The phosphatase inhibitor okadaic acid (100 nM) reduced by 20% the accumulation of L-DOPA at IRU, but not at SS. 5. It is suggested that P-glycoprotein plays a role in regulation of intracellular availability of L-DOPA in renal epithelial cells, and phosphorylation/dephosphorylation of P-glycoprotein may be involved in the regulation of the transporter.
authors.affiliation: Institute of Pharmacology & Therapeutics, Faculty of Medicine, Porto, Portugal.
authors.forename: B
authors.initials: B
authors.lastname: Sampaio-Maia
authors.forename: R
authors.initials: R
authors.lastname: Gomes
authors.forename: P
authors.initials: P
authors.lastname: Soares-da-Silva
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism*; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D004848:Epithelium / Q000378:metabolism*; D006801:Humans; D007668:Kidney / Q000378:metabolism; D007980:Levodopa / Q000378:metabolism*; D019319:Okadaic Acid / Q000494:pharmacology; D010704:Phorbols / Q000494:pharmacology; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D019311:Staurosporine / Q000494:pharmacology; D013997:Time Factors; D014700:Verapamil / Q000494:pharmacology
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D010704:Phorbols; D019319:Okadaic Acid; D007980:Levodopa; D014700:Verapamil; D011493:Protein Kinase C; D010744:Phosphoric Monoester Hydrolases; D019311:Staurosporine; C033085:phorbol