title: Proton-coupled organic cation antiporter-mediated uptake of apomorphine enantiomers in human brain capillary endothelial cell line hCMEC/D3.
abstract: R(-)-Apomorphine is a dopamine agonist used for rescue management of motor function impairment associated with levodopa therapy in Parkinson's disease patients. The aim of this study was to examine the role of proton-coupled organic cation antiporter in uptake of R(-)-apomorphine and its S-enantiomer in human brain, using human endothelial cell line hCMEC/D3 as a model. Uptake of R(-)- or S(+)-apomorphine into hCMEC/D3 cells was measured under various conditions to evaluate its time-, concentration-, energy- and ion-dependency. Inhibition by selected organic cations was also examined. Uptakes of both R(-)- and S(+)-apomorphine increased with time. The initial uptake velocities of R(-)- and S(+)-apomorphine were concentration-dependent, with similar Km and Vmax values. The cell-to-medium (C/M) ratio of R(-)-apomorphine was significantly reduced by pretreatment with sodium azide, but was not affected by replacement of extracellular sodium ion with N-methylglucamine or potassium. Intracellular alkalization markedly reduced the uptake, while intracellular acidification increased it, suggesting that the uptake is driven by an oppositely directed proton gradient. The C/M ratio was significantly decreased by amantadine, verapamil, pyrilamine and diphenhydramine (substrates or inhibitors of proton-coupled organic cation antiporter), while tetraethylammonium (substrate of organic cation transporters (OCTs)) and carnitine (substrate of carnitine/organic cation transporter 2; (OCTN2)) had no effect. R(-)-Apomorphine uptake was competitively inhibited by diphenhydramine. Our results indicate that R(-)-apomorphine transport in human blood-brain barrier (BBB) model cells is similar to S(+)-apomorphine uptake. The transport was dependent on an oppositely directed proton gradient, but was sodium- or membrane potential-independent. The transport characteristics were consistent with involvement of the previously reported proton-coupled organic cation antiporter.
authors.affiliation: Laboratory of Drug Disposition & Pharmacokinetics, Faculty of Pharma-Sciences, Teikyo University.
authors.forename: Takashi
authors.initials: T
authors.lastname: Okura
authors.forename: Kei
authors.initials: K
authors.lastname: Higuchi
authors.forename: Atsushi
authors.initials: A
authors.lastname: Kitamura
authors.forename: Yoshiharu
authors.initials: Y
authors.lastname: Deguchi
mesh_terms: D017920:Antiporters / Q000378:metabolism*; D001058:Apomorphine / Q000493:pharmacokinetics*; D001692:Biological Transport; D001812:Blood-Brain Barrier / Q000378:metabolism*; D002412:Cations / Q000378:metabolism*; D002460:Cell Line; D018491:Dopamine Agonists / Q000493:pharmacokinetics*; D042783:Endothelial Cells / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008564:Membrane Potentials; D027701:Organic Cation Transport Proteins / Q000378:metabolism; D011522:Protons; D012964:Sodium / Q000378:metabolism; D013237:Stereoisomerism
chemical_list: D017920:Antiporters; D002412:Cations; D018491:Dopamine Agonists; D027701:Organic Cation Transport Proteins; D011522:Protons; D012964:Sodium; D001058:Apomorphine