title: Transport of organic cations by a renal epithelial cell line (OK).
abstract: The goal of this study was to determine the mechanisms involved in the transport of the organic cation, tetraethylammonium (TEA), across the apical membrane of OK cells. [14C]TEA accumulated in OK cell monolayers reaching equilibrium in 2 h. The uptake of [14C]TEA at equilibrium was dependent upon temperature and was inhibited by sodium azide and by various organic cations, including N1-methylnicotinamide (NMN), mepiperphenidol, and cimetidine but not by the organic anion, p-aminohippuric acid. The initial uptake of [14C]TEA was characterized by a saturable process. The mean +/- S.D. Km was 27.8 +/- 2.6 microM and the Vmax was 414 +/- 26.5 pmol/mg protein/min. Both an accelerated efflux and influx of [14C]TEA in the presence of a trans-gradient of unlabeled TEA and NMN was observed, whereas a deaccelerated influx and efflux was observed in the presence of a trans-gradient of mepiperphenidol. The mechanism of interaction between NMN and TEA was examined. NMN significantly increased the apparent Km (mean +/- S.D.) of TEA to 82.8 +/- 16.4 microM (p less than 0.001), whereas the Vmax (mean +/- S.D.) was only slightly affected (478 +/- 72 pmol/mg protein/min) suggesting a competitive inhibition. The stimulatory effect of trans-gradients of NMN on TEA transport was due to an increase in the Vmax of TEA suggesting that NMN trans-stimulates TEA transport by increasing the turnover rate of the exchanger. In the presence of an inwardly directed proton gradient, the efflux at 30 s of [14C]TEA from the OK cell monolayers was significantly accelerated (p less than 0.05). Studies with the pH-sensitive fluorescent probe, 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein, suggested that TEA could drive the countertransport of protons. In apical membrane vesicles prepared from OK cells, the uptake of [3H]NMN exhibited an apparent "overshoot phenomenon" in the presence of an initial outwardly directed proton gradient. Protons competitively inhibited TEA uptake suggesting that the proton/organic cation and the organic cation/organic cation self exchange mechanism are the same mechanism. This is the first report describing both TEA self-exchange and proton/TEA exchange in the apical membrane of a continuous cell line. OK cells are an excellent model for the study of organic cation transport across the apical membrane.
authors.affiliation: School of Pharmacy, University of California, San Francisco 94143.
authors.forename: G
authors.initials: G
authors.lastname: Yuan
authors.forename: R J
authors.initials: RJ
authors.lastname: Ott
authors.forename: C
authors.initials: C
authors.lastname: Salgado
authors.forename: K M
authors.initials: KM
authors.lastname: Giacomini
mesh_terms: D000818:Animals; D001386:Azides / Q000494:pharmacology; D001693:Biological Transport, Active / Q000187:drug effects; D002412:Cations; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002474:Cell-Free System; D004848:Epithelium / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007687:Kidney Tubules, Proximal / Q000378:metabolism*; D009536:Niacinamide / Q000031:analogs & derivatives* / Q000378:metabolism; D009893:Opossums; D010880:Piperidines / Q000378:metabolism; D013696:Temperature; D013757:Tetraethylammonium Compounds / Q000378:metabolism*
chemical_list: D001386:Azides; D002412:Cations; D010880:Piperidines; D013757:Tetraethylammonium Compounds; D009536:Niacinamide; C010482:mepiperphenidol; C024058:N(1)-methylnicotinamide