title: Functional and pharmacological mechanisms of nucleoside transport across the basolateral membrane of rabbit tracheal epithelial cells.
abstract: The role of basolateral membrane nucleoside transport in primary cultured rabbit tracheal epithelial cells (RTEC) was studied. Primary cultured RTEC were grown on permeable support at an air-interface. Transport studies were conducted in the uptake, efflux, and transepithelial transport configurations using (3)H-uridine as a model substrate. Time, temperature and concentration dependency of (3)H-uridine transport were evaluated in parallel to the metabolism of this substrate using scintillation counting and thin layer chromatography. Inhibition of (3)H-uridine uptake from basolateral fluid was estimated in presence of all unlabeled natural nucleosides as well as analogs and nucleobases. Functional modulation pathways of (3)H-uridine uptake were studied after treatment of RTEC with pharmacological levels of A23187, forskolin, tamoxifen, H89 and colchicine. The basolateral aspect has a low-affinity and high-capacity transport system that exhibits characteristics of bi-directionality, temperature/concentration dependency, and broad specificity towards purines and pyrimidines without requiring Na(+). Basolateral equilibrative-sensitive/insensitive (es/ei) type transport machinery manifested as a biphasic dose response to nitro-benzyl-mercapto-purine-ribose (NBMPR) inhibition. In addition, a number of therapeutically relevant nucleoside analogs appeared to compete with the uptake of uridine from basolateral fluid. Short-term pre-incubation of primary cultured RTEC with the calcium ionophore A23187 inhibited basolateral uridine uptake without affecting the J(max) and K(m). The inhibitory effect was not reversible with a protein kinase C (PKC) antagonist, tamoxifen. In contrast, basolateral uridine uptake was increased by adenylyl cyclase activator forskolin (reversible with protein kinase A (PKA) inhibitor H89), resulting in a decreased K(m), but a lower J(max). Uridine exit across the basolateral membrane of primary cultured RTEC occurs via a facilitative diffusion carrier, which can be modulated by intracellular Ca(2+) levels and PKA. Information about these carriers will help improve the transportability of antitumor and antiviral nucleoside analogs in the pulmonary setting.
authors.affiliation: Department of Pharmaceutical Sciences, University of Southern California, Los Angeles, CA 90089-9121, USA. sharon.k.wu@cardinal.com
authors.forename: Sharon K
authors.initials: SK
authors.lastname: Wu
authors.forename: Neil R
authors.initials: NR
authors.lastname: Mathias
authors.forename: Kwang-Jin
authors.initials: KJ
authors.lastname: Kim
authors.forename: Vincent H L
authors.initials: VH
authors.lastname: Lee
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D003470:Culture Media; D004305:Dose-Response Relationship, Drug; D004847:Epithelial Cells / Q000378:metabolism*; D008297:Male; D008954:Models, Biological; D033703:Nucleoside Transport Proteins / Q000378:metabolism; D009705:Nucleosides / Q000493:pharmacokinetics*; D011817:Rabbits; D014132:Trachea / Q000166:cytology* / Q000378:metabolism; D014529:Uridine / Q000493:pharmacokinetics
chemical_list: D003470:Culture Media; D033703:Nucleoside Transport Proteins; D009705:Nucleosides; D014529:Uridine