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 3H-uridine as a model substrate. Time, temperature and concentration dependency of 3H-uridine transport were evaluated in parallel to the metabolism of this substrate using scintillation counting and thin layer chromatography. Inhibition of 3H-uridine uptake from basolateral fluid was estimated in presence of all unlabeled natural nucleosides as well as analogs and nucleobases. Functional modulation pathways of 3H-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.