An air-interface rabbit tracheal epithelial cell culture model for the characterization of nucleoside and nucleoside drug transpor

The overall goal of this study was to develop a primary cultured rabbit airway epithelial cell culture model to study the mechanism of nucleoside absorption in the airway epithelium. Rabbit tracheal epithelial cells cultured in an air-interface were found to exhibit morphological and ion transport properties that closely resemble the native epithelium. Permeability characterization studies revealed that epithelial permeability was inversely related to a solute size with a molecular cut-off limit of 20,000 daltons, consistent with an equivalent pore radius of 5 nm. For solutes of the same size, the influence of solute lipophilicity on permeability was best described by a sigmoidal relationship with a half maximal permeability at a log octanol/pH 7.4 buffer partition coefficient of 2.08. In the second phase of the project, using uridine as a model nucleoside, uptake was rapid, temperature and direction dependent. Uridine uptake was strongly dependent on the presence of Na$sp+$, and exhibited saturability with a Km of 3.4 $mu$M and a Vmax of 0.7 pmoles/mg protein/30s. All physiological nucleosides were effective inhibitors of uridine uptake. Substrate selectivity studies indicated that base modified nucleoside analogs interact with the nucleoside transporters, whereas those analogs with substitution on the sugar ring are less likely to interact with the nucleoside transporters. Internalization of nucleoside analogs was confirmed by the direct measurement of $sp3$H-2$spprime$-deoxyadenosine uptake (a transporter substrate). Functional inhibition of $sp3$H-uridine, $sp3$H-thymidine and $sp3$H-inosine with physiological nucleosides suggested a pyrimidine selective (N2) transporter, a broad substrate selective (N4) transporter, and an equilibrative (ei) transporter may be present on the apical membrane. On the basolateral membrane, an ei equilibrative transporter may mediate the translocation of uridine across the membrane. Nucleoside transport function was measured in cells infected with adenovirus. Uridine uptake was slightly reduced but transporter activity was retained under infected conditions. In conclusion, the air-interface airway epithelial cell culture model was successfully applied for the study of ion and drug transport in the airways. The predominant mechanism of nucleoside and antiviral nucleoside analog transport was via specialized nucleoside transporters that accumulate nucleosides intracellularly, thus facilitating the delivery of these otherwise impermeable hydrophilic drugs when locally administered to the airway epithelium.