Transepithelial transport of tyramine across filter-grown MDCK cells via a poly(D-lysine) carrier

Abstract

In order to investigate the advantage of using membrane-adsorptive carriers to mediate drug transport across epithelial tissue, we have prepared disulfide- and thioether-linked conjugates of tyramine (tyn) as a model drug to a cationic, nondegradable carrier, poly(D-lysine) (PDL). The transport properties were evaluated using microporous filter-grown Madin-Darby canine kidney (MDCK, strain I) epithelial cells, and we have determined that: (a) the [125I]tyn-SS-PDL conjugate predominantly transported [125I]tyn in the apical-to-basal direction (20-fold greater transport vs. basal-to-apical); (b) [125I]tyn-SS-PDL elicits a 10-fold greater degree of [125I]tyn transport than [125I]tyn-S-PDL, thus demonstrating the importance of the reducible disulfide linkage for [125I]tyn transport to occur; (c) 125I-radioactivity recovered in the basal media was determined to be 95% [125I]tyn-containing small molecules, thus demonstrating the release of [125I]tyn from its PDL carrier; (d) the apical addition of an anionic species, heparin, completely blocks apical-to-basal transport of [125I]tyn, indicating the importance of PDL-mediated binding to the apical membrane for transport to occur; (e) apical-to-basal transport proceeds via non-lysosomal pathways, as lysosomal inactivation by NH4Cl exposure does not inhibit transport, and (f) the addition of a membrane-impermeable inhibitor of disulfide reduction, bisdithionitrobenzoic acid (DTNB), to the apical media inhibits transport by approximately 70%, indicating the importance of apically-localized disulfide reducing reactions for transport of [125I]tyn. Pulse-chase studies indicate that there is no paracellular leakage of conjugate, and the ratio of recycled:membrane-associated:transported [125I]tyn fragment following chase is 4:2:1.