Abstract 372

We studied the uptake, transport (i.e. passive diffusion versus active transport) and metabolism of cocaine in polarized epithelial monolayers of human colonic T-84 cells, a model of intestinal transport. T-84 monolayers (passage 47-56) were grown in DMEM/Ham's F-12 medium containing 6 % newborn calf serum, on 1.0µm collagen type I coated tissue culture inserts in 24 well plates. Cell confluency was determined by attainment of steady state transepithelial resistance of ≥ 600 ohms cm2. Monolayers were transferred to serum-free media and cocaine HCl (100 to 800 ng) was added to Upper chamber (Uch) only. The cell monolayer therefore forms an effective barrier separating Uch corresponding to the luminal aspect from the lower chamber (Lch) corresponding to the serosal surface. After incubation at 37°C for 30 min or 60 min, samples from Uch and Lch were removed for analysis. GC/MS method was used to analyze cocaine (C), benzoylecgonine (BE), norcocaine and ecgonine methyl ester(EME); the data for 60 min are shown. All studies (n=4) were run in duplicates.

C transport across T-84 monolayers increased linearly with increasing C concentration in Uch, with no significant differences between 30 min and 60 min of exposure. Transepithelial resistance did not change even at 800 ng of C. Significant amounts of BE and EME were detected in Uch. There was no saturation in the levels of C in Lch even when 800 ng was added to Uch. There was no norcocaine in either Uch or Lch. To determine whether BE and EME detected in Uch were accompanied by the metabolizing enzyme. C was added to cell free media, either fresh or exposed previously ("treated") to the monolayer. BE and EME were detected in the "treated" but not in the fresh media. (Figure)

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Summary: (1). T-84 cells vectorically transport and metabolize C from the luminal to the basolateral side. (2). The sum of C & its metabolites in Uch & Lch are less than 58%, which suggests that C is retained in the cell layer. (3). T-84 cells metabolize C, perhaps both within and outside the cell by the extrusion of esterase in the lumen.