High-resolution Radioautography of Phlorizin-3h in Rings of Hamster Intestine

Quantitative light microscope radioautographs of galactose-(3)H and phlorizin-(3)H were prepared from freeze-dried plastic-embedded hamster small intestine incubated in vitro. The usual uphill epithelial cell accumulation of galactose accompanied by a somewhat smaller lamina propria accumulation was observed in control tissue incubated 3 min in 1 mM galactose-(3)H. The addition of 5 x 10(-4)M phlorizin to the medium blocked uphill accumulation, but did not prevent galactose equilibration with the epithelial cells. The galactose content of the lamina propria was considerably less than the galactose content of the epithelial cell. Varying the phlorizin-(3)H content of the medium from 0.6 to 60 microM revealed a brush border binding of phlorizin which followed a Langmuir adsorption isotherm with a half-saturation constant of 13 microM and a maximum binding of 84 micromoles of phlorizin/liter of microvilli or 2.6 x 10(6) sites/epithelial cell. The phlorizin content of the epithelial cell compartment, excluding microvilli, never exceeded 10% that of the medium after 20 min of incubation. These findings directly support the view that phlorizin is a nontransported inhibitor which binds glucose-galactose carriers at the surface of epithelial cell microvilli.