EFFECTS OF HYPOTONIC AND HYPOIONIC MEDIA ON DRUG PUMPING BY P-GLYCOPROTEIN EXPRESSED IN EPITHELIAL AND NONEPITHELIAL CELL-LINES

The effects of anisotonic and anisoionic media on the drug-pumping fun ction of P-glycoprotein (Pgp) were studied in epithelial and nonepithe lial cells. We used HT-29 colon cells (HT-29/Pgp(-)) induced to expres s Pgp and MDR phenotype (HT-29/Pgp(+)) and NIH3T3 (3T3/Pgp(-)) cells w hich were stably transfected with human MDR1 DNA (3T3/Pgp(+)). Intrace llular concentrations of rhodamine 123 (R-123) preloaded into cells we re monitored as a function of time by fluorescence imaging microscopy, while cells were superfused with media of different tonicity and/or i onic strength. Efflux was analyzed by a single exponential decay funct ion. In all media tested efflux was considerably higher in Pgp(+) than Pgp(-) cells. In both HT-29 and 3T3 cells loaded with dye in isotonic conditions, dye efflux was not significantly different whether it was measured in isoionic-isotonic (130 mM NaCl, 300 mOsm), hypoionic-isot onic (87 mM NaCl), or hypoionic-hypotonic (200, 150, or 100 mOsm) medi a throughout the entire experiment or whether the media were changed d uring the experiment. Similar results were obtained when cells were pr eincubated and preloaded with dye under hypotonic conditions. Under ex treme hypotonic and hypoionic challenge (changing from 130 mM NaCl-300 mOsm to 43 mM NaC1-100 mOsm medium), 3T3 cells, but not HT-29 cells, underwent marked shape and size changes which reduced R-123 cell-assoc iated fluorescence. The changes were most conspicuous in Pgp(+) cells, possibly reflecting a Pgp effect on the osmotic or osmoregulatory pro perties of the cells. However, drug-pumping activity remained essentia lly unimpaired even under the most extreme hypotonic/hypoionic conditi ons. (C) 1995 Wiley-Liss, Inc.