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 function of P-glycoprotein (Pgp) were studied in epithelial and nonepithelial cells. We used HT-29 colon cells (HT-29/Pgp(-)) induced to express Pgp and MDR phenotype (HT-29/Pgp(+)) and NIH3T3 (3T3/Pgp(-)) cells which were stably transfected with human MDR1 DNA (3T3/Pgp(+)). Intracellular concentrations of rhodamine 123 (R-123) preloaded into cells were monitored as a function of time by fluorescence imaging microscopy, while cells were superfused with media of different tonicity and/or ionic strength. Efflux was analyzed by a single exponential decay function. 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-isotonic (87 mM NaCl), or hypoionic-hypotonic (200, 150, or 100 mOsm) media throughout the entire experiment or whether the media were changed during the experiment. Similar results were obtained when cells were preincubated and preloaded with dye under hypotonic conditions. Under extreme 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-associated fluorescence. The changes were most conspicuous in Pgp(+) cells, possibly reflecting a Pgp effect on the osmotic or osmoregulatory properties of the cells. However, drug-pumping activity remained essentially unimpaired even under the most extreme hypotonic/hypoionic conditions. (C) 1995 Wiley-Liss, Inc.