EFFECTS OF P-GLYCOPROTEIN EXPRESSION ON CYCLIC-AMP AND VOLUME-ACTIVATED ION FLUXES AND CONDUCTANCES IN HT-29 COLON ADENOCARCINOMA CELLS

The tissue distribution of P-glycoprotein (Pgp) and the structurally r elated cystic fibrosis transmembrane conductance regulator (CFTR) is a pparently mutually exclusive, particularly in epithelia; where one pro tein is expressed the other is not. To study the possible function(s) of Pgp and its potential effects on CFTR expression in epithelia, HT-2 9 colon adenocarcinoma cells, which constitutively ex press CFTR, were pharmacologically adapted to express the classical multidrug resistan ce (MDR) phenotype (Pgp(+)). Concomitant-with the appearance of Pgp an d MDR phenotype (drug resistance, reduced drug accumulation and increa sed drug efflux), CFTR levels and cAMP-stimulated Cl conductances were markedly decreased compared to wild-type HT-29 (Pgp(-)) cells (as sho wn using the whole cell patch clamp technique). Removal of drug pressu re led to the gradual decrease in Pgp levels and MDR phenotype, as evi denced by increased rhodamine 123 accumulation (Pgp-Rev). Concomitantl y, CFTR levels and cAMP-stimulated Cl- conductances increased. The cel l responses of Pgp/Rev cells were heterogeneous with respect to both P gp and CFTR functions. We also stud led the possible contribution of P gp to hypotonically activated (HCS) ion conductances. K+ and Cl- efflu xes from Pgp(-) cells were markedly increased by HCS. This increase wa s twice as high as that induced by the cation ionophore gramicidin; it was blocked by the Cl(-)channel blocker DIDS (4,4'-disothiocyano-2,2' -disulfonic stilbene) and required extracellular Ca2+. In Pgp(+) cells , the HCS-induced fluxes were not significantly different from those o f Pgp(-) cells. Verapamil (10 mu M), wh ich caused 80% reversal of Pgp -associated drug extrusion, failed to inhibit the HCS-evoked Cl- efflu x of Pgp(+) cells. Similarly, HCS increased Cl- conductance to the sam e extent in Pgp(-), Pgp(+) and Pgp-Rev cells. Verapamil (100 mu M), bu t not 1,9-dideoxyforskolin (50 and 100 mu M), partially inhibited the HCS-evoked whole cell current (WCC) in all three lines. Since the inhi bition by verapamil was not detected in the presence of the K+ channel blocker Ba2+ (3 mM), it is suggested that verapamil affects K+ and no t Cl- conductance. We conclude that hypotonically activated Cl- and K conductances are similar in HT-29 cells irrespective of Pgp expressio n. Expression of high levels of Pgp in HT-29 cells confers no physiolo gically significant capacity for cell volume regulation. (C) 1994 Wile y-Liss, Inc.