PROBENECID-RESISTANT J774 CELL EXPRESSION OF ENHANCED ORGANIC ANION TRANSPORT BY A MECHANISM DISTINCT FROM MULTIDRUG-RESISTANCE

Macrophages possess organic anion transporters that carry membrane-imp ermeant fluorescent dyes, such as lucifer yellow (LY) and carboxyfluor escein, from the cytoplasm into endosomes and out of the cells. Proben ecid, an organic anion transport inhibitor, blocks these processes. Pr olonged incubation of J774 cells in medium containing 2.5 mM probeneci d eventually kills most of these cells. To identify J774 variants that express increased organic anion transport activity, we selected probe necid-resistant (PBR) J774 cells by growing them in medium containing increasing concentrations of probenecid. When PBR and unselected J774 cells were loaded with LY by ATP(4-) permeabilization, the amount of L Y accumulated by the PBR cells was about half that in the unselected c ells. This difference was abolished by adding 10 mM probenecid to the medium in which the cells were loaded, suggesting that the diminished LY accumulation in PBR cells was due to enhanced LY secretion and that the PBR cells expressed increased organic anion transport activity. D irect comparison of LY efflux from J774 and PBR J774 cells showed a fa ster initial rate of secretion of LY from PBR J774 cells than from uns elected J774 cells. To determine whether LY efflux is mediated by P-gl ycoprotein, we compared LY efflux in unselected J774 cells, PBR J774 c ells, and multidrug-resistant J774 cells (J7.C1). LY efflux from J7.C1 cells was not sensitive to verapamil, which inhibits multidrug-resist ance transporters, and reverses the multidrug-resistant phenotype of J 7.C1 cells. The rates of LY efflux from unselected J774 and J7.C1 cell s were virtually identical. In addition, Northern blot analysis showed much lower levels of mdr mRNA in PBR J774 cells than in J7.C1 cells. These observations indicate that organic anion transporter activity an d multiple drug resistance are regulated independently of each other i n J774 cells; they also suggest that organic anion transport in macrop hages is mediated by protein(s) other than P-glycoprotein.