The human colon
carcinoma cell line HT29 c1.19A was studied for
organic anion transporter activity by determining intracellular
fluo-3 and
fura-red accumulation and by measuring
fluo-3 efflux. Modulators of organic
anion transport systems were used to identify the transporters that are involved in
dye extrusion. Addition of
probenecid to the
dye-loading medium, containing 10 microM
fluo-3/AM and
fura-red/AM, resulted in a dose-dependent increase in
fluo-3 and
fura-red accumulation in the cells. The increase in
fluo-3 accumulation in the cells in the presence of
probenecid was explained by the inhibitory effect of this compound on
fluo-3 efflux.
Fluo-3 efflux from the cells was also inhibited by
sulfinpyrazone, another inhibitor of organic
anion transport. Substrates of renal
probenecid-sensitive organic
anion exchange mechanisms as well as modulators of
multidrug resistance associated protein (MRP) activity did not influence
fluo-3 extrusion rates. However, reducing intracellular
ATP contents completely blocked
fluo-3 extrusion. Moreover, MK571, an inhibitor of MRP, significantly stimulated
dye accumulation, whereas inhibitors of the multidrug resistance gene (MDR1) product Pglycoprotein,
cyclosporin A and
verapamil, did not. As
probenecid inhibits
fluo-3 efflux across the apical membrane of cells grown on permeable supports, we conclude that a
probenecid-sensitive
organic anion transporter is present in the apical membrane of HT29 c1.19A cells. This organic
anion transport system differs from MDRI and MRP2.