MECHANISM OF INHIBITION OF HEPATIC BILE-ACID UPTAKE BY AMILORIDE AND 4,4'-DIISOTHIOCYANO-2,2'-DISULFONIC STILBENE (DIDS)

The mechanisms by which amiloride and 4,4'-diisothiocyano-2,2'-disulfonic stilbene (DIDS) inhibit hepatic uptake of cholate and taurocholate (TC) were investigated in isolated rat hepatocytes. Amiloride inhibited Na+-dependent uptake of cholate and TC only when hepatocytes were preincubated with amiloride, indicating an indirect effect of amiloride. Time-dependent studies showed that the inhibition of bile acid uptake was associated with a parallel increase in intracellular Na+ concentration ([Na+]i). Although amiloride decreased intracellular pH, this decrease preceded amiloride-induced inhibition of bile acid uptake and increase in [Na+]i. Amiloride inhibited bile acid uptake, decreased membrane potential, and increased [Na+]i with comparable concentration dependency. DIDS inhibited Na+-dependent uptake of cholate and TC non-competitively. Neither DIDS nor amiloride inhibited Na+-independent uptake of cholate and TC. These results indicate that amiloride inhibits Na+-dependent cholate and TC uptake by decreasing the transmembrane Na+-gradient, and further support the hypothesis that two different transporters may be involved in hepatic bile acid uptake by Na+-dependent and Na+-independent mechanisms.