Uptake, accumulation and release of ouabain by isolated rat hepatocytes

We investigated uptake of ouabain into isolated rat hepatocytes and release of ouabain from preloaded hepatocytes, thus assessing separately the two membrane transport steps, involved in biliary elimination of the drug. The following results were obtained: Uptake of ouabain was saturable (Km = 263 + 61 microM, V = 3.3 + 1.0 nmol/mgprot. x min), energy-dependent, sensitive to dinitrofluorobenzene and temperature-dependent (E = 80 - 96 kJ/mol). It had no pH-optimum in the physiological pH-range and was independent of the extracellular cation composition. Uptake of ouabain was competitively inhibited by the cardiac glycoside digitoxin (Ki = 0.3 microM). Uptake was not inhibited in the presence of the glycosidic sugar l-rhamnose, but it was competitively inhibited by the steroid taurocholate (Ki = 6.3 microM). Ouabain was accumulated within hepatocytes 170-fold. The predominant fraction of intracellular ouabain being unbound. Release of ouabain from preloaded cells was energy-independent, independent of the Na+-concentration and not susceptible to inhibition by dinitrofluorobenzene or taurocholate. It is concluded, first that hepatocellular uptake of ouabain is mediated by a carrier for steroids and second that the pathway of release is distinct from that of uptake. We assume, that the high bile/plasma concentration-gradient of ouabain in vivo is generated during active uptake into the cell and not during release into bile.