Potent bile salt and organic anion inhibition of methotrexate uptake and accumulation in the freshly isolated rat hepatocyte.

Influx of [3H]methotrexate into freshly isolated hepatocytes is mediated by highand low-affinity processes, both of which are inhibited by the bile salts, cholate, taurocholate, and de oxycholate, and the organic anions, bromosulfophthalein and rose bengal. At 100 @Mconcentrations, these compounds inhibit 1 @LM [3H]methotrexate influx by 70 to 90%. Dixon plots established a similar K (-@†̃20 @sM) for taurocholate inhibition of the highand low-affinity influx routes for methotrexate, al though the kinetics of this inhibition has not, as yet, been established. Bile salt inhibition of methotrexate influx requires the simultaneous presence of the bile salt and methotrexate at the cell membrane. Hence, pretreatment of hepatocytes with taurocholate, with subsequent removal of the bile salt from the extracellular compartment, does not alter methotrexate influx. Methotrexate at high concentration was found to inhibit the influx of [3HJtaurocholate, suggesting the possibility of shared transport site(s) for bile salts and methotrexate. Influx of the naturally occurring folate, 5-methyltetrahydrofolate, is also in hibited by the bile salts and bromosulfophthalein, although to a lesser extent than influx of methotrexate. Cholate and tau rocholate reduce not only methotrexate influx but also the net level of intracellular methotrexate accumulation, with a propor tional reduction in the synthesis of methotrexate polyglutamate derivatives. Deoxycholate and bromosulfophthalein inhibit methotrexate polyglutamate synthesis to a greater extent than inhibition of total drug uptake. The presence of albumin in the buffer markedly reduces bromosulfophthalein inhibition of MTX influx, but has a much lesser effect on taurocholate inhibition of methotrexate influx, presumably related to the much higher affinity of albumin for bromosulfophthalein than for taurocho