Characterization of a transporting system in rat hepatocytes. Studies with competitive and non-competitive inhibitors of phalloidin transport.

Primary cultures of rat hepatocytes were used for assaying several drugs not previously known for inhibiting the transport of phalloidin. In order to have 50% inhibition (IC50) of the entrance of a tritiated phallotoxin derivative ([3H]demethylphalloin, 1 microM) from the medium into the cells the following concentrations (microM) of the various inhibitors were determined: cyclolinopeptide (0.5), Nocloprost (5.0), Nileprost (7.0), beta-estradiol (42), Verapamil (70). For comparison, the corresponding IC50 values of some known antagonists of phalloidin toxicity were determined by the same method. Moreover, we studied several natural and synthetic phallotoxins and alpha-amanitin for their ability to displace [3H]demethylphalloin from the transporting system. Lineweaver-Burk plots made it obvious that two groups of inhibitors exist. Competitive inhibitors are, for example, antamanide, beta-estradiol, silybin, Nileprost, taurocholate, and the cyclic somatostatin analog cyclo[Phe-Thr-Lys-Trp-Phe-D-Pro], whereas Verapamil and monensin inhibit phallotoxin uptake in a non-competitive way. Considering the very different chemical features of the competitive inhibitors, we tentatively conclude that the phallotoxin transport system selects compounds not on the basis of their chemical features, but rather their physical properties. The physical properties of a typical substrate are low molecular mass, lipophilic nature, and, possibly the presence of rigid ring structures. Negative charges accelerate the transport of a substrate, while positive charges have the opposite effect. The phalloidin-transporting system may represent part of a hepatic equipment which clears portal blood from, for example, bile acids, lipophilic hormones, or xenobiotics. By chance, the transporting system incorporates phallotoxins into the hepatocytes leading to the death of these cells.