phalloidine and desmethylphalloin

Phalloidin, the major phallotoxin of the mushroom Amanita phalloides, enters hepatocytes by a carrier-mediated mechanism. The molecular identity of the transport proteins mediating phalloidin uptake was so far unknown. Earlier studies in rat liver indicated that phalloidin may share a common mechanism of uptake with organic anions like bile salts. In the current study on human transporters, we analyzed the uptake of phalloidin into transfected HEK293 cells stably expressing the recombinant hepatocyte-specific organic anion uptake transporters OATP2 (also termed OATP1B1, OATP-C, LST1, symbol SLC21A6) or OATP8 (OATP1B3 or SLC21A8). Time-dependent uptake of phalloidin was observed with SLC21A6-expressing cells and was inhibited by typical substrates of SLC21A6 such as bromosulfophthalein or cholyltaurine. A K(m) value of 39+/-11 micro M was determined for SLC21A6-mediated phalloidin uptake. Additional inhibitors of phalloidin uptake mediated by SLC21A6 included the immunosuppressive drugs cyclosporin A, FK506, and rapamycin, whereas alpha-amanitin was only a weak inhibitor. Cyclosporin A was a most potent competitive inhibitor for SLC21A6-mediated phalloidin transport with a K(i) value of 51 nM.

Topics: Alkaloids; Biological Transport; Carrier Proteins; Cell Line; Cyclosporine; Humans; Immunosuppressive Agents; Liver; Liver-Specific Organic Anion Transporter 1; Organic Anion Transporters, Sodium-Independent; Phalloidine; Sirolimus; Solute Carrier Organic Anion Transporter Family Member 1B3; Sulfobromophthalein; Tacrolimus; Taurocholic Acid; Time Factors

Cyclosporin A at concentrations of more than 10 nM protects isolated hepatocytes against the action of phalloidin. Cyclosporin A at 100 nM inhibits the uptake of demethyl[3H]phalloin by 50%, and at 5 microM also that of [14C]cholate. This inhibition is independent of the preincubation period and is not reversed by washing the cells. With a 30-60-fold excess of cyclosporin A, affinity labeling of plasma membrane proteins using 12 microM [3H]isothiocyanatobenzamido cholate was reduced to 40-60% of the control. These findings indicate that transport inhibition by cyclosporin A in liver cells cannot be explained by simple competition on the level of the membrane protein(s) involved.

Topics: Affinity Labels; Alkaloids; Animals; Biological Transport; Cell Membrane; Cholic Acid; Cholic Acids; Cyclosporins; Isothiocyanates; Liver; Male; Oligopeptides; Phalloidine; Rats; Rats, Inbred Strains; Thiocyanates

Several earlier studies suggested that the uptake of phallotoxins by liver cells is a carrier mediated process using a transport system normally handling bile acids (see Frimmer 1982). In this study we have shown whether ileal cells, well known to transport bile acids too, are able to take up phallotoxins. Isolated epithelial cells prepared from guinea pig ileum accumulated [14C]-cholate, whereas [3H]-demethylphalloin ([3H]-DMP) was not taken up. The same observation was made with isolated jejunal cells but the uptake of [14C]-cholate was much slower. [3H]-DMP, however, was partly bound to intestinal cells. This process was not inhibited by cholate, iodipamide, oligomycin and carbonylcyano-chlorophenylhydrazone (CCCP), compounds known to decrease the uptake of phallotoxins into liver cells. Substituting Na+ for choline+ and also Cl- for SCN- did not influence the binding of [3H]-DMP. Frozen intestinal cells from the guinea pig bound two time more [3H]-DMP after thawing compared with intact cells. Supplementary uptake experiments on isolated brush border membrane vesicles from rat ileum revealed that phalloidin does not inhibit taurocholate uptake and that taurocholate does not interfere with [3H]-DMP binding. The results suggest that [3H]-demethylphalloin is not recognized by the bile acid carrier of the guinea pig and the rat ileum. It is concluded that the transport system for bile acids present in ileal cell is different from that of liver cells.

Topics: Alkaloids; Amanitins; Animals; Bile Acids and Salts; Cell Membrane; Guinea Pigs; Ileum; In Vitro Techniques; Intestinal Absorption; Intestine, Small; Jejunum; Male; Microvilli; Phalloidine; Rats; Rats, Inbred Strains

The effect of somatostatin in phalloidin-intoxicated rats was studied. Animals were given phalloidin i.p. 1.2 mg/kg (LD 90-100). Somatostatin, 250 microgram/animal, was administered i.p. in saline 5 min prior and s.c. in protamine-sulphate/ZnCl2 suspension 30 min prior and 30 min after intoxication, unless stated otherwise. In vivo and in vitro uptake studies of the toxin were performed. Liver enzymes (GPT, GLDH) and kallikrein-like activities were determined in blood obtained by orbital venipuncture. Light and electron microscopy was carried out. Somatostatin treatment led to an increase in survival rate. Of the 20 treated rats six died whereas of the 20 untreated animals 18 died. A dose dependency was proven effective when half of the initial dose of somatostatin was given. In vivo and in vitro uptake studies of the toxin demonstrate that somatostatin does not alter uptake rate by rat livers. Liver enzymes remained elevated in treated and control rats. Kallikrein-like activities showed a 61% decline in treated animals whereas they rose up to 120% in controls as compared to pretreatment conditions. Light and electron microscopy reveals less severe lesions in somatostatin-treated animals. A possible interaction of somatostatin in shock development is discussed, phalloidin seems to be a suitable tool for further investigations concerning cell protection by somatostatin.

Topics: Alanine Transaminase; Alkaloids; Animals; Dose-Response Relationship, Drug; Liver; Male; Microscopy, Electron; Oligopeptides; Phalloidine; Rats; Somatostatin

Rats made cholestatic by bile duct ligation survive phalloidin poisoning. This protection against phalloidin poisoning is correlated with the rate of the toxin uptake by the liver. [3H]demethylphalloin was used as a tracer for uptake studies. This is justified because there is no significant difference in the rate of uptake of the phallotoxins used. Within 4 h after bile duct ligation phalloidin uptake is reduced to about 25% and after 24 h to about 15% of controls. Isolated perfused livers and isolated hepatocytes from cholestatic rats bind less phalloidin than normal controls. Besides morphological changes serum factors may account for the decreased rate of phallotoxin uptake in cholestatic cells.

Topics: Alkaloids; Animals; Cholestasis; In Vitro Techniques; Liver; Male; Oligopeptides; Phalloidine; Rats

To exclude an involvement of ligandin in the uptake and storage of phalloidin in hepatocytes equilibrium-dialysis studies were made with phalloidin, cholic acid and bromosulfophthalein (BSP). Binding studies with isolated ligandin indicated that the affinity of ligandin for phalloidin is low (KD = 0.8 X 10-3 M). Phalloidin neither displaced BSP (KD = 1.3 X 10-7 M) or cholic acid (KD = 7.6 X 10-5 M) from ligandin, when preloaded with these substrates. Hepatocytes prepared from rats after daily treatment with phenobarbital during 5 days contained 3-4-fold concentrations of ligandin and bound greater amounts of BSP than controls, Nevertheless the velocity of the uptake both of [3H]-demethylphalloin ([3H]-DMP) and of [35S]-BSP was not augmented. Also the sensitivity of liver cells to phalloidin was not drastically modified after induction with phenobarbital and agrees with earlier findings in vivo. We conclude that ligandin plays a negligible role in the uptake and a minor role in a storage of phallotoxins in liver cells.

Topics: Alkaloids; Amanitins; Animals; Cholic Acids; Glutathione Transferase; Liver; Male; Phalloidine; Phenobarbital; Rats; Rats, Inbred Strains; Sulfobromophthalein

Several anionic substances used for cholecystography inhibit the development of protrusions in isolated hepatocytes in response to phalloidin. Drugs from the iopodate family were equieffective with those of th iodipamide type. The above protective effect results from a competitive inhibition of the phallotoxin uptake as shown for iopodate. Cholecystographic agents similarly inhibit the inward transport of cholic acid in a competitive manner. The inhibition of the phallotoxin response is inversely correlated with the uptake of 3H-demethylphalloin (r = 0.94) and with the inward transport of cholate (r = 0.84) at various inhibiting concentrations of iopodate.

Topics: Alkaloids; Animals; Bile Acids and Salts; Binding, Competitive; Cholecystography; Cholic Acids; Dose-Response Relationship, Drug; Iodipamide; Ipodate; Liver; Oligopeptides; Phalloidine; Rats

The uptake of trace amounts of 3H-demethylphalloin (3H-DMP) by isolated hepatocytes was studied in the presence of various concentrations of unlabeled demethylphalloin (DMP), of phalloin and of phalloidin. The addition of phalloidin (or phalloin) reduces the uptake of 3H-DMP more than the addition of the equivalent concentration of DMP. The error caused by dilution of 3H-DMP with phalloin or phalloidin is not constant and depends on the concentration of the unlabeled compound. The relative differences between the uptake of 3H-DMP in the presence of demethylphalloin and in the presence of either phalloin or phalloidin cannot be explained by a competitive model. Some consequences for the use of 3H-DMP in toxicokinetic experiments are discussed.

Topics: Alkaloids; Animals; In Vitro Techniques; Kinetics; Liver; Oligopeptides; Phalloidine; Rats; Time Factors