M. Frimmer

Comparative studies on the uptake of 14C-bile acids and 3H-demethylphalloin in isolated rat liver cells.

The inward transport of bile acids in isolated hepatocytes competes with the uptake of phallotoxins. Cholate, taurocholate and glycocholate added 30 s prior to phallotoxins reduce their uptake in a concentration dependent manner. 100 μM bile acids suppress the uptake of phallotoxins completely. Several compounds known to inhibit the bile acid transport reduce the phallotoxin uptake to a similar degree. Hepatocytes exposed to reagents reacting preferentially with amino groups of proteins lose their uptake of both bile acids and phallotoxins. In hepatocytes isolated from 5 day old rats the uptake of both phallotoxins and cholate is reduced as compared to cells from adult controls. AS-30D ascites hepatoma cells, known to be insensitive to phallotoxins are unable to take up both phallotoxins and cholate. The results are consistent with our working hypothesis of a very similar mechanism for the uptake of bile acids and phallotoxins.

Inhibition of 3H-demethylphalloin uptake in isolated rat hepatocytes under various experimental conditions

Summary3H-Demethylphalloin (3H-DMP) a cyclopeptide very similar to phalloidin is taken up by isolated hepatocytes in vitro. Hepatocytes prepared from newborn animals are less sensitive to phalloidin. Their uptake of 3H-DMP is about one tenth of that of cells from adult animals. Ascites hepatoma cells, known to be insensitive to phalloidin took up negligible amounts of 3H-DMP. Cells prepared from regenerating livers took up insignificantly lower amounts of the toxin than in hepatocytes from adult animals.Treatment of hepatocytes with low concentrations of trypsin was found to switch off the phalloidin sensitivity in a reversible manner. This inhibition is due to a reduced uptake of 3H-DMP. Pretreatment of animals with CCl4, known to reduce the sensitivity to phalloidin, also decreases the uptake of 3H-DMP in isolated hepatocytes.Various agents, drugs and reagents were found to inhibit the response of isolated hepatocytes to phalloidin. All these compounds (bile acids, rifampicin, silybin, DIDS, glutardialdehyde, bromosulphophthalein, fusidic acid, antamanide, novobiocin) inhibit also the uptake of 3H-DMP in isolated hepatocytes. The results confirm our working hypothesis, presented in several previous papers, that decreased sensitivity to phalloidin is probably due to a reduced or blocked uptake of the toxin.

Untersuchungen zum Wirkungsmechanismus des Phalloidins

Summary1. Lysosomal enzymes were rapidly released when phalloidin was added to perfused isolated rat liver.2. Phalloidin has no significant effect on isolated lysosomes from normal bovine leucocytes or from rat liver. Preincubation of microsomes or other subcellular fractions from rat liver with phalloidin does not produce a toxic metabolite for isolated lysosomes. It appears unlikely that phalloidin has a direct effect on the lysosomal membrane.3. Potassium is released from the liver before lysosomal enzymes are liberated, which corresponds the destruction of the cell membranes as seen in the electrone microscope.4. A change of medium of liver perfused for 20 min in the presence of toxin did not reverse the toxic effects.5. Phalloidin does not appear to have a marked affinity for liver tissue.Zusammenfassung1. An der isoliert perfundierten Rattenleber wurde unter der Wirkung von Phalloidin eine rasch einsetzende Freisetzung lysosomaler Enzyme (Kathepsin, β-Glucuronidase, N-Acetylglucosaminidase) gemessen.2. Auf isolierte Lysosomen aus normalen Rinderleukocyten oder Rattenlebern hatte Phalloidin keine nennenswerten Wirkungen. Vorinkubation des Phalloidins mit Mikrosomen oder anderen subcellulären Fraktionen aus Rattenleber führte nicht zur Bildung eines für isolierte Lysosomen toxischen Produktes. Ein primärer Angriffspunkt des Phalloidins an der Lysosomenmembran ist unwahrscheinlich.3. Der Freisetzung lysosomaler Enzyme geht an der perfundierten Rattenleber ein schnell einsetzender Kaliumverlust voraus, dem elektronenoptisch eine Auflösung der Zellgrenzen entspricht.4. Das Schicksal der perfundierten Rattenleber ist 20 min nach der Zugabe des Giftes auch dann entschieden, auch wenn das Perfusionsmedium ausgetauscht wird.5. Anhaltspunkte für eine besonders hohe Affinität der Leber für Phalloidin konnten nicht gewonnen werden.

Driving forces in hepatocellular uptake of phalloidin and cholate

Active uptake of phalloidin and cholate in isolated rat liver cells depends upon both Na+ gradient and membrane potential. Omission of Na+ or inhibition of the (Na+ + K+)-ATPase diminished both phalloidin and cholate uptake. Dissipation of the sodium, potassium or proton gradient by monensin, nigericin, gramicidin and valinomycin blocked phalloidin uptake and also caused reduction of cholate transport. Chelation of Ca2+ and Mg2+ by EGTA or incubation of liver cells with NH4Cl neither influenced phalloidin nor cholate uptake. Hyperpolarization of liver cells by the lipophilic anions NO3- or SCN- enhanced phalloidin but reduced cholate uptake. Depolarization induced by a reversed K+ gradient reduced both kinds of transport. The results indicate that sodium ions and the membrane potential are driving forces for phalloidin and cholate uptake in hepatocytes.

Decreased sensitivity of isolated hepatocytes from baby rats, from regenerating and from poisoned livers to phalloidin

SummaryIsolated hepatocytes, prepared from 5 day old rats, from regenerating livers or from livers after poisoning with carbon tetrachloride, are less sensitive to phalloidin in vitro than hepatocytes from untreated adult controls. The time course of the reduced susceptibility to phalloidin was compared with the ability of hepatocytes to take up bile acids under various conditions. SDS-electrophoresis of cell lysates gave no evidence for decreased levels of actin in cells with reduced sensitivity to phalloidin. In contrast, there was a good relationship between the active uptake of bile acids and the sensitivity of hepatocytes to phalloidin. The decreased response of hepatocytes from baby rats, from regenerating livers or from poisoned livers to phalloidin is more probably related to differences in phalloidin uptake than to a reduced endowment with microfilamentous structures.

The Role of Bile Acids in Phalloidin Poisoning

SummaryGlycocholate and other bile acids inhibit the response of isolated hepatocytes to phalloidin in a concentration dependent manner. It is suggested that the inhibition is due to a block of phalloidin uptake. This interaction might explain the high specificity of phalloidin for liver tissue.

Hepatocellular transport of cyclosomatostatins: evidence for a carrier system related to the multispecific bile acid transporter.

The uptake of the cyclopeptide c(Phe-Thr-Lys-Trp-Phe-D-Pro) (008), an analog of somatostatin with retro sequence, was studied in isolated hepatocytes. 008 is taken up by hepatocytes in a concentration-, time-, energy- and temperature- dependent manner. Since 008 is hydrophobic, it binds rapidly to liver cells. This is evident by the positive intercept at the gamma-axis in the uptake curves. At higher concentrations, a minor part of the transport occurs by diffusion at a rate of 8.307.10(-6) cm/s. This part of diffusion is measured at 4 degrees C and can be subtracted from the uptake at 37 degrees C resulting in the carrier mediated part of uptake which is saturable. Kinetic parameters for the saturable part of uptake are Km 1.5 microM and Vmax 40.0 pmol/mg per min. The transport is decreased in the absence of oxygen and in the presence of metabolic inhibitors. Uptake is accelerated at temperatures above 20 degrees C. The activation energy was determined to be 30.77 kJ/mol. The membrane potential and not a sodium gradient is the main driving force for 008 transport. Cholate (a typical substrate of the multispecific bile acid transporter) and taurocholate are mutual competitive inhibitors of 008 uptake. Phalloidin, antamanide and iodipamide, typical foreign substrates of the transporter, interfere with the uptake of 008. AS 30D ascites hepatoma cells, known to be unable to transport bile acids, phalloidin and iodipamide, are also unfit to transport 008. Interestingly, sulfobromophthalein (BSP) but not rifampicin, both foreign substrates of the bilirubin carrier, inhibits the transport of 008 in a competitive manner.

Properties of the leak permeability induced by a cytotoxic protein from Pseudomonas aeruginosa (PACT) in rat erythrocytes and black lipid membranes

A cytotoxic protein, isolated from Pseudomonas aeruginosa (PACT), was tested on red blood cells of rats and on black lipid membranes for changes of membrane permeability. In rat erythrocytes PACT induces lysis indicative of the formation of a leak permeable to monovalent ions. The dose response curve for the PACT-induced hemolysis demonstrates that the rate of lysis as well as the fraction of lytic cells increases with increasing toxin concentration. Furthermore, the leak pathway discriminates hydrophilic non-electrolytes according to their molecular weight. The findings indicate formation by PACT of a pore with an apparent radius of about 1.2 nm. In pure lipid membranes PACT forms hydrophilic pathways with moderate selectivity for small cations over small anions. The presence of cholesterol is a prerequisite for the occurrence of these PACT-induced permeability changes.

What we have learned from phalloidin

In contrast to popular opinion phallotoxins do not play a role in poisoning with Amanita phalloides when the fungi are ingested orally. All toxic properties of this mushroom are due to amatoxins which, in contrast to the phallotoxins, are absorbed upon ingestion. Nearly all experiments on intact animals were performed by parenteral injection of phalloidin and therefore, most of these are unsuitable for practical consideration. In the present survey, however, a series of important findings are discussed, which provide insight into various functions of liver cells. When present in the blood, phalloidin and other phallotoxins are selectively taken up by hepatocytes. No other types of cells are sensitive to the toxin. No extrahepatic tissue is primarily impaired by phalloidin. Phalloidin cannot be degraded by peptidases or by proteases occurring in animals. Phalloidin is therefore a useful model substance for studies on the uptake of cyclopeptides by liver cells. The carrier system responsible for the active uptake of phalloidin can also translocate antamanide and several cyclic modifications of somatostatin. Phallotoxins bind with high affinity to microfilamentous structures, in particular to F-actin [Govindan et al., Naturwissenschaften, 59 (1972) 521-522] whereas phallotoxins are not bound to the monomer (G-actin). With respect to the strong organotropism of phallotoxins, intravenously injected phalloidin binds preferentially to microfilamentous F-actin of hepatocytes. Phalloidin is therefore a tool for inactivation of microfilamentous functions specifically in liver cells, and is suitable as a prototype of a cholestatic agent. In perfused livers arrest of bile flow is the earliest effect seen after addition of the toxin. In cells from other tissues phalloidin is only toxic when applied by intracellular microinjection. Phalloidin poisoning has been often used as a model for liver damage in the testing of hepatoprotective drugs. This substance is, however, not useful for such studies since the mechanism of phalloidin poisoning is too specific for interpretation in the sense of general liver protection.

Hepatocellular uptake of cyclosporin A by simple diffusion

Cyclosporin A is known to be eliminated mainly via the biliary++ pathway after biotransformation. Whether liver cells take up the drug by simple diffusion across the lipid barrier or by carrier-mediated transport, as shown for some other peptides, was unknown up to the present. Experiments with [3H]cyclosporin A on isolated rat hepatocytes indicate that the uptake of cyclosporin A is neither saturable nor is driven by metabolic energy. Cholestasis caused by cyclosporin A treatment is therefore not the result of mutual competition for a carrier protein. Nevertheless, cyclosporin A interacts with the bile acid transport system by non-competitive inhibition of bile salt uptake.