Comparative studies by scanning electron microscopy of the effect of ricin on the cell membrane of hepatoma cells and isolated hepatocytes.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
E Petzinger
Publications and source records attributed to E Petzinger.
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 completes with the uptake of phallotoxins. Cholate, taurocholate and glycocholate added 30 s prior to phallotoxins reduce their uptake in a concentration dependent manner. 100 microM bile acids suppress the uptake of phallotoxins completely. Several compounds known to inhibit the bile acid transport reduce the phallotoxin uptake to similar degree. Hepatocytes exposed to reagents reacting preferentially with amino groups of proteins lose their up take 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.
Alkaloids↗
Decreased phalloidin response, phallotoxin uptake and bile acid transport in hepatocytes prepared from wistar rats treated chronically with diethylnitrosamine.
Isolated hepatocytes prepared from rats pretreated with diethylnitrosamine (0.5 mg/kg DENA/DAY P.O.) are less sensitive to phalloidin poisoning. They take up lower amounts of both phallotoxins and bile acids than controls. The degree of inhibition depends on the period of pretreatment.
Amanitins↗
Uptake of 3H-demethylphalloin by isolated hepatocytes in the presence of various concentrations of phalloin or phalloidin.
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.
Alkaloids↗
Protective effect of anionic cholecystographic agents against phalloidin on isolated hepatocytes by competitive inhibition of the phallotoxin uptake. Comparison of the influence on the inward transport of 3H-demethylphalloin and of 14C-cholate.
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.
Alkaloids↗
Inhibition of 3H-demethylphalloin uptake in isolated rat hepatocytes under various experimental conditions.
3H-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.
Alkaloids↗
Decreased sensitivity of isolated hepatocytes from baby rats, from regenerating and from poisoned livers to phalloidin.
Isolated hepatocytes, prepared from 5 day old rats, from regenerating livers of 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.
Actins↗
A possible reason for the phalloidin tolerance of hepatoma cells.
In contrast to normal liver cells, AS-30D hepatoma cells are insensitive to phalloidin. The lack of the typical phalloidin response in the latter cells is not due to a deficiency of contractile proteins. Actin prepared from hepatoma cells is able to form filamentous structures and is stabilized in a manner similar to muscle actin. Isolated liver cells were exposed to a medium containing phalloidin and removed after 20 min by centrifugation. The supernatant was incubated again with fresh cells. The procedure was repeated four times. The phalloidin response decreased to about 19% of the control because of the uptake of phalloidin during each incubation. When the same procedure was carried out with AS-30D hepatoma cells, and aliquots of the supernatants were tested with hepatocytes no marked decrease of the phalloidin response was seen. This indicates that hepatoma cells do not consume the toxin as do normal liver cells.
Actins↗
Inhibitory effects of 4,4'-diisothiocyano stilbene-2,2'-disulfonic acid (DIDS) in the response of isolated hepatocytes to phalloidin.
4,4'-Diisothiocyano stilbene-2,2'-disulfonic acid (DIDS) inhibits the typical development of protrusions, regularly seen after treatment of isolated hepatocytes with phalloidin. The degree of inhibition depends on the time of preincubation and on the concentration of DIDS, but not on the concentration of phalloidin. DIDS is more effective than H2DIDS. The inhibition by both compounds is irreversible. The binding capacity of hepatocytes for H2DIDS is much higher than that of the phalloidin-insensitive hepatoma cells. Gel electrophoresis of lysates from cells, pretreated with 3H2DIDS demonstrates that actin binds very little of the inhibitor. Our results suggest that a protein structure on the surface of hepatocytes, needed for the response to phalloidin, is influenced by DIDS or H2DIDS.
Animals↗
The role of bile acids in phalloidin poisoning.
Glycocholate 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.
Animals↗
Trypsin protection of hepatocytes against phalloidin.
Explore the source record for details and available documents.
Animals↗
Scanning electron microscopic studies on the cytolytic effect of phallolysin on isolated rat hepatocytes and AS-30 D hepatoma cells.
Rat hepatocytes and AS-30 D hepatoma cells were treated in vitro with phallolysin, the toxic haemolysin from the death cap fungus Amanita phalloides. Scanning electron microscopy revealed dose-dependent formation of bulky protrusions on the cell surface, which later on burst, expelling intracellular material into the surrounding medium, while the cells became stainable with trypan blue. After 1 haemolytic unit (HU)/ml the protrusions burst within 10 min; after 3 HU/ml all cells were destroyed within 3-5 min. Susceptibility of hepatoma cells did not differ from that of hepatocytes.
Amanitins↗
Comparative studies on isolated rat hepatocytes and AS-30D hepatoma cells with leucocidin from Pseudomonas aeruginosa.
Leucocidin, one of the cytotoxic principles of Pseudomonas aeruginosa induces potassium loss and swelling in isolated hepatocytes and in AS-30D ascites hepatoma cells in a dose and time dependent manner. Hepatoma cells are more sensitive than normal hepatocytes. As shown by scanning electron microscopy the volume increase of both types of cells is accompanied by disappearance of microvilli. In contrast to phalloidin poisoning no protrusions were formed when the cells were exposed to leucocidin under isotonic conditions.
Animals↗
Proceedings: Isolation of phalloidin binding protein fractions from pig liver plasma membranes.
Explore the source record for details and available documents.
Animals↗
[Phalloidin antagonists/3rd communication: effects of antamanide derivatives in isolated perfused rat livers (author's transl)].
The protective effects of antamanide, O-carboxy-methyl-Tyr5-antamanide and of Tyr6-antamanide-monosulfate against phalloidin were tested in perfused rat livers. None of the applied doses was able to inhibit swelling and potassium loss of poisoned livers completely. In the perfused liver antamanide preferentially inhibited swelling but not potassium loss. O-Carboxymethyl-Tyr5-antamanide protected isolated hepatocytes against phalloidin. The different effects of antamanide derivatives in perfused livers and in vivo are discussed.
Animals↗
[Phalloidin antagonists 4th communication: Thioctic acid, SH-compounds, rifampicin, choleretics, dexamethasone, estradiol, unspecific inhibitors, and ineffective compounds (author's transl)].
1. Thioctic acid used clinically in poisoning by A. phalloides, protected perfused livers and also isolated hepatocytes against phalloidin, when given in high concentrations. 2. Some SH-compounds like coenzyme A, dimercaprol, cysteine and cysteamine were found to be protective in different concentrations. 3. Rifampicin protects mice against lethal doses of phalloidin, and inhibits poisoning of isolated hepatocytes at low concentrations. 4. Some choleretic drugs like dehydrocholate, temoebilin (extr. cucumae xanth.), ethacrynic acid influenced phalloidin poisoning by inhibition of binding. 5. Doses of 0.2 to 4.0 mg dexamethasone added to 100 ml of perfusion medium did not protect perfused rat livers against 0.5 mg phalloidin. 6. Pretreatment of female rats with estrogens effected protection against phalloidin in vivo. The same procedure resulted in moderate decrease of phalloidin effects when the livers of pretreated animals were poisoned in vitro. In male rats estrogen pretreatment was less effective. Castration did not augment the protective effect. 7. Secophalloidin, a biologically inactive derivative, did not influence phalloidin poisoning in perfused livers, even when applied in excessive concentrations. 8. Concanavalin A, probably bound in the neighborhood of binding sites for phalloidin, did not protect perfused livers against phalloidin. 9 Diethyldithiocarbamate, a compound protecting livers against carbon tetrachloride and halothane, was ineffective in phalloidin poisoning. 10. Further protective actions of Evans blue, of some phenanthrolines and of EDTA are discussed. 11. Pretreatment of animals with hepatotoxic compounds (CCl4, CHCl3, cinchophen) decreased the toxicity of phalloidin in vivo. Possible mechanisms are discussed.
Animals↗
Transient desensibilization of isolated hepatocytes against phalloidin by treatment with phospholipase A.
The development of typical protrusions in isolated hepatocytes after incubation with phalloidin was prevented by phospholipase A (from bee venom). When cells were preincubated with low concentrations of phospholipase A and the enzyme was removed by washing, the number of cells affected by 10 microgram phalloidin/ml was markedly reduced. If the pretreated cells were allowed to recover after removal of phospholipase, the sensitivity to phalloidin returned to nearly normal values. Transient treatment of hepatocytes with sublytic concentrations of phospholipase A did not destroy cell membranes, whereas 5-fold higher concentrations of the enzyme produced large protrusions quite different from those appearing during phalloidin poisoning. These findings suggest that phosphatides are needed for the recognition of phalloidin by liver cells. A series of marker enzymes were analysed in isolated plasma membranes from rat liver after treatment with phospholipase A. Changes in the activities of K+ Na+-ATPase and of p-nitrophenylphosphatase were observed. Other membranal enzymes were not markedly influenced. The inhibitory effect of phospholipase A on the phalloidin response is discussed in context of earlier findings suggesting an evident role of a membranal protein for the recognition of phalloidin.
Animals↗