Biomedical subjects
Publications and source records attributed to M Frimmer.
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.
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.
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.
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.
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.
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.
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.
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.
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Below 21 degrees C isolated hepatocytes are insensitive to phalloidin. Between 21 and 30 degrees C the amount of affected cells increases to nearly maximal values, whereas a further rise of the temperature has little additional effect. These findings suggest that phase transition of membranal lipids might be responsible for the unusual temperature characteristics. The fluidity of lipids seems to control an early step of the phalloidin response.
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Suspensions of freshly prepared plasma membranes from rat livers were equilibrated with defined mixtures of air with polyhalogenated hydro-carbons (PHHC) at 37 degrees C. During the exposition enzymatic degradation of ATP was inhibited by carbon tetrachloride, chloroform and halothane in a dose-dependent manner. On a molar basis carbon tetrachloride was 5 times more effective than chloroform and halothane. The inhibition was partially reversible. Diethyldithiocarbamate, an agent protecting animals against carbon tetrachloride, was ineffective in this assay.
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.
Leucocidin from Pseudomonas aeruginosa causes cardiovascular failure in rats and mice. The time between i.v. injection and death depends on the dose. After injection of high doses (500 mug/kg) the arterial blood pressure decreases rapidly and cardiac irregularities and AV block occur within about 5 min. In contrast to endotoxin shock no pulmonary hypertension was observed, whereas portal hypertension was seen in our experiments. Injection of lower doses (less than 200 mug/kg) caused peripheral vascular damage with lung oedema, vascular disturbances in various tissues, exudation and bleeding. Finally cardiac insufficiency predominated. Dexamethasone delayed the symptoms but did not prevent death in either rats or mice. Heparin was ineffective in this type of shock.
Hemoglobin-free perfused rat livers were treated with carbon tetrachloride, chloroform, trichloroethylene and halothane by equilibrium of the perfusion medium with the varorous PHHCs. The resulting swelling of liver tissue, the potassium loss and the decrease of the perfusion rates were reduced by DEDTC to a different degree: The decrease of swelling, potassium loss and of the microcirculatory alteration by DEDTC was dose dependent in the case of carbon tetrachloride and halothane, whereas the effects of chloroform and trichloroethylene were not markedly influenced. Glutathione was mostly ineffective when given in similar doses. Pretreatment of rats with phenobarbital for 3 days increased itself swelling, K+-loss and reduction of perfusion rates, when the livers were perfused in absence of PHHCs. The effects of PHHCs in livers from pretreated rats were seldom greater but often lower than additive ones. Chiefly the effects of halothane were not markedly influenced by treatment of rats with phenobarbital. Isolated parenchymal liver cells were exposed to gas mixtures of O2, CO2 and PHHCs under controled conditions. Changes of the shape and staining of the cells were used for additional information. In view of these latter effects chloroform appeared as the most toxic and halothane as a nontoxic agent. DEDTC inhibits all effects of PHHCs demonstrated in isolated hepatocytes.
Leucocidin, a toxic protein obtained from Pseudomonas aeruginosa, was tested in perfused rat livers. Doses of greater than 4 mug/g liver produced a heavy potassium loss and a massive decrease of the perfusion rate but only moderate swelling of the liver at 37 decrees C. All effects depended on the dose. If the perfusions were carried out at 22 or 9 degrees C neither K+-loss nor swelling, and only a moderate increase of the vascular resistance was observed. Swelling at 37 degrees C was stronger at low initial perfusion rates (2.0 ml.min-1.g-1) than at higher ones (4.0 ml.min-1.g-1). The major effect of leucocidin could be prevented when the perfusion medium was changed 30 min after the addition of the toxin. Only small amounts o of 125I-leucocidin were taken up by the liver tissue. The time course and the quantity of nearly all effects of leucocidin were quite different from those of phalloidin.
Microsomes were prepared from perfused rat livers after different perfusion procedures. The yield of microsomal protein and the kinetic data (Km, Vmax) of glucose-6-phosphatase (3.1.3.9) and esterase (3.1.1.1) activities were analysed in each preparation. No marked differences were detected between conventionally prepared liver microsomes and those from livers perfused 1 hr with an erythrocytes-free medium under the conditions of open outflow. If the outflow pressure was increased artificially, the yield of microsomal protein decreased. The Vmax of both enzymes was markedly increased, whereas the Km values remained unchanged. The same microsomal alterations occurred when perfused rat livers were poisoned with phalloidin in vitro under the condition of open outflow. Our findings indicate that microsomal alterations in livers from poisoned animals might be due to microcirculatory disturbances, and not primary effects of the toxin on the endoplasmatic reticulum.