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Biomedical subjects

M Frimmer

Publications and source records attributed to M Frimmer.

At least 37 records · Page 2Linked to original sources
PubMed1986

Effects of phalloidin and cytochalasin B on cytoskeletal structures in cultured rat hepatocytes.

In short-term cultures of rat hepatocytes, bile canaliculi enclosed between unseparated cell couplets are able to perform periodical contractions resulting in expulsion of bile. Pericanalicular cytoskeletal proteins are involved in canalicular contractility: F-actin, myosin and tropomyosin are associated around bile canaliculi, as revealed by staining with tetramethylrhodaminyl-phalloidin and by immunofluorescence. Bile canalicular contractility is distributed by cholestatic agents that are known to interfere with actin polymerization; e.g., phalloidin and also cytochalasin B inhibit canalicular contractility and cause pericanalicular vacuolization and formation of blebs. Whereas the association of the cytoskeletal proteins is not affected by treatment with cytochalasin B, treatment with phalloidin results in dissociation of F-actin and myosin, indicating that binding of phalloidin to F-actin impairs its molecular interaction with myosin.

Animals↗
PubMed1986

Preservation of cellular polarity in isolated hepatocytes. Visualization of cytoskeletal structures by indirect immunofluorescence and fluorescent staining with tetramethylrhodaminyl-phalloidin.

The distribution of actin, myosin and tropomyosin in freshly isolated and short-term cultured rat hepatocytes was investigated by use of both rhodaminyl-phalloidin staining and immunofluorescence techniques. The cytoskeletal proteins were mainly located in distinct areas of the hepatocyte membrane, corresponding to their accumulation in the bile-canalicular region of liver tissue. In freshly prepared cells, these sections resembled sharp, angled or branched bands, similar to the pattern of hemicanaliculi. During incubation in a monolayer culture, these bands were transformed to circular formations. Simultaneously, enclosed bile-canalicular spaces between undissociated hepatocytes were visualized by staining of actin, myosin, and tropomyosin. The preservation of canalicular cytoskeletal structures in isolated hepatocytes is an indication of cellular polarity. Our findings suggest a uniform association of membrane-bound F-actin with myosin and tropomyosin.

Actins↗
PubMed1986

Molecular aspects of cytoprotection by modified somatostatins.

Somatostatin and cyclic modifications of this molecule inhibit the development of protrusions on the surface of isolated hepatocytes in presence of phalloidin. This prevention of phalloidin injury is caused by competitive inhibition of the phallotoxin uptake. Transport inhibition is not a hormonal effect of somatostatin. The concentrations needed are in the micromolar range. The most protective somatostatin modifications lack hormonal activity (GH release). Somatostatin and its analogs are substrates of a hepatocellular transporter which also translocates other cyclopeptides, among them phalloidin, antamanide, and several organic anions, such as iodipamide and fusidic acid. Physiological substrates of this multispecific transport system are bile acids. The protection of phallotoxin injury by somatostatin is a specific mechanism only representative for liver cells. No other cell contains the above multispecific transporter.

Affinity Labels↗
PubMed1985

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.

Animals↗
PubMed1985

Modified somatostatins as inhibitors of a multispecific transport system for bile acids and phallotoxins in isolated hepatocytes.

Somatostatin inhibits the uptake of phallotoxins and of cholic acid in isolated liver cells in a concentration-dependent manner. The inhibition is independent on the preincubation period and fully reversed by switching to a somatostatin-free buffer. Concentrations needed for 50% inhibition decreased 30-80-fold when somatostatin was modified by variation of its amino acid sequence. Some cyclic hexa- or penta-peptides inhibited both kinds of transport more strongly as the original (14 amino acid) somatostatin did. Three of the analogs showed a 2-3-fold higher potency than the others. The most potent compound (cyclo (Phe-Thr-Lys-Trp-Phe-D-Pro) 1 was studied in detail. The IC50 for the initial uptake of phallotoxin (6 microM) or of cholate (6 microM) was 1.5 or 3 microM, respectively. 1 inhibited the uptake of cholate in a competitive manner. The inhibition was independent on the preincubation time, but in contrast to somatostatin not fully reversible after a preincubation of 35 min. Somatostatin as well as its analogs prevented binding of isothiocyanatobenzamido [3H]cholate (an affinity label of the cholate transporter) to isolated plasma membranes from rat liver. The transport inhibition of cholate uptake is unlikely to be a hormonal effect of somatostatin, because the concentrations needed are approx. 1000-fold higher than circulating levels; however, it is apparently possible to increase the inhibitory potency on the tested transport system by modification of the sequence without increase of the well-known hormonal effects (Designing Activity and Receptor-Selectivity in Cyclic Peptide Hormone Analogs, Kessler, H., 18th Ervag Conference, Brussels, 1983).

Amanitins↗
PubMed1985

Crystallization of cytoplasmic actin in complex with deoxyribonuclease I.

Crystals of cytoplasmic (porcine liver) actin in complex with deoxyribonuclease I (DNAase I) were prepared for structural determination by X-ray-diffraction analysis. The crystallization of porcine liver actin-DNAase I complex is preceded by a brief treatment with immobilized trypsin, whereby a C-terminal tri- or di-peptide including cysteine-374 is removed from the actin without any noticeable degradation of both proteins as judged by sodium dodecyl-sulphate/polyacrylamide-gel electrophoresis. Analysis of the crystals obtained does not reveal any differences in the three-dimensional structure of porcine liver actin from its skeletal compartment at up to 0.6 nm resolution. However, in contrast with crystalline skeletal-muscle actin-DNAase I complex, heavy-atom substitution of crystals of porcine liver actin-DNAase I complex could not be achieved with methyl mercuriacetate. Evidence is presented that, in porcine liver actin, the N-terminal cysteine residue is not located at position no. 10, as in skeletal- and smooth-muscle actin, but most probably at position no. 17. Thus, because this site is covered by DNAase I, the cysteine becomes inaccessible to titration with 5,5'-dithiobis-(2-nitrobenzoic acid) after complex-formation with DNAase I.

Actins↗
PubMed1985

Properties of iodipamide uptake by isolated rat hepatocytes.

Exposure of isolated rat hepatocytes to iodipamide resulted in its time dependent accumulation in the cells. No accumulation was observed with rat AS-30D hepatoma cells and isolated jejunal and ileal cells from guinea pig. At concentrations below 75 microM, the iodipamide uptake into the liver cells showed saturation kinetics with a Km of 55 microM and Vmax of 555 pmol/mg cell protein X min. At higher concentrations, a nonsaturable component with a permeability coefficient (P) of 1.02 X 10(-5) cm/s is superimposed on the hepatoselective iodipamide uptake. Uptake in liver cells was partially inhibited by DIDS, an irreversible inhibitor of bile acid and phalloidin uptake in liver cells. Iodipamide uptake was found to be dependent upon Cl- and was slightly reduced in the absence of Na+. Both SCN- and NO3- decreased iodipamide accumulation in liver cells whereas SO4(2-) enhanced the accumulation. As with bile acid and phalloidin uptake, monensin, valinomycin and gramicidin A markedly reduced iodipamide uptake in rat hepatocytes. The results support the hypothesis that the organotropic excretion of iodipamide is partially performed by an energy dependent carrier which is the bile acid transporter of hepatocytes.

Animals↗
PubMed1984

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.

Adenosine Triphosphate↗
PubMed1984

Cyclosporin A protects liver cells against phalloidin. Potent inhibition of the inward transport of cholate and phallotoxins.

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.

Affinity Labels↗
PubMed1984

3'-Isothiocyanatobenzamido[3H]cholate, a new affinity label for hepatocellular membrane proteins responsible for the uptake of both bile acids and phalloidin.

Substitution of the hydroxyl group on C7 of cholic acid by a benzamido group leads to a derivative with inhibiting quality for the inward transport of both bile acids and phallotoxins by isolated liver cells. The tritiated isothiocyanate derivative was prepared (3'- isothiocyanatobenzamido [3H]cholate, [3H] IBCA ) with a specific activity of 70-80 mCi/mmol. The latter compound was used for affinity labeling of liver plasma membranes in order to detect chemically modified proteins involved in the transport of bile acids. [3H] IBCA and the noncovalently binding analogs were recognized by the transport system; they inhibited the uptake of both [14C]cholate and of demethyl[3H] phalloin in vitro. Isothiocyanatobenzamidocholate ( IBCA ) was able to protect isolated hepatocytes against phalloidin. In isolated and purified plasma membranes prepared from liver cells [3H] IBCA binds to saturable sites in an irreversible manner. Micromolar concentrations of unlabeled IBCA or millimolar concentrations of natural substrates prevented [3H] IBCA binding in a concentration dependent manner; some other substrates of the transport system also protected liver membranes against chemical modification. Membranes from AS- 3OD hepatoma cells, well known to transport neither bile acids nor phallotoxins, could not be labeled by [3H] IBCA . The major targets of labeling in hepatocellular plasma membranes were polypeptides with molecular mass of 67, 60, 54, 50, and 37 kDa as shown by SDS-polyacrylamide gel electrophoresis (10% acrylamide). The 67 kDa protein could be found in the aqueous phase after phase separation in Triton X-114. The 54 kDa and 50 kDa proteins remained in the detergent phase and can therefore be regarded as integral membrane proteins.

Affinity Labels↗
PubMed1984

Further characterization of membrane proteins involved in the transport of organic anions in hepatocytes. Comparison of two different affinity labels: 4,4'-diisothiocyano-1,2-diphenylethane-2,2'-disulfonic acid and brominated taurodehydrocholic acid.

4,4'-Diisothiocyano-1,2-diphenylethane-2,2'-disulfonic acid (H2DIDS) known as an irreversible inhibitor of the anion transport in red blood cells (Cabantchik, Z.I. and Rothstein, A. (1972) J. Membrane Biol. 10, 311-330) blocks also the uptake of bile acids and of some foreign substrates in isolated hepatocytes (Petzinger, E. and Frimmer, M. (1980) Arch. Toxicol. 44, 127-135). [3H]H2DIDS was used for labeling of membrane proteins probably involved in anion transport of rat liver cells. The membrane proteins modified in vitro by [3H]H2DIDS were compared with those labeled by brominated taurodehydrocholic acid. The latter is one of a series of suitable taurocholate derivatives, all able to bind to defined membrane proteins of hepatocytes and also known to block the uptake of bile acids as well as of phallotoxins and of cholecystographic agents (Ziegler, K., Frimmer, M., Möller, W. and Fasold, H. (1982) Naunyn-Schmiedeberg's Arch. Pharmacol. 319, 254-261). The radiolabeled proteins were compared after SDS-electrophoresis with and without reducing agent present, solubilization by detergents, two-dimensional electrophoresis and after separation of integral and peripheral proteins. Our results suggest that the anion transport system of liver cells cannot distinguish between bile acids and the anionic stilbene derivative (DIDS). The labeling pattern for both kinds of affinity labels was very similar. Various combinations of separation techniques gave evidence that the radiolabeled membrane proteins are not subunits of a single native channel protein.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1983

Common properties of hepatocellular uptake of cholate, iodipamide and antamanide, as distinct from the uptake of bromosulfophthalein.

The uptake of iodipamide and of the cyclopeptide antamanide by isolated hepatocytes was reduced reversibly in the absence of oxygen as recently shown for the transport of cholate. Oligomycin, antimycin A and carbonylcyano-chlorophenylhydrazone (CCCP) completely blocked the uptake of iodipamide and antamanide whereas the uptake of cholate was only partially decreased. Reduction of ATP in hepatocytes following replacement of glucose by fructose inhibited the uptake of iodipamide, of antamanide, and also of cholate. In contrast, the penetration of bromosulfophthalein remained unaffected under the above conditions. Arrhenius paralysis yielded high apparent activation energies for the uptake of cholate, iodipamide, and antamanide being 89, 77 and 55 kJ/mol respectively but only 22 kJ/mol for bromosulfophthalein. Mutual transport inhibition was found for iodipamide, antamanide and cholate as well as for bromosulfophthalein. Cholate inhibited the uptake of iodipamide and antamanide competitively. In contrast, bromosulfophthalein inhibited iodipamide uptake in a mixed order fashion. The results suggest a common uptake mechanism for cholate, iodipamide and antamanide different from that of bromosulfophthalein.

Absorption↗
PubMed1982

Energy linked uptake of demethylphalloin by isolated rat liver cells.

Isolated hepatocytes accumulate demethylphalloin (DMP) under aerobic conditions. In the absence of oxygen the initial rate of the DMP uptake is reduced to less than 20%, while reoxygenation restores the transport. Liver cells release previously accumulated phallotoxin when the oxygen supply is interrupted. DMP uptake is blocked by oligomycin, antimycin A, carbonylcyano-chlorophenylhydrazon (CCCP) or dinitrophenol and is partially inhibited by carboxyatractyloside. Depletion of ATP in hepatocytes by replacement of glucose by fructose reduces the accumulation of toxin too. Below 22 degrees C no uptake was measurable. Between 22 degrees and 37 degrees C an apparent activation energy of 76.6 kJ/mol toxin and a Q10 of 2.6 was calculated for the carrier mediated uptake of DMP. The results suggest that the uptake of demethylphalloin is an energy dependent substrate transfer very similar to that of cholate.

2,4-Dinitrophenol↗
PubMed1982

Affinity labels for membrane components involved in the uptake of bile acids and of phallotoxins by hepatocytes. Development of covalently binding derivatives of bile acids and of compounds related to cholecystographic agents.

A series of covalently binding derivatives of bile acids, fusidic acid and of compounds similar to cholecystographic agents were synthesized. Nearly all of them inhibited the development of protrusions on the surface of isolated hepatocytes regularly seen after treatment with phalloidin. The same compounds inhibited the uptake of demethylphalloin and of cholate in a concentration dependent manner. Two kinds of effects could be distinguished: The irreversible part of the inhibition depended on the incubation period and could not be removed by washing procedures. The reversible one was independent on the duration of the preincubation. Final results indicated that the tested derivatives inhibited either both transports, and the phalloidin response of liver cells to the same degree and in the same manner, or were found to be ineffective in all tests. The above parallelism supports the hypothesis that phallotoxins may be translocated by a carrier system normally responsible for the uptake of bile acids from the portal blood.

Affinity Labels↗
PubMed1982

Chemical modification of membrane proteins by brominated taurodehydrocholate in isolated hepatocytes; relationship to the uptake of cholate and of phalloidin and to the sensitivity of hepatocytes to phalloidin.

In vitro treatment of isolated rat hepatocytes with brominated taurodehydrocholic acid (BTC) reduced their sensitivity against phalloidin and inhibited the uptake of phalloidin as well as of cholate in an irreversible and concentration dependent manner. BTC was taken up itself by liver cells; this process was inhibited by 4,4'-diisothiocyano 2,2'-stilbene disulfonate (DIDS). When hepatocytes were incubated with 35S-BTC their plasma membranes contained five labeled protein species with molecular weights of 67,000, 49,000, 38,000, 32,000 and 24,000 as shown by SDS-electrophoresis. No marked difference was observed when isolated plasma membranes from livers were directly treated with the affinity label. DIDS suppressed covalent binding of 35S-BTC to membrane components drastically. Incubation of phalloidin insensitive AS-30D ascites hepatoma cells with 35S-BTC did not result in a chemical modification of the above five proteins. This agrees with an earlier observation that hepatoma cells are unable to take up phalloidin and bile acids (Petzinger et al. 1979; Rufeger and Grundmann 1977; Kroker et al. 1978).

Affinity Labels↗
PubMed1981

Is ligandin relevant for the uptake and storage of phallotoxins in liver cells?

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.

Alkaloids↗
PubMed1980

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↗