Kornelia Ziegler

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.

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.

Hepatocellular uptake of peptides by bile acid transporters: relationship of carrier-mediated transport of linear peptides with renin-inhibiting activity to multispecific bile acid carriers

The uptake of a linear peptide with renin-inhibiting activity (code number EMD 51921) was characterized in isolated rat liver cells. Isolated hepatocytes take up EMD 51921 in a time-, concentration-, energy- and temperature-dependent manner. Transport of the peptide follows mixed-type kinetics. Diffusion occurs at a rate of 8.123 x 10(-6) cm/sec at 6 degrees C. For the saturable part of uptake, a Km of 2.0 microM and a Vmax of 160 pmol/mg per min were calculated. Various substrate analogues inhibit the uptake of EMD 51921. Absence of oxygen or decreased cellular ATP content (e.g., by metabolic inhibitors or xylulose) blocks hepatocellular uptake of EMD 51921. Temperatures above 20 degrees C accelerate the uptake. The activation energy was calculated to be 58.3 kJ/mol. The apparently active uptake of EMD 51921 was not sodium dependent. The membrane potential is a driving force for the accumulation of EMD 51921. Mutual competitive transport inhibition of EMD 51921, cholate and taurocholate is indicative of a common transport system. Benzamidotaurocholate and a cyclosomatostatin analog 008, not phalloidin and iodipamide, however, considerably decrease the uptake of EMD 51921. AS 30D ascites hepatoma cells, unable to accumulate bile acids and certain cyclopeptides, also fail to transport EMD 51921. BSP, a foreign substrate of the bilirubin carrier, noncompetitively inhibits the transport of EMD 51921. The inhibition of the uptake of EMD 51921 by rifampicin, a further substrate of the bilirubin carrier, is mixed: competitive at high EMD 51921 concentrations and uncompetitive at low EMD 51921 concentrations. The uptake of rifampicin into isolated rat liver cells, however, is not influenced by EMD 51921. Substrates of the transport systems for cations, amino acids, long chain fatty acids and hexoses did not influence the transport of EMD 51921.

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

SummaryIn 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).

Photoaffinity labeling of whole cells by flashed light: a simple apparatus for high-energy ultraviolet flashes

A simple apparatus for the photolysis of affinity labels is described. A commercial quartz tube produces high-energy flashes (wavelength from 200 to 1000 nm). A single flash is normally sufficient to activate photoaffinity labels in the presence of cells. Flash photolysis has several advantages over continuous irradiation, e.g. there is no need for cooling and photolabeling may be performed after different preincubation periods. The above apparatus is therefore suitable for investigations on time-dependent uptake of substrates by intact cells. Examples are demonstrated by photoaffinity labeling of rat liver cells by [3H]cyclosporin-diaziridine.

Hepatoselective carrier-mediated sodium-independent uptake of pravastatin and pravastatin-lactone.

Pravastatin and pravastatin-lactone are not taken up into extrahepatic cells such as fibroblasts, or hepatoma cells such as AS-30D ascites hepatoma cells or FAO cells. In contrast, pravastatin is taken up into isolated rat hepatocytes by a carrier mediated, saturable, temperature-dependent and energy-dependent mechanism. The kinetic parameters for the saturable uptake are Km 27 microM, Vmax 537 pmol/mg per min. The permeability coefficients were determined to be 9.829 x 10(-7) cm/s at 4 degrees C, 1.76 x 10(-6) cm/s at 7 degrees C, 3.85 x 10(-6) cm/s at 17 degrees C and 5.82 x 10(-6) cm/s at 37 degrees C. The activation energy is 60 kJ/mol for 100 microM pravastatin at 37 degrees C. The Q10 values are between 1.7 and 2.8. In the presence of metabolic inhibitors and in the absence of oxygen, transport is inhibited. Uptake of pravastatin is not dependent on an extracellular to intracellular sodium-gradient. Replacement of chloride by sulfate, nitrate, gluconate or thiocyanate significantly inhibits the uptake of pravastatin. Uptake is competitively inhibited by cholate and taurocholate in the presence and absence of sodium. Pravastatin, however, competitively inhibits the uptake of cholate and taurocholate only in the absence of sodium. In addition, pravastatin-lactone enters liver cells via an energy-dependent, carrier-mediated uptake system. For the saturable energy-dependent part of the hepatocellular uptake a Km value of 9 microM and a Vmax value of 621 pmol/mg per min was determined. The permeability coefficient of pravastatin-lactone uptake is calculated to be 5.41 x 10(-6) cm/s at 37 degrees C. The uptake of pravastatin-lactone is competitively-noncompetitively inhibited by pravastatin and by lovastatin and vice versa. These results indicate that the hepatoselectivity of pravastatin is due to its carrier-mediated uptake into rat hepatocytes via a sodium-independent bile acid carrier. Pravastatin-lactone resembles pravastatin-sodium in its hepatoselectivity.

Identification of cyclosporin binding sites in rat liver plasma membranes, isolated hepatocytes, and hepatoma cells by photoaffinity labeling using [3H]cyclosporin-diaziridine.

[3H]Cyclosporin diaziridine, a new photoaffinity label, enters rat liver cells in the dark. Photoaffinity labeling of isolated rat liver-cell plasma membranes with this probe modifies several polypeptides with molecular mass of 200, 85, 54, 50, 34 kDa. The major labeled protein of 85 kDa represents 2% of the total plasma membrane protein. A 50 kDa protein is heavily labeled in freshly isolated rat hepatocytes at low temperature and after short incubation in the dark. The 85 kDa protein becomes substituted after longer preincubation periods at temperatures above 10 degrees C. This suggests a localisation at the cytoplasmic side of the membrane. Several controls point to a specific interaction with the above mentioned proteins. Comparison of [3H]cyclosporin-diaziridine- and isothiocyanatobenzamido[3H] cholic acid-labeled membrane proteins reveals identity of binding proteins with the exception of the 85 kDa protein. However, the interaction of bile acids with the 85 kDa protein became apparent at higher concentrations as demonstrated by the differential photoaffinity labeling experiments. In the cytosol of rat liver cells, further [3H]cyclosporin-diaziridine binding proteins could be identified. In particular, a 17 kDa polypeptide was found which appears similar to cyclophilin, a protein known to be present in T-lymphocytes (R. Handschumacher et al. (1984) Science 226, 544-547: Cyclophilin. A specific cytosolic binding protein for cyclosporin A). Proteins with molecular mass of 90, 56, 30, 24, 20 kDa are labeled in AS-30D ascites hepatoma cells and those with molecular mass of 200, 150, 80, 70, 42, 25 kDa in Ehrlich ascites tumor cells.

New substrates of the multispecific bile acid transporter in liver cells : interference of some linear renin inhibiting peptides with transport protein(s) for bile acids

Interactions between some stable linear peptides with renin inhibitory activity and a multispecific transport system in the basolateral plasma membrane of liver cells was studied on cell suspensions. The peptides used in our experiments were taken up by liver cells and subsequently eliminated without any biotransformation (e.g., proteolysis). No degradation products could be detected in the extracellular medium by thin-layer chromatography. All peptides tested inhibited the uptake of physiological and of some foreign substrates of the multispecific bile acid transporter (MT). The phalloidin response of liver cells was also inhibited to a similar degree in a concentration-dependent manner. The potency of inhibition did not correlate with the lipophilic properties of the peptides. On the other hand a tight correlation could be documented between the inhibition of cholate transport and that of the phalloidin response. Transport inhibition of typical substrates of the MT by the above renin inhibitors was competitive. In contrast, the transport of a typical substrate of the bilirubin carrier (rifampicin), of amino acids (alpha-aminoisobutyric acid), long chain fatty acids (oleic acid) and cationic compounds (thiamin hydrochloride) was not inhibited by the same renin inhibitors. These results indicate that linear renin inhibiting peptides are taken up into liver cells by carrier proteins related to the MT.

Azidobenzamido-008, a new photosensitive substrate for the ‘multispecific bile acid transporter’ of hepatocytes: evidence for a common transport system for bile acids and cyclosomatostatins in basolateral membranes

Cyclo(-Phe(p-NH[1-14C]Ac)-Thr-Lys-(CO(p-N3)C6H4)-Trp-Phe-DPro++ +), in the following named azidobenzamido-008, was synthesized in order to identify binding sites for c(Phe-Thr-Lys-Trp-Phe-DPro), named 008, (a cyclosomatostatin with retro sequence) in liver cell plasma membranes. In the dark the above photolabel was taken up into isolated hepatocytes, inhibiting the sodium dependent uptake of cholate and taurocholate in a competitive manner (Ki for cholate uptake inhibition = 1 microM; Ki for taurocholate uptake inhibition = 5 microM). When activated by flashed light the inhibition became irreversible (IC50 for cholate uptake inhibition = 2 microM; IC50 for taurocholate uptake inhibition = 9 microM) and the activated cyclopeptide bound chiefly to hepatocellular membrane proteins of 67, 54, 50, 37 kDa. Excess of the initial 008, or of cholate or phalloidin partially protected the above membrane components against labeling with 14C-labeled azidobenzamido-008. In contrast AS 30 D ascites hepatoma cells, known to be deficient in bile acid and cyclosomatostatin transport, could not be specifically labeled by azidobenzamido-008. The membrane proteins preferentially labeled in hepatocytes (50 and 54 kDa) are integral glycoproteins. The 67 kDa protein is a hydrophilic nonglycosylated membrane component. Independent of labeling with 14C-labeled azidobenzamido-008 or with 14C-labeled azidobenzamido-taurocholate, the main radioactive peaks in the pH region of 7, 5.5, 5.25 were identical after solubilization with Nonidet P-40 and subsequent isoelectric focusing. Proteins of 67, 54, 50 and 37 kDa could be enriched by use of 008-containing gels in affinity electrophoresis. Binding sites for 008 were not destroyed by SDS or Nonidet P-40 treatment of plasma membranes.

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.