Properties of iodipamide uptake by isolated rat hepatocytes

Summary 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 M, the iodipamide uptake into the liver cells showed saturation kinetics with a K _m of 55 M and V _max of 555 pmol/mg cell protein x min. At higher concentrations, a nonsaturable component with a permeability coefficient (P) of 1.02×10^–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 was slightly reduced in the absence of Na⁺. Both SCN^– and NO ₃ ^– decreased iodipamide accumulation in liver cells whereas SO ₄ ^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.Abbreviation DIDS 4,4-diisothiocyano stilbene-2,2-disulfonic acid     

Protective effect of anionic cholecystographic agents against phalloidin on isolated hepatocytes by competive inhibition of the phallotoxin uptake

Summary 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 the iodipamide type.The above protective effect results from a competitive inhibition of the phallotoxin uptake as shown for iopodate. Cholecystographic agents similarily 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.This work was supported by the Deutsche Forschungsgemeinschaft     

Interaction of fusidates with bile acid uptake by isolated rat hepatocytes

Summary The interaction of fusidic acid and two of its conjugates with carrier-mediated uptake of bile acids was investigated in isolated rat hepatocytes. All three fusidates inhibited the uptake of both cholate and taurocholate competitively suggesting a direct interaction of fusidates with bile acid carrier. The inhibition constants for all three fusidates for the inhibition of cholate uptake were significantly different from the respective inhibition constants for the inhibition of taurocholate uptake. This would indicate that both cholate and taurocholate are transported by more than one carrier into hepatocytes. The results may also indicate that taurine conjugated bile acids may be transported preferentially by one transport system while unconjugated bile acids may be preferentially transported by another transport system.Part of this publication was presented in the 18. Frühjahrstagung der Deutschen Pharmakologischen Gesellschaft (1977)     

Energy linked uptake of demethylphalloin by isolated rat liver cells

Summary 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°C no uptake was measurable. Between 22° and 37°C an apparent activation energy of 76.6 kJ/mol toxin and a Q ₁₀ 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.Abbreviations DMP demethylphalloin - CCCP carbonylcyano-chlorophenylhydrazon - DNP 2,4-dinitrophenol This work was supported by the Deutsche Forschungsgemeinschaft. Part of it was presented at the 21st Spring Meeting of the German Society of Pharmacology in Mainz 1980 (Petzinger 1980).     

Affinity labels for membrane components involved in the uptake of bile acids and of phallotoxins by hepatocytes

Summary 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.This work was supported by the Deutsche Forschungsgemeinschaft     

Taurolithocholate inhibits taurocholate uptake by isolated hepatocytes at low concentrations

Summary The cholestatic bile acid taurolithocholate inhibits taurocholate uptake by isolated liver cells non-competitively. Inhibition is instantaneous and inversely related to the cell number in the incubate. The K _i amounts to 7 M in the presence of 2 mg cellular protein per ml. Secretion of taurocholate by isolated liver cells is not affected by taurolithocholate up to a concentration of 50 M. This indicates a difference between the carrier for taurocholate uptake and the carrier for taurocholate secretion. Inhibition of bile acid uptake by liver cells may be involved in the pathogenesis of lithocholate-induced cholestasis.     

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 M 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.This work was supported by the Deutsche Forschungsgemeinschaft     

The role of bile acids in phalloidin poisoning

Summary 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.This paper was supported by the Deutsche Forschungsgemeinschaft     

A model describing the disposition of phenytoin in isolated rat hepatocytes

The use of isolated rat hepatocytes in studies of drug metabolism has become well documented in the past few years. However, in part because of modelling difficulties due to the simultaneously occurring substrate transferring processes, its predictability of in vivo situations has not been emphasized. Much controversy surrounds the metabolism of phenytoin (5,5-diphenylhydantoin), a widely used anticonvulsant, and an appropriate pharmacokinetic model to describe the disposition of this drug still lacks general acceptance. In the present study, metabolism of phenytoin in the isolated rat hepatocyte system was followed by assaying either the unchanged drug or the pooled metabolites in both the suspending medium and the cells. A model was developed which can describe the time course of the different species sampled. Inhibition of biotransformation by the major metabolic product [5-(p-hydroxyphenyl)-5-phenylhydantoin or p-HPPH] and the uptake and release of the latter were also studied, in order to elucidate the role of product inhibition in determining the dose-dependent pharmacokinetic behaviour of the drug. The results obtained strongly suggest that only concentrations of p-HPPH higher than the ones attained by phenytoin biotransformation alone can significantly inhibit the main enzymatic reaction.     

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

Summary 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.This work was supported by the Deutsche Forschungsgemeinschaft     

Transport systems of isolated hepatocytes

Hepatocellular transport of some substances with predominantly biliary elimination was studied by use of isolated rat liver cells. Active uptake of the hydrophilic compounds ouabain and cholate proceeded over 10–20 min and the final intracellular concentrations exceeded the extracellular concentrations more than 60-fold. Uptake of both substances was saturable and compatible with carrier-mediation. Uptake of the lipophilic compounds digitoxin and naphthol was not active, terminated within 1 min and compatible with simple diffusion.The velocity of uptake depended on 3 major parameters: substrate features, cellular factors, and experimental conditions. Substrate features included: lipophilicity or hydrophilicity of the compound. Cellular factors included: presence and activity of transport systems. Experimental conditions included: substrate concentration, amount of cells, composition of medium.Intracellular conjugation of cholate and naphthol produced the same polar metabolites which are formed in vivo. These metabolites were then in part released from the isolated hepatocytes into the medium. The transport systems for release are more difficult to characterize than those for uptake, and therefore little is known about their nature, despite their importance in detoxication.If isolated hepatocytes are used in toxicology to assess metabolism or biological effects of foreign compounds, transport across the liver cell membrane is potentially a rate limiting step.     

Transient desensibilization of isolated hepatocytes against phalloidin by treatment with phospholipase A

Summary 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 g 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.Supported by a grant of the Deutsche Forschungsgemeinschaft     

Decreased phalloidin response,phallotoxin uptake and bile acid transport in hepatocytes prepared from wistar rats treated chronically with diethylnitrosamine

Summary 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.This work was supported by the Deutsche Forschungsgemeinschaft.     

Benoxaprofen induced toxicity in isolated rat hepatocytes

The toxicity of benoxaprofen, a non-steroidal anti-inflammatory compound was investigated using rat hepatic microsomal and isolated hepatocyte suspensions. In microsomes, benoxaprofen produced a Type I binding spectra and competitively inhibited (k_i 380 μM) the oxidative metabolism of aminopyrine. Marked toxicity was observed following incubation of benoxaprofen with isolated hepatocytes from either untreated, phenobarbitone (PB) or 3-methylcholanthrene (3-MC) pretreated male rats. In untreated hepatocytes increases in the intracellular lactate/pyruvate (L/P) ratio and alanine aminotransferase (ALT) release were related to the benoxaprofen concentration and duration of incubation. Alterations in L/P ratio preceded the release of cytosolic ALT and at 4 h a well defined dose-response relationship existed between the benoxaprofen concentration and the observed increases in the L/P ratio and ALT release. Pretreatment of animals with either PB or 3-MC did not affect the temporal nature nor the magnitude of the hepatocyte response to benoxaprofen. In addition, inhibitors of cytochrome P-450 isozymes (SKF-525A, metyrapone and -napthoflavone) were ineffective with regard to modifying the observed toxicity. The results of this study suggest that hepatic cytochrome P-450 mediated metabolism may not be implicated in the toxicity of benoxaprofen in isolated hepatocytes. However, alterations in the cellular redox state and evidence of plasma membrane bleb formation suggest that benoxaprofen may uncouple oxidative phosphorylation and disturb intracellular calcium ion homeostasis.     

Temperature dependence of phalloidin response in isolated rat hepatocytes

Summary Below 21° C isolated hepatocytes are insensitive to phalloidin. Between 21 and 30° 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.     

Isolation of hepatocytes from newborn rats

A method is described for the preparation of isolated hepatocytes from 5 day old rats, based on the procedure of Berry and Friend (1969). In contrast to the original procedure the liver of newborn rats was perfused in the reverse direction from the vena cava caudalis (pars thoracalis) to the vena portae.The cells obtained by the above method are morphologically intact as shown by electron micrographs and by phase contrast microscopy. About 85% of the isolated cells excluded trypan blue.Neonatal liver cell preparations were used for dose response studies with phallotoxins and further experimental applications in the field of phalloidin tolerance of newborns are described.     

Effect of nitric oxide on the sinusoidal uptake of organic cations and anions by isolated hepatocytes

The issue of whether or not the presence NOx (NO and oxidized metabolites) in the hepatocytes at pathological levels affects the functional activity of transport systems within the sinusoidal membrane was investigated. For this purpose, the effect of the pretreatment of isolated hepatocytes with sodium nitroprusside (SNP), a spontaneous NO donor, on the sinusoidal uptake of tributylmethylammonium (TBuMA) and triethylmethyl ammonium (TEMA), representative substrates of the organic cation transporter (OCT), and taurocholate, a representative substrate of the Na+/taurocholate cotransporting polypeptide (NTCP), was measured. The uptake of TBuMA and TEMA was not affected by the pretreatment, as demonstrated by the nearly identical kinetic parameters for the uptake (i.e., Vmax, Km and CL(linear)). The uptake of mannitol into hepatocytes was not affected, demonstrating that the membrane integrity remained constant, irregardless of the SNP pretreatment. On the contrary, the uptake of taurocholate was significantly inhibited by the pretreatment, resulting in a significant decrease in Vmax, thus providing a clear demonstration that NOx preferentially affects the function of NTCP rather than OCT on the sinusoidal membrane. A direct interaction between NOx and NTCP or a decrease in Na+/K+ ATPase activity as the result of SNP pretreatment might be responsible for this selective effect of NOx.     

Inhibition of hepatic uptake of bile acids by rifamycins

Summary The effect of rifamycin SV and rifampicin on hepatic bile acid uptake was studied using isolated rat hepatocytes in presence and in absence of albumin. The drugs inhibited cholate uptake more than taurocholate uptake and the inhibition was of non-competitive type. In presence of 3% albumin the inhibitory effect of the drugs was more for cholate and less for taurocholate uptake than in absence of albumin. Neither the binding of bile acids nor that of the drugs to albumin was altered by one another. Thus the effect in presence of albumin cannot be explained by the binding of the drugs and bile acids to albumin alone. It is suggested that albumin interacts with hepatic bile acid uptake process and this interaction with cholate uptake is different from that with taurocholate uptake. This additional and different effect of albumin may explain the effect of the drugs in presence of albumin. The results may be of clinical significance in rifamycins treatments.Part of this publication was presented in the 18th Frühjahrstagung der Deutschen Pharmakologischen Gesellschaft (1977)     

Lipid peroxidation in isolated hepatocytes from rats ingesting ethanol chronically

Summary Isolated hepatocytes from rats consuming ethanol (8.5 g/kg) daily produce malondialdehyde in significantly higher amounts than liver cells from control animals. The release of LDH and the uptake of trypan blue in both types of hepatocytes do not differ during the incubation period of 2 h. GLDH, however, is only set free into the medium from liver cells of ethanol drinking rats, indicating that mitochondrial alterations are involved.Bomotrichloromethane (CBrCl₃) promotes lipid peroxidation in hepatocytes from ethanol drinking rats in a much higher degree than in cells from control rats. The cell damage induced by CBrCl₃ and indicated by a release of LDH and GLDH from the hepatocytes and their uptake of trypan blue is also much more pronounced in liver cells from ethanol drinking animals.The stronger action of CBrCl₃ cannot be explained by an enhanced microsomal metabolism, because no increase of drug metabolizing enzymes could be observed. The relatively low ethanol consumption did not influence body growth and liver weight and did not evoke any triglyceride accumulation.The normal balance between processes favouring lipid peroxidations and reactions protecting the liver cells seems to be shifted to a state during alcohol intake which promotes formation of radicals.