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A method was developed to estimate the relative contributions of paracellular and transcellular pathways to the total biliary clearance of sucrose in isolated perfused rat liver. When livers were perfused with a sucrose-containing medium (1 mM), biliary sucrose concentration reached an equilibrium of 165 +/- 27 microM within 10 min, without further significant change up to 40 min. After removal of sucrose from the perfusate, the decrease of the sucrose concentration in bile was found to obey biphasic first-order kinetics, showing a rapid initial decrease (half-life 3.3 +/- 0.5 min) and then a slower decrease (half-life 29.4 +/- 5.7 min). Both phases of decrease were further characterized. Pretreating rats with the cholestatic agents alpha-naphthyl isothiocyanate (ANIT), oestradiol valerate (OV) and colchicine increased the biliary equilibrium concentration and decreased the half-life of the fast phase of the biliary sucrose elimination. The slow phase was unaffected in the livers of ANIT- and OV-treated rats. The slow phase of biliary sucrose efflux was sensitive to colchicine treatment. A close correlation was observed between the slow-phase fraction of the biliary sucrose and the corresponding sucrose content of the liver. By quantitative analysis of the efflux kinetics the relative contribution of the paracellular pathway to the biliary clearance of sucrose was estimated to be 83 +/- 2% in control livers, which increased to about 90% in livers of pretreated animals. These results are important in view of the use of sucrose in evaluating the paracellular-pathway permeability in intra- and extra-hepatic cholestasis.

Hemoglobin-free perfused rat liver was demonstrated to be a suitable experimental model in studying bile secretion. Bile flow slowly decreased to more than 3 h of perfusion. Despite differences in metabolic states, the bile flow was the same in the recirculating as in the nonrecirculating mode of perfusion. Sulfobromophthalein stimulated bile flow at high rates of infusion. In bile, the ratio conjugated to unconjugated sulfobromophthalein also increased with sulfobromophthalein infusion rate. The access of [14C]insulin, [14C] sucrose, and inorganic [32P] phosphate from perfusate into bile was restricted. Bile flow, secretion of taurocholate and sulfobromophthalein, and bile pressure are compared with values from anesthetized animals and from isolated livers perfused with medium containing erythrocytes.

The need for quick viability tests is stressed. Aas these should achieve more than statically categorizing dead or non-dead cells, several procedures are suggested that picture the energetic state of the cells. The almost classical criterion of this category, namely stimulation of respiration by succinate, must be questioned on the basis of the present results. It is shown, that restricted respiration by succinate is not due to limited permeability of the plasma membrane, but to competition by endogenous substrates for uptake into mitochondria. Distribution equilibria for succinate appear to be according to (delta pH)2 with regard to cytoplasm. They are attained within 5-20 s or faster. Uptake is in part regulated by the surface charge density. Permeability changes caused by effectors of surface charge, such as amphiphilic ions, are examplified for succinate, chloride, phosphate, Na+, K+, and Ca2+. Such changes repeatedly also occur after pulses of BSP. They are counterregulated by the cell within a minute in a manner dependent on BSP concentration and the state of the cells. During the preincubation phase, that is the time of readaptation after transfer of cells from 0 degree C to higher temperature, a special labile state transiently occurs, where cyclic permeability changes for Ca2+, Na+, K+ can be caused by substrate addition, especially succinate, and/or ATP. The extent of these changes and their sequence again depend on the energetic state of the cells. In a probably narrow energetic window a sequence of cation movements reminding of that after depolarization of an excitable cell, is observed. Manipulation of the Na+/K+-ratio by variation of preincubation time and by ouabain shows that this is not simply the denominator for reversible calcium uptake. As the surface charge appears to reflect the energetic state, ANS fluorescence is applied to monitor the state of the plasma membrane, though difficulties arising from a slow ANS permeation are not yet solved.

The presence of pre-S1 proteins in serum and liver of individuals with acute and chronic hepatitis B virus infection was investigated in Western blots using antibodies against a fusion protein, containing amino acids 20-120 of the pre-S region. Pre-S1 proteins were present in 20 of 38 HBsAg-positive sera. All sera positive for pre-S1 proteins were also positive for hepatitis B virus DNA indicating the presence of hepatitis B virions, and 16 of these sera were also positive for HBeAg. In five sera positive for hepatitis B virus DNA, pre-S1 proteins were not found. In an additional study, pre-S1 proteins could be detected in 4 of 6 patients with acute hepatitis B virus infection during the first 2 weeks after admission to the hospital. The presence of pre-S1 proteins showed a good correlation with the detection of hepatitis B virus DNA. After seroconversion from HBeAg to anti-HBe, both hepatitis B virus DNA and pre-S1 proteins were no longer detectable. Pre-S1 proteins were present in three liver tissue specimens from two patients with acute hepatitis B virus infection and from one patient with cirrhosis of the liver. The proteins were not found in the liver of two HBsAg-positive patients with hepatocellular carcinoma (primary liver carcinoma), negative for HBeAg. Pre-S1 proteins can be detected in serum, positive for hepatitis B virus DNA and in liver tissue of hepatitis B virus-infected individuals. The presence of these proteins appears to correspond with the presence of hepatitis B virus DNA, both markers indicating hepatitis B virus replication.

In isolated hepatocytes the influence of cellular glutathione (GSH) on initial influx, net uptake and efflux of methotrexate (MTX) was determined. Endogenous glutathione in rat liver cells was depleted by either fasting of rats or by in vivo administration of phorone prior to cell preparation. The initial rate of influx of MTX was found to be higher in hepatocytes of fasted and phorone-treated rats than in those of untreated, fed control rats. The Km values for the methotrexate influx in GSH-deficient hepatocytes were up to 3 times lower than in normal cells, whereas Vmax remained unchanged. These results disclose an increased efficiency of the MTX transport system in cells with diminished cellular GSH levels. On the other hand, titration of external membrane SH groups by 203Hg p-CMBS revealed up to three times higher amounts of free SH groups on cells from starved and phorone-treated rats than on hepatocytes of fed rats. Increased efficiency of the MTX transport system in GSH-deficient cells may, therefore, be interpreted as increased capacity of the MTX transport carrier for which free membrane SH groups are known to be essential. Despite activation of initial transport of MTX here, later net accumulation of MTX became smaller than in cells with normal GSH levels. Efflux of MTX from liver cells was not influenced by fasting or phorone treatment of rats, however, the "nonexchangeable" pool of MTX was found to be decreased, which indicates inhibition of formation of MTX polyglutamates here. This inhibition was most likely responsible for the decreased amounts of MTX finally accumulated in GSH-deficient hepatocytes.

The C gene of hepatitis B virus (HBV) codes for at least two different proteins (p 21c and p 17e). To investigate the expression of C-gene-encoded proteins in vivo, serum and liver samples from HBsAg-positive patients as well as serial serum samples from an HBV-transfected chimpanzee were studied. Antibodies directed against bacterially synthesized C-fusion proteins were used in Western blots to test for the presence of p 21c and p 17e. In serial serum samples from the chimpanzee, p 21c and p 17e were detected concomitantly during the acute phase of the infection. When sera of patients with chronic HBV infection were studied, all sera containing p 17e were found to be positive also for p 21c. Sera positive for HBV DNA but negative for HBeAg were only positive for p 21c, indicating that HBeAg/p 17e is not an absolutely reliable marker for infectivity. In liver tissue specimens from 20 patients with HBV-related liver diseases, p 21c was detected in five cases, indicating viral replication. The p 17e antigen, however, was present only in low amounts in three of these five, suggesting that synthesis of p 21c and p 17e is not strictly coupled. C/Pol-gene-encoded fusion proteins were found in the liver tissue of only one patient with cirrhosis, supporting our previous finding that detectable levels of these proteins are expressed rarely.

Efflux of taurocholate from isolated rat hepatocytes was studied to characterize the mechanism of bile acid secretion. Cells were incubated with taurocholate for 15 min. The amount of the intracellularly accumulated bile acid was directly related to the concentration in the medium. Transfer of the loaded cells from the incubation medium to a medium without taurocholate led to taurocholate efflux. Efflux was saturable, its activation energy amounted to 12 kcal/mol (50 kJ). It was strongly inhibited by the metabolic inhibitor antimycin A and to a lesser extend by the uncoupler carbonylcyanide-m-chlorophenylhydrazone. Dinitrofluorobenzene and mersalyl, reagents which react with amino acids, inhibited efflux by about 30% when applied at concentrations of 50 muM. Ouabain increased the rate of efflux. The observations indicate that secretory functions are maintained in isolated liver cells.

Under certain modulating conditions the liver of the male Syrian golden hamster is a target organ for the carcinogenic effect of the synthetic estrogen diethylstilbestrol (DES). As a basis for mechanistic studies aimed at elucidating the role of metabolic activation in the process of DES-induced neoplasia, the metabolism of 14C-DES was investigated in freshly isolated hamster hepatocytes. These oxidative metabolites of DES, viz. Z,Z-dienestrol,3'-hydroxy-DES and 1-hydroxy-E-DES, were formed in 14.2, 9.1, and 0.3% yield, respectively, when hepatocytes were incubated with 50 nmol DES/mg cellular protein for 60 min. Glucuronides (4.0%) and sulfates (2.8%) of DES and of the oxidative metabolites were also found, and non-extractable binding of radioactivity to cellular protein was observed indicating the formation of reactive intermediates. The capability of hamster hepatocytes to oxidize and conjugate DES should allow the investigation of the effects of modulators on the metabolic activation of DES in this cellular system in order to help clarify the mechanisms of DES-induced hepatocarcinogenesis.

36.4 +/Various cellular parameters were measured with regard to their usefulness as criteria of viability of isolated cells. Stainability by trypan blue and release of lactate dehydrogenase indicate only severe irreversible damage of cells. Neither endogenous respiration nor even the ATP/ADP ratio is a sensitive criterion of viability. On aging of cells, the ATP/ADP ratio remains high, even though the membrane potential, the intracellular K concentration and the content of adenine nucleotides decrease considerably. A sensitive, easily performed test is the stimulation of cellular respiration by 1mM succinate. Only a damaged plasma membrane allows succinate permeation of a rate sufficient to stimulate respiration. The membrane potential and the intracellular Na and K concentrations are the most sensitive criteria of viability, since they indicate the earliest changes on aging. (For freshly isolated cells, we found a membrane potential of 36.4 "/- 3.4 mv [n = 5], an intracellular K concentration of 109.0 +/- 9.1 mM, and an intracellular Na concentration of 47.0 +/- 13.4mM.) The incorporation of [14C]uridine also sensitively reflects cellular damage.

1. Aerobic incubation of isolated rat liver cells--after dilution from the anaerobic stock suspension--transiently brings about a state, during which a reversible calcium uptake can be observed on addition of a respiratory substrate. Uptake varies greatly and can reach more than 50 nmol/mg protein, but declines to zero on prolonged preincubation, especially at higher temperature. Repeated additions of succinate or 3-hydroxybutyrate evoke new calcium transients. If ATP is simultaneously added, if greatly potentiates succinate-initiated reversible uptake. 2. If rotenone is present during the preincubation phase, calcium transients are strongly enhanced. Uptake is blocked by uncouplers and respiratory inhibitors, indicating the involvement of mitochondria. 3. Calcium uptake is not accompanied by increased oxygen consumption. The actual respiration cannot account sufficiently for the energy need of calcium uptake. Participation of cytoplasmic ATP is likely, as inhibitors of adenine nucleotide translocase affect uptake. 4. Lanthanum enhances calcium uptake in contrast to its action on mitochondria. 5. Pulse-labeling experiments indicate that the calcium taken up is removed from a rapidly exchangeable calcium pool by withdrawal into the mitochondria as a deep compartment. 6. Calcium uptake is accelerated either by increasing the phosphate level or by high temperature. It is prolonged by low temperature, high pH or high ATP concentration. Calcium release accelerates with increasing temperature, decreasing pH and a further rise in phosphate concentration. 7. The dependency on phosphate and temperature reveals a delicately poised equilibrium of uptake and release. At ambient temperature, phosphate increases uptake up to a concentration of 0.5 mM. Higher concentrations accelerate both uptake and release. At lower temperature, the accelerating effect on uptake predominates. A temperature shift during incubation results in adaptation of the calcium equilibrium to the new temperature, i.e. release of calcium at high temperature, uptake at low temperature. 8. Oxidizing metabolites inhibit succinate-stimulated calcium uptake and promote release of previously accumulated calcium. An increased sensitivity to phosphate is established. 9. With respect to isolated mitochondria, isolated liver cells appear to be a more realistic model for studying the physiological mechanism of mitochondrial calcium release, since compartmental constraints and regulations are maintained.

Development of intrahepatic cholestasis induced by alpha-naphthylisothiocyanate was studied in rats. At various times after alpha-naphthylisothiocyanate application, livers were isolated from treated rats and perfused hemoglobin-free to assess cholestatic parameters. Unstimulated bile flow was found to only slightly decrease up to 10 h after alpha-naphthylisothiocyanate administration. In contrast, secretion into bile of sulfobromophthalein and taurocholate declined markedly between 4 and 7 h as their concentrations in the perfusate increased, and stimulation of bile flow by taurocholate decreased. The permeability of the bile-to-perfusate barrier to [14C]sucrose and [32P]orthophosphate increased in parallel with the changes in sulfobromophthalein and taurocholate distributions. This correlation of changes in the distribution of cholephilic substances with biliary accessibility for extracellular markers suggests that, in alpha-naphthylisothiocyanate-induced cholestasis, increased leakage of tight junctions may contribute to regurgitation of bile constituents into the vascular system.

The initial rates of BSP uptake by isolated hepatocytes were compared in cells of good and poor viability. Cells with impaired viability were obtained by ageing or by accident also in fresh preparations. Viability was judged by trypan blue stainability, membrane potential and respiratory parameters indicative for energy state, substrate supply and plasma membrane permeability changes. It was found that concomitant with impaired viability there was a decline of uptake rates at low and an increase at high BSP concentrations with a crossover point at 10 muM as manifest in an increase of Km and V. Simultaneously, the affinity and size of the membrane bound fraction decreases. The results give kinetic support to the supposition that it is the decreased uptake from plasma to liver that is responsible for the prolonged plasma retention times in the liver function test of patients with impaired hepatobiliary function.

The uptake of methotrexate (MTX) by isolated rat hepatocytes and its changes under the influence of exogenous GSH have been studied under various conditions: GSH concentration, pH of incubation medium, preincubation of cells prior to MTX and GSH addition, ionic composition of the incubation medium (standard saline, Na+-free, Na+ and K+-free, or ion-deficient), after prior treatment of cells by membrane -SH blockers (p-CMBS, 4-CMB and DIP2+) and ATP. It was found that GSH strongly accelerated MTX uptake. This effect depended on GSH concentration and on preincubation of cells. The GSH effect was not dependent on medium pH in spite of an observed close relationship between pH of incubate and MTX transport itself. Activation by GSH of MTX transport was connected to an increase in intracellular K+. It was also noted that while blockers of membrane -SH groups like p-CMBS and 4-CMB inhibited MTX uptake and increased the intracellular Na+/K+ ratio, both effects were partially overcome by GSH. After treatment by DIP2+, Na+/K+ ratio was unaffected, but MTX uptake inhibited. Still GSH abolished inhibition. Added ATP also inhibited MTX uptake and caused loss of cellular K+ and accumulation of Na+. Here neither effect could be reversed by GSH; consequently, high cellular amounts of K+ and MTX accumulated by previous action of GSH were depleted on subsequent ATP addition. MTX uptake was low in sucrose medium. But in this ion-deficient medium, GSH had the greatest stimulatory effect on MTX uptake. It is concluded that binding GSH can affect the redox state of the -S-S-/-SH groups of the cellular plasma membrane and that this effect of GSH might demonstrate involvement of the redox state in the control of MTX permeability.

1. The ability of external ATP to induce calcium uptake in isolated rat liver cells was further characterized. Stimulation of calcium uptake was specific for ATP, other nucleotides or ATP metabolites had no comparable effect. ATP was dephosphorylated while stimulating calcium uptake, but there was no stoichiometry between ATP hydrolysis and calcium uptake nor did dephosphorylation depend on calcium concentration. ATP acted from outside and was dephosphorylated by an ecto-ATPase of the cells. 2. In addition to its direct action, ATP enhanced succinate-dependent calcium uptake in a cooperative fashion. This is best explained by different sites of action. ATP increases cell membrane permeability while succinate stimulates uptake into mitochondria. 3. ATP was able to lower Na+ and K+ gradients and the pH gradient between cells and incubation medium. Increasing calcium concentration counteracted this effect though calcium uptake was then stimulated. 4. Succinate alone did not affect monovalent cation gradients but raised the pH gradient. It partially counteracted the ATP effects on these gradients. 5. Since catecholamine-like actions of ATP may be mediated by an increase in cytoplasmic calcium concentration, the action of extracellular ATP can be taken as a model to study the role of calcium as a transmitter of hormone actions. From interdependence between ATP-stimulated and succinate-stimulated calcium uptake, conclusions can be drawn on the resulting cytoplasmic calcium concentration and its effect on plasma membrane permeability.

Binding and transport characteristics for uptake of taurocholic acid by isolated rat liver cells were studied. 1. An adsorption of taurocholate to the cell surface is terminated in less than 15 s. A Ks of 0.55 mM and a total binding capacity of 3.8 nmol/mg cell protein is determined. 2. The rate of uptake of taurocholate follows Michaelis-Menten kinetics with Km = 19 muM and V = 1.7 nmol/mg protein min. 3. There is a broad pH optimum for uptake between pH 6.5 -- 8.0. 4. The activation energy amounts to 29 kcal/mol. At high taurocholate concentration an unusual upward bend is observed in the Arrhenius plot. 5. Taurocholate uptake is competitively inhibited by taurochenodeoxycholate (Ki = 9 muM). It is noncompetitively inhibited by bromosulfophthalein (Ki = 3 muM). 6. At physiological taurocholate concentrations a 200-fold intracellular accumulation of taurocholate is observed. 7. Uptake is inhibited by about 75% by either antimycin A, carbonylcyanide m-chlorophenyl-hydrazone, ouabain. 8. Replacement of extracellular Na+ by either K+ or sucrose results in a 75% decrease of uptake. 9. It is concluded that taurocholate uptake is a carrier-mediated process, and suggested that the energy for intracellular accumulation is made available by cotransport of Na+.

Diurnal variation of the methotrexate (MTX) initial influx and net uptake in isolated rat liver cells was studied in dependence on diurnal variation of cellular glutathione. It was found that the most significant differences concerning the MTX initial transport and accumulation were observed between hepatocytes prepared at 1200 hr when cellular glutathione reached its maximum, and those isolated at 0000 hr when liver glutathione had its minimal concentration. The initial influx of MTX was the biggest in cells isolated at 0000 hr and the smallest in cells prepared at 1200 hr. The Km values in cells with low cellular glutathione (at 1800 and 0000 hr) were about three times smaller than in cells with high glutathione (at 0700 and at 1200 hr), whereas the Vmax value remained unchanged. Titration of external membrane SH groups by [203Hg]p-CMBS revealed a much larger amount of free SH groups on cells having low glutathione level than on those with high glutathione. Despite big initial influx of MTX found in cells with low cellular glutathione, net accumulation of MTX was significantly smaller in these cells as compared with hepatocytes having high glutathione level. In conclusion, the present studies confirmed a statement of the preceding paper on the important role of cellular glutathione played in methotrexate transport and accumulation in rat liver cells.

In the isolated liver transient perfusion without calcium results in cholestasis which was characterized by an increased efflux rate across the sinusoidal membrane and inhibition of the concentrative transport of bromosulfophthalein to the canalicular side of the cell. Cholestasis could not be reversed within the usual duration of liver perfusion.

In isolated perfused rat liver, addition of adrenaline induced a complex response of bile flow including rapid, reversible stimulation (1/2-2 min), reversible inhibition (2-10 min), and prolonged stimulation. Both the reversible stimulation and the inhibition were mimicked by the alpha-sympathomimetic agonist phenylephrine but not by the beta-agonist isoproterenol. The reversible stimulation was a very early effect being terminated prior to all other alpha-adrenergic responses of liver. External ATP considerably lowered bile flow while inducing release of glucose and lactate, inhibition of respiration, and a reversible efflux of Ca2+. Variations of mannitol clearance parallel to those of bile flow indicate a canalicular origin of all changes.

1. The term "unspecific permeability" in mitochondria is defined for describing a rapid equilibration of solutes between the extramitochondrial space and a portion of the intramitochondial space. It appears to be a property of the outer membrane and is restricted only by the size of the molecule (maximal molecular weight of 5,OOO-12,OOO). 2 The unspecifically permeable space varies considerably. It ranges from 20-80% in different preparations of mitochondria. For a single mitochondrial preparation it is shown to be of equal volume for various small molecules such as sucrose, adenine nucleotides, NAD+ and aspartate. 3. The total aqueous phase of rat liver mitochondria in isotonic sucrose medium rages from 2.5-3.4 μl/mg protein; the aqueous part of the impermeable space from 0.8-1.3 &0.8-1.8 μl/mg protein. Only about 1 μl of this portion expands osmotically and represents thesolvent for endogenous mitochondrial solutes. The major part (about 0.4-0.6 μl/mg protein ) of the residual water of the impermeable space may be attributed to the hydrate shell of mitochondria are compared with the morphologically defined inter membrane and matrix space. The tonicity of the incubation medium was varied over a large range for rat liver and rat kidney mitochondria in order to facilitate this comparison. Large variations in the ratio of sucrose permeable to impermeable space (ranging from 1:3 in liver mitochondria to 3:1 in kidney mitochondria) correspond to changes in the ratio of the areas covered by the intermembrane and matrix space in the sections seen in the electron-micrographs. Mitochondria with low unspecific permeability are homogenous, mitochondria with high unspecific permeability show greater individual variability at all tonicities. [ABSTRACT FROM AUTHOR]