Autophagic sequestration of [14C]sucrose introduced into isolated rat hepatocytes by electrical and non-electrical methods

Isolated rat hepatocytes were found to become permeable to [14C]sucrose at 0 degree C under three different conditions: Immediately following their liberation from the collagenase-perfused liver. Following a short incubation under hypoxic conditions. After electropermeabilisation. All three conditions were characterised by the formation of small protuberances (blebs) indicative of localised cell surface damage, and it is possible that the stretched plasma membrane of such blebs acted as a high-permeability region. Disappearance of blebs and restoration of normal plasma membrane impermeability could be achieved by a short (15 min) incubation at 37 degrees C. It could be shown that [14C]sucrose introduced into rat hepatocytes by non-electrical means was autophagically sequestered at the same rate as [14C]sucrose introduced electrically. In both cases the sequestration was inhibited by the specific autophagy inhibitor 3-methyladenine to a similar extent. The subcellular distribution of sequestered isotope in metrizamide/sucrose density gradients was found to be independent of the conditions of its introduction into cells.     

Protection from hypoxic injury in cultured hepatocytes by glycine,alanine, and serine

Summary Isolated hepatocytes from rat liver in primary culture rapidly lost viability under hypoxic conditions. In the presence of glycine, L-alanine or L-serine loss of viability under hypoxic conditions was greatly retarded. Glycine and L-serine already showed significant protection from hypoxic injury at a concentration of 0.1 mM; at 10 mM, all three amino acids offered almost complete protection. Beside these standard amino acids, 1-aminocyclopropane-1-carboxylic acid (ACPC) and sarcosine significantly decreased hypoxic injury of the hepatocytes, although to a lesser extent. Other amino acids tested provided only slight protection or had no effect on hypoxic injury of the hepatocytes. In the presence of the protective amino acids neither the ATP content nor the lactate production of the hypoxic hepatocytes were significantly affected. The addition of glycine, L-alanine and L-serine led to marked membrane alterations (blebs). These alterations, however, occurred without loss of viability and were reversible upon reoxygenation after up to 4 h of hypoxia.Abbreviations LDH lactate dehydrogenase - ACPC 1-amino-cyclopropane-1-carboxylic acid - HEPES 2-(4-(2-hydroxyethyl)-1-piperazinyl)-ethanesulfonic acid     

Autophagic sequestration of [C]sucrose introduced into isolated rat hepatocytes by electrical and non-electrical methods

Isolated rat hepatocytes were found to become permeable to [¹⁴C]sucrose at 0 °C under three different conditions:

1. 1. Immediately following their liberation from the collagenase-perfused liver.

2. 2. Following a short incubation under hypoxic conditions.

3. 3. After electropermeabilisation.

All three conditions were characterised by the formation of small protuberances (blebs) indicative of localised cell surface damage, and it is possible that the stretched plasma membrane of such blebs acted as a high-permeability region. Disappearance of blebs and restoration of normal plasma membrane impermeability could be achieved by a short (15 min) incubation at 37 °C.It could be shown that [¹⁴C]sucrose introduced into rat hepatocytes by non-electrical means was autophagically sequestered at the same rate as [¹⁴C]sucrose introduced electrically. In both cases the sequestration was inhibited by the specific autophagy inhibitor 3-methyladenine to a similar extent. The subcellular distribution of sequestered isotope in metrizamide/sucrose density gradients was found to be independent of the conditions of its introduction into cells.     

Protein-catabolic state of isolated rat hepatocytes

Isolated rat hepatocytes in suspension are in a protein-catabolic state (negative nitrogen balance), as measured by the continuous release of nitrogen in the form of amino acids and urea. The nitrogen loss corresponds to a protein degradation rate of 3–4% per h, while the rate of protein synthesis is negligible. Cells prepared from fasted, fed ot regenerating livers are all highly protein-catabolic.The nitrogen balance is unaffected by insulin or amino acids (physiological mixture), and various metabolites and sera have only moderate effects. However, incubation of the cells for 2–4 h in a tissue culture medium (Dulbecco's) reduces the nitrogen loss dramatically, suggesting the formation of an anticatabolic factor under these conditions.     

The metabolism of halothane by hepatocytes: a comparison between free radical spin trapping and lipid peroxidation in relation to cell damage

The technique of free radical spin trapping has been applied to demonstrate the formation of free radicals produced during the metabolism of halothane by rat liver hepatocytes under hypoxic conditions. The results obtained support previous findings that reported sex differences in the metabolic activation of halothane by rats in vivo. Cell viability under hypoxic conditions, as judged by trypan blue staining and lactate dehydrogenase release, shows a correlation with the extent of metabolism of halothane as measured by electron spin resonance spectroscopy. The extent of lipid peroxidation was measured by diene conjugation, malondialdehyde production and chemiluminescence. The latter technique allowed the demonstration of lipid peroxidation during incubations of hepatocytes under aerobic conditions. The magnitude of the aerobic chemiluminescence showed a similar sex dependency to the extent of free radical formation under hypoxic conditions. Cell viability measurements show that halothane metabolism in both hypoxic and aerobic conditions can lead to cell death. Consequently, oxidative lipid damage could be a cause of cell damage, as judged by cell viability, additional to covalent binding.     

Metabolism of aqueous soluble nitroxides in hepatocytes: effects of cell integrity, oxygen, and structure of nitroxides

The optimum use of nitroxides in viable biological systems, including live animals, requires knowledge of the metabolism of nitroxides by major organ systems, especially the liver. We report here details of the metabolism of several prototypic aqueous soluble nitroxides in suspensions of freshly isolated hepatocytes. The general patterns of metabolism were similar to those observed in other types of cells (previous studies have been done principally in cells from tissue culture, such as CHO cells) including the primary initial reaction being reduction to the hydroxylamine, an increased rate of metabolism of some nitroxides in hypoxic cells, faster rates of reduction of nitroxides on six-membered piperidine rings compared to five-membered pyrrolidine rings, and most metabolism being intracellular. Metabolism in hepatocytes differed from other cell lines in having (1) significant reduction in the extracellular medium due to ascorbate that was released from damaged hepatocytes; (2) decreased rates of metabolism in freeze-thawed cells due to damage to subcellular organelles. These results provide much of the data needed to understand the role of the liver in the metabolism of nitroxides by intact animals and explain some previously puzzling results which indicated an apparent unusually high rate of metabolism of a charged nitroxide (Cat1) by hepatocytes. Our results also indicate that the use of freshly isolated cells or tissue homogenates may introduce experimental artifacts in the study of the metabolism of nitroxides.     

Growth potential of adult hepatocytes in mammals: highly replicative small hepatocytes with liver progenitor-like traits

The liver is one of the few organs that is capable of completely regenerating itself without using a stem cell population. When damaged, growth factors and cytokines are released, stimulating terminally differentiated adult hepatocytes and making them re-enter the cell cycle. We have been developing a series of studies on the growth potential of rat and human hepatocytes to identify a population of hepatocytes that is responsible for the regeneration of the injured liver. For this purpose, we established an appropriate culture method for hepatocytes by which growth and differentiation capacities are practically examined under various experimental conditions. This in vitro assay system allows us to identify small hepatocytes that are prominently replicative compared to large hepatocytes. Non-parenchymal cells play critical roles in the proliferation of small hepatocytes. These hepatocytes are present in both rat and human liver and are located in portal regions there. Phenotypic features were examined at morphological and gene/protein levels in detail, which showed the phenotypic plasticity in vitro. Mammalian liver includes a population of small hepatocytes in normal adults with a minute occupancy rate. We speculate that small hepatocytes play a role in regenerating the injured liver and in compensating for apoptotic hepatocytes in the physiological turnover of hepatocytes.     

A simplified model of hypoxic injury in primary cultured rat hepatocytes

Summary The Anaeropack system for cell culture, which was originally designed for the growth of anaerobic bacteria, was used to produce a hypoxic atmosphere for cultured hepatocytes. We measured changes in the oxygen and carbon dioxide concentrations and the atmospheric temperature in an airtight jar. We also measured changes in the pH of the medium during hypoxia to assess the accuracy of this system. Moreover, we used three durations (2, 3, and 4 h) of hypoxia and 8 h of reoxygenation in cultured rat hepatocytes, and then measured the lactate dehydrogenase (LDH), ketone body concentration (acetoacetate + β-hydroxybutyrate), and the ketone body ratio (KBR: acetoacetate/β-hydroxybutyrate) in the medium in order to assess the suitability of this system as a model for reperfusion following liver ischemia. The oxygen concentration dropped to 1% or less within 1 h. The concentration of carbon dioxide rose to about 5% at 30 min after the induction of the hypoxic conditions, and was maintained at this level for 5 h. No effect of the reaction heat produced by the oxygen absorbent in the jar was recognized. The extent of cell injury produced by changing the hypoxic parameters was satisfactorily reflected by the KBR, the ketone body concentration, and the LDH activity released into the medium. Because this model can duplicate the conditions of the hepatocytes during revascularization following ischemic liver, and the Anaeropack system for cell culture is easy to manipulate, it seems suitable for the experimental study of hypoxic injury and revascularization in vitro.     

Correlation between plasma membrane surface area and transferrin secretion rate in isolated hepatocytes

It is generally considered that in exocytosis the size of the secreting cells does not increase when the membranes of exocytosis vesicles fuse with the plasma membrane. As the factors involved in the regulation of this phenomenon are poorly understood, we thought it worthwhile to investigate the relationship between the plasma membrane surface area and secretory activity. Isolated rat hepatocytes were prepared by liver collagenase perfusion. Secretion of the plasma protein, transferrin (Tf) was detected at the single cell level with specific anti-rat transferrin antibodies using the reverse hemolytic plaque test. Hepatocyte surface and hemolytic ring surface areas were calculated from diameters of hepatocyte and hemolytic plaque measured after 5h of incubation. A highly significant correlation was established between the plaque-forming hepatocyte surface areas and the corresponding hemolytic surface areas. This result was confirmed using an automatic image analysis method. Two-month-old rats were compared to 4-month-old rats. We observed that the ratio of the quantity of transferrin secreted by hepatocytes to the hepatocyte surface area was constant for a given incubation time, whatever the size of the hepatocytes. These results suggest that the plasma membrane surface area of hepatocytes may constitute a limiting factor in Tf secretion.     

FK506, but not cyclosporin A, prevents mitochondrial dysfunction during hypoxia in rat hepatocytes.

Hepatic ischemia/reperfusion injury occurs in the clinical situations including liver transplantation. FK506 and cyclosporin A (CsA) are reported to be hepatotrophic agents in addition to being a powerful immunosuppressive agent. Studies were performed to determine whether the drugs influence a mitochondrial dysfunction under the hypoxic conditions in primary culture model of rat hepatocytes. The Anaeropack system was used for cell culture to create a hypoxia. Cells were treated with FK506 or CsA under the normoxic and hypoxic conditions. Hypoxia markedly decreased intracellular adenosine 5'-triphosphate (ATP) contents and the ketone body ratio (KBR, acetoacetate/beta-hydroxybutyrate) in culture medium as compared with normoxia. FK506 prevented the decreases of ATP contents and the KBR. In contrast, CsA had no effect on either ATP contents or the KBR. FK506, but not CsA, increased the KBR under the normoxic conditions. Under the hypoxic conditions, heat shock protein 70 (Hsp70) was detected after reoxygenation. FK506 enhanced the induction of Hsp70, but CsA again had no effect on Hsp70 induction. These results indicate that FK506 protects the hypoxia injury in part by preventing the mitochondrial dysfunction in concert with the enhancement of heat shock response in hepatocytes.     

Polarised membrane traffic in hepatocytes

The liver was used widely in early studies of polarised transport but has been largely overlooked in recent years, mostly because of the development of epithelial cell lines which provide more tractable experimental systems. The majority of membrane proteins and lipids reach the hepatocyte apical membrane by transcytosis and it remains unclear whether there is a direct route for apical targeting, although the pathways present have yet to be fully characterised. The recent development of systems that allow hepatocyte transport processes to be studied in culture and the observation that transcytosis can be significantly stimulated under physiological conditions suggest that hepatocytes have a role to play in future studies of polarised transport. This review discusses the known features of polarised membrane traffic in hepatocytes and contrasts them with the characteristics of vesicular transport in other epithelial cell types.     

Evaluation of isolated human hepatocytes

This preliminary study reports the functional capacities of freshly isolated human hepatocytes in regard to their energetic metabolism and monooxygenase activities. Incubated for 30 or 60 min, isolated cells maintain their membrane integrity, AIP and reduced glutathione content and redox potential estimated by means of lactate to pyruvate and ß-hydroxypyruvate to acetoacetate ratios. Three monooxygenase activities, supported by different isoenzymes of cytochrome P30 are determined by the accumulation of unconjugated metabolites: their relative magnitudes are similar to those observed in microsomes, indicating a good preservation of hydroxylase activities during cell isolation and incubation. Although incubations did not exceed 60 min, one can conclude that human hepatocytes maintain their viability and metabolic capacities after isolation and might be considered in transplantation process for the treatment of acute hepatic failure. Isolated human hepatocytes might be also used as a tool for studying biochemical and toxicological effects of a drug.     

Prolongation of liver-specific function for primary hepatocytes maintenance in 3D printed architectures

Isolated primary hepatocytes from the liver are very similar to in vivo native liver hepatocytes, but they have the disadvantage of a limited lifespan in 2D culture. Although a sandwich culture and 3D organoids with mesenchymal stem cells (MSCs) as an attractive assistant cell source to extend lifespan can be used, it cannot fully reproduce the in vivo architecture. Moreover, long-term 3D culture leads to cell death because of hypoxic stress. Therefore, to overcome the drawback of 2D and 3D organoids, we try to use a 3D printing technique using alginate hydrogels with primary hepatocytes and MSCs. The viability of isolated hepatocytes was more than 90%, and the cells remained alive for 7 days without morphological changes in the 3D hepatic architecture with MSCs. Compared to a 2D system, the expression level of functional hepatic genes and proteins was higher for up to 7 days in the 3D hepatic architecture. These results suggest that both the 3D bio-printing technique and paracrine molecules secreted by MSCs supported long-term culture of hepatocytes without morphological changes. Thus, this technique allows for widespread expansion of cells while forming multicellular aggregates, may be applied to drug screening and could be an efficient method for developing an artificial liver.     

Variable responses of small and large human hepatocytes to hypoxia and hypoxia/reoxygenation (H-R)

Hypoxia and hypoxia-reoxygenation (H-R) regulate human hepatocyte cell death by mediating the accumulation of reactive oxygen species (ROS). Hepatocytes within the liver are organised into peri-portal (PP) and peri-venous (PV) subpopulations. PP and PV hepatocytes differ in size and function. We investigated whether PP and PV human hepatocytes exhibit differential susceptibility to hypoxic stress. Isolated hepatocytes were used in an in vitro model of hypoxia and H-R. ROS production and cell death were assessed using flow cytometry. PV, and not PP hepatocytes, accumulate intracellular ROS in a mitochondrial dependent manner during hypoxia and H-R. This increased ROS regulates hepatocyte apoptosis and necrosis via a mitochondrial pathway. These findings have implications on the understanding of liver injury and application of potential therapeutic strategies.     

Postthaw viability of precultured hepatocytes

Hepatocytes are being studied for a wide variety of applications, including drug metabolism studies, gene therapy, and use in liver-assist devices for temporary liver support. The ability to cryopreserve isolated hepatocytes would permit the pooling of cells to reach the required therapeutic coordination of the cell supply with patient care regimes and the completion of safety and quality-control testing. The objective of this investigation was to develop a method of cryopreserving isolated hepatocytes that will retain high levels of function and facilitate the use of the cells in different applications. Freshly isolated hepatocytes were cultured in a spinner flask for different periods of time, up to 48 h. The cells were cryopreserved by use of a range of solution concentrations and cooling rates. For fresh, nonfrozen hepatocytes precultured for 24 h prior to being plated on collagen, the albumin secretion rate was 0.88 +/- 0.62 mg/ml/h. When the cells were precultured for 24 h, frozen in a solution containing 10% Me2SO with a cooling rate of 1 degrees C/min, thawed, plated on collagen, and cultured, the albumin secretion rate was 0.21 +/- 0.24 microg/ml/h. In contrast, freshly isolated hepatocytes cryopreserved without preculture and cultured on collagen had an albumin secretion rate of 0.07 +/- 0.08 mg/ml/h. The influences of different solution compositions and cooling rates on postthaw function of precultured hepatocytes were also determined. These results indicate that the use of a preliminary culture step prior to cryopreservation can enhance the postthaw function of hepatocytes.     

The effects of bioregulators upon amino acid transport and protein synthesis in isolated rat hepatocytes

Isolated rat hepatocytes prepared by an enzyme perfusion technique possess a functional amino acid transport system and retain the capacity to synthesize protein. Amino acid transport was studied using the non-metabolizable amino acid analog alpha-aminoisobutyric acid. The transport process was time, temperature and concentration dependent. Similarly, leucine incorporation into protein was time and temperature dependent being optimal at 3m degrees C. Amino acid, fetal calf serum, growth hormone and glucose all produced small, reproducible increases in protein synthesis rates. Bovine serum albumin diminished the uptake of alpha-aminoisobutyric acid and leucine incorporation into protein. The amino acid content on either side of the cell membrane was found to affect transport into or out of the cellular compartment (transconcentration effects). High cell concentrations decreased transport and protein synthesis as a result of isotopic dilution of labelled amino acids with those released by the hepatocytes. This was consistent with the capacity of naturally occurring amino aicds to compete with alpha-aminoisobutyric acid for uptake into the hepatocyte. In order to define more precisely the effects of bioregulators on transport and protein synthesis it will be necessary to define and subfractionate cellular compartments and proteins which are the specific targets of cellular regulation.     

Spontaneous51Cr release by isolated rat hepatocytes: An indicator of membrane damage

Summary Radiochromium uptake and release by isolated rat hepatocytes in suspension was monitored under continuous-labeling conditions. Cell protein remained unchanged during the absorption phase, whereas the release of⁵¹Cr correlated well with the loss of cell viability and release of cytoplasmic protein. The results suggest that under equilibrium conditions,⁵¹Cr release represents an efflux of label from damaged or dying preparations and not an elution of radioisotope from intact cells.     

Enhanced carrier-mediated lactate entry into isolated hepatocytes from starved and diabetic rats

Hepatic plasma membrane lactate transport was studied in isolated hepatocytes prepared from fed, starved, and streptozotocin diabetic rats. Carrier-mediated lactate entry was determined using the lactate transport inhibitors alpha-cyano-3-hydroxycinnamate and D-3-hydroxybutyrate and was significantly greater in hepatocytes from starved compared to fed rats and in hepatocytes from diabetic fed compared to fed rats. The saturable component of lactate entry which corresponds to carrier-mediated transport was higher in the starved than in the fed state with results from diabetic fed being intermediate between the two. Insulin treatment prevented the increment in carrier-mediated lactate transport observed in hepatocytes from diabetic fed rats. The data indicate that hepatic plasma membrane lactate transport is increased under conditions of starvation and diabetes mellitus. This may partly explain the increased gluconeogenic flux under these conditions.     

Glutathione depletion by 2-nitroimidazole-1-acetohydroxamic acid as a radiosensitizer in hypoxic rat hepatocytes

The effects of nitroimidazoles as radiosensitizers on intracellular glutathione (GSH) level were investigated in rat isolated hepatocytes. Dinitroimidazoles have lowered almost completely GSH level during the incubation for 30 min under oxic (95% O2+5% CO2) condition, while mononitroimidazoles had scarcely affected. In the case of hypoxic (95% N2+5% CO2) condition, however, 2-nitroimidazoles, not 4-nitroimidazoles, as well as 2,4- and 4,5-dinitroimidazoles have caused the significant depletion of GSH. This suggests that nitro group in the 2-position of imidazoles may be responsible for the GSH depletion under hypoxia. Especially, 2-nitroimidazole-1-acetohydroxamic acid (KIH-801) was found to be the most potent GSH depletor only under hypoxic, not oxic conditions, and might be useful for the new hypoxic cell radiosensitizer instead of misonidazole.     

Paracetamol-stimulated lipid peroxidation in isolated rat and mouse hepatocytes

Treatment of isolated hepatocytes from 3-methylcholanthrene induced rats with 1 mM paracetamol has been found to greatly decrease cellular reduced glutathione (GSH) content and to promote lipid peroxidation, evaluated as malonaldehyde (MDA) production and conjugated diene absorbance. A similar dosing of hepatocytes from phenobarbital-induced or normal rats is ineffective in that respect. On the other hand, the aspecific stimulation of the cytochrome P-450-mediated paracetamol activation due to acetone addition further increases GSH depletion as well as MDA production.Isolated hepatocytes with basal low GSH content are also more susceptible to paracetamol-induced lipid peroxidation, indicating that the rate of the drug metabolism and the cellular GSH content are critical factors in the determination of such peroxidative attack.In isolated mouse liver cells paracetamol does not require preliminary cytochrome P-450 induction to stimulate MDA formation, even at concentrations ineffective in rat cells.However, 5 mM paracetamol, despite a great depletion of cellular GSH content, does not promote MDA formation either in the rat or in the mouse hepatocytes. This effect may be due to the ability of paracetamol to scavenge lipid peroxides under defined conditions, as tested in various lipid peroxidizing systems.Membrane leakage of lactate dehydrogenase (LDH) is evident in paracetamol treated cells undergoing lipid peroxidation, but not when MDA formation is inhibited by high doses of the drug or by addition of antioxidants such as α-tocopherol and diphenylphenylenediamine (DPPD).Nevertheless in these conditions the covalent binding of activated paracetamol metabolites is not affected, suggesting that lipid peroxidation might play a role in the pathogenesis of liver damage following paracetamol overdose.