Inhibition by cycloserine of mitochondrial and cytosolic aspartate aminotransferase in isolated rat hepatocytes.

Isolated hepatocytes were incubated with L-cycloserine and then treated with digitonin so that mitochondrial and cytosolic fractions were obtained in 5 s. Mitochondrial and total cellular aspartate aminotransferases (EC 2.6.1.1) were inactivated in parallel. The enzyme was also inhibited in isolated mitochondria incubated with L-cycloserine. These results, in contrast with previous reports, indicate that cycloserine reacts equally with mitochondrial and cytosolic aspartate aminotransferases.     

Zinc uptake by isolated rat hepatocytes

Isolated rat hepatocytes were used to investigate the uptake of zinc at early exposure times. Hepatocytes were incubated with ⁶⁵Zn (1–500 μM) and samples were withdrawn at times ranging from 25 s to 60 min. A biphasic pattern of uptake was observed with a rapid first phase of uptake followed by a slower second phase. The relationship between velocity of uptake and substrate concentration for the first phase was nonlinear, while that of the second phase was linear. The presence of 10 μM cadmium produced a decrease in the velocity of uptake of only the first phase. This suggests that the first phase is at least partly carrier mediated, while there is no indication of involvement of a carrier in the second phase. KCN (1 mM) and carbonyl cyanide m-chlorophenylhydrazone (2 μM), did not cause any change in the uptake of ⁶⁵Zn (1 μM), which suggests that there is no active component in the uptake of zinc.     

Tert-butyl hydroperoxide selectively inhibits mitochondrial respiratory-chain enzymes in isolated rat hepatocytes

Sensitivity of various mitochondrial enzymes to oxidative damage was tested on isolated rat liver hepatocytes permeabilized by digitonin. In permeabilized hepatocytes normal respiratory control values were obtained and mitochondrial membranes remained intact. Respiratory rates of NADH-dependent (glutamate + malate, palmitylcarnitine + malate) and flavoprotein-dependent (succinate) substrates were determined in hepatocytes exposed for 5 min to 0.5-3 mM tert-butyl hydroperoxide before addition of digitonin. Our data showed that oxidation of NADH-dependent substrates is much more sensitive to oxidative stress than oxidation of flavoprotein-dependent ones, evidently due to the modification of iron-sulfur clusters or SH groups in the NADH dehydrogenase enzyme complex (Complex I).     

Glycogen autophagy in newborn rat hepatocytes

Glycogen autophagy in newborn rat hepatocytes was studied by using enzyme determinations and electron microscopy. Cyclic AMP induced glycogen autophagy in these cells. Glycogen-hydrolyzing acid glucosidase activity increased whereas acid mannose 6-phosphatase activity decreased in the liver of these animals. Parenteral glucose, which prevents postnatal glucagon secretion and tissue cyclic AMP elevation, and propranolol which antagonizes cyclic AMP, inhibited glycogen autophagy. Glucosidase activity decreased and phosphatase activity increased. These findings raise the possibility that cyclic AMP-induced autophagic mechanisms in newborn rat hepatocytes are associated with changes in the activity of acid mannose 6-phosphatase.     

Energy depletion and autophagy. Cytochemical and biochemical studies in isolated rat hepatocytes

Summary In this paper, data dealing with the sensitivity of autophagy towards partial ATP depletion in isolated rat hepatocytes are reviewed. Partial reduction of intracellular ATP causes: (1) a decrease of proteolytic flux; (2) a decrease in uptake of cytosolic components into the autophagic-lysosomal compartment; (3) either a decrease or no change in the ratio between volume densities of autophagosomes and lysosomes, depending on whether or not the cytosolic phosphate potential is affected; and (4) impairment of the lysosomal proton pump. It is concluded that the consecutive steps of autophagy all respond to relatively small changes of intracellular ATP concentration.     

Plasma protein synthesis by isolated rat hepatocytes

A system of preparation of rat hepatocytes with extended viability has been developed to study the role of hormones and other plasma components upon secretory protein synthesis. Hepatocytes maintained in minimal essential medium reduced the levels of all amino acids in the medium except the slowly catabolized amino acids leucine, isoleucine, and valine, which steadily increase as the result of catabolism of liver protein. Although the liver cells catabolize 10-15% of their own protein during a 20-h incubation, the cells continue to secrete protein in a linear fashion throughout the period. The effects of insulin, cortisol, and epinephrine on general protein synthesis, and specifically on fibrinogen and albumin synthesis, have been tested on cells from both normal rats and adrenalectomized rats. Cells from normal animals show preinduction of tyrosine amino transferase (TAT), having at the time of isolation a high level of enzyme which shows only an increase of approximately 60% upon incubation with cortisol. In contrast, cells from adrenalectomized animals initially have a low level of enzyme which increases fourfold over a period of 9 h. The effects of both epinephrine and cortisol on protein synthesis are also much larger in cells from adrenalectomized animals. After a delay of several hours, cortisol increases fibrinogen synthesis sharply, so that at the end of the 20-h incubation, cells treated with hormone have secreted nearly 2.5 times as much fibrinogen as control cells. The effect is specific; cortisol stimulates neither albumin secretion nor intracellular protein synthesis. The combination of cortisol and epinephrine strongly depresses albumin synthesis in both types of cells. Insulin enhances albumin and general protein synthesis but has little effect on fibrinogen synthesis.     

Free fatty-acid uptake by isolated rat hepatocytes

In isolated rat hepatocytes, the rate of palmitic acid binding and uptake is directly related to the concentration of free fatty acid (FFA) in the medium. After their entry into the cell, FFA are immediately incorporated into cellular phospholipids and triglycerides and no accumulation of free fatty acids can be demonstrated inside the cell. The rate of free fatty-acid uptake remains unchanged after incubation in a 2 mM KCN containing medium, indicating that in the range of fatty-acid concentrations used in this study, this phenomenon does not require energy.     

Mechanism of sucrose uptake by isolated rat hepatocytes

The transport of molecules by nonspecific endocytosis has been described in many cell types, but it has not been characterized in hepatocytes. Because of its central role in the clearance of solutes from portal blood, endocytosis might represent a significant mode of cellular transport. We investigated the mechanism of sucrose uptake in an isolated hepatocyte system. Liver cells were isolated by perfusion and collagenization of rat liver, followed by differential centrifugation. Hepatocytes were then incubated with 14C-sucrose and harvested by spinning through oil in microfuge tubes. Radioactivity was standardized against DNA content. We found that sucrose uptake is concentration-dependent from 5 microM to 100 mM and follows first-order kinetics. Washout studies indicate that exocytosis is responsible for the dynamic equilibrium reached. Arrhenius analysis of temperature dependence yields a linear plot (Ea = 14.2 Kcal/mol). In addition, sucrose uptake is independent of cellular ATP levels. We conclude that sucrose is transported by fluid-phase micropinocytosis in isolated hepatocytes and that this transport mechanism may be important in the uptake of diverse molecules into liver cells.     

Cryopreservation of isolated rat hepatocytes

Summary Isolated parenchymal hepatocytes from adult rats were frozen in media containing 10% glycerol, 10% dimethylsulfoxide (DMSO), or 20% DMSO. Three microsome-associated functions were compared in nonfrozen cells and cells frozen in each of the above cryoprotectant solutions. Freezing in DMSO maintains cytochromes P-450 and b₅ and NADPH-cytochrome C reductase at levels nearer to control values than does freezing in glycerol. Cells frozen and subsequently thawed and cultured for 24 h lose a greater amount of cytochrome P-450 than do nonfrozen cultured cells. The levels of cytochrome b₅ and reductase in frozen-thawed cells remain close to control values. Cell viability (trypan blue dye exclusion and percentage of attached cells) after freezing is maintained better using DMSO as a cryoprotectant. Dimethylsulfoxide protects the hepatocytes from freeze-induced damage to the extent that many viable cells attach to collagen-coated petri dishes, survive for at least 24 h, and still maintain significant levels of enzymes of importance to drug and carcinogen metabolism. This work was supported by Grant CA-30241 from the National Institutes of Health, Bethesda, Maryland.     

Cholesterogenesis and related enzymes in isolated rat hepatocytes during pre- and postnatal life

Cholesterogenesis pathway during pre- and postnatal development was studied in isolated rat hepatocytes. No modified activity of cytosol acetoacetyl coenzyme A (CoA), thiolase, or 3-hydroxy-3-methylglutaryl CoA (HMGCoA) synthase was detectable at the different stages examined. Minimal levels of 1(14)C-acetate incorporation into cholesterol and HMGCoA reductase activity were present at 16 days of fetal development in newborn and suckling rats, whereas both parameters increased rapidly before birth. The pattern of NaF nonsuppressible reductase activity showed a different activation state of the enzyme, suggesting the appearance of a modulation state, probably related to the development of some short-term regulatory mechanisms.     

Induction of cytosolic aspartate aminotransferase by glucagon in primary cultured rat hepatocytes

The activity and the mRNA content of cytosolic aspartate aminotransferase (EC 2.6.1.1) were examined in cultured rat hepatocytes. Addition of glucagon (1 x 10(-7) M) in the presence of dexamethasone (1 x 10(-7) M) caused about 2-fold increase in the activity and mRNA content. Dibutyryl cAMP (1 x 10(-4) M) could replace glucagon for this effect. Maximal induction of cytosolic aspartate aminotransferase mRNA was observed 8 h after their additions. Insulin (1 x 10(-7) M) did not inhibit the enzyme induction by glucagon or dibutyryl cAMP. These results suggest that the cytosolic aspartate aminotransferase gene is regulated by cAMP, and not by insulin.     

Synthesis and secretion of ceruloplasmin by isolated rat hepatocytes

The synthesis and secretion of ceruloplasmin (Cp) by isolated rat hepatocytes were investigated. Cp released by liver cells appeared to have properties similar to those of the blood-circulating protein, i. e. Mr, oxidase activity, immunological specificity and the peptide set of tryptic fingerprints. The polypeptides with Mr of 130,000, 65,000, 48,000 and 18,000 were revealed in Cp isolated from the incubation medium. These results suggest the susceptibility of the single-chain protein molecule (Mr 130,000) to limited proteolysis which is accomplished by the proteases released from the cells. When fresh serum was added to the incubation medium, the proteolytic degradation of Cp proceeded at a much slower rate, which led to an increase in the content of excreted polypeptides with Mr 130,000. The secretion was strongly diminished by the addition of colchicine to the medium. The time of Cp molecule synthesis on membrane-bound polyribosomes (3.5 min) was determined.     

Metabolic activation of 3-hydroxyanisole by isolated rat hepatocytes

A tyrosinase-directed therapeutic approach for malignant melanoma therapy uses the depigmenting phenolic agents such as 4-hydroxyanisole (4-HA) to form cytotoxic o-quinones. However, renal and hepatic toxicity was reported as side effects in a recent 4-HA clinical trial. In search of novel therapeutics, the cytotoxicity of the isomers 4-HA, 3-HA and 2-HA were investigated. In the following, the order of the HAs induced hepatotoxicity in mice, as measured by increased in vivo plasma transaminase activity, or in isolated rat hepatocytes, as measured by trypan blue exclusion, was 3-HA > 2-HA > 4-HA. Hepatocyte GSH depletion preceded HA induced cytotoxicity and a 4-MC-SG conjugate was identified by LC/MS/MS mass spectrometry analysis when 3-HA was incubated with NADPH/microsomes/GSH. 3-HA induced hepatocyte GSH depletion or GSH depletion when 3-HA was incubated with NADPH/microsomes was prevented by CYP 2E1 inhibitors. Dicumarol (an NAD(P)H: quinone oxidoreductase inhibitor) potentiated 3-HA- or 4-methoxycatechol (4-MC) induced toxicity whereas sorbitol (an NADH generating nutrient) greatly prevented cytotoxicity indicating a quinone-mediated cytotoxic mechanism. Ethylendiamine (an o-quinone trap) largely prevented 3-HA and 4-MC-induced cytotoxicity indicating that o-quinone was involved in cytotoxicity. Dithiothreitol (DTT) greatly reduced 3-HA and 4-MC induced toxicity. The ferric chelator deferoxamine slightly decreased 3-HA and 4-MC induced cytotoxicity whereas the antioxidants pyrogallol or TEMPOL greatly prevented the toxicity suggesting that oxidative stress contributed to 3-HA induced cytotoxicity. In summary, ring hydroxylation but not O-demethylation/epoxidation seems to be the bioactivation pathway for 3-HA in rat liver. The cytotoxic mechanism for 3-HA and its metabolite 4-MC likely consists cellular protein alkylation and oxidative stress. These results suggest that 3-HA is not suitable for treatment of melanoma.     

Uptake and metabolism of S-adenosyl-L-methionine by isolated rat hepatocytes

In the present study, direct evidence is given to SAMe capability of crossing the membrane of isolated rat hepatocytes. The kinetics of SAMe uptake is biphasic: a fast phase being completed in less than 15 sec and a slower one with an apparent K_m of 8.33 μM and a V_max of 10.6 pmol/min/mg protein. Both processes are pH and temperature dependent. Analysis of the fast phase by a Scatchard plot discloses two sets of binding sites of high and low affinity, respectively. Experiments carried out incubating isolated hepatocytes with double-labelled SAMe (methyl-³H, carboxyl-¹⁴C) have shown that about 70% of SAMe uptake by the cell is rapidly decarboxylated.     

Subcellular distribution of enzymes determined by rapid digitonin fractionation of isolated hepatocytes

Conditions were determined for rapid separation of cytosolic and mitochondrial compartments by digitonin fractionation of rat hepatocytes. The minimum time required for separation of mitochondrial and cytosolic enzyme markers decreased rapidly with increasing temperature. Kyro EOB, a non-ionic detergent, increases the release of cytosolic enzymes, particularly at lower temperatures. Experimental procedures are described for greater than 90% release of cytosolic enzymes and less than 2% release of mitochondrial enzymes in 3s. By using appropriate concentrations of digitonin and Kyro EOB in a fractionation medium maintained at 1°C and a minimum time of exposure to the medium, nearly separate patterns of release were obtained for enzyme markers for the cytosol, mitochondrial matrix and mitochondrial intermembrane space. The distribution of enzymes that exist in more than one of these compartments was quantified by comparing their rates of release with those of marker enzymes. The cytosol/mitochondrial-matrix distributions for such enzymes in hepatocytes from starved rats were 16%/84% for aspartate aminotransferase, 34%/66% for fumarase and 77%/23% for ATP citrate lyase. In hepatocytes from rats that were induced to synthesize ATP citrate lyase by starvation and re-feeding, the ratio had increased to 95%/5%. The maximum cytosol/intermembrane-space ratio for adenylate kinase was 8%/92%. A procedure is also described for treating commercial digitonin that increases its solubility in water from about 1mg/ml to more than 800mg/ml.     

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.