A morphologic and morphometric study of the mitochondria in several hepatoma cell lines and in isolated hepatocytes.

Some characteristics of the mitochondria of hepatocytes and of three hepatoma cell lines have been compared. By means of stereologic analysis of electron micrographs of cross-sections through cells the volume of mitochondria per unit volume of cell cytoplasm and the surface areas of the mitochondrial envelope and cristae membranes have been measured. The relative mitochondrial volume in the cytoplasm decreases with increasing growth rate but the surface area of outer and cristae membranes per unit volume of mitochondria is not altered. The internal organization of hepatoma mitochondria, however, differs distinctly from that of normal liver mitochondria as evident from electron micrographs; the hepatoma cells contain mitochondria in which parallel cristae appear to cross the whole mitochondrial profile unlike the irregular, short cristae seen in normal liver mitochondria. Furthermore, in the fast-growing hepatoma cells the mitochondrial matrix appears less dense than in the hepatocyte. Hepatoma cells contain less organized rough endoplasmic reticulum than normal liver cells and the spatial relationship of the mitochondria to the rough cisternae, seen in the hepatocyte, is absent in the fast-growing hepatoma cell lines. It is concluded that hepatoma cells have fewer mitochondria than normal liver cells, but that the organelles have a normal content of inner membranes.     

Submicromolar Ca2+ regulates phosphorylating respiration by normal rat liver and AS-30D hepatoma mitochondria by different mechanisms

The stimulation of 2-oxoglutarate and NAD(+)-isocitrate dehydrogenase by Ca2+ in mitochondria from normal tissues has been proposed to mediate partially the activation of oxidative energy metabolism elicited by physiological elevations in cytosolic Ca2+. This mode of regulation may also occur in tumor cells in which several aspects of mitochondrial metabolism are known to be altered. This study provides a comparison of the stimulation by submicromolar concentrations of Ca2+ on the rates of ATP-generating (state 3) respiration under physiologically realistic conditions by mitochondria isolated from normal rat liver and from highly malignant rat AS-30D ascites hepatoma cells. The K0.5 for activation of glutamate-dependent state 3 respiration by Ca2+ in the presence of ATP at 37 degrees C was determined to be 0.70 +/- 0.05 (S.E.) microM for hepatoma mitochondria and 0.90 +/- 0.03 microM for rat liver mitochondria. This activation was also reflected by a Ca2(+)-induced shift in the oxidation-reduction state of hepatoma mitochondrial pyridine nucleotides to a more reduced level and Ca2+ stimulation of 14CO2 production from [1-14C]glutamate. Whereas the Ca2+ sensitivity of state 3 respiration by hepatoma mitochondria can be explained by the activation of 2-oxoglutarate and possibly NAD(+)-isocitrate dehydrogenases, the Ca2+ sensitivity of liver mitochondrial respiration appears to be predominantly mediated by activation of electron flow through ubiquinone and Complex III of the electron transport chain, as indicated by the specificity of the effects of Ca2+ on respiration with different oxidizable substrates. Although rat liver and hepatoma mitochondria employ different modes of Ca2(+)-activated ATP generation, these results support the hypothesis that changes in cytosolic Ca2+ play a significant role in the potentiation of energy production in tumor, as well as normal tissue.     

Inhibition of respiration in mitochondria and in digitonin-treated rat hepatocytes by podophyllotoxin

Summary The effects of the microtubular inhibitor, podophyllotoxin, on mitochondrial respiration were determined using isolated, digitonin-permeabilized hepatocytes and isolated mitochondria. In hepatocytes, podophyllotoxin (1.5 mM) inhibited coupled and uncoupled respiration of both FAD and NAD-linked substrates. In mitochondria, podophyllotoxin inhibited State III respiration, prevented the return to State IV respiration, and inhibited uncoupled respiration. There was no inhibition of ascorbate/TMPD oxidation in either the hepatocytes or the mitochondria. Podophyllotoxin had no effect upon oligomycin inhibition of coupled respiration. Oligomycin had no effect on the podophyllotoxin-inhibition of uncoupled respiration in either hepatocytes or mitochondria. The results indicate that podophyllotoxin alters electron flow at a site early in the electron transport chain.     

The role of mitochondria in modifying the cellular ionic environment. Calcium-induced respiratory activities in mitochondria isolated from various tumour cells

Cyclic stimulation by Ca(2+) of respiration in mitochondria isolated from Ehrlich ascites-tumour cells occurs only when low phosphate concentrations (approx. 0.5mm) are also included in the incubation system. Under these circumstances the extra oxygen consumed is related stoicheiometrically to the amount of Ca(2+) taken up by the mitochondria; the values are similar to those obtained with mitochondria from rat liver in the absence of added phosphate. In contrast with liver mitochondria, up to 280nmol of Ca(2+)/mg of protein can be added to ascites mitochondria without causing any deleterious effect. Respiration in mitochondria isolated from the Yoshida ascites hepatoma (HA 130) and from the Morris hepatomas 5123C and 9618A is also stimulated by Ca(2+) in a cyclic manner. However, that in mitochondria from regenerating rat liver responds to Ca(2+) in the same way as those from normal rat liver. ADP-stimulated respiration in mitochondria from Ehrlich ascites tumour cells, but not from rat liver, is inhibited by low amounts of Ca(2+).     

Mitochondrial thymidine kinase and ribonucleotide reductase from rat liver and rat hepatoma 27]

Fractions of heavy and light mitochondria are isolated from homogenates of homologous rat tissues (intact liver, regenerating liver within 24 hours after hepatectomy and 27 hepatoma) by means of differential centrifugation. It is found that tumour mitochondria have higher heterogeneity and lower buyoant density than mitochondria from normal hepatocytes. The activity of two enzymes of DNA precursors synthesis (ribonucleotide reductase and thymidine kinase) in subcellular fractions is demonstrated to correlate with the tissue growth rate. A single injection of cyclic AMP into hepatectomised rats resulted in the retardation of the regeneration process, and the activity of both enzymes reached its normal level in all the fractions studied after 24 hours after the operation. Thymidine kinase and ribonucleotide reductase are located mainly in the mitochondrial matrix, however, pronounced enzyme activity is observed also in membrane fractions. The activity of the enzymes in the fraction of external mitochondria membranes in rapidly growing tissues is 2--3 times as high as in the same fraction from normal rat liver.     

Molecular mechanism for the selective impairment of cancer mitochondrial function by a mitochondrially targeted vitamin E analogue

The effects of α-tocopheryl succinate (α-TOS), α-tocopheryl acetyl ether (α-TEA) and triphenylphosphonium-tagged vitamin E succinate (mitochondrially targeted vitamin E succinate; MitoVES) on energy-related mitochondrial functions were determined in mitochondria isolated from AS-30D hepatoma and rat liver, bovine heart sub-mitochondrial particles (SMPs), and in rodent and human carcinoma cell lines and rat hepatocytes. In isolated mitochondria, MitoVES stimulated basal respiration and ATP hydrolysis, but inhibited net state 3 (ADP-stimulated) respiration and Ca(2+) uptake, by collapsing the membrane potential at low doses (1-10μM). Uncoupled mitochondrial respiration and basal respiration of SMPs were inhibited by the three drugs at concentrations at least one order of magnitude higher and with different efficacy: MitoVES>α-TEA>α-TOS. At high doses (>10μM), the respiratory complex II (CII) was the most sensitive MitoVES target. Acting as an uncoupler at low doses, this agent stimulated total O(2) uptake, collapsed ?ψ(m), inhibited oxidative phosphorylation and induced ATP depletion in rodent and human cancer cells more potently than in normal rat hepatocytes. These findings revealed that in situ tumor mitochondria are preferred targets of the drug, indicating its clinical relevance.     

The effect of glucagon on hepatic respiratory capacity

Data from numerous laboratories show that mitochondria isolated from livers treated acutely with glucagon have higher rates of state 3 respiration than control mitochondria. The purpose of the present study was to learn whether this phenomenon is an isolation artifact resulting from a stabilization of the mitochondrial membrane or whether it represents a real increase in the maximal respiratory capacity of liver cells due to glucagon treatment. Electron transport was measured through different spans of the electron transport chain by using ferricyanide as an alternate electron acceptor to O2. With isolated intact liver mitochondria, pretreatment with glucagon was found to cause an increase in electron flow, through both Complex I and Complex III, suggesting that the effect of glucagon was not specific for a single site in the electron transport chain. Using intact isolated hepatocytes, different results are obtained. Respiration was measured in isolated hepatocytes after quantitation of the hepatocyte mitochondrial content by assay of citrate synthase. Hepatocyte respiration could therefore be reported per mg of mitochondrial protein. By providing durohydroquinone to the cells, it was possible to measure electron flow from coenzyme Q to O2 in the absence of the physiological regulation of substrate supply. Likewise, the addition of carbonyl cyanide p-trifluoromethoxyphenylhydrazone released the in situ mitochondria from control by the cytosolic ATP/ADP ratio and it was possible to measure maximal electron flow rates through Complex III. In the presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone, electron flow was higher in mitochondria in the cell than in isolated mitochondria. Glucagon caused no increase in mitochondrial respiration in situ either in the presence of the physiological substrates or in the presence of durohydroquinone. The data obtained do not support a role for the electron transport chain as a target of glucagon action in hepatocytes.     

Energy state of hepatocytes of fed rats isolated by the means of EDTA and vibration

The energy state of mitochondria in fed rat hepatocytes isolated by the use of non-enzymatic method including liver perfusion with an EDTA-containing solution with a further mild mechanical effect of tissue fragments by vibration has been studied. The isolation procedure used permits to obtain significant amounts of hepatocytes whose viability is not less than 80%. The endogenous respiration rate (10-15 nm O2/min.mln cells) is slightly stimulated by succinate. In the course of incubation in a balanced salt medium, the cells accumulate ATP and the lipophilic cation, tetraphenylphosphonium. Data from the inhibitory analysis testify to the fact that tetraphenylphosphonium accumulation reflects the membrane potential of intact cell mitochondria, which are in a metabolic state similar to state 3.     

Immunochemical analysis of the membrane proteins of rat liver and Zajdela hepatoma mitochondria

The contents of mitochondrial inner membrane protein complexes were compared in normal liver and in Zajdela hepatoma mitochondria by the immunotransfer technique. Antibodies against core proteins 1 and 2, cytochrome c1, the iron-sulfur protein of Complex III, subunits I and II of cytochrome oxidase, and the alpha and beta subunits of the F1-ATPase were used. In addition, antibodies against a primary dehydrogenase, beta-hydroxybutyrate dehydrogenase, as well as the outer membrane pore protein were used. The results indicate that the components of the cytochrome chain and porin are greatly enriched in hepatoma mitochondria compared to normal rat liver mitochondria. This enrichment was also reflected in the rates of respiration in tumor mitochondria using a variety of substrates. Enrichment of porin may partially account for increased hexokinase binding to tumor mitochondria. In contrast to the respiratory chain components, the F1-ATPase and F0 (measured by DCCD binding) were not increased in tumor mitochondria. Thus, Zajdela hepatoma mitochondria components are nonstoichiometric, being enriched in oxidative capacity but relatively deficient in ATP synthesizing capacity. Finally, beta-hydroxybutyrate dehydrogenase, which is often decreased in hepatoma mitochondria, was shown here by immunological methods to be decreased by only 40%, whereas enzyme activity was less than 5% of that in normal rat liver.     

Ability of different hepatoma cells to metabolize 4-hydroxynonenal

4-Hydroxynonenal (4-HNE), produced during the oxidative lipid breakdown of biological membranes, modulates various biochemical processes in normal liver and in hepatoma cells. It is very probable that the effects of 4-HNE are related to the quantity formed in the cells and the cells' ability to metabolize it. Aldehyde catabolism takes place within the cells through oxidative and reductive enzymes, and through conjugation with intracellular glutathione. In this paper, the various enzymatic pathways involved in the metabolism of 4-HNE were studied in normal hepatocytes and in hepatoma cells. The hepatocyte pathway undergoes a complex variety of change during neoplastic transformation. In hepatoma cells, generally, 4-HNE metabolism was due mainly to aldehyde dehydrogenases, whereas in normal hepatocytes 4-HNE metabolism was mainly due to alcohol dehydrogenase and glutathione-S-transferase. The increase in oxidative enzymes compared to normal tissue was not the same in all types of hepatoma: in HTC hepatoma cells, the enzyme levels were considerably higher; in AH-130 hepatoma cells of Yoshida, they were lower in subcellular particles and similar in the cytosol. Indeed, consumption of externally-added 4-HNE in hepatoma cells was proportional to their content of 4-HNE metabolizing enzymes.     

Codistribution of galactosyl- and sialyltransferase: reorganization of trans Golgi apparatus elements in hepatocytes in intact liver and cell culture

The intracellular distribution of galactosyl- and sialyltransferase was investigated in rat hepatocytes of intact liver, primary monolayer cultures of freshly isolated hepatocytes, in a nontumorigenic hepatocyte cell line and in a hepatoma cell line. The two glycosyltransferases were detected by immunofluorescence using affinity-purified rabbit antibodies. Indirect double immunofluorescence showed that both terminal glycosyltransferases were identically codistributed in the same cell. This codistribution was always observed regardless of the cell type investigated, and in both stationary and migrating cells. The immunofluorescence pattern for both galactosyl- and sialyltransferase was found to be different in hepatocytes in vivo compared to hepatocytes grown in vitro. In hepatocytes of intact liver a spot-like cytoplasmic fluorescence was observed, whereas in cultured normal hepatocytes a perinuclear fluorescence from which an extensive tubular network radiated far into the cytoplasm existed. Cultured hepatoma cells also exhibited an extensive cytoplasmic fluorescence, which in contrast to the normal hepatocytes was rather diffuse. We conclude that (a) galactosyl- and sialyltransferase are codistributed in rat hepatocytes, and (b) a reorganization of (trans) Golgi apparatus elements containing both terminal glycosyltransferases occurs under conditions of in vitro growth and malignant transformation.     

Tissue-specific regulation of rat liver mitochondrial oxidative phosphorylation by a soluble phase of cells from that organ

Tissue specificity of mitochondrial respiration stimulation under the effect of a soluble phase of liver cells (SPC) is preserved by addition to dinitrophenol but is reserved in the presence of oligomycin. Addition of rotenon in the presence of SPC entails a tissue-specific increase in respiration that is proportional to the respective increase in respiration of intact mitochondria in the presence of the inhibitor mentioned. SPC tissue-specifically inhibits ATPase activity of liver mitochondria. This fraction of SPC is capable of recovering the coupling of oxidative phosphorylation of mitochondria whose respiration is inhibited by adding ADP. A conclusion is made that SPC is capable not only to decrease tissue-specifically the coupling of intact mitochondria but also to raise it in mitochondria with deranged oxidative phosphorylation. This assures intratissue organization of liver metabolism by means of tissue-specific stabilization of liver cell energy metabolism.     

Pathway of glutamate oxidation and its regulation in the HuH13 line of human hepatoma cells.

Well-coupled mitochondria were isolated from a HuH13 line of human hepatoma cells and human liver tissue. The liver mitochondria showed a feeble glutamine oxidation activity in contrast to the hepatoma mitochondria, whereas they utilized glutamate well for the oxidative phosphorylation. In the liver mitochondria, glutamate was oxidized via the routes of transamination and deamination. On the other hand, glutamate oxidation was initiated preferentially via transamination pathway in the tumor mitochondria. In the liver mitochondria, bicarbonate nearly at a physiological concentration inhibited oxygen uptake with glutamate as substrate. The interaction of bicarbonate with the pathway of glutamate oxidation occurred primarily at the level of succinate dehydrogenase, due to competitive inhibition of the enzyme by the compound. In contrast to the liver mitochondria, glutamate oxidation was not affected by bicarbonate in the tumor mitochondria. These results indicate that the aberrations in the glutamate metabolism and its regulation observed in the hepatoma mitochondria may be favorable to the respiration utilizing glutamine and/or glutamate as an energy source.     

Effect of aldehydes on polyamine metabolism. III. Inhibition of S-adenosyl methionine decarboxylase (SAMD) by CCl4 and by aldehydes produced during lipid peroxidation

In isolated rat liver cells in which lipid peroxidation is stimulated by CCl4, a strong inhibition of S-adenosylmethionine decarboxylase (SAMD) activity occurs. Some purified aldehydes, which are produced during lipid peroxidation, are able to inhibit SAMD activity in Yoshida hepatoma cells. The most active aldehyde is hydroxypentenal (HPE). It inhibits by 50% SAMD activity at 0.5 mM concentration in entire hepatoma cells, or in hepatoma cell sap, and at 0.1 mM concentration in partially purified hepatoma cell sap fractions.     

The effect of preparations of non-histone chromosomal proteins on the expression of hetero-organic membrane antigens of kidney origin in a primary rat hepatocyte culture

The expression of membrane hetero-organic antigens of kidney origin, associated with the rat hepatomas in primary culture of intact adult rat hepatocytes, was investigated by means of the indirect immunofluorescence method using a specific immune serum. These antigens were observed on the membrane of some hepatocytes after their contact with nonhistone chromosomal proteins (NHCP), which were obtained from the kidney of intact rats from cells of hepatoma 27 and Zajdela hepatoma, or from the carcinogenic liver after a single diethylnitrosamine injection. Negative results were obtained after the incubation of hepatocytes in the medium lacking some of NHCP, or in that with NHCP obtained from the liver of intact rats.     

Hepatic gluconeogenesis and mitochondrial function during hibernation

1. The aim of these studies was to investigate a mitochondrial basis for changes in gluconeogenesis during hibernation. 2. State 3 respiration rates in liver mitochondria from hibernating ground squirrels were reduced by 62-66%. The limiting reaction appeared to be electron transport, particularly in respiratory complex III. 3. The mitochondrial ATP + ADP + AMP content was reduced by 29% during hibernation; cellular adenine nucleotide content was unchanged. 4. Pyruvate carboxylation in intact mitochondria was decreased 75% during hibernation, although total pyruvate carboxylase activity was not lower. 5. Rates of gluconeogenesis in intact hepatocytes isolated from hibernators were lower than in cells from non-hibernators.     

Investigation by amperometric methods of intracellular reduction of 2,6-dichlorophenolindophenol in normal and transformed hepatocytes in the presence of different inhibitors of cellular metabolism

The reduction of 2,6-dichlorophenolindophenol (DCIP) was measured by amperometric methods in Morris hepatoma 3924A cells, normal isolated rat hepatocytes and in mitochondria isolated from normal rat liver. The influence of aerobic and anaerobic atmospheres and of various inhibitors of cellular metabolism, especially of the respiratory chain (KCN, NaN3, oligomycin), on DCIP-reduction were studied using glucose, succinate, beta-hydroxybutyrate, alpha-keto-glutarate and oxalacetate as substrates. Under the influence of KCN and oligomycin the velocity of DCIP-reduction was increased in both cell types. Azide showed a similar effect on tumour cells and to a lower extent on hepatocytes. Using isolated mitochondria total DCIPred was increased by KCN and azide using various mitochondrial metabolites as substrates and with ADP/Pi present. The effects of KCN, azide and oligomycin could be explained by taking DCIP as an artificial coupling site in mitochondria which is only used when oxygen is absent or when the respiratory chain is blocked by inhibitors of cytochrome oxidase. Evaluation of the reaction kinetics revealed differences between normal and transformed cells in terms of the pseudo-first-order rate constants and the activity of overall oxidoreductases. The results apparently reflect quantitative differences in enzymatic equipment and the metabolic pathways predominating in normal and neoplastic cells.     

Structure and properties of an under-sulfated heparan sulfate proteoglycan synthesized by a rat hepatoma cell line

A rat hepatoma cell line was shown to synthesize heparan sulfate and chondroitin sulfate proteoglycans. Unlike cultured hepatocytes, the hepatoma cells did not deposit these proteoglycans into an extracellular matrix, and most of the newly synthesized heparan sulfate proteoglycans were secreted into the culture medium. Heparan sulfate proteoglycans were also found associated with the cell surface. These proteoglycans could be solubilized by mild trypsin or detergent treatment of the cells but could not be displaced from the cells by incubation with heparin. The detergent-solubilized heparan sulfate proteoglycan had a hydrophobic segment that enabled it to bind to octyl- Sepharose. This segment could conceivably anchor the molecule in the lipid interior of the plasma membrane. The size of the hepatoma heparan sulfate proteoglycans was similar to that of proteoglycans isolated from rat liver microsomes or from primary cultures of rat hepatocytes. Ion-exchange chromatography on DEAE-Sephacel indicated that the hepatoma heparan sulfate proteoglycans had a lower average charge density than the rat liver heparan sulfate proteoglycans. The lower charge density of the hepatoma heparan sulfate can be largely attributed to a reduced number of N-sulfated glucosamine units in the polysaccharide chain compared with that of rat liver heparan sulfate. Hepatoma heparan sulfate proteoglycans purified from the culture medium had a considerably lower affinity for fibronectin-Sepharose compared with that of rat liver heparan sulfate proteoglycans. Furthermore, the hepatoma proteoglycan did not bind to the neoplastic cells, whereas heparan sulfate from normal rat liver bound to the hepatoma cells in a time-dependent reaction. The possible consequences of the reduced sulfation of the heparan sulfate proteoglycan produced by the hepatoma cells are discussed in terms of the postulated roles of heparan sulfate in the regulation of cell growth and extracellular matrix formation.