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.     

Non-ohmic proton conductance of the mitochondrial inner membrane in hepatocytes

The mitochondrial membrane potential in isolated hepatocytes was measured using the distribution of the lipophilic cation triphenylmethylphosphonium (TPMP+) with appropriate corrections for plasma membrane potential, cytoplasmic and mitochondrial binding of TPMP+, and other factors. The relationship between mitochondrial membrane potential and respiration rate in hepatocytes was examined as the respiratory chain was titrated with myxothiazol in the presence of oligomycin. This relationship was nonproportional and similar to results with isolated mitochondria respiring on succinate. This shows that there is an increased proton conductance of the mitochondrial inner membrane in situ at high values of membrane potential. From the respiration rate and mitochondrial membrane potential of hepatocytes in the absence of oligomycin, we estimate that the passive proton permeability of the mitochondrial inner membrane accounts for 20-40% of the basal respiration rate of hepatocytes. The relationship between log[TPMP+]tot/[TPMP+]e and respiration rate in thymocytes was also nonproportional suggesting that the phenomenon is not peculiar to hepatocytes. There is less mitochondrial proton leak in hepatocytes from hypothyroid rats. A large proportion of the difference in basal respiration rate between hepatocytes from normal and hypothyroid rats can be accounted for by differences in the proton permeability characteristics of the mitochondrial inner membrane.     

Hepatocyte mitochondrion electron-transport chain alterations in CCl<Subscript>4</Subscript> and alcohol induced hepatitis in rats and their correction with simvastatin

The goal of this study was to examine the state of hepatocyte mitochondrial respiratory chain of rats with toxic hepatitis induced by CCl₄ and ethanol. Oxygen consumption by hepatocytes and mitochondria was determined. Endogenous oxygen consumption by pathological hepatocytes was 1.3-fold higher compared with control. Rotenone resulted in 27% suppression of respiration by pathological hepatocytes whereas 2,4-dinitrophenol produced a 1.4-fold increase of respiration. States 3 and 4 of mitochondrial respiration with malate and glutamate were found to be higher as compared to control. State dinitrophenol and state 3 respirations were similar within every group of animals when being tested with malate and glutamate or succinate. Cytochrome c oxidase activity in hepatitis was 1.8-fold higher compared with control. Simvastatin administration resulted in a decrease in hepatocyte endogenous respiration in hepatitis. The presented data lead to the assumption that the increased oxygen consumption by the respiratory chain of pathological mitochondria to be linked mainly with the altered function of complex I.     

Crabtree effect induced by fructose in hepatocytes isolated from developing rats.

The inhibition of respiration caused by fructose is investigated in hepatocytes isolated from fetal and developing rats. The respiration rate of the hepatocytes increases in the early days after birth. Adult values for oxygen uptake are reached by hepatocytes isolated from 14-day-old rats. After weaning, the Crabtree effect induced by fructose appears simultaneously with changes in energy metabolism.     

Respiratory activity of isolated hepatocytes

Oligomycin and uncoupler of oxidative phosphorylation have been studied for their effect on the respiration activity of hepatocytes in rats. The respiration rate in the presence of oligomycin and uncoupler is higher than it is with the respiration uncoupled in the absence of oligomycin. Exogenic succinate makes endogenic respiration of hepatocytes in the presence of digitonin 5 times more intensive. The obtained results evidence for the fact that the uncoupled respiration is limited by the concentration of substrates able to be oxidized in the respiration chain of mitochondria. Oligomycin induces accumulation of substrates and following addition of the disconnector evokes their fast oxidation.     

The proton leak across the mitochondrial inner membrane

The proton conductance of the mitochondrial inner membrane increases at high protonmotive force in isolated mitochondria and in mitochondria in situ in rat hepatocytes. Quantitative analysis of its importance shows that about 20-30% of the oxygen consumption by resting hepatocytes is used to drive a heat-producing cycle of proton pumping by the respiratory chain and proton leak back to the matrix. The flux control coefficient of the proton leak pathway over respiration rate varies between 0.9 and zero in mitochondria depending on the rate of respiration, and has a value of about 0.2 in hepatocytes. Changes in the proton leak pathway in situ will therefore change respiration rate. Mitochondria isolated from hypothyroid animals have decreased proton leak pathway, causing slower state 4 respiration rates. Hepatocytes from hypothyroid rats also have decreased proton leak pathway, and this accounts for about 30% of the decrease in hepatocyte respiration rate. Mitochondrial proton leak may be a significant contributor to standard metabolic rate in vivo.     

On the thyroid hormone-induced increase in respiratory capacity of isolated rat hepatocytes.

The respiratory capacities of hepatocytes, derived from hypothyroid, euthyroid and hyperthyroid rats, have been compared by measuring rates of oxygen uptake and by titrating components of the respiratory chain with specific inhibitors. Thyroid hormone increased the maximal rate of substrate-stimulated respiration and also increased the degree of ionophore-stimulated oxygen uptake. In titration experiments, similar concentrations of oligomycin or antimycin were required for maximal inhibition of respiration regardless of thyroid state, suggesting that the changes in respiratory capacity were not the result of variation in the amounts of ATP synthase or cytochrome b. However, less rotenone was required for maximal inhibition of respiration in the hypothyroid state than in cells from euthyroid or hyperthyroid rats, implying that hepatocytes from hypothyroid animals contain less NADH dehydrogenase. The concentration of carboxyatractyloside necessary for maximal inhibition of respiration was 100 microM in hepatocytes from hypothyroid rats, but 200 microM and 300 microM in hepatocytes from euthyroid and hyperthyroid rats, respectively, indicating a possible correlation between levels of thyroid hormone and the amount or activity of adenine nucleotide translocase. The increased capacity for coupled respiration in response to thyroid hormone is not associated with an increase in the components of the electron transport chain or ATP synthase, but correlates with an increased activity of adenine nucleotide translocase.     

The mechanism of stimulation of respiration by fatty acids in isolated hepatocytes

Addition of fatty acids to isolated hepatocytes raised respiration rate by 92% and raised mitochondrial membrane potential (delta psi m) in situ from 155 to 162 mV suggesting that the increased fuel supply had a greater effect on respiration rate than any increases in processes that consumed mitochondrial protonmotive force (delta p). The relationship between delta psi m and respiration rate was changed by addition of fatty acids or lactate, showing that there was also stimulation of delta p-consuming reactions. In the presence of oligomycin the relationship between delta psi m and respiration rate was unaffected by substrate addition, showing that the kinetics of delta p consumption by the H+ leak across the mitochondrial inner membrane were unchanged. The stimulation of delta p consumers by fatty acids therefore must be in the pathways of ATP synthesis and turnover. Inhibition of several candidate ATP-consuming reactions had little effect on basal or fatty acid-stimulated respiration, and the nature of the ATP turnover reactions in hepatocytes remains speculative. We conclude that fatty acids (and other substrates) stimulate respiration in hepatocytes in two distinct ways. They provide substrate for the electron transport chain, raising delta p and increasing the non-ohmic proton leak across the mitochondrial inner membrane and the rate of oxygen consumption. They also directly stimulate an unidentified delta p-consuming reaction in the cytoplasm. They do not work by uncoupling or by stimulation of intramitochondrial ATP-turnover reactions.     

Crabtree effect induced by fructose in isolated hepatocytes from fed rats.

In hepatocytes isolated from fed rats, the addition of fructose caused an inhibition of respiration. In hepatocytes isolated from starved rats the Crabtree effect was not observed. No difference in oxygen uptake was found by addition of glucose to hepatocytes from fed or starved animals. The inhibition of respiration was parallel with a rise in the glycolytic flux and the oxidation of the mitochondrial respiratory carriers. The metabolic conditions in which the Crabtree effect can be operative in liver cells are discussed.     

Ethanol suppresses ureagenesis in rat hepatocytes: role of acetaldehyde

We proposed previously that closure of voltage-dependent anion channels (VDAC) in the mitochondrial outer membrane after ethanol exposure leads to suppression of mitochondrial metabolite exchange. Because ureagenesis requires extensive mitochondrial metabolite exchange, we characterized the effect of ethanol and its metabolite, acetaldehyde (AcAld), on total and ureagenic respiration in cultured rat hepatocytes. Ureagenic substrates increased cellular respiration from 15.8 ± 0.9 nmol O(2)/min/10(6) cells (base line) to 29.4 ± 1.7 nmol O(2)/min/10(6) cells in about 30 min. Ethanol (0-200 mM) suppressed extra respiration after ureagenic substrates (ureagenic respiration) by up to 51% but not base line respiration. Urea formation also declined proportionately. Inhibition of alcohol dehydrogenase, cytochrome P450 2E1, and catalase with 4-methylpyrazole, trans-1,2-dichloroethylene, and 3-amino-1,2,3-triazole restored ethanol-suppressed ureagenic respiration by 46, 37, and 66%, respectively. By contrast, inhibition of aldehyde dehydrogenase with phenethyl isothiocyanate increased the inhibitory effect of ethanol on ureagenic respiration by an additional 60%. AcAld, an intermediate product of ethanol oxidation, suppressed ureagenic respiration with an apparent IC(50) of 125 μM. AcAld also inhibited entry of 3-kDa rhodamine-conjugated dextran in the mitochondrial intermembrane space of digitonin-permeabilized hepatocytes, indicative of VDAC closure. In conclusion, AcAld, derived from ethanol metabolism, suppresses ureagenesis in hepatocytes mediated by closure of VDAC.     

The contribution of ATP turnover by the Na+/K+-ATPase to the rate of respiration of hepatocytes. Effects of thyroid status and fatty acids

In less than 1 min ouabain maximally inhibits oxygen consumption due to gramicidin-induced ATP turnover by the Na+/K+-ATPase in hepatocytes. Ouabain rapidly inhibits respiration on palmitate or glucose by only 6-10% indicating that the Na+/K+-ATPase plays a minor role in cell ATP turnover. 29% of the extra oxygen consumption of hepatocytes isolated from hyperthyroid rats was inhibited by ouabain showing that the Na+/K+-ATPase is responsible for some but not the majority of the stimulation of respiration induced by thyroid hormone.     

Decrease in mitochondrial energy coupling by thyroid hormones: a physiological effect rather than a pathological hyperthyroidism consequence

The effect of the in vivo thyroid status on mitochondrial membrane potential (ΔΨ_m) in isolated rat hepatocytes was studies by means of a cytofluorimetric technique and the ΔΨ_m-specific probe JC-1. It is shown that the ΔΨ_m level decreases in the order hypothyroid>euthyroid>hyperthyroid. Polarographic measurement of the hepatocyte respiratory rates revealed an opposite trend of values: the highest respiratory rate in hepatocytes from hyperthyroid animals, the lowest in those from hypothyroid ones. This means that mitochondrial energy coupling is highest in hypothyroid hepatocytes and lowest in hyperthyroid hepatocytes. 6-Ketocholestanol added to hepatocytes failed to counterbalance the uncoupling effect of thyroid hormones on ΔΨ_m and respiration rate. Under the same conditions, 6-ketocholestanol appeared to be effective in recoupling of respiration uncoupled by low concentrations of the artificial protonophore FCCP. The mechanism and possible physiological functions of the thyroid hormone-induced decrease in mitochondrial energy coupling are discussed.     

Radiosensitivity of parenchymal hepatocytes as a function of oxygen concentration

To investigate the mechanism(s) of hepatocyte radioresistance (D0 2.7 Gy), the radiosensitivities of respiring (37 degrees C) and nonrespiring (0 degrees C) hepatocytes were determined as a function of oxygen concentration. Fischer 344 female rat hepatocytes were isolated by liver perfusion, equilibrated in Leibowitz-15 media with different oxygen tensions, and exposed to 60Co radiation at either 37 or 0 degrees C. Cell survival and DNA single-strand breaks were used as the biological end points of radiosensitivity. The K value for respiring hepatocytes (37 degrees C) was 14.3 +/- 0.5 mm Hg O2 (18.8 +/- 0.7 mumol O2/liter), demonstrating that the K value for freshly isolated parenchymal hepatocytes is significantly greater than those previously obtained for cultured cells. In contrast, the K value for nonrespiring hepatocytes (0 degree C) is 1.4 +/- 0.4 mm Hg O2 (3.7 +/- 1.0 mumol O2/liter) indicating that hepatocyte respiration results in a plasma membrane-to-nucleus oxygen gradient of approximately 12.9 +/- 0.6 mm Hg (15.1 +/- 1.2 microns O2/liter). The hypothesis that the hepatic nucleus typically resides in a hypoxic condition, although the liver is uniformly perfused with well-oxygenated blood, is supported by (1) the nonradom perinuclear distribution of the mitochondria, (2) the high cellular respiration rate, and (3) the large intracellular oxygen diffusion distance in hepatocytes (25 microns diameter).     

Endogenous and endobiotic induced reactive oxygen species formation by isolated hepatocytes.

The rat hepatocyte catalyzed oxidation of 2',7'-dichlorofluorescin to form the fluorescent 2,7'-dichlorofluorescein was used to measure endogenous and xenobiotic-induced reactive oxygen species (ROS) formation by intact isolated rat hepatocytes. Various oxidase substrates and inhibitors were then used to identify the intracellular oxidases responsible. Endogenous ROS formation was markedly increased in catalase-inhibited or GSH-depleted hepatocytes, and was inhibited by ROS scavengers or desferoxamine. Endogenous ROS formation was also inhibited by cytochrome P450 inhibitors, but was not affected by oxypurinol, a xanthine oxidase inhibitor, or phenelzine, a monoamine oxidase inhibitor. Mitochondrial respiratory chain inhibitors or hypoxia, on the other hand, markedly increased ROS formation before cytotoxicity ensued. Furthermore, uncouplers of oxidative phosphorylation inhibited endogenous ROS formation. This suggests endogenous ROS formation can largely be attributed to oxygen reduction by reduced mitochondrial electron transport components and reduced cytochrome P450 isozymes. Addition of monoamine oxidase substrates increased antimycin A-resistant respiration and ROS formation before cytotoxicity ensued. Addition of peroxisomal substrates also increased antimycin A-resistant respiration but they were less effective at inducing ROS formation and were not cytotoxic. However, peroxisomal substrates readily induced ROS formation and were cytotoxic towards catalase-inhibited hepatocytes, which suggests that peroxisomal catalase removes endogenous H(2)O(2) formed in the peroxisomes. Hepatocyte catalyzed dichlorofluorescin oxidation induced by oxidase substrates, e.g., benzylamine, was correlated with the cytotoxicity induced in catalase-inhibited hepatocytes.     

The effect of various aldehydes on the respiration of rat liver and hepatoma AH-130 cells

Some aldehydes, produced during lipid peroxidation of liver lipids, are able to inhibit the respiration of mitochondria and of intact cells both in normal hepatocytes and in Yoshida hepatoma. In mitochondria, the respiratory stimulation produced by addition of ADP and dinitrophenol is decreased more in hepatoma than in normal liver. Two- to four-fold higher concentrations of aldehydes are needed to obtain the same degree of inhibition in normal liver mitochondria as in tumorous organs. The effect of aldehydes on intact cell respiration is absent or very low in hepatocytes, but it is consistently observed in hepatoma cells.     

A non-enzymatic method of preparing isolated hepatocytes: assessment of their morpho-functional activity

A method for isolating hepatocytes using the two-step liver perfusion by chelate-containing solutions is described. The hepatocytes obtained are highly inactive for the trypan blue staining test, (not less than 90%), ATP content, the rate of endogenic respiration and its stimulation by succinate, a degree of xenobiotic oxidation.     

Respiration rates, ATP content and ionic regulation in hepatocytes of starving lamprey during the pre-spawning period of their life cycle

During the winter pre-spawning migration, lampreys Lampetra fluviatilis stop feeding, and their liver metabolism is reduced substantially. Aerobic ATP production in hepatocytes decreased to one third and ATP content decreased by 50% as compared with the values in autumn. In spite of the decrease of endogenous and phosphorylating (oligomycin-sensitive) respiration in winter, the oxygen consumption used to drive sodium and potassium pumping through Na,K-ATPase activity (ouabain-sensitive respiration) remained virtually constant. Consequently its share in phosphorylating respiration increased from 16·3% in November to 54·2% in February. Potassium influx was similar within the range of ATP content between 2·5 and 1 nmol 10⁻⁶ cells and decreases only in hepatocytes which contained <0·8 nmol ATP 10⁻⁶ cells.     

A multichannel perifusion system for the continuous monitoring of glycogenolysis and gluconeogenesis in isolated rat hepatocytes

A multichannel perifusion system for isolated rat hepatocytes entrapped in a Sephadex matrix is described and criteria for the choice of matrices are discussed. This system overcomes the usual problem of clogged filters and impaired flow rates encountered in suspension perifusion systems, and is assembled from standard widely available components. Gluconeogenic capability and mitochondrial respiratory control ratios were unaltered. Decreases in trypan blue viability index and respiration rate were small when compared with flask-incubated hepatocytes. The endogenous rate of glycogenolysis was slightly higher in perifused hepatocytes but hormone response, as judged by glucagon stimulation of glycogenolysis, was unimpaired. The potential of this system is indicated by experiments monitoring glycogenolysis and gluconeogenesis in recycling and non-recycling modes.     

Basal reactive oxygen species determine the susceptibility to apoptosis in cirrhotic hepatocytes

Hepatocytes from cirrhotic murine livers exhibit increased basal ROS activity and resistance to TGFbeta-induced apoptosis, yet when ROS levels are decreased by antioxidant pretreatment, these cells recover susceptibility to apoptotic stimuli. To further study these redox events, hepatocytes from cirrhotic murine livers were pretreated with various antioxidants prior to TGFbeta treatment and the ROS activity, apoptotic response, and mitochondrial ROS generation were assessed. In addition, normal hepatocytes were treated with low-dose H(2)O(2) and ROS and apoptotic responses determined. Treatment of cirrhotic hepatocytes with various antioxidants decreased basal ROS and rendered them susceptible to apoptosis. Examination of normal hepatocytes by confocal microscopy demonstrated colocalization of ROS activity and respiring mitochondria. Basal assessment of cirrhotic hepatocytes showed nonfocal ROS activity that was abolished by antioxidants. After pretreatment with an adenovirus expressing MnSOD, basal cirrhotic hepatocyte ROS were decreased and TGFbeta-induced colocalization of ROS and mitochondrial respiration was present. Treatment of normal hepatocytes with H(2)O(2) resulted in a sustained increase in ROS and resistance to TGFbeta apoptosis that was reversed when these cells were pretreated with an antioxidant. In conclusion, cirrhotic hepatocytes have a nonfocal distribution of ROS. However, normal and cirrhotic hepatocytes exhibit mitochondrial localization of ROS that is necessary for apoptosis.