Protein synthesis in a hepatocyte culture in the presence of exogenous adenylic and guanyl nucleotides

Circahoral opposite-in phase fluctuations of protein syntheses and intracellular ATP content have been observed in monolayer hepatocyte cell culture. Peculiarities of protein synthesis in hepatocytes have been studied in vitro in presence of adenylic and guanylic nucleotides. Addition of exogenous ATP leads to the decrease in the level of protein synthesis and smoothing off of the fluctuations. The presence of exogenous ADP leads to the increase in protein synthesis and retaining of the amplitude of fluctuations of this process. Effect of exogenous GTP is similar to that of ATP. Different aspects of action of exogenous NTPs on the rhythms of protein synthesis have been considered.     

Circadian rhythm of tRNA aminoacylation in hepatocyte culture

In the monolayer of rat hepatocytes in vitro, the circadian rhythms were revealed of 3H-leucine incorporation in proteins and in aminoacyl-tRNA(Leu) fraction. The oscillations were mainly synphasic, though coordination between aminoacylation and protein synthesis was not stable in the coarse of time. In cell free system with the excess of ATP and aa-tRNA, the rhythm of 3H-leucine incorporation was also clear. This means, that oscillatory kinetics of the protein synthesis rate is not caused by oscillation of ATP which has been revealed earlier in the hepatocyte monolayer.     

Toxic Effects of Tacrine on Primary Hepatocytes and Liver Epithelial Cells in Culture

Administration of tacrine (THA) for the treatment of Alzheimer's disease results in a reversible hepatotoxicity in 30–50% of patients, as indicated by an increase in transaminase levels. However, the intracellular mechanisms underlying such a toxicity have not yet been elucidated. In this study, we performed short-term and long-term in vitro treatments on primary human and rat hepatocyte cultures as well as on nonparenchymal rat liver epithelial cells (RLEC), known as CYP1A-deficient cells. Cell ultrastructure was analyzed under different conditions and the release of lactate dehydrogenase (LDH) was used to evaluate cytotoxicity. The effects of THA on protein synthesis, intermediary metabolism and reduced glutathione (GSH) level were also determined in rat hepatocytes. THA induced dose-dependent toxic effects in liver parenchymal and nonparenchymal cells, with human hepatocytes being less sensitive. This toxicity appeared to be unrelated to metabolism of THA since similar effects were observed in rat hepatocytes and RLEC, in which THA metabolism was found negligible. Ribosome aggregation appeared only at high concentrations (>1 mmol/L) and was not specific to hepatocytes. Therefore, the THA-induced decrease in protein synthesis observed at lower concentrations was likely not related to this alteration. ATP and glycogen levels as well as GSH content were reduced upon THA. However, while glycogen level decreased at THA doses similar to those inducing an increase in LDH release, the fall in ATP and GSH contents occurred at higher doses. Thus, glycogen level in hepatocytes appeared to be a more sensitive indicator of THA toxicity than were ATP and GSH levels. We also found that protein synthesis started to decrease at THA doses that were still ineffective on LDH release. This might suggest that the decrease in synthesis of one or several proteins upon THA treatment represents the early signal leading cells to death.     

Energy dependence of autophagic protein degradation in isolated rat hepatocytes

The effect of small changes in intracellular ATP on autophagic flux was studied in isolated rat hepatocytes by using inhibitors of ATP production or by varying the metabolic conditions. The following observations were made. There was a linear relationship between endogenous protein degradation and intracellular ATP, the rate of proteolysis declining with decreasing ATP concentrations. 15% of the maximal proteolysis is either independent of ATP or has a very high affinity for this metabolite. There was a linear relationship between the autophagic sequestration of cytosolic [14C]sucrose and intracellular ATP, the sequestration rate decreasing with decreasing ATP concentrations. ATP depletion did not cause release of [14C]sucrose previously sequestered in autophagosomes and lysosomes at high ATP levels. Intracellular accumulation of chloroquine, used as an indicator of the pH inside lysosomes and other acidic cell compartments, diminished with decreasing cellular ATP content. Amino acids inhibited proteolysis without affecting ATP levels or chloroquine accumulation. We conclude from the high sensitivity of autophagy towards relatively small changes in the concentration of intracellular ATP that, besides amino acids, ATP is a very important factor in controlling the rate of autophagy in rat hepatocytes.     

Glutathione synthesis during the rewarming of rat hepatocytes preserved in the University of Wisconsin solution

In this study, we used isolated rat hepatocytes to investigate the effect of nucleoside content of the preserved cells on the ability to synthesize glutathione (GSH) during the rewarming process. We cold-stored hepatocytes in University of Wisconsin (UW) solution (72 h, 0 degrees C, N(2)) without nucleosides and with the addition of 5 mM adenosine or 10 mM ATP. After 72 h of cold storage, we determined the GSH synthesis rate and the ATP content of the cells. We found a GSH synthesis rate similar to that of freshly isolated hepatocytes only in the group of cells cold-stored with 10 mM ATP. When we tested the cellular ATP concentrations, we found that controls and preserved cells with 10 mM ATP showed a similar value of ATP during the rewarming step. Our results suggested that the incorporation of ATP in the UW solution increased the ATP content and the rate of GSH synthesis of cold-stored hepatocytes during rewarming.     

Correlation of the rhythms of protein synthesis and secretion in a hepatocyte monolayer culture

In primary monolayer culture of hepatocytes, circahoralian rhythms of protein synthesis and secretion were discovered. In one of the hepatocyte populations the rhythmic cycle of protein secretion was found to be about twice as long as that of protein synthesis. Combination of different experimental variants such as the impulse and the long-term labelling of proteins or inhibition of protein synthesis, revealed that the secretion rhythms were predetermined by the secretion of both newly formed proteins and those stored in the cell for a long time. Upon the inhibition of protein synthesis with cyclohexeimide, the protein secretion in the monolayer changes its rhythmic pattern for a constant rate secretion. Possible causes of the alteration of circahoralian variations of protein synthesis are discussed.     

Insulin effects on protein synthesis and secretion in primary cultures of amphibian hepatocytes.

The objective of the present study was to determine the effects of insulin on amphibian hepatocytes in primary culture. Hepatocytes were isolated from adult bullfrogs by collagenase perfusion and maintained as monolayers in serum-free medium. Cells cultured in the continuous presence of insulin exhibited a relatively constant rate of protein secretion over the first four to five days, whereas controls showed an almost three-fold decrease over the same time period. The decline in secreted proteins was equally represented in most exported proteins, except that serum albumin secretion showed twice as much of a decrease relative to the other proteins. The maintenance of protein secretion by insulin was the result of its effect on protein synthesis. The rate of protein synthesis was measured by the incorporation of (3H)-leucine into protein using culture medium containing 0.5 mM leucine, a condition where the specific radioactivity of leucyl-tRNA was shown to be equal to that of (3H)-leucine in the medium. Cultures maintained with insulin for 60 hours synthesized protein at two to three times the rate found in non-insulin treated controls whose rate of protein synthesis was first detectably decreased after nine hours of culture in the insulin-free medium. Sedimentation profiles of polyribosomes from hepatocytes maintained for 60 hours without insulin showed proportionately fewer ribosomes in large polysomes and more in monosomes and free ribosomal subunits than ribosomes from cells cultured with insulin. This result suggests that the decrease in protein synthesis found in the absence of insulin is due to a defect in initiation. Insulin does not exert its effect by regulating cellular levels of ATP; no change in ATP content was found in cells maintained with or without insulin. The results show that insulin maintains high levels of protein synthesis and secretion in amphibian hepatocytes. The hepatocytes in monlayer culture provide a system to study the molecular mechanisms involved in the translational control of protein synthesis by insulin.     

Role of the adenylate system and glycolytic flux in the control of protein synthesis in isolated rat lung cells

(1) Glucose stimulates the incorporation of amino acids into protein in lung cells isolated by digestion of the lung stroma with collagenase. This effect reflects mainly an increase in protein synthesis since no effect of glucose had been found to the uptake of amino acid precursors and, although glucose decreases the rate of intracellular proteolysis by 15%, this effect cannot account for the increased incorporation of radioactivity into proteins. Furthermore, glucose did not induce any significant change in the intracellular content of valine. (2) For glucose to act on protein synthesis, it must be glycolyzed since its stereoisomer, L-glucose, which is not metabolized by lung cells, has no effect. (3) The mechanism of glucose action does not seem to be related simply to variations of cellular ATP content or energy charge. The following arguments seem to support this conclusion: (i) glucose does not bring about significant variations in the concentration of reactants of the adenylate system; (ii) the increase in protein synthesis induced by glucose in energy-depleted cells correlates with a rise in ATP content and energy charge; however, adenosine, which increases ATP levels in a form quantitatively similar to glucose, is unable to affect protein synthesis: (iii) glucose also accelerates the incorporation of amino acids into proteins in adenosine-treated lung cells in which the ATP concentration was almost double that of the control and the energy charge was considerably elevated, ruling out the possibility that a rise in the steady-state concentration of ATP and/or energy charge alone could be responsible for the acceleration of protein synthesis. (4) It can be concluded that the effect of glucose in increasing protein synthesis in lung cells is dependent on some signal arising from its breakdown and not to variations in the concentration of reactants or energy charge of the adenylate system.     

Participation of cellular thiol/disulphide groups in the uptake, degradation and bioactivity of insulin in primary cultures of rat hepatocytes.

The effects on the uptake (cell-associated 125I) and degradation (125I-labelled products released into the medium) of 125I-insulin and bioactivity (protein, glycogen and lipid synthesis) of insulin caused by altering the cellular thiol/disulphide status in primary cultures of rat hepatocytes were studied. Incubation of hepatocyte cultures with various exogenous thiol compounds (reduced glutathione, 2-mercaptoethanol, cysteamine, dithiothreitol) resulted in increased insulin binding, but markedly decreased degradation and bioactivity. These effects could be reversed by washing or by the addition of oxidized glutathione, which alone had no effect. When cultures were exposed to certain thiol-modifying reagents (N-ethylmaleimide, p-chloromercuribenzoate, p-chloromercuribenzenesulphonate, iodoacetamide, iodoacetate), some decreases in bioactivity were evident, but the pronounced decrease in insulin degradation observed with the thiol-containing compounds was not observed with this class of compounds. None of the thiol-containing or -modifying agents tested had any significant effect on cellular ATP concentrations, indicating that the effects observed were due to perturbation of the thiol/disulphide status. Depletion of intracellular glutathione by DL-buthionine SR-sulphoximine (a specific inhibitor of glutathionine biosynthesis) decreased the syntheses of glycogen and lipid by about one-half, while having essentially no effect on protein synthesis, ATP concentrations or on the binding and degradation of insulin. The data presented here indicate that although intracellular thiol (glutathione) concentrations may be important for the maintenance of full expression of certain biological activities (glycogen and lipid synthesis), the thiol/disulphide groups on the cell surface and those immediately inside the cell membrane may be more critical in the mediation of insulin action, including the degradation and bioactivity of insulin in primary cultures of rat hepatocytes.     

Spermidine level and protein synthesis are coregulated in nonproliferating hepatocytes

The relationship between polyamines and the rate of protein synthesis was investigated in non-proliferating cells: primary cultures of adult rat hepatocytes maintained in serum-free media, and treated with dexamethasone or dexamethasone + insulin. During the second day of culture, polyamine biosynthesis became induced along with the rate of protein synthesis. While the activity of ornithine decarboxylase and the intracellular concentration of putrescine increased only transiently and that of spermine declined, the rise of the protein synthetic rate was paralleled by that of the intracellular spermidine concentration. The polyamine analogue diamino-propanol specifically decreased spermidine content and the protein synthetic rate. The intracellular concentration of spermidine was found subject to tight homeostatic regulation, e.g. not being altered by the addition of up to 1 mM of this polyamine to the culture medium. In contrast, addition of putrescine or spermine led to an increase in their respective intracellular concentrations. These findings indicate that spermidine specifically of the polyamines is involved in protein synthesis in the intact hepatocyte. Moreover, spermidine may mediate part of the trophic action of dexamethasone and insulin upon cultured hepatocytes.     

Direct cell-cell communication: a new approach derived from recent data on the nature and self-organisation of ultradian (circahoralian) intracellular rhythms

Recent data concerning ultradian (circahoralian) intracellular rhythms are used to assess the biochemical mechanisms of direct cell-cell communication. New results and theoretical considerations suggest a fractal nature of ultradian rhythms and their self-organisation. The fundamental and innate nature of these rhythms relates to their self-similarity at different levels of cell and tissue organisation. They can be detected in cell-free systems as well as in cells and organs in vivo. Such rhythms are a means of finding an optimal state of cell function rather than achieving a state of absolute stability. As a consequence, oscillations, being irregular and numerous by the set of periods, are resilient to functional overload and injury. Recent data on the maintenance of their fractal structure and, especially on the selection of optimal periods are discussed. The positive role of chaotic dynamics is stressed.The ultradian rhythm of protein synthesis in hepatocytes in vitro was used as a marker of direct cell-cell communication. The system demonstrates cell cooperation and synchronisation throughout the cell population, and suggests that the ultradian rhythms are self-organised. These observations also led to the detection of mechanisms of direct cell-cell communication in which extracellular factors have an essential role. Experimental evidence indicated the involvement of gangliosides and/or catecholamines in this large-scale synchronisation of protein synthesis. The response of all, or a major part, of the cell population is important; after the initial trigger effect, a periodic pattern is retained for some time. The influence of Ca2+-dependent protein kinases on protein phosphorylation can be a final step in the phase modulation of rhythms during cell-cell synchronisation.The intercellular medium plays an important role in self-synchronisation of ultradian rhythms between individual cells. Low cooperative activity of hepatocytes of old rats resulted from altered composition of the intercellular medium rather than direct effects of animal and cellular ageing. Similarly, in the whole body, changes in levels of gangliosides and catecholamines in the blood serum, a natural intercellular medium, can be critical events in age-dependent changes of the serum and accordingly cell-cell synchronisation. Hepatocytes of old rats exhibit some of the properties of young cells following an increase in blood serum ganglioside level, as well as, in in vitro conditions, after the addition of gangliosides to the culture medium.Together with data on ultradian functional and metabolic rhythms, all the material reviewed here allows us to propose a mechanism of direct cell-cell cooperation via the medium in which the cells exist, that supplements the nervous and hormonal central regulation of organ functions. Ultradian intracellular rhythms may thus provide a finer framework within which the integrated dynamics of respiration, heart rate, brain activity, and even behavioural patterns, are brought to an optimal functional pattern. Innate and direct cell-cell cooperation may have been employed as a means of intercellular regulation during the course of metazoan evolution, that preceded nervous regulation and is presently retained in mammals.     

Decrease of intracellular ATP content downregulated UCP2 expression in mouse hepatocytes

Mitochondrial uncoupling protein 2 (UCP2) plays an important role in regulating energy metabolism. We previously reported that UCP2 expression in steatotic livers is increased which leads to diminished hepatic ATP stores and renders steatotic hepatocytes vulnerable to ischemic damage. In this study, reagents that inhibit the production of ATP were used to mimic an ischemic state in the liver in order to investigate the effects of decreased intracellular ATP levels on UCP2 expression in a murine hepatocyte cell line (HEP6-16). Carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), an oxidative phosphorylation uncoupler, was found to decrease intracellular ATP levels in a dose- and time-dependent manner. Relatively high concentrations of FCCP from 8 to 80 microM were required to reduce the intracellular concentration of ATP. The inhibitory effect of FCCP on intracellular ATP was significantly potentiated by 2-deoxy-D-glucose, an inhibitor of glycolysis that when administered alone had no negative effect on cellular ATP levels in mouse hepatocytes. Decreased intracellular ATP levels were accompanied by lower UCP2 mRNA expression. Upon removal of FCCP and/or 2-deoxy-D-glucose and reculture with normal medium, ATP and UCP2 mRNA levels returned to normal within a few hours. Mitochondrial membrane potential in HEP6-16 cells was dissipated by 80 microM FCCP but not 8 microM FCCP, suggesting that the downregulation of UCP2 expression by FCCP was not related to mitochondrial potential changes. Consequently, the in vitro manipulation of ATP stores is consistent with the in vivo observations associated with ischemia/reperfusion injury.     

The involvement of cellular ATP in receptor-mediated internalization of epidermal growth factor and hormone-induced internalization of beta-adrenergic receptors

Beta-Adrenergic receptors and epidermal growth factor receptors are both expressed on the cell surface of human astrocytoma cells. Incubation with a catecholamine or epidermal growth factor results in rapid internalization of the respective receptor. The internalized receptors co-migrate in light fractions on sucrose gradients. Astrocytoma cells maintain a constant ATP concentration by either glycolytic or mitochondrial ATP production. When cells are incubated in a medium depleted of substrates for glycolysis and gluconeogenesis, addition of inhibitors of mitochondrial ATP synthesis causes a rapid reduction in cellular ATP content. An immediate return to control ATP levels occurs upon addition of an appropriate nutrient, such as glucose. Decreasing the cellular ATP content to less than 10% of control markedly inhibits internalization of beta-adrenergic receptors and epidermal growth factor. The inhibition of endocytosis is reversed as soon as the intracellular ATP content is restored. Previous work by others (Clarke, B.L., and Weigel, P.H. (1985) J. Biol. Chem. 260, 128-133) suggested that ATP is not required for internalization (per se) of asialoglycoprotein in hepatocytes but was required for recycling of the asialoglycoprotein receptor. In contrast, our results indicate that in astrocytoma cells the process of internalization of epidermal growth factor and beta-adrenergic receptors, per se, is highly ATP dependent.     

Methanogenesis and ATP synthesis in methanogenic bacteria at low electrochemical proton potentials. An explanation for the apparent uncoupler insensitivity of ATP synthesis

The rate of methane formation from H2 and CO2, the intracellular ATP content and the electrochemical proton potential (delta mu H+) were determined in cell suspensions of Methanobacterium thermoautotrophicum, which were permeabilized for K+ with valinomycin (1.2 mumol/mg protein). In the absence of extracellular K+ the cells formed methane at a rate of 4 mumol min-1 (mg protein)-1, the intracellular ATP content was 20 nmol/mg protein and the delta mu H+ was 200 mV (inside negative). When K+ was added to the suspensions the measured delta mu H+ decreased to the value calculated from the [K+]in/[K+]out ratio. Using this method of delta mu H+ adjustment, it was found that lowering delta mu H+ from 200 mV ([K+]in/[K+]out = 1000) to 100 mV ([K+]in/[K+]out = 40) had no effect on the rate of methane formation and on the intracellular ATP content. At delta mu H+ values below 100 mV ([K+]in/[K+]out less than 40) both the rate of methanogenesis and the ATP content decreased. Methanogenesis completely ceased and the ATP content was 2 nmol/mg when delta mu H+ was adjusted to values lower 50 mV ([K+]in/[K+]out less than 7). The data show that methanogenesis from H2 and CO2 and ATP synthesis in M. thermoautotrophicum are possible at relatively low electrochemical proton potentials. Similar results were obtained with Methanosarcina barkeri. Protonophoric uncouplers like 3,5,3',4'-tetrachlorosalicylanilide (TCS) or 3,5-di-tert-butyl-4-hydroxy-benzylidenemalononitrile (SF 6847) were found not to dissipate delta mu H+ below 100 mV in M. thermoautotrophicum even when used at high concentrations (400 nmol/mg protein). This finding explains the observed uncoupler insensitivity of methanogenesis and ATP synthesis in this organism.     

Monitoring the progress of infection and recombinant protein production in insect cell cultures using intracellular ATP measurement

Several monitoring methods used to predict viable cell density have been the subject of extensive studies, including oxygen uptake rate, carbon dioxide evolution rate, optical density, NADH-dependent fluorescence and relative permittivity measurement . We propose intracellular ATP determination by bioluminescence assay to monitor the progress of baculovirus infection and recombinant protein production in insect cell cultures. We found that the ATP content in viable cells increased after virus addition. The increase in the ATP level was observed until the maximum recombinant protein accumulation was reached. At maximum product yield, the specific ATP content significantly decreased. Results obtained in both batch and fed-batch cultures demonstrated that the specific ATP level could be considered as a good indicator of recombinant protein productivity. Monitoring the cellular ATP content after viral infection makes it possible to define the optimum time for product harvest. The main advantage of applying the ATP assay as an index of the progress of infection and recombinant protein synthesis is its short time and sensitivity.     

Regulation of protein synthesis in intact rat liver by calcium mobilizing agents

1. Exposure of intact perfused rat liver to EGTA, vasopressin or phenylephrine resulted in a rapid decrease in polysome formation. Pretreatment with phentolamine, an alpha-adrenergic antagonist, blocked the effect of phenylephrine. 2. Hormonal inhibitions of leucine incorporation into protein in isolated hepatocytes and of polysome formation in perfused liver were reversed in the presence of supraphysiologic extracellular Ca2+ concentrations. 3. The beta-adrenergic agonist isoproterenol exerted minimal effects on polysome content. 4. It is proposed that intracellular Ca2+ stores sensitive to hormonal modulation are necessary for maintenance of protein synthesis in hepatocytes.     

Increased mitochondrial fusion and autophagy help isolated hepatocytes repolarize in collagen sandwich cultures

Freshly isolated, depolarized rat hepatocytes can repolarize into bile canalicular networks when plated in collagen sandwich cultures. We studied the events underlying this repolarization process, focusing on how hepatocytes restore ATP synthesis and resupply biosynthetic precursors after the stress of being isolated from liver. We found that soon after being plated in collagen sandwich cultures, hepatocytes converted their mitochondria into highly fused networks. This occurred through a combination of upregulation of mitochondrial fusion proteins and downregulation of a mitochondrial fission protein. Mitochondria also became more active for oxidative phosphorylation, leading to overall increased ATP levels within cells. We further observed that autophagy was upregulated in the repolarizing hepatocytes. Boosted autophagy levels likely served to recycle cellular precursors, supplying building blocks for repolarization. Repolarizing hepatocytes also extensively degraded lipid droplets, whose fatty acids provide precursors for β-oxidation to fuel oxidative phosphorylation in mitochondria. Thus, through coordination of mitochondrial fusion, autophagy, and lipid droplet consumption, depolarized hepatocytes are able to boost ATP synthesis and biosynthetic precursors to efficiently repolarize in collagen sandwich cultures.     

Importance of glycolysis for the energetics of anoxia-tolerant and anoxia-intolerant teleost hepatocytes

The importance of glycolysis, as an ATP-producing and substrate-providing pathway, was studied in anoxia-tolerant (goldfish) and anoxia-intolerant (trout) hepatocytes. Inhibition of glycolysis with iodoacetic acid (IAA) left aerobic ATP production largely unaffected in hepatocytes from both species but caused a significant decrease of ATP contents in the goldfish cells. Ouabain-sensitive oxygen consumption (osVo2), an estimate of mitochondrial ATP production coupled to ATP consumption by the Na(+) pump, was significantly reduced in IAA-treated goldfish hepatocytes, whereas it was unaltered in trout hepatocytes. Partial reduction of mitochondrial respiration, achieved by titration with cyanide (CN), strongly stimulated glycolytic flux but did not affect ATP contents of hepatocytes from both species. Under these conditions, osVo2 became undetectable. Rb(+)-uptake rates, providing a direct estimate of Na(+)-pump activity, were in good agreement with estimates derived from osVo2 in IAA-treated cells, showing a decrease in goldfish and no change in trout. However, they indicated persistent Na(+)-pump activity despite the lack of osVo2 in CN-treated cells. Overall, these data indicate that in goldfish hepatocytes Na(+)-pump activity is more dependent on glycolytic ATP production as compared to trout hepatocytes. Protein synthesis of goldfish hepatocytes was inhibited in IAA- and CN-treated cells, possibly reflecting the hierarchical organization of energy metabolism. In trout hepatocytes, protein synthesis could be sustained at control levels, given that energetic substrate provision was not limited.