Glutamic Acid Signal Synchronizes Protein Synthesis Kinetics in Hepatocytes from Old Rats for the Following Several Days. Cell Metabolism Memory.

The kinetics of protein synthesis was investigated in primary cultures of hepatocytes from old rats in serum-free medium. The rats were fed mixed fodder supplemented with glutamic acid and then transferred to a regular mixed fodder. The amplitude of protein synthesis rhythm in hepatocytes isolated from these rats increased on average 2-fold in comparison with the rats not receiving glutamic acid supplement. Based on this indicator reflecting the degree of cell-cell interactions, the cells from old rats were not different from those of young rats. The effect was preserved for 3-4 days. These results are discussed in connection with our previous data on preservation of the effect of single administration of gangliosides, noradrenaline, serotonin, and other synchronizers on various cell populations. In contrast to the other investigated factors, glutamic acid is capable of penetrating the blood-brain barrier, which makes its effect possible not only in the case of hepatocytes and other non-brain cells, but also in neurons.

Glutamic Acid as Enhancer of Protein Synthesis Kinetics in Hepatocytes from Old Rats.

Dense cultures of hepatocytes from old rats (~2 years old, body weight 530-610 g) are different from similar cultures of hepatocytes from young rats by the low amplitude of protein synthesis rhythm. Addition of glutamic acid (0.2, 0.4, or 0.6 mg/ml) into the culture medium with hepatocytes of old rats resulted in increase in the oscillation amplitudes of the protein synthesis rhythm to the level of young rats. A similar action of glutamic acid on the protein synthesis kinetics was observed in vivo after feeding old rats with glutamic acid. Inhibition of metabotropic receptors of glutamic acid with α-methyl-4-carboxyphenylglycine (0.01 mg/ml) abolished the effect of glutamic acid. The amplitude of oscillation of the protein synthesis rhythm in a cell population characterizes synchronization of individual oscillations caused by direct cell-cell communications. Hence, glutamic acid, acting as a receptor-dependent transmitter, enhanced direct cell-cell communications of hepatocytes that were decreased with aging. As differentiated from other known membrane signaling factors (gangliosides, norepinephrine, serotonin, dopamine), glutamic acid can penetrate into the brain and thus influence the communications and protein synthesis kinetics that are disturbed with aging not only in hepatocytes, but also in neurons.

Glutamic Acid - Amino Acid, Neurotransmitter, and Drug - Is Responsible for Protein Synthesis Rhythm in Hepatocyte Populations in vitro and in vivo.

Primary cultures of rat hepatocytes were studied in serum-free media. Ultradian protein synthesis rhythm was used as a marker of cell synchronization in the population. Addition of glutamic acid (0.2 mg/ml) to the medium of nonsynchronous sparse cultures resulted in detection of a common protein synthesis rhythm, hence in synchronization of the cells. The antagonist of glutamic acid metabotropic receptors MCPG (0.01 mg/ml) added together with glutamic acid abolished the synchronization effect; in sparse cultures, no rhythm was detected. Feeding rats with glutamic acid (30 mg with food) resulted in protein synthesis rhythm in sparse cultures obtained from the rats. After feeding without glutamic acid, linear kinetics of protein synthesis was revealed. Thus, glutamic acid, a component of blood as a non-neural transmitter, can synchronize the activity of hepatocytes and can form common rhythm of protein synthesis in vitro and in vivo. This effect is realized via receptors. Mechanisms of cell-cell communication are discussed on analyzing effects of non-neural functions of neurotransmitters. Glutamic acid is used clinically in humans. Hence, a previously unknown function of this drug is revealed.

[Blockade of proteasome activity disturbs the rhythm of synthesis of the protein marker of direct cell-cell interactions].

The effect of inhibition of proteasome activity on direct cell-cell interactions in primary hepatocyte cultures was studied. The circahoralian rhythm of protein synthesis was a marker of cell-cell communication. The addition of the proteasome inhibitor MG132 at doses of 10 or 20 µM to the medium with hepatocyte cultures for 19 h resulted in a significant reduction in the total pool of 3H-leucine in cells. The incorporation of leucine into proteins changed slightly or negligibly, whereas the content of free labeled leucine in hepatocytes decreased. The rhythm of protein synthesis was distorted compared to the control. The rhythm was restored by external organizers, such as gangliosides and melatonin, as well as by enhancing the activity of protein kinases--the key factor in the organization of the rhythm of protein synthesis. A short-term (3-h) exposure to MG132 did not change the pool of leucine, but the rhythm of protein synthesis was also disturbed. Thus, protein catabolism affects cell-cell interactions organizing the rhythm of protein synthesis. Another factor of the downregulation of the rhythm of protein synthesis, the secretion of proteins from the hepatocytes in vivo, which was shown in vivo in many studies, was also revealed in our study when measuring the content of proteins stained with Coomassie Brilliant Blue G250 in the medium with hepatocyte cultures.

Administration of dopamine to rats disorganizes the rhythm of protein synthesis in hepatocytes.

Dopamine was injected intravenously (9 µg/kg) or intraperitoneally (15 µg/kg) to Wistar rats (3-4 months, 300-400 g). Hepatocytes were isolated 40 min after dopamine injection. Dense cultures were maintained on collagen-coated glasses. By the 5th hour, the circaholarian rhythm of protein synthesis in hepatocytes cultures was absent in the dopamine group, but was present in cultures from animals receiving physiological saline (NaCl). The rhythm-disorganizing effect of dopamine was reversible. The rhythm was observed in cultures of hepatocytes isolated 1 day after dopamine treatment. The effect of dopamine was abolished by melatonin. The protein synthesis rhythm was revealed in 5-h cultures of hepatocytes from rats receiving melatonin (32 ng/kg) 40 min after intraperitoneal injection of dopamine. The results of our in vitro experiments with addition of dopamine into the medium of cultured hepatocytes [1] suggest that dopamine in vivo produces a direct effect on liver cells. The observed changes are discussed taking into account the biochemical mechanisms for a direct cell-cell interaction and previously unknown properties of catecholamines.

Effect of protein synthesis rhythm-organizing signal persists for a day after single administration of melatonin to rat.

Melatonin administered to rat intraperitoneally organizes ultradian rhythm of protein synthesis in hepatocytes that persists for 1 day after exposure to the synchronizing signal. Hepatocytes were isolated 1 day after melatonin administration and cultured on coverslips in a serum-free medium. In 24 h in culture, the kinetics of protein synthesis was analyzed. In our previous experiments, we detected a rhythm in cells isolated in 1.5 h, but not in 3 days after melatonin administration to the rat. We have found that synchronization of oscillations of the protein synthesis intensity in vivo persists over 1 day after rat exposure to melatonin. Phenylephrine, an efficient synchronizer of protein synthesis in vitro, does not organize the rhythm in vivo.

Dopamine disorganizes the rhythm of protein synthesis disrupting self-organization of hepatocytes in vitro.

We studied dense 24-hour cultures of rat hepatocytes in serum-free medium on collagen-coated slides. As before, a circahoralian rhythm of protein synthesis was observed in control cultures in a fresh medium. No rhythm was found after addition of 1-10 µM dopamine to the medium containing such cultures. The rhythm was observed after addition of 0.3 µM ganglioside to pretreated-dopamine cultures. Dopamine is likely to influence the conditioning of intercellular medium with gangliosides. Deficit of this endogenous synchronizing factor in the intercellular medium blocks self-organization of the protein synthesis rhythm. Thus, in contrast to previously studied norepinephrine and serotonin, as well as gangliosides, which organized the population rhythm of protein synthesis, dopamine disorganized the rhythm, impairing direct intercellular interactions.

[Mesenchymal stromal cells synchronize the rhythm of protein synthesis under the effect of an exogenous signal].

A comparative study was performed of dense 5-hour cultures of rat hepatocytes and equal-density cultures of mesenchymal stromal cells (MSC) isolated from human adipose tissue of rat bone marrow. The cells were grown on collagen-coated class slides in serum-free medium. Unlike in hepatocytes, no rhythm of protein synthesis was initially revealed in MSC, but such a rhythm manifested itself when the culture medium was supplemented with melatonin (2 nM, 5 min). The results of experiments with cytoplasmic calcium chelator BAPTA-AM and protein kinase inhibitor H7 indicate that the mechanism of protein synthesis synchronization in MSC consists in calcium-dependent phosphorylation of cell proteins.

[Self-synchronization of the protein synthesis rhythm in HaCaT cultures of human keratinocytes].

In cultures of human keratinocytes HaCaT contained in a serum-free medium on glass, a circahoralian rhythm of protein synthesis was found similar to the one in hepatocytes in vitro. The intensity of the synthesis was determined by the inclusion of 3H-leucine corrected for the pool of free marked leucine. Rhythm was studied in washed 1- or 2-day cultures after the change of the medium. The medium conditioned with keratinocytes HaCaT synchronized the rarefied hepatocyte cultures nonsynchronous in the control. Therefore, the keratinocytes liberate synchronizing factors into the medium. A BAPTA-AM chelator of calcium ions eliminates the protein synthesis rhythm both in dense hepatocyte cultures synchronous in the control and in the HaCaT keratinocyte cultures. The effect of the H7 inhibitor of protein kinases was analogous. Thus, both in keratinocytes and hepatocytes, self-synchronization of fluctuations of the intensity of protein synthesis takes place. The mechanism of self-synchronization is the calcium-depending phosphorylation of cell proteins.

[Melatonin injected into rat efficiently synchronizes the rhythm of protein synthesis in primary hepatocyte cultures isolated from this rat].

Melatonin injected intraperitoneally into rat synchronizes the ultradian rhythm of protein synthesis after 100 min in primary hepatocyte cultures isolated from this rat, which are studied after 1 or 2 days. The effective synchronization concentrations of melatonin--0.01-0.02 microg per kg of rat weight--are three orders lower than melatonin doses used in clinical practice in human treatment.

[Signaling factors of self-organization of protein synthesis rhythm in hepatocyte cultures--gangliosides and catecholamines function independently].

Considering the data on the low level of self-organization (self-synchronization) of protein synthesis rhythm in aging, we studied the possible interference of the signaling factors of self-organization, gangliosides and catecholamines, as well as catecholamine reception. Experiments were carried out on primary cultures of rat hepatocytes on slides. Inhibited ganglioside synthesis did not prevent the organization of protein synthesis rhythm by the alpha-adrenomimetic agent phenylephrine. Upon the blockade of alpha-receptors by prazosin, the protein synthesis rhythm was observed after the exposure to gangliosides. Alpha-adrenolytic agents prazosin and benoxathian abolished the synchronizing effect of the beta-adrenomimetic isoproterenol. A mixture of alpha- and beta-adrenomimetic agents inhibited the protein synthesis rhythm-organizing effect of noradrenaline. Thus, the signaling molecules of self-organization of protein synthesis function independently via specific receptors.

[Melatonin promotes and synchronizes protein synthesis in hepatocyte culture from old rats].

The effect of 1 to 1000 nM melatonin was studied on daily cultures of rat hepatocytes on slides in serum-free medium. The minimum melatonin concentration (1 nM) proved to synchronize protein synthesis in asynchronous sparse cultures of hepatocytes from rats of different age, and a circahoralian rhythm of protein synthesis was revealed in them. In dense weekly synchronous hepatocytes from old rats (2.5-years-old with the weight of about 600 g), melatonin improved cell synchronization to the level of young animals. Melatonin treatment increased the mean rate of protein synthesis in rats of different age.

[Mechanism of direct cell interactions. Self-organization of protein synthesis rhythm].

Primary 24-hour cultures of hepatocytes on slides in a serum-free medium were studied. Circahoralian rhythm of protein synthesis served as a marker of cell cooperation. Stimulation of protein kinase activities by phorbol 12-myristate 13-acetate at 0.5 or 1.0 microM or forskolin at 10 microM led to visualization of the protein synthesis rhythm in sparse cultures, which were asynchronous in the control and with linear kinetics of protein synthesis. Inhibitors of protein kinase activities H7 (1-(5-isoquinolinylsulfonyl)-5-methylpiperasine dihydrochloride) at 40 microM or H8 (N-(2-[methylamino]ethyl)-5-isoquinolinesulfonamide hydrochloride) at 25 microM eliminated the protein synthesis rhythm in dense cultures, which are normally synchronous with oscillatory kinetics of protein synthesis. After inhibition of the protein kinase activities, gangliosides or phenylephrine did not synchronize the protein synthesis rhythm. Phorbol 12-myristate 13-acetate modulated the protein synthesis rhythm, shifted the rhythm phase, i.e., stimulation of the protein kinase activities, and, correspondingly, protein phosphorylation may be a factor of synchronization of synthesis oscillations in individual cells and of population rhythm formation. Thus, a cascade of processes leading to self-organization of hepatocytes during formation of summarized protein synthesis was revealed in a series of studies: signal of gangliosides or other calcium agonists-->changes in the level of calcium ions in cytoplasm-->increased protein kinase activities-->protein phosphorylation-->modulation of individual oscillations in the intensity of protein synthesis and their coordination in a summarized rhythm. cAMP-dependent protein kinases also affect the protein synthesis rhythm. Protein phosphorylation is a key process. The mechanisms of cell self-organization are similar in vitro and in vivo, specifically in the liver in situ.

[Aftereffect of synchronizers of protein synthesis rhythm in hepatocytes in vitro].

The effect of gangliosides and phenylephrine synchronizing the protein synthesis rhythm was preserved in hepatocytes cultured in the normal serum-free medium for one-two days. Hence, the membrane signal triggers intracellular, as was shown by us earlier, calcium-dependent processes, which regulate the kinetics of protein synthesis for a certain time after the signal perception.

[Age-related features of protein synthesis rhythm in hepatocytes. Effects of extracellular medium].

Cell interactions have been studied in cultures pf hepatocytes from young and old rats. The rhythm of protein synthesis is an index of cell interaction and synchronization in culture, while the amplitude of oscillations characterized cell cooperation in an aggregate rhythm. The mean rhythm amplitude in the culture of hepatocytes from old rats is twice lower than that from young rats. Gangliosides (mixture, bovine brain gangliosides) and alpha1-adrenomimetic phenylephrine enhanced synchronization of cultures of the cells from old rats and increased the amplitude of oscillations to the level of young animals. Addition of rat blood serum (10%) to the medium revealed the rhythm of protein synthesis in the culture, asynchronous in the control, i.e., led to their synchronization. In media with young and old rat blood sera, oscillations were intense, with high amplitudes, and low, respectively. Addition of bovine brain gangliosides to a medium with old rat blood serum increased the amplitudes of oscillations to a level of the rhythm stimulated by the young rat serum. Thus, the cells of old animals can fully perceive synchronizing factors and, in the case of their increased concentration, the rhythm of protein synthesis in old animals did not differ from that in young rats. Current data on biochemical mechanisms underlying intercellular cooperation in the formation of population rhythm of protein synthesis have been discussed.

[Cooperation of the hepatocytes in vitro in the rythm of the protein synthesis is intensified by GM1 ganglioside in vesicles and liposomes]. / Kooperatsiia gepatotsitov in vitro v ritme sinteza belka intensifitsiruetsia gangliozidom GM1 v vezikulakh i liposomakh.

The medium conditioned by dense, self-synchronized hepatocyte cultures was centrifuged at 150,000 g to obtain two fractions. The light fraction (supernatant fluid) contained ganglioside monomers and micelles, and the heavy fraction (pellet) contained gangliosides in the vesicles shed from the cell membrane. In the test populations of hepatocytes, the rhythm of protein synthesis was used as an indicator of cell synchronization resulting from their cooperative activity. Low-density hepatocyte cultures with asynchronous fluctuations of protein synthesis proved to be synchronized by both the initial conditioned medium and its vesicular fraction. Our previous studies have shown that this occurs under the effect of GM1 monosialoganglioside, which is released from cultured cells and accumulates in the conditioned medium. Liposomes consisting of GM1 and phosphatidylcholine from egg yolk (1:19 mol%), compared to free exogenous GM1, synchronized the rhythm of protein synthesis more effectively: synchronization was observed at a GM1 concentration in liposome suspension of only 0.0003 microM, compared to 0.06 microM and higher in the case of free GM1. Thus, GM1 as a component of membranes and monolayer lipid structures proved to be much more effective than free GM1 in promoting hepatocyte cooperation with respect to the rhythm of protein synthesis.

[Changes in the concentration of calcium ions and rhythm of protein synthesis in the culture of hepatocytes]. / Izmeneniia kontsentratsii ionov kal'tsiia i ritm sinteza belka v kul'ture gepatotsitov.

We studied the effects of the chelating agents of extra- and intracellular calcium ions, EGTA and BAPTA-AM, and of the inhibitor of Ca2+ release from the reticulum, TMB-8, in the kinetics of protein synthesis in hepatocyte cultures. We also studied dense cultures capable of self-synchronization of protein synthesis oscillations and diluted cultures, in which synchronization is induced by phenylephrine or gangliosides (standard preparation of total gangliosides from the bovine brain). Preincubation of the diluted or dense cultures in the presence of 2 mM EGTA for 1-2 h with subsequent protein assay in a medium with EGTA did not affect the kinetics of protein synthesis: no rhythm was found in the diluted cultures, while it was preserved in the dense cultures. When the diluted cultures preincubated in the presence of EGTA were placed in a medium with EGTA and 2 microM phenylephrine for 2 min, the rhythm was visualized. The treatment of diluted cultures with 100 microM TMB-8 for 5 or 10 min with subsequent washing and incubation in a medium with 3 microM gangliosides led to visualization of the protein synthesis rhythm, i.e., to the synchronization of oscillations, while no rhythm was found in the standard cultures. Preincubation of the diluted cultures in a medium with 10, 15, or 20 microM BAPTA-AM did not affect the kinetics of protein synthesis. When, after such preincubation, the diluted cultures were placed in the medium with gangliosides, the rhythm was visualized. In the dense cultures, normally capable of self-synchronization, no rhythm of protein synthesis was found after their treatment with 10-20 microM BAPTA-AM for 1 h. The transfer of such cultures in the medium with gangliosides led to visualization of the rhythm. Thus, calcium affects the kinetics of protein synthesis: after the rise of Ca2+ in the cytoplasm was blocked, the rhythm of protein synthesis was not visualized due, supposedly, to disturbed mechanisms of medium conditioning. However, exogenous gangliosides in the dense or diluted cultures preincubated in the presence of BAPTA-AM ore TMB-8 allowed the rhythm visualization, i.e., synchronization may not depend on changes in the intracellular calcium concentration.

Phenotype determination of anti-GM3 positive cells in atherosclerotic lesions of the human aorta. Hypothetical role of ganglioside GM3 in foam cell formation.

Earlier we reported that atherosclerotic plaques contain cells which were specifically and very intensively stained with anti-GM3 antibodies although no GM3 positive cells were detected in the normal non-diseased arterial intima. Because of their lipid inclusions, GM3 positive cells in atherosclerotic lesions seemed to be foam cells but their origin needed clarification. Using an immunohistochemical technique in the present work, we showed that some of these foam cells contained CD68 antigen. However, the most intense accumulation of GM3 occurred in the areas composed of foam cells which did not stain with any cell type-specific antibodies, including antibodies to macrophages (anti-CD68) and smooth muscle cells (anti-smooth muscle alpha-actin), perhaps, because the cell type-specific antigens were lost during the transformation of intimal cells into foam cells. Ultrastructural analysis of the areas where foam cells overexpressed GM3 demonstrated that some foam cells lacked both a basal membrane and myofilaments but contained a large number of secondary lysosomes and phagolysosomes, morphological features which might indicate their macrophage origin. Other foam cells contained a few myofilaments and fragments of basal membrane around their plasmalemmal membrane, suggesting a smooth muscle cell origin. These observations indicate that accumulation of excessive amounts of GM3 occurs in different cell types transforming into foam cells. We suggest that up-regulation of GM3 synthesis in intimal cells might be an essential event in foam cell formation. Shedding of a large number of membrane-bound microvesicles from the cell surface of foam cells was observed in areas of atherosclerotic lesions corresponding to extracellular GM3 accumulation. We speculate that extracellularly localised GM3 might affect the differentiation and modification of intimal cells in atherosclerotic lesions.

[The adrenoreceptor blocker prazosin does not prevent synchronization of the protein biosynthesis rhythm by exogenous gangliosides in the hepatocyte culture]. / Blokator adrenoretseptorov prazozin ne prepiatstvuet sinkhronizatsii ritma sinteza belka ékzogennymi gangliozidami v kul'ture gepatotsitov.

We studied the effect of the alpha 1-adrenolytic prazosine on dense cultures of hepatocytes, which are normally characterized by the protein synthesis rhythm, and diluted cultures, in which such a rhythm is revealed after external synchronization. Exogenous gangliosides (a fraction of the total gangliosides of the bovine brain) then synchronize the rhythm in diluted cultures; this effect is also displayed in the presence of 10(-7) M prazosine. The synchronizing effect of the medium conditioned by dense cultures was also preserved in the presence of prazosine. In the dense cultures that don't normally require external synchronization, prazosine affected intensified the rhythmic patter of changes in the protein synthesis. After a total of 0.3 microM gangliosides were introduced in the medium with prazosine-pretreated dense cultures, the protein synthesis rhythm was visualized. We propose that, while blocking adrenoreceptors, prazosine does not prevent the action of exogenous synchronizing factors on the hepatocytes, but inhibits the release of such factors from the cell.

[Phenylephrine synchromizes rhythm of protein synthesis in hepatocyte culture]. / Feniléfrin sinkhroniziruet ritm sinteza belka v kul'turakh gepatotsitov.

Published data indicate that 1-3 microM adrenomimetic phenylephrine increases the concentration of calcium ions in the cytoplasm of cultured hepatocytes. We studied low-density cultures exhibiting no protein synthesis rhythm in the fresh medium and demonstrated that a 2 min action of 2 microM phenylephrine induces protein synthesis rhythm, i.e., synchronizes the synthesis oscillations in hepatocytes. A similar effect was observed for a selective inhibitor of the reticulum calcium pump, di-tert-butyl-benzohydroquinone, that increases the cytoplasm concentration of calcium ions by a receptor-independent mechanism. A calcium antagonist imipramine obviated the synchronization effect of phenylephrine. We propose that short-term changes in the cytoplasm concentration of calcium ions covering the whole cell population are among intracellular mechanisms of protein synthesis synchronization in hepatocytes in vitro.