[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 modifies the rhythm of protein synthesis.

Melatonin (5 nM) added to medium with primary hepatocyte cultures shifted the phase of circahoralian rhythm of protein synthesis and hence, can be a factor synchronizing fluctuations in protein synthesis and rhythm organizer in the hepatocyte population. Blockade of melatonin receptors with luzindole (20 nM) arrested rhythm organization of protein synthesis by melatonin. Prospects of studying biochemical mechanisms of protein synthesis rhythm organization with other drugs (calcium agonists, similarly to melatonin) are discussed.

[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.

[Disturbed cooperation of hepatocytes in the rhythm of protein synthesis by chelating agent of cytoplasmic calcium BAPTA-AM]. / Narushenie kooperatsii gepatotsitov v ritme sinteza belka khelatorom tsitoplasmaticheskogo kal'tsiia BAPTA-AM.

Previously we demonstrated synchronized oscillations of protein synthesis rate in the hepatocyte cultures upon accumulation of monosialognaglioside GM1 in the medium or after introduction of exogenous GM1 to the medium. The synchronized oscillations of the protein synthesis rate in dense hepatocyte cultures were blocked 30 min after their treatment with 10-20 microM BAPTA-AM--a chelating agent of cytoplasmic calcium. Enzyme immunoassay for GM1 demonstrated similar amounts of GM1 in the medium conditioned for 3 h by dense hepatocyte cultures pretreated with 20 microns]M BAPTA-AM for 1 h and in the medium of normal dense cultures--0.0060 +/- 0.0005 and 0.0055 +/- 0.0005 pmol/1000 cells, respectively. The content of GM1 was also similar in the normal and BAPTA-AM-pretreated hepatocytes--0.158 +/- 0.013 and 0.183 +/- 0.014 pmol/1000 cells, respectively. The synchronized rhythm of protein synthesis has been confirmed in the diluted cultures in the medium conditioned by the normal dense cultures. However, the medium conditioned by the dense cultures pretreated with BAPTA-AM induces no synchronization of the diluted cultures. Since GM1 concentration was normal in this medium, we propose the effect of physicochemical form of the gangliosides accumulated in the medium on their ability to synchronize the rhythm of protein synthesis.

[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.

[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.

[Rhythm of protein synthesis in cultures of hepatocytes from rats of different ages. Norm and effect of the peptide livagen]. / Ritm sinteza belka v kul'turakh gepatotsitov krys raznogo vozrasta. Norma i deistvie peptida livagena.

The circumhoralian rhythm of protein synthesis was determined in a monolayer culture of hepatocytes from rats at the age of 1 to 24 months and weighing from 45 to 480 g, respectively. The peptide lyvagen (Lys-Glu-Asp-Ala) obtained by directed chemical synthesis on the basis of amino acid analysis of the liver polypeptide preparations increased the level of protein synthesis in the hepatocytes from rats of different ages; the highest effect was observed in the cells of old animals. In old rats, lyvagen increased the amplitude of protein synthesis fluctuations. The peptide epitalon (Ala-Glu-Asp-Gly) constructed on the basis of analysis of the epiphysis peptides did not change the intensity of protein synthesis in the cultured hepatocytes.

[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.

Ganglioside-mediated metabolic synchronization of protein synthesis activity in cultured hepatocytes.

An ultradian oscillation of protein synthesis was detected by synchronization of metabolic activity in rat hepatocyte cultures. This oscillation occurs in dense cultures in fresh medium, but not in sparse ones. Metabolic synchronization of sparse cultures, however, was initiated by conditioned medium or addition of 0.3-0.5 microm of a mixture of bovine brain gangliosides to fresh culture medium along with either 0.06-0.2 microm GM1 or 0.1-0.2 microm GDIa. GTIb and GDIb did not produce oscillations, nor did human liver ganglioside GM3. High expression of GM1 ganglioside determinants in hepatocytes maintained in the conditioned medium purified polyclonal antibodies to GM1 was coupled with protein synthetic oscillatory activity, i.e. metabolic synchronization. Incubation of dense cultures with GM1-antibodies for 24 h decreased the amplitude of these oscillations. In sparse cultures maintained in fresh medium where protein synthesis showed no oscillatory pattern, GM1 expression was low.

[The synchronization of the rhythm of protein synthesis in hepatocyte cultures occurs during conditioning of the medium with ganglioside GM1 accumulation in the cells: an immunohistochemical study]. / Sinkhronizatsiia ritma sinteza belka v kul'turakh gepatotsitov proiskhodit pri konditsionirovanii sredy s nakopleniem gangliozida GM1 v kletkakh: immunogistokhimicheskoe issledovanie.

We studied the kinetics of proteins synthesis in the cultures of hepatocytes on collagen-coated slides in medium 199 enriched with 0.2 mg/ml albumin and 0.5 microgram/ml insulin and devoid of bovine serum. Circahoral fluctuations of proteins synthesis intensity were found in the monolayer cultures and sparse cultures in a conditioned medium. No protein synthesis rhythm was expressed in a fresh medium after repeated washing of the cultures. Addition of micromolar concentrations of gangliosides GM1 or GD1a to the medium revealed the rhythm in washed cultures. Addition of GT1b or GM3 to the medium did not synchronize the oscillations: the kinetics of protein synthesis did not differ from that in the control space cultures in a fresh medium without exogenous gangliosides. Accumulation of gangliosides GM1 and GM3 in the hepatocytes in vitro upon conditioning of a serum-free culture medium was shown using the indirect immunocytochemical method. The effect was found in dense monolayer cultures and in cultures with separated cells in a conditioned medium. The protein synthesis rhythm was found in such cultures. Gangliosides are weakly expressed in most cells of a repeatedly washed 24-hour monolayer and washed sparse cultures. No protein synthesis rhythm was found in washed cultures. Similar changes in the dynamics of the culture medium conditioning, accumulation of gangliosides in cells and rhythmic activity of cells population confirm the concept of the synchronizing role of gangliosides.

[Gangliosides synchronize the rhythm of protein synthesis in hepatocytes in vitro]. / Gangliozidy sinkhroniziruiut ritm sinteza belka v gepatotsitakh in vitro.

The kinetics of protein synthesis was studied in the cultures of rat hepatocytes on slides covered with collagen or fibronectin and kept in a serum-free medium 199 complemented by albumin and insulin. No protein synthesis rhythm was found in the cultures with markedly separated cells obtained from a diluted suspension of hepatocytes ("sparse cultures"). After 0.3-0.4 microM total gangliosides were added to the medium with such cultures, the rhythm was found in all experiments. Such an effect was earlier found under the influence of a medium conditioned by a monolayer on the sparse cultures. The results obtained suggest the involvement of gangliosides in synchronization of oscillations of the protein synthesis intensity and call for studies of the conditioned medium properties.

[A serum-free medium that preserves the normal morphology and high protein-synthesis level in hepatocytes in vitro]. / Bessyvorotochnaia sreda, sokhraniaiushchaia normal'nuiu morfologiiu i vysokii uroven' sinteza belka v gepatotsitakh in vitro.

In a culture of rat hepatocytes, kept in a serum-free medium 199, the number of attached cells was decreased and a monolayer was not formed within 24 h. Soon after the cells were attached to collagen, the intensity of protein synthesis in the serum-free medium was decreased due to the smaller number of cells in the culture and was calculated per cell. The properties of cultures, i.e., the number and shape of cells, external appearance of the culture, and intensity of protein synthesis, were similar in a serum-containing medium and in the serum-free medium 199 complemented with 0.2 mg/ml albumin and 5 micrograms/ml insulin. A monolayer typical for the medium with serum was formed in the medium with albumin and insulin within approximately 20 h after attachment of the hepatocytes to the substrate.

[The intensity of protein synthesis in the cells of a diluted hepatocyte culture is higher than in a dense culture]. / Intensivnost' sinteza belka v kletke razvedennoi kul'tury gepatotsitov vyshe, chem v plotnoi kul'ture.

Incorporation of 3H-leucine in proteins and radioactivity of free leucine have been studied in hepatocyte culture on collagen-coated glass. A high density culture obtained from a suspension of isolated rat hepatocytes at 5 x 10(6) cells/ml was compared with low density cultures diluted from the same suspension 10- to 20-fold. The incorporation pool ratio showing the efficiency of the precursor utilization was higher in the low density culture as compared with the high density monolayer. The effect was due to disproportionate low pool in the diluted cultures. The total radioactivity of proteins was lower in the diluted cultures as compared with the monolayer, although the incorporation was not in proportion with the cell density. Specific rate of protein synthesis (per cell) was higher in the diluted cultures than in the monolayer.