[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.

[The rhythm of protein synthesis in denervated rat liver]. / Ritm sinteza belka v denervirovannoi pecheni krysy.

Using the radiochemical method, we found a circahoralian rhythm of protein synthesis in slices of rat liver two weeks after transection of the vagus nerve anterior stem or bilateral subphrenic vagotomy with simultaneous transection of the celiac plexus hepatic branches. The rhythm was similar to those found in slices of normal liver or in a monolayer culture of hepatocytes. The incorporation of leucine in proteins and the pool of free leucine were similar in the denervated and normal liver. The self-synchronization of individual cell oscillations in protein synthesis intensity was shown earlier in experiments performed on cell cultures. A study of protein synthesis in the denervated liver suggests that cell interactions are also the main mechanism underlying the synchronization of oscillating cellular functions in the organ in situ.

[Cellular self-synchronization in a hepatocyte culture with antiphase fluctuations in the intensity of protein synthesis]. / Samosinkhronizatsiia kletok v kul'ture gepatotsitov s protivofaznymi kolebaniiami intensivnosti sinteza belka.

A part of suspension with isolated hepatocytes was cooled and then warmed to normal temperature. In cultures formed with such cells, antiphase oscillations of the protein synthesis rate were detected concerning control cultures from another part the same hepatocyte suspension. Oscillations of protein synthesis were observed in the mixed cultures formed with equal parts of antiphase subpopulations of hepatocytes. The experiments established cell interactions in vitro resulted in synchronization of protein synthesis oscillations in different cells the same population.

[The effect of macromolecular adhesion factors isolated from the blood serum and liver of mammals on the intensity of protein synthesis and the ATP content in hepatocytes in vitro]. / Issledovanie vliianiia makromolekuliarnykh adgezionnykh faktorov, vydelennykh iz syvorotki krovi i pecheni mlekopitaiushchikh, na intensivnost' sinteza belka i soderzhaniia ATF v gepatotsitakh in vitro.

The influence of the extra-low dozes of the macromolecular adhesive factors (MAF) on the protein synthesis and on the ATP contents in the rat hepatocytes on different models in vitro has been investigated. The protein synthesis intensity, the cell penetrability for labelled 3H-leucine and the intracell ATP contents in the experiments with the hepatocyte monolayer in control cells has been showed to be the same as those in the cells treated by MAF-1 and MAF-2. In the liver cells the protein synthesis intensity was depressed essentially under the action of MAF-1 and MAF-2 with respect to the control cells. The penetrability of the liver cells has increased in 50% of experiments after MAF-1 and MAF-2 treatment. The data obtained show the necessity of preservation of morphological structure of organs for the realization of the biological activity of appropriate MAF.

[The periodicity of the metabolic activity of hepatocytes in culture]. / Periodichnost' metabolicheskoi aktivnosti gepatotsitov v kul'ture.

Synchronous changes were found in the intracellular ATP content and in the ratio of "dark" and "light" cells in the monolayer culture of hepatocytes. An attempt has been made to change these ratios by the effect of hormones. The rhythmical processes in the culture are interpreted as a universal cell property.

The rhythm of protein synthesis does not depend on oscillations of ATP level.

A rhythm of the [3H]leucine incorporation rate with a period of about one hour (circahoralian rhythm) has been found in rat hepatocytes grown in vitro as a monolayer and in liver organ culture. The periodicity of the incorporation rate remained after correction for changes in leucine pool size. A similar periodicity of the leucine incorporation rate was detected in a cell-free system prepared from rat hepatocytes. We have also found circahoralian oscillations of the ATP level and similar oscillations of the leucine tRNA aminoacylation rate in a hepatocyte monolayer. The addition of 1 mM ADP to the culture resulted in a considerable increase in the ATP level in the cells, but the rhythm of protein synthesis was retained under these conditions. The conclusion that there is a flexible association between changes in the ATP and GTP levels on the one hand, and oscillations of the protein synthesis rate on the other, is also supported by experiments with a cell-free system, in which the rhythm of protein synthesis rate was observed in the presence of excess ATP and GTP. We propose an hypothesis to explain the fractal pattern of circahoralian metabolic rhythms.

[Changes in the intensity of protein synthesis in the gastric mucosa following the laser irradiation of a duodenal ulcer]. / Izmeneniia intensivnosti sinteza belka v slizistoi zheludka posle lazernogo oblucheniia iazvy dvenadtsatiperstnoi kishki.

The intensity of protein synthesis (incorporation/pool) is usually low in normal stomach mucosa and enhanced in exacerbation of peptic ulcer; following the effective laser therapy it decreases only in one third of cases. Therefore the scarring of ulcer not always coincides with the normalization of protein synthesis. The results characterize also the community of reactions in stomach and duodenal mucose membranes. The changes in protein synthesis intensity following the laser irradiation depended neither on the initial size of ulcer nor on the age of patient and the total duration of disease.