[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.

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 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.

[The action of tiazofurin on the development of sea urchin embryos and larvae]. / Deistvie tiazofurina na razvitie zarodyshei i lichinok morskikh ezhei.

The effects of the antitumor drug tiazofurin on development of sea urchins Sphaerechinus granularis, Paracentrotus lividus, Strongylocentrotus intermedius, and Arbacia lixula were studied. When 0.01-200 microM tiazofurin (TAF) was introduced in the incubation medium (artificial sea water) just after fertilization or at the midblastula stage, the development proceeded quite normally until the beginning of gastrulation. But later TAF blocked gastrulation and induced formation of mobile ball-shaped larvae with normal pigment cells but devoid of the nervous system, skeletal spicules and digestive tract. The threshold TAF concentrations varied from 0.05 microM (S. granularis) to 2-5 microM (all other species). When TAF was introduced during gastrulation and just after gastrulation, the larvae had defective nervous system and skeleton and suppressed expression of gangliosides. The nonhydrolyzable analog of GTP, GTP-gamma-S (5-20 microM), introduced in artificial sea water no later than at the midblastula stage prevented all above mentioned developmental defects.

[The action of serotonin and its analogs on the in vitro maturation of amphibian oocytes]. / Deistvie serotonina i ego analogov na sozrevanie in vitro ootsitov amfibii.

Indolylalkylamines (5-HT, 5-chlorotryptamine, 5-ethyltryptamine) suppress or block the progesterone-induced oocyte maturation in Bufo viridis and Xenopus laevis and disturb cytoskeletal rearrangements associated with oocytes involved, together with the other transmitter systems, in modulation of progesterone effect.

[Effect of cyclic nucleotides on the sensitivity of early sea urchin embryos to cytotoxic neuropharmacological preparations]. / Vliianie tsiklicheskikh nukleotidov na chuvstvitel'nost' rannykh zarodyshei morskikh ezhei k tsitoksicheskim neirofarmakologicheskim preparatam.

It has been shown that cytotoxic action of neuropharmacological drugs (transmitter antagonists) on early urchin embryos is reduced or abolished by cyclic nucleotides and sodium fluoride. Such a protective action differs in cAMP and dibutyryl analogs of cyclic nucleotides, depending on the chemical structure of an embryotoxic substance. It is suggested that, endogenous intracellular "prenervous" transmitters and cyclic nucleotides are interrelated in the regulation of cell division in urchin embryos.

[Action of heavy metal salts on the development of sea urchin embryos and on protein synthesis by the cells of transplantable mouse tumors]. / Deistvie solei tiazhelykh metallov na razvitie zarodyshei morskikh ezhei i na sintez belkov kletkami myshinykh perevivnykh opukholei.

Zu, Cu, Cd, Pb and Hg salts blocked the cleavage divisions in the sea urchin embryos and, in lesser concentrations, affected gastrulation and induced postgastrulation defects. The early embryo- and cytotoxic effects of Zn salts appear to be based on the inhibition of protein synthesis. Unithiol exerted a protective effect. All salts under study inhibited protein synthesis in the Ehrlich's ascite carcinoma and ascite hepatoma 22a cells; unithiol and other suldhydryl compounds exerted in this case a good protective effect against Cd salts only.