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.

Ultradian protein synthesis rhythm was used as a marker of cell cooperation in synchronous dense and non-synchronous sparse hepatocyte cultures. Phenylephrine (2 microM, 2 min), an alpha (1)-adrenoreceptor agonist, which exerts [Ca(2+)](cyt)elevation from intracellular stores, affected protein synthesis rhythm in sparse cultures, i.e. initiated cooperative activity of the cells. The same effect was produced by 2,5-di(tertiary-butyl)-1,4-benzohydroquinone (10 microM, 2 min), which increases [Ca(2+)](cyt)by a non-receptor pathway. Pretreatment of dense cultures with the intracellular calcium chelator, 1,2-bis (2-aminophenoxy) ethane-N,N,N',N'- tetraacetic acid (acetoxymethyl) ester (BAPTA-AM) at 10-20 microM for, 30-60 min resulted in loss of the rhythm of protein synthesis, i.e. loss of cooperative activity between the cells. The medium conditioned by control dense cultures initiated rhythm in sparse cultures, whereas the conditioned medium of cultures pretreated with BAPTA-AM did not. [Ca(2+)](cyt)increase is known to occur with monosialoganglioside GM1 treatment. By ELISA estimation, the GM1 content in 3 h conditioned medium was similar in control dense cultures to that in cultures pretreated with BAPTA-AM. Bearing in mind data on the Ca(2+)-dependence of vesicle formation and shedding, the conditioned medium was separated by 150000 g centrifugation to supernatant containing monomers and micelles, and a pellet containing vesicular form of gangliosides. Only the latter initiated cooperative activity of the cells of sparse cultures. These cultures were also synchronized by GM1-containing liposomes at lower concentrations than added free GM1, 0.0003 and 0.06 microM respectively. Thus, GM1 and calcium are both involved in cell-cell synchronization. Activation of gangliosides, including GM1 and elevation of [Ca(2+)](cyt,)is known to lead to changes of protein kinase activity and protein phosphorylation resulting in modulation of oscillations in protein metabolism.

Pretreatment of hepatocyte cultures with 1 microM d-l-threo-1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol-HCL (PPPP) for 24 h decreased the ganglioside GM1 content of the cells by approximately 50% and that of the conditioned medium by 90%. No rhythm in the rate of protein synthesis was detected in dense cultures pretreated with PPPP, but was observed in control dense cultures. Conditioned medium from control dense cultures induced synchrony in sparse cultures, which were non-synchronous in their own medium. In contrast, conditioned medium from dense cultures pretreated with PPPP did not synchronize sparse cultures. Since protein synthesis rhythm is a marker of cell synchronization, i.e. their co-operative activity, then non-oscillatory behavior means loss of cell co-operation. The protein synthesis rhythm was restored 24 h after hepatocytes were transferred to PPPP-free medium. Restoration was more rapid when 0.9 microM gangliosides (standard mixture from bovine brain) were added to the medium just after the withdrawal of PPPP. These novel results concerning the loss of rhythm of protein synthesis in low GM1 ganglioside medium support the conclusion that ganglioside is implicated in the regulation of cell co-operative activity.

Primary cultures of rat hepatocytes were studied in serum-free medium. Ultradian protein synthesis rhythm was used as a marker of overall cell synchronization and cooperation amongst the population. The level of synchronization was determined by amplitudes of the rhythm. Low synchronization of old rat hepatocytes can be enhanced by addition of either gangliosides or phenylephrine to the medium. Incubation of cultures with gangliosides lasted for 2.5 h, while action of phenylephrine was only for 2 min. The amplitude of protein synthesis rhythm was increased 1.5-2 times. In cultures transferred to a fresh normal medium, this increased amplitude was observed for at least 2-3 days. Thus, both gangliosides and phenyleprine are triggers, which, as shown earlier, initiated calcium-dependent processes in the cytoplasm. The results are discussed in the light of concept of the cell self-organization by a direct cell-cell communication.

Circahoralian, or ultradian, intracellular rhythms, including protein synthesis rhythm, were described in various cells both in situ and in vitro (for reviews see Brodsky, 1975; Ultradian Rhythms …, 1992). They have been studied for a long time in the cultures of hepatocytes as well. Visualization of any rhythm in a cell culture is only possible when oscillations are synchronized due to intercellular cooperation. The rhythm expression depends on the degree of synchrony, i.e., on cooperative activity of cell in the formation of an aggregate rhythm of a cell population. The amplitude of rhythm, or range of oscillations, is a visible reflection of this cooperation. In the case when each individual cell has a rhythm, but oscillations in different cells are antiphasic, the aggregate kinetics of a population will not be oscillatory. When the phases of oscillations correspond to each other, a rhythm is visualized and, the closer the phases of oscillations of individual cells, the higher the amplitude of the aggregate rhythm.We have already shown that the amplitude of oscillations of protein synthesis intensity in the cultures of hepatocytes from old rats is lower than that from young animals (Brodsky et al. , 2001), which characterized a low cooperative activity of the cells of old animals. However, these data were preliminary: we assayed the kinetics of protein synthesis only in two old rats. In order to confirm the first observations, it was necessary to carry out experiments of a larger material and, in case of a positive result, clarify possible causes of agerelated changes and establish to what extent they are reversible.When studying intercellular cooperation, we investigated dense, fast self-synchronizing in a fresh medium, hepatocytes and sparse asynchronous cultures (Brodsky et al. , 1994(Brodsky et al. , , 1996a. We used the synchronizers we found earlier: gangliosides (Brodsky et al. , 1996a(Brodsky et al. , , 1997Zvezdina et al. , 2000). Gangliosides are cell membrane glycolipids; they are constantly secreted in extracellular medium and incorporated in other cells and affect many intracellular processes (for reviews see Hakomori, 1981Hakomori, , 2000Tettamanti and Riboni, 1994;Yates and Rampersaud, 1998). In our experiments, asynchronous sparse cultures of hepatocytes were synchronized soon after addition of a standard mix of bovine brain gangliosides (BBG: 20% GM1, 40% GD1a, 16% GD1b, and ca. 4% of others) or individual fractions (GM1 or GD1a). The synchronizing effect of gangliosides can be reproduced by a medium conditioned by dense cultures (Brodsky et al. , 1995(Brodsky et al. , , 2000b.In order to simulate the influence of gangliosides on intracellular processes, we studied their well known effect on the concentration of calcium ions in the cytoAbstract -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 i...