Involvement of protein kinases in self-organization of the rhythm of protein synthesis by direct cell-cell communication.

Primary cultures of rat hepatocytes grown on slides were studied in serum-free medium. Ultradian protein synthesis rhythm was used as a marker of synchronization of individual oscillations, resulting in the formation of a common rhythm of the cell population, i.e. cell-cell self-organization. Dense synchronous and sparse non-synchronous cultures were used to estimate effect of protein kinase activity on the kinetics of protein synthesis. Treatment of dense cultures with the inhibitors H7 (40 microM) or H8 (25 microM) resulted in a loss of the protein synthesis rhythm, a suppression of the cell-cell self-organization. Stimulation of protein kinase activity with either 0.5 or 1.0 microM phorbol 12-miristate-13-acetate (PMA) or 10 microM forskolin caused the appearance of the synthetic rhythm in non-synchronous sparse cultures under otherwise normal conditions. Inhibition of protein kinase activity with H7 resulted in signal factors, such as gangliosides and phenylephrine, failing to initiate this rhythm in sparse cultures. Activation of protein kinase activity with PMA shifted the phase pattern of the protein synthesis rhythm. Thus, according to our previous and the new data, protein kinase activity and consequently protein phosphorylation is the crucial step of sequence of processes resulting in synchronization during self-organization of cells in producing a common rhythm in the population. The general pathway can be presented as follows: signaling of gangliosides or other calcium agonists-->efflux of calcium ion from intracellular stores, with elevation of calcium concentration in the cytoplasm-->activation of protein kinases-->protein phosphorylation-->synchronization of individual oscillations in protein synthesis rates-->induction of a common rhythm throughout this population. The data have been discussed concerning similarity of the direct cell-cell communication and the cell self-organization in cultures and in organism.

Small cooperative activity of old rat hepatocytes may depend on composition of the intercellular medium.

Ultradian oscillations of protein synthesis were used as a marker of hepatocyte synchronous cooperative activity producing a common rhythm in vitro; amplitude of the rhythm defines expression of the cell cooperation. Dense synchronous and sparse non-synchronous rat hepatocyte cultures on slides in a serum-free incubation medium 199 supplemented with 0.2 mg/ml albumin and 0.5 microg/ml insulin have been studied. The amplitude of the rhythm averaged approximately 2x in dense cultures of young (3 month old) rats than in old (2 year old) rats. But some cultures of young rats had the amplitude patterns similar to cultures of old rats, and vice versa. Addition to the medium of either 0.3 microM bovine brain gangliosides or 2 microM phenylephrine resulted in increase of the oscillation amplitude in dense cultures of old rats to the level inherent in young ones. Addition to the medium of 10% rat blood serum in non-synchronous sparse cultures from young rats resulted in detection of a protein synthetic rhythm. Although after serum from young rats, the rhythm expression was high, the rhythm after serum from old rats had been given was weak. Addition of gangliosides to old-rat serum resulted in synchronization of sparse cultures with amplitudes inherent of young-rat serum. The data tend to the conclusion that cell cooperation depends to a greater extent on the composition of the medium rather than on the age of the cell or animal.

Calcium ions as a factor of cell-cell cooperation in hepatocyte cultures.

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.