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.