Mechanism of Direct Cell Interactions. Self-Organization of Protein Synthesis Rhythm

摘要

Primary 24-hour cultures of hepatocytes on slides in a serum-free medium were studied. Circaho-ralian 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 uM or forskolin at 10uM 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 (l-(5-isoquinolinylsulfonyl)-5-methylpiperasine dihydro-chloride) at 40 uM or H8 (N-(2-[methylamino]ethyl)-5-isoquinolinesulfonamide hydrochloride) at 25uM 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. cAMP-dependent protein kinases also affectthe protein synthesis rhythm. 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. Protein phosphorylation isa key process. The mechanisms of cell self-organization are similar in vitro and in vivo, specifically in the liver in situ.