Changes in Concentration of Calcium Ions and the Rhythm of Protein Synthesis in the Culture of Hepatocytes

Abstract

We studied the effects of the chelating agents of extra- and intracellular calcium ions, EGTA and BAPTA-AM, and of the inhibitor of Ca²⁺release from the reticulum, TMB-8, in the kinetics of protein synthesis in hepatocyte cultures. We also studied dense cultures capable of self-synchronization of protein synthesis oscillations and diluted cultures, in which synchronization is induced by phenylephrine or gangliosides (standard preparation of total gangliosides from the bovine brain). Preincubation of the diluted or dense cultures in the presence of 2 mM EGTA for 1–2 h with subsequent protein assay in a medium with EGTA did not affect the kinetics of protein synthesis: no rhythm was found in the diluted cultures, while it was preserved in the dense cultures. When the diluted cultures preincubated in the presence of EGTA were placed in a medium with EGTA and 2 μM phenylephrine for 2 min, the rhythm was visualized. The treatment of diluted cultures with 100 μM TMB-8 for 5 or 10 min with subsequent washing and incubation in a medium with 3 μM gangliosides led to visualization of the protein synthesis rhythm, i.e., to the synchronization of oscillations, while no rhythm was found in the standard cultures. Preincubation of the diluted cultures in a medium with 10, 15, or 20 μM BAPTA-AM for 1 h did not affect the kinetics of protein synthesis. When, after such preincubation, the diluted cultures were placed in the medium with gangliosides, the rhythm was visualized. In the dense cultures, normally capable of self-synchronization, no rhythm of protein synthesis was found after their treatment with 10–20 μM BAPTA-AM for 1 h. The transfer of such cultures in the medium with gangliosides led to visualization of the rhythm. Thus, calcium affects the kinetics of protein synthesis: after the rise of Ca²⁺in the cytoplasm was blocked, the rhythm of protein synthesis was not visualized due, supposedly, to disturbed mechanisms of medium conditioning. However, exogenous gangliosides in the dense or diluted cultures preincubated in the presence of BAPTA-AM ore TMB-8 allowed the rhythm visualization, i.e., synchronization may not depend on changes in the intracellular calcium concentration.