Calcium ions as a factor of cell—cell cooperation in hepatocyte cultures

Abstract

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.

Loss of hepatocyte co‐operative activity after inhibition of ganglioside GM1 synthesis and shedding

“…We have also observed a protein synthesis rhythm after addition of phenylephrine, a calcium agonist, to non-synchronous sparse cultures (Brodsky et al, 2003). BAPTA-AM, an intracellular Ca 2+ chelator that abrogates calcium changes in the cytoplasm, affected the loss of protein synthesis rhythm in dense cultures, which are self-synchronized in controls.…”

Involvement of protein kinases in self‐organization of the rhythm of protein synthesis by direct cell—cell communication

“…According to the data (Brodsky et al, 2000(Brodsky et al, , 2003a, ganglioside signaling is at the beginning of a chain of processes leading to cellecell self-organization in the induction of the common protein synthesis rhythm in a certain cell population. The second step is induction of cytoplasmic calcium ion [Ca 2þ cyt ] efflux from intracellular stores resulting in [Ca 2þ cyt ] elevation (Brodsky et al, 2003b).…”

“…Ultradian rhythms are an innate cell property presumably of a fractal nature (Barnsley et al, 1987;Brodsky, 1992Brodsky, , 2006Lloyd, 1992Lloyd, , 2005Lloyd, 1993, 1995). In a series of articles already published in this journal (Brodsky et al, 2000(Brodsky et al, , 2003a(Brodsky et al, ,b, 2004(Brodsky et al, , 2005, some signal factors that promoted synchronization of protein synthesis rhythm were described. Depending on cell density, two types of primary hepatocyte cultures on slides in the serum-free medium were obtained, dense cultures with closely arranged cells, and sparse cultures with widely separated cells.…”

Prion Protein Signaling in the Nervous System—A Review and Perspective

“…Any role of PrP C may act through an interaction between PrP C and ganglioside GM1. 88,165 Genes involved in sleep regulation include melanocortin genes such as proopiomelanocortin (POMC)-tyrosinase, which also affect breathing rhythms, pain, cardiac regulation, and autonomic response. 166 The daily light-dark cycle is known to be the primary signal that entrains circadian rhythms to the earth's rotation.…”