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.
Brodsky
Vsevolod Y
VY
Institute of Developmental Biology, Russian Academy of Sciences, 26 Vavilov Street, 117808, Moscow, Russia. brodsky@proxima.idb.ac.ru
Zvezdina
Natalia D
ND
Nechaeva
Natalia V
NV
Avdonin
Pavel V
PV
Novikova
Tatjana E
TE
Gvasava
Inessa G
IG
Fateeva
Valentina I
VI
Malchenko
Ludmila A
LA
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D002614:Chelating Agents / Q000494:pharmacology; D017077:Culture Media, Conditioned / Q000378:metabolism; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D005677:G(M1) Ganglioside / Q000378:metabolism; D022781:Hepatocytes / Q000378:metabolism / Q000473:pathology*; D006801:Humans; D006873:Hydroquinones / Q000494:pharmacology; D007477:Ions; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism; D010656:Phenylephrine / Q000494:pharmacology; D010766:Phosphorylation; D013997:Time Factors
D000975:Antioxidants; D002614:Chelating Agents; D017077:Culture Media, Conditioned; D016895:Culture Media, Serum-Free; D006873:Hydroquinones; D007477:Ions; D008081:Liposomes; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D010656:Phenylephrine; C019359:2,5-di-tert-butylhydroquinone; D005677:G(M1) Ganglioside; D004533:Egtazic Acid; D002118:Calcium