Phosphatidylserine synthesis in glioma C6 cells is inhibited by Ca2+ depletion from the endoplasmic reticulum: effects of 2,5-di-tert-butylhydroquinone and thimerosal.

The effects of 2,5-di-tert-butylhydroquinone (DBHQ) and thimerosal on phosphatidylserine synthesis by the base exchange reaction and on calcium mobilization in intact glioma C6 cells were compared with that of thapsigargin, a selective inhibitor of the endoplasmic reticulum Ca(2+)-ATPase. It has been found that all these agents inhibit phosphatidylserine synthesis by 70%, but their effectiveness are different. The data show that this inhibition is caused by Ca2+ depletion of the endoplasmic reticulum, indicating that phosphatidylserine synthesis requires high concentration of Ca2+ within this structure. On this basis and on literature data, a new model for the localization of the serine base exchange enzyme in the endoplasmic reticulum membrane is proposed.

Sphingosine stimulates calcium mobilization and modulates calcium signals evoked by thapsigargin in glioma C6 cells.

The effect of sphingosine on intracellular calcium signalling in glioma C6 cells was studied with Fura-2 video imaging technique. Sphingosine had a direct effect on changes in cytosolic Ca2+ concentration only when applied at high concentration of 100 microM, causing the cytosolic Ca2+ level to rise. However, at a much lower concentration of 15 microM sphingosine diminished calcium responses triggered by thapsigargin (a specific inhibitor of calcium pump in the endoplasmic reticulum) and ionomycin (calcium ionophore). Since responses to thapsigargin and ionomycin were blocked in Ca(2+)-free medium, we postulate that sphingosine is acting on the intracellular calcium stores. Additionally, sphingosine (at 15 microM and 100 microM) markedly decreases thapsigargin-induced sustained elevation in cytosolic Ca2+ concentration, indicating its inhibitory effect on thapsigargin-evoked Ca2+ influx. Sphingosine is a known inhibitor of protein kinase C and the involvement of this enzyme is postulated in the modulatory effects of sphingosine on intracellular calcium dynamics.

Phosphatidylserine synthesis in phorbol ester treated glioma C6 cells.

Phosphatidylserine (PS) synthesis was studied in glioma C6 cells with [14C]serine and in the presence or absence of the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA). It was found that incubation of the cells with 10 nM or 100 nM TPA for 1 h inhibited PS formation by 30% and 60%, respectively. Long-term (18 h) treatment of the cells with 100 nM TPA diminished PS formation and further addition of TPA to down-regulated cells did not affect PS synthesis. The data show that the changes in PS synthesis can be associated with alterations in morphology of cell and the actin cytoskeleton organization. The role of protein kinase C in this process is discussed.