Sphingosine at the concentration of 100 muM is depleting ER calcium stores, causing a rise in intracellular calcium ranging from 50 to 100 nM. It also decreases Ca2+ response caused by 100 nM thapsigargin. Preincubation with 10 mM neomycin, a high affinity ligand for PIP2, which inhibits PLC and PLD activitity in brain, partially eliminates the effect of sphingosine on thapsigargin elicited Ca2+ signal. This effect is not observed after preincubation with 150 mM ethanol, which acts as a substrate for PLD, blocking its normal activity by production of phosphatidylethanol instead phosphatidic acid. These results suggest that sphingosine may act on intracellular Ca2+ stores and stimulate Ca2+ mobilization via process mediated rather by PLC than by PLD activation.ion.
The effect of sphingosine on intracullular 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 concentation of 100 muM, causing the cytosolic Ca2+ level to rise.However, at a much lower concentration of 15 muM sphingosine diminished calcium responses triggered by thapsigargin and ionomycin (calcium ionophore).Since responses to thapsigargin and ionomycin were blocked in Ca2+ -free medium, we postulate that sphingosine is acting on the intracellular calcium stores.Additionally, sphingosine (at 15muM and 100muM)markedly decreases thapsigargin-induced sustained elevation in cytosolic Ca2+ concentation, indicating its inhibitory effect in thapsigargin-evoked Ca2+ influx.Sphingosine is known inhibitor of protein kinase C and the involvment of this enzyme is postulated in the modulatory effects of spingosine on intracellular calcium dynamics.
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