Sphingosine 1-phosphate generated in the endoplasmic reticulum membrane activates release of stored calcium

Tarun K. Ghosh, Junhui Bian, Donald Gill

Research output: Contribution to journalArticle

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Abstract

Sphingosine and sphingosine derivatives induce Ca2+ release from inositol 1,4,5-trisphosphate (InsP3)-sensitive Ca2+ pools in permeabilized cells (Ghosh, T. K., Bian, J., and Gill, D. L. (1990) Science 248, 1653- 1656). To further assess the mechanism of sphingoid base-mediated Ca2+ release, the effects of sphingosine and sphingosine derivatives on Ca2+ fluxes were characterized using a microsomal membrane vesicle fraction (B3) enriched in rough endoplasmic reticulum (ER) prepared from cells of the DDT1MF-2 cell smooth muscle line (Ghosh, T. K., Mullaney, J. M., Tarazi, F. I., and Gill, D. L. (1989) Nature 340, 236-239). Addition of 15 μM sphingosine to Ca2+ pump-loaded B3 vesicles induced a delayed but thereafter rapid Ca2+ release from vesicles which was dependent on the presence of ATP and was blocked by ADP. Sphingosylphosphorylcholine (SPC) released Ca2+ to the same extent (more than 80% of pumped Ca2+), but in contrast to sphingosine, there was no lag and the effect was independent of ATP or ADP. The EC50 for sphingosine and SPC in activating Ca2+ release was 1 and 3 μM, respectively. Such observations are consistent with the view that sphingosine, unlike SPC, must be modified by an ATP-requiring kinase activity located within the ER membrane. Sphingoid bases do not appear to release Ca2+ through InsP3 receptors since heparin had no effect on sphingoid base-mediated Ca2+ release. Sphingosine 1-phosphate (sph-1-P), the likely active Ca2+-releasing derivative of sphingosine, was synthesized by phospholipase D-catalyzed cleavage of SPC, purified, and tested for Ca2+-releasing activity. sph-1-P at 10 μM induced Ca2+ release from both B3 vesicles and permeabilized DDT1MF-2 cells to exactly the same extent as sphingosine. Unlike sphingosine, the effect of sph-1-P was immediate and not blocked by ADP. Using B3 membrane vesicles incubated with [γ-32P]ATP and sphingosine under the same conditions as Ca2+ flux studies, a labeled band was detected on TLC which ran identically with authentic sph-1-P. Formation of this labeled product was prevented by removal of exogenous sphingosine and blocked by ADP. Sphingosine- but not SPC-mediated Ca2+ release was blocked by 10 mM oxalate. 10 mM oxalate also blocked the formation of 32P-labeled sph-1-P indicating that it is an inhibitor of sph-1-P formation. The studies establish that the ER membrane contains the necessary kinase to convert sphingosine to sph-1-P which functions as a powerful mediator of Ca2+ release through a non-InsP3 receptor-mediated mechanism in the same ER membrane, perhaps reflecting a novel Ca2+ signaling pathway.

Original languageEnglish (US)
Pages (from-to)22628-22635
Number of pages8
JournalJournal of Biological Chemistry
Volume269
Issue number36
StatePublished - Sep 9 1994

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Sphingosine
Endoplasmic Reticulum
Calcium
Membranes
Adenosine Diphosphate
Adenosine Triphosphate
Oxalates
sphingosine 1-phosphate
Derivatives
Phosphotransferases
Fluxes
Phospholipase D
Inositol 1,4,5-Trisphosphate
Rough Endoplasmic Reticulum

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

@article{4e3d6441ea24455f978217c90789a9a9,
title = "Sphingosine 1-phosphate generated in the endoplasmic reticulum membrane activates release of stored calcium",
abstract = "Sphingosine and sphingosine derivatives induce Ca2+ release from inositol 1,4,5-trisphosphate (InsP3)-sensitive Ca2+ pools in permeabilized cells (Ghosh, T. K., Bian, J., and Gill, D. L. (1990) Science 248, 1653- 1656). To further assess the mechanism of sphingoid base-mediated Ca2+ release, the effects of sphingosine and sphingosine derivatives on Ca2+ fluxes were characterized using a microsomal membrane vesicle fraction (B3) enriched in rough endoplasmic reticulum (ER) prepared from cells of the DDT1MF-2 cell smooth muscle line (Ghosh, T. K., Mullaney, J. M., Tarazi, F. I., and Gill, D. L. (1989) Nature 340, 236-239). Addition of 15 μM sphingosine to Ca2+ pump-loaded B3 vesicles induced a delayed but thereafter rapid Ca2+ release from vesicles which was dependent on the presence of ATP and was blocked by ADP. Sphingosylphosphorylcholine (SPC) released Ca2+ to the same extent (more than 80{\%} of pumped Ca2+), but in contrast to sphingosine, there was no lag and the effect was independent of ATP or ADP. The EC50 for sphingosine and SPC in activating Ca2+ release was 1 and 3 μM, respectively. Such observations are consistent with the view that sphingosine, unlike SPC, must be modified by an ATP-requiring kinase activity located within the ER membrane. Sphingoid bases do not appear to release Ca2+ through InsP3 receptors since heparin had no effect on sphingoid base-mediated Ca2+ release. Sphingosine 1-phosphate (sph-1-P), the likely active Ca2+-releasing derivative of sphingosine, was synthesized by phospholipase D-catalyzed cleavage of SPC, purified, and tested for Ca2+-releasing activity. sph-1-P at 10 μM induced Ca2+ release from both B3 vesicles and permeabilized DDT1MF-2 cells to exactly the same extent as sphingosine. Unlike sphingosine, the effect of sph-1-P was immediate and not blocked by ADP. Using B3 membrane vesicles incubated with [γ-32P]ATP and sphingosine under the same conditions as Ca2+ flux studies, a labeled band was detected on TLC which ran identically with authentic sph-1-P. Formation of this labeled product was prevented by removal of exogenous sphingosine and blocked by ADP. Sphingosine- but not SPC-mediated Ca2+ release was blocked by 10 mM oxalate. 10 mM oxalate also blocked the formation of 32P-labeled sph-1-P indicating that it is an inhibitor of sph-1-P formation. The studies establish that the ER membrane contains the necessary kinase to convert sphingosine to sph-1-P which functions as a powerful mediator of Ca2+ release through a non-InsP3 receptor-mediated mechanism in the same ER membrane, perhaps reflecting a novel Ca2+ signaling pathway.",
author = "Ghosh, {Tarun K.} and Junhui Bian and Donald Gill",
year = "1994",
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volume = "269",
pages = "22628--22635",
journal = "Journal of Biological Chemistry",
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}

Sphingosine 1-phosphate generated in the endoplasmic reticulum membrane activates release of stored calcium. / Ghosh, Tarun K.; Bian, Junhui; Gill, Donald.

In: Journal of Biological Chemistry, Vol. 269, No. 36, 09.09.1994, p. 22628-22635.

Research output: Contribution to journalArticle

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T1 - Sphingosine 1-phosphate generated in the endoplasmic reticulum membrane activates release of stored calcium

AU - Ghosh, Tarun K.

AU - Bian, Junhui

AU - Gill, Donald

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N2 - Sphingosine and sphingosine derivatives induce Ca2+ release from inositol 1,4,5-trisphosphate (InsP3)-sensitive Ca2+ pools in permeabilized cells (Ghosh, T. K., Bian, J., and Gill, D. L. (1990) Science 248, 1653- 1656). To further assess the mechanism of sphingoid base-mediated Ca2+ release, the effects of sphingosine and sphingosine derivatives on Ca2+ fluxes were characterized using a microsomal membrane vesicle fraction (B3) enriched in rough endoplasmic reticulum (ER) prepared from cells of the DDT1MF-2 cell smooth muscle line (Ghosh, T. K., Mullaney, J. M., Tarazi, F. I., and Gill, D. L. (1989) Nature 340, 236-239). Addition of 15 μM sphingosine to Ca2+ pump-loaded B3 vesicles induced a delayed but thereafter rapid Ca2+ release from vesicles which was dependent on the presence of ATP and was blocked by ADP. Sphingosylphosphorylcholine (SPC) released Ca2+ to the same extent (more than 80% of pumped Ca2+), but in contrast to sphingosine, there was no lag and the effect was independent of ATP or ADP. The EC50 for sphingosine and SPC in activating Ca2+ release was 1 and 3 μM, respectively. Such observations are consistent with the view that sphingosine, unlike SPC, must be modified by an ATP-requiring kinase activity located within the ER membrane. Sphingoid bases do not appear to release Ca2+ through InsP3 receptors since heparin had no effect on sphingoid base-mediated Ca2+ release. Sphingosine 1-phosphate (sph-1-P), the likely active Ca2+-releasing derivative of sphingosine, was synthesized by phospholipase D-catalyzed cleavage of SPC, purified, and tested for Ca2+-releasing activity. sph-1-P at 10 μM induced Ca2+ release from both B3 vesicles and permeabilized DDT1MF-2 cells to exactly the same extent as sphingosine. Unlike sphingosine, the effect of sph-1-P was immediate and not blocked by ADP. Using B3 membrane vesicles incubated with [γ-32P]ATP and sphingosine under the same conditions as Ca2+ flux studies, a labeled band was detected on TLC which ran identically with authentic sph-1-P. Formation of this labeled product was prevented by removal of exogenous sphingosine and blocked by ADP. Sphingosine- but not SPC-mediated Ca2+ release was blocked by 10 mM oxalate. 10 mM oxalate also blocked the formation of 32P-labeled sph-1-P indicating that it is an inhibitor of sph-1-P formation. The studies establish that the ER membrane contains the necessary kinase to convert sphingosine to sph-1-P which functions as a powerful mediator of Ca2+ release through a non-InsP3 receptor-mediated mechanism in the same ER membrane, perhaps reflecting a novel Ca2+ signaling pathway.

AB - Sphingosine and sphingosine derivatives induce Ca2+ release from inositol 1,4,5-trisphosphate (InsP3)-sensitive Ca2+ pools in permeabilized cells (Ghosh, T. K., Bian, J., and Gill, D. L. (1990) Science 248, 1653- 1656). To further assess the mechanism of sphingoid base-mediated Ca2+ release, the effects of sphingosine and sphingosine derivatives on Ca2+ fluxes were characterized using a microsomal membrane vesicle fraction (B3) enriched in rough endoplasmic reticulum (ER) prepared from cells of the DDT1MF-2 cell smooth muscle line (Ghosh, T. K., Mullaney, J. M., Tarazi, F. I., and Gill, D. L. (1989) Nature 340, 236-239). Addition of 15 μM sphingosine to Ca2+ pump-loaded B3 vesicles induced a delayed but thereafter rapid Ca2+ release from vesicles which was dependent on the presence of ATP and was blocked by ADP. Sphingosylphosphorylcholine (SPC) released Ca2+ to the same extent (more than 80% of pumped Ca2+), but in contrast to sphingosine, there was no lag and the effect was independent of ATP or ADP. The EC50 for sphingosine and SPC in activating Ca2+ release was 1 and 3 μM, respectively. Such observations are consistent with the view that sphingosine, unlike SPC, must be modified by an ATP-requiring kinase activity located within the ER membrane. Sphingoid bases do not appear to release Ca2+ through InsP3 receptors since heparin had no effect on sphingoid base-mediated Ca2+ release. Sphingosine 1-phosphate (sph-1-P), the likely active Ca2+-releasing derivative of sphingosine, was synthesized by phospholipase D-catalyzed cleavage of SPC, purified, and tested for Ca2+-releasing activity. sph-1-P at 10 μM induced Ca2+ release from both B3 vesicles and permeabilized DDT1MF-2 cells to exactly the same extent as sphingosine. Unlike sphingosine, the effect of sph-1-P was immediate and not blocked by ADP. Using B3 membrane vesicles incubated with [γ-32P]ATP and sphingosine under the same conditions as Ca2+ flux studies, a labeled band was detected on TLC which ran identically with authentic sph-1-P. Formation of this labeled product was prevented by removal of exogenous sphingosine and blocked by ADP. Sphingosine- but not SPC-mediated Ca2+ release was blocked by 10 mM oxalate. 10 mM oxalate also blocked the formation of 32P-labeled sph-1-P indicating that it is an inhibitor of sph-1-P formation. The studies establish that the ER membrane contains the necessary kinase to convert sphingosine to sph-1-P which functions as a powerful mediator of Ca2+ release through a non-InsP3 receptor-mediated mechanism in the same ER membrane, perhaps reflecting a novel Ca2+ signaling pathway.

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