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 microM 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 microM, 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 Ca(2+)-releasing derivative of sphingosine, was synthesized by phospholipase D-catalyzed cleavage of SPC, purified, and tested for Ca(2+)-releasing activity. sph-1-P at 10 microM 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 [gamma-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.
authors.affiliation: Department of Biological Chemistry, University of Maryland School of Medicine, Baltimore 21201.
authors.forename: T K
authors.initials: TK
authors.lastname: Ghosh
authors.forename: J
authors.initials: J
authors.lastname: Bian
authors.forename: D L
authors.initials: DL
authors.lastname: Gill
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D006224:Cricetinae; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism*; D006493:Heparin / Q000494:pharmacology; D015544:Inositol 1,4,5-Trisphosphate / Q000494:pharmacology; D007700:Kinetics; D008246:Lysophospholipids; D008861:Microsomes / Q000378:metabolism*; D008954:Models, Biological; D009130:Muscle, Smooth; D010070:Oxalates / Q000494:pharmacology; D019815:Oxalic Acid; D015398:Signal Transduction; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology
chemical_list: D008246:Lysophospholipids; D010070:Oxalates; C060506:sphingosine 1-phosphate; D000001:Calcimycin; D000244:Adenosine Diphosphate; D015544:Inositol 1,4,5-Trisphosphate; D000255:Adenosine Triphosphate; D006493:Heparin; D019815:Oxalic Acid; D013110:Sphingosine; D002118:Calcium