CALCIUM SIGNALING BY G-PROTEIN-COUPLED SPHINGOLIPID RECEPTORS IN BOVINE AORTIC ENDOTHELIAL-CELLS

Besides its role as a putative second messenger releasing Ca2+ from in tracellular stores, sphingosine-1-phosphate (SPP) has recently been id entified as an extracellularly acting ligand activating a high affinit y G protein-coupled membrane receptor in various cell types. Since SPP can be released from activated platelets, we examined in the present study whether endothelial cells express receptors for SPP and related sphingolipids. In bovine aortic endothelial cells loaded with fura-2, addition of SPP caused a rapid and transient increase in intracellular Ca2+ concentration ([Ca2+](i)), amounting to maximally about 230 nM. Removal of extracellular Ca2+ revealed that SPP-induced [Ca2+](i) elev ations were due to both release of Ca2+ from intracellular stores and influx of extracellular Ca2+. Pretreatment of the cells with pertussis toxin inhibited the SPP-induced increase in [Ca2+](i) by 83%, in line with the previously reported involvement of G proteins of the G(i/o) family in SPP signalling in other cell types. In contrast to other [Ca 2+](i)-elevating agonists, e.g., ATP and bradykinin, SPP did not activ ate phospholipase C in bovine aortic endothelial cells, suggesting the involvement of a novel, unidentified signalling pathway in SPP-induce d release of intracellular Ca2+. Furthermore, SPP also did not cause a ctivation of either phospholipase D or A(2). Out of various related sp hingolipids studied, only sphingosylphosphorylcholine (SPPC) induced a similar maximal increase in [Ca2+](i) as SPP, and its effect was also fully pertussis toxin-sensitive. However, the potencies of the two sp hingolipids to increase [Ca2+](i) differed by more than two orders of magnitude, with the EC(50) values being 0.8 nM and 260 nM for SPP and SPPC, respectively These results identify SPP and SPPC as novel and po tent endothelial agonists, inducing calcium signalling by activation o f a G(i/o) protein-coupled receptor(s). Given the recently reported re lease of SPP from thrombin-activated platelets, SPP may represent a no vel mediator of platelet-endothelial cell interactions.