calcimycin and sphingosine-phosphorylcholine

Sphingolipid-gated Ca2+ signaling is mediated through Ca(2+)-permeable channels. In this report, we characterize the properties of the channel in a human endothelial cell line (EA.hy926). Ca2+ release from intracellular stores is not antagonized by nifedipine, omega conotoxin G-VIa, or heparin. To further characterize the molecular properties of the channel, we developed a novel assay to directly measure efflux of Ca2+ from intracellular stores of permeabilized Xenopus oocytes. Following size fractionation by sucrose gradient, poly(A)+ RNA from EA.hy926 cells is microinjected into oocytes of Xenopus laevis. We find that the mRNA encoding Ca2+ release activity is approximately 1.5-2.0 kilobases in length. The sphingolipid-gated Ca(2+)-permeable channel is thus likely to be a novel Ca(2+)-permeable channel distinct from other characterized intracellular Ca2+ channels such as the ryanodyne receptor and the inositol 1,4,5-trisphosphate receptor. The method described here provides a new approach to further characterizing this channel and other intracellular Ca2+ channels.

Topics: Animals; Calcimycin; Calcium; Calcium Channel Blockers; Calcium Channels; Cell Line; Endothelium, Vascular; Female; Heparin; Humans; In Vitro Techniques; Ion Channel Gating; Kinetics; Nifedipine; omega-Conotoxin GVIA; Oocytes; Peptides; Phosphorylcholine; RNA, Messenger; Sphingolipids; Sphingosine; Time Factors; Xenopus laevis

Soluble and hydrophobic lipid breakdown products have a variety of important signaling roles in cells. Here sphingoid bases derived in cells from sphingolipid breakdown are shown to have a potent and direct effect in mediating calcium release from intracellular stores. Sphingosine must be enzymically converted within the cell to a product believed to be sphingosine-1-phosphate, which thereafter effects calcium release from a pool including the inositol 1,4,5-trisphosphate-sensitive calcium pool. The sensitivity, molecular specificity, and reversibility of the effect on calcium movements closely parallel sphingoid base-mediated inhibition of protein kinase C. Generation of sphingoid bases in cells may activate a dual signaling pathway involving regulation of calcium and protein kinase C, comparable perhaps to the phosphatidylinositol and calcium signaling pathway.

Topics: Adenosine Diphosphate; Animals; Calcimycin; Calcium; Cell Line; Kinetics; Phosphoric Monoester Hydrolases; Phosphorylcholine; Protein Kinase C; Second Messenger Systems; Sphingosine; Thermodynamics