title: Sphingosine 1-phosphate evokes calcium signals in C2C12 myoblasts via Edg3 and Edg5 receptors.
abstract: Sphingosine 1-phosphate (SPP) is a bioactive lipid that exerts multiple biological effects in a large variety of cell types, acting as either an intracellular messenger or an extracellular ligand coupled to Edg-family receptors (where Edg stands for endothelial differentiation gene). Here we report that in C(2)C(12) myoblasts SPP elicited significant Ca(2+) mobilization. Analysis of the process using a confocal laser-scanning microscope showed that the Ca(2+) response occurred in a high percentage of cells, despite variations in amplitude and kinetics. Quantitative analysis of SPP-induced Ca(2+) transients performed with a spectrophotofluorimeter showed that the rise in Ca(2+) was strictly dependent on availability of extracellular Ca(2+). Cell treatment with pertussis toxin partially prevented the Ca(2+) response induced by SPP, indicating that G(i)-coupled-receptors were involved. Indeed, SPP action was shown to be mediated by agonist-specific Edg receptors. In particular, suramin, an antagonist of the SPP-specific receptor Edg3, as well as down-regulation of Edg3 by cell transfection with antisense oligodeoxyribonucleotides (ODN), significantly reduced agonist-mediated Ca(2+) mobilization. Moreover, an antisense ODN designed to inhibit Edg5 expression also decreased the SPP-induced rise in Ca(2+), although to a lesser extent than that observed by inhibiting Edg3. On the contrary, the SPP response was unaffected in myoblasts loaded with antisense ODN specific for Edg1. Remarkably, the concomitant inhibition of Edg3 and Edg5 receptors abolished the SPP-induced Ca(2+) increase, supporting the notion that Ca(2+) mobilization in C(2)C(12) cells induced by SPP is a receptor-mediated process that involves Edg3 and Edg5, but not Edg1.
authors.affiliation: Dipartimento di Scienze Biochimiche, Università degli Studi di Firenze, Viale G.B. Morgagni 50, 50134 Florence, Italy.
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Meacci
authors.forename: Francesca
authors.initials: F
authors.lastname: Cencetti
authors.forename: Lucia
authors.initials: L
authors.lastname: Formigli
authors.forename: Roberta
authors.initials: R
authors.lastname: Squecco
authors.forename: Chiara
authors.initials: C
authors.lastname: Donati
authors.forename: Bruno
authors.initials: B
authors.lastname: Tiribilli
authors.forename: Franco
authors.initials: F
authors.lastname: Quercioli
authors.forename: Sandra
authors.initials: S
authors.lastname: Zecchi Orlandini
authors.forename: Fabio
authors.initials: F
authors.lastname: Francini
authors.forename: Paola
authors.initials: P
authors.lastname: Bruni
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects* / Q000502:physiology; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004533:Egtazic Acid / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D020672:I-kappa B Proteins; D007700:Kinetics; D008246:Lysophospholipids; D018482:Muscle, Skeletal / Q000187:drug effects / Q000502:physiology*; D000072000:NF-KappaB Inhibitor alpha; D037342:Pertussis Toxin; D011493:Protein Kinase C / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled; D049312:Receptors, Lysophospholipid; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
chemical_list: D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D020672:I-kappa B Proteins; D008246:Lysophospholipids; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D049312:Receptors, Lysophospholipid; D010566:Virulence Factors, Bordetella; D000072000:NF-KappaB Inhibitor alpha; C060506:sphingosine 1-phosphate; D004533:Egtazic Acid; D037342:Pertussis Toxin; D011493:Protein Kinase C; D013110:Sphingosine; D002118:Calcium