Sphingosine 1-phosphate induces Ca2+ transients and cytoskeletal rearrangement in C2C12 myoblastic cells.
In many cell systems, sphingosine 1-phosphate (SPP) increases cytosolic Ca2+ concentration ([Ca2+]i) by acting as intracellular mediator and extracellular ligand. We recently demonstrated (Meacci E, Cencetti F, Formigli L, Squecco R, Donati C, Tiribilli B, Quercioli F, Zecchi-Orlandini S, Francini F, and Bruni P. Biochem J 362: 349-357, 2002) involvement of endothelial differentiation gene (Edg) receptors (Rs) specific for SPP in agonist-mediated Ca2+ response of a mouse skeletal myoblastic (C2C12) cell line. Here, we investigated the Ca2+ sources of SPP-mediated Ca2+ transients in C2C12 cells and the possible correlation of ion response to cytoskeletal rearrangement. Confocal fluorescence imaging of C2C12 cells preloaded with Ca2+ dye fluo 3 revealed that SPP elicited a transient Ca2+ increase propagating as a wave throughout the cell. This response required extracellular and intracellular Ca2+ pool mobilization. Indeed, it was significantly reduced by removal of external Ca2+, pretreatment with nifedipine (blocker of L-type plasma membrane Ca2+ channels), and inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]-mediated Ca2+ pathway inhibitors. Involvement of EdgRs was tested with suramin (specific inhibitor of Edg-3). Fluorescence associated with Ins(1,4,5)P3Rs and L-type Ca2+ channels was evident in C2C12 cells. SPP also induced C2C12 cell contraction. This event, however, was unrelated to [Ca2+]i increase, because the two phenomena were temporally shifted. We propose that SPP may promote C2C12 cell contraction through Ca2+-independent mechanisms.
Formigli
Lucia
L
Department of Anatomy, Histology, and Forensic Medicine, University of Florence, 50134 Florence, Italy.
Francini
Fabio
F
Meacci
Elisabetta
E
Vassalli
Massimo
M
Nosi
Daniele
D
Quercioli
Franco
F
Tiribilli
Bruno
B
Bencini
Chiara
C
Piperio
Claudia
C
Bruni
Paola
P
Orlandini
Sandra Zecchi
SZ
D000814:Aniline Compounds; D000818:Animals; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002121:Calcium Channel Blockers / Q000494:pharmacology; D020746:Calcium Channels, L-Type / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects* / Q000502:physiology; D002460:Cell Line; D003599:Cytoskeleton / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors; D004075:Diglycerides / Q000096:biosynthesis; D005110:Extracellular Space / Q000378:metabolism; D005456:Fluorescent Dyes; D020672:I-kappa B Proteins; D015544:Inositol 1,4,5-Trisphosphate / Q000096:biosynthesis / Q000494:pharmacology; D007424:Intracellular Fluid / Q000378:metabolism; D008246:Lysophospholipids; D051379:Mice; D018613:Microscopy, Confocal; D009119:Muscle Contraction / Q000187:drug effects; D018482:Muscle, Skeletal / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D000072000:NF-KappaB Inhibitor alpha; D011188:Potassium / Q000494:pharmacology; D049312:Receptors, Lysophospholipid; D019837:Ryanodine Receptor Calcium Release Channel / Q000187:drug effects; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D013498:Suramin / Q000494:pharmacology; D014966:Xanthenes
D000814:Aniline Compounds; D002121:Calcium Channel Blockers; D020746:Calcium Channels, L-Type; D004268:DNA-Binding Proteins; D004075:Diglycerides; D005456:Fluorescent Dyes; D020672:I-kappa B Proteins; D008246:Lysophospholipids; C000606205:Nfkbia protein, mouse; D049312:Receptors, Lysophospholipid; D019837:Ryanodine Receptor Calcium Release Channel; D014966:Xanthenes; D000072000:NF-KappaB Inhibitor alpha; C059715:Fluo-3; C060506:sphingosine 1-phosphate; D002110:Caffeine; D013498:Suramin; D015544:Inositol 1,4,5-Trisphosphate; D013110:Sphingosine; D011188:Potassium; D002118:Calcium