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Am J Physiol Cell Physiol 282: C1361-C1373, 2002. First published January 30, 2002; doi:10.1152/ajpcell.00378.2001
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Vol. 282, Issue 6, C1361-C1373, June 2002

Sphingosine 1-phosphate induces Ca2+ transients and cytoskeletal rearrangement in C2C12 myoblastic cells

Lucia Formigli1, Fabio Francini2, Elisabetta Meacci3, Massimo Vassalli4, Daniele Nosi1, Franco Quercioli4, Bruno Tiribilli4, Chiara Bencini2, Claudia Piperio2, Paola Bruni3, and Sandra Zecchi Orlandini1

Departments of 1 Anatomy, Histology, and Forensic Medicine, 2 Physiological Sciences, and 3 Biochemical Sciences, University of Florence, 50134 Florence; and 4 Biophotonics Lab, National Institute of Applied Optics, 50125 Florence, Italy

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.

calcium ion transients; cytoskeleton; cell contraction; confocal microscopy


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