Effects of S1P on myoblastic cell contraction: possible involvement of Ca-independent mechanisms.
Sphingosine-1-phosphate (S1P) is a lipid mediator, which affects many essential processes such as cell proliferation, differentiation and contraction in many cell types. We have previously demonstrated that the lipid mediator elicits Ca(2+) transients in a myoblastic cell line (C2C12) by interacting with its specific receptors (S1PR(s)). In the present study, we wanted to correlate the Ca(2+) response with activation of myoblastic cell contractility. C2C12 cells were first investigated for the expression and cellular organization of cytoskeletal proteins by immunoconfocal microscopy. We found that myoblasts exhibited a quite immature cytoskeleton, with filamentous actin dispersed as a web-like structure within the cytoplasm. To evaluate intracellular Ca(2+) mobilization, the cells were loaded with a fluorescent Ca(2+) indicator (Fluo-3), stimulated with S1P and simultaneously observed with differential interference contrast and fluorescence optics. Exogenous S1P-induced myoblastic cell contraction was temporally unrelated to S1P-induced intracellular Ca(2+) increase; cell contraction occurred within 5-8 s from stimulation, whereas intracellular Ca(2+) increase was evident only after 15-25 s. To support the Ca(2+) independence of myoblastic cell contraction, the cells were pretreated with a Ca(2+) chelator, BAPTA/AM, prior to stimulation with S1P. In these experimental conditions, the myoblasts were still able to contract, whereas the S1P-induced Ca(2+) transients were completely abolished. On the contrary, when C2C12 cells were induced to differentiate into skeletal myotubes, they responded to S1P with a rapid cell contraction concurrent with an increase in the intracellular Ca(2+). These data suggest that Ca(2+)-independent mechanism of cell contraction may be replaced by Ca(2+)-dependent ones during skeletal muscle differentiation.
Nosi
D
D
Department of Anatomy, Histology and Forensic Medicine, University of Florence, Viale Morgagni, 85, IT-50134 Florence, Italy.
Vassalli
M
M
Polidori
L
L
Giannini
R
R
Tani
A
A
Chellini
F
F
Paternostro
F
F
D000199:Actins / Q000187:drug effects; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects*; D002460:Cell Line; D048429:Cell Size / Q000187:drug effects; D002614:Chelating Agents / Q000494:pharmacology; D003599:Cytoskeleton / Q000187:drug effects; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D019084:Fluorescent Antibody Technique, Indirect; D005456:Fluorescent Dyes; D007091:Image Processing, Computer-Assisted; D007700:Kinetics; D008246:Lysophospholipids / Q000494:pharmacology*; D051379:Mice; D018613:Microscopy, Confocal; D009119:Muscle Contraction / Q000187:drug effects*; D032446:Myoblasts; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000494:pharmacology*
D000199:Actins; D002614:Chelating Agents; D005456:Fluorescent Dyes; D008246:Lysophospholipids; C060506:sphingosine 1-phosphate; D004533:Egtazic Acid; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D013110:Sphingosine; D002118:Calcium