Effects of S1P on myoblastic cell contraction:: Possible involvement of Ca2+-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 Ca2+ transients in a myoblastic cell line (C2C12) by interacting with its specific receptors (S1PRs). In the present study, we wanted to correlate the Ca2+ 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 Ca2+ mobilization, the cells were loaded with a fluorescent Ca2+ 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 Ca2+ increase; cell contraction occurred within 5 - 8 s from stimulation, whereas intracellular Ca2+ increase was evident only after 15 - 25 s. To support the Ca2+ independence of myoblastic cell contraction, the cells were pretreated with a Ca2+ chelator, BAPTA/AM, prior to stimulation with S1P. In these experimental conditions, the myoblasts were still able to contract, whereas the S1P-induced Ca2+ 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 Ca2+. These data suggest that Ca2+-independent mechanism of cell contraction may be replaced by Ca2+-dependent ones during skeletal muscle differentiation. Copyright (C) 2004 S. Karger AG, Basel.