Bencini Chiara, Squecco Roberta, Piperio Claudia, Formigli Lucia, Meacci Elisabetta, Nosi Daniele, Tiribilli Bruno, Vassalli Massimo, Quercioli Franco, Bruni Paola, Zecchi Orlandini Sandra, Francini Fabio
Department of Physiological Sciences, University of Florence, Viale GB Morgagni 64, 1-50134 Florence, Italy.
J Muscle Res Cell Motil. 2003;24(8):539-54. doi: 10.1023/b:jure.0000009898.02325.58.
Sphingosine 1-phosphate (S1P) activates a subset of plasma membrane receptors of the endothelial differentiation gene family (EdgRs) in many cell types. In C2C12 myoblasts, exogenous S1P elicits Ca2+ transients by activating voltage-independent plasma membrane Ca2+ channels and intracellular Ca2+-release channels. In this study, we investigated the effects of exogenous S1P on voltage-dependent L-type Ca2+ channels in skeletal muscle fibers from adult mice. To this end, intramembrane charge movements (ICM) and L-type Ca2+ current (I(Ca)) were measured in single cut fibers using the double Vaseline-gap technique. Our data showed that submicromolar concentrations of S1P (100 nM) caused a approximately 10-mV negative shift of the voltage threshold and transition voltages of q(gamma) and q(h) components of ICM, and of I(Ca) activation and inactivation. Biochemical studies showed that EdgRs are expressed in skeletal muscles. The involvement of EdgRs in the above S1P effects was tested with suramin, a specific inhibitor of Edg-3Rs. Suramin (200 microM) significantly reduced, by approximately 90%, the effects of S1P on ICM and I(Ca), suggesting that most of S1P action occurred via Edg-3Rs. Moreover, SIP at concentration above 10 microM elicited intracellular Ca2+ transients in muscle fibers loaded with the fluorescent Ca2+ dye Fluo-3, as detected by confocal laser scanning microscopy.
鞘氨醇-1-磷酸（S1P）可激活多种细胞类型中内皮分化基因家族（EdgRs）的一部分质膜受体。在C2C12成肌细胞中，外源性S1P通过激活电压非依赖性质膜Ca2+通道和细胞内Ca2+释放通道引发Ca2+瞬变。在本研究中，我们研究了外源性S1P对成年小鼠骨骼肌纤维中电压依赖性L型Ca2+通道的影响。为此，使用双凡士林间隙技术在单根切断的纤维中测量膜内电荷运动（ICM）和L型Ca2+电流（I(Ca)）。我们的数据表明，亚微摩尔浓度的S1P（100 nM）使ICM的q(γ)和q(h)成分以及I(Ca)激活和失活的电压阈值和转折电压产生约10 mV的负向偏移。生化研究表明，EdgRs在骨骼肌中表达。用Edg-3Rs的特异性抑制剂苏拉明测试了EdgRs在上述S1P效应中的作用。苏拉明（200 μM）使S1P对ICM和I(Ca)的影响显著降低约90%，表明大多数S1P作用是通过Edg-3Rs发生的。此外，通过共聚焦激光扫描显微镜检测，浓度高于10 μM的SIP在装载有荧光Ca2+染料Fluo-3的肌肉纤维中引发细胞内Ca2+瞬变。
