Faculty of Bioscience and Technology for Food Agriculture and Environment, University of Teramo, 64100 Teramo, Italy.
Department of Experimental and Clinical Biomedical Sciences Mario Serio, University of Florence, 50121 Firenze, Italy.
Int J Mol Sci. 2022 Sep 21;23(19):11103. doi: 10.3390/ijms231911103.
The sphingosine 1-phosphate (S1P) and endocannabinoid (ECS) systems comprehend bioactive lipids widely involved in the regulation of similar biological processes. Interactions between S1P and ECS have not been so far investigated in skeletal muscle, where both systems are active. Here, we used murine C2C12 myoblasts to investigate the effects of S1P on ECS elements by qRT-PCR, Western blotting and UHPLC-MS. In addition, the modulation of the mitochondrial membrane potential (ΔΨm), by JC-1 and Mitotracker Red CMX-Ros fluorescent dyes, as well as levels of protein controlling mitochondrial function, along with the oxygen consumption were assessed, by Western blotting and respirometry, respectively, after cell treatment with methanandamide (mAEA) and in the presence of S1P or antagonists to endocannabinoid-binding receptors. S1P induced a significant increase in TRPV1 expression both at mRNA and protein level, while it reduced the protein content of CB2. A dose-dependent effect of mAEA on ΔΨm, mediated by TRPV1, was evidenced; in particular, low doses were responsible for increased ΔΨm, whereas a high dose negatively modulated ΔΨm and cell survival. Moreover, mAEA-induced hyperpolarization was counteracted by S1P. These findings open new dimension to S1P and endocannabinoids cross-talk in skeletal muscle, identifying TRPV1 as a pivotal target.
鞘氨醇 1-磷酸(S1P)和内源性大麻素(ECS)系统包含广泛参与调节相似生物过程的生物活性脂质。迄今为止,在骨骼肌中尚未研究 S1P 和 ECS 之间的相互作用,而这两个系统在骨骼肌中均活跃。在这里,我们使用鼠 C2C12 成肌细胞通过 qRT-PCR、Western blot 和 UHPLC-MS 来研究 S1P 对 ECS 元件的影响。此外,通过 JC-1 和 Mitotracker Red CMX-Ros 荧光染料评估线粒体膜电位(ΔΨm)的调制,以及通过 Western blot 和呼吸计分别评估控制线粒体功能的蛋白质水平和耗氧量,在用 S1P 或内源性大麻素结合受体的拮抗剂处理细胞后和在 mAEA 存在下。S1P 在 mRNA 和蛋白水平上均诱导 TRPV1 表达显著增加,而 CB2 的蛋白含量降低。证明了 mAEA 对 ΔΨm 的剂量依赖性影响,由 TRPV1 介导;具体而言,低剂量导致 ΔΨm 增加,而高剂量则负调节 ΔΨm 和细胞存活。此外,S1P 拮抗了 mAEA 诱导的超极化。这些发现为 S1P 和内源性大麻素在骨骼肌中的相互作用开辟了新的维度,确定 TRPV1 为关键靶点。