The TRPV1 Receptor Is Up-Regulated by Sphingosine 1-Phosphate and Is Implicated in the Anandamide-Dependent Regulation of Mitochondrial Activity in C2C12 Myoblasts

Abstract

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.

Sphingosine Kinases at the Intersection of Pro-Inflammatory LPS and Anti-Inflammatory Endocannabinoid Signaling in BV2 Mouse Microglia Cells

“…The present study aimed to investigate potential crosstalk between the eCB and the S1P systems in an inflammation model of murine BV2 microglia cells. Such crosstalk has been observed for example in skeletal muscle cells [36] and in the regulation of the vascular tone [37,38]. Here, we show that both SphK1 and SphK2 were induced by LPS and were required for pro-inflammatory cytokine production in BV2 cells, and that the FAAH inhibitor URB597, and CB 2 receptor agonist reverted both LPS-induced cytokine production and SphK1/2 induction, thus placing SphKs at the intersection of pro-inflammatory LPS and anti-inflammatory eCBs signaling in these cells.…”

“…Due to the unprecedented evidence of the influence of the eCB system on the S1P system, and vice versa, in cultured murine skeletal-muscle C2C12 cells [36], in the regulation of rat coronary artery reactivity [37] and in the mediation of the blood pressure changes in mice [38], in this study the possible functional interplay between the S1P and the eCB systems has been explored in inflamed murine microglial BV2 cells.…”

“…Absorbance was detected by Enspire multimode plate reader (Perkin Elmer, Waltham, MA, USA). The cell viability was calculated by subtracting the 630 nm OD background from the 570 nm OD total signal of the cell-free blank of each sample and was expressed as a percentage of controls set to 100% [36].…”

Deciphering Complex Interactions in Bioactive Lipid Signaling

“…Recent studies implicated a regulatory function of sphingolipids in prostaglandin production, and sphingosine-1-phosphate (S1P) has been shown to induce COX-2 expression [ 78 ]. In line with this, S1P has been recently demonstrated to activate TRPV1, with an impact on AEA-dependent regulation of mitochondrial activity [ 79 ]; conversely, RvD1 activates the S1P signaling pathway [ 80 ]. Moreover, steroid hormones also can contribute to modulating AA/PUFA-derived lipid signaling, for instance, at the level of gene expression.…”