Photolysis of intracellular caged sphingosine‐1‐phosphate causes Ca²⁺ mobilization independently of G‐protein‐coupled receptors

Abstract

Sphingosine-1-phosphate (S1P), the product of sphingosine kinase, activates several widely expressed G-proteincoupled receptors (GPCR). S1P might also play a role as second messenger, but this hypothesis has been challenged by recent ¢ndings. Here we demonstrate that intracellular S1P can mobilize Ca 2+ in intact cells independently of S1P-GPCR. Within seconds, S1P generated by the photolysis of caged S1P raised the intracellular free Ca 2+ concentration in HEK-293, SKNMC and HepG2 cells, in which the response to extracellularly applied S1P was either blocked or absent. Ca 2+ transients induced by photolysis of caged S1P were caused by Ca 2+ mobilization from thapsigargin-sensitive stores. These results provide direct evidence for a true intracellular action of S1P.

Cis-4-methylsphingosine is a sphingosine-1-phosphate receptor modulator

“…It is concluded that the cis-4M-S1P that was produced by SphK1 mobilized Ca A c c e p t e d M a n u s c r i p t 14 the stores as previously demonstrated for S1P [12], and that relatively high concentrations of cis-4M-Sph are required for this effect. However, another mechanism, for example activation not of S1P receptors or G i -coupled receptors but other plasma membrane receptors, cannot be fully excluded.…”

“…Finally we wondered whether acute [Ca 2+ ] i increases by cis-4M-Sph were due to activation of G-protein-coupled S1P receptors. Although it is well known that S1P-induced [Ca 2+ ] i increases in HEK-293 cells are fully blocked by PTX and thus mediated by G i -coupled receptors [12], [Ca 2+ ] i increases by cis-4M-Sph were not at all affected by PTX (Fig. 3E) and thus not mediated by G-protein-coupled S1P receptors in these cells.…”

“…There was specificity so that [Ca 2+ ] i increases by agonists other than S1P were not affected to a similar degree, which would be the case if cis-4M-Sph acted for example as an activator of protein kinases C that can lead to receptor desensitization and shutdown of Ca and not observed at lower concentrations of the compound. We think that this activity of cis-4M-Sph is not related to internalization and desensitization of G-protein-coupled S1P receptors since it was independent of G i proteins, which otherwise mediate S1P-induced [Ca 2+ ] i increases in these cells [12], and caused by a distinct pool of cis-4M-S1P. The data show that S1P receptor desensitization was strongly dependent on expression of SphK2, while Ca 2+ mobilization rather required SphK1.…”

“…S1P furthermore has intracellular target sites as it directly inhibits histone deacetylases [11] or mobilizes stored Ca 2+ independently of G-protein-coupled S1P receptors [12]. FTY720 is used by the S1P signalling toolkit as a surrogate for sphingosine.…”

Sphingosine kinase 1 and 2 regulate the capacity of mesangial cells to resist apoptotic stimuli in an opposing manner

“…So far, most evidence in the literature supports S1P receptor-mediated activation of the PI3K/PKB cascade. However, intracellular S1P can activate signal transduction and acutely triggers intracellular Ca 2q mobilisation (Meyer zu Heringdorf et al, 2003) and stimulates cell proliferation of fibroblasts (van Brocklyn et al, 1998).…”