Sphingosine 1-Phosphate Has Dual Functions in the Regulation of Endothelial Cell Permeability and Ca Metabolism

Ca signaling plays an important role in endothelial cell (EC) functions including the regulation of barrier integrity. Recently, the endogenous lipid derivative, sphingosine-1-phosphate (S1P), has emerged as an important modulator of EC barrier function. We investigated the role of endogenously generated S1P in Ca metabolism and barrier function in human umbilical endothelial cells (HUVECs) stimulated by thrombin, histamine, or other agonists. Barrier function was assessed by dextran diffusion through HUVEC monolayers, and Ca transients were measured using a fluoroprobe. Thrombin or histamine increased Ca release from the endoplasmic reticulum (ER) and Ca entry through store-operated channels (SOCs) that was accompanied by increased EC permeability. Inhibition of S1P synthesis by a specific sphingosine kinase inhibitor (SKI) decreased thrombin or histamine-induced increased permeability and decreased Ca entry via SOC in a concentrationdependent fashion. SKI had minuscule effects on thrombin or histamine-induced Ca release from ER. SKI also inhibited thapsigargin or ionomycin-induced Ca entry via SOC without affecting Ca release from the ER. In contrast to the effects of endogenously generated S1P, when S1P was administered externally, it initiated Ca release from ER similar to thrombin and histamine while decreasing EC permeability. These observations indicate that after agonist-induced conditions, endogenously generated S1P functions as a positive modulator of Ca entry via SOC and a mediator of increased cell permeability. In contrast, extracellular exposure to S1P has different signaling mechanisms and effects. Thus, the potential dual roles of endogenous and exogenous S1P on EC function need to be considered in pharmacological studies targeting sphingosine metabolism. Alterations in endothelial cell (EC) barrier integrity are important determinants of organ pathology following inflammation, sepsis, or septic shock (Hallström et al., 1991). For example, increases in EC permeability is one of the underlying causative mechanisms of the adult respiratory distress syndrome, which is one of the most frequent life-threatening clinical complication following severe injuries or infection. Ca signaling plays an important role in EC functions, including the regulation of cell permeability and the maintenance of EC barrier integrity (Malik et al., 1989; Lum and Malik, 1994; Tiruppathi et al., 2002; Ahmmed and Malik, 2005; Mehta and Malik, 2006). However, the exact mechanisms leading to the disintegration of EC barrier function under pathological conditions have been only partially elucidated. In this context, thrombin and histamine, acting through G-protein-coupled receptors, are well known stimulators of Ca release and influx and are important mediators of EC permeability changes under physiological as well as pathological conditions (Tiruppathi et al., 2001). Other inflammatory mediators including cytokines, nitric oxide, activated complement proteins, or reactive oxidant species may also contribute to changes in EC Ca signaling and associated changes in permeability and barrier function (Hasleton