Sphingosine 1-Phosphate Has Dual Functions in the Regulation of Endothelial Cell Permeability and Ca2+Metabolism

Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ transients were measured using a fluoroprobe. Thrombin or histamine increased Ca²⁺ release from the endoplasmic reticulum (ER) and Ca²⁺ 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 Ca²⁺ entry via SOC in a concentration-dependent fashion. SKI had minuscule effects on thrombin or histamine-induced Ca²⁺ release from ER. SKI also inhibited thapsigargin or ionomycin-induced Ca²⁺ entry via SOC without affecting Ca²⁺ release from the ER. In contrast to the effects of endogenously generated S1P, when S1P was administered externally, it initiated Ca²⁺ 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 Ca²⁺ 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.