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

Abstract

Ca2ϩ 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 2ϩ 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 2ϩ transients were measured using a fluoroprobe. Thrombin or histamine increased Ca 2ϩ release from the endoplasmic reticulum (ER) and Ca 2ϩ 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 2ϩ entry via SOC in a concentrationdependent fashion. SKI had minuscule effects on thrombin or histamine-induced Ca 2ϩ release from ER. SKI also inhibited thapsigargin or ionomycin-induced Ca 2ϩ entry via SOC without affecting Ca 2ϩ release from the ER. In contrast to the effects of endogenously generated S1P, when S1P was administered externally, it initiated Ca 2ϩ 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 2ϩ 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.

Sphingosine kinase regulates oxidized low density lipoprotein-mediated calcium oscillations and macrophage survival

“…4A ), in that it also decreased survival in control cells incubated without ox-LDL. Dantrolene inhibits Ca 2+ release from RyR channels signifi cant amounts of S1P ( 55 ), does not elicit a Ca 2+ response similar to S1P, suggesting that endogenous production, perhaps in the plasma membrane, may be required to induce calcium oscillation ( 56 ). Our results do not exclude a role for other components of oxLDL, such as oxysterols, in stimulating intracellular [Ca 2+ ] i oscillations.…”

Endothelial Angiogenesis and Barrier Function in Response to Thrombin Require Ca2+ Influx through the Na+/Ca2+ Exchanger

“…In Vitro Endothelial Barrier Function Assays-Endothelial barrier function in vitro was assessed by determining the passage of FITC-labeled dextran through a confluent endothelial monolayer according to a published method (22) with minor modifications. HUVECs (1 ϫ 10 4 ) were seeded into modified Boyden transwell inserts (Costar, 0.4 m pore size) precoated with 50 g/ml rat tail collagen-I (BD Biosciences) and cultured in complete medium for 2 days until confluent.…”

“…2E). Conversely, when HUVECs were loaded, under similar conditions, with the "fast" Ca 2ϩ chelator BAPTA-AM, which suppresses localized Ca 2ϩ signals (22), ERK1/2 activation was inhibited (Fig. 1A).…”

Lipopolysaccharide and Sphingosine-1-Phosphate Cooperate To Induce Inflammatory Molecules and Leukocyte Adhesion in Endothelial Cells

“…P]ATP and D-erythro-sphingosine using a protocol described for endothelial cells (25). Samples (10-20 mg) were incubated with 200 mM ATP and 2 mCi of g-[ 32 P]ATP for 30 min at 37˚C in the SphK assay buffer (20 mM Tris, pH 7.4, 20% glycerol, 1 mM 2-mercaptoethanol, 1 mM EDTA, 1 mM Na 3 VO 4 , 15 mM NaF, and 0.5 mM 4-deoxypyridoxine).…”

“…Samples (10-20 mg) were incubated with 200 mM ATP and 2 mCi of g-[ 32 P]ATP for 30 min at 37˚C in the SphK assay buffer (20 mM Tris, pH 7.4, 20% glycerol, 1 mM 2-mercaptoethanol, 1 mM EDTA, 1 mM Na 3 VO 4 , 15 mM NaF, and 0.5 mM 4-deoxypyridoxine). Lipid extraction and liquid scintillation counting was performed as described (25).…”