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CELLULAR AND MOLECULAR

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

Department of Surgery, Beth Israel Deaconess Medical Center, Boston, Massachusetts (K.I., C.J.H.); Department of Pharmacology, Pennsylvania State College of Medicine, Hershey, Pennsylvania (J.K.Y., J.A.H.); and Department of Surgery, University of Medicine and Dentistry of New Jersey, Newark, New Jersey (A.Y., Z.S., E.A.D.)

Received April 10, 2007; accepted July 11, 2007.

	Abstract

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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.

In recent years, the endogenous lipid derivative, sphingosine-1-phosphate (S1P), has emerged as a potentially important regulator of EC barrier function (Itagaki and Hauser, 2003; McVerry and Garcia, 2004; Finigan et al., 2005; Seol et al., 2005; Zheng et al., 2006). Upon a variety of stimuli leading to protein C activation, S1P is generated from sphingosine through the action of ubiquitous sphingosine kinase (SK). The newly produced intracellular S1P may act on the Ca²⁺ signaling machinery through not yet identified intracellular receptors or by interacting with the components of Ca²⁺ transporters. Furthermore, S1P can be released from platelets or white blood cells at the sites of EC injury, thrombus formation, or neutrophil attachment, thereby targeting ECs from the extracellular space (Itagaki and Hauser, 2003). Because exogenous administration of S1P has been shown to enhance EC barrier integrity, it is believed that S1P released into the blood stream supports endothelial function during inflammatory states (McVerry and Garcia, 2004; Finigan et al., 2005; Seol et al., 2005). Despite these observations, the exact role of S1P in the regulation of Ca²⁺-mediated EC permeability has not been fully elucidated.

Therefore, in the current study, we investigated the relationship among S1P metabolism, Ca²⁺ signaling, and EC permeability utilizing two physiologically relevant mediators, thrombin and histamine. We also employed a recently developed highly specific sphingosine kinase inhibitor (SKI) to modulate intracellular S1P levels and test associated changes in cellular Ca²⁺ signaling. We hypothesized that S1P generated intracellularly by SK or administered exogenously has distinct and different effects on Ca²⁺ signaling and endothelial cell permeability.

	Materials and Methods

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Materials. The SKI, 2-(p-hydroxyanilino)-4-(p-chlorophenyl) thiazole, which was formally named SKI-II, was developed and synthesized by our group as described previously (French et al., 2003) and was used as a specific sphingosine kinase inhibitor previously (Lee et al., 2004, 2005; Francy et al., 2007; Jung et al., 2007). In this article, we described SKI-II as SKI for simplicity. S1P, thrombin, histamine, and thapsigargin were purchased from Sigma-Aldrich (St. Louis, MO). Fura-2-acetoxymethyl ester was from Molecular Probes (Eugene, OR). Ionomycin was purchased from Calbiochem (La Jolla, CA). All other chemicals and reagents were from Sigma-Aldrich.

Cell Culture. Human umbilical vein endothelial cells (HUVECs) were from BioWhittaker (Walkersville, MD) and were cultured to confluence at 37°C with 95% O₂/5% CO₂ in endothelial cell basal media. Only cells of low passage (three to five) were used for assays. SKI was administered in the concentration range of 10 to 40 µMin the presence of vehicle. SKI together with DMSO caused no cytotoxicity up to 40 µM.

EC Permeability Assay. EC permeability was measured as described previously (Magnotti et al., 1998; Deitch et al., 2001, 2004). In brief, HUVECs were seeded at 20,000 cells per insert (0.33 cm²) on type I rat-tailed collagen-coated membranes (pore size, 3 µm) contained on the apical chamber of a two-chambered transwell system (Costar, Cambridge, MA). As described previously, the EC monolayers had become confluent by 72 h after seeding. After experimental treatments, a 0.05% solution of a 40-kDa dextran rhodamine permeability probe (Molecular Probes) was added to the apical chamber of the transwell system. After an additional 1-h incubation period, medium in the basal chamber was removed, and the amount of dextran rhodamine present was determined spectrophotometrically at 555/580 nm. Permeability of the EC monolayers was expressed as clearance relative to control conditions.

Ca²⁺ Transient Measurements. Intracellular Ca²⁺ transients were measured as described previously (Itagaki and Hauser, 2003). In brief, ECs were incubated with 2 µM Fura-2-acetoxymethyl ester at 37°C for 30 min. Cells were divided into aliquots of 0.3 x 10⁶ and placed on ice in the dark until ready for use. Just before each experiment, individual aliquots were incubated at 37°C for 5 min. Cells were then pelleted by centrifugation at 4500 rpm for 5 s in a microcentrifuge and resuspended in 3 ml of Hanks' balanced salt solution in the cuvette. Experiments were generally begun in nominally Ca²⁺-free medium containing 0.3 mM EGTA. Intracellular Ca²⁺ was monitored by measuring Fura fluorescence at 505 nm, using 340/380-nm dual-wavelength excitation in a Fluoromax-3 spectrofluorometer (Jobin Yvon-Spex, Edison, NJ). Cuvette temperatures were kept at 37°C with constant stirring. Calibration was performed at the end of each experiment by the addition of 100 µM digitonin (Molecular Probes) for R_MAX and then 15 mM EGTA for R_MIN. The autofluorescence of a sample cell suspension treated with 100 µM digitonin and 2 µM MnCl₂ was subtracted from total fluorescence. The intracellular Ca²⁺ concentration was then calculated from the 340-/380-nm fluorescence ratio (K_d = 220 nM) as per the methods of Grynkiewicz et al. (1985).

Sphingosine Kinase Activity Assays. To assess the catalytic activity of SK activity, the formation of sphingosine-1-³²P from [-³²P]ATP and D-erythro-sphingosine was determined by liquid scintillation counting assays as described previously (French et al., 2003; Itagaki and Hauser, 2003). In brief, whole-cell extracts (20 µg) were combined with 50 µM D-erythro-sphingosine and 200 µM ATP + 2 µCi of [-³²P]ATP in a 100-µl final reaction volume of sphingosine kinase activity assay buffer (20 mM Tris, pH 7.4, 20% glycerol, 1 mM -mercaptoethanol, 1 mM EDTA, 1 mM Na₃VO₄, 15 mM NaF, and 0.5 mM 4-deoxypyridoxine) for 30 min at 37°C with shaking. Reactions were terminated by the addition of 10 µl of 6 N HCl, and labeled lipids were extracted by the addition of 400 µl of chloroform/MeOH/HCl (100:200:1), 125 µl of chloroform, and 125 µl of 1 M KCl. The organic phase was removed and dried under nitrogen. Dried lipids were then resuspended in chloroform/methanol (2:1), and S1P was separated by TLC (Silica gel G60) in an n-butanol/water/acetic acid (3:1:1) solvent system. In place of S1P separation by TLC, we also developed a protocol to determine S1P formation by terminating the reactions by the addition of 10 µl of 6 N HCl and extracting the labeled lipids by the addition of 400 µl of chloroform/MeOH/HCl (100:200:1), 125 µl of chloroform, and 125 µl of 1 M KCl. The organic phase was removed and dried on filter paper squares before liquid scintillation counting. The relative efficiency of this protocol was comparable with the TLC separation method for determining SK activity (J. K. Yun, unpublished data).

Statistics. Results were analyzed using analysis of variance followed by Student's t test for pair-wise comparisons or Tukey-Kramer's test for multiple comparisons. Data are expressed as mean ± S.E. Statistically significant difference was concluded at p < 0.05.

	Results

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SKI Prevents Thrombin- and Histamine-Induced Increase in EC Permeability. Figure 1A shows that thrombin significantly increased clearance of 40-kDa dextran rhodamine through a confluent monolayer of ECs. SKI caused a dose-dependent attenuation of thrombin-induced increase in EC permeability. Likewise, histamine caused an increase in EC permeability, and SKI abrogated the histamine-induced effect at 30 µM SKI (Fig. 1B). SKI alone did not cause any change in EC permeability, indicating that endogenous S1P activity does not significantly contribute to EC permeability under nonstimulated "baseline" conditions. Because SKI was dissolved in DMSO, we tested its effect on EC permeability. We found that DMSO had no effect on EC permeability at concentrations up to 0.5%. Taken together, these results indicate that S1P has a role in G-protein-coupled agonist-induced changes in EC permeability. We next examined the cellular mechanisms of S1P effects on EC permeability.

View larger version (17K): [in this window] [in a new window]   Fig. 1. SKI promotes HUVEC barrier integrity upon thrombin or histamine challenge. EC permeability change was assessed by clearance of 40-kDa dextran rhodamine through a confluent monolayer after various treatments. A, effects of SKI at 10 or 40 µM on 1 U/ml thrombin-induced increased EC permeability. Data are expressed as percentage of control (medium) value. EC permeability was not altered with 0.1% DMSO (vehicle). B, effects of SKI 30 µMon100 µM histamine-induced changes in EC permeability. Mean ± S.E. from eight to nine (A) or three to six (B) independent incubations in each treatment groups. *, statistically significant difference as compared with all groups except 10 µM SKI.

Effects of SKI on Thrombin- and Histamine-Induced Increase in Intracellular Ca²⁺ Concentration. An increase in cytosolic Ca²⁺ has been shown as the initial pivotal signal for agonist-induced increase in EC permeability (Mehta and Malik, 2006). To determine whether SKI effects on EC permeability are associated with changes in intracellular Ca²⁺ concentration, ECs were loaded with Fura-2, and intracellular Ca²⁺ concentration was measured. Figure 2 shows the effects of thrombin and histamine on intracellular Ca²⁺ transients. Consistent with earlier observations (Mehta and Malik, 2006), upon EC stimulation with thrombin (Fig. 2A) or histamine (Fig. 2B), an increase in cytosolic Ca²⁺ concentration is apparent. There are two phases of Ca²⁺ transients. The initial peak is the result of Ca²⁺ release from the endoplasmic reticulum (ER) Ca²⁺ store, which is coupled to inositol 1,4,5-triphosphate receptors. The second, more sustained response is secondary to Ca²⁺ entry through nonselective cation channels known as store-operated Ca²⁺ channels (SOCs) (Mehta and Malik, 2006). Figure 2, A and B, show that thrombin and histamine caused immediate increase in the initial phase of the Ca²⁺ transient, presumably due to stimulation of Ca²⁺ release from the intracellular ER store (Itagaki et al., 2002; Itagaki and Hauser, 2003). When extracellular Ca²⁺ (1.8 mM) was added to the bath medium, the sustained component of Ca²⁺ transient was evident. Both agents induced dose-dependent increase in both phases of Ca²⁺ transients.

View larger version (21K): [in this window] [in a new window]   Fig. 2. Effects of thrombin and histamine on Ca2+ transients in HUVEC. Ca2+ release from the ER and Ca2+ influx from extracellular space via SOC were determined. Thrombin (0.1–1 U/ml; A) or histamine (0–100 µM; B) was applied at t = 30 s in Ca2+-free medium to detect Ca2+ depletion from the ER, and then 1.8 mM Ca2+ was added at t = 250 s (A) or t = 200 s (B) to detect Ca2+ influx. Results indicate a typical finding from at least three independent experiments.

View larger version (28K): [in this window] [in a new window]   Fig. 3. SKI inhibits thrombin- or histamine-induced Ca2+ influx in HUVEC. Cells were pretreated with SKI for 3 min before the start of the experiment (t = 0) at the indicated concentrations. At t = 30 s, 1 U/ml thrombin (A) or 100 µM histamine (B) was applied in Ca2+-free medium, and then 1.8 mM Ca2+ was added at t = 250 s (A) or at t = 200 s (B). Results indicate a typical finding from at least three independent experiments.

Effects of SKI on Thapsigargin- and Ionomycin-Induced SOC Activation in EC. The effects of SKI on Ca²⁺ transients were also assessed after release of Ca²⁺ from stores with the Ca²⁺ pump blocker, thapsigargin (TG), which depletes Ca²⁺ from the ER as a slow leak in the absence of external Ca²⁺ (Fig. 4A). Reintroduction of Ca²⁺ to the bath solution results in Ca²⁺ influx via SOC activation. Pretreatment with SKI blocked the second phase of Ca²⁺ entry without changing the initial phase of Ca²⁺ transients (Fig. 4A). We also conducted this experiment after store depletion with a Ca²⁺ ionophore, ionomycin (to eliminate Ca²⁺ release through reactivated inositol 1,4,5-triphosphate). Ionomycin induced a more rapid and complete release of stored Ca²⁺ (compared with Fig. 3 or 4A). Readdition of external Ca²⁺ resulted in SOC-mediated Ca²⁺ entry. Preincubation of EC with SKI reduced ionomycin-induced SOC activation during reintroduction of external Ca²⁺ without significant effects on the initial Ca²⁺ transients (Fig. 4B). These results suggest that the effects of SKI on agonist-induced increases in EC permeability are mediated by Ca²⁺ entry via SOC but not Ca²⁺ released from ER. Our results also suggest that ER Ca²⁺ stores and SOC are not coupled to regulate EC permeability.

View larger version (22K): [in this window] [in a new window]   Fig. 4. Effects of SKI on TG- or ionomycin-induced Ca2+ influx via SOC in HUVEC. Cells were treated with 10 or 30 µM SKI for 3 min before the start of the experiment (t = 0). At t = 30 s, 1 µM TG (A) or 100 nM ionomycin (B) was applied in Ca2+-free condition, and then 1.8 mM Ca2+ was added at t = 400 s (A) or at t = 200 s (B). Results indicate a typical finding from at least three independent experiments.

Effects of SKI on Thrombin-Induced S1P Synthesis. We next measured thrombin-induced S1P synthesis and the effects of SKI to confirm that SKI's effects are mediated by alterations of intracellular S1P levels through the action of SK. Figure 5 shows the effects of thrombin on S1P production and the effects of SKI. Upon thrombin receptor stimulation, S1P synthesis was significantly increased in HUVECs. Pretreatment with SKI inhibited S1P synthesis to a value comparable with the control level.

View larger version (18K): [in this window] [in a new window]   Fig. 5. Effects of SKI on thrombin-induced SK activity in HUVEC. Cells were treated with SKI (30 µM) or thrombin alone or in combination as indicated. Whole-cell lysates were prepared and assayed for SK activity as described under Materials and Methods. Results indicate mean ± S.E. from three independent experiments. *, statistically significant difference compared with thrombin treatment.

Effects of Extracellularly Applied S1P on EC Permeability and Ca²⁺ Signaling. The presented findings indicate that inhibition of intracellular S1P production supports EC barrier function by decreasing agonist-induced Ca²⁺ influx through store-operated calcium entry. These observations however may be interpreted as contradictory to previous findings indicating that exogenously administered S1P improves EC barrier function (Mehta et al., 2005). Therefore, in the last series of experiments, we tested the effects of extracellular administration of S1P on EC permeability as well as cellular Ca²⁺ transients. In accordance with previous investigations by others, exogenous S1P administration to endothelial monolayers markedly decreased basal permeability, a finding that is consistent with enhanced barrier function (Fig. 6A). Furthermore, as shown in Fig. 6B, S1P administration to EC induced Ca²⁺ transients with very similar kinetics to that reported previously (Mehta et al., 2005).

View larger version (16K): [in this window] [in a new window]   Fig. 6. Effects of extracellularly applied S1P on HUVEC permeability and Ca2+ transients. A, EC permeability was determined as described for Fig. 1 under control conditions (medium) and after the treatment with 1 µMS1P for 1 h. B, Ca2+ transients were determined in the presence of increasing concentrations of S1P administered to the cells.

	Discussion

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The relationship between Ca²⁺ signaling and EC barrier function is not yet fully elucidated. Independent investigations demonstrated that inflammatory agents including thrombin and histamine markedly increase endothelial permeability (Mehta and Malik, 2006). These agents initiate characteristic Ca²⁺ kinetics with an early transient release from the ER followed by a more sustained Ca²⁺ entry via SOC (Mehta and Malik, 2006). Thrombin-induced EC permeability requires Ca²⁺ entry from the extracellular space as well as Ca²⁺ depletion from the ER (Ahmmed and Malik, 2005; Mehta and Malik, 2006). The reduction of EC permeability involves the reduction of transient receptor potential canonical proteins such as transient receptor potential canonical 1 and 4 (TRPC1 and 4) (Tiruppathi et al., 2001, 2002; Ahmmed and Malik, 2005). It was also suggested that increases in EC Ca²⁺ result in the phosphorylation of myosin light chain through Rho kinase, which consequently increases EC permeability (Ahmmed and Malik, 2005). These data strongly suggest that regulation of Ca²⁺ influx is a key process in maintaining EC permeability.

Interestingly, extracellular application of S1P initiates a Ca²⁺ response that is similar to that caused by thrombin or histamine, albeit the SOC phase is less pronounced. These observations are consistent with earlier reports (Mehta et al., 2005). However, despite the similar Ca²⁺ responses after thrombin (or histamine) and extracellular S1P administrations, these agents have opposing effects on EC barrier function. This discrepancy between Ca²⁺ responses and the parallel changes in cell permeability indicate that thrombin, histamine, and S1P alter cell permeability through different signaling pathways. Thus, based on these observations, the finding that exogenous or endogenous S1P has different effects on cell permeability also suggests the potential existence of different extra- and intracellular S1P receptors and the involvement of different signaling pathways mediating these S1P effects. The potential dual roles of endogenous and exogenous S1P as a critical lipid messenger molecule regulating EC function are consistent with previous observations on other cells and experimental settings as summarized in recent reviews (Young and Nahorski, 2001; Sanchez and Hla, 2004; Rosen and Goetzl, 2005; Milstien et al., 2007).

It is also important to mention that although the phosphorylated form of sphingosine may remain imbedded in membranes through its hydrophobic portion, it is likely that the presence of charged phosphorylated groups on S1P prevents its free transit through the cell membranes. Therefore, the exogenous S1P released from blood cells targeting ECs externally or the S1P generated within ECs is expected to remain compartmentalized and initiate endothelial responses through different receptor targets and signaling mechanisms.

Our observation that the inhibition of endogenous S1P synthesis down-regulated store-operated Ca²⁺ entry initiated by all of the investigated Ca²⁺-mobilizing agonists has a particular importance. These agents initiate Ca²⁺ mobilization through different mechanisms; thrombin and histamine acting through their distinct surface receptors and TG directly targeting the ER, causing slow Ca²⁺ release, whereas ionomycin is a general Ca²⁺ ionophore. Thus, the fact that after administration of all of the employed agonists SKI decreased SOC similarly, whereas it minimally affected Ca²⁺ release from the ER, suggests that intracellular S1P modulate SOC at downstream targets of signaling cascades. Because sphingosine is a membrane-associated lipid, its phosphorylation by SKI may alter its assembly within membrane rafts with consequent changes in the interaction between S1P and membrane-associated proteins in the vicinity. Thus, we propose that endogenously generated S1P may modulate Ca²⁺ entry via SOC by direct interactions with components of the transport protein complex. It is evident that the elucidation of the specific mechanisms responsible for the observed effects of endogenous S1P needs further investigations. In summary, our observations indicate an important role of endogenously generated S1P in the modulation of agonist-induced Ca²⁺ signaling and accompanied changes in EC permeability. Endogenous S1P functions as a positive modulator of Ca²⁺ uptake via SOC, resulting in increased cell permeability upon a variety of agonist-induced conditions including thrombin and histamine. These observations also suggest that S1P targeting ECs from the extracellular space exerts it effects through different receptor and signaling mechanisms than those of S1P generated intracellularly. The potential dual roles of endogenous and exogenous S1P need to be considered in pharmacological interventions aiming to prevent EC dysfunction under pathological conditions.

	Footnotes

 
This work was supported by National Institutes of Health-National Institute of General Medical Sciences Grants GM59841 and 69790 (to E.A.D.), GM69861 (to Z.S.), and GM059179 (to C.J.H.).

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.107.121210.

ABBREVIATIONS: EC, endothelial cell; S1P, sphingosine 1-phosphate; SK, sphingosine kinase; SKI, sphingosine kinase inhibitor; HUVEC, human umbilical vein endothelial cell; DMSO, dimethyl sulfoxide; TLC, thin layer chromatography; ER, endoplasmic reticulum; SOC, store-operated Ca²⁺ channel; TG, thapsigargin.

Address correspondence to: Kiyoshi Itagaki, Beth Israel Deaconess Medical Center/Harvard Medical School, 330 Brookline Avenue, ST-8M10A, Boston, MA 02215. E-mail: kitagaki{at}bidmc.harvard.edu

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