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Research Paper

Journal of Pharmacy And Pharmacology

Sphingosine 1-phosphate increases an intracellular Ca2+ concentration via S1P3 receptor in cultured vascular smooth muscle cells Kazumi Fujii, Takuji Machida, Kenji Iizuka and Masahiko Hirafuji Department of Pharmacological Sciences, School of Pharmaceutical Sciences, Health Sciences University of Hokkaido, Ishikari-Tobetsu, Hokkaido, Japan

Keywords intracellular Ca2+ concentration; L-type voltage-dependent Ca2+ channel; S1P3 receptor; sphingosine 1-phosphate; vascular smooth muscle cells Correspondence Takuji Machida, Department of Pharmacological Sciences, School of Pharmaceutical Sciences, Health Sciences University of Hokkaido, Ishikari-Tobetsu, Hokkaido 061-0293, Japan. E-mail: [email protected] Received August 2, 2013 Accepted December 7, 2013 doi: 10.1111/jphp.12214

Abstract Objective We investigated the effect of sphingosine 1-phosphate (S1P) on intracellular Ca2+ dynamics in rat vascular smooth muscle cells (VSMCs). Methods Intracellular Ca2+ concentration ([Ca2+]i) was determined using a fluorescence dye fura-2/AM. Small interfering RNAs (siRNA) were transfected into VSMCs to deplete the expression of S1P2 and S1P3 receptors. Key findings S1P induced a rapid and transient elevation in [Ca2+]i, which was maximal 1 min after the stimulation, followed by a sustained increase. When extracellular Ca2+ was removed, a decrease in resting level and a small and transient increase in [Ca2+]i by S1P stimulation were observed. siRNA targeted for the S1P3 receptor almost completely inhibited the S1P-induced increase in [Ca2+]i. The rapid and transient increase in [Ca2+]i was significantly inhibited by diltiazem at a high concentration. Pertussis toxin and a phospholipase C (PLC) inhibitor inhibited the S1P-induced increase in [Ca2+]i regardless of the presence of extracellular Ca2+. Furthermore, S1P activated store-operated and receptoroperated Ca2+ entry. Conclusions These results suggest that S1P increases [Ca2+]i via the S1P3 receptor by inducing an influx of extracellular Ca2+ partially through the voltage-dependent Ca2+ channels, as well as by mobilizing Ca2+ from its intracellular stores. S1P3 receptor-coupled Gi/o protein and PLC activation mediate the mechanisms.

Introduction There is increasing evidence that sphingosine 1-phosphate (S1P), a bioactive lipid mediator, exerts potent and diverse biological effects on a variety of cell types, including vascular endothelial cells and vascular smooth muscle cells (VSMCs). In the vascular system, the major source of S1P is plasma, and S1P is stored abundantly in hematopoietic cells such as red blood cells and activated platelets, which lack the S1P-degrading enzyme S1P lyase.[1–4] Five S1P receptor subtypes (S1P1, S1P2, S1P3, S1P4 and S1P5) have been identified in a wide variety of cells. These receptors are coupled to multiple pertussis toxin (PTX)-sensitive and PTXinsensitive G proteins.[5] Rat VSMCs have been shown to express high levels of S1P2 and S1P3 mRNA, and under certain conditions low levels of S1P1 mRNA.[6] The S1P2 and S1P3 receptors have common signalling characteristics, and 802

couple to Gq, Gi/o and G12/13 proteins.[5] Through these cascades, S1P dramatically affects the functions of VSMCs, such as proliferation, inhibition or stimulation of migration, and vasoconstriction or release of vasoactive mediators.[6–8] The intracellular Ca2+ concentration ([Ca2+]i) in VSMCs plays a regulatory role as a second messenger in the mechanism of cellular functions. A number of mechanisms regulate intracellular Ca2+ dynamics induced by various vasoactive agonists in VSMCs. S1P induces an increase of [Ca2+]i in many cell types, including VSMCs.[9] Recent reports suggest that activation of the S1P3 receptor seems to play an important role in Ca2+ mobilization. S1P activates Rho, phospholipase C (PLC) and intracellular Ca2+ mobilization in mouse embryonic fibroblasts, and the deletion of

© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 66, pp. 802–810

Kazumi Fujii et al.

the S1P3, but not the S1P2, receptor leads to marked inhibition of PLC activation and S1P induction of [Ca2+]i.[10] Furthermore, S1P-induced vasoconstriction in cerebral arteries isolated from S1P3 receptor-null mice is significantly inhibited.[11] Previously, we have reported that S1P induces cyclooxygenase-2 (COX-2) expression and subsequent prostaglandin I2 production, a potent vasodilator, via Ca2+dependent pathway in rat primary VSMCs.[6] The COX-2 expression is completely and partially abolished by inhibiting S1P3 receptor and Gi/o protein, respectively. On the other hand, we also demonstrated that S1P strongly suppresses the other potent vasodilator, nitric oxide (NO) production and inducible NO synthase (iNOS) expression induced by interleukin-1β stimulation in rat primary VSMCs.[7] The iNOS inhibition is regulated by both Ca2+-dependent and Ca2+-independent pathways. Thus, S1P-induced increase in [Ca2+]i has important and pivotal roles for producing vasoactive mediators in VSMCs by regulating its signal transduction. VSMCs are well known to undergo phenotypic modulation during cell culturing, that is switch from a contractile to a more synthetic phenotype.[12,13] Furthermore, the expression pattern of S1P receptor subtype changes depending on culture passages.[14] Although the effect of S1P on [Ca2+]i in VSMCs has been reported in other studies, the Ca2+ dynamics by S1P stimulation may be affected by conditions of isolation or cell culture. In this study, using small interfering RNA (siRNA) targeted for specific S1P receptors, we investigated the mechanisms of an S1P-induced increase in [Ca2+]i in rat primary VSMCs. We found that S1P increases [Ca2+]i via the S1P3, but not the S1P2, receptor by inducing an influx of extracellular Ca2+ partially through the voltage-dependent Ca2+ channels, including L-type Ca2+ channels, as well as by mobilizing Ca2+ from its intracellular stores. S1P3 receptorcoupled Gi/o protein and PLC activation are involved in the mechanisms.

Materials and Methods Cell culture VSMCs were enzymatically isolated from the aortic media of 6- to 7-week-old Wistar rats using collagenase and elastase described previously.[15] Cells were suspended in Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal calf serum (FCS), 100 U/ml penicillin and 100 μg/ml streptomycin, and cultured until confluence at 37°C in 95% air plus 5% CO2 with medium changes every 2–3 days (6–8 days). Primary cultured VSMCs were used throughout the experiments. This study was conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals by the Animal Research Committee of Health Sciences University of Hokkaido.

S1P increases [Ca2+]i via S1P3 receptor

Measurement of [Ca2+]i [Ca2+]i was measured as described previously.[16,17] Cells seeded and grown on cover glasses (8 × 16 mm) were loaded with 5 μm fura-2 acetoxymethyl ester (fura-2/AM) for 45 min at 37°C in Hank’s balanced salt solution containing 0.1% bovine serum albumin and 10 mM HEPES (HBSS; pH 7.4). Fluorescence at 505 nm emission wavelength alternately excited at 340 and 380 nm was measured using a fluorescence spectrophotometer (Hitachi F-4000, Tokyo, Japan). Cells were perfused continuously at 1 ml/ min with HBSS, to which test drugs were added. Rmax, the maximal fluorescence ratio, was measured by exposing cells to 10 μm ionomycin in the presence of 5 mM Ca2+, followed by perfusion with Ca2+-free HBSS containing 1 mM EGTA to obtain Rmin, the minimum ratio. The cells were finally exposed to 0.05% Triton X-100 to obtain the autofluorescence. After the subtraction of auto fluorescence, [Ca2+]i was calibrated according to the equation of Grynkiewicz et al., assuming the Kd of the Ca2+–fura-2 interaction to be 225 nM in the cytosolic environment.[18]

Transfection of small interfering RNAs siRNAs for the S1P2 and S1P3 receptors were introduced into cells using Lipofectamine 2000 (Life Technologies) according to the manufacturer’s instruction. The cells were further cultured for 72 h. siRNA cocktail targeting for rat S1P2 and siRNA combo targeting for rat S1P3 were designed and synthesized by B-Bridge International Inc. (Cupertino, CA, USA). The three-target siRNAs for S1P2 used in the experiments were as follows: (1) 5′-GUG CCA UCG UGG UGG AGA A-3′ (sense), 5′-UUC UCC ACC ACG AUG GCA C-3′ (antisense), (2) 5′-CAA CAU UUC UGG AGG GCA A −3′ (sense), 5′-UUG CCC UCC AGA AAU GUU G-3′ (antisense), and (3) 5′-GGG CAG AUG UUG AGG AGA A-3′ (sense), 5′-UUC UCC UCA ACA UCU GCC C-3′ (antisense); and the three-target siRNAs for S1P3 were as follows: (1) 5′-GCA UAG CCU ACA AGG UCA A-3′ (sense), 5′-UUG ACC UUG UAG GCU AUG C-3′ (antisense), (2) 5′-GGA ACU GCC UGG AGA ACU U-3′ (sense), 5′-AAG UUC UCC AGG CAG UUC C-3′ (antisense), and (3) 5′-CAG CCA UUC UGG UGA CCA U-3′ (sense), 5′-AUG GUC ACC AGA AUG GCU G-3′ (antisense). The nonsilencing siRNA was obtained from Thermo Scientific, Dharmacon Division (Lafayette, CO, USA).

Quantitative reverse transcription-polymerase chain reaction The mRNA expression levels for S1P2 receptor and S1P3 receptor were determined by quantitative reverse transcription-polymerase chain reaction (RT-PCR) with total RNA. Total RNA in isolated cultured VSMCs was

© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 66, pp. 802–810

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extracted with TRI reagent (Sigma-Aldrich) according to the manufacturer’s instruction. RT and PCR reactions were carried out using Superscript First-Strand Synthesis System (Life Technologies) and SYBR Green real-time PCR master mix (Toyobo, Osaka, Japan), respectively. The quantitative PCR was carried out using a 7500 Fast Real-Time PCR system (Applied Biosystems, Life Technologies). The following primer sets were used for quantitative RT-PCR: S1P2, 5′-ATT GGG GAG CAG AGA GGA AT-3′ (forward) and 5′-CGC CTC CCC TAG AGT TTT CT-3′ (reverse); S1P3, 5′-CCT TCT GAT TGG GAT GTG CT-3′ (forward) and 5′-GGC TGT GAA GAT GCT GAT GA-3′ (reverse); GAPDH, 5′-ATG ACT CTA CCC ACG GCA AG-3′ (forward) and 5′-TCC ACG ACA TAC TCA GCA CC-3′ (reverse). Concurrent RT-PCR amplification of the GAPDH message was performed as the internal control.

Materials FCS, penicillin, streptomycin and DMEM were obtained from Life Technologies (Grand Island, NY, USA). S1P was from Cayman Chemicals (Ann Arbor, MI, USA). PTX was from List Biological Laboratories (Campbell, CA, USA). Fura-2/AM and EGTA were from Dojindo (Kumamoto, Japan). Diltiazem hydrochloride was from Research Biochemicals (Natick, MA, USA). PTX was from List Biological Laboratories. U 73 122 was from Sigma-Aldrich (St. Louis, MO, USA). Y 27 632 and ionomycin were from Calbiochem (San Diego, CA, USA). All other agents were purchased from standard suppliers.

Statistical analysis Statistical analysis of the results was performed using Student’s unpaired t-test for unpaired data and analysis of variance, followed by Dunnett’s test for multiple comparisons. P values less than 0.05 were considered significant.

Results S1P increases [Ca2+]i by extracellular Ca2+ influx and Ca2+ release from internal stores Figure 1a demonstrates representative tracings showing the S1P-induced increase in [Ca2+]i in cultured VSMCs isolated from rat aortic media. S1P at 1 μm induced a rapid and transient elevation, which was maximal by 1 min after stimulation followed by the sustained increase in [Ca2+]i. Figure 1b summarizes the concentration-dependent effect of S1P on peak [Ca2+]i and [Ca2+]i 5 min after stimulation in the presence of extracellular Ca2+. In the 0.1–10 μm range, S1P significantly increased both peak and sustained [Ca2+]i. The peak [Ca2+]i and [Ca2+]i 5 min after stimulation by S1P 10 μm were 1450.97 ± 109.13 nM and 265.51 ± 9.96 nM, respectively (mean ± SEM values). When extra804

cellular Ca2+ (1.3 mM) was removed, the basal level of [Ca2+]i decreased, and a small transient increase by S1P stimulation was observed (Figure 1c). As shown in Figure 1d, the peak [Ca2+]i was significantly inhibited by removing extracellular Ca2+ (control vs extracellular Ca2+ depletion: 1226.72 ± 230.64 vs 316.35 ± 91.37 nM, (mean ± SEM values) n = 5–6 experiments). When cells were pretreated by thapsigargin to deplete Ca2+ in the sarcoplasmic reticulum (SR) in the absence of extracellular Ca2+, S1P had no effect (Figure 1e).

S1P increases in [Ca2+]i via S1P3 receptor We previously reported that rat VSMCs express both the S1P2 and S1P3 receptors.[6] Thus, we investigated which S1P receptor is responsible for intracellular Ca2+ mobilization. To elucidate this, cells transfected by the siRNA specific to the S1P2 receptor and the S1P3 receptor were used. As shown in Figure 2a, transfection of siRNA of the S1P2 receptor and siRNAs of both the S1P2 and S1P3 receptors to the cells significantly decreased S1P2 receptor mRNA expression (by 71% and 64%, respectively), whereas transfection of siRNA of the S1P3 receptor to the cells had no effect. On the other hand, transfection of siRNA of the S1P3 receptor and siRNAs of both the S1P2 and S1P3 receptors to the cells significantly decreased S1P3 receptor mRNA expression (by 59% and 78%, respectively), whereas transfection of siRNA of the S1P2 receptor to the cells had no effect (Figure 2b). Using these cells, the effect of S1P on intracellular Ca2+ mobilization was investigated. As shown in Figure 2c, S1P-induced increases in peak and sustained [Ca2+]i were significantly inhibited in the cells transfected with siRNA of the S1P3 receptor (control vs siRNA of S1P3 receptor: 1534.57 ± 224.50 vs 533.49 ± 39.52 nM for peak [Ca2+]i, 274.23 ± 13.59 vs 164.16 ± 10.09 nM for sustained [Ca2+]i, n = 10 experiments) and siRNAs of both the S1P2 and S1P3 receptors (siRNA of both the S1P2 and S1P3 receptor: 749.36 ± 40.82 nM for peak [Ca2+]i, 196.19 ± 8.20 nM for sustained [Ca2+]i, n = 11 experiments).

Involvement of the L-type voltage-dependent Ca2+ channels, Gi/o protein and phospholipase C in S1P-induced increase in [Ca2+]i Figure 3 demonstrates the effect of diltiazem, an L-type voltage-dependent Ca2+ channel blocker, on peak and sustained [Ca2+]i in VSMCs. Diltiazem at a high concentration of 10 μm significantly suppressed the peak, but did not affect the sustained increase [Ca2+]i (control vs 10 μm diltiazem: 1081.43 ± 180.87 vs 426.85 ± 12.13 nM for peak [Ca2+]i, 176.88 ± 12.71 vs 149.83 ± 14.63 nM for sustained [Ca2+]i, n = 4–5 experiments). One micromolar of diltiazem also tended to suppress the peak [Ca2+]i, but it was not

© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 66, pp. 802–810

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S1P increases [Ca2+]i via S1P3 receptor

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Figure 1 S1P increases in [Ca2+]i in vascular smooth muscle cells. (a) A representative result. Cells were stimulated with S1P (1 μM). (b) S1P concentration-dependent increase of [Ca2+]i in vascular smooth muscle cells. Cells were stimulated with the indicated concentration of S1P. Peak: peak [Ca2+]i after stimulation; at 5 min: [Ca2+]i 5 min after stimulation. (c) A representative result. Cells were preincubated with Ca2+-free buffer (+1 mM EGTA) and then stimulated with S1P (1 μM). (d) Effect of extracellular Ca2+ depletion on S1P-induced increase in [Ca2+]i in vascular smooth muscle cells. Cells were preincubated without (open column) or with (hatched column) Ca2+-free buffer (+1 mM EGTA) and then stimulated with S1P (1 μM). Basal: basal [Ca2+]i before stimulation; peak: peak [Ca2+]i after stimulation. (e) A representative result. Cells were preincubated with Ca2+free buffer (+1 mM EGTA) in the presence of thapsigargin (TG: 1 μM) and then stimulated with S1P (1 μM). Symbols are the mean ± SEM of (n) experiments for (b), n = 5–6 experiments for (d). *P < 0.01, **P < 0.001 vs basal. When not visible, error bars are included in the symbol. © 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 66, pp. 802–810

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Figure 3 The voltage-dependent Ca2+ channel mediates the S1Pinduced increase in [Ca2+]i in vascular smooth muscle cells. Cells were pretreated with the indicated concentrations of diltiazem and then stimulated with S1P (1 μM). Basal: basal [Ca2+]i before stimulation; peak: peak [Ca2+]i after stimulation; at 5 min: [Ca2+]i 5 min after stimulation. Bars show mean ± SEM values (n = 4–6 experiments). *P < 0.01 vs control.

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Figure 2 S1P increases in [Ca2+]i via S1P3 receptor. (a) and (b) mRNA expression of the S1P2 (a) and S1P3 (b) receptor. Cells were transfected either with siRNA of the S1P2 receptor, siRNA of the S1P3 receptor or siRNA of both receptors. (c) Effect of S1P on [Ca2+]i in vascular smooth muscle cells transfected with siRNA of the S1P receptor. Cells were stimulated with S1P. Basal: basal [Ca2+]i before stimulation; peak: peak [Ca2+]i after stimulation; at 5 min: [Ca2+]i 5 min after stimulation. Bars show mean ± SEM values (n = 3 experiments for (a) and (b), n = 10–11 experiments for (c)). *P < 0.001 vs scramble.

increase in [Ca2+]i (control vs Y 27 632: 810.63 ± 197.54 vs 916.21 ± 79.32 nM for peak [Ca2+]i, n = 7–8 experiments).

S1P activates store-operated Ca2+ entry and receptor-operated Ca2+ entry As shown in Figure 5a, the addition of extracellular Ca2+ to the Ca2+-free condition caused an increase in [Ca2+]i by Ca2+ influx. Similarly, in Figure 1c, a small transient increase by S1P stimulation was observed under the Ca2+-free condition, suggesting that the increased [Ca2+]i was derived from SR (Figure 5b). However, the Ca2+ influx by adding extracellular Ca2+ to the Ca2+-free condition was increased by pretreatment of S1P (Figure 5b), indicating that S1Pinduced Ca2+ depletion in SR caused store-operated Ca2+ entry (SOCE). As shown in Figure 5c, the increase in [Ca2+]i by adding extracellular Ca2+ to the Ca2+-free condition was notably augmented under the S1P receptor stimulation.

Discussion significant. The effect of PTX, an inhibitor of Gi/o protein activation, on the S1P-induced increase in [Ca2+]i was then investigated. As shown in Figure 4a and 4b, when VSMCs were pretreated with 200 ng/ml PTX for 24 h before S1P stimulation, the S1P-induced increase in [Ca2+]i was partially but significantly suppressed in the absence or presence of extracellular Ca2+. As shown in Figure 4c and 4d, U 73 122, a PLC inhibitor, also suppressed the S1P-induced increase in [Ca2+]i in the absence or presence of extracellular Ca2+. On the other hand, Y 27 632 (10 μm), a Rho kinase inhibitor, had no effect on the S1P-induced 806

In this study, we first showed that S1P at more than 0.1 μm induced a biphasic change in intracellular Ca2+ dynamics, that is a transient peak increase followed by a smaller sustained increases in [Ca2+]i in rat aortic VSMCs. This biphasic action has also been shown in human embryonic kidney cells.[19] Since both increases in [Ca2+]i were significantly suppressed by removing extracellular Ca2+, these increases are due mainly to the extracellular Ca2+ influx. Furthermore, by using thapsigargin, we demonstrated that the small S1P-induced increases in [Ca2+]i under the Ca2+free condition were derived from SR.

© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 66, pp. 802–810

Kazumi Fujii et al.

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Figure 4 Activation of Gi/o protein and phospholipase C mediate the S1P-induced increase in [Ca2+]i in vascular smooth muscle cells. (a) and (b): Cells were pretreated with pertussis toxin (200 ng/ml) for 24 h and preincubated without (a) or with (b) Ca2+-free buffer (+1 mM EGTA) and then stimulated with S1P (1 μM). (c) and (d): Cells were preincubated without (c) or with (d) Ca2+-free buffer (+1 mM EGTA) and pretreated with U 73 122 (10 μM) for 4 min and then stimulated with S1P (1 μM). Basal: basal [Ca2+]i before stimulation; peak: peak [Ca2+]i after stimulation. Bars show mean ± SEM values (n = 7–8 experiments for (a), n = 6 experiments for (b), n = 5 experiments for (c), n = 4 for (d)). *P < 0.001 vs control.

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Figure 5 Effect of S1P on store-operated Ca2+ entry and receptor-operated Ca2+ entry in vascular smooth muscle cells. Representative results. Cells were preincubated with Ca2+-free buffer (+1 mM EGTA) in the absence (a) or presence of S1P (1 μM, (b) and (c)) and changed the buffer containing 1.3 mM Ca2+ in the absence (b) or presence of S1P (1 μM, (c)).

We previously showed that rat VSMCs express a high level of S1P2 and S1P3 receptor mRNA.[6] To elucidate which subtype of S1P receptor is responsible for intracellular Ca2+ mobilization, we used three different types of cells by

transfecting siRNAs, namely (1) S1P2 receptor-deficient cells, (2) S1P3 receptor-deficient cells, and (3) both S1P2 and S1P3 receptor-deficient cells. Using three-target siRNAs for the S1P2 and S1P3 receptors, significant and specific

© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 66, pp. 802–810

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inhibition of the S1P receptor in rat primary VSMCs was observed. The result clearly shows that the transient and the sustained increases in [Ca2+]i are both mediated by S1P3 receptor activation. Consistent with this finding, deletion of the S1P3 but not the S1P2 receptor in mouse embryonic fibroblasts led to marked inhibition of the S1P-induced increase in [Ca2+]i.[10] Furthermore, Murakami et al.[20] reported that TY-52 156, which is a selective S1P3 receptor antagonist developed by their laboratories, prevented the S1P-induced increase in [Ca2+]i in human coronary artery smooth muscle cells. The results are also consistent with our previous observation that S1P induces COX-2 expression via S1P3 receptor through mechanisms involving [Ca2+]i.[6] One micromolar of diltiazem tended to suppress S1Pinduced peak [Ca2+]i, although it was not statistically significant. This tendency may be largely due to the block of the L-type Ca2+ channels, since diltiazem has been reported to block L-type voltage-dependent Ca2+ currents with an IC50 of 3 μm.[21] However, diltiazem at a higher concentration also blocks low-voltage-activated T-type Ca2+ currents.[21] Therefore, we consider that the significant suppression by 10 μm of diltiazem is due to the block of T-type Ca2+ channels in addition to L-type Ca2+ channels. Future studies with more specific channel blockers will be required to clarify the Ca2+ influx pathways by S1P. The peak [Ca2+]i suppressed by 10 μm of diltiazem was smaller than the peak [Ca2+]i suppressed by S1P stimulation under the Ca2+-free condition (Figure 1d). Thus, Ca2+ influx by S1P stimulation is due not only to the voltage-dependent Ca2+ channels, including L-type Ca2+ channels, but also to other pathways such as receptor-operated Ca2+ entry (ROCE) or non-selective cation channels. Taken together with the finding that the sustained [Ca2+]i was almost eliminated by removing extracellular Ca2+ (Figure 2a), the sustained [Ca2+]i is due to Ca2+ influx, but not through the voltagedependent Ca2+ channels. The S1P2 and S1P3 receptors are coupled to multiple G proteins, Gq, G12/13 and Gi/o proteins, to activate the PLC pathway and the Rho pathway, as well as the Ras-mitogenactivated protein kinase (MAPK) and phosphoinositide 3-kinase-Akt pathways.[5] We showed that PTX suppressed the S1P-induced increase in the peak [Ca2+]i in the absence or the presence of extracellular Ca2+. However, even 200 ng/ml of PTX, which is believed to inhibit Gi/o protein sufficiently,[6,22,23] did not suppress the peak [Ca2+]i completely. In contrast with PTX, U 73 122 almost completely suppressed the S1P-induced increase in the peak [Ca2+]i in the absence or the presence of extracellular Ca2+. A similar result to the complete and partial inhibition of Ca2+ responses by U 73 122 and PTX, respectively, was found in rat HTC4 hepatoma cells transfected with the S1P2 receptor and the S1P3 receptor.[24] These results suggest that S1P3 receptor-mediated PLC activation and intracellular Ca2+ 808

mobilization in VSMCs may be only partially mediated through Gi/o proteins. PLC is also activated by Gq protein. Although the members of the Gqα class (Gqα, G11α, G14α and G15/16α) activate distinct signalling pathways, all Gqα class members commonly activate PLCβ in VSMCs.[25] Thus, the reason for the potent inhibition by U 73 122 was due to the downstream inhibition of both Gq protein and Gi/o protein activation. The result further suggests that Ca2+ release from the SR is due mainly to PLC activation. Activation of the S1P3 receptor has been shown to activate Rho kinase, which is activated by PTX-insensitive G12/13 protein.[6,20,22] However, Rho kinase seems not to be involved in S1P-induced intracellular Ca2+ mobilization, since Y 27 632 showed no effect on the S1P-induced increase in [Ca2+]i. It is reported that both extracellular and intracellular S1P are able to activate SOCE in VSMCs.[26] In agreement with this, we confirmed that S1P activates SOCE in VSMCs. SOCE is considered to be involved in the pathogenesis of various vascular responses, such as vessel constriction and VSMCs proliferation.[26] Furthermore, under S1P receptor stimulation, the increase in [Ca2+]i by adding extracellular Ca2+ was notably augmented, suggesting that S1P causes not only SOCE but also ROCE. Although the actual molecular mechanisms of SOCE and ROCE have not been conclusively identified, transient receptor potential canonical (TRPC) proteins are now believed to have an important role in SOCE and ROCE.[27,28] In fact, TRPC1 has been shown to contribute to both SOCE and ROCE in rat primary cultured VSMCs stimulated with endothelin-1.[29] Further studies are required to clarify the roles and involvement of TRPC proteins in SOCE and ROCE induced by S1P stimulation. The immune modulator FTY720 has been recently approved as treatment for multiple sclerosis. FTY720 is phosphorylated by sphingosine kinase, and the phosphorylated compound is a potent agonist at four sphingosine 1-phophate receptors.[30] Phosphorylated FTY720 (FTY720-P) may be useful to elucidate the signal transduction by the S1P3 receptor activation in rat cultured VSMCs, since FTY720-P does not activate S1P2 receptor.[30] In fact, FTY720-P induces an increase in [Ca2+]i in Chinese hamster ovary K1 cells that stably expressed human S1P3 receptors.[20] Furthermore, not only FTY720-P but also various selective S1P receptor agonists have become commercially available, and they could be useful tools to further elucidate the physiological role of S1P receptors in vascular cells.[31] Further studies are required to clarify the precise mechanism of S1P-induced [Ca2+]i dynamics using such pharmacological tools. The plasma concentrations of S1P in healthy human are between 0.2 μm and 1 μm.[32,33] Although S1P tightly binds to plasma components, such as lipoproteins,[34–36] S1P

© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 66, pp. 802–810

Kazumi Fujii et al.

S1P increases [Ca2+]i via S1P3 receptor

concentration could possibly attain even higher levels of vascular injury where platelet activation or thrombus formation occurs. Therefore, the present findings may contribute to the understanding of basic mechanisms underlying the pathophysiological effects of S1P at the site of a local vascular injury where endothelial function is lacking.

partially through the voltage-dependent Ca2+ channels, including L-type Ca2+ channels, as well as by mobilizing Ca2+ from its intracellular stores. S1P3, but not S1P2, receptor-coupled Gi/o protein and PLC activation mediate the mechanisms.

Declarations Conclusions

Conflict of interest 2+

The present findings suggest that S1P increases [Ca ]i via the S1P3 receptor by inducing an influx of extracellular Ca2+

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The Author(s) declare(s) that they have no conflicts of interest to disclose.

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© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 66, pp. 802–810

Sphingosine 1-phosphate increases an intracellular Ca(2+) concentration via S1P3 receptor in cultured vascular smooth muscle cells.

We investigated the effect of sphingosine 1-phosphate (S1P) on intracellular Ca(2+) dynamics in rat vascular smooth muscle cells (VSMCs)...
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