© 2003 by European Society of Cardiology
Copyright © 2003, European Society of Cardiology
Sphingosine 1-phosphate induces contraction of coronary artery smooth muscle cells via S1P2
aDepartment of Laboratory Medicine, Yamanashi Medical University, Nakakoma, Yamanashi 409-3898, Japan
bDepartment of Gastroenterology, University of Tokyo School of Medicine, Tokyo, Japan
* Corresponding author. Tel.: +81-55-273-9694; fax: +81-55-273-6924. yatomiy{at}res.yamanashi-med.ac.jp
Received 17 September 2002; accepted 23 December 2002
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Abstract |
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 Top Abstract 1 Introduction2 Methods3 Results4 DiscussionReferences |
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Objectives: Sphingosine 1-phosphate (Sph-1-P), a bioactive lipid derived from activated platelets, may play an important role in coronary artery spasm and hence the pathogenesis of ischemic heart diseases, since we reported that a decrease in coronary blood flow was induced by this lysophospholipid in an in vivo canine heart model [Cardiovasc. Res. 46 (2000) 119]. In this study, metabolism related to and cellular responses elicited by Sph-1-P were examined in human coronary artery smooth muscle cells (CASMCs). Methods and results: [3H]Sphingosine (Sph), incorporated into CASMCs, was converted to [3H]Sph-1-P intracellularly, but its stimulation-dependent formation and extracellular release were not observed. Furthermore, the cell surface Sph-1-P receptors of S1P family (previously called EDG) were found to be expressed in CASMCs. Accordingly, Sph-1-P seems to act as an extracellular mediator in CASMCs. Consistent with Sph-1-P-elicited coronary vasoconstriction in vivo, Sph-1-P strongly induced CASMC contraction, which was inhibited by JTE-013, a newly-developed specific antagonist of S1P2 (EDG-5). Furthermore, C3 exoenzyme or Y-27632 inhibited the CASMC contraction induced by Sph-1-P, indicating Rho involvement. Finally, exogenously-added [3H]Sph-1-P underwent a rapid degradation. Since lipid phosphate phosphatases, ectoenzymes capable of dephosphorylating Sph-1-P, were expressed in CASMCs, Sph-1-P may be dephosphorylated by the ectophosphatases. Conclusions: Sph-1-P, derived from platelets and dephosphorylated on the cell surface, may induce the contraction of coronary artery smooth muscle cells through the S1P2/Rho signaling.
KEYWORDS Lipid metabolism; Receptor; Signal transduction; Smooth muscle; Vasoconstriction/dilation
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1 Introduction |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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Sphingosine 1-phosphate (Sph-1-P) is a bioactive lysophospholipid capable of inducing a wide spectrum of biological responses, including cell growth, differentiation, survival, and motility [1,2]. Originally, it was reported that Sph-1-P can serve as an intracellular second messenger regulating intracellular Ca2+ mobilization and cell growth and survival [3,4]. Furthermore, the dynamic balance between the intracellular levels of ceramide (Cer) and Sph-1-P, with the consequent regulation of opposing signaling pathways, was proposed to be an important factor that determined the cell fate [5]. However, recent evidence has indicated that Sph-1-P also acts as an intercellular mediator, interacting with the S1P (also called endothelial differentiation gene (EDG)) family of G protein-coupled receptors [1,2,6]. S1P1 (EDG-1), S1P2 (EDG-5), S1P3 (EDG-3), S1P4 (EDG-6), and S1P5 (EDG-8) exhibit overlapping, as well as distinct patterns of expression in various tissues as Sph-1-P receptors; the eventual cellular responses to extracellular Sph-1-P depend on the types of S1P receptors expressed [6,7]. Accordingly, Sph-1-P is now considered to be a unique lipid mediator that has dual actions, signaling inside and outside of the cell.
Blood platelets are unique in that they store Sph-1-P abundantly (possibly due to the existence of highly active sphingosine (Sph) kinase and to a lack of Sph-1-P lyase) and release this bioactive lipid extracellularly upon stimulation [8,9]. This is consistent with the fact that Sph-1-P is a normal constituent of plasma and serum; the Sph-1-P levels of the latter are higher [10]. In view of the diverse biological effects of Sph-1-P, including those toward vascular cells, Sph-1-P released from activated platelets may be involved in a variety of physiological and pathophysiological processes, in which critical platelet–vascular cell interactions (including thrombosis, hemostasis, angiogenesis, atherosclerosis, and ischemia) occur.
Platelet-derived mediators play an important role in coronary artery constriction and hence the pathogenesis of ischemic heart diseases, and inhibition of the spastic effects of these mediators would be useful therapeutically. We recently reported that in a canine isolated, blood-perfused papillary muscle preparation, which is a well-established in vivo model, a decrease in the coronary blood flow and the resultant negative inotropic effect were induced by Sph-1-P [11]. In this study, metabolism related to and cellular responses elicited by Sph-1-P were examined in human coronary artery smooth muscle cells (CASMCs) to provide an insight into the in vivo responses produced by Sph-1-P.
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2 Methods |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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2.1 Materials
Recombinant Clostridium botulinum C3 exoenzyme was prepared as described previously [12], and kindly donated by Dr. S. Narumiya (Department of Pharmacology, Kyoto University Faculty of Medicine). Y-27632, a specific Rho kinase inhibitor [13], was a gift from Welfide (Osaka, Japan).
The following materials were obtained from the indicated suppliers: anti-RhoA monoclonal antibody (MoAb) (Santa Cruz Biotech, Santa Cruz, CA, USA); glutathione-Sepharose 4B (Amersham Pharmacia Biotech, Buckinghamshire, UK); tetramethyl rhodamine isothiocyanate-phalloidin, suramin, serotonin, endothelin-1, and D-erythro-Sph (Sigma, St. Louis, MO, USA); Sph-1-P (Biomol, Plymouth Meeting, PA, USA); pertussis toxin (Kaken Pharmaceutical, Tokyo, Japan); recombinant human platelet-derived growth factor-BB (PDGF) (Genzyme-TECHNE, Cambridge, MA, USA); thrombin (Mochida Pharmaceutical, Tokyo, Japan); angiotensin II (Bachem California, Torrance, CA, USA); [Arg8]-vasopressin (Seikagaku, Tokyo, Japan); isoproterenol (Wako Pure Chemical Industries, Tokyo, Japan); U46619 [GenBank] (Calbiochem-Novabiochem, CA, USA); D-erythro-[3-3H]Sph and [3-3H]Sph-1-P (DuPont NEN, Boston, MA, USA).
2.2 Characterization of the S1P2 antagonist JTE-013
The pyrazolopyridine derivative JTE-013 was a gift from the Central Pharmaceutical Research Institute, Japan Tobacco Incorporation, Osaka, Japan. JTE-013 is a specific S1P2 antagonist (PCT (WO) Patent: publication number, WO 01/98301; publication date, December 27, 2001). It was confirmed that JTE-013 inhibited a specific binding of radio-labeled Sph-1-P to the cell membranes of Chinese hamster ovary (CHO) cells stably transfected with human S1P2 and rat S1P2, with IC50 values of 17±6 nmol/l and 22±9 nmol/l, respectively. In contrast, this compound at concentrations up to 10 µmol/l did not affect the Sph-1-P binding to the cell membranes of CHO cells stably transfected with human S1P1. Furthermore, only 4.2% inhibition was observed with 10 µmol/l JTE-013 when the effect of this compound was examined on the Sph-1-P binding to the CHO cell membranes expressing S1P3.
2.3 Cell culture
Human CASMCs were purchased from the Applied Cell Biology Research Institute (Kirkland, WA, USA), and maintained in Dulbecco's modified Eagle's medium with 20% fetal bovine serum (ICN Biomedicals, Aurora, OH, USA), 10 ng/l of recombinant human basic fibroblast growth factor (bFGF) (Becton Dickinson Labware, Lincoln Park, NJ, USA), penicillin G (100 U/ml), and streptomycin sulfate (100 µg/ml) at 37°C under an atmosphere of 5% CO2 and 95% room air. The cells were not used after the seventh passage.
2.4 Metabolism of [3H]Sph or [3H]Sph-1-P
CASMCs were incubated with 1 µM (0.2 µCi) [3-3H]Sph or 100 nM (0.2 µCi) [3-3H]Sph-1-P. Lipids were extracted from the cells and medium separately, and then analyzed for [3H]sphingolipid metabolism as described previously [14]. Finally, portions of the extracted lipids were applied to silica gel high-performance thin layer chromatography (TLC) plates (Merck, Darmstadt, Germany), and the plates were then developed in butanol/acetic acid/water (3:1:1), followed by autoradiography.
2.5 RNA isolation, Northern blot analysis, and RT-PCR
Total RNA was prepared from CASMCs with a total RNA isolation system (Isogen, Wako Pure Chemical Industries, Osaka, Japan), and the isolation of mRNA was performed with OligotexTM-dT30
Super
(Takara Biomedicals, Tokyo, Japan), according to the manufacturer's instructions.
For Northern analysis, total RNAs were separated on a 1% agarose gel and transferred to a Hybond N+TM nylon filter (Amersham Pharmacia Biotech). The membranes were probed with PCR products from cDNA of S1P1–4, or β-actin, which had been labeled with digoxigenin using a PCR DIG Probe Synthesis KitTM (Boehringer Mannheim, Mannheim, Germany). The probe binding was detected using alkaline phosphatase-conjugated anti-digoxigenin antibody and visualized with CSPDTM according to the manufacturer's recommendations (Boehringer Mannheim). Quantitative analysis was performed using a PDI400oe Scanner and Quantity One 2.5a software for Macintosh.
For RT-PCR, the isolated mRNA was reverse transcribed using a SuperScriptTM Preamplification System (Gibco BRL, Life Technologies, Rockville, MD, USA). Reverse transcribed cDNA was amplified in a Perkin-Elmer 9600R thermal cycler (Perkin-Elmer, Norwalk, CT, USA) using Takara TaqTM (rTaq DNA polymerase) (Takara Biomedicals).
The oligonucleotide primer pairs used for lipid phosphate phosphatase (LPP)-1, 2, and 3 were:
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Amplification was conducted with 35 cycles of 1 min at 94°C, 1 min at 60°C, and 1 min at 72°C. The absence of contaminating DNA was confirmed by control reactions with RNA that had not been reverse transcribed. The PCR products (5 µl) were resolved by electrophoresis on a 2% agarose gel in TBE buffer (90 mM Tris–borate, 2 mM EDTA, pH 8.3) and stained with ethidium bromide. The PCR products were cut from the gels, solubilized, and sequenced with Dye Terminator Cycle Sequencing FS Ready Reaction Kits (Perkin-Elmer) and analyzed using an ABI PRISM 310 Genetic Analyzer (Perkin-Elmer) according to the supplied protocols.
2.6 Cell contraction assay
In order to visualize agonist-induced contractility, suspensions of CASMCs were seeded onto dishes coated with 0.2% gelatin, and the cells were allowed to adhere to and spread on the substrate for 24 h. The cells were serum-starved for 3 h, and then stimulated with the indicated agonist for 30 min. The cells were fixed with 3% paraformaldehyde for 40 min, and then permeabilized with 0.2% Triton X-100 for 10 min. Actin filaments were detected with 0.1 µg/ml of tetramethyl rhodamine isothiocyanate-phalloidin. Cell morphology was observed with a confocal microscope, and the images were digitalized with a digital scanner (Canoscan D2400U, Canon, Tokyo, Japan). For quantitative evaluation, the maximal cord length of the cells was measured and was defined as the cell length. To preclude the possibility that the reduced viability of the cells accounted for the observed effects, we performed the following control experiments. For every batch, the cells challenged with the contractile agonist were subsequently exposed to 10 µmol/l isoproterenol. Only those batches of CASMCs that reduced their maximum cell length in response to the agonist and relaxed after the β-adrenergic receptor stimulation were used for the analysis.
2.7 Rho activity assay
Rho activity was assayed by the detection of cellular GTP-Rho. The coding sequence for the Rho-binding domain of Rho kinase (amino acids 759–1097) was amplified by PCR, and then cloned into a pGEX-2T vector (Amersham Pharmacia Biotech). The construct was transformed into E. coli, and the GST fusion protein was purified according to the manufacturer's recommendations. Affinity-precipitation of cellular GTP-Rho was performed with the GST fusion protein as described previously [15]. When incubated with lysates from COS cells expressing a HA-tagged mutant of Rho, this GST fusion protein was confirmed to precipitate recombinant V14Rho (GTP bound) but not N19 Rho (GDP-bound) (data not shown).
2.8 Statistics
When indicated, statistical analysis was performed by Student's t-test, and P<0.05 was considered significant.
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3 Results |
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3.1 Sph metabolism and S1P expression in CASMCs
Sph-1-P is a bioactive sphingolipid, acting as an intracellular second messenger in some cells and as an extracellular mediator in others. In this context, we first checked the intracellular Sph-1-P formation (from Sph) and S1P expression in CASMCs.
[3H]Sph incorporated into CASMCs was phosphorylated by Sph kinase to [3H]Sph-1-P (Fig. 1A). The [3H]Sph-1-P formation was transient, possibly due to degradation by Sph-1-P lyase, and was not affected by the established SMC agonists such as angiotensin II, endothelin-1, PDGF, vasopressin, and thrombin (Fig. 1B). Accordingly, it is unlikely that Sph-1-P acts as an intracellular second messenger in CASMCs. Furthermore, [3H]Sph-1-P was not detected in the medium under these conditions (Fig. 1B), indicating that stimulation-dependent extracellular Sph-1-P release does not occur in these cells.
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We then checked the expression of the cell surface Sph-1-P receptors. Northern blot analysis of CASMC RNA showed that S1P3 was abundantly expressed, while S1P1 and S1P2 transcripts were expressed at lower levels in these cells; S1P4 was not expressed (Fig. 2). Accordingly, it was confirmed that CASMCs express cell surface Sph-1-P receptors. The facts that stimulation-dependent Sph-1-P formation was not observed and that the cell surface Sph-1-P receptors are expressed on CASMCs suggest Sph-1-P acting as an extracellular mediator in these cells.
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3.2 Sph-1-P-induced CASMC contraction via S1P2
We next examined the actions of Sph-1-P in CASMCs to provide an insight into the mechanism by which Sph-1-P exerted the response in an in vivo heart model (see Introduction). We evaluated CASMC contraction by examining the cell shape change. As shown in Figs. 3A and 4A
, Sph-1-P strongly induced CASMC contraction.
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Of the S1P receptors expressed in CASMCs, S1P1 is known to activate only members of the Gi family, while S1P2 and S1P3 have a broader coupling profile than S1P1 and can communicate with several G proteins, including G12/13 [1,2,6]. We first examined the effect of JTE-013, a specific and potent S1P2 antagonist, to check S1P2 involvement in the Sph-1-P-induced CASMC contraction. This pyrazolopyridine derivative strongly and concentration-dependently inhibited Sph-1-P-induced CASMC contraction (Fig. 3A and B). On the other hand, this S1P2 antagonist failed to affect CASMC contraction induced by serotonin or U46619 [GenBank] (a stable thromboxane A2 analogue) (Fig. 3C). Although S1P3 is abundantly expressed in CASMCs (see Fig. 2), the S1P3 antagonist suramin [16] did not inhibit Sph-1-P-induced CASMC contraction (data not shown); the data, however, should be interpreted cautiously because of its low specificity. Furthermore, pertussis toxin, which inactivates Gi, had no effect (Fig. 4B), excluding S1P1 involvement.
These results indicate that Sph-1-P-induced CASMC contraction is mediated specifically by S1P2.
3.3 Involvement of the Rho/Rho kinase pathway in Sph-1-P-induced CASMC contraction
A guanine nucleotide exchange factor for Rho, p115RhoGEF, has been identified as a target for the 
subunit of the G12/13 family, with which S1P2 communicate [1,2,6]. Furthermore, the small GTPase Rho and its downstream targets Rho kinase (and myosin light chain phosphatase) play an important role in phosphorylation of myosin light chain and thereby induce actomyosin contractile force [17,18]. Accordingly, the involvement of this signaling pathway was examined. As expected, pretreatment of CASMCs with the specific Rho inactivator C3 exoenzyme [12] or the Rho kinase inhibitor Y-27632 [13] reduced the CASMC contraction induced by Sph-1-P (Fig. 4B), indicating the importance of the Rho/Rho kinase pathway in Sph-1-P-induced CASMC contraction.
We next checked Rho activation in CASMCs stimulated with Sph-1-P. The Rho activity increased as early as 1 min after the Sph-1-P stimulation, and was sustained for at least 30 min, when it was assayed by an affinity-precipitation of cellular GTP-Rho (Fig. 5A). As expected, the Rho activation induced by Sph-1-P was abolished by pretreatment with C3 exoenzyme (Fig. 5B). Pertussis toxin did not inhibit the Rho activation induced by Sph-1-P; rather, it enhanced the Sph-1-P effect (Fig. 5B) for unknown reasons. These data further strengthened the idea that the Sph-1-P-elicited CASMC response observed is mediated via Rho.
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3.4 Degradation of exogenously-added Sph-1-P in the presence of CASMCs
We finally evaluated the fate of Sph-1-P in the presence of CASMCs by pursuing the metabolic changes of [3-3H]Sph-1-P, which should retain the radioactivity after its dephosphorylation. When [3H]Sph-1-P was extracellularly added to CASMCs, [3H]Sph-1-P associated with CASMCs underwent a marked metabolism; instead, [3H]Sph, [3H]Cer, and [3H]sphingomyelin were formed (Fig. 6A). These results can be best explained by the idea that non-polar [3-3H]Sph, formed from polar [3-3H]Sph-1-P by ectophosphatase activity [19], is incorporated into CASMCs and then converted to [3-3H]Cer (and then to [3-3H]sphingomyelin) intracellularly; Sph (but not Sph-1-P) is hydrophobic and easily passes through the lipid bilayer. In fact, [3H]Sph, added exogenously, was incorporated into CASMCs and converted to [3H]Cer and [3H]sphingomyelin (see Fig. 1A).
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Recently, several isoenzymes of mammalian lipid phosphate phosphatase (LPP) (type 2 phosphatidic acid phosphatase) have been cloned [20], and they are believed to act at the outer leaflet of the cell surface bilayer, accounting for the ecto-lipid phosphate phosphatase activities (toward Sph-1-P, lysophosphatidic acid, or phosphatidic acid) previously described [21]. Accordingly, we finally checked the expression of LPPs by RT-PCR. RNA from CASMCs was prepared and reverse transcribed, followed by PCR amplification of specific transcripts. As demonstrated in Fig. 6B, CASMCs were found to express mRNA transcripts for LPP-1, 2, and 3; the sequence of this PCR product was confirmed (data not shown).
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4 Discussion |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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As expected from our previous in vivo study showing that Sph-1-P induces coronary artery vasoconstriction [11], this lysophospholipid was found to induce contraction of CASMCs. The facts that stimulation-dependent Sph-1-P formation from Sph was not affected upon activation and that the cell surface Sph-1-P receptors (EDG-1, 3, and 5) were expressed suggest Sph-1-P acting as an extracellular mediator in CASMCs. Furthermore, CASMCs themselves failed to release Sph-1-P extracellularly and hence are not a likely source for extracellular Sph-1-P. It can be speculated that these cells may receive this lysophospholipid mediator from the outside, probably from platelets; Sph-1-P is abundantly stored in platelets and released extracellularly upon stimulation [8,9]. In vivo, Sph-1-P derived from platelets has to pass through vascular endothelial cells to interact with CASMCs, as is the case with thromboxane A2. Although further studies are needed to solve this problem, not only endothelial cells but also smooth muscle cells are likely to be exposed to significant levels of Sph-1-P under the conditions in which the integrity of vascular endothelial cells is disturbed.
As for the signaling pathway involved in this Sph-1-P-induced response, the Rho/Rho kinase pathway seems to be important, based on the inhibitory effects by C3 exoenzyme and Y-27632. In fact, Sph-1-P was confirmed to activate Rho, a member of the small GTPase protein. Rho is converted to an active form by Rho-GEF through the activation of G12/13 [17,18], and plays important roles in a variety of cellular functions, including cytoskeletal reorganization, integrin activation, and gene expression [17,18]. Rho, through its target Rho kinase (and the myosin binding subunit of myosin phosphatase) [17,18], regulates myosin light chain phosphorylation, and hence induces smooth muscle contraction in a Ca2+-independent manner [22]. Recently, hydroxyfasudil, an inhibitor of Rho kinase, was shown to inhibit serotonin-induced coronary spasm in a porcine model both in vivo and in vitro [23,24], and RT-PCR analysis revealed that the expression of Rho kinase mRNA was significantly increased in the spastic segments of coronary arteries [23]. These results indicate that Rho kinase is a key molecule in coronary artery hypercontraction induced by vasospastic agents (including Sph-1-P).
Platelets release a variety of vasospastic agents upon their activation, including serotonin and thromboxane A2, and hence are involved in coronary artery vasoconstriction. Sph-1-P should be added to the list of such vasospastic substances released from activated platelets. In CASMCs, the Sph-1-P receptors S1P1, S1P2, and S1P3 were confirmed to be expressed. Of these, S1P2, which has been shown to preferentially activate G12/13 and hence Rho, is the most probable candidate receptor for transducing the Sph-1-P effect on CASMC contraction because of the specific inhibition of the Sph-1-P-induced response by JTE-013, an S1P2 antagonist. This is compatible with the facts that Sph-1-P-induced CASMC contractile response is not inhibited when pertussis toxin inactivates Gi, only with which S1P1 couples [16,25] and that the S1P3 antagonist suramin fails to affect the response.
Finally, Sph-1-P degradation in the presence of CASMCs may be important when the in vivo effects of Sph-1-P are evaluated. Although the possibility of Sph-1-P degradation occurring after its intracellular uptake by S1P-mediated and ligand-dependent recycling [26] cannot be ruled out, it is most likely that Sph-1-P is metabolized at the cell surface by LPPs, which were confirmed to be expressed in CASMCs. Based on the previous results, Kds for S1P receptors are much lower than the concentrations of Sph-1-P in the plasma [2,6,10]. Probably, due to Sph-1-P dephosphorylation at the cell surface, the concentrations of Sph-1-P interacting with S1P receptors may be lower than the plasma Sph-1-P levels. This is also consistent with a recent in vitro transfection study showing that LPPs may limit the bioactivity of Sph-1-P by regulating its concentration (interacting with S1P receptors) [27]. The Sph-1-P-induced effects on CASMCs in vivo may depend on various factors, including Sph-1-P release from platelets, S1P expression on CASMCs, and LPP activity on CASMCs.
Sph-1-P is attracting much attention as a bioactive sphingolipid released from platelets. Considering the great variety of responses induced by Sph-1-P, the development of its receptor agonists/antagonists of the S1P family may lead to a new therapeutic approach to regulate various diseases. Very recently, it was shown that lymphocyte trafficking is altered by Sph-1-P and by its related synthetic compounds and that the inhibition of lymphocyte recirculation by the Sph-1-P receptor agonists may result in therapeutically useful immunosuppression [28]. In the present study, we have shown for the first time that Sph-1-P induces CASMC contraction through S1P2; antagonists of this Sph-1-P receptor may have potential as drugs to control vascular diseases.
Time for primary review 27 days.
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Acknowledgements |
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This study was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan. The authors are thankful to Dr. S. Narumiya, Welfide Corporation, and the Central Pharmaceutical Research Institute, Japan Tobacco Incorporation for valuable materials. The authors also thank Drs. A. Wada and Y. Igarashi (Hokkaido University) for their helpful discussion and the experiments using COS cells expressing the mutant of Rho.
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A. Skoura and T. Hla Regulation of vascular physiology and pathology by the S1P2 receptor subtype Cardiovasc Res, May 1, 2009; 82(2): 221 - 228. [Abstract] [Full Text] [PDF]
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 H. Ikeda, N. Watanabe, I. Ishii, T. Shimosawa, Y. Kume, T. Tomiya, Y. Inoue, T. Nishikawa, N. Ohtomo, Y. Tanoue, et al. Sphingosine 1-phosphate regulates regeneration and fibrosis after liver injury via sphingosine 1-phosphate receptor 2 J. Lipid Res., March 1, 2009; 50(3): 556 - 564. [Abstract] [Full Text] [PDF]
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 J.-F. Lee, S. Gordon, R. Estrada, L. Wang, D. L. Siow, B. W. Wattenberg, D. Lominadze, and M.-J. Lee Balance of S1P1 and S1P2 signaling regulates peripheral microvascular permeability in rat cremaster muscle vasculature Am J Physiol Heart Circ Physiol, January 1, 2009; 296(1): H33 - H42. [Abstract] [Full Text] [PDF]
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 A. Kimura, T. Ohmori, Y. Kashiwakura, R. Ohkawa, S. Madoiwa, J. Mimuro, K. Shimazaki, Y. Hoshino, Y. Yatomi, and Y. Sakata Antagonism of Sphingosine 1-Phosphate Receptor-2 Enhances Migration of Neural Progenitor Cells Toward an Area of Brain Infarction * Supplemental Materials and Methods Stroke, December 1, 2008; 39(12): 3411 - 3417. [Abstract] [Full Text] [PDF]
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 M. Hashimoto, X. Wang, L. Mao, T. Kobayashi, S. Kawasaki, N. Mori, M. L. Toews, H. J. Kim, D. R. Cerutis, X. Liu, et al. Sphingosine 1-Phosphate Potentiates Human Lung Fibroblast Chemotaxis through the S1P2 Receptor Am. J. Respir. Cell Mol. Biol., September 1, 2008; 39(3): 356 - 363. [Abstract] [Full Text] [PDF]
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 K. Takabe, S. W. Paugh, S. Milstien, and S. Spiegel "Inside-Out" Signaling of Sphingosine-1-Phosphate: Therapeutic Targets Pharmacol. Rev., June 1, 2008; 60(2): 181 - 195. [Abstract] [Full Text] [PDF]
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K. M. Argraves and W. S. Argraves HDL serves as a S1P signaling platform mediating a multitude of cardiovascular effects J. Lipid Res., November 1, 2007; 48(11): 2325 - 2333. [Abstract] [Full Text] [PDF]
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 M. Osada, Y. Yatomi, T. Ohmori, S. Aoki, S. Hosogaya, and Y. Ozaki Involvement of Sphingosine 1-Phosphate, a Platelet-Derived Bioactive Lipid, in Contraction of Mesangium Cells J. Biochem., September 1, 2007; 142(3): 351 - 355. [Abstract] [Full Text] [PDF]
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 D. Leiber, Y. Banno, and Z. Tanfin Exogenous sphingosine 1-phosphate and sphingosine kinase activated by endothelin-1 induced myometrial contraction through differential mechanisms Am J Physiol Cell Physiol, January 1, 2007; 292(1): C240 - C250. [Abstract] [Full Text] [PDF]
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 J. N. Lorenz, L. J. Arend, R. Robitz, R. J. Paul, and A. J. MacLennan Vascular dysfunction in S1P2 sphingosine 1-phosphate receptor knockout mice Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2007; 292(1): R440 - R446. [Abstract] [Full Text] [PDF]
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 W. Hu, S. Mahavadi, J. Huang, F. Li, and K. S. Murthy Characterization of S1P1 and S1P2 receptor function in smooth muscle by receptor silencing and receptor protection Am J Physiol Gastrointest Liver Physiol, October 1, 2006; 291(4): G605 - G610. [Abstract] [Full Text] [PDF]
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R. Schubert Non-capacitative calcium entry-Extension of the possibilities for calcium entry in vascular tissue Cardiovasc Res, October 1, 2005; 68(1): 5 - 7. [Full Text] [PDF]
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S. Aoki, Y. Yatomi, M. Ohta, M. Osada, F. Kazama, K. Satoh, K. Nakahara, and Y. Ozaki Sphingosine 1-Phosphate-Related Metabolism in the Blood Vessel J. Biochem., July 1, 2005; 138(1): 47 - 55. [Abstract] [Full Text] [PDF]
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 M. Tolle, B. Levkau, P. Keul, V. Brinkmann, G. Giebing, G. Schonfelder, M. Schafers, K. v. W. Lipinski, J. Jankowski, V. Jankowski, et al. Immunomodulator FTY720 Induces eNOS-Dependent Arterial Vasodilatation via the Lysophospholipid Receptor S1P3 Circ. Res., April 29, 2005; 96(8): 913 - 920. [Abstract] [Full Text] [PDF]
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 A. Damirin, H. Tomura, M. Komachi, M. Tobo, K. Sato, C. Mogi, H. Nochi, K. Tamoto, and F. Okajima Sphingosine 1-Phosphate Receptors Mediate the Lipid-Induced cAMP Accumulation through Cyclooxygenase-2/Prostaglandin I2 Pathway in Human Coronary Artery Smooth Muscle Cells Mol. Pharmacol., April 1, 2005; 67(4): 1177 - 1185. [Abstract] [Full Text] [PDF]
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 K. Lockman, J. S. Hinson, M. D. Medlin, D. Morris, J. M. Taylor, and C. P. Mack Sphingosine 1-Phosphate Stimulates Smooth Muscle Cell Differentiation and Proliferation by Activating Separate Serum Response Factor Co-factors J. Biol. Chem., October 8, 2004; 279(41): 42422 - 42430. [Abstract] [Full Text] [PDF]
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 M. Forrest, S.-Y. Sun, R. Hajdu, J. Bergstrom, D. Card, G. Doherty, J. Hale, C. Keohane, C. Meyers, J. Milligan, et al. Immune Cell Regulation and Cardiovascular Effects of Sphingosine 1-Phosphate Receptor Agonists in Rodents Are Mediated via Distinct Receptor Subtypes J. Pharmacol. Exp. Ther., May 1, 2004; 309(2): 758 - 768. [Abstract] [Full Text] [PDF]
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H. Zhou and K. S. Murthy Distinctive G protein-dependent signaling in smooth muscle by sphingosine 1-phosphate receptors S1P1 and S1P2 Am J Physiol Cell Physiol, May 1, 2004; 286(5): C1130 - C1138. [Abstract] [Full Text] [PDF]
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J. D. Saba and T. Hla Point-Counterpoint of Sphingosine 1-Phosphate Metabolism Circ. Res., April 2, 2004; 94(6): 724 - 734. [Abstract] [Full Text] [PDF]
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M. G. Sanna, J. Liao, E. Jo, C. Alfonso, M.-Y. Ahn, M. S. Peterson, B. Webb, S. Lefebvre, J. Chun, N. Gray, et al. Sphingosine 1-Phosphate (S1P) Receptor Subtypes S1P1 and S1P3, Respectively, Regulate Lymphocyte Recirculation and Heart Rate J. Biol. Chem., April 2, 2004; 279(14): 13839 - 13848. [Abstract] [Full Text] [PDF]
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