Sphingosine 1-Phosphate (S1P) Regulates Vascular Contraction via S1P3 Receptor: Investigation Based on a New S1P3 Receptor Antagonist □S

Sphingosine 1-phosphate (S1P) induces diverse biological responses in various tissues by activating specific G proteincoupled receptors (S1P1–S1P5 receptors). The biological signaling regulated by S1P3 receptor has not been fully elucidated because of the lack of an S1P3 receptor-specific antagonist or agonist. We developed a novel S1P3 receptor antagonist, 1-(4chlorophenylhydrazono)-1-(4-chlorophenylamino)-3,3-dimethyl2-butanone (TY-52156), and show here that the S1P-induced decrease in coronary flow (CF) is mediated by the S1P3 receptor. In functional studies, TY-52156 showed submicromolar potency and a high degree of selectivity for S1P3 receptor. TY-52156, but not an S1P1 receptor antagonist [(R)-phosphoric acid mono-[2-amino-2-(3-octyl-phenylcarbamoyl)-ethyl] ester; VPC23019] or S1P2 receptor antagonist [1-[1,3-dimethyl-4-(2methylethyl)-1H-pyrazolo[3,4-b]pyridin-6-yl]-4-(3,5-dichloro-4pyridinyl)-semicarbazide; JTE013], inhibited the decrease in CF induced by S1P in isolated perfused rat hearts. We further investigated the effect of TY-52156 on both the S1P-induced increase in intracellular calcium ([Ca ]i) and Rho activation that are responsible for the contraction of human coronary artery smooth muscle cells. TY-52156 inhibited both the S1P-induced increase in [Ca ]i and Rho activation. In contrast, VPC23019 and JTE013 inhibited only the increase in [Ca ]i and Rho activation, respectively. We further confirmed that TY-52156 inhibited FTY-720-induced S1P3 receptor-mediated bradycardia in vivo. These results clearly show that TY-52156 is both sensitive and useful as an S1P3 receptor-specific antagonist and reveal that S1P induces vasoconstriction by directly activating S1P3 receptor and through a subsequent increase in [Ca ]i and Rho activation in vascular smooth muscle cells. Sphingosine 1-phosphate (S1P) is a bioactive lysophospholipid mediator that is mainly released from activated platelets and induces many biological responses, including angiogenesis, vascular development, and cardiovascular function (Siess, 2002; Yatomi, 2006; Takuwa et al., 2008). A wide variety of biological cellular responses to S1P have been ascribed to the presence of five S1P receptors, S1P1 to S1P5 receptors, that belong to the family of G protein-coupled receptors (GPCRs). Furthermore, a variation of heterotrimeric G protein downstream of S1P receptors accounts for the diversity of cellular responses to S1P (Rosen et al., 2009). In addition to the coupling of S1P receptors and G proteins, the expression of the combiThis work was supported by Research on Health Science Focusing on Drug Innovation from the Japan Health Sciences Foundation [Grant KHC1016]. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.109.061481. □S The online version of this article (available at http://molpharm. aspetjournals.org) contains supplemental material. ABBREVIATIONS: S1P, sphingosine 1-phosphate; GPCR, G protein-coupled receptor; TY-52156, 1-(4-chlorophenylhydrazono)-1-(4-chlorophenylamino)-3,3-dimethyl-2-butanone; MAPK, mitogen-activated protein kinase; CF, coronary flow; HUVEC, human umbilical vein endothelial cell; HCASMC, human coronary artery smooth muscle cell; SBP, systemic blood pressure; HR, heart rate; MBP, mean blood pressure; JTE013, 1-[1,3-dimethyl-4-(2-methylethyl)-1H-pyrazolo[3,4-b]pyridin-6-yl]-4-(3,5-dichloro-4-pyridinyl)-semicarbazide; VPC23019, (R)-phosphoric acid mono-[2-amino-2-(3-octyl-phenylcarbamoyl)-ethyl] ester; SD, Sprague-Dawley; IkAch, cardiac G protein-gated potassium channel; Eu-GTP, europium-GTP; LC/MS/MS, liquid chromatography/tandem mass spectrometry; U46619, 9,11-dideoxy-9 ,11 -methanoepoxy prostaglandin F2 ; FTY-720, fingolimod; Y-27632, (R)-( )-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride monohydrate; SEW2871, 5-[4-phenyl-5-(trifluoromethyl)-2-thienyl]-3-[3-(trifluoromethyl)phenyl]-1,2,4-oxadiazole. 0026-895X/10/7704-704–713$20.00 MOLECULAR PHARMACOLOGY Vol. 77, No. 4 Copyright © 2010 The American Society for Pharmacology and Experimental Therapeutics 61481/3574034 Mol Pharmacol 77:704–713, 2010 Printed in U.S.A. 704 http://molpharm.aspetjournals.org/content/suppl/2010/01/22/mol.109.061481.DC1 Supplemental material to this article can be found at: at A PE T Jornals on Jne 3, 2017 m oharm .aspeurnals.org D ow nladed from nation of S1P receptors determines multiple cellular responses. To identify the signaling that is specific for each receptor, S1P receptor antagonists have been developed and have contributed to our understanding of S1P-mediated signaling (Huwiler and Pfeilschifter, 2008). S1P1 to S1P5 receptors couple to different G proteins upon binding to S1P. Whereas S1P1, S1P4, and S1P5 receptors mainly couple to Gi, S1P2 and S1P3 receptors couple to Gi, Gq, and G12/13 (Rosen et al., 2009). The signal that converges from Gi-coupled S1P receptors inhibits the activation of adenylate cyclase and induces the activation of p44/p42 mitogen-activated protein kinase (MAPK). Although the S1P1 receptor slightly increases intracellular calcium ([Ca ]i) through G , S1P2 and S1P3 receptors mainly increase [Ca ]i through the activation of phospholipase C from Gq (Watterson et al., 2005). The deletion of S1P3 but not S1P2 receptor in mouse embryonic fibroblasts led to the marked inhibition of S1P-induced phospholipase C activation, which suggests that S1P3 receptor plays an important role in the S1P-induced increase in [Ca ]i (Ishii et al., 2002). S1P2 and S1P3 receptors also couple to G12/13 protein to activate a small GTPase, Rho, which is involved in the regulation of actin-cytoskeleton (Ryu et al., 2002; Sugimoto et al., 2003). Rho kinase is activated by Rho through the G12/13-Rho guanine nucleotide exchange factor family. S1P-induced Rho activation has been shown to be significantly reduced in S1P2 but not S1P3 receptor-null mouse embryonic fibroblasts (Ishii et al., 2002). Meanwhile, an association between S1P3 receptor and Rho activation has been reported in cells expressing S1P3 receptor (Sugimoto et al., 2003). An S1P-induced contraction of vascular smooth muscle cells has been ascribed to an increase in [Ca ]i and Rho activation (Ohmori et al., 2003; Watterson et al., 2005). S1P-induced vasoconstriction is significantly inhibited in cerebral arteries isolated from S1P3 receptornull mice but not in those from S1P2 receptor-null mice (Salomone et al., 2008). In addition, Y-27632, a selective Rho kinase inhibitor, inhibits S1P-induced vasoconstriction in canine cerebral arteries (Tosaka et al., 2001), indicating that S1P3 receptor plays an indispensable role in S1P-induced vasoconstriction mediated by Rho-Rho kinase signaling. Although S1P decreases coronary flow (CF) in isolated perfused canine heart, the receptor subtype that is responsible for the S1P-induced reduction of CF has not yet been fully identified. To distinguish S1P3 receptordependent signal from S1P2 receptor-dependent signal, an S1P3 receptor-specific antagonist has been needed. We have developed an S1P3 receptor antagonist, TY52156. By confirming that TY-52156 has a selective antagonistic effect toward S1P3 receptor, we can delineate the role of S1P3 receptor-specific signaling in vascular contraction. Moreover, the effectiveness of TY-52156 in vivo was bolstered by evidence that S1P3 receptor-dependent bradycardia was suppressed by the oral administration of TY-52156. Materials and Methods