Abstract
Sphingosine 1-phosphate (S1P) induces diverse biological responses in various tissues by activating specific G protein-coupled 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-(4-chlorophenylhydrazono)-1-(4-chlorophenylamino)-3,3-dimethyl- 2-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-(2-methylethyl)-1H-pyrazolo[3,4-b]pyridin-6-yl]-4-(3,5-dichloro-4-pyridinyl)-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 ([Ca2+]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 [Ca2+]i and Rho activation. In contrast, VPC23019 and JTE013 inhibited only the increase in [Ca2+]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 [Ca2+]i and Rho activation in vascular smooth muscle cells.
Footnotes
↵ The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
This 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.
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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-9α,11α-methanoepoxy prostaglandin F2α
- 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.
- Received October 13, 2009.
- Accepted January 19, 2010.
- Copyright © 2010 The American Society for Pharmacology and Experimental Therapeutics
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