title: Sphingosine 1-phosphate (S1P) regulates vascular contraction via S1P3 receptor: investigation based on a new S1P3 receptor antagonist.
abstract: Sphingosine 1-phosphate (S1P) induces diverse biological responses in various tissues by activating specific G protein-coupled receptors (S1P(1)-S1P(5) receptors). The biological signaling regulated by S1P(3) receptor has not been fully elucidated because of the lack of an S1P(3) receptor-specific antagonist or agonist. We developed a novel S1P(3) 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 S1P(3) receptor. In functional studies, TY-52156 showed submicromolar potency and a high degree of selectivity for S1P(3) receptor. TY-52156, but not an S1P(1) receptor antagonist [(R)-phosphoric acid mono-[2-amino-2-(3-octyl-phenylcarbamoyl)-ethyl] ester; VPC23019] or S1P(2) 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 ([Ca(2+)](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(2+)](i) and Rho activation. In contrast, VPC23019 and JTE013 inhibited only the increase in [Ca(2+)](i) and Rho activation, respectively. We further confirmed that TY-52156 inhibited FTY-720-induced S1P(3) receptor-mediated bradycardia in vivo. These results clearly show that TY-52156 is both sensitive and useful as an S1P(3) receptor-specific antagonist and reveal that S1P induces vasoconstriction by directly activating S1P(3) receptor and through a subsequent increase in [Ca(2+)](i) and Rho activation in vascular smooth muscle cells.
authors.affiliation: Drug Research Department, Tokyo Research Laboratories, TOA EIYO Ltd., 2-293-3 Amanuma, Omiya, Saitama 330-0834, Japan. murakami.akira@toaeiyo.co.jp
authors.forename: Akira
authors.initials: A
authors.lastname: Murakami
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Takasugi
authors.forename: Shinya
authors.initials: S
authors.lastname: Ohnuma
authors.forename: Yuuki
authors.initials: Y
authors.lastname: Koide
authors.forename: Atsuko
authors.initials: A
authors.lastname: Sakurai
authors.forename: Satoshi
authors.initials: S
authors.lastname: Takeda
authors.forename: Takeshi
authors.initials: T
authors.lastname: Hasegawa
authors.forename: Jun
authors.initials: J
authors.lastname: Sasamori
authors.forename: Takashi
authors.initials: T
authors.lastname: Konno
authors.forename: Kenji
authors.initials: K
authors.lastname: Hayashi
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Watanabe
authors.forename: Koji
authors.initials: K
authors.lastname: Mori
authors.forename: Yoshimichi
authors.initials: Y
authors.lastname: Sato
authors.forename: Atsuo
authors.initials: A
authors.lastname: Takahashi
authors.forename: Naoki
authors.initials: N
authors.lastname: Mochizuki
authors.forename: Nobuyuki
authors.initials: N
authors.lastname: Takakura
mesh_terms: D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D002536:Cerebral Arteries / Q000187:drug effects / Q000502:physiology; D003326:Coronary Circulation / Q000187:drug effects; D006224:Cricetinae; D003412:Cricetulus; D004285:Dogs; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D000068876:Fingolimod Hydrochloride; D006339:Heart Rate / Q000187:drug effects; D006835:Hydrazones / Q000494:pharmacology*; D008246:Lysophospholipids / Q000494:pharmacology*; D008297:Male; D010766:Phosphorylation; D011409:Propylene Glycols / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D049349:Receptors, Lysosphingolipid / Q000037:antagonists & inhibitors* / Q000502:physiology*; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D014661:Vasoconstriction / Q000187:drug effects*; D054460:rho-Associated Kinases / Q000502:physiology
chemical_list: C548679:1-(4-chlorophenylhydrazono)-1-(4-chlorophenylamino)-3,3-dimethyl-2-butanone; D006835:Hydrazones; D008246:Lysophospholipids; D011409:Propylene Glycols; D049349:Receptors, Lysosphingolipid; C060506:sphingosine 1-phosphate; D054460:rho-Associated Kinases; D048049:Extracellular Signal-Regulated MAP Kinases; D000068876:Fingolimod Hydrochloride; D013110:Sphingosine; D002118:Calcium