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

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 ([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 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 [Ca2+](i) and Rho activation in vascular smooth muscle cells.