Drug Research Department, Tokyo Research Laboratories, TOA EIYO Ltd., 2-293-3 Amanuma, Omiya, Saitama 330-0834, Japan.
Mol Pharmacol. 2010 Apr;77(4):704-13. doi: 10.1124/mol.109.061481. Epub 2010 Jan 22.
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+)) 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+) and Rho activation. In contrast, VPC23019 and JTE013 inhibited only the increase in Ca(2+) 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+) and Rho activation in vascular smooth muscle cells.
鞘氨醇 1-磷酸（S1P）通过激活特定的 G 蛋白偶联受体（S1P（1）-S1P（5）受体）在各种组织中诱导多种生物学反应。由于缺乏 S1P（3）受体特异性激动剂或拮抗剂，因此尚未完全阐明 S1P（3）受体调节的生物学信号。我们开发了一种新型的 S1P（3）受体拮抗剂，1-（4-氯苯腙基）-1-（4-氯苯氨基）-3,3-二甲基-2-丁酮（TY-52156），并在此表明 S1P 诱导的冠状动脉流量（CF）减少是由 S1P（3）受体介导的。在功能研究中，TY-52156对 S1P（3）受体表现出亚微摩尔效力和高度选择性。TY-52156（而非 S1P（1）受体拮抗剂[（R）-磷酸单-[2-氨基-2-（3-辛基-苯甲酰胺基）-乙基]酯；VPC23019]或 S1P（2）受体拮抗剂[1-[1,3-二甲基-4-（2-甲基乙基）-1H-吡唑并[3,4-b]吡啶-6-基]-4-（3,5-二氯-4-吡啶基）-半卡巴肼；JTE013]）抑制了 S1P 在分离灌注的大鼠心脏中引起的 CF 减少。我们进一步研究了 TY-52156 对 S1P 诱导的人冠状动脉平滑肌细胞内钙（[Ca（2+）]（i））增加和 Rho 激活的影响，这是收缩的原因。TY-52156 抑制 S1P 诱导的[Ca（2+）]（i）增加和 Rho 激活。相反，VPC23019 和 JTE013 分别仅抑制[Ca（2+）]（i）增加和 Rho 激活。我们进一步证实，TY-52156 抑制了 FTY-720 诱导的体内 S1P（3）受体介导的心动过缓。这些结果清楚地表明，TY-52156 既是敏感的又是有用的 S1P（3）受体特异性拮抗剂，并且表明 S1P 通过直接激活 S1P（3）受体并随后增加血管平滑肌细胞中的[Ca（2+）]（i）和 Rho 激活来引起血管收缩。
