Lu Rong, Alioua Abderrahmane, Kumar Yogesh, Kundu Pallob, Eghbali Mansoureh, Weisstaub Noelia V, Gingrich Jay A, Stefani Enrico, Toro Ligia
Department of Anaesthesiology, Division of Molecular Medicine, University of California, Los Angeles, Los Angeles, CA 90095-7115, USA.
J Physiol. 2008 Aug 15;586(16):3855-69. doi: 10.1113/jphysiol.2008.153593. Epub 2008 Jul 3.
Serotonin (5-hydroxytryptamine, 5-HT) receptors (5-HTRs) play critical roles in brain and cardiovascular functions. In the vasculature, 5-HT induces potent vasoconstrictions, which in aorta are mainly mediated by activation of the 5-HT(2A)R subtype. We previously proposed that one signalling mechanism of 5-HT-induced vasoconstriction could be c-Src, a member of the Src tyrosine kinase family. We now provide evidence for a central role of c-Src in 5-HT(2A)R-mediated contraction. Inhibition of Src kinase activity with 10 mum 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) prior to contraction resulted in approximately 90-99% inhibition of contractions induced by 5-HT or by alpha-methyl-5-HT (5-HT(2)R agonist). In contrast, PP2 pretreatment only partly inhibited contractions induced by angiotensin II and the thromboxane A(2) mimetic, U46619, and had no significant action on phenylephrine-induced contractions. 5-Hydroxytryptamine increased Src kinase activity and PP2-sensitive tyrosine-phosphorylated proteins. As expected for c-Src identity, PP2 pretreatment inhibited 5-HT-induced contraction with an IC(50) of approximately 1 mum. Ketanserin (10 nm), a 5-HT(2A) antagonist, but not antagonists of 5-HT(2B)R (100 nm SB204741) or 5-HT(2C)R (20 nm RS102221), prevented 5-HT-induced contractions, mimicking PP2 and implicating 5-HT(2A)R as the major receptor subtype coupled to c-Src. In HEK 293T cells, c-Src and 5-HT(2A)R were reciprocally co-immunoprecipitated and co-localized at the cell periphery. Finally, 5-HT-induced Src activity was unaffected by inhibition of Rho kinase, supporting a role of c-Src upstream of Rho kinase. Together, the results highlight c-Src activation as one of the early and pivotal mechanisms in 5-HT(2A)R contractile signalling in aorta.
血清素（5-羟色胺，5-HT）受体（5-HTRs）在大脑和心血管功能中发挥着关键作用。在血管系统中，5-HT可诱导强烈的血管收缩，在主动脉中，这主要是由5-HT（2A）R亚型的激活介导的。我们之前提出，5-HT诱导血管收缩的一种信号传导机制可能是c-Src，它是Src酪氨酸激酶家族的一员。我们现在提供证据表明c-Src在5-HT（2A）R介导的收缩中起核心作用。在收缩前用10 μmol 4-氨基-5-（4-氯苯基）-7-（叔丁基）吡唑并[3,4-d]嘧啶（PP2）抑制Src激酶活性，可导致5-HT或α-甲基-5-HT（5-HT（2）R激动剂）诱导的收缩受到约90-99%的抑制。相比之下，PP2预处理仅部分抑制血管紧张素II和血栓素A2模拟物U46619诱导的收缩，对去氧肾上腺素诱导的收缩无显著作用。5-羟色胺增加了Src激酶活性和PP2敏感的酪氨酸磷酸化蛋白。正如对c-Src特性的预期，PP2预处理以约1 μmol的IC50抑制5-HT诱导的收缩。酮色林（10 nM），一种5-HT（2A）拮抗剂，但不是5-HT（2B）R拮抗剂（100 nM SB204741）或5-HT（2C）R拮抗剂（20 nM RS102221），可阻止5-HT诱导的收缩，类似于PP2，并表明5-HT（2A）R是与c-Src偶联的主要受体亚型。在HEK 293T细胞中，c-Src和5-HT（2A）R相互免疫共沉淀并在细胞周边共定位。最后，5-HT诱导的Src活性不受Rho激酶抑制的影响，支持c-Src在Rho激酶上游的作用。总之，这些结果突出了c-Src激活是主动脉中5-HT（2A）R收缩信号传导的早期关键机制之一。
