c-Src tyrosine kinase, a critical component for 5-HT2A receptor-mediated contraction in rat aorta

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-HT2AR 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-HT2AR-mediated contraction. Inhibition of Src kinase activity with 10 mu M 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) prior to contraction resulted in similar to 90-99% inhibition of contractions induced by 5-HT or by alpha-methyl-5-HT (5-HT2R 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 IC50 of similar to 1 mu M. Ketanserin (10 nM), a 5-HT2A antagonist, but not antagonists of 5-HT2BR (100 nM SB204741) or 5-HT2CR (20 nM RS102221), prevented 5-HT-induced contractions, mimicking PP2 and implicating 5-HT2AR as the major receptor subtype coupled to c-Src. In HEK 293T cells, c-Src and 5-HT2AR 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-HT2AR contractile signalling in aorta.