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-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.
Lu
Rong
R
Department of Anaesthesiology, Division of Molecular Medicine, University of California, Los Angeles, Los Angeles, CA 90095-7115, USA.
Alioua
Abderrahmane
A
Kumar
Yogesh
Y
Kundu
Pallob
P
Eghbali
Mansoureh
M
Weisstaub
Noelia V
NV
Gingrich
Jay A
JA
Stefani
Enrico
E
Toro
Ligia
L
D000818:Animals; D001011:Aorta / Q000502:physiology*; D008297:Male; D009200:Myocardial Contraction / Q000502:physiology*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D051381:Rats; D011916:Rats, Inbred F344; D017207:Rats, Sprague-Dawley; D044402:Receptor, Serotonin, 5-HT2A / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*
D044402:Receptor, Serotonin, 5-HT2A; D011505:Protein-Tyrosine Kinases