Abstract

Vascular 5-Hydroxytryptamine_2A (5-HT_2A) receptor signaling and contraction has been associated with the activation of L-type calcium channels, phospholipase C (PLC) and, as we previously demonstrated, tyrosine kinase activation. We hypothesize the 5-HT_2A receptor activates all three pathways independently to elicit contraction and that one of the tyrosine kinases activated by 5-HT is mitogen-activated protein kinase kinase (MEK). Endothelium-denuded rat thoracic aorta was mounted into isolated tissue baths for measurement of isometric contractile force. 5-HT, α-methyl-5-HT and 2,5-dimethoxy-4-iodoamphetamine all contracted the rat aorta, whereas the 5-HT_2A receptor antagonist ketanserin (30 nM) blocked contraction to 5-HT. The tyrosine kinase inhibitor genistein (5 μM) shifted contraction to 5-HT, α-methyl-5-HT and DOI ∼10-fold to the right, whereas daidzein (5 μM), the inactive isomer of genistein, was unable to shift 5-HT-induced contraction. PD098059 (10 μM), an inhibitor of MEK, shifted contraction to 5-HT ∼7-fold to the right. We next examined the integration of tyrosine kinase activation in 5-HT_2Areceptor signaling. 5-HT-induced contraction was reduced individually by the PLC inhibitor 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate (NCDC; 100 μM) or the Ca⁺⁺ channel inhibitor nifedipine (50 nM); the remaining response to 5-HT was reduced by further addition of either genistein or PD098059. When nifedipine and NCDC were used in combination, a part of the contraction to 5-HT remained; this contraction was further reduced by genistein or PD098059. In cultured aortic smooth muscle cells, 5-HT (0.01–100 μM) stimulated tyrosyl-phosphorylation of 42- and 44-kDa proteins identified as Erk MAPKs; this phosphorylation was reduced by PD098059 (10 μM). Neither nifedipine nor NCDC reduced 5-HT (1 μM)-stimulated Erk MAPK tyrosyl-phosphorylation, but the combination of nifedipine, NCDC and PD098059 abolished 5-HT (1 μM)-stimulated Erk MAPK tyrosyl-phosphorylation. Taken together, these studies indicate that stimulation of a vascular 5-HT_2A receptor activates Ca⁺⁺ channels and PLC as well as MEK to cause rat aortic contraction and that MEK activation is at least partially independent of the two pathways classically associated with 5-HT_2Areceptors.