title: Integration of mitogen-activated protein kinase kinase activation in vascular 5-hydroxytryptamine2A receptor signal transduction.
abstract: Vascular 5-Hydroxytryptamine2A (5-HT2A) 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-HT2A 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, alpha-methyl-5-HT and 2,5-dimethoxy-4-iodoamphetamine all contracted the rat aorta, whereas the 5-HT2A receptor antagonist ketanserin (30 nM) blocked contraction to 5-HT. The tyrosine kinase inhibitor genistein (5 microM) shifted contraction to 5-HT, alpha-methyl-5-HT and DOI approximately 10-fold to the right, whereas daidzein (5 microM), the inactive isomer of genistein, was unable to shift 5-HT-induced contraction. PD098059 (10 microM), an inhibitor of MEK, shifted contraction to 5-HT approximately 7-fold to the right. We next examined the integration of tyrosine kinase activation in 5-HT2A receptor signaling. 5-HT-induced contraction was reduced individually by the PLC inhibitor 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate (NCDC; 100 microM) 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 microM) stimulated tyrosyl-phosphorylation of 42- and 44-kDa proteins identified as Erk MAPKs; this phosphorylation was reduced by PD098059 (10 microM). Neither nifedipine nor NCDC reduced 5-HT (1 microM)-stimulated Erk MAPK tyrosyl-phosphorylation, but the combination of nifedipine, NCDC and PD098059 abolished 5-HT (1 microM)-stimulated Erk MAPK tyrosyl-phosphorylation. Taken together, these studies indicate that stimulation of a vascular 5-HT2A 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-HT2A receptors.
authors.affiliation: Department of Pharmacology and Toxicology, Michigan State University, East Lansing, USA.
authors.forename: J A
authors.initials: JA
authors.lastname: Florian
authors.forename: S W
authors.initials: SW
authors.lastname: Watts
mesh_terms: D000818:Animals; D015220:Calcium Channels / Q000502:physiology; D020746:Calcium Channels, L-Type; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000502:physiology*; D004789:Enzyme Activation; D019833:Genistein / Q000494:pharmacology; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000187:drug effects* / Q000378:metabolism; D010766:Phosphorylation; D051381:Rats; D017207:Rats, Sprague-Dawley; D044402:Receptor, Serotonin, 5-HT2A; D011985:Receptors, Serotonin / Q000502:physiology*; D012701:Serotonin / Q000494:pharmacology; D015398:Signal Transduction; D010738:Type C Phospholipases / Q000502:physiology; D014661:Vasoconstriction / Q000187:drug effects
chemical_list: D015220:Calcium Channels; D020746:Calcium Channels, L-Type; D044402:Receptor, Serotonin, 5-HT2A; D011985:Receptors, Serotonin; D012701:Serotonin; D019833:Genistein; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D010738:Type C Phospholipases