Mechanisms of 5-Hydroxytryptamine2A Receptor Activation of the Mitogen-Activated Protein Kinase Pathway in Vascular Smooth Muscle1
Abstract
5-Hydroxytryptamine (5-HT) activates the extracellular signal-regulated kinase (Erk) mitogen-activated protein kinases (MAPKs) in the vasculature, resulting in contraction. The mechanisms by which this occurs are unclear. G protein-coupled receptors can activate Erk MAPK pathways through a variety of mechanisms, including stimulation of Src, phosphoinositide-3 kinase (PI-3-K), protein kinase C (PKC), or the epidermal growth factor (EGF) receptor tyrosine kinase. We hypothesize that 5-HT uses one or more of these pathways. In isolated strips of rat aorta, the MAPK/Erk kinase inhibitor U0126 (50 μM), Src inhibitor PP1 (0.5 μM), PKC inhibitors calphostin C (1 μM) and chelerythrine (10 μM), and the PI-3-K inhibitor LY294002 (1–20 μM) reduced 5-HT-induced contraction. The EGF receptor tyrosine kinase inhibitor AG1478 (0.25–1 μM) was without effect. Thus, 5-HT activates PKC, Src, and possibly PI-3-K to result in contraction. In rat aortic myocytes, 5-HT (1 μM) activated Erk MAPK proteins 2- to 3-fold over basal values; activation was reduced by U0126, PP1, and LY294002 and unaffected by calphostin C or chelerythrine, wortmannin, or AG1478. The lack of effect of EGF receptor tyrosine kinase and PI-3-K inhibitors was confirmed in that the EGF receptor immunoprecipitated from 5-HT-exposed cells did not display an increase in autophosphorylation, nor did 5-HT significantly increase activation of Akt/protein kinase B, a downstream substrate for PI-3-K. These data suggest that the rat aortic 5-HT2A receptor uses Src but not PKC, PI-3-K, or the EGF receptor tyrosine kinase in stimulating Erk MAPK activation.
Footnotes
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Send reprint requests to: Stephanie W. Watts, Ph.D., B445 Life Sciences Building, Department of Pharmacology & Toxicology, Michigan State University, East Lansing, MI 48824-1317. E-mail:wattss{at}pilot.msu.edu
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↵1 This study was supported by a grant from the National Institutes of Health (HL58489) and a National Institutes of Health Pharmacological Sciences training grant.
- Abbreviations:
- 5-HT
- 5-hydroxytryptamine
- EGF
- epidermal growth factor
- Erk
- extracellular signal-regulated kinase
- MAPK
- mitogen-activated protein kinase
- MAPKK
- mitogen-activated protein kinase kinase
- PI-3-K
- phosphoinositide-3-kinase
- PKB
- protein kinase B
- PKC
- protein kinase C
- PDBu
- phorbol-12,13-dibutyrate
- PLC
- phospholipase C
- PP1
- 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine
- TBS
- Tris-buffered saline
- TBS-T
- Tris-buffered saline and 0.1% Tween-20
- U0126
- 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene
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- Received February 10, 1999.
- Accepted August 25, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
