Research article

Role of ERK/MAPK in endothelin receptor signaling in human aortic smooth muscle cells

Qing-wen Chen^1,2 , Lars Edvinsson^1,2 and Cang-Bao Xu¹

¹Division of Experimental Vascular Research, Institute of Clinical Science in Lund, Lund University, Lund, Sweden

²Department of Clinical and Experimental Research, Glostrup Hospital, Copenhagen University, Copenhagen, Denmark

author email corresponding author email

BMC Cell Biology 2009, 10:52doi:10.1186/1471-2121-10-52

Published:	3 July 2009

Abstract

Background

Endothelin-1 (ET-1) is a potent vasoactive peptide, which induces vasoconstriction and proliferation in vascular smooth muscle cells (VSMCs) through activation of endothelin type A (ET_A) and type B (ET_B) receptors. The extracellular signal-regulated kinase 1 and 2 (ERK1/2) mitogen-activated protein kinases (MAPK) are involved in ET-1-induced VSMC contraction and proliferation. This study was designed to investigate the ET_A and ET_B receptor intracellular signaling in human VSMCs and used phosphorylation (activation) of ERK1/2 as a functional signal molecule for endothelin receptor activity.

Results

Subconfluent human VSMCs were stimulated by ET-1 at different concentrations (1 nM-1 μM). The activation of ERK1/2 was examined by immunofluorescence, Western blot and phosphoELISA using specific antibody against phosphorylated ERK1/2 protein. ET-1 induced a concentration- and time- dependent activation of ERK1/2 with a maximal effect at 10 min. It declined to baseline level at 30 min. The ET-1-induced activation of ERK1/2 was completely abolished by MEK1/2 inhibitors U0126 and SL327, and partially inhibited by the MEK1 inhibitor PD98059. A dual endothelin receptor antagonist bosentan or the ET_A antagonist BQ123 blocked the ET-1 effect, while the ET_B antagonist BQ788 had no significant effect. However, a selective ET_B receptor agonist, Sarafotoxin 6c (S6c) caused a time-dependent ERK1/2 activation with a maximal effect by less than 20% of the ET-1-induced activation of ERK1/2. Increase in bosentan concentration up to 10 μM further inhibited ET-1-induced activation of ERK1/2 and had a stronger inhibitory effect than BQ123 or the combined use of BQ123 and BQ788. To further explore ET-1 intracellular signaling, PKC inhibitors (staurosporin and GF109203X), PKC-delta inhibitor (rottlerin), PKA inhibitor (H-89), and phosphatidylinositol 3-kinase (PI3K) inhibitor (wortmannin) were applied. The inhibitors showed significant inhibitory effects on ET-1-induced activation of ERK1/2. However, blockage of L-type Ca²⁺ channels or calcium/calmodulin-dependent protein kinase II, chelating extracellular Ca²⁺ or emptying internal Ca²⁺ stores, did not affect ET-1-induced activation of ERK1/2.

Conclusion

The ET_A receptors predominate in the ET-1-induced activation of ERK1/2 in human VSMCs, which associates with increments in intracellular PKC, PKA and PI3K activities, but not Ca²⁺ signalling.