Role of ERK/MAPK in endothelin receptor signaling in human aortic smooth muscle cells.
muscle cells (VSMCs) through activation of endothelin type A (ETA) and type B (ETB) 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 ETA and ETB 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 microM). 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 ETA antagonist BQ123 blocked the ET-1 effect, while the ETB antagonist BQ788 had no significant effect. However, a selective ETB 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 microM 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.


CONCLUSIONS
The ETA 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.
Chen
Qing-wen
QW
Division of Experimental Vascular Research, Institute of Clinical Science in Lund, Lund University, Lund, Sweden. cherqw@hotmail.com
Edvinsson
Lars
L
Xu
Cang-Bao
CB
D001011:Aorta / Q000166:cytology*; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D019332:Endothelin-1 / Q000378:metabolism; D004789:Enzyme Activation; D006801:Humans; D020930:MAP Kinase Kinase Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism*; D020559:Monomeric GTP-Binding Proteins / Q000378:metabolism; D032389:Myocytes, Smooth Muscle / Q000378:metabolism*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D044022:Receptor, Endothelin A / Q000378:metabolism*; D044023:Receptor, Endothelin B / Q000378:metabolism*
D019332:Endothelin-1; D044022:Receptor, Endothelin A; D044023:Receptor, Endothelin B; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020930:MAP Kinase Kinase Kinases; D020559:Monomeric GTP-Binding Proteins; D002118:Calcium