Antioxidants inhibit endothelin-1 (1-31)-induced proliferation of vascular smooth muscle cells via the inhibition of mitogen-activated protein (MAP) kinase and activator protein-1 (AP-1).
We previously found that human chymase cleaves big endothelins (ETs) at the Tyr(31)-Gly(32) bond and produces 31-amino acid ETs (1-31), without any further degradation products. In the present study, we investigated the effects of various antioxidants on the ET-1 (1-31)-induced change in intracellular signaling and proliferation of cultured rat aortic smooth muscle cells (RASMC). ET-1 (1-31) stimulated rapid and significant activation of the mitogen-activated protein (MAP) kinase family, i.e. extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun NH(2)-terminal kinase (JNK), and p38 MAPK, in RASMC to an extent similar to that of ET-1. All of the antioxidants examined, i.e. N-acetyl-L-cysteine (NAC), diphenyleneiodonium chloride (DPI), and L-(+)-ascorbic acid (ascorbic acid), inhibited both ET-1 (1-31)- and ET-1-induced JNK and p38 MAPK activation but not ERK1/2 activation. Electron paramagnetic resonance (EPR) spectroscopy measurements revealed that NAC, DPI, and ascorbic acid inhibited xanthine oxidase-induced superoxide (O(2)(.-)) generation in a cell-free system. ET-1 (1-31) in addition to ET-1 increased the generation of cellular reactive oxygen species (ROS) in RASMC. ET-1 (1-31)- and ET-1-induced cellular ROS generation was inhibited similarly by NAC, DPI, and ascorbic acid in RASMC. Gel-mobility shift analysis showed that ET-1 (1-31) and ET-1 caused an increase in activator protein-1 (AP-1)-DNA binding activity in RASMC that was inhibited by the above three antioxidants. ET-1 (1-31) increased [3H]thymidine incorporation into cells to an extent similar to that of ET-1. This ET-1 (1-31)-induced increase in [3H]thymidine incorporation was also inhibited by NAC and DPI, but not by ascorbic acid. These results suggest that antioxidants inhibit ET-1 (1-31)-induced RASMC proliferation by inhibiting ROS generation within the cells. The underlying mechanisms of the inhibition of cellular proliferation by antioxidants may be explained, in part, by the inhibition of JNK activation and the resultant inhibition of AP-1-DNA binding.
Kyaw
Moe
M
Department of Pharmacology, The University of Tokushima School of Medicine, 3-18-15 Kuramoto, Tokushima 770-8503, Japan.
Yoshizumi
Masanori
M
Tsuchiya
Koichiro
K
Kirima
Kazuyoshi
K
Suzaki
Yuki
Y
Abe
Shinji
S
Hasegawa
Toyoshi
T
Tamaki
Toshiaki
T
D000111:Acetylcysteine / Q000494:pharmacology; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology*; D001205:Ascorbic Acid / Q000494:pharmacology; D002455:Cell Division / Q000187:drug effects; D003497:Cyclic N-Oxides / Q000494:pharmacology; D004247:DNA / Q000187:drug effects / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D019332:Endothelin-1 / Q000494:pharmacology / Q000031:analogs & derivatives; D016232:Endothelins / Q000494:pharmacology*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D048031:JNK Mitogen-Activated Protein Kinases; D008297:Male; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects*; D009861:Onium Compounds / Q000494:pharmacology; D010446:Peptide Fragments / Q000494:pharmacology*; D011725:Pyridines / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013997:Time Factors; D018808:Transcription Factor AP-1 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases
D000975:Antioxidants; D003497:Cyclic N-Oxides; D019332:Endothelin-1; D016232:Endothelins; D004791:Enzyme Inhibitors; D007093:Imidazoles; D009861:Onium Compounds; D010446:Peptide Fragments; D011725:Pyridines; D018808:Transcription Factor AP-1; C114932:endothelin-1 (1-31); C007517:diphenyleneiodonium; C017245:5,5-dimethyl-1-pyrroline-1-oxide; D004247:DNA; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; C093642:SB 203580; D001205:Ascorbic Acid; D000111:Acetylcysteine