OBJECTIVE: The role of reactive oxygen species (ROS) in mitogen-activated protein kinase (MAPK) signaling by angiotensin (Ang) II and endothelin-1 (ET-1) in human vascular smooth muscle cells (VSMC) was investigated. DESIGN: VSMCs were derived from resistance arteries from healthy subjects. MAPK activity was assessed using phospho-specific antibodies. ROS generation was measured by CMH2DCFDA fluorescence and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity by lucigenin chemiluminescence. RESULTS: Ang II and ET-1 increased MAPK phosphorylation (P < 0.01). Pre-treatment with Tiron and Tempol, *O2 scavengers, attenuated agonist-stimulated phosphorylation of p38MAPK, c-Jun N-terminal kinases (JNK) and ERK5, but not of ERK1/2 (extracellular signal-regulated kinases). Apocynin and diphenylene iodinium (DPI), NAD(P)H oxidase inhibitors, decreased Ang II-induced responses 60-70%. ET-1-mediated MAPK phosphorylation was unaffected by apocynin but was reduced (> 50%) by thenoyltrifluoroacetone (TIFT) and carboxyl cyanide-m-chlorophenylhydrazone (CCCP), mitochondrial inhibitors. Allopurinol and N-nitro-l-arginine methyl ester (l-NAME), xanthine oxidase and nitric oxide synthase (NOS) inhibitors, respectively, did not influence MAPK activation. Intracellular ROS generation, was increased by Ang II and ET-1 (P < 0.01). DPI inhibited Ang II- but not ET-1-mediated ROS production. Expression of p22phox and p47phox and activation of NAD(P)H oxidase were increased by Ang II but not by ET-1. CCCP and TIFT significantly attenuated ET-1-mediated ROS formation (P < 0.05), without influencing Ang II effects. CONCLUSIONS: Ang II activates p38MAPK, JNK and ERK5 primarily through NAD(P)H oxidase-generated ROS. ET-1 stimulates these kinases via redox-sensitive processes that involve mitochondrial-derived ROS. These data suggest that redox-dependent activation of MAPKs by Ang II and ET-1 occur through distinct ROS-generating systems that could contribute to differential signaling by these agonists in VSMCs.
OBJECTIVE: The role of reactive oxygen species (ROS) in mitogen-activated protein kinase (MAPK) signaling by angiotensin (Ang) II and endothelin-1 (ET-1) in human vascular smooth muscle cells (VSMC) was investigated. DESIGN: VSMCs were derived from resistance arteries from healthy subjects. MAPK activity was assessed using phospho-specific antibodies. ROS generation was measured by CMH2DCFDA fluorescence and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity by lucigenin chemiluminescence. RESULTS: Ang II and ET-1 increased MAPK phosphorylation (P < 0.01). Pre-treatment with Tiron and Tempol, *O2 scavengers, attenuated agonist-stimulated phosphorylation of p38MAPK, c-Jun N-terminal kinases (JNK) and ERK5, but not of ERK1/2 (extracellular signal-regulated kinases). Apocynin and diphenylene iodinium (DPI), NAD(P)H oxidase inhibitors, decreased Ang II-induced responses 60-70%. ET-1-mediated MAPK phosphorylation was unaffected by apocynin but was reduced (> 50%) by thenoyltrifluoroacetone (TIFT) and carboxyl cyanide-m-chlorophenylhydrazone (CCCP), mitochondrial inhibitors. Allopurinol and N-nitro-l-arginine methyl ester (l-NAME), xanthine oxidase and nitric oxide synthase (NOS) inhibitors, respectively, did not influence MAPK activation. Intracellular ROS generation, was increased by Ang II and ET-1 (P < 0.01). DPI inhibited Ang II- but not ET-1-mediated ROS production. Expression of p22phox and p47phox and activation of NAD(P)H oxidase were increased by Ang II but not by ET-1. CCCP and TIFT significantly attenuated ET-1-mediated ROS formation (P < 0.05), without influencing Ang II effects. CONCLUSIONS: Ang II activates p38MAPK, JNK and ERK5 primarily through NAD(P)H oxidase-generated ROS. ET-1 stimulates these kinases via redox-sensitive processes that involve mitochondrial-derived ROS. These data suggest that redox-dependent activation of MAPKs by Ang II and ET-1 occur through distinct ROS-generating systems that could contribute to differential signaling by these agonists in VSMCs.
Authors: Talin Ebrahimian; Melissa Wei Li; Catherine A Lemarié; Stefania M C Simeone; Patrick J Pagano; Matthias Gaestel; Pierre Paradis; Sven Wassmann; Ernesto L Schiffrin Journal: Hypertension Date: 2010-12-20 Impact factor: 10.190
Authors: Augusto C Montezano; Dylan Burger; Tamara M Paravicini; Andreia Z Chignalia; Hiba Yusuf; Mahmoud Almasri; Ying He; Glaucia E Callera; Gang He; Karl-Heinz Krause; David Lambeth; Mark T Quinn; Rhian M Touyz Journal: Circ Res Date: 2010-03-25 Impact factor: 17.367
Authors: Imad Al Ghouleh; Nicholas K H Khoo; Ulla G Knaus; Kathy K Griendling; Rhian M Touyz; Victor J Thannickal; Aaron Barchowsky; William M Nauseef; Eric E Kelley; Phillip M Bauer; Victor Darley-Usmar; Sruti Shiva; Eugenia Cifuentes-Pagano; Bruce A Freeman; Mark T Gladwin; Patrick J Pagano Journal: Free Radic Biol Med Date: 2011-06-14 Impact factor: 7.376