Reactive oxygen species mediate ET-1-induced activation of ERK1/2 signaling in cultured feline esophageal smooth muscle cells.
Reactive oxygen species (ROS) have been shown to play a critical role in propagating the signals of several growth factors, peptide hormones, and cytokines, such as epidermal growth factor, insulin, and interleukin-1. We investigated a possible role for ROS generation in mediating the action of ET-1 on activation of ERK1/2 in cultured feline esophageal smooth muscle cells (ESMC). Confluent layers of ESMC were stimulated by 10nM ET-1; activation of ERK was examined by western blot analysis with phospho-specific antibodies of ERKs. ET-1 induced ERK1/2 phosphorylation in a dose- and time- dependent manner. ERK1/2 activation by ET-1 reached the maximal levels at 5min showing slight activation up to 20min, and then slowly declined. It was confirmed that the activation of ERK1/2 was reduced by MEK inhibitor PD98059. We observed the dose-dependent inhibitory effect of diphenyleneiodonium (DPI), an inhibitor of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase on the ET-1-enhanced ERK1/2 phosphorylation in ESMC. Pretreatment of ESMC with N-acetylcysteine, a ROS scavenger, also attenuated the ET-1-induced ERK1/2 activation. In addition, DPI significantly inhibited the ET-1- induced ROS production when ROS was measured as a function of DCF fluorescence. The results suggest that ROS might be critical mediators of the ET-1-induced ERK1/2 signaling events in ESMC.
Song
Hyun Ju
HJ
Department of Pharmacology, College of Pharmacy, Chung Ang University, Seoul 156-756, Korea.
Kim
Ji Soo
JS
Lee
Myong Jae
MJ
Nam
Yoon Sung
YS
Sohn
Uy Dong
UD
D000111:Acetylcysteine / Q000494:pharmacology; D000818:Animals; D002415:Cats; D019332:Endothelin-1 / Q000494:pharmacology*; D004947:Esophagus / Q000187:drug effects* / Q000378:metabolism; D005260:Female; D020935:MAP Kinase Signaling System / Q000502:physiology*; D008297:Male; D019950:Mitogen-Activated Protein Kinase 1 / Q000502:physiology*; D048052:Mitogen-Activated Protein Kinase 3 / Q000502:physiology*; D032389:Myocytes, Smooth Muscle / Q000187:drug effects* / Q000378:metabolism; D019255:NADPH Oxidases / Q000502:physiology; D009861:Onium Compounds / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism*
D019332:Endothelin-1; D009861:Onium Compounds; D017382:Reactive Oxygen Species; C007517:diphenyleneiodonium; D019255:NADPH Oxidases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D000111:Acetylcysteine