pISSN 1226-4512 eISSN 2093-3827

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Korean J Physiol Pharmacol 2004; 8(6): 339-344

Published online December 30, 2004

Copyright © Korean J Physiol Pharmacol.

MAPK Activation and Cell Viability after H2O2 Stimulation in Cultured Feline Ileal Smooth Muscle Cells

Hyun Ju Song, Ji Hoon Jeong, Dong Kyu Lee, Tai Sang Lee, Young Sil Min, and Uy Dong Sohn

Department of Pharmacology, College of Pharmacy, Chung Ang University, Seoul 156-756, Korea

Abstract

Recent data have shown the importance of oxidative stresses in the pathogenesis of inflammatory bowel disease, crohn's disease and ulcerative colitis. H2O2, reactive oxygen species (ROS) donor, has been reported to act as a signaling molecule involved in a variety of cellular functions such as apoptosis and proliferation. In the present study, we investigated viability of cultured ileal smooth muscle cells (ISMC) after stimulation with H2O2. Trypan blue method revealed that the cell viability of ISMC treated with 1 mM H2O2 was not different from that of controls at up to 2 h time point, while treatment of ISMC with 1 mM H2O2 for 48 h finally induced significant decrease in the cell viability. Therefore, we evaluated whether H2O2 was capable of ERKs activation in ISMC for the short-term exposure and examined whether tyrosine kinase was involved in the process of ERK activation by H2O2 in ISMC. We also investigated the effects of H2O2 on activation of SAPK/JNK and p38 MAP kinase in ISMC. Thus, ISMC were cultured and exposed to H2O2, and western blot analysis was performed with phospho- specific MAP kinase antibodies. Robust activation of ERK occurred within 30 min of 1 mM H2O2 treatment. H2O2-induced ERK activation was attenuated by a tyrosine kinase inhibitor, genistein, indicating that tyrosine kinase was probably involved in the ERK activation by H2O2. H2O2 was a moderate activator of SAPK/JNK, while p38 MAP kinase was not activated by H2O2. We suggest that ERK activation induced by short-term H2O2 treatment plays a critical role in cellular protection in the early stage of response to oxidative stress. The present study suggests the necessity of identification of MAPK signaling pathways affected by ROS, since it could ultimately elucidate cellular consequences involved in initiation and perpetuation of intestinal tissue damage in the diseases such as crohn's disease and ulcerative colitis, resulted from excessive ROS.

Keywords: H2O2, ERKs, SAPK/JNK, Cell viability, Ileal smooth muscle cells