Received for publication July 16, 2004.
Revised August 17, 2004.
Accepted for publication August 20, 2004.

Hydrogen Peroxide-induced ERK Activation in Cultured Feline Ileal Smooth Muscle Cells

Abstract

H₂O₂ has been shown to act as a signaling molecule involved in many cellular functions such as apoptosis and proliferation. In the present study, we characterized the effects of H₂O₂ on activation of MAP kinases and examined the factors involved in the process of ERK activation by H₂O₂ in ileal smooth muscle cells (ISMC). ISMC were cultured and exposed to H₂O₂. Western blot analysis was performed with phospho-specific MAP kinase antibodies. Potent activation of ERK and moderate activation of SAPK/JNK occurred within 30min of 1mM H₂O₂ treatment. However, p38 MAP kinase was not activated by H₂O₂. The activation of ERK by H2O2 was reduced by MEK inhibitor PD98059, Ras inhibitor FTS, removal of extracellular Ca²⁺, depletion of the intracellular Ca²⁺ pool by thapsigargin, or pretreatment of ISMC with the calmodulin antagonist W-7. Also, H₂O₂-induced ERK activation was attenuated by a receptor tyrosine kinase inhibitor, tyrphostin 51, but not by downregulation of protein kinase C (PKC) with phorbol-12-myristate-13-acetate (PMA) or by a PKC inhibitor, GF109203X. Growth factor receptor antagonist suramin pretreatment inhibited H₂O₂-induced ERK activation, highlighting a role for growth factor receptors in this activation. Further, the ERK activation by H₂O₂ was blocked by pretreatment with either N-acetyl-cysteine, o-phenanthroline, or mannitol, indicating that metal-catalyzed free radical formation may mediate the initiation of signal transduction by H₂O₂. These data suggest that short-term stimulation with H₂O₂ activates the signaling pathways of cell mitogenic effects which are thought to be a protective response against intestinal oxidative stress.

Key words: Antioxidants, ERK, Growth factor receptor, Hydrogen Peroxide, Ileal smooth muscle cells, Ras

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