Hydrogen peroxide-induced extracellular signal-regulated kinase 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 the activation of mitogen-activated protein ( MAP) kinases and examined the factors involved in the process of extracellular signal-regulated kinase (ERK) activation by H2O2 in ileal smooth muscle cells (ISMC). ISMC were cultured and exposed to H2O2. Western blot analysis was performed with phosphospecific MAP kinase antibodies. Potent activation of ERK and moderate activation of stress-activated protein kinase/c-Jun NH2-terminal kinase occurred within 30 min of 1 mM H2O2 treatment. However, p38 MAP kinase was not activated by H2O2. The activation of ERK by H2O2 was reduced by the mitogen-activated/ ERK-activating kinase inhibitor PD98059 [2-(2-amino-3-methoxyphenyl)- 4H-1-benzopyran-4-one], Ras inhibitor S-farnesylthiosalicylic acid, 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 down-regulation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate or by a PKC inhibitor, GF109203X [3-[1-(dimethylaminopropyl) indol-3- yl]-4-(indol-3-yl) maleimide hydrochloride]. Growth factor receptor antagonist suramin pretreatment inhibited H2O2-induced ERK activation, highlighting a role for growth factor receptors in this activation. Furthermore, 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.