Extracellular signal-regulated kinase (ERK) is involved in LPS-induced disturbances in intestinal motility S. GONZALO, L. GRASA, M. P. ARRUEBO, M. A ´ . PLAZA & M. D. MURILLO Departamento de Farmacologı ´a y Fisiologı ´a, Unidad de Fisiologı ´a, Facultad de Veterinaria, Universidad de Zaragoza, Zaragoza, Spain Abstract Background Lipopolysaccharide (LPS) is a causative agent of sepsis. A relationship has been described between LPS, free radicals, and cyclooxygenase-2 (COX-2). Here, we investigate the role of extracellular signal-regulated kinase (ERK) mitogen-activated pro- tein kinases (MAPK) in the effect of LPS on intestinal motility, oxidative stress status, and COX-2 expres- sion. Methods Rabbits were injected with (i) saline, (ii) LPS, (iii) U0126, an ERK MAPK inhibitor, or (iv) U0126+LPS. Duodenal contractility was studied in an organ bath with acetylcholine, prostaglandin E 2 , and KCl added. Neuromuscular function was assessed by electrical field stimulation (EFS). Neurotransmitter blockers were used to study the EFS-elicited contrac- tile response. The formation of products of oxidative damage to proteins (carbonyls), lipids, [malondialde- hyde (MDA), and 4-hydroxyalkenals (4-HDA)] was quantified in plasma and intestine. The protein expression of phospho-ERK (p-ERK), total ERK, and COX-2 in the intestine was measured by western blot, and p-ERK was localized by immunohistochemistry. Key Results Acetylcholine, prostaglandin E 2 , and KCl-induced contractions decreased with LPS. Elec- trical field stimulation induced a neurogenic contrac- tion that was reduced by LPS. Lipopolysaccharide increased p-ERK and COX-2 expression and the levels of carbonyls and MDA+4-HDA. U0126 blocked the effect of LPS on acetylcholine, prostaglandin E 2 , KCl, and EFS-induced contractions, the levels of carbonyls and MDA+4-HDA and p-ERK and COX-2 expression. Phospho-ERK was detected mostly in the neurons of the myenteric and submucosal ganglia. Conclusions & Inferences We can suggest that ERK is involved in the mechanism of action of LPS in the intestine. Keywords cyclooxygenase-2, ERK MAPK, free radicals, gastrointestinal motility, lipopolysaccharide. INTRODUCTION Cells recognize and respond to extracellular stimuli by engaging specific intracellular programs, such as the signaling cascade that leads to the activation of mitogen- activated protein kinases (MAPKs). The most exten- sively studied groups of vertebrate MAPKs to date are the ERK1/2, JNKs, and p38 kinases. 1 Each family of MAPKs is composed of a set of three evolutionarily conserved, sequentially acting kinases: a MAPK, a MAPK kinase (MAPKK), and a MAPKK kinase. The ERK pathway is upregulated via Ras. Upon activation, Ras in turn activates the serine–threonine kinase Raf-1, which then phosphorylates the MAPKK, and MEK. MEK, a dual-specific kinase, activates ERK by phosphory- lating it on two critical residues, Tyr183 and Thr185. ERK then translocates into the nucleus and regulates the activities of several nuclear transcription factors. 2 In this study, we examined the effects of blocking the ERK pathway by using an inhibitor, U0126 that specifically inhibits the ability of MEK to phosphory- late ERK without affecting JNK and p38 pathways. 3 Many studies have shown that LPS activates ERK1/2 in monocytes/macrophages. 4–6 Lipopolysaccharide (LPS), a major structural and functional component of Gram-negative bacteria, is believed to be responsible for the initiation of Gram- negative bacteria-induced endotoxic shock. 7 Many alterations such as intestinal motility disturbances and oxidative stress have been attributed to LPS, 8–10 but the intracellular mechanism of action of LPS needs further investigation. Previous studies by our group Address for Correspondence Dr Marı ´a Divina Murillo, Departamento de Farmacologı ´a y Fisiologı ´a, Unidad de Fisiologı ´a, Facultad de Veterinaria, Universidad de Zaragoza, c/Miguel Servet 177, 50013 Zaragoza, Spain. Tel: +34 976 761652; fax: +34 976 761612; e-mail: dmurillo@unizar.es Received: 20 July 2010 Accepted for publication: 20 October 2010 Neurogastroenterol Motil (2011) 23, e80–e90 doi: 10.1111/j.1365-2982.2010.01632.x Ó 2010 Blackwell Publishing Ltd e80