Mitogen activated protein kinases blockade improves lipopolysaccharide-induced ileal motor disturbances
El bloqueo de las proteínas cinasas activadas por mitógenos mejora las alteraciones motoras inducidas por el lipopolisacárido en íleon
Sergio Gonzalo, Laura Grasa, Ligia Verónica Hernández, María Pilar Arruebo, Miguel Ángel Plaza and María Divina Murillo
Department of Pharmacology and Physiology. Unit of Physiology. Facultad de Veterinaria. Universidad de Zaragoza. Zaragoza, Spain
This work was presented, in part, at the "LXVII Congreso Anual de la Sociedad Española de Patología Digestiva (SEPD), Sitges-Spain June 2008".
This work was funded by the Ministerio de Ciencia y Tecnología, España (Dirección General de Investigación AGL2006-04317 and ERDF), the Gobierno de Aragón (B61/2008-2010) and the Universidad de Zaragoza (UZ2009-BIO-03). An individual grant to Sergio Gonzalo was provided by Ministerio de Educación y Ciencia, España (BES-2007-156469).
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ABSTRACT
Background: several diseases such as sepsis can affect the ileum. Lipopolysaccharide (LPS), an endotoxin present in the cell wall of gram negative bacteria, is a causative agent of sepsis.
Objectives: the aims of this study were: a) to investigate the role of mitogen activated protein kinases (MAPKs) in the effect of LPS on the acetylcholine-induced contractions of rabbit ileum; and b) to study the localization of MAPKs in the ileum.
Material and methods: ileal contractility was studied in an organ bath and MAPKs were localized by immunohistochemistry.
Results: acetylcholine-induced contractions decreased with LPS. SB203580, SP600125 and U0126 blocked the effect of LPS on the acetylcholine-induced contractions. Phosphorylated p38 and ERK were detected in neurons of myenteric plexus and phosphorylated p38 and JNK in smooth muscle cells of ileum.
Conclusion: we can suggest that p38, JNK, and ERK MAPKs are involved in the mechanism of action of LPS in the ileum.
Key words: Intestinal contractility. LPS. p38. ERK. JNK. Myenteric plexus. Smooth muscle.
Introducción: varias enfermedades como la sepsis pueden afectar al íleon. El lipopolisacárido (LPS), una endotoxina presente en la pared celular de las bacterias gram-negativas, es un agente causal de la sepsis.
Objetivos: los objetivos del presente estudio fueron: a) investigar el papel de las proteína cinasas activadas por mitógenos (MAPKs) en los efectos del LPS en las contracciones inducidas por acetilcolina en el íleon de conejo; y b) estudiar la localización de las MAPKs en el íleon.
Material y métodos: la contractilidad ileal se estudió en un baño de órganos y las MAPKs se localizaron mediante inmunohistoquímica.
Resultados: el LPS disminuyó las contracciones inducidas por acetilcolina. El SB203580, el SP600125 y el U0126 bloquearon los efectos del LPS sobre las contracciones inducidas por acetilcolina. La p38 y la ERK fosforiladas se detectaron en las neuronas del plexo mientérico y la p38 y la JNK fosforiladas en las células del músculo liso del íleon.
Conclusión: concluimos que la p38, la JNK y la ERK MAPKs parecen estar involucradas en el mecanismo de acción del LPS en el íleon.
Palabras clave: Contractilidad intestinal. LPS. p38. ERK. JNK. Plexo mientérico. Músculo liso.
Introduction
Gastrointestinal stasis is a common complication of sepsis (1,2). Lipopolysaccharide (LPS), an endotoxin present in the cell wall of gram negative bacteria, is widely used to reproduce sepsis symptoms. For example, LPS administration results in a decrease in contractility of ileal longitudinal smooth muscle (3).
]]> Mitogen activated protein kinases (MAPKs) are a family of protein kinases involved in many cellular programs such as cell proliferation, cell differentiation and cell death and it has been observed an abnormal activation of MAPKs in pathological circumstances such as sepsis. The most important groups of MAPKs are the p38, c-Jun-N-terminal kinase (JNK1/2) and extracellular signal-regulated kinase (ERK1/2) MAPK(4).The aims of this study were: (1) to investigate the role of MAPKs in the effect of LPS on the acetylcholine-induced contractions of rabbit ileum and (2) to study the localization of MAPKs in the ileum.
Materials and methods
Drugs and solutions
Acetylcholine (ACh) and lipopolysaccharide (LPS, from Escherichia coli serotype 0111:B4) were purchased from Sigma (Madrid, Spain). 4-[5-(4-Fluorophenyl)-2-[4-(methylsulphonyl)phenyl]-1H-imidazol-4-yl]pyridinehydrochloride (SB-203580), anthra[1-9-cd]pyrazol-6(2H)-one (SP-600125), and 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene (U-0126) were acquired from Tocris (Madrid, Spain).
Animals
All experimental protocols were approved by the Ethics Committee of the University of Zaragoza (Spain). Male New Zealand rabbits weighing 2-2.25 kg were kept with standard rabbit fodder and free access to water.
Rabbits were divided into 8 groups and were injected i.v. with either 1) saline; 2) LPS (0.2 µg kg-1); 3) SB203580 (30 µg kg-1); 4) SB203580 + LPS; 5) SP600125 (25 µg kg1); 6) SP600125 + LPS; 7) U0126 (30 µg kg-1) or U0126 + LPS. SB203580, SP600125 or U0126 were administered 15 minutes before LPS or saline. Animals were humanely killed by a blow to the head 90 minutes after LPS or saline injection.
Functional studies: muscle contractility
]]> Pieces of rabbit ileum, were removed, washed and freed from mesenteric attachment. Isometric recordings of ileal contractility were studied in an organ bath as previously described (5). Each protocol was performed on 8 segments of ileum (4 longitudinal and 4 circular muscle) taken from the same rabbit, and repeated in three or four different animals. After the equilibration period, acetylcholine (10-7 to 10-3 mol L-1) was added to the bath for 3 min in a non-cumulative manner. We increased concentrations at 20 min intervals.Immunohistochemistry
The protocol was carried out following the Vectastain ABC kit instructions (Vector Laboratories, CA, U.S.A.) as previously described (6). Sections were incubated at 4 oC overnight with mouse antibody anti-phosphorylated p38 (p-p38) (1:100), anti-phosphorylated JNK (p-JNK) (1:2000) or anti-phosphorylated ERK (p-ERK) (1:100) diluted in normal serum (10%). The p-p38, p-JNK and p-ERK antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA, SC-7973, SC-6254 and SC-7383, respectively).
Data analysis and statistics
The acetylcholine motor responses (MR) were measured and expressed as mN s-1 mm-2 as previously described (5).
Results are expressed as mean ± s.e.m. Comparisons between means were made using one-way analysis of variance (ANOVA) tests and p-values were determined using the Bonferroni's post hoc test. Differences with p-values < 0.05 were considered statistically significant.
Results
Effects of SB203580, SP600125 and U0126 on LPS altered ileal contractility
The treatment with LPS (0.2 µg kg-1, 90 min) decreased the acetylcholine induced response in longitudinal and circular muscle of rabbit ileum, as was previously reported in part by our laboratory (7). The treatment with SB203580 (Fig. 1A, B), SP600125 (Fig. 1C, D) or U0126 (Fig. 2E, F) blocked the effect of LPS on acetylcholine-induced contractions both in longitudinal and circular muscles. MAPKs inhibitors had no effect per se in the acetylcholine-induced contractions.
]]> ImmunohistochemistryThe expression of p-p38, p-JNK and p-ERK1/2 was found both in saline and LPS-treated rabbits. p-p38 was detected in the neurons of the myenteric plexus and smooth muscle cells of the ileum (Fig. 2A, B). p-JNK was detected in the smooth muscle cells (Fig. 2D, E). p-ERK was detected mainly in the neurons of the myenteric ganglia and slightly in the smooth muscle cells (Fig. 2G, H).
The specificity of the staining obtained was controlled by omitting the primary antibody, with a clear suppression of staining being observed in all cases (Fig. 2C, F, I).
Discussion
LPS is one of the most common models used for decades to study sepsis, still one of the great challenges of medicine. Previous studies of our group show that LPS alters intestinal contractility (7). In this study we demonstrate that blockade of p38, JNK1/2 or ERK1/2 pathways abolished the effect of LPS on the acetylcholine-induced contractions in rabbit ileum. We have shown similar results in rabbit duodenum (6,8,9) although there are some differences. The effect of LPS in the longitudinal layer is higher in the duodenum than in the ileum (58 vs. 43% of reduction), while the effect of LPS is similar in the circular layer of duodenum and ileum.
In this study p-p38 was detected in the neurons of the myenteric plexus and smooth muscle cells, p-JNK in the smooth muscle cells and p-ERK mainly in the neurons of the myenteric ganglia and slightly in the smooth muscle cells of ileum. In previous studies we have described a similar localization of these MAPKs in the rabbit duodenum, although the expression of p-p38 and p-ERK1/2 in the smooth muscle of duodenum was lower than in ileum (6,8,9).
In conclusion, our results suggest that p38, JNK1/2 and ERK1/2 MAPKs are involved in the effects of LPS in the ileum and, therefore, the blockade of MAPKs could be a target to avoid the intestinal motor disturbances observed in sepsis.
References
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9. Gonzalo S, Grasa L, Arruebo MP, Plaza MA, Murillo MD. Lipopolysaccharide-induced intestinal motility disturbances are mediated by c-Jun NH2-terminal kinases. Dig Liver Dis 2011;43(4):277-85. [ Links ]
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Correspondence: Received: 22-11-2011 ]]> Accepted: 03-03-2012
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