In-vitro toxicity of carbon nanotube/polylysine colloids to colon cancer cells.

Single-walled carbon nanotubes (SWCNTs) are thoroughly purified and dispersed in an aqueous solution of high molecular weight poly-L-lysine (pLlys). Human intestinal epithelial Caco-2/TC7 cells are incubated with the SWCNT dispersions in pLlys, and their effects on cell viability are studied by image flow cytometry. No significant changes are observed in the cell culture wells up to pLlys concentrations of 10 µg ml-1. However, high mortality is detected at pLlys concentrations of 100 µg ml-1. The presence of oxygen-free SWCNTs does not modify the effects of pLlys on cell cultures at any of the tested concentrations (≤1 µg ml-1). In addition, SWCNTs having an 8 wt.% of surface oxygen are tested with identical results. Thus, purified SWCNTs, even bearing oxygen functional groups, act as inert particles in the cell culture medium. This result supports the applicability of SWCNTs as carriers in pharmacological formulations against digestive tract diseases.

Spasmolytic effect of Jasonia glutinosa on rodent intestine.

INTRODUCTION: Jasonia glutinosa is an endemic plant species of the Iberian Peninsula and Southern France traditionally used in infusions as a spasmolytic; this plant is also known as "té de roca" (rock tea) but there is no scientific evidence about the effects of this plant. AIM: To evaluate the spasmolytic effect of rock tea. METHODS: We have studied the in vitro effect of a rock tea extract on rat duodenum spontaneous contractions and the in vivo effect on mice gastrointestinal transit. RESULTS: Rock tea extract reduced the spontaneous contractions of rat duodenal smooth muscle, inhibited KCl-induced contractions and blocked the contractions invoked by both extracellular Ca2+ and the agonist of L-type calcium channels Bay K8644. This inhibitory effect was similar to the one observed after the addition of the antagonist of L-type calcium channels verapamil. Rock tea did not modify gastrointestinal transit in healthy mice. However, after the treatment with dextran sulfate sodium, an inducer of colitis, rock tea extract reverted the increase in the gastrointestinal transit associated with this treatment. CONCLUSION: Rock tea extract relaxed duodenal smooth muscle via L-type calcium channels and normalized gastrointestinal transit in a model of colitis. These results may validate the traditional use of Jasonia glutinosa in patients with gastrointestinal alterations. Thus, rock tea may be used as a spasmolytic agent to treat gastrointestinal disorders.

Antispasmodic Effects and Action Mechanism of Essential Oil of Chrysactinia mexicana A. Gray on Rabbit Ileum.

The Chrysactinia mexicana A. Gray (C. mexicana) plant is used in folk medicine to treat fever and rheumatism; it is used as a diuretic, antispasmodic; and it is used for its aphrodisiac properties. This study investigates the effects of the essential oil of C. mexicana (EOCM) on the contractility of rabbit ileum and the mechanisms of action involved. Muscle contractility studies in vitro in an organ bath to evaluate the response to EOCM were performed in the rabbit ileum. EOCM (1-100 µg·mL(-1)) reduced the amplitude and area under the curve of spontaneous contractions of the ileum. The contractions induced by carbachol 1 µM, potassium chloride (KCl) 60 mM or Bay K8644 1 µM were reduced by EOCM (30 µg·mL(-1)). Apamin 1 µM and charybdotoxin 0.01 µM decreased the inhibition induced by EOCM. The d-cAMP 1 µM decreased the inhibition induced by EOCM. l-NNA 10 µM, Rp-8-Br-PET-cGMPS 1 µM, d,l-propargylglycine 2 mM, or aminooxyacetic acid hemihydrochloride 2 mM did not modify the EOCM effect. In conclusion, EOCM induces an antispasmodic effect and could be used in the treatment of intestinal spasms or diarrhea processes. This effect would be mediated by Ca(2+), Ca(2+)-activated K⁺ channels and cAMP.

Rock Tea extract (Jasonia glutinosa) relaxes rat aortic smooth muscle by inhibition of L-type Ca2+ channels

In traditional herbal medicine, Rock Tea (Jasonia glutinosa) is known for its prophylactic and therapeutic value in various disorders including arterial hypertension. However, the mechanism by which Rock Tea exerts blood pressure-lowering actions has not been elucidated yet. Our aim was to demonstrate vasorelaxing effects of Rock Tea extract and to reveal its possible action mechanism. Isometric myography was conducted on high-K+-precontracted rings from rat thoracic aorta and tested extracts at concentrations of 0.5-5 mg/ml. Whole-cell patch-clamp experiments were performed in rat aortic vascular smooth muscle cells (line A7r5) to determine blocking effects on L-type Ca2+ channels. Rock Tea extract relaxed the aorta contracted by high [K+] concentration dependently with an EC50 of ≈2.4 mg/ml and produced ≈75 % relaxation at the highest concentration tested. The L-type Ca2+ channel blocker, verapamil (10−6 M), had similar effects. Rock Tea extract had no effect in nominally Ca2+-free high-K+ buffer but significantly inhibited contractions to re-addition of Ca2+. Rock Tea extract inhibited the contractions induced by the L-type Ca2+ channel activator Bay K 8644 (10−5 M) and by phenylephrine (10−6 M). Rock Tea extract and Y-27632 (10−6 M), Rho-kinase inhibitor, had similar effects and the respective effects were not additive. Patch-clamp experiments demonstrated that Rock Tea extract (2.5 mg/ml) virtually abolished L-type Ca2+ currents in A7r5. We conclude that Rock Tea extract produced vasorelaxation of rat aorta and that this relaxant effect is mediated by inhibition of L-type Ca2+ channels. Rock Tea extracts may be of phytomedicinal value for prevention and adjuvant treatment of hypertension and other cardiovascular diseases

Neomycin and bacitracin reduce the intestinal permeability in mice and increase the expression of some tight-junction proteins.

BACKGROUND: Tight-junction (TJ) proteins regulate paracellular permeability. Gut permeability can be modulated by commensal microbiota. Manipulation of the gut microbiota with antibiotics like bacitracin and neomycin turned out to be useful for the treatment of diarrhoea induced by Clostridium difficile or chemotherapy drugs. AIM: To evaluate the effects of the microbiota depletion evoked by the oral administration of neomycin and bacitracin on the intestinal permeability and expression of TJ proteins in mice. METHODS: Mice received neomycin and bacitracin orally for 7 days. Intestinal permeability was measured by the fluorescein-isothiocyanate-dextran (FITC-dextran) method. The gene expression of TJ proteins in the intestine was determined by real time-PCR. RESULTS: FITC-dextran levels in serum were reduced by half in antibiotic-treated mice, indicating a reduction of intestinal permeability. Antibiotics increased the expression of zonula occludens 1 (ZO-1), junctional adhesion molecule A (JAM-A, and occludin in the ileum and ZO-1, claudin-3, and claudin-4 in the colon. CONCLUSION: The combination of neomycin and bacitracin reduce intestinal permeability and increase the gene expression of ZO-1, junctional adhesion molecule A (JAM-A), and occludin in the ileum and ZO-1, claudin-3, and claudin-4 in the colon.

La neomicina y bacitracina disminuyen la permeabilidad intestinal en ratones e incrementan la expresión de algunas proteínas de unión estrecha / Neomycin and bacitracin reduce the intestinal permeability in mice and increase the expression of some tight-junction proteins

ANTECEDENTES: las proteínas de unión estrecha (UE) regulan la permeabilidad paracelular. La permeabilidad intestinal puede estar modulada por la microbiota comensal. Las manipulaciones de la microbiota intestinal con antibióticos como la bacitracina y neomicina han resultado ser útiles para el tratamiento de la diarrea inducida por Clostridium difficile o los fármacos quimioterápicos. OBJETIVOS: evaluar los efectos de la depleción de la microbiota mediante la administración oral de bacitracina y neomicina sobre la permeabilidad intestinal y la expresión de las proteínas de UE en ratón. MÉTODOS: los ratones recibieron por vía oral la combinación de neomicina y bacitracina durante 7 días. La permeabilidad intestinal se cuantificó con el método del dextrano marcado con isotiocianato de fluoresceína (FITC-dextrano). La expresión de las proteínas de UE en el intestino se determinó mediante PCR a tiempo real. RESULTADOS: los niveles de FITC-dextrano en suero se redujeron a la mitad en los ratones tratados con antibióticos, indicando una reducción de la permeabilidad intestinal. Los antibióticos incrementaron la expresión de zónula occludens 1 (ZO- 1), molécula de adhesión de unión A (JAM-A) y ocludina en íleon y de ZO-1, claudina-3 y claudina-4 en colon. CONCLUSIONES: la combinación de neomicina y bacitracina reduce la permeabilidad intestinal e incrementa la expresión de ZO-1, JAM-A y ocludina en íleon y ZO-1, claudina-3 y claudina-4 en colon

BACKGROUND: Tight-junction (TJ) proteins regulate paracellular permeability. Gut permeability can be modulated by commensal microbiota. Manipulation of the gut microbiota with antibiotics like bacitracin and neomycin turned out to be useful for the treatment of diarrhoea induced by Clostridium difficile or chemotherapy drugs. AIM: To evaluate the effects of the microbiota depletion evoked by the oral administration of neomycin and bacitracin on the intestinal permeability and expression of TJ proteins in mice. METHODS: Mice received neomycin and bacitracin orally for 7 days. Intestinal permeability was measured by the fluorescein-isothiocyanate-dextran (FITC-dextran) method. The gene expression of TJ proteins in the intestine was determined by real time-PCR. RESULTS: FITC-dextran levels in serum were reduced by half in antibiotic-treated mice, indicating a reduction of intestinal permeability. Antibiotics increased the expression of zonula occludens 1 (ZO-1), junctional adhesion molecule A (JAM-A, and occludin in the ileum and ZO-1, claudin-3, and claudin-4 in the colon. CONCLUSION: The combination of neomycin and bacitracin reduce intestinal permeability and increase the gene expression of ZO-1, junctional adhesion molecule A (JAM-A), and occludin in the ileum and ZO-1, claudin-3, and claudin-4 in the colon

Rock Tea extract (Jasonia glutinosa) relaxes rat aortic smooth muscle by inhibition of L-type Ca(2+) channels.

In traditional herbal medicine, Rock Tea (Jasonia glutinosa) is known for its prophylactic and therapeutic value in various disorders including arterial hypertension. However, the mechanism by which Rock Tea exerts blood pressure-lowering actions has not been elucidated yet. Our aim was to demonstrate vasorelaxing effects of Rock Tea extract and to reveal its possible action mechanism. Isometric myography was conducted on high-K+-precontracted rings from rat thoracic aorta and tested extracts at concentrations of 0.5-5 mg/ml. Whole-cell patch-clamp experiments were performed in rat aortic vascular smooth muscle cells (line A7r5) to determine blocking effects on L-type Ca(2+) channels. Rock Tea extract relaxed the aorta contracted by high [K+] concentration dependently with an EC50 of ≈2.4 mg/ml and produced ≈75 % relaxation at the highest concentration tested. The L-type Ca(2+) channel blocker, verapamil (10(-6) M), had similar effects. Rock Tea extract had no effect in nominally Ca(2+)-free high-K(+) buffer but significantly inhibited contractions to re-addition of Ca(2+). Rock Tea extract inhibited the contractions induced by the L-type Ca(2+) channel activator Bay K 8644 (10(-5) M) and by phenylephrine (10(-6) M). Rock Tea extract and Y-27632 (10(-6) M), Rho-kinase inhibitor, had similar effects and the respective effects were not additive. Patch-clamp experiments demonstrated that Rock Tea extract (2.5 mg/ml) virtually abolished L-type Ca(2+) currents in A7r5. We conclude that Rock Tea extract produced vasorelaxation of rat aorta and that this relaxant effect is mediated by inhibition of L-type Ca(2+) channels. Rock Tea extracts may be of phytomedicinal value for prevention and adjuvant treatment of hypertension and other cardiovascular diseases.

Different mechanisms of actions of genistein, quercetin on spontaneous contractions of rabbit duodenum.

Flavonoids are known to relax precontracted intestinal smooth muscle and delay intestinal transit or intestinal peristalsis. The aim of this study was to determine the effects of genistein and quercetin on spontaneous contractions of rabbit duodenum in vitro in an organ bath. Genistein and quercetin (0.1-10µM) reduced the amplitude of spontaneous contractions in the longitudinal and circular smooth muscle of rabbit duodenum, but they did not modify the frequency. Bay K8644 (L-type Ca2+ channel activator), apamin, charybdotoxin, and tetraetylammonium (K+ channel blockers) reverted the inhibition of amplitude of spontaneous contractions induced by genistein in longitudinal and circular smooth muscle. H-89 (protein kinase A inhibitor) antagonized the reduction of the amplitude of spontaneous contractions induced by quercetin in longitudinal and circular smooth muscle of duodenum, while 2,5-dideoxiadenosine (adenylyl cyclase inhibitor) reverted only the reduction of the amplitude in circular smooth muscle. In conclusion, genistein and quercetin reduce the spontaneous contractions in the duodenum by different mechanisms of actions. The effect of genistein would be mediated by Ca2+ and K+ channels, while the effect of quercetin would be mediated by cAMP and protein kinase A.

Antibiotic-Induced Depletion of Murine Microbiota Induces Mild Inflammation and Changes in Toll-Like Receptor Patterns and Intestinal Motility.

We examine the impact of changes in microbiota induced by antibiotics on intestinal motility, gut inflammatory response, and the function and expression of toll-like receptors (TLRs). Alterations in mice intestinal microbiota were induced by antibiotics and evaluated by q-PCR and DGGE analysis. Macroscopic and microscopic assessments of the intestine were performed in control and antibiotic-treated mice. TLR expression was determined in the intestine by q-RT-PCR. Fecal parameter measurements, intestinal transit, and muscle contractility studies were performed to evaluate alterations in intestinal motility. Antibiotics reduced the total bacterial quantity 1000-fold, and diversity was highly affected by treatment. Mice with microbiota depletion had less Peyer's patches, enlarged ceca, and mild gut inflammation. Treatment with antibiotics increased the expression of TLR4, TLR5, and TLR9 in the ileum and TLR3, TLR4, TLR6, TLR7, and TLR8 in the colon, and it reduced the expression of TLR2, TLR3, and TLR6 in the ileum and TLR2 and TLR9 in the colon. Antibiotics decreased fecal output, delayed the whole gut and colonic transit, and reduced the spontaneous contractions and the response to acetylcholine (ACh) in the ileum and colon. Activation of TLR4 by lipopolysaccharide (LPS) reverted the reduction of the spontaneous contractions induced by antibiotics in the ileum. Activation of TLR4 by LPS and TLR5 by flagellin reduced the response to ACh in the ileum in control mice. Our results confirm the role of the microbiota in the regulation of TLRs expression and shed light on the microbiota connection to motor intestinal alterations.

A novel pan-negative-gating modulator of KCa2/3 channels, fluoro-di-benzoate, RA-2, inhibits endothelium-derived hyperpolarization-type relaxation in coronary artery and produces bradycardia in vivo.

Small/intermediate conductance KCa channels (KCa2/3) are Ca(2+)/calmodulin regulated K(+) channels that produce membrane hyperpolarization and shape neurologic, epithelial, cardiovascular, and immunologic functions. Moreover, they emerged as therapeutic targets to treat cardiovascular disease, chronic inflammation, and some cancers. Here, we aimed to generate a new pharmacophore for negative-gating modulation of KCa2/3 channels. We synthesized a series of mono- and dibenzoates and identified three dibenzoates [1,3-phenylenebis(methylene) bis(3-fluoro-4-hydroxybenzoate) (RA-2), 1,2-phenylenebis(methylene) bis(3-fluoro-4-hydroxybenzoate), and 1,4-phenylenebis(methylene) bis(3-fluoro-4-hydroxybenzoate)] with inhibitory efficacy as determined by patch clamp. Among them, RA-2 was the most drug-like and inhibited human KCa3.1 with an IC50 of 17 nM and all three human KCa2 subtypes with similar potencies. RA-2 at 100 nM right-shifted the KCa3.1 concentration-response curve for Ca(2+) activation. The positive-gating modulator naphtho[1,2-d]thiazol-2-ylamine (SKA-31) reversed channel inhibition at nanomolar RA-2 concentrations. RA-2 had no considerable blocking effects on distantly related large-conductance KCa1.1, Kv1.2/1.3, Kv7.4, hERG, or inwardly rectifying K(+) channels. In isometric myography on porcine coronary arteries, RA-2 inhibited bradykinin-induced endothelium-derived hyperpolarization (EDH)-type relaxation in U46619-precontracted rings. Blood pressure telemetry in mice showed that intraperitoneal application of RA-2 (≤100 mg/kg) did not increase blood pressure or cause gross behavioral deficits. However, RA-2 decreased heart rate by ≈145 beats per minute, which was not seen in KCa3.1(-/-) mice. In conclusion, we identified the KCa2/3-negative-gating modulator, RA-2, as a new pharmacophore with nanomolar potency. RA-2 may be of use to generate structurally new types of negative-gating modulators that could help to define the physiologic and pathomechanistic roles of KCa2/3 in the vasculature, central nervous system, and during inflammation in vivo.

Roles of Toll-Like Receptor 4, IκB Kinase, and the Proteasome in the Intestinal Alterations Caused by Sepsis.

BACKGROUND: Lipopolysaccharide decreases intestinal contractility and induces the production of cytokines, which play an important role in the pathogenesis of sepsis. AIM: The objective of the present study was to examine the role of Toll-like receptor 4, IκB kinase, and the proteasome in the intestinal alterations induced by lipopolysaccharide. METHODS: Sepsis was induced in rabbits by intravenous injection of lipopolysaccharide. Contractility studies of rabbit duodenum were performed in an organ bath. Expressions of interleukin-1ß, interleukin-6, interleukin-8, interleukin-10, IκB kinase-α, IκB kinase-ß, IκB kinase-γ, and the proteasome mRNA were determined by RT-PCR on rabbit duodenum. RESULTS: Neomycin and polymyxin B (Toll-like receptor 4 inhibitors), IKK NBD peptide (IκB kinase complex inhibitor), and MG-132 (proteasome inhibitor) blocked partially the effects of lipopolysaccharide on the acetylcholine-, prostaglandin E2-, substance P-, and KCl-induced contractions in the longitudinal and circular smooth muscle of rabbit duodenum. Lipopolysaccharide increased the mRNA expression of interleukin-6 and interleukin-8 in duodenal tissue, and this effect was partly reversed by neomycin, polymyxin B, IKK NBD peptide, and MG-132. IκB kinase-α, IκB kinase-ß, IκB kinase-γ, and the proteasome mRNA expressions was not affected by lipopolysaccharide treatment. CONCLUSIONS: Toll-like receptor 4, the IκB kinase complex, and the proteasome could be therapeutic targets in the treatment of sepsis symptoms in the intestine.

Single-walled carbon nanotubes (SWCNTs) enhance KCl-, acetylcholine-, and serotonin-induced contractions and evoke oxidative stress on rabbit ileum.

We examined the effects of intravenous administration of purified arc-discharge single-walled carbon nanotubes (SWCNTs) on rabbit ileum to establish the possibility of using these SWCNTs as cell markers or drug carriers for the treatment of intestinal diseases. The SWCNT purification process eliminated carbonaceous impurities and decreased the amount of metals. SWCNTs increased the contractile responses induced by KCl, acetylcholine (ACh), and serotonin (5-HT) in rabbit ileum. Verapamil, apamin, glibenclamide, quinine and charybdotoxin reduced the contractile responses induced by ACh and 5-HT in ileum from rabbits treated with SWCNTs, indicating that voltage-dependent Ca2+ channels and small, intermediate, and large-conductance Ca(2+)-activated, ATP-sensitive, and voltage-dependent K+ channels are involved in these effects. Atropine and hexamethonium reduced the ACh response, indicating that muscarinic and nicotinic receptors are involved in this effect. Ondansetron and GR 113808 reduced the 5-HT response, indicating that serotonin 5-HT3 and 5-HT4 receptors are involved in this effect. SWCNTs increased the malondialdehyde plus 4-hydroxyalkenals and carbonyl levels in rabbit plasma and ileum, indicating that SWCNTs produce oxidative stress. SWCNTs did not produce relevant histological changes or modify the levels of the inflammatory mediators iNOS and COX-2 in the ileum. In conclusion, this study demonstrates that the intravenous administration of SWCNTs can evoke oxidative stress and affect contractility in rabbit ileum. These effects could reduce the possibility of using the arc-discharge SWCNTs as cell markers or drug carriers to treat intestinal diseases.

Novel phenolic inhibitors of small/intermediate-conductance Ca²âº-activated K⁺ channels, KCa3.1 and KCa2.3.

BACKGROUND: KCa3.1 channels are calcium/calmodulin-regulated voltage-independent K(+) channels that produce membrane hyperpolarization and shape Ca(2+)-signaling and thereby physiological functions in epithelia, blood vessels, and white and red blood cells. Up-regulation of KCa3.1 is evident in fibrotic and inflamed tissues and some tumors rendering the channel a potential drug target. In the present study, we searched for novel potent small molecule inhibitors of KCa3.1 by testing a series of 20 selected natural and synthetic (poly)phenols, synthetic benzoic acids, and non-steroidal anti-inflammatory drugs (NSAIDs), with known cytoprotective, anti-inflammatory, and/or cytostatic activities. METHODOLOGY/PRINCIPAL FINDINGS: In electrophysiological experiments, we identified the natural phenols, caffeic acid (EC50 1.3 µM) and resveratrol (EC50 10 µM) as KCa3.1 inhibitors with moderate potency. The phenols, vanillic acid, gallic acid, and hydroxytyrosol had weak or no blocking effects. Out of the NSAIDs, flufenamic acid was moderately potent (EC50 1.6 µM), followed by mesalamine (EC50≥10 µM). The synthetic fluoro-trivanillic ester, 13b ([3,5-bis[(3-fluoro-4-hydroxy-benzoyl)oxymethyl]phenyl]methyl 3-fluoro-4-hydroxy-benzoate), was identified as a potent mixed KCa2/3 channel inhibitor with an EC50 of 19 nM for KCa3.1 and 360 pM for KCa2.3, which affected KCa1.1 and Kv channels only at micromolar concentrations. The KCa3.1/KCa2-activator SKA-31 antagonized the 13b-blockade. In proliferation assays, 13b was not cytotoxic and reduced proliferation of 3T3 fibroblasts as well as caffeic acid. In isometric vessel myography, 13b increased contractions of porcine coronary arteries to serotonin and antagonized endothelium-derived hyperpolarization-mediated vasorelaxation to pharmacological KCa3.1/KCa2.3 activation. CONCLUSIONS/SIGNIFICANCE: We identified the natural phenols, caffeic acid and resveratrol, the NSAID, flufenamic acid, and the polyphenol 13b as novel KCa3.1 inhibitors. The high potency of 13b with pan-activity on KCa3.1/KCa2 channels makes 13b a new pharmacological tool to manipulate inflammation and cancer growth through KCa3.1/KCa2 blockade and a promising template for new drug design.

Mitogen activated protein kinases blockade improves lipopolysaccharide-induced ileal motor disturbances.

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.

El bloqueo de las proteínas cinasas activadas por mitógenos mejora las alteraciones motoras inducidas por el lipopolisacárido en íleon / Mitogen activated protein kinases blockade improves lipopolysaccharide-induced ileal motor disturbances

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(AU)

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 therole 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(AU)

Nuclear factor κB is a key transcription factor in the duodenal contractility alterations induced by lipopolysaccharide.

Alterations in intestinal motility are one of the features of sepsis induced by lipopolysaccharide (LPS). This study investigated the role of the nuclear transcription factor κB (NF-κB) in the LPS-induced duodenal contractility alterations, generation of reactive oxygen species (ROS) and production of cytokines in rabbit duodenum. Rabbits were treated with saline, LPS, sulfasalazine + LPS, pyrrolidinedithiocarbamate (PDTC) + LPS or RO 106-9920 + LPS. Contractility studies were performed in an organ bath. The formation of products of oxidative damage to proteins (carbonyls) and lipids (malondialdehyde and 4-hydroxyalkenals) was quantified in intestinal tissue and plasma. The protein expression of NF-κB was measured by Western blot. The DNA binding activity of NF-κB was evaluated by transcription factor activity assay. The expression of interleukin-1ß, tumour necrosis factor α (TNF-α), interleukin-6, interleukin-10 and interleukin-8 mRNA was determined by RT-PCR. Sulfasalazine, PDTC and RO 106-9920 blocked the inhibitory effect of LPS on contractions induced by ACh in the longitudinal smooth muscle of rabbit duodenum. Sulfasalazine, PDTC and RO 106-9920 reduced the increased levels of malondialdehyde and 4-hydroxyalkenals and the carbonyls induced by LPS in plasma. Lipopolysaccharide induced the activation, translocation to the nucleus and DNA binding of NF-κB. Lipopolysaccharide increased the mRNA expression of interleukin-6 and TNF-α in duodenal tissue, and this effect was partly reversed by PDTC, sulfasalazine and RO 106-9920. In conclusion, NF-κB mediates duodenal contractility disturbances, the generation of ROS and the increase in the expression of interleukin-6 and TNF-α induced by LPS. Sulfasalazine, PDTC and RO 106-9920 may be therapeutic drugs to reduce these effects.

Role of TLR4 and MAPK in the local effect of LPS on intestinal contractility.

OBJECTIVES: Lipopolysaccharide (LPS) has been shown to alter intestinal contractility. Toll-like receptor 4 (TLR4), K(+) channels and mitogen-activated protein kinases (MAPKs) have been proposed to be involved in the mechanism of action of LPS. The aim of this study was to determine the role of TLR4, K(+) channels and MAPKs (p38, JNK and MEK1/2) in the local effect of LPS on the acetylcholine (ACh)-induced contractions in rabbit small intestine in vitro. METHODS: Segments of rabbit duodenum were suspended in the direction of longitudinal or circular smooth muscle fibres in a thermostatically controlled organ bath. KEY FINDINGS: LPS (0.3 µg/ml) reduced the contractions induced by ACh (100 µm) in the longitudinal and circular smooth muscle of the duodenum after 90 min of incubation. Polymyxin (TLR4 inhibitor), SB203580 (p38 MAPK inhibitor), SP600125 (JNK1/2 inhibitor) and U0126 (MEK1/2 inhibitor) antagonized the effects of the LPS on ACh-induced contractions in duodenal smooth muscle. Incubation with the blockers of K(+) channels, TEA, apamin, charybdotoxin, iberiotoxin, glibenclamide or quinine, did not reverse the effect of LPS on ACh-induced contractions. CONCLUSIONS: These results suggest that the effect of LPS on ACh-induced contractions in the rabbit duodenum might be mediated by TLR4 and p38, JNK1/2 and MEK1/2 MAPKs.

Contractile effect of tachykinins on rabbit small intestine.

AIM: To study the role of the tachykinin receptors in spontaneous contractions of longitudinal and circular smooth muscle from rabbit small intestine and to determine the mechanism of action of Substance P (SP). METHODS: Rabbit duodenum, jejunum and ileum segments were prepared. The spontaneous contractions of longitudinal and circular smooth muscle were recorded using a computer via an isometric force transducer. The specific agonists and antagonists of tachykinin receptors were added into the organ bath. RESULTS: The agonists of tachykinin NK1 receptor (SP and [Sar9] SP), NK2 receptor (NKA and (ß-Ala8)-NKA), and NK3 receptor (NKB and Senktide) all induced contractions in the small intestine. The contractions were diminished by NK1 receptor antagonist L-733,060, NK2 receptor antagonist GR-94800, and NK3 receptor antagonist SB 218795. Contractions caused by SP were also reduced by atropine, verapamil, PKC inhibitor staurosporine, and PLC inhibitor U73122. CONCLUSION: Ttachykinin NK1, NK2, and NK3 receptors mediate the contractions of the smooth muscle in rabbit intestine. Furthermore, SP acts directly on smooth muscle cells through the tachykinin NK1 receptor.

Lipopolysaccharide-induced intestinal motility disturbances are mediated by c-Jun NH2-terminal kinases.

BACKGROUND: Lipopolysaccharide (LPS) is a causative agent of sepsis. Many alterations, such as intestinal motility disturbances, have been attributed to LPS. AIMS: Here we investigated the role of c-Jun NH(2)-terminal kinases (JNK) in the effect of LPS on intestinal motility, the oxidative stress status and the cyclooxygenese-2 (COX-2) expression. METHODS: Rabbits were injected with either (1) saline, (2) LPS, (3) SP600125, a specific JNK inhibitor, or (4) SP600125+LPS. Duodenal contractility was studied in an organ bath. The formation of products of oxidative damage to proteins (carbonyls) and lipids [malondialdehyde (MDA) and 4-hydroxyalkenals (4-HDA)] was quantified by spectrophotometry in the intestine and plasma. The protein expression of p-JNK, total JNK, and COX-2 was measured by Western blot, and p-JNK was localized by immunohistochemistry. RESULTS: LPS decreased the contractions evoked by acetylcholine and prostaglandin E(2) and KCl-induced contractions. LPS increased phospho-JNK and COX-2 expressions and the levels of carbonyls and MDA+4-HDA. SP600125 blocked the effect of LPS on the acetylcholine, prostaglandin E(2), and KCl-induced contractions, the levels of carbonyls and MDA+4-HDA, and the p-JNK and COX-2 expressions. p-JNK was detected in the smooth muscle cells of duodenum. CONCLUSION: Our results suggest that JNK is involved in the mechanism of action of LPS in the intestine.

Intestinal effects of lipopolysaccharide in rabbit are mediated by cyclooxygenase-2 through p38 mitogen activated protein kinase.

The mediators of the pathophysiological symptoms of septic shock are not completely understood. The intracellular signalling mechanisms of lipopolysaccharide (LPS)-induced effects need further investigation. This study investigates (1) the role of COX-2 in the effect of LPS on (a) the KCl, acetylcholine and prostaglandin E2-induced contractions of rabbit duodenum and (b) the oxidative stress status in plasma and intestine and (2) the relationship between p38 MAPK and COX-2 expression in rabbit duodenum. Rabbits were injected i.v. with either (1) saline, (2) LPS, (3) SB203580, a p38 MAPK inhibitor, (4) SB203580+LPS, (5) NS-398, a COX-2 inhibitor or (6) NS-398+LPS. Contractility studies were performed by suspending pieces of duodenum in an organ bath in the direction of longitudinal and circular smooth muscle fibres. The formation of products of oxidative damage to proteins (carbonyls) and lipids [malondialdehyde (MDA) and 4-hydroxyalkenals (4-HDA)] was quantified in intestinal tissue and plasma. The protein expression of COX-2 was measured by western blot. The KCl, acetylcholine and prostaglandin E2-induced contractions decreased with LPS. In addition, LPS increased the levels of carbonyls and MDA+4-HDA in plasma and duodenum as well as COX-2 expression in duodenal tissue. All these effects were blocked by NS-398. The p38 MAPK inhibitor SB203580 blocked the effect of LPS on COX-2 expression. These results suggest that the effect of LPS on KCl, acetylcholine and prostaglandin E2-induced contractions in the rabbit duodenum and oxidative stress might be mediated by an increase in COX-2 expression through the p38 MAPK pathway.