María Divina Murillo

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.

Novel Phenolic Inhibitors of Small/Intermediate-Conductance Ca2+-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 Ca2+-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.

Melatonin and Trolox ameliorate duodenal LPS-induced disturbances and oxidative stress

BACKGROUND AND AIMS Lipopolysaccharide evokes gastrointestinal motility disturbances and oxidative stress. The aims of the present study were to investigate the effect of melatonin and Trolox in the actions of lipopolysaccharide on duodenal contractility and on lipid peroxidation in rabbit duodenum. METHODS The in vitro duodenal contractility studies were carried out in organ bath and the levels of malondialdehyde were assayed by spectrophotometry. Duodenal segments were incubated with lipopolysaccharide (0.3 microg mL(-1)). RESULTS Lipopolysaccharide decreased acetylcholine-induced contractions and increased malondialdehyde and 4-hydroxyalkenals concentrations in homogenates of duodenum. Melatonin reduced the amplitude of spontaneous contractions in duodenal muscle. Acetylcholine-induced contractions were not altered by melatonin in longitudinal and circular muscles. Trolox decreased the amplitude of spontaneous contractions of duodenal muscle. Trolox (1.2 or 4 mM) did not alter acetylcholine-induced contractions in duodenal muscle, but the concentration of 12 mM diminished the frequency of contractions and acetylcholine-induced contractions. Melatonin (0.3 mM) or Trolox (4 mM) diminished malondialdehyde and 4-hydroxyalkenals levels induced by lipopolysaccharide in the duodenum. CONCLUSIONS Melatonin and Trolox reduce oxidative stress induced by lipopolysaccharide and ameliorate the effect of lipopolysaccharide on duodenal contractility.

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 Ca2+/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 Ca2+ 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.

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.

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.

Effects of 5-hydroxytryptamine agonists on myoelectric activity of the forestomach and antroduodenal area in sheep

To increase knowledge of the role of 5‐hydroxytryptamine (5‐HT) receptors in the regulation of reticuloruminal, omasal and antroduodenal myoelectric activity in sheep, the effects of 5‐HT agonists on forestomach and antroduodenal myoelectric activity have been investigated in conscious sheep. 5‐Carboxamidotryptamine, methysergide, α‐methyl‐5‐HT, 2‐methyl‐5‐HT, cisapride, zacopride or metoclopramide were infused intravenously for 5 min and myoelectric recordings were obtained from electrodes chronically implanted in the reticulum, rumen (dorsal sac), omasal body, abomasal antrum and duodenal bulb.

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‐1β, 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.

The effect of Ca2+ antagonists on spontaneous motility from sheep duodenum

Abstract— Longitudinal smooth muscle of the sheep duodenum showed a rhythmic spontaneous activity with an average frequency of 5·6 ± 0·55 phasic movements min−1 and a mean value of the amplitude of phasic contractions of 0·956 ± 0·1 g. When the strips were incubated in Ca2+‐free medium, the spontaneous motility amplitude (SMA) was reduced to 37 ± 8·2% of control values. In Ca2+‐free medium plus EDTA (1 or 2 Mm), the SMA was strongly reduced to 21·9 ± 8·3 and 1·8 ± 1·8%, respectively. Verapamil, nifedipine and diltiazem diminished the SMA. The EC50 value for verapamil was 10−9 M., whereas that for diltiazem was 2 × 10−9 M and for nifedipine was 3 × 10−14 M. Trifluoperazine and TMB‐8 reduced the SMA with EC50 values of 7 × 10−6and 3 × 10−5 m, respectively. The spontaneous activity in the sheep duodenum seemed to be mediated by influx extracellular Ca2+, which enters through potential‐dependent channels and intracellular Ca2+ release.

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.