Miguel Ángel Plaza

Effect of lipopolysaccharide on rabbit small intestine muscle contractility in vitro: role of prostaglandins

Abstract The purpose of this study was to investigate the effect of lipopolysaccharide (LPS) on spontaneous contractions and acetylcholine (ACh) induced contractions of rabbit intestinal segments in vitro, with two different protocols: intestinal segments isolated from LPS‐treated rabbits and intestinal segments incubated with LPS. The frequency of spontaneous movements decreased significantly in LPS‐treated rabbits at 2 μg kg−1 in the duodenum and 20 μg kg−1 in the duodenum, jejunum and ileum. LPS (0.2 μg kg−1) reduced significantly the ACh contractions (10−6 mol L−1) in the duodenum (61%), jejunum (48%) and ileum (21%). Indomethacin (1, 5 and 10 mg kg−1) administered 15 min before LPS (0.2 μg kg−1) antagonized the LPS effects on the ACh‐induced contractions. Prostaglandin (PG)E2 (8 μg kg−1) inhibited significantly the frequency of spontaneous contractions in the ileum and reduced the ACh‐induced contractions in the three segments, mimicking the LPS effects. The amplitude and frequency of contractions in rabbit intestinal segments previously incubated with LPS (0.03, 0.3, 3 and 30 μg mL−1) were not modified with respect to the control. The ACh‐induced contractions (10−4 mol L−1) were significantly reduced after 90 min of incubation with LPS. The inhibition of LPS (0.3 μg mL−1) was 43% in the duodenum, 35% in the jejunum and 17% in the ileum. Indomethacin added before LPS blocked the effect of LPS on the ACh‐induced contractions in the duodenum, jejunum and ileum. These results show that LPS decreases intestinal contractility in rabbits and suggest that PGs are implicated in these actions.

Role of Central Interleukin-1β in Gastrointestinal Motor Disturbances Induced by Lipopolysaccharide in Sheep

Cytokines are involved in the symptoms of theacute phase response induced by infectious diseases inhumans as well as in animals, and interleukin-1β(IL-1 ) has a pivotal role in these changes. The role of central IL-1β in the gastrointestinalhypomotility and fever evoked by intravenousadministration of lipopolysaccharide (LPS) and themechanisms involved, were investigated in sheep as anexperimental model. LPS (0.1 μg/kg, intravenously)induced gastrointestinal hypomotility and fever thatwere significantly reduced by priorintracerebroventricular administration of IL-1βreceptor antagonist protein (IL-1ra, 2 μg/kg). The effects of LPS were mimickedby intracerebroventricular IL-1β (50 ng/kg),whereas IL-1β injected intravenously at the samedose only caused a slight and transient fever withoutmodifying the gastrointestinal motility. Priorintracerebroventricular administration of thecyclooxygenase inhibitor indomethacin (100 μg/kg) butnot the corticotropin-releasing factor (CRF) receptorantagonist α-helical CRF9-41 (5 μg/kg) blocked alleffects evoked by both LPS and IL-1β. These resultssuggest that in sheep, LPS induces digestive motordisturbances through a central release of IL-1β andprostaglandins.

Inhibition of p38 MAPK improves intestinal disturbances and oxidative stress induced in a rabbit endotoxemia model

Background  Lipopolysaccharide (LPS) decreases intestinal contractility and induces the release of reactive oxygen species, which play an important role in the pathogenesis of sepsis. p38 mitogen‐activated protein kinase (MAPK) can be activated by a variety of stimuli such as LPS. The aims of this study were: (i) to investigate the role of p38 MAPK in the effect of LPS on (a) the acetylcholine, prostaglandin E2 and KCl‐induced contractions of rabbit duodenum and (b) the oxidative stress status; (ii) to localize the active form of p38 in the intestine.

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.

Central tumour necrosis factor-alpha mediates the early gastrointestinal motor disturbances induced by lipopolysaccharide in sheep.

Cytokines are involved in fever and other symptoms of the acute phase response induced by endotoxins. The aim of this work was to study the involvement of central tumour necrosis factor‐α (TNF‐α) in the changes induced by lipopolysaccharide (LPS) on gastrointestinal (GI) motility in sheep. Body temperature and myoelectric activity of the antrum, duodenum and jejunum was recorded continuously. Intravenous (i.v.) administration of LPS (0.1 μg kg−1)‐induced hyperthermia, decreased gastrointestinal myoelectric activity and increased the frequency of the migrating motor complex (MMC). These effects started 40–50 min after LPS and lasted for 6–7 h. TNF‐α (50 and 100 ng kg−1) mimicked these effects when injected intracerebroventricularly (i.c.v.) but not i.v. Pretreatment with soluble recombinant TNF receptor (TNFR:Fc, 10 μg kg−1, i.c.v.) abolished the TNF‐induced actions and reduced those evoked by LPS. Furthermore, the effects induced by either LPS or TNF were suppressed by prior i.c.v. injection of indomethacin (100 μg kg−1). In contrast, the i.v. injections of TNFR:Fc or indomethacin were ineffective. Our data suggest that LPS disturbs GI motility in sheep through a central pathway that involves TNF‐α and prostaglandins sequentially.

5-Hydroxytryptamine induces forestomach hypomotility in sheep through 5-HT4 receptors

The effects evoked by 5‐hydroxytryptamine (5‐HT; serotonin) on forestomach myoelectric activity were investigated in conscious sheep. Myoelectric signals were recorded with electrodes chronically implanted in the reticulum, rumen (dorsal sac) and omasal body, and were analysed by a computer‐based method. The 5‐HT receptors and the neuronal pathways involved in these actions were studied. The intravenous (i.v.) infusion of 5‐HT (8 micrograms kg‐1 min‐1 for 5 min) evoked an inhibition of activity of the whole forestomach. Methiothepin, injected i.v. at 0.1 mg kg‐1, inhibited rumen secondary contractions and omasum activity. However, forestomach activity remained unchanged after the administration of 0.2 mg kg‐1 of ketanserin, ondansetron, tropisetron, GR‐113808, phentolamine, propranolol, domperidone and naloxone. Atropine (0.2 mg kg‐1), hexamethonium (2 mg kg‐1) or haloperidol (0.1 mg kg‐1) abolished rumen secondary cycles and inhibited omasum activity. In addition, atropine also suppressed primary cycles. GR‐113808 blocked all 5‐HT‐induced effects. Furthermore, atropine or hexamethonium prevented the 5‐HT‐evoked inhibition of reticulorumen primary cycles. In contrast, the remaining antagonists did not alter the 5‐HT‐evoked forestomach hypomotility. In conclusion, 5‐HT induces inhibition of forestomach myoelectric activity through 5‐HT4 receptors, these actions being mediated by cholinergic neural pathways involving muscarinic and nicotinic receptors. However, adrenergic, dopaminergic or opiate pathways are not implicated.

K+ channels involved in contractility of rabbit small intestine

Most excitable cells, including gastrointestinal smooth muscle cells, express several types of K+ channels. The aim of this study was to examine the types of K+ channels involved in the contractility of longitudinal smooth muscle of rabbit small intestinein vitro. Spontaneous contractions and KCl-stimulated contractions were reduced by atropine, phentolamine, propranolol, suramin, tetrodotoxin and indomethacin. The amplitude and tone of spontaneous contractions were increased by apamin, charybdotoxin, iberiotoxin, E4031, tetraetylammonium (TEA) and BaCl2. The frequency of contractions was reduced in the presence of apamin and TEA and increased by charybdotoxin. It was found that 4-aminopyridine increased the tone of spontaneous contractions and reduced the amplitude and frequency of contractions. Glibenclamide did not modify the amplitude, frequency or tone of contractions. KCl-stimulated contractions were increased by E4031, were not modified by apamin, glibenclamide, NS1619 or diazoxide, and were reduced by charybdotoxin, TEA, 4-aminopyridine or BaCl2. These results suggest that both Ca2+-activated K+ channels of small and high conductance, and HERG K+ channels and inward rectifier K+ channels participate in spontaneous contractions of small intestine. On the other hand, voltage-dependent K+ channels, HERG K+ channels, inward rectifier K+ channels and high conductance Ca2+-activated K+ channels are involved in KCl-stimulated contractions.ResumenLa mayoría de las células excitables, incluyendo las células lisas gastrointestinales, expresan varios tipos de canales de K+. El objetivo de este estudio es examinar los tipos de canales de K+ que están involucrados en la contractilidad del músculo liso longitudinal del intestino delgado de conejoin vitro. Las contracciones espontáneas y las producidas por KCl se redujeron por atropina, fentolamina, propranolol, suramina, tetrodotoxina e indometacina. La amplitud y tono de las contracciones espontáneas aumentaron por apamin, charybdotoxina, iberiotoxina, E4031, tetraetilamonio (TEA) y BaCl2, mientras que la frecuencia de las contracciones se redujo en presencia de apamin, charybdotoxina y TEA. La 4-aminopiridina aumentó el tono de las contracciones espontáneas y redujo la amplitud y frecuencia de las contracciones. La glibenclamida no modificó la amplitud, frecuencia y tono de las contracciones. Las contracciones producidas por el KCl aumentaron en presencia de E4031, no fueron modificadas por el apamin, glibenclamida, NS1619 o diazóxida y disminuyeron en presencia de la charybdotoxina, TEA, 4-aminopiridina o BaCl2. Estos resultados sugieren que los canales de K+ activados por Ca2+ de pequeña y gran conductancia, canales de K+ HERG canales de K+ rectificadores de entrada participan en las contracciones espontáneas del intestino delgado. Por otra parte, los canales de K+ voltaje-dependientes, canales de K+ HERG, canales de K+ rectificadores de entrada y canales de K+ activados por Ca2+ de gran conductancia están implicados en las contracciones producidas por el KCl.

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.

Involvement of neuronal nitric oxide synthase (nNOS) in the regulation of migrating motor complex (MMC) in sheep.

The objectives of this study were to evaluate the role of nitric oxide (NO) synthase isoforms (nNOS, eNOS, and iNOS) in the regulation of the migrating motor complex (MMC) in sheep using electromyography and their expression in the gastrointestinal (GI) tract by Western blot (WB) and immunohistochemistry. Intravenous administration of L-NAME or the nNOS inhibitor 7-nitroindazole (7-NI) decreased the MMC interval. Myoelectric activity of intestinal phase II was increased, whereas antral activity was reduced. These effects were blocked by L-arginine. Inhibitors of either iNOS (aminoguanidine and S-methylisothiourea) or eNOS (L-NIO) were ineffective. The NO donor sodium nitroprusside decreased GI myoelectric activity, inhibited the MMC pattern, and prevented the effects induced by L-NAME and 7-NI in the intestine. Intracerebroventricular administration of these agents did not modify GI motility. In the rumen, abomasal antrum, duodenum, and jejunum, WB showed three bands at about 155, 145, and 135kDa corresponding to nNOS, and a 140-kDa band (eNOS); however iNOS was not detected. Positive nNOS immunostaining was observed in neurons of the myenteric and submucous plexus of all GI tissues, while eNOS was found in the endothelial cells, ruminal and intestinal epithelium, as well as in some enteric neurons and in endocrine-like cells of the duodenal Brunners glands. In contrast, only weak iNOS immunoreactivity was found in ruminal epithelium. Taken together, our results suggest that NO, synthesized at a peripheral level by nNOS, is tonically inhibiting the MMC pattern and intestinal motility in sheep.