Hemin reduces postoperative ileus in a heme oxygenase 1-dependent manner while dimethyl fumarate does without heme oxygenase 1-induction.

BACKGROUND: Postoperative ileus (POI), the impairment of gastrointestinal motility after abdominal surgery, is mainly due to intestinal muscular inflammation. Carbon monoxide (CO)-releasing compounds were shown to exert an anti-inflammatory effect in murine POI partially through induction of heme oxygenase-1 (HO-1). The influence of hemin and dimethyl fumarate (DMF), currently used for multiple sclerosis (MS), was therefore tested in murine POI. METHODS: C57BL/6J mice were anesthetized and after laparotomy, POI was induced via intestinal manipulation (IM). Animals were treated with either 30 mg kg-1 hemin intraperitoneally (ip), 30 mg kg-1 DMF ip, or 100 mg kg-1 intragastrically (ig) 24 hours before IM. Intestinal transit was assessed 24 hours postoperatively and mucosa-free muscularis or whole segments of the small intestine were stored for later analysis. Intestinal HO-1 protein expression was studied at 6, 12, and 24 hours after administration of hemin or DMF in non-manipulated mice. KEY RESULTS: Pretreatment with hemin and DMF, both ig and ip, prevented the delayed transit seen after IM. Concomitantly, both hemin and DMF significantly reduced the increased interleukin-6 levels and the elevated leukocyte infiltration in the muscularis. Hemin but not DMF caused a significant increase in intestinal HO-1 protein expression and co-administration of the HO-1 inhibitor chromium mesoporphyrin abolished the protective effects of hemin on POI; DMF reduced the IM-induced activation of NF-κB and ERK 1/2. CONCLUSIONS AND INFERENCES: Both hemin and DMF improve the delayed transit and inflammation seen in murine POI, but only hemin does so in a HO-1-dependent manner.

The H2S-Releasing Naproxen Derivative ATB-346 and the Slow-Release H2S Donor GYY4137 Reduce Intestinal Inflammation and Restore Transit in Postoperative Ileus.

Objective: Intestinal inflammation triggers postoperative ileus (POI), commonly seen after abdominal surgery and characterized by impaired gastrointestinal transit; when prolonged, this leads to increased morbidity. Hydrogen sulfide (H2S) is recognized as an important mediator of many (patho)physiological processes, including inflammation, and is now investigated for anti-inflammatory application. Therefore, the aim of this study was to investigate the effect of the H2S-releasing naproxen derivative ATB-346, developed to reduce gastrointestinal injury by naproxen, and the slow-release H2S donor GYY4137 on intestinal inflammation and delayed gastrointestinal transit in murine POI. Methods: C57Bl6J mice were fasted for 6 h, anesthetized and after laparotomy, POI was induced by compressing the small intestine with two cotton applicators for 5 min (intestinal manipulation; IM). GYY4137 (50 mg/kg, intraperitoneally), ATB-346 (16 mg/kg, intragastrically) or naproxen (10 mg/kg, intragastrically) were administered 1 h before IM. At 24 h postoperatively, gastrointestinal transit was assessed via fluorescent imaging, and mucosa-free muscularis segments were prepared for later analysis. Inflammatory parameters and activity of inducible nitric oxide synthase (iNOS) and cyclo-oxygenase (COX)-2 were measured. Histological examination of whole tissue sections was done on hematoxylin-eosin stained slides. Results: Pre-treatment with GYY4137 (geometric center; GC: 7.6 ± 0.5) and ATB-346 (GC: 8.4 ± 0.3) prevented the delayed transit induced by IM (GC: 3.6 ± 0.5 vs. 9.0 ± 0.4 in non-operated controls) while naproxen only partially did (GC: 5.9 ± 0.5; n = 8 for all groups). GYY4137 and ATB-346 significantly reduced the IM-induced increase in muscular myeloperoxidase (MPO) activity and protein levels of interleukin (IL)-6, IL-1ß and monocyte chemotactic protein 1; the reduction by naproxen was less pronounced and only reached significance for MPO activity and IL-6 levels. All treatments significantly reduced the increase in COX-2 activity caused by IM, whereas only GYY4137 significantly reduced the increase in iNOS activity. Naproxen treatment caused significant histological damage of intestinal villi. Conclusion: The study shows that naproxen partially prevents POI, probably through its inhibitory effect on COX-2 activity. Both ATB-346 and GYY4137 were more effective, the result with GYY4137 showing that H2S per se can prevent POI.

Investigation of orally delivered carbon monoxide for postoperative ileus.

Endogenously produced carbon monoxide (CO) has antioxidant and anti-inflammatory effects which is why CO has been investigated as a possible therapeutic agent for inflammatory disorders in different body systems, including the gastrointestinal (GI) tract. In an effort to develop an easy to use platform for CO delivery to the GI tract, we recently introduced the Oral CO Release System (OCORS) and demonstrated its preventive effect for experimental colitis in a rodent model. Building off on a comprehensive preclinical dataset on efficacy of inhaled and intraperitoneal CO in reducing postoperative ileus (POI), which is being defined as GI transit retardation after abdominal surgery, we evaluated an adapted OCORS platform to ameliorate POI by local CO delivery to the murine small intestine. To match design characteristics of OCORS with the murine physiology we developed a miniaturized version of the OCORS and tailored its release pattern to release CO for 2 h following first order kinetics. Upon intragastric gavage of 20 tablets, 55% of the tablets reached the murine small intestine after 1 h while triggering a blood carboxyhemoglobin rise to 5.2%. Although this is in line with previous systemic CO dosing protocols, GI muscular inflammation and transit retardation by small intestinal manipulation, performed at 1 h after gavage of 20 tablets, was not prevented while the positive control - intravenous nitrite - prevented POI. The results show that local CO treatment of POI is insufficient - suggesting a strong systemic component for effective therapy - thereby providing critical insight into effective design of CO drug delivery in POI.

cAMP Catalyzing Phosphodiesterases Control Cholinergic Muscular Activity But Their Inhibition Does Not Enhance 5-HT4 Receptor-Mediated Facilitation of Cholinergic Contractions in the Murine Gastrointestinal Tract.

Background: As the signal transduction of 5-HT4 receptors on cholinergic neurons innervating smooth muscle is controlled by phosphodiesterase (PDE) 4 in porcine stomach and colon, and human large intestine, the in vivo gastroprokinetic effects of a 5-HT4 receptor agonist might be enhanced by combination with a selective PDE4 inhibitor. The presence of 5-HT4 receptors on cholinergic neurons towards murine gastrointestinal circular muscle was recently shown. If the control of this receptor pathway by PDE4 is also present in mice, this might be a good model for in vivo testing of the combination therapy. Therefore this study investigates the role of cAMP catalyzing PDEs in smooth muscle cell activity and in the intraneuronal signal transduction of the 5-HT4 receptors in the gastrointestinal tract of C57Bl/6J mice. Methods: In circular smooth muscle strips from murine fundus, jejunum, and colon, submaximal cholinergic contractions were induced by either electrical field stimulation (EFS) or by carbachol (muscarinic receptor agonist). The influence of the PDE inhibitors IBMX (non-selective), vinpocetine (PDE1), EHNA (PDE2), cilostamide (PDE3), and rolipram (PDE4) was tested on these contractions and on the facilitating effect of a submaximal concentration of prucalopride (5-HT4 receptor agonist) on EFS-induced contractions. Results: In the three gastrointestinal regions, IBMX and cilostamide concentration-dependently decreased carbachol- as well as EFS-induced contractions. Some inhibitory effect was also observed with rolipram. In the fundus a non-significant trend for an enhancement of the facilitating effect of prucalopride on EFS-induced contractions was observed with IBMX, but none of the selective PDE inhibitors enhanced the facilitating effect of prucalopride in fundus, jejunum or colon. Conclusion: In analogy with the porcine gastrointestinal tract, in murine fundus, jejunum, and colon circular smooth muscle PDE3 is the main regulator of the cAMP turnover, with some contribution of PDE4. In contrast to the porcine gastrointestinal tract, the in vitro facilitation of electrically induced cholinergic contractions by 5-HT4 receptor stimulation could not be enhanced by specific PDE inhibition. The C57Bl/6J murine model is thus not suitable for in vivo testing of a 5-HT4 receptor agonist combined with a selective PDE4 inhibitor.

Differential Effects of CORM-2 and CORM-401 in Murine Intestinal Epithelial MODE-K Cells under Oxidative Stress.

Carbon monoxide (CO)-releasing molecules (CO-RMs) are intensively studied to provide cytoprotective and anti-inflammatory effects of CO in inflammatory conditions including intestinal inflammation. The water-soluble CORM-A1 reduced apoptosis and NADPH oxidase (NOX)-derived reactive oxygen species (ROS) induced by tumor necrosis factor (TNF)-α/cycloheximide (CHX) in mouse MODE-K intestinal epithelial cells (IECs), without influencing TNF-α/CHX-induced mitochondrial superoxide anion ([Formula: see text]). The aim of the present study in the same model was to comparatively investigate the influence of lipid-soluble CORM-2 and water-soluble CORM-401, shown in vitro to release more CO under oxidative conditions. CORM-2 abolished TNF-α/CHX-induced total cellular ROS whereas CORM-401 partially reduced it, both partially reducing TNF-α/CHX-induced cell death. Only CORM-2 increased mitochondrial [Formula: see text] production after 2 h of incubation. CORM-2 reduced TNF-α/CHX-, rotenone- and antimycin-A-induced mitochondrial [Formula: see text] production; CORM-401 only reduced the effect of antimycin-A. Co-treatment with CORM-401 during 1 h exposure to H2O2 reduced H2O2 (7.5 mM)-induced ROS production and cell death, whereas CORM-2 did not. The study illustrates the importance of the chemical characteristics of different CO-RMs. The lipid solubility of CORM-2 might contribute to its interference with TNF-α/CHX-induced mitochondrial ROS signaling, at least in mouse IECs. CORM-401 is more effective than other CO-RMs under H2O2-induced oxidative stress conditions.

Synergistic effect between 5-HT4 receptor agonist and phosphodiesterase 4-inhibitor in releasing acetylcholine in pig gastric circular muscle in vitro.

5-HT4 receptor agonists have a gastroprokinetic effect by facilitating acetylcholine release from cholinergic nerves innervating gastrointestinal smooth muscle. The role of phosphodiesterase (PDE) 4 in the signal transduction pathway of the 5-HT4 receptors located on the cholinergic neurons towards the circular muscle layer in pig stomach was investigated by analysis of acetylcholine release. Circular muscle strips were prepared from pig proximal stomach and tritium outflow, induced by electrical field stimulation, was studied as a marker for acetylcholine release after incubation with [(3)H]-choline. The PDE4-inhibitor roflumilast concentration-dependently (0.1-1µM) enhanced the facilitating effect of a submaximally effective concentration of the 5-HT4 receptor agonist prucalopride (0.01µM) on electrically induced acetylcholine release. Roflumilast (0.3µM) enhanced acetylcholine release per se but in the combined presence of roflumilast and prucalopride, acetylcholine release was enhanced more than the sum of the effect of the 2 compounds alone. The 5-HT4 receptor agonist velusetrag concentration-dependently (0.01-0.1µM) enhanced acetylcholine release; the effect of the minimally effective concentration (0.01µM) was significantly enhanced by 1µM of the PDE4-inhibitor rolipram, again to a level higher than the sum of the effect of the 2 compounds alone. The synergistic effect between 5-HT4 receptor agonists and PDE4-inhibitors demonstrates that the intracellular pathway of the 5-HT4 receptors located on cholinergic neurons towards pig gastric circular muscle is controlled by PDE4. Combining a 5-HT4 receptor agonist with a PDE4-inhibitor might thus enhance its gastroprokinetic effect.

Presynaptic M3 muscarinic cholinoceptors mediate inhibition of excitatory synaptic transmission in area CA1 of rat hippocampus.

Acetylcholine can modulate hippocampal network function through activation of both nicotinic and muscarinic acetylcholine receptors (mAChRs). All five mAChR subtypes have been identified in the hippocampus. Besides by their involvement in excitability of hippocampal cells, synaptic plasticity and memory, a large body of research has demonstrated the involvement of presynaptic mAChRs in the inhibition of glutamatergic transmission in the hippocampus. Over the years, however, pharmacological and molecular genetic studies have yielded quite contradictory results regarding the mAChR subtype(s) involved. In this study, multi-electrode array technology was used for the pharmacological elucidation of the subtype of mAChR mediating the depression of excitatory synaptic transmission at the SC-CA1 synapse. Using selective antagonists (VU0255035, MT7, tripinamide, MT3) and allosteric potentiators (VU 10010, VU 0238429) the involvement of M1, M2, M4, and M5 subtypes was ruled out thereby implying a major modulatory role for M3 receptors in the inhibition of excitatory synaptic transmission in area CA1 of rat hippocampus.

Antioxidant potential of CORM-A1 and resveratrol during TNF-α/cycloheximide-induced oxidative stress and apoptosis in murine intestinal epithelial MODE-K cells.

Targeting excessive production of reactive oxygen species (ROS) could be an effective therapeutic strategy to prevent oxidative stress-associated gastrointestinal inflammation. NADPH oxidase (NOX) and mitochondrial complexes (I and II) are the major sources of ROS production contributing to TNF-α/cycloheximide (CHX)-induced apoptosis in the mouse intestinal epithelial cell line, MODE-K. In the current study, the influence of a polyphenolic compound (resveratrol) and a water-soluble carbon monoxide (CO)-releasing molecule (CORM-A1) on the different sources of TNF-α/CHX-induced ROS production in MODE-K cells was assessed. This was compared with H2O2-, rotenone- or antimycin-A-induced ROS-generating systems. Intracellular total ROS, mitochondrial-derived ROS and mitochondrial superoxide anion (O2(-)) production levels were assessed. Additionally, the influence on TNF-α/CHX-induced changes in mitochondrial membrane potential (Ψm) and mitochondrial function was studied. In basal conditions, CORM-A1 did not affect intracellular total or mitochondrial ROS levels, while resveratrol increased intracellular total ROS but reduced mitochondrial ROS production. TNF-α/CHX- and H2O2-mediated increase in intracellular total ROS production was reduced by both resveratrol and CORM-A1, whereas only resveratrol attenuated the increase in mitochondrial ROS triggered by TNF-α/CHX. CORM-A1 decreased antimycin-A-induced mitochondrial O2(-) production without any influence on TNF-α/CHX- and rotenone-induced mitochondrial O2(-) levels, while resveratrol abolished all three effects. Finally, resveratrol greatly reduced and abolished TNF-α/CHX-induced mitochondrial depolarization and mitochondrial dysfunction, while CORM-A1 only mildly affected these parameters. These data indicate that the cytoprotective effect of resveratrol is predominantly due to mitigation of mitochondrial ROS, while CORM-A1 acts solely on NOX-derived ROS to protect MODE-K cells from TNF-α/CHX-induced cell death. This might explain the more pronounced cytoprotective effect of resveratrol.

Mitochondria and NADPH oxidases are the major sources of TNF-α/cycloheximide-induced oxidative stress in murine intestinal epithelial MODE-K cells.

TNF-α/cycloheximide (CHX)-induced apoptosis of the mouse intestinal epithelial cell line MODE-K corresponds with the production of reactive oxygen species (ROS). The aim of the study is to investigate the sources of ROS production contributing to apoptotic cell death during TNF-α/CHX-induced oxidative stress in MODE-K cells. Total ROS or mitochondrial superoxide anion production was measured simultaneously with cell death in the absence or presence of pharmacological inhibitors of various ROS-producing systems, and of ROS scavengers/antioxidants. The influence of TNF-α/CHX on mitochondrial membrane potential (Ψ(m)) and cellular oxygen consumption was also studied. TNF-α/CHX time-dependently increased intracellular total ROS and mitochondrial superoxide anion production in MODE-K cells, starting from 2h. Inhibition of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) by a pan-NOX inhibitor (VAS-2870) and a specific inhibitor of Rac1 (NSC23766) significantly reduced TNF-α/CHX-induced total ROS and cell death levels. The mitochondrial electron transport chain inhibitors, amytal (IQ site of complex I) and TTFA (Qp site of complex II) showed a pronounced decrease in TNF-α/CHX-induced total ROS, mitochondrial superoxide anion and cell death levels. TNF-α/CHX treatment caused an immediate decrease in mitochondrial respiration, and a loss of Ψ(m) and increase in mitochondrial dysfunction from 1 h on. The results suggest that mitochondria and NOX are the two major sources of ROS overproduction during TNF-α/CHX-induced cell death in MODE-K cells, with superoxide anions being the major ROS species. Particularly, the quinone-binding sites of mitochondrial complex I (site I(Q)) and complex II (site Qp) seem to be the major sites of mitochondrial ROS production.

Influence of cinaciguat on gastrointestinal motility in apo-sGC mice.

BACKGROUND: Cinaciguat (BAY 58-2667), an NO- and heme-independent sGC activator, was shown to be more effective when the heme-group of sGC is oxidized in vascular tissue. In apo-sGC mice (sGCß1 (His105Phe) knockin) both sGC isoforms (sGCα1 ß1 and sGCα2 ß1 ) are heme-deficient and can no longer be activated by NO; these mice, showing decreased gastrointestinal nitrergic relaxation and decreased gastric emptying, can be considered as a model to study the consequence of heme-oxidation in sGC. Our aim was to compare the influence of cinaciguat, on in vitro muscle tone of gastrointestinal tissues, and on gastric emptying in WT and apo-sGC mice. METHODS: Gastrointestinal smooth muscle strips were mounted in organ baths for isometric force recording and cGMP levels were determined by enzyme immunoassay. Protein levels of sGC subunits were assessed by immunoblotting. Gastric emptying was determined by phenol red recovery. KEY RESULTS: Although protein levels of the sGC subunits were lower in gastrointestinal tissues of apo-sGC mice, cinaciguat induced concentration-dependent relaxations and increased cGMP levels in apo-sGC fundus and colon to a similar or greater extent than in WT mice. The sGC inhibitor ODQ increased cinaciguat-induced relaxations and cGMP levels in WT fundus and colon. In apo-sGC antrum, pylorus and jejunum, cinaciguat was not able to induce relaxations. Cinaciguat did not improve delayed gastric emptying in apo-sGC mice. CONCLUSIONS & INFERENCES: Cinaciguat relaxes the fundus and colon efficiently when sGC is in the heme-free condition; the non-effect of cinaciguat in pylorus explains its inability to improve the delayed gastric emptying in apo-sGC mice.

Investigation of 5-HT4 receptors in bronchial hyperresponsiveness in cigarette smoke-exposed mice.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) arises from an interaction between genetic host factors and environmental exposures (mainly cigarette smoke (CS)). Genome Wide Association studies have demonstrated that genetic variations in the gene encoding 5-hydroxytryptamine 4 receptors (5-HT(4)R), HTR4, were associated with measures of airway obstruction and with COPD. We hypothesised that 5-HT(4) receptors, in addition to 5-HT2AR and muscarinic receptors, contribute to the pathogenesis of COPD by facilitating cholinergic bronchoconstriction. METHODS: The levels of pulmonary 5-HT(4)R mRNA were measured in CS-exposed mice by qRT-PCR. We investigated the effect of CS exposure on bronchial hyperresponsiveness (BHR) to 5-HT and evaluated the contribution of 5-HT2AR, muscarinic receptors and 5-HT(4)R in the response to 5-HT by using the corresponding antagonists and 5-HT(4)R knockout (KO) mice. RESULTS: The 5-HT(4)R mRNA levels were significantly elevated upon acute (3 days), subacute (4 weeks) and chronic (24 weeks) CS exposure. Both acute and subacute CS exposure significantly increased BHR to 5-HT. Antagonism of 5-HT2AR abolished the CS-induced BHR to 5-HT, and antagonism of muscarinic receptors significantly reduced the response to 5-HT. However, pre-treatment with GR113808, a specific 5-HT(4)R antagonist, did not alter the response to 5-HT in CS-exposed mice. Accordingly, the CS-induced BHR to 5-HT was not different between wild-type and 5-HT(4)R KO mice. CONCLUSION: CS increased the levels of 5-HT(4)R mRNA in the lungs, concomitantly with bronchial responsiveness to 5-HT. Our in vivo data using pharmacologic and genetic approaches suggest that 5-HT(4) receptors are not involved in the BHR to 5-HT in CS-exposed mice.

Predominant mucosal expression of 5-HT4(+h) receptor splice variants in pig stomach and colon.

AIM: To investigate cellular 5-HT4(-h/+h) receptor distribution, particularly in the epithelial layer, by laser microdissection and polymerase chain reaction (PCR) in porcine gastrointestinal (GI) tissues. METHODS: A stepwise approach was used to evaluate RNA quality and to study cell-specific 5-HT4 receptor mRNA expression in the porcine gastric fundus and colon descendens. After freezing, staining and laser microdissection and pressure catapulting (LMPC), RNA quality was evaluated by the Experion automated electrophoresis system. 5-HT4 receptor and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expressions were examined by endpoint reverse transcription (RT)-PCR in mucosal and muscle-myenteric plexus (MMP) tissue fractions, in mucosal and MMP parts of hematoxylin and eosin (HE) stained tissue sections and in microdissected patches of the epithelial and circular smooth muscle cell layer in these sections. Pig gastric fundus tissue sections were also stained immunohistochemically (IHC) for enterochromaffin cells (EC cells; MAB352); these cells were isolated by LMPC and examined by endpoint RT-PCR. RESULTS: After HE staining, the epithelial and circular smooth muscle cell layer of pig colon descendens and the epithelial cell layer of gastric fundus were identified morphologically and isolated by LMPC. EC cells of pig gastric fundus were successfully stained by IHC and isolated by LMPC. Freezing, HE and IHC staining, and LMPC had no influence on RNA quality. 5-HT4 receptor and GAPDH mRNA expressions were detected in mucosa and MMP tissue fractions, and in mucosal and MMP parts of HE stained tissue sections of pig colon descendens and gastric fundus. In the mucosa tissue fractions of both GI regions, the expression of h-exon containing receptor [5-HT4(+h) receptor] mRNA was significantly higher (P < 0.01) compared to 5-HT4(-h) receptor expression, and a similar trend was obtained in the mucosal part of HE stained tissue sections. Large microdissected patches of the epithelial and circular smooth muscle cell layer of pig colon descendens and of the epithelial cell layer of pig gastric fundus, also showed 5-HT4 receptor and GAPDH mRNA expression. No 5-HT4 receptor mRNA expression was detected in gastric LMPC-isolated EC cells from IHC stained tissues, which cells were positive for GAPDH. CONCLUSION: Porcine GI mucosa predominantly expresses 5-HT4(+h) receptor splice variants, suggesting their contribution to the 5-HT4 receptor-mediated mucosal effects of 5-HT.

Influence of phosphodiesterases and cGMP on cAMP generation and on phosphorylation of phospholamban and troponin I by 5-HT4 receptor activation in porcine left atrium.

Our objective was to investigate the role of phosphodiesterase (PDE)3 and PDE4 and cGMP in the control of cAMP metabolism and of phosphorylation of troponin I (TnI) and phospholamban (PLB) when 5-HT4 receptors are activated in pig left atrium. Electrically paced porcine left atrial muscles, mounted in organ baths, received stimulators of particulate guanylyl cyclase (pGC) or soluble guanylyl cyclase (sGC) and/or specific PDE inhibitors followed by 5-HT or the 5-HT4 receptor agonist prucalopride. Muscles were freeze-clamped at different moments of exposure to measure phosphorylation of the cAMP/protein kinase A targets TnI and PLB by immunoblotting and cAMP levels by enzyme immunoassay. Corresponding with the functional results, 5-HT only transiently increased cAMP content, but caused a less quickly declining phosphorylation of PLB and did not significantly change TnI phosphorylation. Under combined PDE3 and PDE4 inhibition, the 5-HT-induced increase in cAMP levels and PLB phosphorylation was enhanced and sustained, and TnI phosphorylation was now also increased. Responses to prucalopride per se and the influence thereupon of PDE3 and PDE4 inhibition were similar except that responses were generally smaller. Stimulation of pGC together with PDE4 inhibition increased 5-HT-induced PLB phosphorylation compared to 5-HT alone, consistent with functional responses. sGC stimulation hastened the fade of inotropic responses to 5-HT, while cAMP levels were not altered. PDE3 and PDE4 control the cAMP response to 5-HT4 receptor activation, causing a dampening of downstream signalling. Stimulation of pGC is able to enhance inotropic responses to 5-HT by increasing cAMP levels, while sGC stimulation decreases contraction to 5-HT cAMP independently.

Influence of phosphodiesterases on basal and 5-HT4 receptor facilitated cholinergic contractility in pig descending colon.

This study in pig colon descendens circular muscle investigated the possible role of phosphodiesterases (PDEs) (1) in the control of smooth muscle activity and (2) in the signal transduction of the 5-HT4 receptors located on the cholinergic neurons. Submaximal cholinergic contractions were electrically induced in colonic circular muscle strips and the influence of the non-selective PDE inhibitor 3-isobutyl-1-methyl-xanthine (IBMX) and selective inhibitors for the 5 classic PDE families (1-5) vinpocetine (PDE1), EHNA (PDE2), cilostamide (PDE3), rolipram (PDE4) and zaprinast (PDE5) was evaluated. IBMX and cilostamide concentration-dependently reduced the amplitude of the cholinergic contractions, as good as abolishing them at 30 and 0.3 µM respectively. EHNA only reduced the contractions significantly at the highest concentration tested (30 µM). IBMX and cilostamide also concentration-dependently inhibited submaximal cholinergic contractions induced with the muscarinic receptor agonist carbachol. The 5-HT4 receptor agonist prucalopride (1 µM) significantly enhanced the electrically induced cholinergic contractions. IBMX, vinpocetine and EHNA did not influence the facilitating effect of prucalopride but rolipram tended to enhance it. When rolipram was added after prucalopride, the facilitating effect of prucalopride was significantly enhanced. These results suggest that PDE3 is the main regulator of circular smooth muscle activity and that the signal transduction of 5-HT4 receptors on the cholinergic nerves towards the circular muscle layer is regulated by PDE4 in pig colon descendens.

TNF-α/cycloheximide-induced oxidative stress and apoptosis in murine intestinal epithelial MODE-K cells.

In the mouse postoperative ileus model, we have shown an increase in oxidative stress after intestinal manipulation occurring earlier in the mucosa than in the muscular layer, which might contribute to epithelial barrier dysfunction. To address these findings in vitro, we assessed TNF-α/cycloheximide (CHX)-induced oxidative stress and apoptosis in a mouse intestinal epithelial cell line, MODE-K. The influence of heme oxygenase (HO)-1-related products and agents known to reduce reactive oxygen species (ROS) production on TNF-α/CHX-induced oxidative stress and apoptosis were investigated. MODE-K cells were exposed to different concentrations of TNF- α/CHX in the absence/presence of the test agents. Cell viability, caspase-3/7 activity, apoptosis, reduced glutathione level (GSH) and intracellular ROS production were measured. TNF-α/CHX decreased cell viability, increased caspase-3/7 activity, induced apoptosis, reduced the GSH level and increased ROS production in a concentration-dependent manner in MODE-K cells. All these effects of TNF- α/CHX were partially prevented by pretreatment with a carbon monoxide-releasing agent (CORM-A1) and nitrite. The antioxidant resveratrol abolished TNF-α/CHX-induced increase in ROS production and caspase-3/7 activity, but apoptosis was only partially prevented. MODE-K cells are sensitive to TNF-α-induced apoptosis in the presence of CHX, which is associated with increased intracellular ROS production and caspase-3/7 activation. The effects were partially mitigated by CORM-A1, nitrite and resveratrol. Thus, these agents could be of potential use in protecting the epithelial barrier against oxidative stress during intestinal ischemia/reperfusion injury.

Mechanism of relaxation and interaction with nitric oxide of the soluble guanylate cyclase stimulator BAY 41-2272 in mouse gastric fundus and colon.

BAY 41-2272 is a heme-dependent nitric oxide-independent soluble guanylate cyclase (sGC) stimulator, but its relaxant effect in vascular, respiratory and urogenital tissue is only partially dependent on sGC activation. As its mechanism of action has not been studied in the gastrointestinal tract, it was investigated in mouse gastric fundus and colon. Circular smooth muscle strips were mounted in organ baths under non-adrenergic non-cholinergic (NANC) conditions for isometric force recording and cGMP levels were determined using an enzyme immunoassay kit. BAY 41-2272 induced concentration-dependent relaxation in both tissues and increased cGMP levels. The sGC inhibitor ODQ totally inhibited this BAY 41-2272-induced increase of cGMP, but only partially reduced the corresponding relaxation. The PDE-5 inhibitor sildenafil had no effect on BAY 41-2272-induced responses. The NO synthase inhibitor L-NAME caused a significant decrease in BAY 41-2272-induced responses in colonic strips. Electrical field stimulation in the presence of BAY 41-2272 induced increased NANC relaxation in fundus, while in colon, rebound contraction at the end of the stimulation train was no longer visible. This suggests synergy with endogenously released NO. Responses to BAY 41-2272 were not significantly influenced by apamin, charybdotoxin or ouabain, excluding interaction with small, intermediate and large conductance Ca(2+)-activated K(+) channels and with Na(+)-K(+)-ATPase. Under depletion of intracellular calcium, CaCl(2)-induced contractions were significantly reduced by BAY 41-2272 in an ODQ-insensitive way. The present study demonstrates that BAY 41-2272 exerts its relaxing effect in mouse gastric fundus and colon partially through a cGMP-dependent mechanism and at least one additional cGMP-independent mechanism involving Ca(2+)-entry blockade.

The facilitating effect of prucalopride on cholinergic neurotransmission in pig gastric circular muscle is regulated by phosphodiesterase 4.

The influence of the selective 5-HT(4) receptor agonist prucalopride on acetylcholine release from cholinergic nerve endings innervating pig gastric circular muscle and the possible regulation of this effect by phosphodiesterases (PDEs) was investigated, as PDEs have been shown to control the response to 5-HT(4) receptor activation in pig left atrium. Circular muscle strips were prepared from pig proximal stomach and either submaximal cholinergic contractions or tritium outflow after incubation with [(3)H]-choline, induced by electrical field stimulation, were studied. Prucalopride concentration-dependently increased the amplitude of submaximal cholinergic contractions and of acetylcholine release induced by electrical field stimulation. The effect of the highest concentration tested (0.3 µM) on cholinergic contractions was antagonized by the selective 5-HT(4) receptor antagonist GR113808 but not by granisetron or methysergide; the antagonism of prucalopride by GR113808 was confirmed in the release assay. The non-selective PDE-inhibitor 3-isobutyl-methyl-xanthine (IBMX) concentration-dependently reduced the amplitude of the cholinergic contractions; 3 µM IBMX reduced the cholinergic contractions maximally by 16% but it enhanced the facilitating effect of prucalopride from 51 to 83%. IBMX (10 µM) induced and enhanced the facilitating effect of prucalopride on electrically induced acetylcholine release. The selective inhibitors vinpocetine (PDE1), EHNA (PDE2) and cilostamide (PDE3) did not influence the effect of prucalopride on acetylcholine release but the PDE4-inhibitor rolipram (1 µM) enhanced the facilitating effect of prucalopride to the same extent as IBMX. These results demonstrate that 5-HT(4) receptors are present on the cholinergic nerves towards the pig gastric circular muscle, facilitating acetylcholine release; the intracellular transduction pathway of this facilitation is regulated by PDE4. Combination of a 5-HT(4) receptor agonist with selective inhibition of the PDE involved in this regulation of transmitter release might enhance the prokinetic effect of the 5-HT(4) receptor agonist.

The influence of peptide impurity profiles on functional tissue-organ bath response: the 11-mer peptide INSL6[151-161] case.

Bioactive peptides have great pharmaceutical potential as nutraceuticals, diagnostics, and therapeutic drugs in several clinical areas. Thus, the search for novel lead peptides with a biological function has attracted renewed interest. Crude peptide material (i.e., ~70% purity) of INSL6[151-161] (NH2-FRSLFWGNHSQ-COOH) was found to trigger a contractile response in guinea pig ileum longitudinal smooth muscle preparations using tissue-organ baths. However, the purified peptide (i.e., ≥ 95% purity) had no effect on this model. Further investigation with crude materials from other suppliers, with purities ranging between 50% and 80%, indicated that the crude products gave a false-positive functional tissue-organ bath conclusion. These observations question the functionality conclusions when using crude-purity peptide materials; during the initial research or discovery phase, peptide quality is generally neglected, possibly leading to misinterpretation of biological results due to by-products from peptide synthesis and, thus, wrong fail/pass decisions. Therefore, we strongly recommend appropriate quality control testing before using any peptides for initial biomedical research or discovery purposes.

In-vitro acetylcholine release is not a straightforward model to study hippocampal 5-HT4 receptors.

5-HT4 receptor (5-HT4R) activation induces procognitive effects. This might be related to stimulation of hippocampal acetylcholine release, which has been shown for 5-HT4R agonists in in-vivo models. We investigated the influence of the 5-HT4R agonists, prucalopride and BIMU-8, on acetylcholine release in rat hippocampal brain slices. In contrast to the report by Siniscalchi et al., no facilitating effect of 5-HT4R agonists on electrically evoked acetylcholine could be shown. The in our hands absence of an effect by 5-HT4R agonists illustrates that an in-vitro evaluation of 5-HT4R agonists on hippocampal acetylcholine release is not a straightforward model to study the relationship between hippocampal 5-HT4Rs and hippocampal acetylcholine release.

Investigation of neurogenic excitatory and inhibitory motor responses and their control by 5-HT(4) receptors in circular smooth muscle of pig descending colon.

The aim of this study was to investigate whether the pig colon descendens might be a good model for the responses mediated via the different locations of human colonic 5-HT(4) receptors. The intrinsic excitatory and inhibitory motor neurotransmission in pig colon descendens was therefore first characterized. In circular smooth muscle strips, electrical field stimulation (EFS) at basal tone induced only in the combined presence of the NO synthase inhibitor N(ω)-nitro-L-arginine methyl ester hydrochloride (L-NAME) and the SK channel blocker apamin voltage-dependent on-contractions. These on-contractions were largely reduced by the neuronal conductance blocker tetrodotoxin (TTX) and by the muscarinic receptor antagonist atropine, illustrating activation of cholinergic neurons. The 5-HT(4) receptor agonist prucalopride facilitated submaximal EFS-evoked cholinergic contractions and this effect was prevented by the 5-HT(4) receptor antagonist GR113808, supporting the presence of facilitating 5-HT(4) receptors on the cholinergic nerve endings innervating circular muscle in pig colon descendens. Relaxations were induced by EFS in strips pre-contracted with substance P in the presence of atropine. The responses at lower stimulation voltages were abolished by TTX. L-NAME or apamin alone did not influence or only moderately reduced the relaxations, but L-NAME plus apamin abolished the relaxations at lower stimulation voltages, suggesting that NO and ATP act as inhibitory neurotransmitters in a redundant way. Prucalopride did not influence the EFS-induced relaxations at lower stimulation voltage, nor did it per se relax contracted circular muscle strips. No evidence for relaxing 5-HT(4) receptors, either on inhibitory neurons or on the muscle cells was thus obtained in pig colon descendens circular muscle.