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.

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.

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.

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.

Peroxisome proliferator-activated receptor gamma activation alleviates postoperative ileus in mice by inhibition of Egr-1 expression and its downstream target genes.

Postoperative ileus, a major cause of morbidity after abdominal surgery, is characterized by intestinal dysmotility and a complex inflammatory cascade within the intestinal muscularis. Treatment with carbon monoxide (CO)--inhaled or intraperitonea--has been shown to ameliorate bowel dysmotility caused by surgical manipulation of the gut in experimental animals. Recent evidence indicates that CO exerts its anti-inflammatory effects through the induction of peroxisome proliferator-activated receptor (PPAR)-gamma, a nuclear receptor whose activation has been linked to several physiological pathways, including those related to the regulation of intestinal inflammation. The purpose of this study was to evaluate pharmacological activation of PPARgamma in a murine model of postoperative ileus by use of the PPARgamma agonist rosiglitazone. Postoperative bowel dysmotility was induced by surgical manipulation of the colon. The functional severity of postoperative ileus was significantly ameliorated in mice pretreated with rosiglitazone (0.3 to 10 mg/kg i.p.); this was associated with a down-regulation of pro-inflammatory cytokines/chemokines, inducible nitric oxide synthase activity, cyclooxygenase-2 activity, as well as a decrease in leukocyte recruitment into the muscularis of both colon and jejunum. These anti-inflammatory effects were preceded by a PPARgamma-dependent down-regulation of early growth response (Egr)-1, a key regulator of inflammatory gene expression. In conclusion, these results indicate that rosiglitazone significantly attenuates postoperative ileus in mice by suppression of the muscularis inflammatory cascade through a PPARgamma-dependent down-regulation of Egr-1 and encourage the further clinical evaluation of synthetic PPARgamma agonists as pharmacological tool to prevent this postoperative event.

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.

Alternative splicing and exon duplication generates 10 unique porcine 5-HT 4 receptor splice variants including a functional homofusion variant.

5-HT(4) receptors are present in human and porcine atrial myocytes while they are absent from the hearts of small laboratory animals. The pig is therefore the only available nonprimate animal model in which to study cardiac 5-HT(4) receptor function under physiological conditions. While several human splice variants of the 5-HT(4) receptor have been described, the splicing behavior of this receptor in porcine tissue is currently unknown. Here we report on the identification of nine novel COOH-terminal splice variants of the porcine 5-HT(4) receptor, which were named 5-HT(4(b2, j, k, l, m, o, p, q, r)). The internal h-variant was found in combination with several COOH-terminal exons. In addition, splice variants were found that comprised duplicated exons fused to the common region of the 5-HT(4) receptor, thereby providing evidence for a duplication of the porcine HTR4 gene. One of these variants putatively encoded a nine transmembrane-spanning domain homofusion receptor, 5-HT(4(9TM)); also the other variants with a duplicated region might translate into functional, transcriptionally fused dimeric 5-HT(4) receptor variants. The elucidation of the genomic context confirmed that the variants were not genomic artefacts but originated from alternative splicing. This was further corroborated by a functional analysis of the variants 5-HT(4(a)), 5-HT(4(r)), and 5-HT(4(9TM)). To our knowledge, our data are the first to report on a functional GPCR with more than seven predicted transmembrane domains. These findings urge for caution when interpreting data on 5-HT(4) receptor-related pharmacology obtained in the pig; validation at the molecular level might be needed before extrapolating results to human.

Investigation of a possible interaction between the heme oxygenase/biliverdin reductase and nitric oxide synthase pathway in murine gastric fundus and jejunum.

This study investigated the possible interaction between the heme oxygenase (HO)/biliverdin reductase (BVR) and nitric oxide synthase (NOS) pathway in murine gastric fundus and jejunum, since previous studies have shown that both HO-2 and BVR are expressed in interstitial cells of Cajal (ICCs) and co-localized with neuronal NOS in a large proportion of myenteric neurons along the gastrointestinal tract. Neither HO inhibition by chromium mesoporphyrin (CrMP) nor co-incubation with CO or biliverdin/bilirubin affected nitrergic neurotransmission - i.e. relaxations induced by non-adrenergic non-cholinergic (NANC) nerve stimulation or exogenous NO - under normal physiological conditions. However, biliverdin/bilirubin reversed the inhibitory effect of the superoxide generator LY83583 on exogenous NO-induced relaxations in both tissues. When gastric fundus muscle strips were depleted of the endogenous antioxidant Cu/Zn superoxide dismutase (SOD) by the Cu-chelator DETCA, electrically induced NANC relaxations were also affected by LY82583; however, biliverdin/bilirubin could not substitute for the loss of Cu/Zn SOD when this specific antioxidant enzyme was depleted. In jejunal muscle strips, the combination DETCA plus LY83583 nearly abolished contractile phasic activity and, hence, did not allow studying nitrergic relaxation in these experimental conditions. In conclusion, this study does not establish a role for HO/CO in inhibitory NANC neurotransmission in murine gastric fundus and jejunum under normal physiological conditions. However, the antioxidants biliverdin/bilirubin might play an important role in the protection of the nitrergic neurotransmitter against oxidative stress.

Involvement of soluble guanylate cyclase alpha(1) and alpha(2), and SK(Ca) channels in NANC relaxation of mouse distal colon.

In distal colon, both nitric oxide (NO) and ATP are involved in non-adrenergic non-cholinergic (NANC) inhibitory neurotransmission. The role of the soluble guanylate cyclase (sGC) isoforms alpha(1)beta(1) and alpha(2)beta(1), and of the small conductance Ca(2+)-dependent K(+) channels (SK(Ca) channels) in the relaxation of distal colon by exogenous NO and by NANC nerve stimulation was investigated, comparing wild type (WT) and sGCalpha(1) knockout (KO) mice. In WT strips, the relaxation induced by electrical field stimulation (EFS) at 1 Hz but not at 2-8 Hz was significantly reduced by the NO-synthase inhibitor L-NAME or the sGC inhibitor ODQ. In sGCalpha(1) KO strips, the EFS-induced relaxation at 1 Hz was significantly reduced and no longer influenced by L-NAME or ODQ. The SK(Ca) channel blocker apamin alone had no inhibitory effect on EFS-induced relaxation, but combined with ODQ or L-NAME, apamin inhibited the relaxation induced by EFS at 2-8 Hz in WT strips and at 8 Hz in sGCalpha(1) KO strips. Relaxation by exogenous NO was significantly attenuated in sGCalpha(1) KO strips, but could still be reduced further by ODQ. Basal cGMP levels were lower in sGCalpha(1) KO strips but NO still significantly increased cGMP levels versus basal. In conclusion, in the absence of sGCalpha(1)beta(1), exogenous NO is able to partially act through sGCalpha(2)beta(1). NO, acting via sGCalpha(1)beta(1), is the principal neurotransmitter in EFS-evoked responses at 1 Hz. At higher stimulation frequencies, NO, acting at sGCalpha(1)beta(1) and/or sGCalpha(2)beta(1), functions together with another transmitter, probably ATP acting via SK(Ca) channels, with some degree of redundancy.

Influence of soluble guanylate cyclase inhibition on inflammation and motility disturbances in DSS-induced colitis.

Nitric oxide (NO) has been associated with a spectrum of harmful to protective roles in inflammatory bowel disease. The involvement of soluble guanylate cyclase (sGC)--the downstream effector of NO--in the negative effect of NO in inflammatory models has been proposed but this has not been evaluated in inflammatory bowel diseases. The present study investigates therefore the influence of colonic inflammation on sGC activity, as well as the effect of in vivo sGC inhibition on colonic inflammation and on in vitro changes in colonic motility in the dextran sulfate sodium (DSS)-model of colitis in rat. Administration of 7% DSS in the drinking water for 6 days resulted in colonic inflammation as judged from histology and myeloperoxidase activity, accompanied by weight loss and bloody stools. Plasma and colonic tissue cyclic guanosine 3',5'-monophosphate (cGMP) levels were decreased in DSS-treated rats. Colonic levels of neuronal NO synthase (nNOS) mRNA and immunoreactivity were not influenced, while those of inducible NO synthase (iNOS) and colonic nitrite/nitrate levels were increased by DSS exposure. Circular muscle strips from inflamed distal colon showed decreased inhibitory responses towards electrical field stimulation and exogenous NO, while methacholine-induced phasic activity was suppressed. Inhibition of sGC by in vivo treatment with ODQ further reduced cGMP levels but did not prevent the inflammation and motility alterations. These results suggest that DSS-induced colitis in rats is accompanied by a reduced sensitivity of sGC, leading to reduced basal cGMP levels and decreased colonic responsiveness towards nitrergic stimuli, but pharmacological reduction of cGMP generation does not prevent the development of DSS-induced colitis.

Role of the soluble guanylyl cyclase alpha1/alpha2 subunits in the relaxant effect of CO and CORM-2 in murine gastric fundus.

Carbon monoxide (CO) has been shown to cause enteric smooth muscle relaxation by activating soluble guanylyl cyclase (sGC). In gastric fundus, the sGCalpha1beta1 heterodimer is believed to be the most important isoform. The aim of our study was to investigate the role of the sGCalpha1/alpha2 subunits in the relaxant effect of CO and CORM-2 in murine gastric fundus using wild-type (WT) and sGCalpha1 knock-out (KO) mice. In WT mice, CO (bolus)-induced relaxations were abolished by the sGC inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ), while CORM-2- and CO (infusion)-induced relaxations were only partially inhibited by ODQ. In sGCalpha1 KO mice, relaxant responses to CO and CORM-2 were significantly reduced when compared with WT mice, but ODQ still had an inhibitory effect. The sGC sensitizer 1-benzyl-3-(5'-hydroxymethyl-2'-furyl-)-indazol (YC-1) was able to potentiate CO- and CORM-2-induced relaxations in WT mice but lost this potentiating effect in sGCalpha1 KO mice. Both in WT and sGCalpha1 KO mice, CO-evoked relaxations were associated with a significant cGMP increase; however, basal and CO-elicited cGMP levels were markedly lower in sGCalpha1 KO mice. These data indicate that besides the predominant sGCalpha1beta1 isoform, also the less abundantly expressed sGCalpha2beta1 isoform plays an important role in the relaxant effect of CO in murine gastric fundus; however, the sGC stimulator YC-1 loses its potentiating effect towards CO in sGCalpha1 KO mice. Prolonged administration of CO-either by the addition of CORM-2 or by continuous infusion of CO-mediates gastric fundus relaxation in both a sGC-dependent and sGC-independent manner.

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.

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.

Mechanisms of relaxation by carbon monoxide-releasing molecule-2 in murine gastric fundus and jejunum.

This study investigated the effects and mechanisms of action of carbon monoxide-releasing molecule-2 (CORM-2), compared to those of carbon monoxide (CO), in murine gastric fundus and jejunal circular smooth muscle. Functional in vitro experiments and cGMP measurements were conducted. In both tissues, CO and CORM-2 induced concentration-dependent relaxations. CO-induced relaxations were abolished by the soluble guanylyl cyclase (sGC) inhibitor ODQ, while CORM-2-evoked inhibitory responses were only partly prevented by ODQ. Relaxations elicited by CO (300 microM) were associated with a significant increase in cGMP levels, whereas for CORM-2 (300 microM) no significant increase in cGMP levels could be measured. The sGC sensitizer YC-1 was able to accelerate and potentiate both CO- and CORM-2-induced relaxations. Furthermore, the intermediate- and large-conductance Ca2+-activated K+ (IKCa-BKCa) channel blocker charybdotoxin significantly reduced CO- and CORM-2-induced relaxations in jejunal tissue; this same effect was observed with the BKCa channel blocker iberiotoxin. The combination of apamin plus charybdotoxin significantly reduced relaxations in gastric fundus and had synergistic inhibitory effects in jejunum. The NOS inhibitor L-NAME had no effect on the induced relaxations in gastric fundus, but significantly reduced CO- and CORM-2-evoked relaxations in jejunum. In conclusion, these results demonstrate that CO and CORM-2 produce relaxation in gastric fundus and jejunum via sGC and activation of KCa channels, and a nitric oxide (NO)-mediated amplification of CO signaling in jejunum is suggested.

Determination of lactate concentrations in blood plasma and peritoneal fluid in horses with colic by an Accusport analyzer.

BACKGROUND: Intestinal hypoperfusion can lead to increased lactate concentrations in plasma and peritoneal fluid of horses with colic. HYPOTHESIS: The purposes of this study were to (1) evaluate the reliability of the Accusport analyzer to assess peritoneal fluid lactate (PFL) concentrations in healthy horses and those with colic, (2) identify clinical features associated with abnormal blood plasma lactate (BPL) and PFL concentrations, and (3) evaluate the prognostic value of BPL and PFL. ANIMALS: BPL and PFL were determined in 20 healthy horses and in 106 horses with colic. RESULTS: The Accusport was reliable for determining BPL concentrations < 13 mM and PFL concentrations < 20 mM. Multivariate analysis indicated that PCV and the need for intestinal resection were independently associated with the BPL; pulse, PCV, venous pO2, the presence of necrotic intestine, an increased amount of peritoneal fluid, and fluid total protein content were independently associated with PFL. With a 1 mM increase in BPL or PFL, the respective odds ratios for required abdominal surgery increase to 1.23 (BPL) and 1.58 (PFL), odds ratios for a required intestinal resection increase to 1.20 (BPL) and 1.41 (PFL), and odds ratios for developing ileus increase by 1.33 (BPL) and 1.36 (PFL). PFL concentrations of 1, 6, 12, and 16 mM correspond to a probability of death of 11, 29, 63, and 82%, respectively, in horses without strangulating obstruction and of 25, 52, 82, and 92%, respectively, in horses with strangulating obstruction. CONCLUSION: PFL is more useful and sensitive than BPL for prognostic purposes in horses with colic.

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.

Contractile effects of 5-hydroxytryptamine and 5-carboxamidotryptamine in the equine jejunum.

The use of human prokinetic drugs in colic horses leads to inconsistent results. This might be related to differences in gastrointestinal receptor populations. The motor effects of 5-hydroxytryptamine (5-HT; serotonin) on the equine mid-jejunum were therefore studied. Longitudinal muscle preparations were set up for isotonic measurement. 5-HT induced tonic contractions with superimposed phasic activity; these responses were not influenced by tetrodotoxin and atropine, suggesting a non-neurogenic, non-cholinergic pathway. The 5-HT receptor antagonists GR 127935 (5-HT(1B,D)), ketanserin (5-HT(2A)), SB 204741 (5-HT(2B)), RS 102221 (5-HT(2C)), granisetron (5-HT(3)), GR 113808 (5-HT(4)) and SB 269970 (5-HT(7)) had no influence on the 5-HT-induced response; the 5-HT(1A) receptor antagonists NAN 190 (pK(b)=8.13+/-0.06) and WAY 100635 (pK(b)=8.69+/-0.07), and the 5-HT(1,2,5,6,7) receptor antagonist methysergide concentration-dependently inhibited the 5-HT-induced contractile response. The 5-HT(1,7) receptor agonist 5-carboxamidotryptamine (5-CT) induced a contractile response similar to that of 5-HT; its effect was not influenced by tetrodotoxin and atropine, and SB 269970, but antagonised by WAY 100635. 8-OHDPAT, buspiron and flesinoxan, which are active at rat and human 5-HT(1A) receptors, had no contractile influence. These results suggest that the contractile effect of 5-HT in equine jejunal longitudinal muscle is due to interaction with muscular 5-HT receptors, which cannot be characterised between the actually known classes of 5-HT receptors.

Porcine left atrial and sinoatrial 5-HT(4) receptor-induced responses: fading of the response and influence of development.

1.--In this study, we aimed to characterize in vitro the effects of the benzofuran 5-HT(4) receptor agonists prucalopride, R149402 and R199715 and the indolic agents tegaserod and 5-HT in the atria of young pigs (10-11 weeks) and newborn piglets. 2.--In the paced left atrium of young pigs, only 5-HT results in positive inotropic responses when administered cumulatively (maximal effect relative to isoprenaline=53%, pEC(50)=6.8); however, all agonists showed lusitropic effects. Noncumulative administration results in greater positive inotropic responses for 5-HT and induces moderate positive inotropic responses for the other agonists; these responses fade. 3.--Phosphodiesterase (PDE) enzyme inhibition with 3-isobutyl-1-methylxanthine (IBMX; 20 microM) enhances the responses to cumulatively administered 5-HT (maximal effect=89%, pEC(50)=7.7) and reveals clear positive inotropic effects for prucalopride, tegaserod, R149402 and R199715; fading is abolished. The maximal effect of the benzofurans is less pronounced than that of the indoles. 4.--In the spontaneously beating right atrium of young pigs, all agonists show chronotropic activity when administered cumulatively in the absence of IBMX, without fade. Benzofurans behaved as partial agonists compared to 5-HT (maximal effect=54%, pEC(50)=6.5). 5.--In newborns, the inotropic activity of the agonists in the IBMX-treated left atrium was less pronounced than in the young pig; the same applied for the chronotropic response in the right atrium, except for 5-HT. 6.--In conclusion, the atrial responses to 5-HT(4) receptor activation increase in the first months of life; the inotropic response is regulated by PDEs. Prucalopride, R149402 and R199715 are partial agonists compared to 5-HT.

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.

Nitric oxide relaxes circular smooth muscle of rat distal colon through RhoA/Rho-kinase independent Ca2+ desensitisation.

1. The aim of this study in circular smooth muscle of rat distal colon was to determine whether Ca(2+) desensitisation, in addition to mechanisms lowering cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)), was involved in the relaxation elicited by nitric oxide (NO). Changes in isometric tension and [Ca(2+)](cyt) were recorded simultaneously in fura-2-loaded strips. 2. In methacholine (10(-5) M)-precontracted preparations, exogenous NO (10(-4) M), adenosine 5'-triphosphate (ATP; 10(-3) M) and electrical field stimulation (EFS; 1 ms, 40 V, 4 Hz, 1 min) induced a decrease in smooth muscle tension, which was accompanied by a fall in [Ca(2+)](cyt). 3. The sarcoplasmic/endoplasmic reticulum Ca(2+) ATP-ase (SERCA) inhibitor thapsigargin (10(-6) M) did not exert an influence on the decrease in tension produced by exogenous NO, but significantly attenuated the fall in [Ca(2+)](cyt). Both the relaxation and the fall in [Ca(2+)](cyt) to ATP and EFS were unaffected by thapsigargin. 4. Calyculin-A (10(-6) M), a myosin light chain phosphatase (MLCP) inhibitor, significantly reduced the decrease in tension elicited by exogenous NO, but did not alter the fall in [Ca(2+)](cyt) to exogenous NO. Inactivating RhoA by exoenzyme C3 (2 mug ml(-1)) or inhibiting Rho-kinase with (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632; 10(-5) M) had no effect on the decrease of both tension and [Ca(2+)](cyt) generated by exogenous NO. 5. This paper demonstrates that a RhoA/Rho-kinase independent Ca(2+) desensitisation pathway contributes to the relaxation by NO in circular smooth muscle strips of rat distal colon.