Inhibiting endocannabinoid biosynthesis: a novel approach to the treatment of constipation.

BACKGROUND AND PURPOSE: Endocannabinoids are a family of lipid mediators involved in the regulation of gastrointestinal (GI) motility. The expression, localization and function of their biosynthetic enzymes in the GI tract are not well understood. Here, we examined the expression, localization and function of the enzyme diacylglycerol lipase-α (DAGLα), which is involved in biosynthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG). EXPERIMENTAL APPROACH: Cannabinoid CB1 receptor-deficient, wild-type control and C3H/HeJ mice, a genetically constipated strain, were used. The distribution of DAGLα in the enteric nervous system was examined by immunohistochemistry. Effects of the DAGL inhibitors, orlistat and OMDM-188 on pharmacologically induced GI hypomotility were assessed by measuring intestinal contractility in vitro and whole gut transit or faecal output in vivo. Endocannabinoid levels were measured by mass spectrometry. KEY RESULTS: DAGLα was expressed throughout the GI tract. In the intestine, unlike DAGLß, DAGLα immunoreactivity was prominently expressed in the enteric nervous system. In the myenteric plexus, it was colocalized with the vesicular acetylcholine transporter in cholinergic nerves. In normal mice, inhibiting DAGL reversed both pharmacologically reduced intestinal contractility and pharmacologically prolonged whole gut transit. Moreover, inhibiting DAGL normalized faecal output in constipated C3H/HeJ mice. In colons incubated with scopolamine, 2-AG was elevated while inhibiting DAGL normalized 2-AG levels. CONCLUSIONS AND IMPLICATIONS: DAGLα was expressed in the enteric nervous system of mice and its inhibition reversed slowed GI motility, intestinal contractility and constipation through 2-AG and CB1 receptor-mediated mechanisms. Our data suggest that DAGLα inhibitors may be promising candidates for the treatment of constipation.

AM841, a covalent cannabinoid ligand, powerfully slows gastrointestinal motility in normal and stressed mice in a peripherally restricted manner.

BACKGROUND AND PURPOSE: Cannabinoid (CB) ligands have been demonstrated to have utility as novel therapeutic agents for the treatment of pain, metabolic conditions and gastrointestinal (GI) disorders. However, many of these ligands are centrally active, which limits their usefulness. Here, we examine a unique novel covalent CB receptor ligand, AM841, to assess its potential for use in physiological and pathophysiological in vivo studies. EXPERIMENTAL APPROACH: The covalent nature of AM841 was determined in vitro using electrophysiological and receptor internalization studies on isolated cultured hippocampal neurons. Mouse models were used for behavioural analysis of analgesia, hypothermia and hypolocomotion. The motility of the small and large intestine was assessed in vivo under normal conditions and after acute stress. The brain penetration of AM841 was also determined. KEY RESULTS: AM841 behaved as an irreversible CB1 receptor agonist in vitro. AM841 potently reduced GI motility through an action on CB1 receptors in the small and large intestine under physiological conditions. AM841 was even more potent under conditions of acute stress and was shown to normalize accelerated GI motility under these conditions. This compound behaved as a peripherally restricted ligand, showing very little brain penetration and no characteristic centrally mediated CB1 receptor-mediated effects (analgesia, hypothermia or hypolocomotion). CONCLUSIONS AND IMPLICATIONS: AM841, a novel peripherally restricted covalent CB1 receptor ligand that was shown to be remarkably potent, represents a new class of potential therapeutic agents for the treatment of functional GI disorders.

International Harmonization of Nomenclature and Diagnostic Criteria (INHAND): Progress to Date and Future Plans.

The International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice proposal (INHAND) has been operational since 2005. A Global Editorial Steering Committee manages the overall objectives of the project, and the development of harmonized terminology for each organ system is the responsibility of the Organ Working Groups, drawing upon experts from North America, Europe, and Japan. Great progress has been made with 9 systems published to date--respiratory, hepatobiliary, urinary, central/peripheral nervous systems, male reproductive and mammary, zymbals, clitoral, and preputial glands in Toxicologic Pathology and the integument and soft tissue and female reproductive in the Journal of Toxicologic Pathology as supplements and on a Web site--www.goReni.org. INHAND nomenclature guides offer diagnostic criteria and guidelines for recording lesions observed in rodent toxicity and carcinogenicity studies. The guides provide representative photomicrographs of morphologic changes, information regarding pathogenesis, and key references. The purpose of this brief communication is to provide an update on the progress of INHAND.

Endogenous cellular prion protein regulates contractility of the mouse ileum.

BACKGROUND: Cellular prion protein (PrP(C) ) is expressed in the enteric nervous system (ENS), however, its physiological role has not been identified. Studies suggest that PrP(C) can function as a metal-binding protein, as absence of the protein has been linked to altered copper metabolism and atypical synaptic activity. Because copper is known to modulate smooth muscle relaxation, we tested the hypothesis that PrP(C) deficiency would alter intestinal contractility. METHODS: We examined electrically evoked ileal contractility in Prnp(-/-) or wild type littermate mice and the effects of copper or copper chelation. PrP(C) expression was studied in whole mount ileal preparations of mice and guinea pigs by immunohistochemistry. KEY RESULTS: Relative to wild type mice, ileal tissues of Prnp(-/-) mice exhibited reduced electrical field stimulation (EFS)-evoked contractility. Furthermore, EFS-induced relaxation, as a percentage of that induced by a nitric oxide donor, was enhanced. Addition of a copper donor to the organ bath increased, whereas the addition of a copper chelator inhibited, nitric oxide donor-induced ileal relaxation in Prnp(-/-) mice. PrP(C) was expressed on nerve fibers or terminals, and some cell bodies in the myenteric and submucosal plexuses of wild type mice. PrP(C) colocalized with a neuron-specific ectonucleotidase, nucleoside triphosphate diphosphohydrolase 3 (NTPDase3), but to only a limited extent with GFAP, a marker of enteric glia. Guinea pigs expressed PrP(C) in nerve fibers or terminals and enteric glia in the myenteric and submucosal plexuses. CONCLUSIONS & INFERENCES: Our findings suggest that PrP(C) , which is abundant in the ENS, has a role in the regulation of ileal contractility.

Ochronosis in a murine model of alkaptonuria is synonymous to that in the human condition.

OBJECTIVE: Alkaptonuria (AKU) is a rare genetic disease which results in severe early onset osteoarthropathy. It has recently been shown that the subchondral interface is of key significance in disease pathogenesis. Human surgical tissues are often beyond this initial stage and there is no published murine model of pathogenesis, to study the natural history of the disease. The murine genotype exists but it has been reported not to demonstrate ochronotic osteoarthropathy consistent with the human disease. Recent anecdotal evidence of macroscopic renal ochronosis in a mouse model of tyrosinaemia led us to perform histological analysis of tissues of these mice that are known to be affected in human AKU. DESIGN: The homogentisate 1,2-dioxygenase Hgd(+/)(-)Fah(-)(/)(-) mouse can model either hereditary tyrosinaemia type I (HT1) or AKU depending on selection conditions. Mice having undergone Hgd reversion were sacrificed at various time points, and their tissues taken for histological analysis. Sections were stained with haematoxylin eosin (H&E) and Schmorl's reagent. RESULTS: Early time point observations at 8 months showed no sign of macroscopic ochronosis of tissues. Macroscopic examination at 13 months revealed ochronosis of the kidneys. Microscopic analysis of the kidneys revealed large pigmented nodules displaying distinct ochre colouration. Close microscopic examination of the distal femur and proximal fibula at the subchondral junctions revealed the presence of numerous pigmented chondrocytes. CONCLUSIONS: Here we present the first data showing ochronosis of tissues in a murine model of AKU. These preliminary histological observations provide a stimulus for further studies into the natural history of the disease to provide a greater understanding of this class of arthropathy.

Inhibiting fatty acid amide hydrolase normalizes endotoxin-induced enhanced gastrointestinal motility in mice.

BACKGROUND AND PURPOSE: Gastrointestinal (GI) motility is regulated in part by fatty acid ethanolamides (FAEs), including the endocannabinoid (EC) anandamide (AEA). The actions of FAEs are terminated by fatty acid amide hydrolase (FAAH). We investigated the actions of the novel FAAH inhibitor AM3506 on normal and enhanced GI motility. EXPERIMENTAL APPROACH: We examined the effect of AM3506 on electrically-evoked contractility in vitro and GI transit and colonic faecal output in vivo, in normal and FAAH-deficient mice treated with saline or LPS (100 µg·kg(-1), i.p.), in the presence and absence of cannabinoid (CB) receptor antagonists. mRNA expression was measured by quantitative real time-PCR, EC levels by liquid chromatography-MS and FAAH activity by the conversion of [(3)H]-AEA to [(3)H]-ethanolamine in intestinal extracts. FAAH expression was examined by immunohistochemistry. KEY RESULTS: FAAH was dominantly expressed in the enteric nervous system; its mRNA levels were higher in the ileum than the colon. LPS enhanced ileal contractility in the absence of overt inflammation. AM3506 reversed the enhanced electrically-evoked contractions of the ileum through CB(1) and CB(2) receptors. LPS increased the rate of upper GI transit and faecal output. AM3506 normalized the enhanced GI transit through CB(1) and CB(2) receptors and faecal output through CB(1) receptors. LPS did not increase GI transit in FAAH-deficient mice. CONCLUSIONS AND IMPLICATIONS: Inhibiting FAAH normalizes various parameters of GI dysmotility in intestinal pathophysiology. Inhibition of FAAH represents a new approach to the treatment of disordered intestinal motility.

Differential effects of CB(1) neutral antagonists and inverse agonists on gastrointestinal motility in mice.

BACKGROUND: Cannabinoid type 1 (CB(1)) receptors are involved in the regulation of gastrointestinal (GI) motility and secretion. Our aim was to characterize the roles of the CB(1) receptor on GI motility and secretion in vitro and in vivo by using different classes of CB(1) receptor antagonists. METHODS: Immunohistochemistry was used to examine the localization of CB(1) receptor in the mouse ileum and colon. Organ bath experiments on mouse ileum and in vivo motility testing comprising upper GI transit, colonic expulsion, and whole gut transit were performed to characterize the effects of the inverse agonist/antagonist AM251 and the neutral antagonist AM4113. As a marker of secretory function we measured short circuit current in vitro using Ussing chambers and stool fluid content in vivo in mouse colon. We also assessed colonic epithelial permeability in vitro using FITC-labeled inulin. KEY RESULTS: In vivo, the inverse agonist AM251 increased upper GI transit and whole gut transit, but it had no effect on colonic expulsion. By contrast, the neutral antagonist AM4113 increased upper GI transit, but unexpectedly reduced both colonic expulsion and whole gut transit at high, but not lower doses. CONCLUSIONS & INFERENCES: Cannabinoid type 1 receptors regulate small intestinal and colonic motility, but not GI secretion under physiological conditions. Cannabinoid type 1 inverse agonists and CB(1) neutral antagonists have different effects on intestinal motility. The ability of the neutral antagonist not to affect whole gut transit may be important for the future development of CB(1) receptor antagonists as therapeutic agents.

The identification of peroxisome proliferator-activated receptor alpha-independent effects of oleoylethanolamide on intestinal transit in mice.

Oleoylethanolamide (OEA) is an endogenous lipid produced in the intestine that mediates satiety by activation of peroxisome proliferator-activated receptor alpha (PPARalpha). OEA inhibits gastric emptying and intestinal motility, but the mechanism of action remains to be determined. We investigated whether OEA inhibits intestinal motility by activation of PPARalpha. PPARalpha immunoreactivity was examined in whole mount preparations of mouse gastrointestinal (GI) tract. The effect of OEA on motility was assessed in wildtype, PPARalpha, cannabinoid CB(1) receptor and CB(2) receptor gene-deficient mice and in a model of accelerated GI transit. In addition, the effect of OEA on motility was assessed in mice injected with the PPARalpha antagonist GW6471, transient receptor potential vanilloid 1 antagonist SB366791 or the glucagon-like peptide 1 antagonist exendin-3(9-39) amide. PPARalpha immunoreactivity was present in neurons in the myenteric and submucosal plexuses throughout the GI tract. OEA inhibited upper GI transit in a dose-dependent manner, but was devoid of an effect on whole gut transit or colonic propulsion. OEA-induced inhibition of motility was still present in PPARalpha, CB(1) and CB(2) receptor gene-deficient mice and in the presence of GW6471, SB366791 and exendin-3(9-39) amide, suggesting neither PPARalpha nor the cannabinoids and other likely receptors are involved in mediating the effects of OEA. OEA blocked stress-induced accelerated upper GI transit at a dose that had no effect on physiological transit. We show that PPARalpha is found in the enteric nervous system, but our results suggest that PPARalpha is not involved in the suppression of motility by OEA.

The expression and role of Fas ligand in intestinal inflammation.

Fas ligand (FasL) is involved in the pathogenesis of inflammatory diseases and immune privilege. We examined the expression of FasL in the enteric nervous system (ENS) in murine colitis and guinea-pig ileitis. We studied FasL immunoreactivity, functional integrity of the ENS, severity of colitis, and distribution of neutrophils in wild type and B6/gld mice that lack functional FasL. In ileitis, the distribution of FasL, CD4+ and CD8+ T cells was examined. FasL expression was increased in the ENS of wild type mice with colitis, but decreased labelling of nerve fibres was noted in B6/gld mice. Neutrophils were more abundant and widely distributed in B6/gld mice. Colitis was more severe and persistent in B6/gld mice 7 days after induction. Functional parameters of intestinal secretion and motility in B6/gld mice were the same as controls. In ileitis, FasL expression was increased in the guinea-pig ENS and returned to control levels following the resolution of inflammation. While T cells were not present in the ENS of controls, they were observed during inflammation, but were excluded from ganglia. The number of enteric neurons was unchanged over the course of inflammation. The expression of FasL is altered in intestinal inflammation and contributes to its resolution in experimental colitis.

Immediate-early gene expression in the inferior mesenteric ganglion and colonic myenteric plexus of the guinea pig.

Activation of neurons in the inferior mesenteric ganglion (IMG) was assessed using c-fos, JunB, and c-Jun expression in the guinea pig IMG and colonic myenteric plexus during mechanosensory stimulation and acute colitis in normal and capsaicin-treated animals. Intracolonic saline or 2% acetic acid was administered, and mechanosensory stimulation was performed by passage of a small (0.5 cm) balloon either 4 or 24 hr later. Lower doses of capsaicin or vehicle were used to activate primary afferent fibers during balloon passage. c-Jun did not respond to any of the stimuli in the study. c-fos and JunB were absent from the IMG and myenteric plexus of untreated and saline-treated animals. Acetic acid induced acute colitis by 4 hr, which persisted for 24 hr, but c-fos was found only in enteric glia in the myenteric plexus and was absent from the IMG. Balloon passage induced c-fos and JunB in only a small subset of IMG neurons and no myenteric neurons. However, balloon passage induced c-fos and JunB in IMG neurons (notably those containing somatostatin) and the myenteric plexus of acetic acid-treated animals. After capsaicin treatment, c-fos and JunB induction by balloon passage was inhibited in the IMG, but there was enhanced c-fos expression in the myenteric plexus. c-fos and JunB induction by balloon stimulation was also mimicked by acute activation of capsaicin-sensitive nerves. These data suggest that colitis enhances reflex activity of the IMG by a mechanism that involves activation of both primary afferent fibers and the myenteric plexus.

Reproduction study with dibasic esters following inhalation in the rat.

Groups of 20 male and 20 female Crl:CD(SD)BR rats were exposed to Dibasic Esters (DBE) at concentrations of 0 (control), 0.16, 0.40 (maximum attainable vapor), or 1.0 mg/L (aerosol). Exposures were conducted for 6 hours/day, 5 days/week for 14 weeks (pre-breeding) then 7 days/week for 8 weeks (through breeding, gestation, and lactation). The exposures were interrupted for female rats between gestation day 19 and postpartum day 3. Gestation day 1 was defined as the day a copulatory plug was found, postpartum day 1 was defined as the day of birth. No significant differences were observed between control and test rats with respect to mating performance, fertility, length of gestation, or progeny numbers, structure, and viability. Body weights of parental rats and of their offspring were reduced at 1.0 mg/L. The only histopathologic changes detected were in the nasal tissues of the parental rats, where an exposure-related increase in squamous metaplasia in the olfactory epithelium was observed. There was an increase in liver-to-body weight ratios in the two higher parental exposure groups and an increase in the lung-to-body weight ratio also seen at 1.0 mg/L. It is concluded that reproduction in rats was not altered by repeated inhalation exposure to up to 1.0 mg/L DBE, a concentration that produced both body weight and histologic effects in parental rats.

Prostaglandin E2 activation of VIP secretomotor neurons in the guinea pig ileum.

The effect of prostaglandin E2 (PGE2) on the activity-related expression of the proto-oncogene c-fos in specific populations of enteric neurons was investigated. Segments of guinea-pig ileum were incubated in vitro in the presence or absence of PGE2, and whole mounts of the myenteric and submucosal plexus were prepared for immunocytochemical localization of Fos, VIP and NPY. Control tissues exhibited a low number of Fos-immunoreactive (Fos-IR) neurons (7 +/- 2% of total). Incubation of the tissues with 10-1000 nM PGE2 for 30 min caused a concentration-dependent increase in Fos-IR submucosal neurons (maximum at 100 nM; 39 +/- 6%), which was not inhibited by TTX. PGE2 did not evoke an increase in Fos-IR myenteric neurons. In double labeling experiments, Fos colocalized exclusively with VIP in the submucosal plexus, and not with NPY. Exposure of stripped segments of guinea pig ileum in Ussing chambers to 100 nM PGE2 evoked an increase in short circuit current (20 +/- 7 microA/cm2), of which the initial rapid phase could be abolished by TTX, and not by atropine and hexamethonium. It is concluded that PGE2 can activate VIP non-cholinergic secretomotor neurons.

Biological consequences of thrombin receptor deficiency in mice.

The thrombin receptor (ThrR) is a membrane-bound, G-protein-coupled receptor for the serine protease thrombin. This receptor is expressed in a wide variety of cells and tissues, and elicits a range of physiological responses associated with tissue injury, inflammation, and wound repair. To achieve a better understanding of the physiological role of the ThrR, we have employed homologous recombination to create mice with a disrupted ThrR gene. Following heterozygous (+/-) intercrosses, a total of 351 surviving offspring were genotyped. Only 7% of these offspring were identified as homozygous (-/-) for the disrupted allele, indicating a profound effect on embryonic development. Paradoxically, adult ThrR-/- mice appeared to be normal by anatomical and histological analysis, including their platelet number and function. Similarly, ThrR deficiency had no detectable effect in adult ThrR-/- mice on basal heart rate, arterial blood pressure, vasomotor responses to angiotensin II and acetycholine, and coagulation parameters, even though the ThrR is expressed in many cardiovascular tissue types. In addition, the loss of ThrR function in the peripheral vasculature of adult ThrR-/- mice was confirmed by the absence of various standard hemodynamic effects of the ThrR-activating peptides SFLLRN-NH2 and TFLLRNPNDK-NH2. Our results indicate that ThrR deficiency has a strong impact on fetal development; however. ThrR-/- mice that proceed to full development display surprisingly little change in phenotype compared to the wild-type.

Preclinical toxicity evaluation of tepoxalin, a dual inhibitor of cyclooxygenase and 5-lipoxygenase, in Sprague-Dawley rats and beagle dogs.

Tepoxalin [5- (4-chlorophenyl)-N-hydroxy-1-(4-methoxyphenyl)-N-methyl-1H-pyrazole -3-propanamide] is an orally active anti-inflammatory agent, which inhibits both cyclooxygenase and 5-lipoxygenase activities. The oral toxicity of tepoxalin was evaluated in 1- and 6-month rat (up to 50 mg/kg/day) and dog (up to 150 mg/kg bid) studies. In rats, increased liver weight, centrilobular hypertrophy, and hepatic necrosis were observed at dosages >/=20 mg/kg/day. Renal changes indicative of analgesic nephropathy syndrome (i.e., papillary edema or necrosis, cortical tubular dilatation) were seen at >/=15 mg/kg. In rats treated for 1 month, these hepatic and renal effects were largely reversible after a 1-month recovery period. Gastrointestinal erosions and ulcers were seen in female rats given 40 mg/kg/day for 6 months. Changes in clinical pathology parameters included decreases in red blood cell count, hemoglobin, and hematocrit mean values; elevation in platelet counts; and an increase in prothrombin and activated partial thromboplastin times. Mild increases in alanine aminotransferase, aspartate aminotransferase, and cholesterol were also noted in rats. Decreased erythrocyte parameters, increased leukocyte counts, and decreased total protein, albumin, and/or calcium were noted in some dogs in the 300 mg/kg/day group following 6 months of dosing. Small pyloric ulcerations were seen at 100 and 300 mg/kg/day dosages for up to 6 months. In both rats and dogs, no accumulation of tepoxalin or its carboxylic acid metabolite was detected in plasma following multiple dosing over a range of 5 to 50 mg/kg/day for rats and 20 to 300 mg/kg/day for dogs. Plasma concentrations of the carboxylic acid metabolite were severalfold higher than those of the parent compound. The no-effect dosages in rats (5 mg/kg/day) and dogs (20 mg/kg/day) were approximately one and six times the ED50 (3.5 mg/kg), respectively, for inhibition of inflammatory effects in the adjuvant arthritic rat without gastric mucosal damage. In terms of severity, the relative lack of gastrointestinal side effects, within the estimated therapeutic dose range, distinguishes tepoxalin from most marketed anti-inflammatory drugs.

Pharmacological investigation of the role of leukotrienes in the pathogenesis of experimental NSAID gastropathy.

The role of leukotrienes in the pathogenesis of acute gastric ulceration induced by nonsteroidal antiinflammatory drugs was investigated using a rat model. One part of the study involved oral pretreatment with a leukotriene synthesis inhibitor 1 h prior to administration of indomethacin (20 mg/kg per os). Three hours after indomethacin, the extent of macroscopically visible gastric damage was determined, and gastric LTB4 synthesis was determined. The compounds tested were PF-5901, A-64077, nordihydroguaiaretic acid, and L-698,037. Each compound produced dose-related inhibition of gastric LTB4 synthesis and a parallel reduction in the severity of indomethacin-induced damage. The antioxidant properties of these compounds was assessed using an in vitro assay. There was no correlation between the antioxidant properties of the compounds and their ability to reduce the severity of indomethacin-induced gastric damage. In the second part of the study, the effects of intravenous, administration of LTD4 and LTB4 receptor antagonists on indomethacin-induced gastric epithelial damage (measured by permeability to [51Cr]EDTA) were assessed. The two LTD4 receptor antagonists (MK-571 and ICI-204,219) significantly reduced the permeability changes induced by indomethacin, while the two LTB4 antagonists (SC-41930 and LY-255,283) were without significant effect. Despite the reduction of gastric epithelial injury, blockade of LTD4 receptors did not markedly affect the extent of macroscopically visible injury. These data are consistent with the hypothesis that leukotrienes contribute to the epithelial injury and macroscopically visible damage induced by NSAIDs. However, it remains unclear to what extent leukotrienes are involved in the initiation of the injury, as opposed to its amplification.

Mechanisms underlying the protective effects of interleukin 1 in experimental nonsteroidal anti-inflammatory drug gastropathy.

Interleukin 1 (IL-1) has been shown to reduce the severity of experimental gastroduodenal ulceration, but the mechanism of action is unclear. The present study examined the possibility that the mechanism underlying the protective effects of IL-1 in experimental nonsteroidal anti-inflammatory drug (NSAID)-induced gastropathy is related to effects on gastric acid secretion, on prostaglandin synthesis, and/or on neutrophil function. IL-1 alpha and IL-1 beta dose-dependently (1-10 micrograms/kg) reduced the severity of gastric damage induced by indomethacin, whereas tumor necrosis factor alpha (1-10 micrograms/kg) had no effect. These effects of IL-1 were not completely attributable to a reduction in the volume or acidity of gastric secretion during the 1-hour pretreatment period. Whereas IL-1 alpha and IL-1 beta significantly inhibited pentagastrin-stimulated acid secretion, the dose-response relationship and time course of actions suggested that effects on acid secretion did not fully account for the ability of these agents to reduce indomethacin-induced gastric injury. The maximally effective dose of IL-1 beta (10 micrograms/kg) in terms of reduction of indomethacin-induced gastric injury did not significantly affect gastric prostaglandin synthesis. Neutrophil function was assessed using two in vivo assays. IL-1 beta inhibited migration of neutrophils in response to intradermal injections of N-formyl-methionyl-leucyl-phenylalanine and leukotriene B4 (LTB4) and dose-dependently (0.1-10 micrograms/kg) inhibited LTB4-induced neutropenia. These effects could be mimicked by dexamethasone (1 mg/kg SC), which inhibited the neutropenic response to LTB4 and significantly (P less than 0.001) reduced the severity of indomethacin-induced gastric damage. Both IL-1 beta and dexamethasone could significantly reduce the extent of histologically detectable leukocyte margination within the gastric mucosal microcirculation after indomethacin administration. The results of this study suggest that effects of IL-1 on gastric acid secretion or prostaglandin synthesis do not fully account for its ability to reduce the severity of experimental NSAID-induced gastropathy, whereas inhibitory effects of IL-1 on neutrophil function may contribute significantly to its protective actions.

Exacerbation of experimental colitis by nonsteroidal anti-inflammatory drugs is not related to elevated leukotriene B4 synthesis.

The ability of nonsteroidal anti-inflammatory drugs to exacerbate experimental colitis, and the possible contributions of the "shunting" of arachidonate via the 5-lipoxygenase pathway, were investigated using a rat model in which colitis was induced by intracolonic administration of trinitrobenzene sulfonic acid in a vehicle of 50% ethanol. Twice daily treatment with indomethacin (0.1-1 mg/kg SC) during the first week after trinitrobenzene sulfonic acid/ethanol administration resulted in dose-dependent increases in the severity of colitis and in the incidence of mortality. Mortality was not observed in vehicle-treated colitic rats or in normal rats treated with indomethacin. Similar exacerbation of colitis was observed in rats treated with naproxen (5 mg/kg). Whereas treatment with a 5-lipoxygenase inhibitor, PF-5901 (100 mg/kg PO), resulted in a significant reduction of the severity of colitis, concomitant administration of PF-5901 and indomethacin (0.5 mg/kg SC) did not inhibit the exacerbative effects of the indomethacin in this model. In separate studies, administration of indomethacin was found to significantly increase colonic myeloperoxidase activity (a measure of tissue granulocyte numbers) and suppress colonic prostaglandin E2 synthesis, while not significantly affecting colonic leukotriene B4 synthesis. The effect on myeloperoxidase activity was seen during the period 21-24 hours after trinitrobenzene sulfonic acid ethanol administration, but not during the period 45-48 hours after induction of colitis. In in vitro studies using samples of inflamed colon and in vivo studies in which colonic eicosanoid production was measured by colonic dialysis, inhibition of prostaglandin E2 synthesis was not accompanied by significant changes in leukotriene B4 synthesis. These results suggest that inhibitors of colonic prostaglandin synthesis can markedly exacerbate colitis, and that this effect is unrelated to alterations in colonic leukotriene B4 synthesis. Endogenous prostaglandins may exert anti-inflammatory effects during the acute stages of colitis.

Contrasting effects of PGE2 and PGD2: ion transport in the canine proximal colon.

Our earlier study [Am. J. Physiol. 256 (Gastrointest. Liver Physiol. 19): G673-G679, 1989] showed that the isomers prostaglandin E2 and D2 (PGE2 and PGD2) had contrasting effects on the canine proximal colonic epithelium in vitro. Whereas PGE2 produced sharp increases in short-circuit current (Isc), PGD2 rapidly reversed these changes. We report here that PGD2 reversed increases in Isc produced by other agonists (forskolin, carbachol) and was thus not a specific antagonist to PGE2. The transient increase in Isc and conductance produced by PGE2 were accompanied by alterations in Cl- but not Na+ fluxes. These were partitioned into early and late phases. In the early phase, net Cl- flux (JCl-net) decreased due to a reduction in Cl- mucosal to serosal flux (JCl-m----s) and increases in JCl-s----m. In the later phase, these changes appeared to revert to prestimulation values. Because forskolin produced more sustained increases in Isc, we used it as an agonist to define the effects of PGD2 on ion fluxes. Forskolin produced a marked reduction in JCl-net due to a decrease in JCl-m----s and an increase in JCl-s----m. PGD2 not only reversed the increases in Isc and conductance produced but also reversed the changes in Cl- flux.