Catherine M. Keenan

Targeting endocannabinoid degradation protects against experimental colitis in mice: involvement of CB1 and CB2 receptors

The endocannabinoid (EC) system mediates protection against intestinal inflammation. In this study, we investigated the effects of blocking EC degradation or cellular reuptake in experimental colitis in mice. Mice were treated with trinitrobenzene-sulfonic acid in presence and absence of the fatty acid amide hydrolase (FAAH) blocker URB597, the EC membrane transport inhibitor VDM11, and combinations of both. Inflammation was significantly reduced in the presence of URB597, VDM11, or both as evaluated by macroscopic damage score, myeloperoxidase levels, and colon length. These effects were abolished in CB1- and CB2-receptor-gene-deficient mice. Quantitative reverse transcription polymerase chain reaction after induction of experimental colitis by different pathways showed that expression of FAAH messenger RNA (mRNA) is significantly reduced in different models of inflammation early in the expression of colitis, and these return to control levels as the disease progresses. Genomic DNA from 202 patients with Crohn’s disease (CD) and 206 healthy controls was analyzed for the C385A polymorphism in the FAAH gene to address a possible role in humans. In our groups, the C385A polymorphism was equally distributed in patients with CD and healthy controls. In conclusion, drugs targeting EC degradation offer therapeutic potential in the treatment of inflammatory bowel diseases. Furthermore, reduction of FAAH mRNA expression is involved in the pathophysiological response to colitis.

Cannabinoid CB2 receptors in the enteric nervous system modulate gastrointestinal contractility in lipopolysaccharide-treated rats

Enhanced intestinal transit due to lipopolysaccharide (LPS) is reversed by cannabinoid (CB)2 receptor agonists in vivo, but the site and mechanism of action are unknown. We have tested the hypothesis that CB2 receptors are expressed in the enteric nervous system and are activated in pathophysiological conditions. Tissues from either saline- or LPS-treated (2 h; 65 microg/kg ip) rats were processed for RT-PCR, Western blotting, and immunohistochemistry or were mounted in organ baths where electrical field stimulation was applied in the presence or absence of CB receptor agonists. Whereas the CB2 receptor agonist JWH133 did not affect the electrically evoked twitch response of the ileum under basal conditions, in the LPS-treated tissues JWH133 was able to reduce the enhanced contractile response in a concentration-dependent manner. Rat ileum expressed CB2 receptor mRNA and protein under physiological conditions, and this expression was not affected by LPS treatment. In the myenteric plexus, CB2 receptors were expressed on the majority of neurons, although not on those expressing nitric oxide synthase. LPS did not alter the distribution of CB2 receptor expression in the myenteric plexus. In vivo LPS treatment significantly increased Fos expression in both enteric glia and neurons. This enhanced expression was significantly attenuated by JWH133, whose action was reversed by the CB2 receptor antagonist AM630. Taking these facts together, we conclude that activation of CB2 receptors in the enteric nervous system of the gastrointestinal tract dampens endotoxin-induced enhanced intestinal contractility.

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-inducec 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 visibls 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.

Effects of Leukotrienes on Susceptibility of the Rat Stomach to Damage and Investigation of the Mechanism of Action

The ability of various leukotrienes to alter the susceptibility of the rat gastric mucosa to injury by 20% ethanol and the possible mechanism of action were examined using an ex vivo gastric chamber preparation. Intraarterial infusions of leukotriene B4 or N-acetyl leukotriene E4 (0.01-1.0 microgram/kg per min for 10 min) had no significant effect on the extent of damage induced by topically applied 20% ethanol. However, infusion of leukotriene C4, D4, or E4 (1.0 micrograms/kg per min) significantly increased ethanol-induced damage, as measured macroscopically, histologically, and functionally. Lower doses of leukotriene C4, D4, or E4 were without significant effect in this model. The increase in damage induced by these three leukotrienes could be blocked by pretreatment with either of two structurally unrelated leukotriene D4 antagonists (L-649,923 or L-660,711). The augmentation of damage by leukotriene C4 was not affected by pretreatment with indomethacin or with a specific thromboxane A2-receptor antagonist (L-670,596). At the dose that increased ethanol-induced damage, none of the leukotrienes tested significantly altered gastric vascular permeability, as measured by Evans blue leakage. However, using laser-Doppler flowmetry, leukotrienes C4 and D4 were found, when administered intraarterially at doses in the 0.05-1.0 micrograms/kg per min range, to produce dose-dependent reductions of gastric blood flow while N-acetyl leukotriene E4 was without effect and leukotriene B4 induced slight increases. The effects of leukotrienes C4 and D4 on gastric blood flow could be inhibited by the two leukotriene D4 antagonists but not by the thromboxane antagonist. These results demonstrate that although they do not produce damage by themselves, leukotrienes C4, D4, and E4 are capable of augmenting ethanol-induced injury to the gastric mucosa. Changes in vascular permeability do not appear to play a role in the mechanism of action of the leukotrienes, while their effects on gastric blood flow are likely to be important. Under certain condition it is therefore possible that local release of leukotrienes could, at least in part through reducing vascular perfusion, predispose the surrounding tissue necrosis.

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

Abstract  Oleoylethanolamide (OEA) is an endogenous lipid produced in the intestine that mediates satiety by activation of peroxisome proliferator‐activated receptor alpha (PPARα). 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 PPARα. PPARα immunoreactivity was examined in whole mount preparations of mouse gastrointestinal (GI) tract. The effect of OEA on motility was assessed in wildtype, PPARα, cannabinoid CB1 receptor and CB2 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 PPARα antagonist GW6471, transient receptor potential vanilloid 1 antagonist SB366791 or the glucagon‐like peptide 1 antagonist exendin‐3(9‐39) amide. PPARα 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 PPARα, CB1 and CB2 receptor gene‐deficient mice and in the presence of GW6471, SB366791 and exendin‐3(9‐39) amide, suggesting neither PPARα 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 PPARα is found in the enteric nervous system, but our results suggest that PPARα is not involved in the suppression of motility by OEA.

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.

Leukotriene B4 potentiates colonic ulceration in the rat

The ability of various leukotrienes, platelet-activating factor andN-formyl-methyl-leucyl-phenylalanine to augment colonic damage induced by 30% ethanol was investigated in the rat. Each of the mediators was tested at a dose of 2 nmol, administered intracolonically in the ethanol vehicle. When colonic damage was assessed 72 hr later, only leukotriene B4 significantly augmented damage compared to the controls. The incidence of ulcers increased from 35% in the control group to 90% in the group receiving leukotriene B4. Leukotriene B4 administration also resulted in significant increases in colonic myeloperoxidase activity and colonic leukotriene B4 synthesis. To assess the possible contribution of infiltrating neutrophils to the increase in colonic leukotriene B4 synthesis that accompanies colonic inflammation, colitis was induced in normal and neutropenic rats by intracolonic administration of trinitrobenzene sulfonic acid. Neutropenia was achieved by treatment with an antineutrophil serum. In the neutropenic animals killed 4 hr after induction of colitis significant changes in leukotriene B4 synthesis were not observed, whereas a fourfold increase was observed in the controls. From these studies we conclude the following: (1) leukotriene B4, at a dose of 2 nmol, can significantly potentiate the colonic ulceration induced by 30% ethanol; (2) this action of leukotriene B4 is not shared by the same dose of the other inflammatory mediators tested; and (3) infiltrating neutrophils are the major source of colonic leukotriene B4 synthesis in a rat model of colitis.

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

Background  Cannabinoid type 1 (CB1) receptors are involved in the regulation of gastrointestinal (GI) motility and secretion. Our aim was to characterize the roles of the CB1 receptor on GI motility and secretion in vitro and in vivo by using different classes of CB1 receptor antagonists.

Reduction of the severity of experimental gastric and duodenal ulceration by interleukin-1β

Interleukin-1 beta (IL-1 beta) has been reported to stimulate prostaglandin synthesis by the rat stomach in vitro and to inhibit gastric acid secretion in vivo. We have therefore tested the hypothesis that IL-1 beta might have protective actions in experimental models of gastroduodenal ulceration. IL-1 beta, given i.p., dose and time dependently reduced the severity of ethanol-induced gastric damage. A pretreatment time of 90 min was found to produce the greatest reduction of damage, while doses of 0.1 micrograms/kg or greater were found to produce significant effects. The protective actions of IL-1 beta were abolished by prior boiling or by pretreatment of the animals with indomethacin, and were not shared by the nonapeptide fragment 163-171. IL-1 beta also reduced the severity of gastric damage induced by indomethacin and the duodenal ulceration induced by cysteamine. The results indicate that IL-1 beta has protective actions in three separate experimental models of gastroduodenal ulceration. The mechanism of action of IL-1 beta is not entirely clear, but contributions of endogenous prostaglandin synthesis and inhibition of gastric acid secretion cannot be excluded.

Comparison of the effects of endothelin-1 and endothelin-3 on the rat stomach

The effects of systemic administration of endothelin-1 and endothelin-3 on the susceptibility of the stomach to injury were compared in the anesthetized rat, as were their effects on gastric vascular tone, systemic blood pressure and hematocrit. When infused at concentrations in the 10(-7)-10(-6) M range, endothelin-1 was a far more potent hypertensive agent than endothelin-3. Endothelin-1 caused significant hemoconcentration, while endothelin-3 did not Endothelin-1 was approximately 5- to 10-times more potent as a vasoconstrictor in the stomach and a similar difference in potencies was observed when the ability of these peptides to increase the susceptibility of the stomach to ethanol-induced damage was compared. The two peptides were equipotent in producing gastric mucosal hemorrhage in the absence of any exogenous irritant. These results demonstrate that like endothelin-1, endothelin-3 has ulcerogenic and vasoconstriction actions in the stomach. While there are very large differences in the potencies of the two peptides in terms of producing systemic hypertension and hemoconcentration, the differences in the potency of the gastric ulcerogenic and vasoconstrictor effects are much less marked.