Oleoylethanolamine and palmitoylethanolamine modulate intestinal permeability in vitro via TRPV1 and PPARα

Cannabinoids modulate intestinal permeability through cannabinoid receptor 1 (CB1). The endocannabinoid-like compounds oleoylethanolamine (OEA) and palmitoylethanolamine (PEA) play an important role in digestive regulation, and we hypothesized they would also modulate intestinal permeability. Transepithelial electrical resistance (TEER) was measured in human Caco-2 cells to assess permeability after application of OEA and PEA and relevant antagonists. Cells treated with OEA and PEA were stained for cytoskeletal F-actin changes and lysed for immunoassay. OEA and PEA were measured by liquid chromatography–tandem mass spectrometry. OEA (applied apically, logEC50 −5.4) and PEA (basolaterally, logEC50 −4.9; apically logEC50 −5.3) increased Caco-2 resistance by 20–30% via transient receptor potential vanilloid (TRPV)-1 and peroxisome proliferator-activated receptor (PPAR)-α. Preventing their degradation (by inhibiting fatty acid amide hydrolase) enhanced the effects of OEA and PEA. OEA and PEA induced cytoskeletal changes and activated focal adhesion kinase and ERKs 1/2, and decreased Src kinases and aquaporins 3 and 4. In Caco-2 cells treated with IFNγ and TNFα, OEA (via TRPV1) and PEA (via PPARα) prevented or reversed the cytokine-induced increased permeability compared to vehicle (0.1% ethanol). PEA (basolateral) also reversed increased permeability when added 48 or 72 h after cytokines (P < 0.001, via PPARα). Cellular and secreted levels of OEA and PEA (P < 0.001–0.001) were increased in response to inflammatory mediators. OEA and PEA have endogenous roles and potential therapeutic applications in conditions of intestinal hyperpermeability and inflammation

The role of CB1 in intestinal permeability and inflammation

The endocannabinoid system has previously been shown to play a role in the permeability and inflammatory response of the human gut. The goal of our study was to determine the effects of endogenous anandamide (AEA) and 2-arachidonoyl glycerol (2-AG) on the permeability and inflammatory response of intestinal epithelium under normal, inflammatory, and hypoxic conditions. Human intestinal mucosa was modeled using Caco-2 cells. Human tissue was collected from planned colorectal resections. Accumulation of AEA and 2-AG was achieved by inhibiting their metabolizing enzymes URB597 (a fatty acid amide hydrolase inhibitor) and JZL184 (a monoacylglycerol lipase inhibitor). Inflammation and ischemia were simulated with TNF-a and IFN-g and oxygen deprivation. Permeability changes were measured by transepithelial electrical resistance. The role of the CB1 receptor was explored using CB1-knockdown (CB1Kd) intestinal epithelial cells. Endocannabinoid levels were measured using liquid chromatography–mass spectrometry. Cytokine secretion was measured using multiplex and ELISA. URB597 and JZL184 caused a concentration-dependent increase in permeability via CB1 (P < 0.0001) and decreased cytokine production. Basolateral application of JZL184 decreased permeability via CB1 (P < 0.0001). URB597 and JZL184 increased the enhanced (worsened) permeability caused by inflammation and hypoxia (

Cannabidiol and palmitoylethanolamide are anti-inflammatory in the acutely inflamed human colon

Objective: We sought to quantify the anti-inflammatory effects of two cannabinoid drugs, cannabidiol (CBD) and palmitoylethanolamide (PEA), in cultured cell lines and compared this effect with experimentally inflamed explant human colonic tissue. These effects were explored in acutely and chronically inflamed colon, using inflammatory bowel disease and appendicitis explants. Design: Caco-2 cells and human colonic explants collected from elective bowel cancer, inflammatory bowel disease (IBD) or acute appendicitis resections, and were treated with the following drug treatments: vehicle, an inflammatory protocol of interferon γ (IFNγ) and tumour necrosis factor α (TNFα; 10 ng/ml), inflammation and PEA (10 µM), inflammation and CBD (10 µM), and PEA or CBD alone, CBD or vehicle were added simultaneously with IFNγ. Nine intracellular signalling phosphoproteins were determined by multiplex. Inflammatory cytokine secretion was determined using ELISA. Receptor mechanisms were investigated using antagonists for CB1, CB2, PPARα, PPARγ, TRPV1 and GPR55. Results: IFNγ and TNFα treatment increased phosphoprotein and cytokine levels in Caco-2 cultures and colonic explants. Phosphoprotein levels were significantly reduced by PEA or CBD in Caco-2 cultures and colonic explants. CBD and PEA prevented increases in cytokine production in explant colon, but not in Caco-2 cells. CBD effects were blocked by the CB2 antagonist AM630 and TRPV1 antagonist SB366791. PEA effects were blocked by the PPARα antagonist GW6471. PEA and CBD were anti- inflammatory in IBD and appendicitis explants. Conclusion: PEA and CBD are anti-inflammatory in the human colon. This effect is not seen in cultured epithelial cells. Appropriately sized clinical trials should assess their efficac

Endocannabinoids and endocannabinoid-like compounds modulate hypoxia-induced permeability in CaCo-2 cells via CB1, TRPV1, and PPARα

Background and purpose We have previously reported that endocannabinoids modulate permeability in Caco-2 cells under inflammatory conditions and hypothesised in the present study that endocannabinoids could also modulate permeability in ischemia/reperfusion. Experimental approach Caco-2 cells were grown on cell culture inserts to confluence. Trans-epithelial electrical resistance (TEER) was used to measure permeability. To generate hypoxia (0% O2), a GasPak™ EZ anaerobe pouch system was used. Endocannabinoids were applied to the apical or basolateral membrane in the presence or absence of receptor antagonists. Key results Complete hypoxia decreased TEER (increased permeability) by ~35% after 4 h (recoverable) and ~50% after 6 h (non-recoverable). When applied either pre- or post-hypoxia, apical application of N-arachidonoyl-dopamine (NADA, via TRPV1), oleamide (OA, via TRPV1) and oleoylethanolamine (OEA, via TRPV1) inhibited the increase in permeability. Apical administration of anandamide (AEA) and 2-arachidonoylglycerol (2-AG) worsened the permeability effect of hypoxia (both via CB1). Basolateral application of NADA (via TRPV1), OA (via CB1 and TRPV1), noladin ether (NE, via PPARα), and palmitoylethanolamine (PEA, via PPARα) restored permeability after 4 h hypoxia, whereas OEA increased permeability (via PPARα). After 6 h hypoxia, where permeability does not recover, only basolateral application PEA sustainably decreased permeability, and NE decreased permeability. Conclusions and implications A variety of endocannabinoids and endocannabinoid-like compounds modulate Caco-2 permeability in hypoxia/reoxygenation, which involves multiple targets, depending on whether the compounds are applied to the basolateral or apical membrane. CB1 antagonism and TRPV1 or PPARα agonism may represent novel therapeutic targets against several intestinal disorders associated with increased permeability

Endocannabinoids and endocannabinoid-like compounds modulate hypoxia-induced permeability in CaCo-2 cells via CB1, TRPV1, and PPAR alpha

Background and purpose: We have previously reported that endocannabinoids modulate permeability in Caco-2 cells under inflammatory conditions and hypothesised in the present study that endocannabinoids could also modulate permeability in ischemia/reperfusion. Experimental approach: Caco-2 cells were grown on cell culture inserts to confluence. Trans-epithelial electrical resistance (TEER) was used to measure permeability. To generate hypoxia (0% O2), a GasPak™ EZ anaerobe pouch system was used. Endocannabinoids were applied to the apical or basolateral membrane in the presence or absence of receptor antagonists. Key results: Complete hypoxia decreased TEER (increased permeability) by ~35% after 4 h (recoverable) and ~50% after 6 h (non-recoverable). When applied either pre- or post-hypoxia, apical application of N-arachidonoyl-dopamine (NADA, via TRPV1), oleamide (OA, via TRPV1) and oleoylethanolamine (OEA, via TRPV1) inhibited the increase in permeability. Apical administration of anandamide (AEA) and 2-arachidonoylglycerol (2-AG) worsened the permeability effect of hypoxia (both via CB1). Basolateral application of NADA (via TRPV1), OA (via CB1 and TRPV1), noladin ether (NE, via PPARα), and palmitoylethanolamine (PEA, via PPARα) restored permeability after 4 h hypoxia, whereas OEA increased permeability (via PPARα). After 6 h hypoxia, where permeability does not recover, only basolateral application PEA sustainably decreased permeability, and NE decreased permeability. Conclusions and implications: A variety of endocannabinoids and endocannabinoid-like compounds modulate Caco-2 permeability in hypoxia/reoxygenation, which involves multiple targets, depending on whether the compounds are applied to the basolateral or apical membrane. CB1 antagonism and TRPV1 or PPARα agonism may represent novel therapeutic targets against several intestinal disorders associated with increased permeability

Interferon γ treatment increases endocannabinoid and related N -acylethanolamine levels in T84 human colon carcinoma cells : NAEs in IFNγ-treated T84 cells

BACKGROUND AND PURPOSE: Endocannabinoids and related N-acylethanolamines (NAEs) are involved in regulation of gut function, but relatively little is known as to whether inflammatory cytokines such as IFNγ affect their levels. We have investigated this in vitro using cultures of T84 colon cancer cells. EXPERIMENTAL APPROACH: T84 cells, when cultured in monolayers, differentiate to form adult colonic crypt-like cells with excellent permeability barrier properties. The integrity of the permeability barrier in these monolayers was measured using transepithelial electrical resistance (TEER). NAE levels were determined by ultra-performance liquid chromatography-tandem mass spectrometric analysis. Expression of the enzymes involved in NAE and 2-arachidonoylglycerol (2-AG) turnover were assessed with qPCR. KEY RESULTS: IFNγ treatment for 8 or 24 h increased levels of both endocannabinoids (anandamide and 2-AG) and the related NAEs. The treatment did not affect the rate of hydrolysis of either anandamide or palmitoylethanolamide by intact cells, and in both cases, fatty acid amide hydrolase (FAAH) rather than NAE-hydrolysing acid amidase (NAAA) was mainly responsible for the hydrolysis of these NAEs. IFNγ treatment reduced the TEER of the cells in a manner that was not prevented by inhibition of either FAAH or NAAA but was partially reversed by apical administration of the NAE palmitoylethanolamide. CONCLUSION AND IMPLICATIONS: IFNγ treatment mobilized endocannabinoid and related NAE levels in T84 cells. However, blockade of anandamide or NAE hydrolysis was insufficient to negate the deleterious effects of this cytokine upon the permeability barrier of the cell monolayers