Cannabinoids modulate intestinal permeability through cannabinoid receptor 1 (CB). 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, logEC -5.4) and PEA (basolaterally, logEC -4.9; apically logEC -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.-Karwad, M. A., Macpherson, T., Wang, B., Theophilidou, E., Sarmad, S., Barrett, D. A., Larvin, M., Wright, K. L., Lund, J. N., O'Sullivan, S. E. Oleoylethanolamine and palmitoylethanolamine modulate intestinal permeability in vitro via TRPV1 and PPARα.

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-α and IFN-γ and oxygen deprivation. Permeability changes were measured by transepithelial electrical resistance. The role of the CB receptor was explored using CB-knockdown (CBKd) 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 CB ( < 0.0001) and decreased cytokine production. Basolateral application of JZL184 decreased permeability CB ( < 0.0001). URB597 and JZL184 increased the enhanced (worsened) permeability caused by inflammation and hypoxia ( < 0.0001 and < 0.05). CBKd cells showed reduced permeability response to inflammation ( < 0.01) but not hypoxia. 2-AG levels were increased in response to inflammation and hypoxia in Caco-2 cells. In human mucosal tissue, inflammation increased the secretion of granulocyte macrophage-colony stimulating factor, IL-12, -13, and -15, which was prevented with treatment with URB597 and JZL184, and was inhibited by a CB antagonist. The results of this study show that endogenous AEA and 2-AG production and CB activation play a key modulatory roles in normal intestinal mucosa permeability and in inflammatory and hypoxic conditions.-Karwad, M. A., Couch, D. G., Theophilidou, E., Sarmad, S., Barrett, D. A., Larvin, M., Wright, K. L., Lund, J. N., O'Sullivan, S. E. The role of CB in intestinal permeability and inflammation.

Background and purpose: We have previously reported that endocannabinoids modulate intestinal permeability in Caco-2 cells under inflammatory conditions and hypothesised in the present study that endocannabinoids could also modulate intestinal permeability in ischemia/reperfusion. Experimental approach: Caco-2 cells were grown on cell culture inserts to confluence. Transepithelial electrical resistance (TEER) was used to measure permeability. To generate hypoxia (0% O 2), 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-aArachidonoylglycerol (2-AG) worsened the permeability effect of hypoxia (both via CB 1). Basolateral application of NADA (via TRPV1), OA (via CB 1 and TRPV1), noladin ether (NE, via PPAR), 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 intestinal Caco-2 permeability in hypoxiischemia/reoxygenationreperfusion, which involves multiple targets, depending on whether the compounds are applied to the basolateral or apical membrane. CB 1 antagonism and TRPV1 or PPAR agonism may represent novel therapeutic targets against several intestinal disorders associated with increased intestinal permeability.