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% O-2), a GasPak (TM) 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 similar to 35% after 4 h (recoverable) and similar to 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 PPAR alpha), and palmitoylethanolamine (PEA, via PPAR alpha) restored permeability after 4 h hypoxia, whereas OEA increased permeability (via PPAR alpha). 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 PPAR alpha agonism may represent novel therapeutic targets against several intestinal disorders associated with increased permeability.

BACKGROUND AND PURPOSE Activation of cannabinoid receptors decreases emesis, inflammation, gastric acid secretion and intestinal motility. The ability to modulate intestinal permeability in inflammation may be important in therapy aimed at maintaining epithelial barrier integrity. The aim of the present study was to determine whether cannabinoids modulate the increased permeability associated with inflammation in vitro . EXPERIMENTAL APPROACH Confluent Caco‐2 cell monolayers were treated for 24 h with IFNγ and TNFα (10 ng·mL −1 ). Monolayer permeability was measured using transepithelial electrical resistance and flux measurements. Cannabinoids were applied either apically or basolaterally after inflammation was established. Potential mechanisms of action were investigated using antagonists for CB 1 , CB 2 , TRPV1, PPAR γ and PPAR α . A role for the endocannabinoid system was established using inhibitors of the synthesis and degradation of endocannabinoids. KEY RESULTS Δ 9 ‐Tetrahydrocannabinol (THC) and cannabidiol accelerated the recovery from cytokine‐induced increased permeability; an effect sensitive to CB 1 receptor antagonism. Anandamide and 2‐arachidonylglycerol further increased permeability in the presence of cytokines; this effect was also sensitive to CB 1 antagonism. No role for the CB 2 receptor was identified in these studies. Co‐application of THC, cannabidiol or a CB 1 antagonist with the cytokines ameliorated their effect on permeability. Inhibiting the breakdown of endocannabinoids worsened, whereas inhibiting the synthesis of endocannabinoids attenuated, the increased permeability associated with inflammation. CONCLUSIONS AND IMPLICATIONS These findings suggest that locally produced endocannabinoids, acting via CB 1 receptors play a role in mediating changes in permeability with inflammation, and that phytocannabinoids have therapeutic potential for reversing the disordered intestinal permeability associated with inflammation. LINKED ARTICLES This article is part of a themed section on Cannabinoids in Biology and Medicine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue‐8 . To view Part I of Cannabinoids in Biology and Medicine visit http://dx.doi.org/10.1111/bph.2011.163.issue‐7

Activation of cannabinoid receptors decreases emesis, inflammation, gastric acid secretion, and intestinal motility. However, the effects of cannabinoids on intestinal permeability have not yet been established. The aim of the present study is to examine the effects of cannabinoids on intestinal permeability in an in vitro model. Caco-2 cells were grown until fully confluent on inserts in 12-well plates. Transepithelial electrical resistance (TEER) measurements were made as a measure of permeability. EDTA (50 μM) was applied to reversibly increase permeability (reduce TEER). The effects of cannabinoids on permeability in combination with EDTA, or alone, were assessed. Potential target sites of action were investigated using antagonists of the cannabinoid (CB)1 receptor, CB2 receptor, transient receptor potential vanilloid subtype 1 (TRPV1), peroxisome proliferator-activated receptor (PPAR)γ, PPARα, and a proposed cannabinoid receptor. When applied to the apical or basolateral membrane of Caco-2 cells, Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) enhanced the speed of recovery of EDTA-induced increased permeability. This effect was sensitive to cannabinoid CB1 receptor antagonism only. Apical application of endocannabinoids caused increased permeability, sensitive to cannabinoid CB1 receptor antagonism. By contrast, when endocannabinoids were applied basolaterally, they enhanced the recovery of EDTA-induced increased permeability, and this involved additional activation of TRPV1. All cannabinoids tested increased the mRNA of the tight junction protein zona occludens-1, but only endocannabinoids also decreased the mRNA of claudin-1. These findings suggest that endocannabinoids may play a role in modulating intestinal permeability and that plant-derived cannabinoids, such as THC and CBD, may have therapeutic potential in conditions associated with abnormally permeable intestinal epithelium.

The emerging potential for the cannabinoid (CB) system in modulating gastrointestinal inflammation has gained momentum over the last few years. Traditional and anecdotal use of marijuana for gastrointestinal disorders, such as diarrhoea and abdominal cramps is recognized, but the therapeutic benefit of cannabinoids in the 21st century is overshadowed by the psychoactive problems associated with CB1 receptor activation. However, the presence and function of the CB2 receptor in the GI tract, whilst not yet well characterized, holds great promise due to its immunomodulatory roles in inflammatory systems and its lack of psychotropic effects. This review of our current knowledge of CB2 receptors in the gastrointestinal tract highlights its role in regulating abnormal motility, modulating intestinal inflammation and limiting visceral sensitivity and pain. CB2 receptors represent a braking system and a pathophysiological mechanism for the resolution of inflammation and many of its symptoms. CB2 receptor activation therefore represents a very promising therapeutic target in gastrointestinal inflammatory states where there is immune activation and motility dysfunction.

Intestinal myofibroblasts have been implicated in the pathogenesis of chronic inflammatory conditions such as Crohn's disease via interactions with an elaborate network of cytokines, growth factors, and other inflammatory mediators. CXCR3 is a Galpha(i) protein-coupled receptor that binds the proinflammatory chemokines CXCL9, CXCL10, and CXCL11, which are released from the intestinal epithelium. The three CXCR3 ligands shared the ability to activate biochemical (e.g., PI3K and MAPK activation) and functional events (actin reorganization) in intestinal myofibroblasts. However, CXCL11 is unique in its ability to elevate intracellular calcium. Surprisingly, although CXCR3 mRNA is detectable in these myofibroblasts, there is no detectable surface expression of CXCR3. Furthermore, the biochemical responses and actin reorganization stimulated by the CXCR3 ligands in intestinal myofibroblasts are insensitive to the Galpha(i) inhibitor, pertussis toxin. This suggests either the existence of differential receptor coupling mechanisms in myofibroblasts for CXCR3 that are distinct from those observed in PBLs and/or that these cells express a modified or variant CXCR3 compared with the CXCR3 expressed on PBLs.

Ligands of peroxisome proliferator-activated receptor-gamma (PPAR(gamma)) are thought to possess anti-inflammatory properties mediated via both PPAR(gamma) dependent and independent mechanisms. This work investigates the effects of PPAR(gamma) ligands on the regulation of cyclooxygenase-2 (COX-2) in the human lung epithelial cell line, A549. The synthetic ligand troglitazone activated the phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase pathway (MAPK), whereas the endogenous ligand, 15-deoxy-Delta(12,14)-prostaglandin J2 (15d-PGJ2), only activated the PI3K pathway. 15d-PGJ2 had no detectable effects on COX-2, mPGES expression, or PGE2 production. However, troglitazone induced time-dependent COX-2 expression, which was insensitive to PPAR(gamma) antagonists, but was abrogated by inhibitors of PI3K and the ERK MAP kinase pathway. Furthermore, troglitazone induced mPGES expression and PGE2 production. Neither troglitazone nor 15d-PGJ2 was able to convincingly activate NF-kappaB in A549 cells. Further heterogeneity in the responses to troglitazone and 15d-PGJ2 was observed in the regulation of gene expression as assessed by microarray analysis. In summary, this study provides compelling evidence that troglitazone (like 15d-PGJ2) can exert functional effects independently of actions via PPAR(gamma). Moreover, we have identified unique biochemical and functional actions of troglitazone that are not shared by 15d-PGJ2, which may influence the therapeutic potential of this compound in inflammatory settings.

Cyclooxygenase (COX)‐2 expression and activity in response to pro‐inflammatory cytokines TNF α and IFN γ was evaluated in the colonic epithelial cell line HT29 and the airway epithelial cell line A549. TNF α induced concentration‐ and time‐dependent upregulation of COX‐2 mRNA, protein and prostaglandin (PG)E 2 synthesis. Co‐stimulation of TNF α with IFN γ resulted in reduced COX‐2 mRNA and protein expression. IFN γ had no effect on the stability of TNF α ‐induced COX‐2 mRNA. TNF α ‐induced PGE 2 biosynthesis was significantly enhanced by the simultaneous addition of IFN γ and was COX‐2 dependent. The combination of IFN γ and TNF α induced the microsomal prostaglandin E synthase (mPGES), comensurate with the enhanced PGE 2 synthesis. These results suggest that, in terms of PGE 2 biosynthesis, IFN γ plays a negative regulatory role at the level of COX‐2 expression and a positive regulatory role at the level of mPGES expression. This may have important implications for the clinical use of IFN γ in inflammatory diseases. British Journal of Pharmacology (2004) 141 , 1091–1097. doi: 10.1038/sj.bjp.0705719

Stromal cell-derived factor-1 (SDF-1) and its receptor CXCR4 are a multifunctional chemokine/receptor system with essential roles in the development of the immune system and other aspects of embryogenesis, including vascularization and organ development. SDF-1 is also a potent chemoattractant for T cells and has roles in both inflammation and immune homeostasis. Our group has previously demonstrated that phosphoinositide 3-kinase (PI 3-kinase) is activated in SDF-1-stimulated T cells and is indeed required for SDF-1-mediated chemotaxis. In this study Jurkat clones were established, stably expressing dominant negative constructs of class IA and class IB PI 3-kinases under the control of the tetracycline off inducible gene system, to determine the relative roles of these PI 3-kinases in SDF-1 signaling. Our results show. that expression of either kinase-dead PI3Kgamma (KD-PI3Kgamma) or Deltap85 (a construct unable to bind class I-A p110alpha, -beta, or -delta) leads to a partial inhibition of SDF-1-stimulated protein kinase B phosphorylation, but had no effect on SDF-1-induced phosphorylation of the mitogen-activated protein kinase ERK1/2. Functional studies demonstrated that expression of KD-PI3Kgamma markedly inhibited SDF-1-mediated chemotaxis, typically eliciting 40-60% inhibition. Interestingly, the expression of Deltap85 also leads to inhibition of the SDF-1-mediated chemotactic response, albeit to a much lesser extent than achieved with the KD-PI3Kgamma mutant, typically in the range of 20-40% inhibition. Furthermore, the inhibition of chemotaxis by the expression of dominant negative class IA or class IB PI 3-kinases could be enhanced by the presence of the PI 3-kinase inhibitor LY294002. Together, these results demonstrate that optimal chemotactic response of leukemic T cells to SDF-1 requires the activation of both class IA and class IB PI 3-kinases.

The leukemic T cell line Jurkat is deficient in protein expression of the lipid phosphatases Src homology 2 domain containing inositol polyphosphate phosphatase (SHIP) and phosphatase and tensin homolog deleted on chromosome ten (PTEN). We examined whether the lack of expression of SHIP-1 and PTEN is shared by other leukemic T cell lines and PBLs. Analysis of a range of cell lines and PBLs revealed that unlike Jurkat cells, two other well-characterized T cell lines, namely CEM and MOLT-4 cells, expressed the 5'-phosphatase SHIP at the protein level. However, the 3-phosphatase PTEN was not expressed by CEM or MOLT-4 cells or Jurkat cells. The AUT78 cell line and PBLs expressed both SHIP and PTEN. Jurkat cells exhibited high basal levels of phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P-3; the lipid substrate for both SHIP and PTEN) as well as saturated protein kinase B (PKB) phosphorylation. Lower levels of PI(3,4,5)p(3) and higher levels' of phosphatidylinositol 3,4-bisphosphate (PI(3,4)P-2) as well as unsaturated constitutive phosphorylation of PKB were observed in CEM and MOLT-4 cells compared with Jurkat cells. In PBLs and HUT78 cells which express both PTEN and SHIP-1, there was no constitutive PI(3,4,5)p3 or PKB phosphorylation, and receptor stimuli were able to elicit robust phosphorylation of PKB. Expression of a constitutively active SHIP-1 protein in Jurkat cells was sufficient to reduce both constitutive PKB membrane localization and PKB phosphorylation. Together, these data indicate important differences between T leukemic cells as well as PBLs, regarding expression of key lipid phosphatases. This study provides the first evidence that SHIP-1 can influence the constitutive levels of PI(3,4,5)P3 and the activity of downstream phosphoinositide 3-kinase effectors in T lymphocytes.

BACKGROUND AND AIMS:Cyclooxygenase (COX)-2 is up-regulated in most colonic cancers and in inflammatory bowel disease in which tumor necrosis factor (TNF)-alpha is believed to play a central role. There has been recent speculation on the activation of phosphatidylinositol 3-kinase (PI 3-kinase) by TNF-alpha and its role in the regulation of genes controlled by NF-kappaB. We investigated the regulatory role of PI 3-kinase on COX-2 expression in colonic epithelial cells.METHODS:In HT-29 and Caco-2 colonic epithelial cells, COX-2 expression was induced by either TNF-alpha or interleukin (IL)-1alpha as observed by Northern and Western analyses. COX-2 activity was assessed by measuring prostaglandin E(2) (PGE2) production by enzyme-linked immunosorbent assay. NF-kappaB binding activity was assessed by electrophoretic mobility shift assay. PI 3-kinase activity was measured by quantifying the accumulation of PI 3-kinase-dependent D-3 lipid products by high-performance liquid chromatography.RESULTS:The PI 3-kinase inhibitor wortmannin up-regulated induced COX-2 expression in a concentration-dependent manner in both HT-29 and Caco-2 cells. An alternative PI 3-kinase inhibitor, LY294002, caused up-regulation of induced COX-2 messenger RNA (mRNA) in HT-29 cells at concentrations of < or =1 micromol/L. IL-4 and IL-13, which are known to activate PI 3-kinase, down-regulated HT-29 COX-2 mRNA, protein, and PGE2 production. NF-kappaB binding activity was unaltered by PI 3-kinase inhibition in HT-29 cells, in which TNF-alpha was shown to activate PI 3-kinase directly.CONCLUSIONS:COX-2 is negatively regulated by PI 3-kinase; we propose that the inhibitory effect of IL-4 and IL-13 is mediated via a PI 3-kinase-dependent pathway. This mechanism does not appear to involve NF-kappaB because PI 3-kinase inhibition did not alter NF-kappaB binding activity. TNF-alpha can activate PI 3-kinase directly in addition to inducing COX-2.

of IL-8 mRNA.This synergy was IL-8 specific, since TNF-oLinduced claudin-2 mRNA expression was inhibited in the presence of MG-132.Neither MG-132 nor lactacystin decreased NF-KB binding activity induced by TNF-o~.In contrast, MG-132, but not lactacystin induced a caspase-3 dependent activation of NF-KB, which correlated with an increase of p65 and IKB/~, but decrease of IKB~ expression.In addition, MG-132 induced the strong activation of ERK1/2-, p38-and JNK1/2-MAP-Kinases.However, prevention of NF-KB activation with the caspase-3 inhibitor zDEVD-FMK, inhibition of p38 with SB203580 or ERK activation with PD98059, did not prevent MG-132 induced IL-8 mRNA expression.Instead, the activation of JNK1/2 by MG-132 resulted in the phosphorylation of c-Jun and the subsequent activation AP-1 consensus binding activity.Conclusions: IL-8 mRNA expression can be regulated independently of NF-KB, ERK-, and p38-MAP-Kinases in T-84 cells.MG-132 can act as a strong inducer of IL-8 mRNA expression, independent of its function as proteosome inhibitor or activator of caspase-3.MG-132 induced IL-8 mRNA expression may involve JNK1/2 dependent AP-1 activation and mRNA stabilization by a p38 independent pathway.

BACKGROUND/AIMS:Nitric oxide synthesis is increased in rectal biopsies from patients with ulcerative colitis and colonic epithelial cells are considered to be a major source of nitric oxide in intestinal inflammation.METHODOLOGY:Human colonic biopsies from normal bowel mucosa and colonic epithelial cell line HT-29 were cultured in the presence of the inflammatory cytokines IL-1 alpha + TNF-alpha + IFN-alpha added after 1 hour pretreatment with vehicle or Interleukin-13. Nitrite levels were determined at 30 hours in culture supernatants by a fluorometric assay.RESULTS:Unstimulated human colonic biopsies and HT-29 cells produced a basal amount of nitrite. Stimulation with IL-1 alpha + TNF-alpha + IFN-alpha induced a significant (P < 0.001) increase of nitrite generation by both human colonic biopsies and HT-29 cells. The presence of Interleukin-13 produced a significant (P < 0.001) suppression of the cytokine-induced nitrite generation from both colonic biopsies and HT-29 cells.CONCLUSIONS:Nitric oxide generation in human colonic mucosa is susceptible to manipulation by proinflammatory cytokines. Interleukin-13 has an inhibitory effect on cytokine induced nitrite production in colonic mucosa and could play an anti-inflammatory role in intestinal inflammation.