Proteasome inhibitors exacerbate interleukin-8 production induced by protease-activated receptor 2 in intestinal epithelial cells.

Protease activated receptors (PARs) and the ubiquitin-proteasome system (UPS) regulate inflammatory response in intestinal cells. We aimed to elucidate putative connections between PARs and UPS pathways in intestinal epithelial cells. Caco-2 cells were treated by agonist peptides of PARs and/or IL-1ß and/or proteasome inhibitors, bortezomib or MG132. Inflammatory response was evaluated by measuring IL-8 production. Proteasome activities were also evaluated. We showed that PAR-1 and -2 activation increased release of IL-8 compared with vehicle and independently of IL-1ß. In contrast, PAR-4 agonist peptide had no effect. Caspase-like and chymotrypsin-like proteasomal activities were increased by PAR-2 activation only in the presence of IL-1ß. Interestingly, in polarized Caco-2 cells, the release of IL-8 was predominantly upregulated in the side where PAR-2 agonist peptide was added, apical or basalolateral. In contrast, proteasome activities were only affected when PAR-2 agonist peptide was added in the apical side. Proteasome inhibitors, bortezomib and MG132, enhanced IL-8 production in both sides, apical and basolateral. In conclusion, PAR-2 activation alone did not affect proteasome but needed inflammatory stimulus IL-1ß to synergistically increase chymotrypsin-like activity in intestinal epithelial cells. However, proteasome inhibition led to exacerbate inflammatory response induced by PAR-2 activation.

Glutamine Restores Tight Junction Protein Claudin-1 Expression in Colonic Mucosa of Patients With Diarrhea-Predominant Irritable Bowel Syndrome.

BACKGROUND: Recent studies showed that patients with diarrhea-predominant irritable bowel syndrome (IBS-D) had an increased intestinal permeability as well as a decreased expression of tight junctions. Glutamine, the major substrate of rapidly dividing cells, is able to modulate intestinal permeability and tight junction expression in other diseases. We aimed to evaluate, ex vivo, glutamine effects on tight junction proteins, claudin-1 and occludin, in the colonic mucosa of patients with IBS-D. MATERIALS AND METHODS: Twelve patients with IBS-D, diagnosed with the Rome III criteria, were included (8 women/4 men, aged 40.7 ± 6.9 years). Colonic biopsy specimens were collected and immediately incubated for 18 hours in culture media with increasing concentrations of glutamine from 0.6-10 mmol/L. Claudin-1 and occludin expression was then measured by immunoblot, and concentrations of cytokines were assessed by multiplex technology. Claudin-1 expression was affected by glutamine (P < .05, analysis of variance). In particularly, 10 mmol/L glutamine increased claudin-1 expression compared with 0.6 mmol/L glutamine (0.47 ± 0.04 vs 0.33 ± 0.03, P < .05). In contrast, occludin expression was not significantly modified by glutamine. Interestingly, glutamine effect was negatively correlated to claudin-1 (Pearson r = -0.83, P < .001) or occludin basal expression (Pearson r = -0.84, P < .001), suggesting that glutamine had more marked effects when tight junction protein expression was altered. Cytokine concentrations in culture media were not modified by glutamine treatment. CONCLUSION: Glutamine increased claudin-1 expression in the colonic mucosa of patients with IBS-D. In addition, glutamine effect seems to be dependent on basal expression of tight junction proteins.

Glutamine and arginine improve permeability and tight junction protein expression in methotrexate-treated Caco-2 cells.

BACKGROUND & AIMS: Chemotherapy induces an increase of intestinal permeability that is partially related to an alteration of tight junction proteins, occludin and zonula occludens-1 (ZO-1). Protective effects of glutamine on intestinal barrier function have been previously shown but the effects of other amino acids remained poorly documented. Thus, we aimed to evaluate the effects of nine amino acids on intestinal permeability during methotrexate (MTX) treatment in Caco-2 cells. METHODS: Caco-2 cells were incubated in culture medium supplemented with glutamine, arginine, glutamate, leucine, taurine, citrulline, glycine, histidine or cysteine during 24 h and then treated with MTX (100 ng/ml). The dose of each amino acid was 16.6 fold the physiological plasma concentrations. Barrier function was assessed by transepithelial electrical resistance (TEER), FITC-dextran paracellular flux, occludin and ZO-1 expression and localization. Signaling pathways were also studied. RESULTS: Only glutamine, glutamate, arginine and leucine reversed the decrease of TEER observed after MTX treatment (P < 0.05). Interestingly, the addition of 6-diazo-5-oxo-1-norleucine, an inhibitor of glutaminase, blunted the effect of glutamine on MTX-treated cells (P < 0.05). Glutamine and arginine combination restored TEER and FITC-dextran flux to a similar extent than glutamine alone. In addition, pretreatment of Caco-2 cells with glutamine and arginine, alone or combined, differently limited the decrease of ZO-1 and occludin expression (P < 0.05) and the alteration of their cellular distribution, through c-Jun N-terminal kinase (JNK), Extracellular signal-regulated kinase (ERK) and nuclear factor kappa B (NF-κB) pathways. CONCLUSIONS: Glutamine prevented MTX-induced barrier disruption in Caco-2 cells. Arginine also had protective effects but in a lesser extent. The effect of glutamine and arginine should be evaluated in vivo.

Gastric electrical stimulation decreases gastric distension-induced central nociception response through direct action on primary afferents.

BACKGROUND & AIMS: Gastric electrical stimulation (GES) is an effective therapy to treat patients with chronic dyspepsia refractory to medical management. However, its mechanisms of action remain poorly understood. METHODS: Gastric pain was induced by performing gastric distension (GD) in anesthetized rats. Pain response was monitored by measuring the pseudo-affective reflex (e.g., blood pressure variation), while neuronal activation was determined using c-fos immunochemistry in the central nervous system. Involvement of primary afferents was assessed by measuring phosphorylation of ERK1/2 in dorsal root ganglia. RESULTS: GES decreased blood pressure variation induced by GD, and prevented GD-induced neuronal activation in the dorsal horn of the spinal cord (T9-T10), the nucleus of the solitary tract and in CRF neurons of the hypothalamic paraventricular nucleus. This effect remained unaltered within the spinal cord when sectioning the medulla at the T5 level. Furthermore, GES prevented GD-induced phosphorylation of ERK1/2 in dorsal root ganglia. CONCLUSIONS: GES decreases GD-induced pain and/or discomfort likely through a direct modulation of gastric spinal afferents reducing central processing of visceral nociception.