Surgical intestinal manipulation increases gene expression of TrkA, CGRP, and PAR-2 IN dorsal root ganglia in the rat.

BACKGROUND: Surgical handling of the bowel evokes degranulation of peritoneal mast cells (PMC). Nonetheless, role of PMCs in postoperative ileus (POI) is somewhat controversial. We aimed to investigate if intestinal manipulation elicits changes in afferent mediators related to MC activation and alteration of gastrointestinal (GI) motility. METHODS: Postoperative ileus was induced by intestinal manipulation in Sprague-Dawley rats. Additionally, compound 48/80 (C48/80) and ketotifen were used to modulate MC activity. Rat mast cell protease 6 (RMCP-6, ELISA) release was determined in peritoneal lavage 20 min after intestinal manipulation. At 24 h, GI transit was determined. Gene expression of calcitonin gene-related peptide (CGRP), protease-activated receptor-2 (PAR-2), nerve growth factor (NGF), and TrkA receptor was determined (PCR) in dorsal root ganglia (DRG). Ileal wall inflammation was assessed by myeloperoxidase (MPO) activity, interleukin-6 expression (IL-6). KEY RESULTS: Intestinal manipulation and exposure to C48/80-induced degranulation of PMCs delayed GI transit and up-regulated IL-6 and MPO activity. Intestinal manipulation, but not C48/80, up-regulated CGRP, PAR-2, and NGF/TrkA in DRGs. Ketotifen only improved gastric emptying and fecal output. Up-regulation of CGRP and TrkA expression in DRG was not prevented by ketotifen. CONCLUSIONS & INFERENCES: Postoperative ileus is accompanied by activation of CGRP, NGF-TrkA, and PAR-2 in DRGs. Our results suggest that these mediators could be a target in further POI studies in order to find new therapeutic targets for this medical condition.

Mast cell degranulation inhibits motor patterns of human ileum and sigmoid colon in vitro: relevance for postoperative ileus.

BACKGROUND: Local release of mast cell proteases during gastrointestinal surgery is associated with the inhibition of motility and postoperative ileus (POI). We determined whether activation of intramuscular mast cell affects the motor patterns of the human ileum and colon and whether proteases are involved. METHODS: Motor response of ileal and colonic circular muscle strips was measured in organ bath. Mast cell degranulation was induced by compound 48/80 (c48/80; 25-675 µg/mL). Motor response was quantified as tone, rhythmic phasic contractions (RPCs) and contractions to electric field stimulation (EFS; 40 Hz), and bethanechol-evoked contractions. Ketotifen (10(-6) mol/L) and a protease inhibitor cocktail (P8340) were used to evaluate the role of mast cell mediators. KEY RESULTS: (a) c48/80 impaired the spontaneous and the electrically evoked motor response in small bowel and colonic strips (sigmoid colon EC50 : 460.0 µg/mL for RPCs and 8.9 µg/mL for electrically evoked contraction amplitudes) and bethanechol-evoked contractions. (b) Preincubation with ketotifen (10(-6) mol/L, 1 h) prevented the impairment of RPCs and EFS-evoked contractions in the sigmoid colon and ileum but not in the right colon. (c) Preincubation with P8340 also prevented the impairment of contractions in the sigmoid colon but not in the ileum or the right colon. CONCLUSIONS & INFERENCES: Mast cell degranulation by c48/80 inhibits the spontaneous and the nerve-mediated motor response in the human ileum and colon. The effect is partially mediated by mast cell proteases and could be relevant in the pathophysiology of POI.

Peritoneal mast cell degranulation and gastrointestinal recovery in patients undergoing colorectal surgery.

BACKGROUND: Degranulation of peritoneal mast cells (MCs) induced by intestinal manipulation has been proposed as a pathophysiological factor in postoperative ileus (POI). We aimed to explore the relationship between peritoneal and colonic MC degranulation and gastrointestinal (GI) recovery following colectomy. METHODS: Patients undergoing elective laparoscopic cholecystectomy (using a laparoscope and small abdominal incisions, n = 14), and elective laparoscopic (n = 32) or open partial colectomy (through a large abdominal incision, n = 10) were studied. MC protease tryptase and chymase were studied in peritoneal fluid at the beginning, middle, and end of each surgical intervention. Density of MCs in colectomy samples were examined and oro-caecal transit time by breath test, GI function recovery by clinical composite endpoint GI-2 and association between MC proteases and clinical recovery. KEY RESULTS: Open and laparoscopic colectomy caused greater peritoneal release of tryptase and chymase (323.0 ng/mL [IQR: 53.05-381.4] and 118.6 ng/mL [IQR: 53.60-240.3]), than cholecystectomy (41.64 ng/mL [IQR: 11.17-90.93]) at the end of the surgical intervention. However, there were no differences between laparoscopic and open colectomy. Increased peritoneal protease release during surgery was observed in patients who developed POI after colectomy. CONCLUSIONS & INFERENCES: Colorectal surgery causes protease release from peritoneal MCs. Protease release does not differ between both types of colectomy (laparoscopy vs laparotomy). However, MC activation is increased in colectomy patients developing POI. Therefore, degranulation of peritoneal MCs as a factor contributing to human POI after colectomy might be considered in future studies as a target to avoid POI.

CGRP1 receptor activation induces piecemeal release of protease-1 from mouse bone marrow-derived mucosal mast cells.

BACKGROUND: The parasitized or inflamed gastrointestinal mucosa shows an increase in the number of mucosal mast cells (MMC) and the density of extrinsic primary afferent nerve fibers containing the neuropeptide, calcitonin gene-related peptide (CGRP). Currently, the mode of action of CGRP on MMC is unknown. METHODS: The effects of CGRP on mouse bone marrow-derived mucosal mast cells (BMMC) were investigated by measurements of intracellular Ca(2+)[Ca(2+)](i) and release of mMCP-1. KEY RESULTS: Bone marrow-derived mucosal mast cells responded to the application of CGRP with a single transient rise in [Ca(2+)](i). The proportion of responding cells increased concentration-dependently to a maximum of 19 ± 4% at 10(-5)mol L(-1) (mean ±SEM; C48/80 100%; EC(50)10(-8) mol L(-1) ). Preincubation with the CGRP receptor antagonist BIBN4096BS (10(-5) mol L(-1)) completely inhibited BMMC activation by CGRP [range 10(-5) to 10(-11) mol L(-1); analysis of variance (ANOVA) P < 0.001], while preincubation with LaCl(3) to block Ca(2+) entry did not affect the response (P = 0.18). The presence of the CGRP1 receptor on BMMC was confirmed by simultaneous immunofluorescent detection of RAMP1 or CRLR, the two components of the CGRP1 receptor, and mMCP-1. Application of CGRP for 1 h evoked a concentration-dependent release of mMCP-1 (at EC(50) 10% of content) but not of ß-hexosaminidase and alterations in granular density indicative of piecemeal release. CONCLUSIONS & INFERENCES: We demonstrate that BMMC express functional CGRP1 receptors and that their activation causes mobilization of Ca(2+) from intracellular stores and piecemeal release of mMCP-1. These findings support the hypothesis that the CGRP signaling from afferent nerves to MMC in the gastrointestinal wall is receptor-mediated.

Impairment of intestinal barrier and secretory function as well as egg excretion during intestinal schistosomiasis occur independently of mouse mast cell protease-1.

Deposition of Schistosoma mansoni eggs in the intestinal mucosa is associated with recruitment of mucosal mast cells (MMC) expressing mouse mast cell protease-1 (mMCP-1). We investigated the involvement of mMCP-1 in intestinal barrier disruption and egg excretion by examining BALB/c mice lacking mMCP-1 (Mcpt-1(-/-)). Tissue and faecal egg counts from 6 weeks until 12 weeks post-infection (w p.i.) revealed no differences between wild type (WT) and Mcpt-1(-/-)mice. Using chamber experiments on ileal tissue revealed that at 8 w p.i., the epithelial barrier and secretory capacity were severely impaired, whereas no difference was found between WT and Mcpt-1(-/-)mice in this respect. However, a fragmented distribution of the tight junction (TJ) protein occludin, but not of claudin-3 or ZO-1, was observed in WT mice at 8 w p.i., while no changes in TJ integrity were seen in Mcpt-1(-/-)mice. Therefore, we conclude that in contrast to the situation in Trichinella spiralis-infected mice, in schistosomiasis, mMCP-1 is not a key mediator in egg excretion or impairment of the intestinal barrier. The marked decrease in ileal secretory capacity during S. mansoni egg excretion suggests that the mechanisms facilitating the passage of schistosoma eggs through the gut wall are directed more particularly at the epithelial cells.