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From the Center for Ulcer Research and Education, Digestive Diseases Research Center,* Division of Digestive Diseases, Departments of Medicine and Neurobiology, David Geffen School of Medicine University of California, Los Angeles, California; the Veterans Affairs Greater Los Angeles Health System, Los Angeles, California; the Department of Pharmacology and Therapeutics, University of Calgary, Calgary, Alberta, Canada; and the Department of Pharmacological, Biological and Applied Chemical Sciences, University of Parma, Parma, Italy
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Abstract |
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70%).1
Intestinal ischemia occurs in a wide variety of clinical manifestations, including mesenteric vascular occlusion, neonatal necrotizing enterocolitis, abdominal angina, and Crohn’s disease.2-9
Graft ischemia is also a serious complication of small bowel transplantation.10,11
Ischemic injury due to severe loss in intestinal blood flow can result in many clinical consequences ranging from bleeding, intestinal perforation, and peritonitis to more serious systemic conditions, including myocardial and renal failure, sepsis, multiple organ dysfunction syndrome, and death.12
Intestinal ischemia and reperfusion induce an acute inflammatory response that is associated with enhanced generation and release of proteinases from different sources, including inflammatory cells, like mast cells and neutrophils, and the coagulation cascade, in addition to digestive and bacterial proteinases normally present in the lumen.13-17
Furthermore, breakdown of the gut barrier occurs with bacterial translocation18
; thus, luminal digestive and pancreatic proteinases may penetrate through the mucosa and the muscle layers of the intestine. These enzymes are potential activators of proteinase-activated receptors (PARs), a family of G-protein-coupled receptors that are activated by proteolytic cleavage within the amino terminus exposing a tethered ligand domain that binds and activates the receptors.17
Trypsin and mast cell tryptase are considered as the most likely activators of proteinase-activated receptor-2 (PAR2) in the gut.19,20
PAR2 is abundantly expressed in the gastrointestinal tract, where it is localized to epithelial, endothelial, muscle, neuronal, and immune cells.21-23
PAR2 modulates several gastrointestinal functions, including motility and secretion.17,24,25
In addition, PAR2 agonists have been reported to either have a pro-inflammatory or anti-inflammatory role in intestinal inflammation17,26,27
depending on the model system, the time-course administration, and the cell targets. Intestinal ischemia with reperfusion induces mast cell degranulation that triggers inflammatory infiltrates associated with increased mucosal permeability, thus resulting in mucosal dysfunction.28 In the gut, mast cells are often in close vicinity to visceral afferents that express PAR2.23 These observations provided the background for our hypothesis that PAR2 modulates intestinal injuries induced by intestinal ischemia/reperfusion through the involvement of mast cells and visceral afferents. To test this hypothesis, we used a model of intestinal ischemia developed in rats by reversible occlusion of the superior mesenteric artery for 1 hour followed by 6 hours of reperfusion. This experimental procedure induces transient mucosal damage and alterations of motor activity.8,29 The aims of the study were to investigate: 1) whether PAR2 activation with a selective PAR2 agonist affects gastrointestinal motility impairment and mucosal damage in rats with intestinal ischemia followed by reperfusion (I/R) compared with sham-operated (SO) mice and in mice with or without deletion of the PAR2 gene (PAR2–/– and PAR2+/+); 2) the expression of PAR2 mRNA in the intestine of I/R rats following PAR2 activation; 3) whether the effects of PAR2 activation on motility and tissue damage in I/R involve mast cells and extrinsic primary afferent nerves; and 4) the role of calcitonin gene-related peptide (CGRP) and substance P (SP), peptides that control inflammation and pain and colocalize with PAR2 in visceral afferents,27,30,31 in mediating PAR2 effects in intestinal ischemia.
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Materials and Methods |
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Animal care and procedures were in accordance with the National Institutes of Health recommendations for the humane use of animals. All experimental procedures were reviewed and approved by the appropriate Animal Use Committee of the University of California, Los Angeles and the Veterans Affairs Greater Los Angeles Health System (VAGLAHS, Los Angeles, CA), and the University of Calgary (Calgary, AB, Canada). Male Wistar rats (175 to 200 g) were purchased from Harlan World Headquarters (Indianapolis, IN). C57BL6 mice, either female or male, (20 to 25 g) were obtained from Charles River Laboratories (Montreal, QC, Canada), and PAR2–/– and wild-type littermates were obtained from Johnson & Johnson Pharmaceutical Research Institute (Spring House, PA). Animals were fasted with free access to water for 12 to 18 hours before experimental procedures. Rats were anesthetized with sodium pentobarbital (Nembutal; Abbott Laboratories, North Chicago, IL) (40 mg/kg; intraperitoneally). Following abdominal laparotomy, the small bowel was retracted to the right and the superior mesenteric artery was identified. The superior mesenteric artery was occluded just proximal to the right colic artery, causing ischemia to the small bowel for 1 hour. Intestinal ischemia was followed by 6 hours of reperfusion (I/R). This time of reperfusion was chosen to avoid interference of anesthesia with gastrointestinal transit measurement and to allow the development of acute inflammation. SO animals, in which abdominal laparotomy and artery isolation were performed without occlusion of the vessel, served as controls. The survival rate of animals in each experimental group was 100%. To determine the effect of PAR2 stimulation on intestinal ischemia and reperfusion, the selective PAR2 agonist SLIGRL-NH2 or the inactive, scrambled control peptide LRGILS-NH2 was used. Peptides were synthesized by Byo-Synthesis Inc. (Lewisville, TX) and by the peptide synthesis facility of the University of Calgary. Their concentration, purity, and composition were determined by high performance liquid chromatography, mass spectrometry, and quantitative amino acid analysis. Peptides were administered intraduodenally (3.5 mg/kg in 1 ml/kg) at the beginning of the reperfusion in I/R rats and following surgery in SO animals. The aminopeptidase inhibitor amastatin (1.25 mg/kg, intraduodenally; Sigma-Aldrich, St. Louis, MO) was simultaneously administered to avoid peptide degradation. The midline incision of the abdominal wall was then closed by two-layer sutures. Following recovering from anesthesia, animals were returned to their cages. Similar procedures were used for both the PAR2+/+ and PAR2–/– mice (C57BL6 strain), except that the superior mesenteric artery was occluded for 30 minutes followed by 6 hours of reperfusion. Animals were euthanized at the end of the experiments by deeply anesthetizing them with an overdose of sodium pentobarbital (50 mg/kg, intraperitoneally) followed by thoracotomy.
Cromolyn Pretreatment
To determine the role of mast cells in intestinal ischemia with and without PAR2 activation, the mast cell stabilizer cromolyn (20 mg/kg; Sigma-Aldrich)32 was administered through the tail vein 1 hour before the induction of ischemia or sham operation in I/R and SO rats with or without intraduodenal treatment with PAR2 agonist. All chemicals were dissolved in sterile saline.
Ablation of Extrinsic Visceral Afferent Neurons
To determine the involvement of extrinsic visceral afferents in I/R-induced alterations with and without PAR2 activation, chemical ablation of extrinsic primary afferent nerves was induced by treatment with the neurotoxin capsaicin (125 mg/kg; Sigma-Aldrich). The total dose of capsaicin was divided into three injections (25 mg/kg in the morning, 50 mg/kg after 6 hours, and 50 mg/kg after 32 hours) and administered subcutaneously (as an emulsion of 12.5 mg/ml capsaicin in a medium containing 10% ethanol, 10% of Tween 80, and 80% of saline) in rats under isofluorane (0.5 to 3%) anesthesia. Animals were then left for 10 days, and I/R or sham operation was performed with or without intraduodenal administration of PAR2 agonist. To evaluate the effectiveness of capsaicin pretreatment, we used the eye-wipe response to a diluted (0.01%) capsaicin solution.
Pretreatments with CGRP and SP Receptor Antagonists
To determine the involvement of the neuropeptides CGRP and SP, both coexpressed with PAR2 by a large proportion of primary sensory visceral afferents,27,30,31 in exogenous stimulation of PAR2 in I/R-induced responses, animals were pretreated with either CGRP receptor antagonist CGRP8–3733-35 or RP67580, a selective antagonist for the preferred SP receptor neurokinin-1 (NK1).36 CGRP8–37 was administered at a dose of 150 µg/kg in the tail vein 15 minutes before the induction of ischemia and then every 2 hours (for a total of four injections), because this compound degrades. RP67580 (1 mg/kg) was administered in the tail vein 15 minutes before the ischemia followed by another injection at 0.5 mg/kg 3 hours later.35
Upper Gastrointestinal Transit (GIT)
A nonabsorbable black marker (10% charcoal suspension in 5% gum arabic; Sigma-Aldrich) was administered by gavage to conscious animals 5 hours and 15 minutes after the beginning of reperfusion as described elsewhere with minor modifications.25,37 Forty-five minutes later, animals were euthanized with sodium pentobarbital, and the small bowel was removed. The distance traveled by the marker was expressed as a percentage of the total length of the small intestine from pylorus to cecum.
Assessment of Tissue Damage: Microscopic Damage Score
Specimens of the distal ileum were collected from the different groups of animals at the end of the perfusion period to determine the level of tissue damage. Following overnight fixation in 10% formalin, specimens of the ileum were embedded in paraffin. Sections (5 µm) were stained with hematoxylin and eosin. Microscopic histological damage score was evaluated by a person unaware of the treatments and was based on a semiquantitative scoring system in which the following features were graded: extent of destruction of normal mucosal architecture (0, normal; 1, 2, and 3, mild, moderate, and extensive damage, respectively), presence and degree of cellular infiltration (0, normal; 1, 2, and 3, mild, moderate, and transmural infiltration), extent of muscle thickening (0, normal; 1, 2, and 3, mild, moderate, and extensive thickening), presence or absence of crypt abscesses (0, absent; 1, present), and presence or absence of goblet cell depletion (0, absent; 1, present). The scores for each feature were then summed with a maximum possible score of 11 as previously described.27,30,38,39
SYBR Green Real-Time RT-PCR
PAR2 mRNA expression was determined by SYBR Green I real-time quantitative polymerase chain reaction (PCR) using an Mx3000P real-time PCR detection system (Stratagene, LA Jolla, CA). Total mRNA was extracted from duodenum and ileum using the absolutely RNA® RT-PCR Miniprep kit (Stratagene) as previously described.40 After verification of its integrity, RNA was quantified spectrophotometrically, and 1 µg was processed for complementary DNA (cDNA) synthesis using SuperScript II reverse transcriptase (Invitrogen Corp., Carlsbad, CA). Specific primers for PAR2 gene were designed by using Primer3 software. The sequences of the primers used were: PAR2, sense: 5'-AAC ATC ACC ACC TGT CAC GA-3'; antisense: 5'-CAC GTA GGC AGA CGC AGT AA-3'; ß-actin, used as a housekeeping gene, sense: 5'-TCA TGA AGT GTG ACG TTG ACA TCC GT-3'; antisense: 5'-CTT AGA AGC ATT TGC GGT GCA CGA TG-3' (Promega Corp., Madison, WI).
The efficiency of the real-time RT-PCR primer pairs was determined by amplifying serial dilutions of cDNA. The real-time RT-PCR was performed using Platinum® SYBR® Green qPCR SuperMix UDG (Invitrogen). Each cycle consisted of three steps: denaturation for 30 seconds at 95°C, annealing for 30 seconds at 55°C, and 30 seconds of elongation at 72°C. The data acquired from each sample were normalized to those of ß-actin. The specificity of the real-time reverse transcriptase (RT)-PCR was further confirmed by a regular RT-PCR followed by agarose gel electrophoretic analysis to verify the presence of a single band corresponding to the size predicted for the amplicon. Relative quantification was performed by using the comparative cycle threshold method as described elsewhere (User Bulletin #2, ABI Prism 7700 Sequence Detection System, December 11, 1997).
Statistical Analysis
Differences among groups that underwent I/R and SO were analyzed using two-way analysis of variance, followed by the Bonferroni post-test. Differences between treatments within the same experimental conditions (ie, I/R or SO) were evaluated using one-way analysis of variance, followed by Dunnett’s post-test. The same test was used to compare differences between mRNA measurements obtained with real-time RT-PCR. Values are expressed as means ± SE. A P value of <0.05 was required to consider group differences as significant, and a P value of <0.01 was considered highly significant.
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Results |
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Gastrointestinal transit was significantly delayed in I/R compared with SO rats (42.7 ± 3.6 vs. 56.8 ± 3.2, P < 0.05) (Figure 1)
. Intraduodenal administration of SLIGRL-NH2 with amastatin significantly accelerated the gastrointestinal transit in I/R rats (77 ± 3.9 vs. 42.7 ± 3.6, P < 0.001) but not in SO animals, indicating a stimulatory role of PAR2 on motility in ischemic conditions. The inactive control peptide LRGILS-NH2 with amastatin or amastatin alone did not affect GIT in either I/R or SO rats, supporting the specificity of the effect detected.
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In PAR2–/– mice, there was a significant delay in GIT in both I/R and SO animals without PAR2 agonist pretreatment, compared with the wild-type groups (I/R: 26.1 ± 2.3 vs. 38.5 ± 1.9, P < 0.001; SO 37.2 ± 2.6 vs. 56.9 ± 1.6, P < 0.001) (Figure 2)
. These data suggest that endogenous activation of PAR2 is involved in the regulation of gastrointestinal transit under normal conditions and potentially under ischemic conditions as well. Treatment with SLIGRL-NH2 significantly accelerated gastrointestinal transit in I/R PAR2+/+ (60.0 ± 5.1 vs. 38.5 ± 1.8, P < 0.001) but not in PAR2–/– mice, further supporting the specificity of PAR2 agonist treatment in PAR2+/+.
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Pretreatment with cromolyn did not significantly affect the GIT in I/R and SO rats but prevented the acceleration induced by SLIGRL-NH2 in I/R rats (51.7 ± 2.6 in cromolyn treated vs. 77.0 ± 3.9 in non-cromolyn treated; P < 0.001) (Figure 3)
. This suggests that in I/R rats PAR2 activation mediates the increase in GIT through degranulation of mast cells.
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. This inhibitory effect suggests that visceral afferents participate to the PAR2 action on GIT in intestinal ischemia with reperfusion. In addition, in capsaicin-treated animals, I/R did not induce a significant delay in GIT as compared to SO animals, suggesting a role of visceral afferent neurons in the control of the gastrointestinal motility in intestinal ischemia followed by reperfusion.
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Antagonism of CGRP receptor by pretreatment with CGRP8–37 or antagonism of NK1 receptor by RP67580 prevented the SLIGRL-NH2-induced increase in GIT in I/R rats (38.4 ± 8.9 in CGRP8–37-treated and 43.1 ± 6.0 in RP67580-treated vs. 77.0 ± 3.9 in non-treated; P < 0.001) (Figures 5)
. CGRP and NK1 receptor blockade did not significantly modify the GIT in I/R and SO animals in the absence of SLIGRL-NH2 (see Figure 5
; compare to Figure 1
). However, blockade of either receptor prevented the GIT delay induced by I/R as observed with capsaicin treatment.
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SLIGRL-NH2 Improves Ischemia-Induced Mucosal Damage in Rats
Figure 6
illustrates the total microscopic damage scores (white bars) and the leukocyte recruitment (gray bars) in each group of rats from the first set of experiments (no pretreatment with cromolyn, capsaicin, or CGRP and NK1 receptor antagonists). Signs of inflammation were observed in I/R animals treated with saline, control peptide LRGILS-NH2, or amastatin as indicated by the significant increase in both total microscopic damage score (white bars; P < 0.001) and leukocytes infiltration (gray bars; P < 0.05) when compared with the corresponding SO groups. By contrast, in the I/R group that received treatment with SLIGRL-NH2 (plus amastatin), total microscopic damage was significantly decreased when compared with I/R controls (1.29 ± 0.29 vs. 4.57 ± 0.65; P < 0.001), whereas leukocyte recruitment was not significantly different. These results suggest that most of the tissue damage induced by ischemia followed by reperfusion is not due to inflammatory cell infiltrates and that the protective effect of PAR2 exogenous activation on ischemia-induced mucosal damage affects parameters different from leukocyte recruitment. The inactive control peptide LRGILS-NH2 or amastatin alone did not affect the damage score in I/R rats, further suggesting that the protective effects of SLIGRL-NH2 are specific of PAR2 activation. In SO animals, PAR2-activating peptide SLIGRL-NH2 caused a significant increase in the inflammatory response, mostly due to a significant increase in leukocytes infiltration, consistent with previous reports27,35
(Figure 6)
. The major differences between SO and I/R groups consisted of changes to the mucosal architecture, massive cellular infiltration, and severe muscle thickening (see Figure 7
). The protective effects of SLIGRL-NH2 were mostly represented by a reduction of mucosal erosion and by less muscle thickening compared with the I/R rats that did not receive SLIGRL-NH2 (see Figure 7
).
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Figure 8
shows the microscopic damage scores in cromolyn-pretreated rats. In these rats, the mucosal damage score, but not the level of leukocytes infiltration, was significantly higher in I/R groups than in the SO groups. SLIGRL-NH2 administration did not cause a significant reduction in the inflammatory response in cromolyn-treated I/R rats compared with I/R rats treated with saline (Figure 8)
. These data suggest that the protective effect of SLIGRL-NH2 treatment is partially mediated by mast cell degranulation and release of their mediators.
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. In animals with visceral afferents ablation, the level of mucosal damage (but not of cell infiltration) in the I/R saline group was significantly higher than in the SO saline group. In capsaicin-treated rats, SLIGRL-NH2 administration did not cause a significant reduction in either mucosal damage or in leukocytes recruitment in I/R rats compared with saline I/R rats (Figure 9)
. These observations suggest that sensory afferents are involved in the exogenous PAR2 activation protective effect in I/R-induced inflammation.
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As shown in Figure 10
, antagonism of CGRP receptor or NK1 receptor did not significantly modify the inflammatory response in I/R saline rats when compared with the corresponding I/R group that was not pretreated with CGRP8–37 or RP67580 (as shown in Figure 6
). Mucosal damage and cell infiltrates were significantly increased in the I/R saline group compared with the SO group pretreated with CGRP8–37 or RP67580. Interestingly, whereas NK1 receptor antagonism reversed the protective effect of SLIGRL-NH2 on mucosal damage (but not leukocytes recruitment) in I/R rats, CGRP receptor blockade did not, as indicated by comparable levels of mucosal damage and cell infiltrates in SLIGRL-NH2 I/R rats with or without CGRP8–37 pretreatment (compare Figure 10
with Figure 6
).
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PAR2 mRNA Levels Are Not Modified in Ischemic Rats
To investigate whether an up-regulation of PAR2 expression could account for the SLIGRL-NH2-induced effects in I/R rats, we measured the levels of PAR2 mRNA with real-time RT-PCR in specimens of the duodenum (Figure 11A)
and ileum (Figure 11B)
. Intestinal tissues were excised from rats subjected to ischemia and no reperfusion (I/R0) and ischemia followed by 6 hours of reperfusion (I/R6) with and without SLIGRL-NH2 treatment. As shown in Figure 11
(A and B), there was no significant difference in the levels of PAR2 mRNAs among groups, suggesting that reperfusion with or without SLIGRL-NH2 treatment did not affect the baseline PAR2 mRNA expression in ischemic rats.
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Discussion |
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Intestinal ischemia with reperfusion leads rapidly to a disruption of the mucosal barrier, which normally protects the tissues from luminal content. Loss of mucosal integrity has been implicated in the pathogenesis of the multiple organ dysfunction syndrome, a life-threatening complication of intestinal ischemia, which may be caused by a hyper-inflammatory response and by the loss of autoregulation of the normal inflammatory response.18 Alterations of gastrointestinal motility,8,41,42 which can be partly due to structural changes and neuronal plasticity occurring within the enteric nervous system,29,43 may also contribute to the development of bacterial overgrowth with subsequent bacterial translocation. Mast cells degranulation has been recognized as an important factor in mediating mucosal damage and motor alterations in small bowel.28,44 There is increasing evidence that PAR2 plays an important role in inflammation acting either as a pro- or anti-inflammatory agent, depending on the activated cells or the type of inflammation27,30,35,45 Indeed, PAR2 agonist induces or exacerbates inflammation by a neurogenic mechanism in rat paw and mice colon by local administration.31,32,46 By contrast, PAR2 activation appears to play a protective role against inflammation in an experimental colitis model by decreasing the production of pro-inflammatory cytokines.26,47 This protective effect also includes a neurogenic component, because it is prevented by ablation of sensory nerves and by antagonism of CGRPs receptor.26 Furthermore, PAR2 has been reported to have a dual role in acute pancreatitis with a local protection but an aggravation of systemic complications.48
In our experimental model, the mucosal damage, but not the leukocyte recruitment, induced by intestinal ischemia/reperfusion was significantly reduced by the intraduodenal administration of the PAR2-selective agonist SLIGRL-NH2 providing evidence for a protective effect of PAR2 exogenous activation. This protective effect also seems to be partially dependent on mast cells and primary sensory afferents, because it was not observed in rats pretreated with cromolyn, a mast cell stabilizer, and chronic capsaicin, which ablates visceral afferents. As it was observed in a model of chronic colonic inflammation,26 it appears from our study that activation of PAR2 on sensory nerves can exert protective effects on intestinal mucosa.49-51 This is in accord with other studies that have shown that activation of capsaicin-sensitive neurons plays an important role in protecting ischemic bowel viability.52-54 Because blockade of NK1 receptor, but not CGRP receptor, reversed the protective effect on mucosal damage induced by exogenous activation of PAR2, it is reasonable to suggest that the PAR2-protective effect is at least in part mediated by SP released from visceral afferents. By contrast, CGRP does not appear to play an important role in this protective effect. It is likely that SP, which is overexpressed in inflammatory conditions,55 when released by visceral afferents activates mast cells that are located in close vicinity to visceral afferents at the level of the mesentery and of the gastrointestinal mucosa,50,56-59 thus resulting in mast cells degranulation, which accentuates the inflammatory response.60 CGRP mediates afferent neuron mucosal protection in experimental gastritis50 and in experimental colitis61 by modulating blood flow and vascular tone with a local action that induces nitric oxide release.51 CGRP has also been reported to exert chemotactive effect and promote mast cells recruitment.62 In our study, CGRP blockade does not reverse the protective effect of exogenous PAR2 activation in intestinal ischemia, suggesting rather a pro-inflammatory role of CGRP.31 By contrast, in view of the increased damage score in SO rats pretreated with CGRP8–37 compared with saline SO rats, we cannot exclude a protective role of CGRP in mild inflammatory status, as induced by laparotomy and intestinal manipulation. Because PAR2 activation reversed the GIT delay induced by intestinal I/R, it is reasonable to think that PAR2 agonist could exert its protective role by modulating gastrointestinal motility. The fact that both cromolyn and capsaicin treatments inhibit both parameters (SLIGRL-NH2-induced changes in GIT and mucosal damage) further supports this hypothesis. Because the stimulatory effect of PAR2 activation on GIT in intestinal ischemia followed by reperfusion is not observed following blockade of either CGRP or NK1 receptor, it is likely that visceral afferents mediate PAR2 effect on GIT via the release of CGRP and SP.
Reperfusion of ischemic tissue results in increased production of oxidants and radicals that in turn cause mast cells activation and degranulation. These phenomena play a critical role in the cascade of events leading to I/R-induced granulocyte infiltration and mucosal barrier dysfunction.28 Mast cell degranulation has been recognized as an important factor in the mediation of mucosal damage44 and of motility alterations in the small bowel,63 and it has been implicated in the pathogenesis of numerous gastrointestinal diseases, including inflammatory bowel disease, celiac disease, and food allergy.63,64 Moreover, mast cells activation leads to changes in intestinal motility through a neuroimmune mechanism mediated by the enteric nervous system.57 Mast cells are the most abundant immunocytes in the gut wall, and they degranulate upon activation, releasing a multifaceted spectrum of inflammatory mediators and tryptase, a neutral serine proteinase that can activate PAR2.65 PAR2 is found on enteric neurons, therefore mast cell degranulation and tryptase release may activate, in a paracrine manner, directly enteric neurons contributing to intestinal motility dysfunction and hypersecretion during intestinal inflammation.57 However, mast cells also seem to be implicated downstream from PAR2 activation, as suggested by the observation that pretreatment with cromolyn, a mast cell stabilizer that prevents degranulation,32,66 reverses the accelerating effect of PAR2 activation on GIT in I/R animals but does not affect GIT in SO animals. This supports the concept that mast cell degranulation is one of the mechanisms involved in the PAR2-protective effect of PAR2 agonist on gastrointestinal motility impairment resulting from I/R.
Up-regulation of PAR2 has been reported in response to PAR2 activation in different experimental models of inflammation,27,67 which prompted our analysis of PAR2 mRNA levels in our experimental conditions. The lack of differences in the PAR2 mRNA levels with or without PAR2 activation suggests that the protective effect of PAR2 on ischemia injuries is not due to PAR2 up-regulation, even though this cannot be ruled out. It must be pointed out that we measured PAR2 mRNA levels 6 hours following PAR2 stimulation, whereas in other studies, PAR2 mRNA levels were measured 10 to 20 hours following stimulation,27,67 which could explain this apparent discrepancy. Indeed, PAR2 activation results in an early down-regulation, probably due to endocytosis and degradation of the receptor, followed by an up-regulation of mRNA and protein reflecting increase of exocytosis.27
In conclusion, the present findings indicate that PAR2 activation plays a protective role on I/R-induced impairment of gastrointestinal motility and mucosal damage through different mechanisms. The stimulatory effect of PAR2 agonist on GIT in I/R is likely to be mediated by the activation of mast cells and primary visceral afferent neurons, with the involvement of both neuropeptides CGRP and SP. The inflammation caused by I/R might induce degranulation of mast cells with release of proteases that in turn activates PAR2 directly and/or through activation of visceral afferents, thus contributing to the maintenance of regular GIT. The mechanism underlying the protective effects of PAR2 activation on mucosal damage appears to be mediated by mast cells degranulation and sensory afferents, with a mechanism that involves at least in part SP release. PAR2 might be an important player affecting gastrointestinal transit under stress conditions such as ischemia/reperfusion. PAR2-related drugs could constitute useful therapeutic approaches for ischemia/reperfusion-induced impairment of gastrointestinal motility.
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Acknowledgements |
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Footnotes |
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Supported by grants from the National Institutes of Health (DK 51455, 57037, and 41301 to C.S.), the Crohn’s and Colitis Foundation of Canada, the Canadian Association of Gastroenterology, and the Canadian Institute for Health Research (to N.V. and N.C.), and the Ministry of University, Scientific Research and Technology, Italy (COFIN 2002 to E.B. and M.I.).
Accepted for publication April 11, 2006.
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