The enteric nervous system in inflammation and pain : The role of proteinase-activated receptors

Department of Pharmacology and Therapeutics, University of Calgary, Calgary, Alberta. Correspondence: Dr Nathalie Vergnolle, Department of Pharmacology and Therapeutics, University of Calgary, 3300 Hospital Drive Northwest, Calgary, Alberta T2N 4N1. Telephone 403-220-4588, fax 403-210-8195, e-mail nvergnol@ucalgary.ca Received for publication August 19, 2003. Accepted August 26, 2003 N Vergnolle. The enteric nervous system in inflammation and pain: The role of proteinase-activated receptors. Can J Gastroenterol 2003;17(10):589-592.

T he gut is extensively innervated: the enteric nervous sys- tem (ENS) contains approximately 10 8 neurons (as many as in the entire spinal cord).The ENS is characterized by an extensive and elaborate network of nerves extending from the esophagus to the anal sphincter.Intrinsic neurons have cell bodies within the submucous and myenteric plexuses, while the cell bodies of extrinsic sensory nerves are in the dorsal root ganglia of the spinal cord and in the nodose ganglia.The ENS controls motility, secretion, absorption, microcirculation, sensation and immune function in the gut.The present review focuses on the role of proteinases as signalling molecules for both the extrinsic and intrinsic nerves of the ENS, through the activation of proteinase-activated receptors (PARs).

NEUROGENIC INFLAMMATION IN THE GUT
The ENS modulates inflammatory processes in several distinct ways.One way is through the release of neuropeptides upon stimulation of enteric nerves by noxious agents.Intrinsic and extrinsic enteric nerves contain neuropeptides, such as substance P, calcitonin gene-related peptide (CGRP) and vasoactive intestinal peptide (VIP), that modulate various components of the inflammatory process.The release of neuropeptides from both intrinsic and extrinsic enteric neurons can initiate neurogenic inflammation.
Vasodilation and increased vascular permeability are observed in response to CGRP and substance P release, through direct endothelial activation of the type I CGRP receptor and the neurokinin-1 receptor (NK-1), respectively.Activation of endothelial receptors for CGRP and NK-1 causes not only extravasation of plasma from the blood into the tissues, but also facilitates the recruitment of inflammatory cells (1) (Figure 1).Moreover, enteric neurons can stimulate mast cell degranulation by the release of substance P (Figure 1).Mast cell degranulation causes the release of numerous inflammatory mediators: cytokines, proteinases, nitric oxide and lipid mediators of inflammation, including prostaglandins, leukotrienes and thromboxane.Those molecules stimulate other inflammatory cells, such as macrophages, neutrophils and lymphocytes, which can then further promote the release of neuropeptides from enteric nerves, thereby amplifying the inflammatory response (Figure 1).
The ENS also influences vasodilation and vascular permeability indirectly, through the release of mast cell mediators (1).Because enteric neurons are closely associated with macrophages and lymphocytes in the gut wall, the release of VIP from those neurons seems to aggravate inflammation by stimulating cytokine release from B and T lymphocytes, as well as by increasing immunglobulin A synthesis by B-lymphocytes (1).Thus, by releasing proinflammatory neuropeptides, the ENS is capable of initiating and amplifying inflammatory reactions.What could initiate a neurogenic mechanism of inflammation by triggering activation of the ENS and subsequent release of neuropeptides?We propose that PARs act as important mediators of ENS activation.

PARS IN THE ENS
PARs constitute a group of seven transmembrane G proteincoupled receptors that are activated by the proteolytic cleavage of their N-terminal domain.Proteolysis releases a new N-terminal domain, which acts as a tethered ligand, binding and activating the receptor itself (2).Small synthetic peptides, corresponding to the tethered ligand domains released by proteolysis, are selective agonists for these receptors, and thus serve as useful pharmacological tools for understanding the physiology of these receptors.Three PARs (PAR 1 , PAR 3 and PAR 4 ) are activated by thrombin, and have been cloned in the course of studies that aimed at understanding the role of thrombin in platelet activation (3).A fourth receptor (PAR 2 ) can be activated by trypsin and mast cell tryptase, as well as by pathogen proteinases, but not by thrombin (2,4,5).
The four PARs are expressed throughout the gastrointestinal tract in different cell types (2).For example, immunoreactive PAR 1 and PAR 2 have been detected in more than 60% of neurons from the myenteric plexus of the guinea pig small intestine, both in primary cultures and in whole mounts of myenteric tissues (6).A large proportion of neurons expressing substance P, a primary excitatory neuropeptide, also expressed PAR 1 (89%) and PAR 2 (50%).Similarly, a large percentage of neurons expressing VIP, which acts as an inhibitory motor transmitter, also expressed PAR 1 (44%) and PAR 2 (90%) (6,7).More than 50% of neurons expressing PAR 1 also expressed PAR 2 (6).
PAR 1 and PAR 2 are also expressed by extrinsic enteric neurons.PAR 2 -like immunoreactivity and PAR 2 mRNA expression have also been detected in rat isolated dorsal root ganglia neurons (8).Among the sensory neurons expressing PAR 2 , approximately 40% coexpressed CGRP and 30% coexpressed substance P (8).PAR 1 is also located in dorsal root ganglia neurons, where it often coexpressed with substance P (9).PAR 1 and PAR 2 are also functional in ENS cells.Linden and colleagues (10) have shown that PAR 2 agonists signal guinea pig myenteric neurons, inducing depolarization and increasing the number of action potentials in isolated cells.Reed and colleagues (7) have shown that PAR 2 agonists evoked depolarization and long term excitation in submucosal neurons isolated from the guinea pig ileum.Both PAR 1 and PAR 2 agonists stimulate calcium mobilization in isolated guinea pig myenteric neurons ( 6) and dorsal root ganglia neurons (8,9).Although the presence of PAR 1 , PAR 2 and PAR 4 was not clearly established, a study by Gao et al (11) suggested that those receptors act on neurons that regulate enteric motor function in the guinea pig small intestine.
This large expression of PARs strongly suggests that they play a crucial role in the physiology and pathophysiology of the ENS.Among the functions of the ENS, it appears that proteinases and PARs are especially likely to regulate inflammation and pain.

PAR 2 TRIGGERS ENTERIC NERVES TO CAUSE INFLAMMATION
It is known that all the hallmarks of inflammation -swelling, increased blood flow and granulocyte infiltration -are induced by exposure of various tissues (paw, airways, skin, etc) to PAR 2 agonists (12)(13)(14).Because proteinases that are able to activate PAR 2 (such as trypsin and mast cell tryptase) are particularly abundant in the gut of patients with inflammatory bowel diseases (IBD) (15)(16)(17), we hypothesized that high concentrations of proteinases in the lumen of the gut might induce intestinal inflammation.We have shown that, in the rat colon, the intracolonic injection of a selective PAR 2 agonist (PAR 2 -activating peptide), as well as of trypsin and mast cell tryptase, caused edema, granulocyte infiltration and intestinal barrier breakdown, as demonstrated by bacterial translocation from the gut to peripheral organs (18).Furthermore, we have shown that this PAR 2 agonist-mediated colitis could be inhibited by a pretreatment with capsaicin, a neurotoxin that depletes sensory neurons of their neuropeptide content, or pretreatment with NK-1 or CGRP receptor antagonists (19).These results showed that the intestinal inflammation induced by PAR 2 agonists is under neural control, and is affected by the release of neuropeptides such as substance P and CGRP.The release of substance P and CGRP, as a direct result of activation of PAR 2 on ENS neurons, was not demonstrated in this study.Evidence supporting this idea include the facts that functional PAR 2 has been identified on ENS neurons that also expressed substance P and CGRP, and that isolated sensory afferents can release substance P and CGRP upon direct application of PAR 2 agonists.Taken together, these results showed that, upon PAR 2 activation, the ENS releases neuropeptides (substance P and CGRP) that initiate neurogenic inflammation, characterized by edema and granulocyte infiltration (19) (Figure 2).An important step in understanding the role of PAR 2 in the gut would be to investigate to what extent PAR 2 -induced ENS activation is associated with bowel disease.Because inflammation driven by enteric infections has been shown to be largely mediated by a neurogenic mechanism involving extrinsic sensory nerves, substance P and CGRP receptors (20), it could be hypothesized that pathogens release proteinases that activate PAR 2 on enteric neurons, and that this activation leads to mucosal inflammation.This hypothesis is supported by the fact that PAR 2 can be activated by proteinases from pathogens such as dust mites (5) or Porphyromonas gingivalis (4).PAR 2 activation by pathogens would support a proinflammatory role for PAR 2 in enteric infections and would suggest that PAR 2 antagonists might be helpful in the treatment of enteric infections.In IBD models, however, activation of the ENS and further release of neuropeptides have been shown to be protective (21).Thus, in the setting of chronic intestinal inflammation, PAR 2 -induced ENS activation might have beneficial and protective effects.This hypothesis is further supported by the findings of Fiorucci and colleagues (22), who have observed that daily systemic treatment with a PAR 2 agonist was protective in a model of trinitrobenzene sulfonic acid-induced colitis.The lack of readily available PAR 2 antagonists has hampered progress in this field.The availability of PAR 2 -deficient mice should help, in the very near future, to clarify this hypothesis.

DOES PAR 2 TRIGGER ENTERIC NERVES TO CAUSE VISCERAL HYPERSENSITIVITY?
Afferent sensory fibres of the ENS not only release inflammatory neuropeptides, but they also convey sensory data.One hypothesis is that activation of PARs, and particularly PAR 2 , on enteric afferent sensory fibres might also send a nociceptive signal to the central nervous system.It has been shown that subinflammatory doses of PAR 2 agonists (PAR 2 -activating peptides but also trypsin and tryptase), when injected into the rat or mouse paw, provoked nociceptor activation at a spinal level, together with severe and prolonged hyperalgesia (23).Within 2 h after their intracolonic administration, PAR 2 agonists (PAR 2 -activating peptides and trypsin) also caused nociceptor activation at a spinal level, as demonstrated by increased fos protein expression in the superficial laminae of the spinal dorsal horn (24).Subinflammatory doses of PAR 2 agonists from the colonic lumen provoked a significant increase in the number of abdominal contractions in response to rectal distension, which is characteristic of visceral hyperalgesia (24).Here again, these experiments could not unequivocally show that these pro-nociceptive and hyperalgesic effects of PAR 2 agonists were due to a direct activation of PAR 2 on the ENS.The study by Reed and colleagues (7), however, showed that transient exposure of enteric submucosal neurons to PAR 2 agonists evoked long term hyperexcitability.Similar hyperexcitability of extrinsic afferent neurons could be responsible for the PAR 2 -induced hyperalgesia we observed in vivo in a rat model of visceral nociception (23).
Recent studies have shown that patients with the irritable bowel syndrome (IBS) exhibit an increased spontaneous release of mast cell tryptase from colonic tissues (25).It could be hypothesized that tryptase-induced PAR 2 activation of enteric sensory nerves induces a long term hyperexcitability of these neurons, resulting in the visceral pain and hyperalgesia experienced by IBS patients.Although a clear role for PAR 2 activation has been demonstrated in the establishment of mast cell degranulation-induced somatic hyperalgesia, using PAR 2deficient mice (23), such a role in visceral hyperalgesia has yet to be demonstrated.

THE ROLES OF OTHER PARS IN THE ENS
Although a neurogenic mechanism involving NK-1 receptor activation has been demonstrated for PAR 1 agonist-induced paw edema (9), there is currently no evidence that such a mechanism also occurs in the gut.Contrary to the situation with PAR 2 , subinflammatory doses of PAR 1 agonists did not induce hyperalgesia after intraplantar injection, but they increased nociceptive threshold in rats and significantly inhibited inflammatory hyperalgesia induced by carrageenan (26).Here again, there is no evidence yet for such a mechanism in the gastrointestinal tract.However, other neurally controlled functions of the gut seem to be regulated by PAR 1 activation on enteric neurons.Buresi and colleagues (27) have recently shown that PAR 1 activation in mouse intestinal tissues, mounted in Ussing chambers, decreased secretory responses to neural stimulation.This suggests that PAR 1 could contribute to disorders of secretory function associated with the development of colitis.It is interesting to note that PAR 2 agonists also regulate intestinal secretion, but, unlike PAR 1 , PAR 2 agonists have been shown to stimulate chloride secretion by a neuro- Figure 2) Proteinase-activated receptor-2 (PAR 2 ) activation causes neurogenic inflammation in the mouse colon.Upon PAR 2 activation, the enteric nervous system releases substance P (SP) and calcitonin gene-related peptide (CGRP), two proinflammatory neuropeptides, which induce increased vascular permeability and vasodilation, thereby causing edema.PAR 2 -induced SP release, through the activation of neurokinin-1 receptors, is involved in granulocyte recruitment genic process (28).No studies have yet demonstrated a role for PAR 3 or PAR 4 in neurally evoked intestinal functions.

CONCLUSIONS
This review summarized recent evidence that proteinases, through the activation of PARs, are able to interact with the ENS, thereby affecting neurally evoked intestinal functions.Inflammation and pain perception appear to be two major functions that are regulated by neuronal PARs.There has been considerable interest recently in the development of agents that modify these processes.PARs that are expressed on nerves represent, together with the proteinases that activate them, exciting new targets for therapeutic intervention.

Figure 1 )
Figure1) Interactions of the enteric nervous system (ENS) with components of the inflammatory process.Upon activation by noxious agents, the ENS (both extrinsic and intrinsic enteric nerves) release neuropeptides (substance P [SP] and calcitonin gene-related peptide[CGRP]) that, by acting on vascular endothelium, cause vasodilation and increased vascular permeability, two major features of the inflammatory reaction.Neuropeptides such as SP also provoke mast cell activation/degranulation, which subsequently activates the other actors of inflammation: vascular endothelium, macrophages, lymphocytes and neutrophils, through the release of various inflammatory mediators (cytokines, prostaglandins [PGs], leukotrienes [LTs], proteinases and nitric oxide [NO]).Inflammatory cells could, in turn, further induce the ENS to release neuropeptides, thus amplifying the inflammatory reaction.Through the release of vasoactive intestinal peptide (VIP), enteric neurons can also interact with inflammatory cells.Activation of nociceptive enteric neurons also conveys nociceptive information, especially pain, to the central nervous system