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Proteases and protease-activated receptors in the gut

  1. Top of page
  2. Proteases and protease-activated receptors in the gut
  3. Altered intestinal protease activity in irritable bowel syndrome
  4. PAR-mediated mechanisms in the generation of IBS symptoms
  5. Clinical relevances and perspectives
  6. Conclusions
  7. References

Proteases are present in great amount in the gastrointestinal tract. Trypsin is released in its proactive form trypsinogen in the upper gastrointestinal tract lumen and in the pancreatic duct, both playing a role in the digestive process. Different extrapancreatic cell types including endothelium and epithelial cells synthesize trypsinogen. Many other serine proteases are present in the gut lumen most of them been secreted by resident immunocytes. Among them, tryptase is released by mast cells and cathepsin G is released by neutrophils upon activation. Proteases of the coagulation cascade such as thrombin, factor VIIa and Xa are also potentially present in the gastrointestinal tract during inflammation or tissue trauma. In addition to endogenous proteases, the gastrointestinal mucosa is constantly exposed to bacterial proteases from normal flora and pathogen microorganisms. In the gut, the balance between proteolytic activities in the lumen and the presence of protease inhibitors at mucosal surfaces ensure efficient digestive processes and mucosal protection.

However, in addition to their digestive role in protein degradation, proteases play a role as signalling molecules regulating cell functions by cleaving protease-activated receptors (PARs). Protease-activated receptors belong to a family of seven transmembrane domain G-protein-coupled receptors that are activated by cleavage of their N-terminal domain by selective proteolytic enzymes.1 Four PARs have been identified in human tissues (PAR1, -2, -3 and -4) and PARs are expressed throughout the gastrointestinal tract on several cell types, as enterocytes, mast cells, smooth muscle cells, myenteric neurons and on colonic epithelial cells.2 Protease-activated receptors are activated by a variety of proteases3 and the gut is particularly abundant in PAR1 and PAR2, which receptors play extensive roles in modulating gastrointestinal functions (see rev3).

Altered intestinal protease activity in irritable bowel syndrome

  1. Top of page
  2. Proteases and protease-activated receptors in the gut
  3. Altered intestinal protease activity in irritable bowel syndrome
  4. PAR-mediated mechanisms in the generation of IBS symptoms
  5. Clinical relevances and perspectives
  6. Conclusions
  7. References

Irritable bowel syndrome (IBS) is a chronic gastrointestinal disorder characterized by continuous or remittent abdominal pain, bloating and altered defecation. The pathogenesis of IBS remains partially understood but recent investigations have confirmed the presence of a micro-inflammation of the colonic mucosa. Our recent studies suggest that increased fecal serine-protease activity is a characteristic of IBS patients with diarrhoea and that such elevated activity may contribute to a PAR2-mediated increased colonic paracellular permeability and subsequent visceral hypersensitivity.4,5 Colonic biopsies from IBS patients also release proteolytic activity that may trigger visceral hyperalgaesia in mice.6

Extrapancreatic form of trypsin, trypsin IV produced by intestinal epithelial cells, is a novel agonist of PAR2 and PAR4, which induce PAR2-mediated inflammation, thermal and mechanical hyperalgaesia in mouse paw.7 In the present issue of Neurogastroenterology and Motility, Kerckhoffs et al.8 report on a study in which they found enhanced mRNA expression of trypsinogen IV in small intestinal mucosal biopsy specimens of IBS patients compared to healthy subjects, while they did not observe any differences in mucosal PAR2 expression. Unfortunately, the number of patients did not permit an IBS subgroup analysis but the data clearly indicate that the distribution of values in each class (IBS-C, IBS-D and IBS-A) is not similar and that the highest overexpression is observed in IBS-D patients. Nevertheless, as the visceral hypersensitivity in IBS patients is not restricted to the colorectal region, the authors propose that elevated trypsinogen IV expression could be responsible for altered pain perception in all IBS patients by activation of PAR2 on peripheral nerve terminals in the small intestine lamina propria or by increasing the luminal pool of serine-proteases susceptible to generate symptoms through different PAR2-mediated pathways.

PAR-mediated mechanisms in the generation of IBS symptoms

  1. Top of page
  2. Proteases and protease-activated receptors in the gut
  3. Altered intestinal protease activity in irritable bowel syndrome
  4. PAR-mediated mechanisms in the generation of IBS symptoms
  5. Clinical relevances and perspectives
  6. Conclusions
  7. References

Altered visceral nociception

Visceral hypersensitivity is a major pathophysiological component of IBS. There is growing evidence for an important role of PARs in the development of altered visceral nociception. In guinea pigs, activation by mast cell tryptase of PAR2 located at the enteric nerves causes neuronal hyperexcitability.9 In rats, intracolonic but not systemic infusion of subinflammatory doses of PAR2 agonist activates spinal afferent neurons and produces delayed rectal hyperalgaesia that involves changes in intestinal permeability and the activation of NK1 receptors.10 In mice, colonic PAR2 activation causes delayed facilitation of capsaicin-evoked visceral nociception and stimulation of colonic PAR2 with endogen activator, as well as PAR2-activating peptide, results in colonic hypersensitivity to capsaicin in wild type but not in PAR2 knock-out (KO) mice.11 Direct PAR2-induced mechanical hyperalgaesia and excitation of colonic afferent neurons are mediated by transient receptor potential vanilloid 4.12

Altered mucosal permeability

Intestinal permeability plays a key role in the pathogenesis of IBS. Both postinfectious and sporadic IBS are associated with an increase in intestinal permeability.13 A recent study emphasizes that the increase in intestinal permeability is more pronounced in IBS patients with diarrhoea than in other subgroups of IBS.14 Incubation of epithelial cells with PAR1 agonists induces apoptosis and increases epithelial permeability in a caspase-3-dependent manner in mice.15 In a murine model, low dose of PAR2-activating peptide infused intracolonically activates PAR2 and increases paracellular permeability to macromolecules in vivo through calmodulin and subsequent MLCK activation provoking tight junction opening by perijunctional ring myosin phosphorylation.16 In mice, fecal supernatants from diarrhoeic IBS patients increase colonic paracellular permeability through activation of PAR2 as these effects are prevented by administration of serine-protease inhibitors and are absent in colonic strips of PAR2 deficient mice.5 Such alterations are coupled with the redistribution of tight junction proteins both in vivo in colonocytes and in vitro in tissue cultures.

Mucosal inflammation

There is recent evidence for an important role of colonic mucosal micro-inflammation in the pathogenesis of IBS. Irritable bowel syndrome symptoms are common in patients in remission from ulcerative colitis (UC)17 and in those having had gastrointestinal infection.18 Increased numbers of inflammatory cells are characteristic in the colonic mucosa of IBS patients.19,20 In mice, intracolonic administration of PAR2-activating peptide causes colonic inflammation partly dependent of paracellular permeability and mediated by capsaicin-sensitive neurons and nitric oxide.21 Citrobacter rodentium infection induces substantially reduced level of murine infectious colitis in both soybean trypsin inhibitor-treated wild-type mice and untreated PAR2-deficient mice suggesting an important role for both pathogen-induced host serine proteases and PAR2 in the development of colonic inflammation.22

Altered intestinal motility

Protease-activated receptors have regulatory effect on intestinal motility, which is an essential pathogenetic factor in both diarrhoea and constipation subtypes of IBS. In mice, intraperitoneal administration of PAR1 and PAR2 agonists facilitates gastrointestinal transit. The effect of PARs on gut transit is modulated by apamin-sensitive K+ channels and dependent on activation of L-type Ca2+ channels, but independent of tyrosine kinase.23 In rats, both PAR1 and PAR2 agonists induce different effects in the two colonic muscular layers: a reduction of spontaneous contractions in the circular muscle and contraction or biphasic effect, relaxation followed by contraction, in longitudinal muscle.24 In rat longitudinal muscle, PAR1 and PAR2 activation could evoke relaxation through the production of nitric oxide or contraction through the release of tachykinins from sensory nerves.25

Altered ion transport

Functional basolateral and apical PAR2 are present in mouse distal colon and their activation stimulate Cl and K+ secretion while inhibiting baseline Na+ absorption.26 In human mucosa samples obtained by rectal biopsies or in colonic epithelial monolayers, basolateral, but not luminal administration of PAR2 agonist induces chloride secretion in Ussing chamber.27 Stimulation of chloride secretion is an important pathophysiological factor in the development of diarrhoea which is a characteristic clinical symptom of one subtype of IBS. Kerckhoffs et al.8 in the present issue of Neurogastroenterology and Motility, are unable to identify changes in PAR2 expression in duodenal biopsies taken from IBS patients that in addition to the overexpression of trypsinogen may amplify the secretory response; but no subgroup analysis was performed in this study to confirm this point for diarrhoeic IBS patients. Such lack of changes in expression may also support the hypothesis that trypsin IV is not released from epithelial cell.

Clinical relevances and perspectives

  1. Top of page
  2. Proteases and protease-activated receptors in the gut
  3. Altered intestinal protease activity in irritable bowel syndrome
  4. PAR-mediated mechanisms in the generation of IBS symptoms
  5. Clinical relevances and perspectives
  6. Conclusions
  7. References

The work of Kerckhoffs et al.8 adds to several new data suggesting that luminal and/or parietal proteases play a role in the genesis of hyperalgaesia and permeability defect described in IBS. The fecal serine-protease activity, reflecting the intestinal and colonic levels, is elevated in IBS patients with diarrhoea similarly to that seen in patents with UC, however, this is not associated with increased levels of inflammatory mediators including tryptase.5 These observations let us speculate that in absence of increased inflammatory markers in the stool, elevated fecal serine-protease activity could have a diagnostic potential in diarrhoea predominant IBS. Further studies in greater patient population are required to define the possible use of fecal serine-protease measurement as a diagnostic biomarker of IBS.

The possible role of proteases in the development of IBS symptoms raises the potential use of protease inhibitors in the treatment of IBS. Protease inhibitors are safely used in the therapy of several gastrointestinal diseases. Camostat, a serine-protease inhibitor, improves reflux oesophagitis after gastrectomy27 and induces and maintains remission in UC patients.28 Bowman-Birk inhibitor concentrate, a soy extract with high protease inhibitor activity, is safely and effectively used in the treatment of active UC.29 Up to now, no studies have reported the therapeutic use of protease inhibitors in IBS. In the absence of available PAR-antagonists in the clinical practice, protease inhibitors could represent new agents for IBS therapy in the near future.

Conclusions

  1. Top of page
  2. Proteases and protease-activated receptors in the gut
  3. Altered intestinal protease activity in irritable bowel syndrome
  4. PAR-mediated mechanisms in the generation of IBS symptoms
  5. Clinical relevances and perspectives
  6. Conclusions
  7. References

Despite growing number of evidence for a role of proteases and PARs in the development of IBS symptoms, clinical relevance of this phenomenon is still upon to question. Further investigations into the role of luminal vs mucosal proteases in the pathogenesis of IBS are warranted. The possible diagnostic use of elevated protease activity in the intestinal mucosa or in the stool samples of IBS patients, and potential therapeutic use of protease inhibitors in IBS need extended clinical studies. The study by Kerckhoffs et al.8 in this issue of Neurogastroenterology and Motility provides new evidence for a possible role of altered epithelial synthesis and secretion of endogenous serine-proteases in the development of IBS symptoms, as it identifies enhanced mRNA expression of trypsinogen IV in small intestinal mucosal biopsy specimens of IBS patients. Further clinical investigations into the possible differences in trypsinogen IV expression among different subtypes of IBS and into the correlation between elevated trypsinogen IV expression, PAR function and altered visceral sensitivity could elucidate the potential clinical relevance of this novel observation.

References

  1. Top of page
  2. Proteases and protease-activated receptors in the gut
  3. Altered intestinal protease activity in irritable bowel syndrome
  4. PAR-mediated mechanisms in the generation of IBS symptoms
  5. Clinical relevances and perspectives
  6. Conclusions
  7. References