Abstract
Trypsin and mast cell tryptase cleave within the extracellular N terminus of proteinase-activated receptor-2 (PAR-2), exposing a tethered ligand (SLIGRL) that binds and activates the cleaved receptor. We examined the neuronal expression of PAR-2 and its role in intestinal ion transport. Short-circuit current elevations in response to trypsin or the receptor-activating peptide SLIGRL-NH2 were measured in sheets of mucosa-submucosa from porcine ileum. SLIGRL-NH2 or trypsin rapidly elevated short-circuit current after their contraluminal application with respective 50% effective concentrations of 184 and 769 nM. Their actions were attenuated after contraluminal administration of the neuronal conduction blocker saxitoxin (0.1 μM); the cyclooxygenase inhibitor indomethacin (10 μM); or the Na+/K+/Cl− cotransport inhibitor furosemide (10 μM), but not by atropine (0.1 μM), a muscarinic cholinergic antagonist. In addition, soybean trypsin inhibitor (5 μg/ml) reduced mucosal responses to trypsin. The δ-opioid agonist [d-Pen2,5]-enkephalin (0.1 μM) inhibited trypsin action, an effect that was prevented by naltrindole (0.1 μM), a δ-opioid antagonist. PAR-2 immunofluorescence was localized in the mucosa using a receptor-specific antibody. PAR-2-like immunoreactivity was detected in myenteric and submucosal neurons, nerve fibers innervating ileal smooth muscle and mucosa, and in enteroendocrine cells. Some neurons coexpressed PAR-2- and choline acetyltransferase-like immunoreactivity. These results indicate that PAR-2 is expressed on cholinergic and noncholinergic submucosal neurons in porcine ileum. PAR-2 agonists stimulate active anion secretion by a neurogenic mechanism that is modulated by prostanoids and opioids. These receptors may have a potentially important role in intestinal neuroimmunomodulation.
Footnotes
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Send reprint requests to: David R. Brown, Ph.D., Department of Veterinary PathoBiology, University of Minnesota, 1988 Fitch Ave., St. Paul, MN55108-6010. E-mail: brown013{at}tc.umn.edu
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↵1 This study was funded in part by National Institutes of Health Grant DA-10200 (to D.R.B.) and NIH Grants DK-57840, DK-39957, and DK-43207, and an award from the Crohn's and Colitis Foundation of America (to N.W.B). Salary support for B.T.G. and A.K-N. was provided by Alcohol, Drug Abuse, and Mental Health Administration/National Institute on Drug Abuse Psychoneuroimmunology and Substance Abuse training Grant T32 DA07239.
- Abbreviations:
- GI
- gastrointestinal
- PAR
- proteinase-activated receptor
- DPDPE
- [d-Pen2,5]-enkephalin
- Isc
- short-circuit current
- Gt
- tissue conductance
- SBTI
- soybean trypsin inhibitor
- ChAT
- choline acetyltransferase
- PGP9.5
- protein gene product 9.5
- Received April 11, 2000.
- Accepted June 9, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
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