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Agonists of proteinase-activated receptor 2 excite guinea pig ileal myenteric neurons

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Abstract

The effects of proteinase-activated receptor 2 (PAR2) agonists on the electrical properties of intact guinea pig ileal myenteric neurons were measured with intracellular microelectrodes. Approximately 52% of AH neurons and 41% of S neurons responded to pressure ejection of SLIGRL-NH2 or trypsin with a prolonged depolarization that was often accompanied by increased excitability. When added to the bathing solution, trypsin caused a concentration-dependent depolarization of responding neurons with an estimated EC50 value of 87 nM. Collectively, these novel observations indicate that PAR2 excites a proportion of myenteric neurons, which may contribute to dysmotility during intestinal inflammation.

Introduction

Proteinase-activated receptor 2 (PAR2) is a member of a family of G-protein coupled receptors that is activated through the proteolytic cleavage of the extracellular amino terminus by serine proteases. The resulting new amino terminus then becomes a tethered ligand that binds to and activates the receptor. PAR2 is cleaved by trypsin, a digestive serine protease derived from pancreatic trypsinogen, and tryptase, a serine protease that is stored in and released from mast cell granules Dery et al., 1998, Vergnolle, 2000.

A potential target for these serine proteases is the myenteric plexus of the gastrointestinal tract, which contains neurons that are responsible for regulating motor activities of the bowel (Furness and Costa, 1987). A subset of ileal myenteric neurons express PAR2 and respond to receptor activation with an increase in intracellular calcium concentration (Corvera et al., 1999). Although functional PAR2 exists on myenteric neurons, nothing is known about the effect of PAR2 activation on the electrical properties of myenteric neurons. To investigate this issue, intracellular recordings were obtained from individual guinea pig ileal myenteric plexus neurons in intact whole mount preparations exposed to exogenously applied activators of PAR2.

Section snippets

Tissue preparation

All methods used in this study were approved by the University of Vermont Animal Care and Use Committee. Adult guinea pigs (either sex, 250 to 300 g) were anesthetized with isoflurane and exsanguinated. The terminal ileum was removed and placed in iced Krebs solution (in mM: NaCl, 121; KCl, 5.9; CaCl2, 2.5; MgCl2, 1.2; NaHCO3, 25; NaH2PO4, 1.2; and glucose, 8; aerated with a 95%O2:5%CO2). Nifedipine (5 mM) and atropine (200 nM) were also added to eliminate smooth muscle contraction. The mucosa,

Results

The effects of PAR2 agonists on passive and active electrical properties were measured in a total of 92 neurons from 64 preparations. Using the criteria described previously Bornstein et al., 1994, Wood, 1994, the neurons were classified as AH [35 neurons; resting membrane potential (RMP): −68±3 mV; input resistance: 105±10 MΩ] or S (57 neurons; RMP: −56±2 mV; input resistance: 141±13 MΩ). In responding cells, activation of PAR2 with SLIGRL-NH2 or trypsin elicited a prolonged depolarization

Discussion

The data presented here indicate that activation of PAR2 elicits a prolonged depolarization and excitation of myenteric neurons in the guinea pig ileum. This observation is novel, and expands our understanding of the role that PAR2 plays in the physiology and pathophysiology of the alimentary canal. Because PAR2 is expressed on a subset of myenteric neurons (Corvera et al., 1999) and tetrodotoxin does not alter the response to SLIGRL-NH2, it is likely that the action of this synthetic peptide

Acknowledgements

This work was supported by NIH grants NS26995, DK45410, DK57840 and DK43207, and an R.W. Johnson Focused Giving Grant.

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