INTESTINE INFLAMMATION

Evidence for the presence of functional protease activated receptor 4 (PAR₄) in the rat colon

F Mulè¹, R Pizzuti², A Capparelli², N Vergnolle³

¹ Dipartimento di Biologia cellulare e dello Sviluppo, Università di Palermo, Palermo, Italy
² Dipartimento Farmaco-Biologico, Università della Calabria, Arcavacata di Rende (Cs), Italy
³ Department of Pharmacology and Therapeutics, University of Calgary, AB, Canada

Correspondence to:
Correspondence to:
Professor F Mulè
Dipartimento di Biologia cellulare e dello Sviluppo, Laboratorio di Fisiologia generale, Università di Palermo, Viale delle Scienze, 90128 Palermo, Italy; fmule{at}unipa.it

Background and aims: Protease activated receptors (PARs) have been postulated to play a role during intestinal inflammation. The presence and role played by PAR₄ in gastrointestinal functions have not been fully clarified. The aims of this study were: (i) to examine expression of PAR₄ in rat proximal colon; (ii) to determine the mechanical effects induced by PAR₄ activation in longitudinal muscle; and (iii) to characterise the underlying mechanisms.

Methods: PAR₄ expression was determined by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. Mechanical activity was recorded as changes in isometric tension.

Results: A PCR product corresponding to the predicted size of the PAR₄ signal was amplified from RNA prepared from the colon of rats, showing the presence of PAR₄ in those tissues. Immunohistochemistry revealed that PAR₄ protein was expressed on epithelial surfaces and submucosa. PAR₄ activating peptides, GYPGKF-NH₂ and AYPGKG-NH₂, produced concentration dependent contractile effects on longitudinal muscle. Tetrodotoxin (TTX) or atropine significantly reduced the contractile responses to AYPGKG-NH₂, and atropine after TTX did not cause any further reduction. NK₁ receptor antagonist, SR140333, or NK₂ receptor antagonist, SR48968, alone or in combination, produced a reduction in PAR₄ induced contractile effect, and when coadministered with TTX abolished it. Capsaicin markedly reduced the contractions evoked by AYPGKG-NH₂.

Conclusions: The present results suggest that PAR₄ is functionally expressed in rat colon and its activation induces contraction of the longitudinal muscle both through TTX sensitive release of acetylcholine and release of tachykinins, probably from sensory nerves. These actions may contribute to motility disturbances during intestinal trauma and inflammation.

Keywords: protease activated receptors; enteric nervous system; intestinal smooth muscle; PAR4; tachykinins

Abbreviations: PARs, protease activated receptors; PBS, phosphate buffer saline; TTX, tetrodotoxin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; EC₅₀, half maximal contractile concentration; RT-PCR, reverse transcription-polymerase chain reaction

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