Impairment of PAR‐2‐mediated relaxation system in colonic smooth muscle after intestinal inflammation

Abstract

Protease‐activated receptor (PAR)‐2 plays important roles in intestinal inflammatory responses. Changes in PAR‐2‐mediated smooth muscle function may contribute pathophysiologically to the intestinal motility disorders often observed in inflammatory bowel disease (IBD). Stimulation of PAR‐2 by trypsin‐induced relaxation of carbachol‐ and KCl‐induced contractions in normal rat colonic smooth muscle was completely resolved by tissue pretreatment with apamin, but not by pretreatment with l‐NMMA or a cocktail of neuronal blockers (tetrodotoxin, hexamethonium and propranolol). In colon inflamed by dextran sodium sulphate (DSS), trypsin‐induced inhibitory effects were significantly reduced. Relaxation induced by SLIGRL‐NH2, a selective PAR‐2‐activating peptide, was also reduced in DSS‐treated rat colon. However, inhibitory effects of 1‐ethylbenzimidazolin‐2‐one, an activator of small conductance Ca2+‐activated K+ channel, were unaffected. Expression of PAR‐2 mRNA in colonic muscularis externa was significantly lower in DSS‐treated rats than in control rats. These results suggest that the PAR‐2 mediated relaxation system in colonic smooth muscle is suppressed in this experimental colitis rat model, and may contribute to motility disorders in IBD. British Journal of Pharmacology (2006) 148, 200–207. doi:

Proinflammatory Cytokines Suppress the Expression Level of Protease-Activated Receptor-2 through the Induction of iNOS in Rat Colon

“…Induction of colitis and tissue preparation: Colonic inflammation was induced in male Sprague Dawley rats (7 to 12 weeks old) by administering 5% (wt/vol) DSS (molecular weight, 36,000-50,000) in drinking water for 0, 5 or 7 days (DSS-treated rats), as previously reported [26]. Procedures and care were approved by the Animal Care and Use Committee of Yamaguchi University.…”

“…Both profiles used a total of 27 cycles, followed by a final extension step of 5 min at 72C. The following oligonucleotide primers were designed [10,26]: PAR-2, 5'-CACCAGTAAGGGAGAAG TCT-3'(sense) and 5'-GGGCAGCACGTCGTGACAGGT-3' (antisense); IL-1, 5'-TCCATGAGCTTTGTACAAGG-3' (sense), 5'-GGTGCTGATGTACCAGTTGG-3' (antisense); TNF-, 5'-AAATGGGCTCCCTCTCATCA-3'(sense), 5'-AGCCTTGTCCCTTGAAGAGA-3' (antisense); iNOS, 5'-CTACCTACCTGGGGAACACCTGGG-3' (sense) and 5'-GGAGGAGCTGATGGAGTAGTAGCG G-3' (antisense); and -actin, 5'-CGTGGGCCGCCCTAG-GCACCA-3' (sense) and 5'-TTGGCCTTAGGG TTCAGG GGG-3' (antisense). After PCR products in each cycle were electrophoresed on 2% agarose gel, the products were stained using ethidium bromide and visualized using an ultraviolet transilluminator (UVP, Cambridge, UK).…”

“…All of these mRNA levels were significantly upregulated in smooth muscle layers of rat colon treated with DSS for 5 or 7 days as compared to control rat colon at a constant level of housekeeping gene -actin. We have previously reported that expression levels of -actin mRNA in DSS-treated rat colon are no different from those in control rat colon [26].…”

“…We have previously reported that trypsin-induced relaxation in rat colonic smooth muscle is attenuated by the induction of inflammation [26]. To determine the direct effects of proinflammatory cytokines on PAR-2-mediated relaxation, we adapted an organ culture method.…”

“…We have already reported that stimulation of PAR-2 with trypsin induces relaxation in rat colon by the activation of small conductance K + -channels, and we suggested that the PAR-2-mediated relaxation system in colonic smooth muscle is inhibited by the attenuation of PAR-2 mRNA expression in an experimental colitis rat model. However, we did not analyse the mechanisms of PAR mRNA downregulation, although we speculated that an excess of protease-mediated inflammatory responses would be counterbalanced by downregulation of PAR-2 [26].…”