Role of calcitonin receptor-like receptor in colonic motility and inflammation

Abstract

A. Role of calcitonin receptor-like receptor in colonic motility and inflammation. Am J Physiol Gastrointest Liver Physiol 293: G36-G44, 2007. First published March 15, 2007 doi:10.1152/ajpgi.00464.2006.-Calcitonin gene-related peptide (CGRP) mediates neurogenic inflammation and modulates intestinal motility. The CGRP receptor is a heterodimer of calcitonin receptor-like receptor (CLR) and receptor-associated modifying protein 1. We used RNA interference to elucidate the specific role of CLR in colonic motility and inflammation. Intramural injection of double-stranded RNA (dsRNA) against CLR (dsCLR) into the colonic wall at two sites caused the spatial and temporal downregulation of CLR in the colon within 1 day of dsRNA injection. Knockdown of CLR persisted for 7-9 days, and the effect of knockdown spread to ϳ2 cm proximal and distal to the injection sites, whereas control dsRNA injection did not affect CLR expression. Measurement of isometric contractions of isolated colonic muscle segments revealed that in control dsRNA-injected rats, CGRP abrogated contractions entirely and decreased resting muscular tone, whereas in dsCLRinjected rats, CGRP decreased muscle tone but slow-wave contractions of varying amplitude persisted. In trinitrobenzene sulfonic acidinduced colitis, rats with knockdown of CLR displayed a significantly greater degree of edema and necrosis than saline-or control dsRNAinjected rats. Levels of the proinflammatory cytokines TNF-␣ and IL-6 were markedly upregulated by trinitrobenzene sulfonic acid treatment. TNF-␣ mRNA levels were further increased in CLR knockdown rats, whereas levels of IL-6 were unaltered. Thus this study demonstrates that CLR is a functional receptor for CGRP.calcitonin gene-related peptide; RNA interference; trinitrobenzene sulfonic acid; cytokines; colon-specific; tumor necrosis factor-␣ CALCITONIN GENE-RELATED PEPTIDE (CGRP) is widely distributed in both the central and peripheral nervous systems. In the mammalian gastrointestinal (GI) tract, CGRP immunoreactivity is present in the myenteric and submucosal plexuses as well as in the sensory neurons, whose cell bodies are located in the nodose and dorsal root ganglia (13, 30). The functional CGRP receptor has been proposed to be a heterodimer of calcitonin receptor-like receptor (CLR) and receptor-associated modifying protein (RAMP) 1. Association of CLR with RAMP1 or RAMP2 confers specificity for binding either CGRP or adrenomedullin, respectively (23). Previous colocalization studies have demonstrated the presence of CLR and RAMP1 in enteric neurons, including the neurons in the myenteric plexus (5). Additionally, nerve fibers containing CGRP are found in close association with the CLR-positive neurons, suggesting that the effects of CGRP may be mediated via the CLR-RAMP1 heterodimer complex (5).CLR knockout mice are embryonic lethal with severe cardiovascular defects (6). Pharmacological antagonists for CLR have been reported, but systemic administration of antagonists does not result in tissue-specific inhibition of r...

Local injection of dsRNA targeting calcitonin receptor-like receptor (CLR) ameliorates Clostridium difficile toxin A-induced ileitis

“…As CGRP has diverse roles in different tissues (23)(24)(25), a targeted local delivery of dsRNA for CLR or CGRP is preferable. In agreement with previous findings, our dsRNA was not proinflammatory, because it did not alter weight gain or basal levels of intestinal secretion or cytokines and MPO activity (20,26) and, importantly, CLR dsRNA specifically ameliorated TxA-induced ileitis. Because local injection of CLR dsRNA universally improved all end points of TxA-induced ileitis examined, our current findings suggest efficacy for encapsulated or luminal delivery of dsRNA to alter local inflammation in the gastrointestinal tract.…”

“…We previously showed that CLR is required for functional responses to CGRP (20). CLR and RAMP1 comprise the CGRP receptor (6), and are colocalized throughout the gastrointestinal tract in nerve fibers and soma that may participate in TxA enteritis (7).…”

“…dsRNA for CLR and control (globin) was prepared as described (20). Cloned cDNAs for CLR and globin were used as templates to transcribe sense and antisense RNAs in vitro using a MEGAscript RNA Kit (Ambion) as specified.…”

“…The terminal ileum was exteriorized and a marking suture was placed in the ileum wall (2 cm from the cecum). dsRNA (20 μg) for either β-globin (non-CLR dsRNA) or CLR (CLR dsRNA) was mixed with 1.5 μL of Lipofectamine 2000 (Invitrogen) and injected into the lumen as described by us previously (20).…”

Highlights in inflammatory bowel disease – from bench to bedside

“…Pharmacological blockage of the SP receptor neurokinin (NK)1 was benefi cial in TNBS and DSS colitis (57,58) . Recent studies on SP, CGRP and double knockout mice confi rmed the pro-infl ammatory effects of SP in experimental colitis and demonstrated the protective effects of CGRP (56,59) , which had been suggested previously (60,61) . Several in vitro studies showed that the effects of SP and CGRP on the immune system are very complex and are more extensive than those of classical neurogenic infl ammation, which comprises vasodilatation and plasma extravasation.…”