It is becoming increasingly evident that the urocortins (Ucns) and their receptors are involved in the initiation and development of inflammation in the gastrointestinal (GI) tract. There has not been a systematic study of the basal expression of Ucns or their receptors in the GI tract. Here, we examined basal expression of Ucn 2 and its high-affinity receptor, CRF-R2 in the rat GI tract. Ucn 2 mRNA was expressed throughout the small and large intestine. Surprisingly, CRF-R2 mRNA expression was detected in only a subset of GI regions that expressed Ucn 2. Immunohistochemical study showed that both Ucn 2 immuno-reactivity (Ucn 2-IR) and CRF-R2-IR were consistently seen in the neurons of the myenteric plexus and the nerve fibers innervating the circular muscle. By and large, Ucn 2-IR was detected in all layers, including the mucosal and the submucosal layers throughout the GI regions. In contrast, CRF-R2-IR was very low or undetectable in the mucosal layers of all regions examined. The role of Ucn 2 and CRF-R2 was then examined in a rat model of chemically-induced colitis. In the early phase of colitis, Ucn 2 mRNA levels peaked, whereas, in striking contrast, CRF-R2 mRNA expression decreased ∼2.5-fold below control levels. At the peptide level, Ucn 2-IR was specifically induced in a large population of immune cells that infiltrated the lamina propria and submucosa of the distal colon, whereas CRFR2-IR was detected in only a small fraction of infiltrated immune cells. CRF-R2-IR was dramatically reduced in the neurons of the myenteric plexus. Thus, we show, for the first time, that in the acute phase of inflammation, Ucn 2 levels are increased whereas expression levels of its only identified receptor, CRF-R2, are decreased. This suggests that Ucn 2 exerts its effects only in part via CRF-R2.

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...

Circadian behavioral rhythms in mammals are controlled by a central clock located in the suprachiasmatic nucleus (SCN). PER2, the protein product of the clock gene, Period 2 (Per2), is expressed rhythmically in the SCN [Beaule C, Houle LM, Amir S (2003) Expression profiles of PER2 immunoreactivity within the shell and core regions of the rat suprachiasmatic nucleus: Lack of effect of photic entrainment and disruption by constant light. J Mol Neurosci 21:133-148] and has been implicated in the control of circadian behavioral rhythms based on the evidence that genetic mutations in Per2 abolish free running locomotor activity rhythms in mice [Zheng B, Larkin DW, Albrecht U, Sun ZS, Sage M, Eichele G, Lee CC, Bradley A (1999) The mPer2 gene encodes a functional component of the mammalian circadian clock. Nature 400:169-173; Bae K, Jin X, Maywood ES, Hastings MH, Reppert SM, Weaver DR (2001) Differential functions of mPer1, mPer2, and mPer3 in the SCN circadian clock. Neuron 30:525-536]. Such mutations eradicate PER2 expression in the SCN and disrupt the SCN molecular clockwork, however, they also affect PER2 in the rest of the brain and body leaving open the possibility that the changes in behavioral rhythms might be influenced, at least in part, by disruptions in PER2 functioning outside the SCN. We used RNA interference-mediated transient knockdown of Per2 to study the effect of selective suppression of PER2 expression in the SCN, per se, on behavioral circadian rhythms. We found that transient suppression of PER2 in the SCN disrupted free running locomotor activity rhythms for up to 10 days in rats. Infusions of control dsRNA into the SCN or infusions of dsRNA to Per2 immediately dorsal to the SCN had no effect. These results constitute evidence for a direct link between PER2 expression in the SCN and the expression of behavioral circadian rhythms in mammals.

No abstract

No abstract