Tomofumi Amano

Activation of Corticotropin-Releasing Factor Receptor 2 Mediates the Colonic Motor Coping Response to Acute Stress in Rodents

BACKGROUND & AIMS Corticotropin-releasing factor receptor-1 (CRF(1)) mediates the stress-induced colonic motor activity. Less is known about the role of CRF(2) in the colonic response to stress. METHODS We studied colonic contractile activity in rats and CRF(2)-/-, CRF-overexpressing, and wild-type mice using still manometry; we analyzed defecation induced by acute partial-restraint stress (PRS), and/or intraperitoneal injection of CRF ligands. In rats, we monitored activation of the colonic longitudinal muscle myenteric plexus (LMMP) neurons and localization of CRF(1) and CRF(2) using immunohistochemical and immunoblot analyses. We measured phosphorylation of extracellular signal-regulated kinase 1/2 by CRF ligands in primary cultures of LMMP neurons (PC-LMMPn) and cyclic adenosine monophosphate (cAMP) production in human embryonic kidney-293 cells transfected with CRF(1) and/or CRF(2). RESULTS In rats, a selective agonist of CRF(2) (urocortin 2) reduced CRF-induced defecation (>50%), colonic contractile activity, and Fos expression in the colonic LMMP. A selective antagonist of CRF(2) (astressin(2)-B) increased these responses. Urocortin 2 reduced PRS-induced colonic contractile activity in wild-type and CRF-overexpressing mice, whereas disruption of CRF(2) increased PRS-induced colonic contractile activity and CRF-induced defecation. CRF(2) colocalized with CRF(1) and neuronal nitric oxide synthase in the rat colon, LMMP, and PC-LMMPn. CRF-induced phosphorylation of extracellular signal-regulated kinase in PC-LMMPn; this was inhibited or increased by a selective antagonist of CRF(1) (NBI35965) or astressin(2)-B, respectively. The half maximal effective concentration, EC(50), for the CRF-induced cAMP response was 8.6 nmol/L in human embryonic kidney-293 cells that express only CRF(1); this response was suppressed 10-fold in cells that express CRF(1) and CRF(2). CONCLUSIONS In colon tissues of rodents, CRF(2) activation inhibits CRF(1) signaling in myenteric neurons and the stress-induced colonic motor responses. Disruption of CRF(2) function impairs colonic coping responses to stress.

T1756 CRF2 Receptor Activation Enhances Nerve-Mediated Relaxation of the Rat Internal Anal Sphincter

Background Corticotropin-releasing factor (CRF) and Urocortins (Ucn) are stress-related peptides. Central or peripheral administration of these peptides has been shown to increase in colonic motor activity via CRF1 receptor. We have reported that CRF1 receptor is present in the enteric nervous system (ENS) in rat colon and direct activation of CRF1 receptor in the ENS induces colonic muscle contraction without CNS pathway (Am J Physiol 2007; 293: G903-10). In contrast to CRF1 receptor, role of CRF2 receptor in the colon has not been well studied. Aim To study whether CRF2 receptor activation cause relaxation of the colonic muscle and whether CRF2 receptor is present in the ENS. Methods Internal anal sphincter (IAS) of the rat was used in this study because IAS has resting tension and its relaxation is clearly observed. IAS circular muscle strips were suspended in an organ bath and its resting tension and electrical field stimulation (EFS) -induced relaxation were examined. The effects of Ucn 2 (selective CRF2 receptor agonist) on them were studied. To study the mechanism of action of Ucn 2, we examined these effects in the presence of tetorodotoxin (TTX), CRF1 receptor antagonist antalarmin, CRF2 receptor antagonist astressin2-B, and LNAME. The localization of Ucn 2, CRF2 receptor, and NOS in the rat IAS was investigated by immunohistochemistry using confocal laser microscope.Results IASmuscle strips showed resting tension and EFS-induced relaxation in a frequency-dependent manner. Ucn 2 caused significant decrease in resting tension of IAS dose-dependently (1 nM ~ 1 μM). In the presence of TTX, astressin2-B, and L-NAME, the effect of Ucn 2 on resting tension were abolished. Ucn 2 caused significant enhancement of EFS-induced relaxation of IAS dosedependently (1 nM ~ 10 nM). L-NAME decreased the EFS-induced relaxation, and this residual relaxation was not augmented by Ucn 2. Ucn 2 and CRF2 receptor were present in the myenteric plexus of rat IAS. Ucn 2 and NOS were colocalized in some of the myenteric neuronal cells. Discussion CRF2 receptor activation caused decrease in resting tension and augmentation of EFS-induced relaxation of IAS. These mechanisms are suggested to be through nitrergic neuronal pathway. Like anxiety or depression in CNS, CRF1 and CRF2 receptors seem to act oppositely in the gut motor activity. Conclusion CRF2 receptor activation enhances nerve-mediated relaxation of the rat IAS.