Differential Actions of Peripheral Corticotropin-Releasing Factor (CRF), Urocortin II, and Urocortin III on Gastric Emptying and Colonic Transit in Mice: Role of CRF Receptor

Peripheral CRF inhibits gastric emptying and stimulates colonic motor function in rats. We investigated the role of CRF1 and CRF2 receptors in i.p. CRF-induced alterations of gut transit in conscious mice using selective CRF1 and CRF2 ligands injected i.p. Gastric emptying 2 h after ingestion of a solid chow meal and colonic transit (time to expel a bead inserted into the distal colon) were determined simultaneously. Rat/human (r/ h)CRF, which has CRF1 CRF2 binding affinity, decreased distal colonic transit time at lower doses (6–12 g/kg) than those inhibiting gastric emptying (20–60 g/kg). Ovine CRF, a preferential CRF1 receptor agonist (6–60 g/kg), reduced significantly the colonic transit time without altering gastric emptying, whereas the selective CRF2 receptor agonists mouse urocortin II (20–60 g/kg) and urocortin III (120 g/kg) inhibited significantly gastric emptying without modifying colonic transit. The CRF1/CRF2 receptor antagonist, astressin (30–120 g/kg), dose dependently prevented r/hCRF (20 g/kg)-induced inhibition of gastric emptying and reduction of colonic transit time. The selective CRF1 receptor antagonists, NBI-27914 (C18H20Cl4N4C7H8O3S) and CP-154,526 (butyl-[2,5-dimethyl7-(2,4,6-trimethylphenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl]ethylamine) (5–30 mg/kg), dose dependently blocked r/hCRF action on the colon without influencing the gastric response, whereas the CRF2 receptor antagonist, antisauvagine-30 (30–100 g/ kg), dose dependently abolished r/hCRF-induced delayed gastric emptying and had no effect on colonic response. These data show that i.p. r/hCRF-induced opposite actions on upper and lower gut transit in conscious mice are mediated by different CRF receptor subtypes: the activation of CRF1 receptors stimulates colonic propulsive activity, whereas activation of CRF2 receptors inhibits gastric emptying. The 41-amino acid peptide corticotropin-releasing factor (CRF) was isolated from ovine hypothalamus and structurally characterized in 1981, as a novel hypothalamic releasing factor stimulating the release of pituitary proopiomelanocortin peptides (Vale et al., 1981). Recently, in addition to CRF, three other mammalian CRF-related peptides, namely, urocortin (Ucn), urocortin II (Ucn II) and urocortin III (Ucn III), have been identified in humans and rodents (Donaldson et al., 1996; Vaughan et al., 1996; Lewis et al., 2001; Reyes et al., 2001). Rat urocortin (rUcn) is a 40-amino acid peptide which shares 45% homology with rat/human (r/h)CRF (Vaughan et al., 1996). Human Ucn II and Ucn III have been discovered by sequence homology to r/hCRF from the Human Genome Database and the mouse orthologs have been cloned (Lewis al., 2001; Reyes et al., 2001). Mouse (m)Ucn II and mUcn III display 34% and 26% sequence homology with r/hCRF and 42% and 18% identity with rUcn, respectively (Lewis et al., 2001; Reyes et al., 2001). Two distinct seven-transmembrane domain Gs proteincoupled receptors, designated CRF1 and CRF2, each encoded by distinct genes, have been cloned in mammals (Perrin and Vale, 1999). Alternative splicing of the CRF2 receptor leads to CRF2 , CRF2 , and CRF2 variants in humans, which differ in their amino terminus, whereas in mice only the CRF2 form has been identified (Perrin and Vale, 1999). Although CRF1 and CRF2 receptors have considerable sequence similarity, they display distinct affinities for the four mammalian This work was supported by the Department of Veterans Affairs Merit Review (Y.T.) and National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-57238 (Y.T.) and DK-41301 (Animal Core, Y.T.), and DK-26741 (J.R.). V. Martı́nez was partially supported by the Conselleria de Cultura Educació i Ciència de la Generalitat Valenciana (Spain). ABBREVIATIONS: CRF, corticotropin-releasing factor; DMSO, dimethyl sulfoxide; oCRF, ovine CRF; r/hCRF, rat/human CRF; Ucn, urocortin; mUcn II, mouse urocortin II; mUcn III, mouse urocortin III; rUcn, rat urocortin. 0022-3565/02/3012-611–617$7.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 301, No. 2 Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics 4741/979885 JPET 301:611–617, 2002 Printed in U.S.A. 611 at A PE T Jornals on A ril 9, 2017 jpet.asjournals.org D ow nladed from members of the CRF family characterized so far (Perrin and Vale, 1999; Lewis et al., 2001; Reyes et al., 2001). In vitro studies established that Ucn binds with high affinity to both CRF receptor subtypes, whereas r/hCRF exhibits higher affinity to CRF1 than to CRF2 receptors (Perrin and Vale, 1999). In contrast, human and mUcn II and mUcn III bind selectively to CRF2 receptors, representing potential endogenous ligands for the receptor subtype 2 (Lewis et al., 2001; Reyes et al., 2001). In addition to their distinct pharmacological profiles, CRF1 and CRF2 receptors have different patterns of distribution in both the brain (Van Pett et al., 2000) and periphery (Baigent and Lowry, 2000; Muramatsu et al., 2000), indicating that they may subserve different biological functions. Previous functional studies established that peripherally administered r/hCRF, rUcn, or the nonmammalian CRFrelated peptides sauvagine and urotensin-I inhibit gastric emptying in rats, mice, and dogs (Pappas et al., 1985; Asakawa et al., 1999; Nozu et al., 1999; Wang et al., 2001). In contrast, r/hCRF and rUcn injected peripherally exert a stimulatory effect on colonic motor function as shown by the induction of spike burst activity in the proximal colon, acceleration of transit, and induction of defecation in rats (Williams et al., 1987; Maillot et al., 2000). The use of the nonselective CRF1/CRF2 receptor antagonists, -helical CRF9–41 and astressin (Gulyas et al., 1995), showed that peripheral r/hCRF-induced delayed gastric emptying in rats and mice and accelerated colonic motor function in rats are CRF receptor-mediated (Williams et al., 1987; Shelton et al., 1990; Martı́nez et al., 1999; Nozu et al., 1999; Maillot et al., 2000). However, the CRF receptor subtype(s) mediating alterations of gastrocolonic motor activity induced by peripheral CRF remains to be ascertained. We recently reported that the selective CRF1 receptor antagonist, CP-154,526 (Schulz et al., 1996), injected subcutaneously, prevented i.p. r/hCRFinduced clustered spike burst activity in the proximal colon and defecation in rats, indicative of a CRF1 receptor-mediated action (Maillot et al., 2000). In contrast, the CRF1 receptor antagonists antalarmin and NBI-27914 (Schulz et al., 1997; Maciejewski-Lenoir et al., 2000) administered intravenously in rats did not influence intravenous r/hCRFand rUcn-induced delay of gastric emptying of a liquid meal, suggesting a possible mediation through CRF2 receptors of the latter response (Nozu et al., 1999). Up to now, the lack of selective CRF2 receptor agonists and antagonists has hampered the direct pharmacological assessment of CRF2 receptors in the actions of CRF. Recently, the highly specific CRF2 antagonist antisauvagine-30 has been developed (Eckart et al., 2001; Higelin et al., 2001), and the novel endogenous CRF2-related peptides, Ucn II and Ucn III, have selective in vitro binding affinity to CRF2 receptors (Hsu and Hsueh, 2001; Lewis et al., 2001; Reyes et al., 2001). The aim of the present study was to establish the CRF receptor subtypes mediating peripheral r/hCRF-induced opposite actions on the upper and lower gut transit in mice. We first assessed whether r/hCRF and ovine CRF (oCRF), which have preferential binding affinity to the CRF1 receptor (Behan et al., 1996), and mUcn II and mUcn III, which exhibit selective affinity to the CRF2 receptor (Lewis et al., 2001; Reyes et al., 2001), exert differential effects on postprandial gastric and colonic transit. In addition, we investigated the blockade of r/hCRF actions by the nonselective CRF1/CRF2 receptor antagonist, astressin (Gulyas et al., 1995), the CRF1 receptor antagonists NBI-27914 and CP-154,526 (Schulz et al., 1997; Maciejewski-Lenoir et al., 2000), and the CRF2 receptor antagonist antisauvagine-30 (Eckart et al., 2001; Higelin et al., 2001). Gastric and colonic transits were monitored using a method that we developed to measure simultaneously the gastric emptying of a solid meal and the distal colonic propulsion in conscious mice. Materials and Methods