Central CRF, urocortins and stress increase colonic transit via CRF1 receptors while activation of CRF2 receptors delays gastric transit in mice

Abstract

Recently characterized selective agonists and developed antagonists for the corticotropin releasing factor (CRF) receptors are new tools to investigate stress-related functional changes. The influence of mammalian CRF and related peptides injected intracerebroventricularly (i.c.v.) on gastric and colonic motility, and the CRF receptor subtypes involved and their role in colonic response to stress were studied in conscious mice. The CRF(1)/CRF(2) agonists rat urocortin 1 (rUcn 1) and rat/human CRF (r/h CRF), the preferential CRF(1) agonist ovine CRF (oCRF), and the CRF(2) agonist mouse (m) Ucn 2, injected i.c.v. inhibited gastric emptying and stimulated distal colonic motor function (bead transit and defecation) while oCRF(9-33)OH (devoid of CRF receptor affinity) showed neither effects. mUcn 2 injected peripherally had no colonic effect. The selective CRF(2) antagonist astressin(2)-B (i.c.v.), at a 20 : 1 antagonist: agonist ratio, blocked i.c.v. r/hCRF and rUcn 1 induced inhibition of gastric transit and reduced that of mUcn 2, while the CRF(1) antagonist NBI-35965 had no effect. By contrast, the colonic motor stimulation induced by i.c.v. r/hCRF and rUcn 1 and 1h restraint stress were antagonized only by NBI-35965 while stimulation induced by mUcn 2 was equally blocked by both antagonists. None of the CRF antagonists injected i.c.v. alone influenced gut transit. These data establish in mice that brain CRF(1) receptors mediate the stimulation of colonic transit induced by central CRF, urocortins (1 and 2) and restraint stress, while CRF(2) receptors mediate the inhibitory actions of these peptides on gastric transit.

Figure 1. Dose-related effects of i.c.v. r/hCRF and CRF-related peptides on fecal pellet output in conscious mice

Under short-duration enflurane anaesthesia, mice fed ad libitum were injected i.c.v. with vehicle (saline solution, 5μl), r/hCRF (0.01, 0.1, 0.5 or 1.0μg), oCRF (0.01, 0.1 or 0.5μg), rat urocortin 1 (rUcn 1: 0.01, 0.1 or 0.5μg), mouse urocortin 2 (mUcn 2: 0.01, 0.1 or 0.5μg), mouse urocortin 3 (mUcn 3: 0.1, 0.5 or 1.0μg) or oCRF_9–33OH (0.5μg). Each point represents the mean ±s.e. of cumulative number of pellets for 1h after i.c.v. injection (n= 5–12mice/group). *P<0.05 versus vehicle-treated group (ANOVA); ^#P<0.05 versus r/hCRF at the same dose.

Figure 2. Time course of i.c.v. r/hCRF- (A), rUcn 1- (B) and mUcn 2- (C) induced fecal pellet output in mice

Under enflurane anaesthesia, mice fed ad libitum were injected i.c.v. with vehicle (saline solution, 5μl), r/hCRF (0.01, 0.1, 0.5 or 1.0μg), rat urocortin 1 (rUcn 1: 0.01, 0.1 or 0.5μg) or mouse urocortin 2 (mUcn 2: 0.01, 0.1 or 0.5μg). Each point represents the mean ±s.e. of number of pallets monitored at each 15min interval for 60min (n= 5–12mice/group). *P<0.05 versus vehicle-treated group (ANOVA).

Figure 3. Effects of i.c.v. CRF receptor antagonists on i.c.v. r/hCRF- (A), rUcn 1- (B) and mUcn 2- (C) induced stimulation of fecal pellet output in conscious mice

Under enflurane anaesthesia, mice fed ad libitum were injected i.c.v. with vehicle (distilled water, 2.5μl), the non-selective CRF₁/CRF₂ antagonist astressin (10μg), the selective CRF₁ antagonist NBI-35965 (50 or 100μg), or the selective CRF₂ antagonist astressin₂-B (10μg). Immediately thereafter vehicle (saline solution, 2.5μl), r/hCRF (0.5μg), rat urocortin 1 (rUcn 1, 0.5μg) or mouse urocortin 2 (mUcn 2, 0.5μg) was administered i.c.v. Each point represents the mean ±s.e. of cumulative number of pellets for 1h after i.c.v. injection (n= 4–6mice/group). *P<0.05 versus vehicle+vehicle- or antagonist+vehicle-treated groups; ^#P<0.05 versus vehicle+respective peptide-treated groups (ANOVA).

Figure 4. Effects of i.c.v. injection of r/hCRF and CRF-related peptides on gastric emptying of a solid nutrient meal (A, C) and distal colonic transit time (B, D) monitored simultaneously in conscious mice

Groups of fasted mice were given chow ad libitum for 1 h, then under short-duration enflurane anaesthesia were injected i.c.v. with either saline (5μl), r/hCRF or mouse urocortin 2 (mUcn 2, 0.01–0.5μg), oCRF, rat urocortin 1 (rUcn 1), mouse urocortin 3 (mUcn 3) or oCRF_9-33 0H (0.5μg) and a glass bead was inserted into the distal colon 2 cm proximal from the anus. Gastric emptying of the ingested meal 2h after peptide administration (A, C) and the time for bead expulsion (B, D) were monitored in the same animal. *P<0.05 versus vehicle-treated group (ANOVA); ^#P<0.05 versus r/hCRF at the same dose.

Figure 5. Effects of i.c.v. CRF receptor antagonists on i.c.v. r/hCRF-, rUcn 1- and mUcn 2-induced inhibition of gastric emptying and stimulation of distal colonic transit in conscious mice

Groups of fasted mice were given chow ad libitum for 1 h, then under short-duration enflurane anaesthesia were injected i.c.v. with distilled water (2.5μl), the selective CRF₁ antagonist NBI-35965 (50μg) or the selective CRF₂ antagonist astressin₂-B (10μg). Immediately thereafter, saline (2.5μl), r/hCRF, rat urocortin 1 (rUcn 1) or mouse urocortin 2 (mUcn 2, 0.5μg) was injected i.c.v. and a glass bead was inserted into the distal colon 2 cm proximal from the anus. Gastric emptying of the ingested meal 2h after peptide administration (left axis) and the time for bead expulsion (right axis) were monitored in the same animal. The dashed lines represent the mean gastric emptying rate (41.2±5.0%) and bead latency time (11.8±1.2min) in animals treated with vehicles or antagonists+vehicle. *P<0.05 versus vehicle-treated group (ANOVA).

Figure 6. Effects of i.c.v. CRF receptor antagonists on restraint stress-induced defecation in mice

Groups of mice fed ad libitum were injected i.c.v., under short-duration enflurane anaesthesia, with distilled water (5μl), the non-selective CRF₁/CRF₂ antagonist astressin (10μg), the selective CRF₁ antagonist NBI-35965 (50 or 100μg) or the selective CRF₂ antagonist astressin₂-B (10μg). Thereafter mice were subjected to a 1h session of stress (restraint in a cylinder) or left undisturbed in their home cages (non-stress). Pellet output was monitored at 15min intervals for the following 60min. A, cumulative pellet output for the 1h experimental time. B, time course changes in fecal pellet output at 15min intervals. *P<0.05 versus non-stress; ^#P<0.05 versus vehicle+stress group (ANOVA).