We characterized the influence of the selective corticotropin-releasing factor 2 (CRF2) receptor agonist human urocortin 2 (Ucn 2), injected intracisternally, on gastric emptying and its mechanism of action compared with intracisternal CRF or urocortin (Ucn 1) in conscious rats. The methylcellulose phenol red solution was gavaged 20 min after peptide injection, and gastric emptying was measured 20 min later. The intracisternal injection of Ucn 2 (0.1 and 1 μg) and Ucn 1 (1 μg) decreased gastric emptying to 37.8 ± 6.9%, 23.1 ± 8.6%, and 21.6 ± 5.9%, respectively, compared with 58.4 ± 3.8% after intracisternal vehicle. At lower doses, Ucn 2 (0.03 μg) and Ucn 1 (0.1 μg) had no effect. The CRF2 antagonist astressin2-B (3 μg ic) antagonized intracisternal Ucn 2 (0.1 μg) and CRF (0.3 μg)-induced inhibition of gastric emptying. Vagotomy enhanced intracisternal Ucn 2 (0.1 or 1 μg)-induced inhibition of gastric emptying compared with sham-operated group, whereas it blocked intracisternal CRF (1 μg) inhibitory action (45.5 ± 8.4% vs. 9.7 ± 9.7%). Sympathetic blockade by bretylium prevented intracisternal and intracerebroventricular Ucn 2-induced delayed gastric emptying, whereas it did not influence intravenous Ucn 2-, intracisternal CRF-, and intracisternal Ucn 1-induced inhibition of gastric emptying. Prazosin abolished the intracisternal Ucn 2 inhibitory effect, whereas yohimbine and propranolol did not. None of the pretreatments modified basal gastric emptying. These data indicate that intracisternal Ucn 2 induced a central CRF2-mediated inhibition of gastric emptying involving sympathetic α1-adrenergic mechanisms independent from the vagus contrasting with the vagal-dependent inhibitory actions of CRF and Ucn 1.
- adrenergic receptors
- corticotropin-releasing hormone receptor
corticotropin-releasing factor (CRF) is a 41-amino acid peptide first isolated from ovine hypothalamus that plays a central role in the stress-related stimulation of the hypothalamic-pituitary-adrenal (HPA) axis (2). Recently, three novel mammalian CRF-related peptides have been characterized (17). Rat (rUcn 1) and human urocortin 1 (hUcn 1) are identical 40-amino acid peptides that share 43% identity with r/hCRF (17). The two novel putative Ucn isoforms, mouse Ucn 2 (mUcn 2) and mUcn 3 are 38-amino acid peptides that are more distantly related to r/hCRF with 34% and 26% homology, respectively (17). The hUcn 2 counterpart, also named urocortin-related peptide, shares 76% homology with mUcn 2 and is homologous to the postulated NH2 terminally extended 43-amino acid peptide stresscopin-related peptide (17).
CRF ligands mediate their biological actions through interaction with two distinct receptors, subtypes 1 (CRF1) and 2 (CRF2), cloned from distinct genes (2). Both CRF1 and CRF2 receptors belong to the class B subfamily of seven-transmembrane receptors coupled to GS proteins (2). Radioreceptor and functional assays have demonstrated that CRF1 and CRF2 receptors differ considerably in their binding characteristics to natural CRF ligands (17). The CRF1 receptor shows no appreciable binding to Ucn 2 and Ucn 3 but binds with high affinity to Ucn 1, CRF, and the amphibian CRF-related peptide sauvagine (17). In contrast, the CRF2 receptor binds to Ucn 1, Ucn 2, Ucn 3, and sauvagine with a greater affinity than to CRF, making this receptor subtype highly selective for mammalian urocortin signaling (17).
Activation of brain CRF receptors results in a number of stresslike responses (2) of which the inhibition of gastric transit under various experimental conditions has been reproducibly documented (42). In particular, initial reports showed that CRF, injected intracerebroventricularly or intracisternally, inhibits gastric emptying of a nonnutrient viscous methylcellulose solution in conscious female or male rats (43, 49). Other studies established that CRF injected into the cerebrospinal fluid at the level of the lateral or 4th ventricle or the cisterna magna inhibits gastric emptying of saline, glucose, acid, or a peptone liquid meal delivered intragastrically as well as gastric emptying of a physiological meal (ingestion of solid chow) in conscious rats (reviewed in Ref. 42). Likewise in other species, intracerebroventricular injection of CRF inhibits gastric emptying of a nonnutrient liquid and an ingested solid chow meal in mice (31, 40) and the total gastric emptying time of a solid nutrient meal in dogs (24).
Consistent sets of observations established that the inhibition of gastric emptying induced by intracisternal or intracerebroventricular injection of CRF in various species is not related to the activation of the HPA axis and is mediated by the autonomic nervous system. Neither hypophysectomy nor acute adrenalectomy altered intracerebroventricular CRF-induced delayed gastric emptying in rats (6, 25, 43), whereas the ganglionic blockade by chlorisondamine abolished intracerebroventricular CRF action in rats and mice (25, 40). Several reports showed that vagal-dependent mechanisms are involved in central CRF-induced delayed gastric emptying of a liquid noncaloric meal in rats and caloric meal in dogs (6, 24, 43). In contrast, two reports indicate the importance of sympathetic pathways in mediating intracisternal or intracerebroventricular injection of CRF-induced delayed gastric emptying in rats (25, 34).
Less is known on the central action of Ucns in inhibiting gastric motor function. A dose-related inhibition of gastric emptying of a solid meal has been reported after the intracisternal injection of Ucn 1 in rats (10) and intracerebroventricular injection of Ucn 2 in mice (31). However, the pathways through which central injection of Ucn 1 and Ucn 2 inhibit gastric emptying are still to be determined. Therefore, the aim of the present study was to characterize the central action of Ucn 2 on gastric emptying in conscious rats and to compare it with that of CRF1/CRF2 agonists, Ucn 1, or CRF. We assessed the CRF2 receptor-mediated action of intracisternal Ucn 2, Ucn 1, and CRF using the selective CRF2 antagonist astressin2-B (38). Autonomic pathways through which intracisternal and intracerebroventricular Ucn 2 and intracisternal Ucn 1 inhibit gastric emptying were delineated using surgical (subdiaphragmatic vagotomy) and pharmacological (noradrenergic, α1-, α2-, and β-adrenergic receptor blockade) approaches.
MATERIALS AND METHODS
Adult male Sprague-Dawley rats (Harlan, San Diego, CA) weighing 250–300 g were housed in group cages under controlled illumination (12:12-h light-dark cycle starting at 6 AM), humidity, and temperature (21–23°C) and had free access to tap water and Purina rat chow. Rats were deprived of food but had free access to tap water for 16–18 h before the experiments, except as otherwise stated. Protocols were approved by the University of California Los Angeles and Veteran Affairs Greater Los Angeles Healthcare System Animal Research Committees (protocol no. 99127–07).
r/hCRF, r/hUcn 1, hUcn 2 (Ucn 2), and astressin2-B (Peptide Biology Laboratory, The Salk Institute, La Jolla, CA) were synthesized using the solid-phase approach and the Boc-strategy (38). Peptides were stored in powder form at −80°C. Immediately before the experiments, CRF, Ucn 1 and Ucn 2 were dissolved in saline and astressin2-B in double-distilled water (pH 7.0). Bretylium tosylate and propranolol hydrochloride (both from Sigma, St. Louis, MO) were stored at room temperature, and yohimbine hydrochloride (Sigma) and prazosin hydrochloride (Pfizer, Croton, CT) were stored at −20°C. Drugs were dissolved in sterile saline just before use, except yohimbine hydrochloride that was dissolved in 5% DMSO and 95% distilled water. Phenol red and methylcellulose (both from Sigma) were stored at room temperature and prepared on the day of experiments. Ketamine hydrochloride (Ketaset, Fort Dodge Laboratories, Fort Dodge, IA) and xylazine (Rompun, Mobay, Shawnee, KS) were used to anesthetize rats undergoing surgery.
Intracisternal injections were performed as previously reported (43) under short isoflurane anesthesia (2–3 min, 5% vapor concentration in oxygen; VSS, Rockmart, GA). The occipital membrane was punctured with a 50-μl Hamilton syringe using stereotaxic equipment. Presence of cerebrospinal fluid into the Hamilton syringe on aspiration before injection ensured the right needle tip position into the cisterna magna. The volume of injections was 10 μl for CRF, Ucn 1, and Ucn 2 and 5 μl for astressin2-B.
Intracerebroventricular injections were performed as previously described (30) in rats equipped with a chronic intracerebroventricular cannula. Animals were anesthetized with an intraperitoneal injection of a mixture of ketamine hydrochloride (75 mg/kg) and xylazine (5 mg/kg). A guide cannula (22 gauge, Plastic One Products, Roanoke, VA) was implanted into the right lateral brain ventricle according to the following coordinates (mm from bregma: antero-posterior, −0.8; lateral, −1.5; dorsoventral, −3.5). The guide cannula was maintained in place by dental cement anchored by four stainless steel jewelry screws fixed to the skull. After the surgery, animals were housed individually with direct bedding. Experiments were performed in conscious rats starting 10 days after the intracerebroventricular cannulation. During this time, rats were habituated to the manipulation of the cannula and handled daily for a period of about 5 min. The intracerebroventricular injection was performed in lightly restrained rats. A 28-gauge injection cannula, 1 mm longer than the guide cannula, was then connected to a 50-μl Hamilton syringe by a PE-50 catheter (Intramedic Polyethylene Tubing, Clay Adams, Sparks, MD) filled with distilled water. A small air bubble (1 μl) was drawn at the distal end of the PE-50 catheter to separate the injected solution from the water and for visual inspection of the 10-μl injection, which was performed slowly over 30 s. At the end of the experiments, the correct location of the cannula into the lateral ventricle was verified by injecting 10 μl of dye (0.1% toluidine blue). Visualization of dye on the wall of the lateral ventricle indicates correctness of the intracerebroventricular injections.
Intravenous injections (0.1 ml) were performed into the right jugular vein after a skin incision under short (2–5 min) isoflurane anesthesia. Wounds were closed with sterile silk (2.0 metric, Ethicon, Somerwille, NJ). Intraperitoneal (0.3 ml) and subcutaneous (0. 1 ml) injections were done in conscious rats.
Subdiaphragmatic vagotomy was performed by circular seromuscular myotomy of the esophagus 2 cm proximal from the gastroesophageal junction in fasted rats under ketamine hydrochloride (75 mg/kg ip) and xylazine (5 mg/kg ip) anesthesia. Sham vagotomy consisted of a laparotomy and similar manipulation of the esophagus and stomach without the myotomy under anesthesia. Rats that underwent subdiaphragmatic vagotomy or sham vagotomy were maintained with a liquid diet (Ensure; Ross Products Division, Abbott Laboratories, Columbus, OH) for 24 h postsurgery, then deprived of liquid diet, but not water, for 12 h before the gastric emptying measurements.
Measurement of Gastric Emptying
Gastric emptying of a nonnutrient viscous meal was determined by the phenol red method as described previously (43). The noncaloric meal consisted of a viscous suspension of continuously stirred 1.5% methylcellulose (wt/vol) containing phenol red (50 mg/100 ml) given intragastrically through a stainless steel gavage tube (in 1.5-ml volume) to conscious rats. At 20 min after the administration of the solution, rats were euthanized by CO2 inhalation. The abdominal cavity was opened, the gastroesophageal junction and the pylorus were clamped, and the stomach was isolated and rinsed in 0.9% saline. The stomach was placed into 100 ml 0.1 N NaOH and homogenized (Polytron; Brinkman Instruments, Westbury, NY). The suspension was allowed to settle for 60 min at room temperature, then 5 ml supernatant was added to 0.5 ml of 20% trichloroacetic acid (wt/vol) and centrifuged at 3,000 rpm at 4°C for 20 min. After the supernatant was mixed with 4 ml 0.5 N NaOH, the absorbance of the sample was read at 560 nm (Shimazu 260 Spectrophotometer). The absorbance of the phenol red recovered from animals euthanized immediately after gavage of the liquid meal was taken as a standard 0% emptying. The percentage of emptying during the 20-min period was calculated with the following formula: percent emptying = (1 − absorbance of test sample/absorbance of standard) × 100.
Effect of intracisternal Ucn 2, Ucn 1, and CRF alone or with CRF2 receptor antagonist on gastric emptying.
Fasted rats were briefly anesthetized with isoflurane and injected intracisternally with saline (10 μl), Ucn 2 (0.03, 0.1, or 1 μg), or Ucn 1 (0.1 or 1 μg). In another set of experiments, fasted rats were injected intracisternally (5 μl) with double-distilled water or astressin2-B (1, 3, or 10 μg) just before intracisternal injection of saline (10 μl), Ucn 2 (0.1 μg), or CRF (0.3 μg). At 20 min after intracisternal injection, the phenol red methylcellulose solution was delivered intragastrically, and gastric emptying was determined 20 min later.
Effect of subdiaphragmatic vagotomy on intracisternal injection of Ucn 2- and CRF-induced delayed gastric emptying.
Sham or subdiaphragmatic vagotomy rats fasted for 12 h were injected intracisternally with saline (10 μl), CRF (1 μg), or Ucn 2 (0.1 or 1 μg), and 20 min later, animals received gastric gavage of 1.5 ml phenol red methylcellulose solution. Gastric emptying was determined 20 min later.
Effect of bretylium pretreatment on intracisternal, intracerebroventricular, and intravenous Ucn 2-, intracisternal Ucn 1-, and intracisternal CRF-induced delayed gastric emptying.
Bretylium tosylate (25 mg/kg) or saline was injected intraperitoneally 60 min before Ucn 2 (0.1 μg ic, 1 μg icv, or 15 μg/kg iv), Ucn 1 (1 μg ic), CRF (0.3 μg ic), or saline (ic, icv, or iv). After 20 min, the 1.5 ml phenol red methylcellulose solution was given by gavage and gastric emptying was monitored 20 min later. The dose of bretylium was based on a previous study showing complete noradrenergic blockade (25). The doses of Ucn 2 injected intracisternally, intracerebroventricularly, and intravenously were based on dose-response studies (present studies and data not shown) inducing a similar suppression of gastric emptying of a nonnutrient meal.
Effect of α1-, α2-, and β-adrenergic blockade on intracisternal Ucn 2-induced delayed gastric emptying.
Prazosin (1 mg/kg), propranolol (1 mg/kg), or saline (0.3 ml) was injected intraperitoneally 30 min before saline or Ucn 2 (0.1 μg ic). In other studies, yohimbine or vehicle (5% DMSO/95% distilled water) was injected subcutaneously 30 min before intracisternal injection of saline or Ucn 2 (0.1 μg). Twenty minutes later, rats were gavaged with 1.5 ml phenol red methylcellulose solution and euthanized after 20 min for gastric emptying determination. Doses of prazosin, yohimbine, and propranolol were based on previous studies showing complete α1-, α2-, and β-adrenergic receptor blockade, respectively, of peptide action on gut function (12, 29).
All results are expressed as means ± SEM. One-way ANOVA followed by Student-Newman-Keuls multiple comparison test was performed for comparison between groups. P values <0.05 were considered statistically significant.
Ucn 2 and Ucn 1 Injected Intracisternally Inhibit Gastric Emptying in Conscious Rats
Ucn 2, injected intracisternally at 0.03, 0.1, and 1 μg, dose dependently inhibits gastric emptying of a viscous noncaloric solution in conscious rats (Fig. 1). At 0.1 μg, intracisternal Ucn 2 significantly reduced gastric emptying to 37.8 ± 6.9% compared with 58.4 ± 3.8% in the intracisternal saline-treated group, whereas Ucn 1 had no effect (56.3 ± 0.5%). At 1 μg, both Ucn 2 and Ucn 1 injected intracisternally induced a similar inhibition of gastric transit (23.1 ± 8.6% and 21.6 ± 5.9%, respectively, P < 0.05 compared with intracisternal saline; Fig. 1).
Astressin2-B Injected Intracisternally Prevents Intracisternal Ucn 2- and Intracisternal CRF-Induced Delayed Gastric Emptying
Astressin2-B, injected at 3 and 10 μg, completely prevented intracisternal Ucn 2-induced inhibition of gastric emptying (51.6 ± 2.8% and 54.3 ± 7.2%, respectively, vs. 36.6 ± 6.2%, P < 0.05), whereas at 1 μg ic, astressin2-B was ineffective (27.9 ± 4.5%; Fig. 2). Similarly, astressin2-B injected intracisternally at 3 μg completely antagonized intracisternal CRF (0.3 μg)-induced inhibition of gastric emptying (52.0 ± 4.6% vs. 18.9 ± 4.8%; P < 0.05). Astressin2-B (3 μg) injected intracisternally alone did not influence basal gastric emptying of a nonnutrient viscous meal (51.3 ± 6.6%; Fig. 2).
Vagotomy Blocked Intracisternal CRF but not Intracisternal Ucn 2-Induced Inhibition of Gastric Emptying
Ucn 2, injected intracisternally at 0.1 μg, did not significantly delay gastric emptying in sham-vagotomized rats (29.0 ± 9.5% vs. 34.6 ± 3.8%, respectively), whereas there was a significant reduction in vagotomized rats (15.6 ± 3.2% vs. 33.4 ± 5.9%). At 1 μg, intracisternal Ucn 2 significantly reduced gastric emptying in the sham group (16.9 ± 2.9% vs. 34.6 ± 3.8%), and the inhibitory effect was significantly enhanced in vagotomized (6.3 ± 3.0%) compared with sham (16.9 ± 2.9%) groups (Fig. 3). In sham-vagotomized rats, intracisternal injection of CRF (1 μg) significantly delayed gastric emptying (9.7 ± 9.7%) compared with vehicle (34.6 ± 3.8%). Subdiaphragmatic vagotomy completely prevented intracisternal CRF-induced delayed gastric emptying (45.5 ± 8.4%; Fig. 3). Subdiaphragmatic vagotomy did not influence basal gastric emptying of viscous solution (33.4 ± 5.9%) compared with sham-operated rats (34.6 ± 3.8%; Fig. 3).
It is of note that sham and vagotomized intracisternal vehicle groups, fed with a liquid diet for 24 h postsurgery followed by a 12-h fast, had lower gastric emptying compared with other nonoperated intracisternal vehicle groups (Fig. 3 vs. Figs. 1, 2–4, 5) fasted for 16–18 h. The shorter fasting time period along with the surgery, which is known to influence gastric emptying (30, 48), may have contributed to the lower gastric emptying in operated rats.
Bretylium Prevents Intracisternal and Intracerebroventricular Ucn 2-, but not Intravenous Ucn 2-, Intracisternal CRF-, or Intracisternal Ucn 1-Induced Inhibition of Gastric Emptying
Ucn 2, injected intracisternally (0.1 μg) in rats under short anesthesia or intracerebroventricularly (1 μg) in lightly restrained conscious rats, significantly delayed gastric emptying (27.1 ± 7.3% and 22.7 ± 5.7%, respectively) compared with groups injected with saline either intracisternally (55.8 ± 6.8%) or intracerebroventricularly (67.6 ± 13.5%; Fig. 4). The inhibitory effect of both intracisternal and intracerebroventricular Ucn 2 was completely prevented by intraperitoneal bretylium tosylate (56.1 ± 4.4% and 57.0 ± 8.7%, respectively; Fig. 4). In contrast, neither intracisternal CRF- (0.3 μg) nor intracisternal Ucn 1 (1 μg)-induced delayed gastric emptying (25.3 ± 4.0% and 19.7 ± 5.7%, respectively) was altered by pretreatment with intraperitoneal bretylium tosylate (18.5 ± 4.9% and 11.9 ± 4.4%, respectively; Fig. 4). In addition, the intravenous Ucn 2-induced significant suppression of gastric emptying (9.0 ± 3.2%) was not modified by bretylium tosylate pretreatment (5.0 ± 3.5%; Fig. 4C). In intracisternal, intracerebroventricular, or intraperitoneal saline-treated groups, intraperitoneal bretylium tosylate did not modify gastric emptying compared with intraperitoneal vehicle (Fig. 4, A-C).
Prazosin Prevents Intracisternal Ucn 2-Induced Inhibition of Gastric Emptying, Whereas Yohimbine and Propranolol had no Effect
Pretreatment with the α1-adrenergic receptor blocker prazosin (1 mg/kg ip) completely abolished the delayed gastric emptying induced by intracisternal Ucn 2 compared with the vehicle-pretreated group (56.9 ± 9.3% vs. 29.2 ± 3.0%). In contrast, the α2-adrenergic receptor blocker yohimbine (4 mg/kg sc) and the β-adrenergic receptor blocker propranolol (1 mg/kg ip) did not modify Ucn 2 (0.1 μg ic)-induced delayed gastric emptying (Fig. 5; Table 1). Prazosin, yohimbine, and propranolol injected alone with intracisternal saline did not significantly alter the 20-min basal gastric emptying values (Fig. 5; Table 1).
The present study demonstrates that the newly characterized CRF2-selective agonist, Ucn 2 injected intracisternally, inhibits gastric emptying of a viscous noncaloric solution in a dose-dependent manner in conscious fasted rats. Ucn 2 injected intracisternally is more potent than Ucn 1 as shown by the significant inhibition of gastric emptying induced by Ucn 2 at 0.1 μg (24 pmol), whereas Ucn 1 (0.1 μg, 21 pmol) had no effect under these conditions. However, at 1 μg, both Ucn 1 and Ucn 2 exert a similar 60% and 63% reduction of gastric emptying, respectively. These observations expend previous reports showing that Ucn 1, injected intracisternally at 0.3 μg (63 pmol), did not alter gastric emptying of a solid meal, whereas inducing a dose-related inhibition at intracisternal doses of 0.6 and 1 μg (10). Likewise, CRF injected intracisternally at 0.1 μg (21 pmol) had no effect on gastric emptying of a methylcellulose solution, whereas it significantly suppressed gastric emptying when injected intracisternally at 0.3–1 μg in conscious rats (28, 43) (present study). Although gastric emptying was monitored in fasted rats briefly anesthetized with isoflurane for the duration of the puncture of the cisterna magna and intracisternal injection, it is unlikely that the peptide inhibitory action represents an interaction with the anesthesia or other stressful aspects of peptide injection. We observed a similar delay in gastric emptying in response to Ucn 2 injected in conscious fasted rats through a chronically implanted intracerebroventricular cannula (present study) or in fed or fasted conscious rats injected intracerebroventricularly with CRF (25, 28).
The selective CRF2 antagonist astressin2-B (38), injected intracisternally, completely prevented both intracisternal Ucn 2- and intracisternal CRF-induced delayed gastric emptying of a liquid nonnutrient meal (present study) and intracisternal Ucn 1-induced delayed gastric emptying of a chow meal (10). The delayed gastric emptying induced by intracisternal injection of the selective CRF2 agonist Ucn 2 and the prevention of intracisternal Ucn 2, intracisternal CRF, and intracisternal Ucn 1 inhibitory action by the selective CRF2 antagonist provides direct pharmacological evidence that the CRF2 receptor is involved in intracisternal CRF and urocortin action. Before the identification of CRF2 agonists and antagonists, indirect evidence was indicative of a role of brain CRF2 receptors based on the rank order of potency of intracisternal sauvagine > urotensin-I > CRF to inhibit gastric emptying of a solid meal in rats (28), which was in line with their differential affinity to CRF2 receptors (sauvagine > urotensin-I > CRF) (35).
Convergent evidence indicates that sympathetic pathways and α1-adrenergic receptors are involved in the inhibition of gastric emptying induced by central injection of Ucn 2. First, intraperitoneal injection of bretylium, an adrenergic neuronal blocking agent taken up selectively at peripheral adrenergic nerve terminals and blocking transmitter released from sympathetic postganglionic nerve terminals (7), abolished both intracisternal and intracerebroventricular Ucn 2-induced decreased gastric emptying. Second, subdiaphragmatic vagotomy did not block, but significantly enhanced, the inhibitory action of Ucn 2 injected intracisternally at 0.1 and 1 μg resulting in a 56% and 82% suppression of gastric emptying, respectively. The increased inhibitory effect of intracisternal Ucn 2 in vagotomized rats may be consistent with the sympathetic mediated inhibitory mechanisms that are no longer restrained by vagal cholinergic tone (1). The main neurotransmitters/neuromodulators in postganglionic sympathetic nerves are noradrenaline, ATP, and neuropeptide Y (8). In the present study, pharmacological blockade of α1-adrenergic receptors by prazosin mimicked the effects of bretylium, whereas α2- and β-adrenergic blockade by yohimbine and propranolol, respectively, had no effect. Such a reversal of intracisternal Ucn 2 action occurs under conditions where sympathetic or adrenergic blockade did not alter basal gastric emptying as previously reported for gastric emptying and motility in fasted and fed-state rats (22, 25). In rats, norepinephrine injected intravenously induced fundic relaxation (3) and the α1-agonist l-phenylephrine reduced the amplitude of gastric phasic contractions, whereas β-agonists prenalterol or salbutamol did not (4). Gastric relaxation and α1 receptor-mediated inhibition of phasic gastric contraction may play a role in the sympathetic α1-adrenergic-mediated delayed gastric emptying induced by intracisternal Ucn 2.
Ucn 2 injected intravenously significantly suppressed gastric emptying of a liquid nonnutrient meal (present study) and of a solid meal in conscious rats (33). However, the inhibitory action of Ucn 2 injected intravenously was not altered by bretylium pretreatment under conditions blocking intracisternal or intracerebroventricular Ucn 2-induced delayed gastric emptying. These results established that the systemic inhibitory action of Ucn 2 is mediated through distinct mechanisms from those elicited by central administration. In addition, these data indicate that intracisternal or intracerebroventricular Ucn 2 did not act by leaking into the periphery but centrally through stimulation of sympathetic pathways. In this regard, because prazosin after its peripheral injection can penetrate the blood-brain barrier and decrease the sympathetic nerve activity in rats (39), it is possible that prazosin in the present study also acts in the brain to block the sympathetic-mediated action of intracisternal Ucn 2. So far, two reports indicate that the activation of brain CRF2 receptors influences other visceral functions through sympathetic pathways (11, 50). The selective CRF2 agonist urocortin 3 (17) increases mean arterial blood pressure, heart rate, and plasma epinephrine release in conscious rats consistent with activation of sympathetic outflow (11). The intracisternal injection of Ucn 2 was also recently shown to decrease hepatic surface perfusion and elevate portal pressure through CRF2 receptors and sympathetic noradrenergic mechanisms in anesthetized rats (50). Interestingly, consistent reports indicate that Ucn 2, injected intracerebroventricularly at 1 μg, displays satiation-like properties that occurred 3–6 h after peptide injection in conscious rats (20). In our study, the inhibition of gastric emptying induced by intracerebroventricular Ucn 2 at 1 μg was observed within the first hour. Whether gastric fullness associated with altered gastric emptying underlies the delayed inhibition of feeding behavior in response to intracerebroventricular Ucn 2 may need to be considered. Previous observations indicate that gastric distension and the presence of food in the stomach act as satiety signals (36).
The exact brain sites at which intracisternal or intracerebroventricular Ucn 2 induces a centrally mediated sympathetic inhibition of gastric motor function are still to be established. In the medulla oblongata, specific CRF2 binding sites detected by autoradiography are restricted to the nucleus of the solitary tract (NTS) and area postrema, and mRNA encoding the CRF2 receptor is also densely expressed in both of these structures (27, 45). In addition, intracerebroventricular injection of hUcn 2 at 1 μg in conscious rats gives rise to Fos expression, indicative of neuronal activation in the NTS, the paraventricular nucleus of the hypothalamus (PVN), and central amygdala, whereas no Fos induction was observed in the locus ceruleus, dorsal motor nucleus (DMN), or area postrema (20, 37). On the basis of these neuroanatomical and neurofunctional studies, it is likely that intracisternal and intracerebroventricular Ucn 2 act at these responsive brain sites to influence sympathetic outflow and inhibit gastric motor function (13, 15).
The present studies also provide evidence that intracisternal CRF and Ucn 1 inhibit gastric emptying of a nonnutrient meal through distinct neural pathways than Ucn 2. Bretylium, injected under conditions that completely antagonized intracisternal or intracerebroventricular Ucn 2 action, did not alter either CRF or Ucn 1 injected intracisternally at doses resulting in a similar percentage of gastric emptying as with intracisternal Ucn 2. Moreover, subdiaphragmatic vagotomy prevented intracisternal CRF-induced inhibition of gastric emptying, but not that of intracisternal Ucn 2 action. Subdiaphragmatic vagotomy did not significantly alter the transit of a liquid caloric meal compared with sham surgery in intracisternal vehicle-injected rats as previously reported (25, 43). Consistent with the vagal pathway involved in intracisternal CRF and Ucn 1 inhibitory action, previous studies showed that subdiaphragmatic vagotomy blocked intracisternal or intracerebroventricular CRF-induced delayed gastric emptying of a liquid nonnutrient meal in rats (6, 43). Vagotomy also blocked gastric motility changes induced by CRF injected into the DMN in fasted rats or intracerebroventricularly in fed dogs (24, 26), whereas surgical sympathectomy had no effect in fed or fasted rats (22). In addition, CRF injected intracisternally or into the DMN inhibits gastric motility stimulated by vagal-dependent thyrotropin releasing hormone (TRH) excitatory action on DMN neurons (10, 19). In support of vagal inhibitory pathways are also the observations that intracisternal CRF decreased gastric vagal efferent discharge (23) and that intracerebroventricular CRF blocked the activation of DMN neurons and gastric function induced by endogenous TRH released by acute cold exposure (47). In contrast, an in vitro patch-clamp study performed on coronal sections containing the dorsal vagal complex of 25-day-old rats showed that superfusion of CRF increases discharge rate and membrane depolarization of gastric projecting DMN neurons (26). Age difference and in vitro vs. in vivo experimental conditions recruiting different circuitries could account for these divergent results. Interestingly, there are two reports of a sympathetic- mediated central action of CRF to suppress gastric emptying when injected intracisternally before the ingestion of a solid meal (34) or intracerebroventricularly before a liquid noncaloric meal in conscious rats (25). Whether vagal vs. sympathetic actions of the CRF are related to different experimental conditions or dose-related effects mimicking the sympathetic-mediated action of Ucn 2 under these conditions needs to be further ascertained.
Although CRF displays high affinity to CRF1 and a 40-fold lower affinity to CRF2 receptors and Ucn 1 has a high affinity to both CRF receptor subtypes (17), the selective CRF2 antagonist astressin2-B antagonized intracisternal CRF- and Ucn 2-induced inhibition of gastric emptying of a liquid meal (present study) and intracisternal Ucn 1-induced inhibition of a solid nutrient meal (10). We previously established the biological CRF2 selectivity of astressin2-B in rats by showing that the peptide did not influence the established CRF1 receptor-mediated action of intravenous CRF on the stimulation of colonic motor function under conditions antagonizing the CRF2-mediated effect of intravenous CRF on the stomach (33). These data support that intracisternal CRF and Ucn 1 interact with CRF2 receptors to induce a vagally mediated inhibition of gastric motor function. However, it cannot be discounted that the astressin2-B-sensitive effect of CRF/Ucn 1 may reflect coactivation of CRF1 and CRF2 receptors or initial activation of CRF1 pathways that recruit brain medullary CRF2 receptors.
The underlying mechanisms whereby preferential vagal vs. sympathetic pathways are recruited through activation of brain CRF2 receptors by different members of the CRF family are still to be defined. Ucn 2 and CRF/Ucn 1 display differential chemical properties that may play a role in the recruitment of different pathways (5). For instance, hUcn 2 binds with high and selective affinity to CRF2 receptors, low affinity to CRF binding protein, and lacks affinity to the recently cloned soluble splice variant, sCRF2α, identified in rat and mice brain, including the pons/medulla (9, 18, 21). In contrast, Ucn 1 and CRF bind to both CRF receptor subtypes and show subnamolar affinity to the CRF binding protein and sCRF2α (9, 21, 46), which may decrease their availability to CRF2 receptors located at more distant sites of injection. Recent studies showed that Ucn 2 injected intracerebroventricularly activates serotonergic neurons in the dorsal region of the mid-rostrocaudal and caudal subdivision of the dorsal raphe nucleus in close proximity of the aqueduct (41), where CRF2 receptors are abundantly expressed (16). Ucn 2 injected intracerebroventricularly and intracisternally may have a wide spread distribution on dorsal raphe neurons and induce sympathetic-mediated inhibition of gastric motor function as demonstrated for the sympathetic-mediated vascular response to activation of similar regions of dorsal raphe neurons (32). The possibility of a third CRF receptor that is recognized by astressin2-B and mediates the differential mechanisms of action of CRF/Ucn 1 and Ucn 2 cannot be ruled out. The notion of additional CRF receptor subtypes has been brought forward previously on the basis of distinct biological responses to CRF linked with differential antagonist:agonist ratios (14). In the present study, the antagonist:agonist ratio of 30:1 was required for astressin2-B to block intracisternal Ucn 2 action, while a 10:1 ratio was ineffective. In contrast, at a 10:1 ratio, astressin2-B completely antagonized the inhibitory effect of intracisternal CRF (present study) and intracisternal Ucn 1 (10).
Brain Ucn 2 may play a role in stress-related alterations of gastric motor function. First, we observed a potent action of intracisternal Ucn 2 to suppress gastric emptying. Second, there is a similar central CRF2 receptor-mediated sympathetic inhibition of gastric emptying induced by intracerebroventricular or intracisternal Ucn 2 (present study) and restraint stress (25, 44). Lastly, recent reports showed the presence of Ucn 2 mRNA in stress-responsive hypothalamic and brainstem nuclei (37), along with dramatic induction of Ucn 2 mRNA in the parvocellular part of the PVN by restraint (44).
In summary, the present study established that intracisternal or intracerebroventricular injection of the selective CRF2 agonist, Ucn 2, acts centrally on CRF2 receptors to inhibit gastric emptying of a noncaloric liquid meal through sympathetic pathways and α1-adrenoreceptor-dependent mechanisms. This was shown by the blockade of the gastric inhibitory response by intracisternal injection of the selective CRF2 antagonist, astressin2-B, and peripheral injection of adrenergic blockers, bretylium and α1 antagonist, prazosin, while vagotomy, propranolol or yohimbine did not prevent intracisternal Ucn 2 action. CRF and Ucn 1 also inhibit gastric emptying through activation of CRF2 receptors, however, their action is vagal-dependent and not altered by bretylium. Ucn 2 action is more potent than Ucn 1 and requires a 3-fold higher antagonist:agonist ratio. These data provide evidence for a differential sympathetic α1-adrenergic pathway recruited by the selective CRF2 agonist, Ucn 2 injected intracerebroventricularly or intracisternally, compared with vagal-dependent mechanisms involved in the intracisternal CRF1/CRF2 agonists, CRF and Ucn 1, to inhibit gastric emptying of a liquid non-caloric meal.
This work was supported by National Institutes of Health Grant R01 DK-33061 and Center Grant DK-41301 (Animal Core) and a Veterans Affairs Career Scientist and Merit Award.
We thank Dr. Jean Rivier (Salk Institute, La Jolla, CA) for the generous supply of peptides and Teresa Olivas for help in the preparation of the manuscript. Dr. Jozsef Czimmer was supported partly by the B-142 Ph.D. program of the Faculty of Medicine of the University of Pecs, Pecs, Hungary.
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