Corticotropin-releasing factor inhibits gastric emptying in dogs
Ted Pappas, Haile Debas and Yvette Tach6
Center for Ulcer Research and Education, VA Wadsworth Medical Center, University of California, Los Angeles, CA 90073, U.S.A.
(Received 8 February 1985; revised manuscript received 8 April 1985; accepted for publication 10 April 1985)
Summary
The purpose of the present study was to evaluate the effect of ovine corticotropin- releasing factor (CRF) on the gastric emptying of a saline meal in conscious dogs. Intravenous infusion of CRF (220-880 pmol • kg-1 . h-1), induced a significant linear dose dependent inhibition of gastric emptying (16-71%). CRF action was not modified by naloxone and not associated with vomiting or other side effects• Intra- venous infusion of sulfated cholecystokinin octapeptide (CCK-8, 50-200 pmol. kg- 1 • h-1) inhibited gastric emptying by 29-52%. The relative potency of CRF with respect to CCK-8 is 4 times less. These studies demonstrated that CRF given intra- venously in picomolar amount inhibits gastric emptying of a liquid meal in dogs through a mechanism unrelated to opiates. The role of endogenous CRF in stress- induced inhibition of gastric emptying needs to be investigated•
In 1981, Vale et al. [1] reported the characterization of a 41-residue peptide CRF (corticotropin-releasing factor), isolated from ovine hypothalamus. Subsequently, the structures of rat, human, bovine and caprine hypothalamic CRF were established [2-5]. The amino acid sequence of caprine CRF is identical to that of ovine CRF [1,5]. Also the structures of human and rat CRF are the same but differ by seven
Address for correspondence: Yvette Tach6, Ph.D., CURE/VA Wadsworth, Building 115, Room 203, Los Angeles, CA 90073, U.S.A. Telephone: (213) 478-3711 ext. 2490.
residues from that of the ovine species [2,3]. Anatomic and biologic studies demon- strated that CRF is a physiologic factor regulating the pituitary secretion of pro- opiomelanocortin-derived under basal and stress conditions [1,6,7]. In addi- tion to its pituitary effects, CRF acts within the brain to modify ingestive [8], sexual [9] and locomotor [10] behaviors, cardiovascular function [11], and gastric secretion [12]. The overall changes are compatible with the concept that CRF may be an important regulatory peptide coordinating the endocrine, behavioral and autonomic responses to stress [1,8-12].
Recent immunohistochemistry, immunoaffinity chromatography and radioimmu- noassay studies have revealed the presence of CRF-like immunoreactivity not only in the brain [13] but also in peripheral tissues, namely the adrenals and the gastroin- testinal tract (pancreas, duodenum, and stomach) of a variety of vertebrate species, including man [14-17]. These findings support a possible peripheral source and role for CRF. Systemic administration of CRF has been reported to inhibit gastric acid secretion [18], to dilate selectively the dog mesenteric circulation [19], and to increase plasma levels of insulin and pancreatic polypeptide, without modifying other circu- lating gut peptides [20]. Nothing is known of the influence of CRF on gut motor activity. In the present study, we report that intravenous CRF potently inhibits the emptying of a liquid meal in dogs.
Materials and Methods
Animal preparations Six adult mongrel dogs (25 kg) were surgically prepared with chronic gastric fis-
tulas drained by Thomas cannulas. The cannula was placed in the dependent portion of the gastric corpus on the greater curvature. The dogs were allowed to recover at least 3 weeks after surgery and 48 h between tests. They were fed Purina Laboratory Chow, and food but not water was withheld for 18 h before each study. Experiments were performed in dogs lightly restrained on Pavlov stands.
Determination of gastric emptying The methodology used to measure gastric emptying was essentially as previously
described [21]. A 300-ml saline (0.9%), phenol red (60 mg/l) meal was placed in the stomach for 10 min. Phenol red served as a non-absorbable marker. At the end of the 10 rain period, the volume left in the stomach was removed and recorded. Gastric emptying was determined by the following equation:
300 ml - Vo [1 - (Ai -- Ao)] = volume emptied in 10 min
where 1Io = volume remaining in the stomach after 10 min; Ai = absorbance of meal placed in stomach; and Ao = absorbance of meal removed from stomach.
The absorbance of the solution put into and removed from the stomach was mea- sured as follows: 1 ml of the phenol red solution was placed in a 10-ml volume flask. 2 ml of a base (Na3PO~, 27 mg/l) and 7 ml of water were added to make a total volume of 10 ml. The absorbance was read at 550 nm on a Zeiss spectrophotometer.
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Peptide administration Ovine CRF (oCRF) in lyophilized form (supplied by Dr. J. Rivier, the Salk Insti-
tute, La Jolla, CA) was dissolved in 0.1% bovine serum albumin, saline solution before each experiment. Sulfated cholecystokinin octapeptide-(26-33) (CCK-8; Pen- insula Laboratories, Inc., Belmont, CA) was dissolved in 0.5% bovine serum albu- min, 0.01% acetic acid stock solution (1 mg/ml). Naloxone hydrochloride solution (NARCAN, Endo Pharmaceuticals Inc., Manati, Puerto Rico) was diluted with 0.1% bovine serum albumin, saline solution. Drugs were infused continuously at a rate of 30 ml/h through a polyethylene tubing inserted into hind limb veins. Intravenous infusions of control solution of peptides were started 45 min (CRF) or 20 min (CCK-8) before the saline meal and continued during the 10 min that the meal re- mained in the stomach. The infusion times were calculated according to the published half life for each peptide [22,23]. CRF administration was well tolerated in all dogs and produced no adverse reactions.
Statistics Data were analyzed by analysis of variance with contrasts of Student's paired
two-tailed t-test. Differences were considered statistically significant if the P value was less than 0.05.
Results
Intravenous infusion of CRF (220-880 pmol. kg-1 . h-1) inhibited gastric empty- ing of a saline meal in a significant linear dose-related manner. The highest dose of CRF inhibited gastric emptying by 71 4- 10% (Fig. 1). Naloxone (28 #g • kg - t • h-1) infused simultaneously with CRF (880 pmol • kg - t • h-1) did not modify the inhibitory effect of the peptide (Table I).
Intravenous infusion of CCK-8 (50, 100, 200 pmol • kg-1 . h-1) inhibited gastric emptying of saline meal by 28 + 18, 38 + 10 and 52 4- 14%, respectively. The dogs that received the highest dose of CCK-8 occasionally demonstrated retching and vomiting.
Fig. I. Dose-dependent inhibitory effect of intravenous infusion of oCRF or CCK-8 on gastric emptying of a saline meal in dogs. Each point represents the mean -4- S.E.M. of six dogs. Analysis of variance with contrasts, significant linear dose-response for CRF P < 0.05.
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TABLE I
Effects of naloxone on intravenous CRF-induced inhibition of gastric emptying
a 18 h fasted dogs were infused intravenously either with control solution (0.1% bovine serum albumin, saline) or CRF (880 pmol • kg- 1 . h- 1) alone or combined with naloxone (28 ~g • kg- 1. h- 1), for 45 min before and 10 re_in after the saline meal. Gastric emptying was muasured 10 min after instillation.
b Mean 4- S.E.M. * P < 0.05. Student's paired two-tailed t-test.
Discussion
Intravenous infusion of CRF inhibited gastric emptying of a saline meal in dogs. A significant dose dependent linear inhibitory effect was observed in doses ranging from 220 to 880 pmol • kg- 1. h - 1. The highest dose suppressed gastric emptying by 71% without associated behavioral change, retching or vomiting. This represents the first evidence that peripherally administered CRF influences gastric emptying in dogs. A structurally related peptide, sauvagine, isolated from the skin of the frog Phylo- medusa sauvagei, was reported to delay gastric emptying when injected subcutaneous- ly in doses ranging from 0.5 to 4.3 nmol/kg in rats [24]. Unrelated peptides, such as somatostatin or peptide YY, inhibit gastric emptying in dogs ([25], unpublished ob- servations), however the magnitude of the inhibitory effect elicited by maximal in- fusion doses of these peptides is greater for CRF. The inhibition of gastric emptying by CCK was demonstrated to be a physiologic action of the peptide [21]. In agree- ment with a previous report [21], the EDso for CCK-8 induced inhibition of gastric emptying was 180 pmol • kg - 1 . h - 1, which is lower than that of CRF (720 pmol • k g - 1 . h-1). However, CCK-8 is more likely to induce retching and vomiting at the doses tested.
The mechanism by which CRF produced its inhibitory effect on gastric emptying is unknown. CRF is well established to release fl-endorphin in the circulation [1]. Opiates have been shown to exert antipropulsive effects on the gastrointestinal tract which are reversed by the antagonist, naloxone [26]. The activity of CRF was not modified by naloxone given at a dose shown to block opiate induced changes into gastric acid secretion [27,28]. These results suggest that CRF action is not mediated through endogenous opiate. Sauvagine induced inhibition of gastric emptying in rats also was unmodified by naloxone (1 mg/kg) [24]. It is unlikely that CRF acts through the release of other intestinal peptides known to influence gastric emptying [29]. This assumption is based on a recent report that CRF (21 nmol) given intravenously in humans did not modify plasma levels of insulin, glucagon, gastrin, somatostatin, motilin, neurotensin, gastric inhibitory peptide (GIP), CCK and Met-enkephalin [20]. To what extent CRF has a direct effect on motor activity of the proximal stomach and duodenum responsible for gastric emptying of liquids [30,3 l] or is acting through
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modification of the autonomic nervous system [11,12] regulating the contractile ac- tivity of the stomach [30] needs to be investigated further.
The physiologic or pathophysiologic significance of intravenous CRF induced in- hibition of gastric secretion cannot be ascertained without further work. CRF-like immunoreactivity has been detected in extrahypothalamic tissues including the ad- renals [14,17], lung [17], pancreas [15,17], stomach and duodenum [15,17]. Stress has been shown to releas~ CRF-like immunoreactivity into the cerebrospinal fluid [32] and the hypophyseal portal circulation [33]. High levels of CRF-like immunoreac- tivity have been detected in the plasma of women in the third trimester of pregnancy, which may originate from the placenta [34]. Interestingly many studies have shown a delay in gastric emptying following a variety of stressful stimuli including the post- surgical state [35,36] as well as during pregnancy, most commonly in the last trimes- ter [37].
In conclusion CRF, dose-dependently inhibits the gastric emptying of a liquid meal in the dog. The doses of the peptide required to inhibit gastric emptying are 4 times higher than those of CCK-8, but doses of CRF that potently inhibit gastric emptying do not cause vomiting as does CCK-8. The action of CRF on gastric emptying does not appear to be opiate mediated. CRF-like immunoreactivity has been reported to be elevated in conditions associated with the inhibition of gastric emptying. Further studies are necessary to determine if CRF plays a pathophysiologic role in delaying gastric emptying in conditions such as pregnancy and stress.
Acknowledgements
This work was funded by the National Institutes of Health NIADDK (Grants AM 33061 and 17238). The authors thank Dr. J.E. Elashoff for statistical evaluation of the data, Mr. R. Melendez for his excellent technical assistance as well as Ms. A. Starlight for helping in the preparation of the manuscript. We also greatly acknowl- edge Dr. J. Rivier (The Salk Institute, La Jolla, CA) for his kind donation of ovine CRF.
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