Cholecystokinin inhibits gastric emptying and contracts the pyloric sphincter in rats by interacting with low affinity CCK receptor sites

ELSEVIER

Regulatory Peptides 52 (1994) 165-172

Cholecystokinin inhibits gastric emptying and contracts the pyloric sphincter in rats by interacting with low affinity CCK receptor sites Timothy H. Moran*, Rebecca Kornbluh, Kimberly Moore, Gary J. Schwartz Department of Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, 720 Rutland A venue, Baltimore, MD 21205, USA Received 30 January 1994; revised version received 5 April 1994; accepted 3 May 1994

Abstract

The aim of these experiments was to characterize the receptor affinity state through which CCK produces pyloric contraction and inhibits gastric emptying in the rat using the novel CCK heptapeptide analog CCK-JMV-180. CCKJMV-180 has been demonstrated to act as a functional agonist at high affinity pancreatic CCK A receptors but as a functional antagonist at CCK A low affinity receptors. CCK-8 (1, 3.2 and 10 nM) induced dose dependent tension increases in isolated pyloric segments. CCK-JMV-180 (3.2/aM) or vehicle failed to mimic this action when administered alone but blocked the ability of CCK-8 (3.2 nM) to induce tension increases. CCK-8 (2/~g/kg) also inhibited the gastric emptying of physiological saline. CCK-JMV-180 (320 and 1000/~g/kg) failed to inhibit emptying when administered alone but dose dependently antagonized CCK induced inhibition of gastric emptying. Thus, in both preparations CCK-JMV-180 acted as a functional CCK antagonist. This profile is consistent with the interpretation that the actions of CCK in pyloric contraction and the inhibition of gastric emptying are mediated through CCK's interactions with receptors functionally similar to pancreatic low affinity sites. Key words." C C K - J M V - 1 8 0 ; Neuropeptide

I. Introduction

The brain/gut peptide cholecystokinin (CCK) has a variety of actions in the gastrointestinal tract following peripheral administration. In rats, these actions include modulation of pancreatic enzyme re* Corresponding author. Fax: + l (410) 6140013. 0167-0115/94/$7.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 1 6 7 - 0 1 1 5 ( 9 4 ) 0 0 0 3 9 - Z

lease [1,2] and the inhibition of feeding [3] and gastric emptying [4,5]. The particular receptor subtypes mediating these various actions have received considerable investigation. Experiments with C C K antagonists have demonstrated that CCK stimulates pancreatic amylase release [6] and inhibits feeding [ 7,8 ] and gastric emptying [ 9] through the activation of C C K A receptors.

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CCK's ability to stimulate pancreatic amylase secretion follows an inverted U shaped dose response curve [10,11]. The upstroke of this curve reflects CCK's occupation of a high affinity CCK receptor stimulating amylase release, while the downstroke of the curve represents occupation of low affinity CCK receptors producing an inhibition of amylase release [10,11]. Use of a C-terminal heptapeptide CCK analog (B OC-Tyr(SO3)-Nle-Gly-Trp-Nle-Asp-2phenylethyl ester) (CCK-JMV-180) provides a functional discrimination of these two sites [ 12]. CCKJMV-180 is an agonist at the high affinity site, in that it mimics the actions of CCK and stimulates amylase secretion at low concentrations. However, in contrast to CCK, at higher concentrations CCKJMV-180 does not inhibit amylase release. In fact, CCK-JMV- 180 blocks the inhibitory actions of high concentrations of CCK on amylase release. Thus, CCK-JMV-180 has the unique profile of being an agonist at C C K A high affinity sites while being an antagonist at C C K A low affinity sites in rats [ 13,14]. CCK-JMV-180 has also been used to identify the receptor affinity state through which CCK affects food intake in the rat [15,16]. CCK-JMV-180 administered alone had no effect on intake but acted as a CCK antagonist when given in combination with CCK in that it dose dependently blocked the ability of CCK to inhibit food intake. The present experiments employ CCK-JMV-180 to examine the receptor affinity state through which CCK exerts its effects on pyloric contraction and gastric emptying.

2. Materials and methods 2.1. In vitro pyloric contraction

Subjects were adult male Sprague-Dawley rats weighing between 250-300 g. Animals were food deprived for 24 h, killed by decapitation, and the stomach and proximal duodenum were rapidly removed. The pylorus including a proximal cuff of

antrum and a distal cuff of duodenum was dissected out and placed in Tyrode buffer (140 mM NaCI, 5 mM KC1, 1 mM MgSO4, 0.7 mM NaH2PO 4, 12 mM NaHCO3, 1.8 mM CaC12 and 0.1~o glucose acidified to a pH of 6.5). Any luminal contents were gently removed by rinsing with buffer. Pyloric samples were attached in a longitudinal orientation to a force transducer (Harvard Instruments) and placed in a siliconized 50 ml organ bath containing Tyrode buffer maintained at 37 °C. Tissue samples were stretched to a baseline tension of 1.0 g. Samples were allowed to equilibrate for 15-20 min prior to testing during which the buffer solution was changed three times. Following equilibration, the contractile response to a concentration range of CCK-8 was assessed (1.0, 3.2 and 10 n M - final bath concentration) in an ascending order. 3 min after the addition of each dose of CCK, the tissue was rinsed twice with fresh buffer. 10 min of equilibration preceded the administration of the next CCK concentration. Following assessment of responses to CCK, the response to the addition of 160/~1 CCK-JMV-180 (3.2 #M final bath concentration) or vehicle (dimethyl sulfoxide (DMSO)) in the absence of CCK were obtained. Tissues were again rinsed and reequilibrated for 10 min. Finally, the effect of 3.2 #M CCK-JMV-180 or D M S O vehicle on 3.2 nM CCK's ability to contract the pylorus was assessed. The responses of a total of 12 samples were examined. Six samples were taken through the sequence receiving CCK-JMV180 and six receiving the D M S O vehicle. Due to variability between samples in the absolute magnitude of tension changes, data were analyzed by ANOVA as a percentage of the tension produced by the 10 nM concentration of CCK, the maximal dose used. 2.2. Gastric emptying

Adult male Sprague Dawley rats weighing between 250 and 400 g served as the experimental subjects. Rats were housed in individual hanging

T.H. Moran et al. / Regulatory Peptides 52 (1994) 165-172

wire mesh cages in rooms kept at 22°C on a 12/12-h light/dark cycle. Rats had access to Purina rat chow from 11.00 to 17.00 h daily, and tap water was available ad libitum. Gastric emptying tests were conducted each day between 9.00 and 11.00 h following an overnight deprivation. Prior to testing, rats were adapted to daily intragastric tubing using an 8-Fr polyethylene intragastric feeding tube. The tube was inserted orally and advanced 5 cm down the esophagus into the stomach. Rats adapted to this procedure rapidly, accepting the tube readily after a few days. The gastric emptying of 5 ml loads of physiological saline over a 5 min emptying period was assessed under a variety of conditions. The ability of two doses of CCK-JMV180 (320 ttg/kg (n = 6) and 1.0 mg/kg (n = 8)) dissolved in 50~o D M S O and 50~o H 2 0 to affect gastric emptying alone or to affect the ability of 2/~g/kg CCK-8 to inhibit gastric emptying were assessed. For each dose of CCK-JMV-180, four testing conditions were used: (1) CCK-JMV-180 vehicle (50~o DMSO, 50~o H20)/CCK-8 vehicle (H20), (2) CCK-JMV-180/H20, (3) 50~o DMSO, 50~o H 2 0 / CCK-8, (4) CCK-JMV-180/CCK-8. A single condi-

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tion was tested for an individual animal on a single test day. CCK-JMV-180 or vehicle was administered IP, 2 min prior to instillation of the gastric load. CCK-8 or vehicle was administered IP 1 min prior to instillation of the gastric load. Gastric emptying was assessed using a modification of the phenol red dye dilution method of Hunt and Spurrel [ 17]. Briefly, the instilled 5 ml saline test meal containing 0.006 ~o phenol red as a marker was left in place for 5 min. At the end of the 5 min emptying period a 5 ml bolus of warmed 37°C unmarked physiological saline was infused into the stomach over 2 s. The stomach was then emptied and the withdrawn gastric contents were centrifuged at 10,000 rpm for 5 min to precipitate out any particulate matter. A 1 ml sample from the centrifuged remaining gastric volume was buffered with 24 ml of 0.014 M NaaPOa-12H20 and the spectrophotometric absorbance of each buffered sample was compared to that of a 1 ml buffered sample from the original instilled phenol red saline solution to determine the volume of the original test meal remaining in the stomach at the end of the 5 min emptying period.

10 nM CCK-8

3.2 nM CCK-8 3.2 ~M

CCK-JMV-180

1 mln Fig. 1. Response of pyloric segment to three concentrations of CCK-8 and the blockade of the response to 3.2 nM CCK-8 by 3.2/~M CCK-JMV-180.

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Data for volumes remaining in the stomach at the end of the 5 min emptying period were analyzed by a 2 way repeated measures ANOVA's for the factors of CCK-8 and CCK-JMV-180 for the two doses. Planned t comparisons using the MS from the error terms were used for comparing individual means. 2.3. Materials

CCK-8 used in both the pyloric contraction and gastric emptying experiments was obtained from Bachem, Torrance, CA. The CCK-JMV-180 was synthesized by Dr. Shian-Jan Shiuey and Irina Kulesha of Hoffmann-LaRoche, Department of Medicinal Chemistry II and was a gift from HoffmannLaRoche, Nutley, NJ.

3. Results

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CCK-8 reliably induced concentration related tension increases in isolated pyloric segments, F(2,11) = 18.768,P< 0.001 (Figs. 1 and 2,top panel). Neither CCK-JMV-180 nor D M S O vehicle produced any contraction when administered alone (Fig. 2, bottom panel), but 3.2/~M CCK-JMV-180 completely eliminated the response to 3.2 nM CCK, while the D M S O vehicle had no significant effect on the response to CCK, F(1,10)= 16.864, P < 0 . 0 1 (Fig. 2, bottom panel). 3.2. Gastric emptying

Peripheral administration of 2 #g/kg of CCK-8 caused a reliable inhibition of the emptying of physiological saline from the rats stomach. Administration of CCK-JMV-180 alone did not affect emptying but CCK-JMV-180 resulted in a dose related blockade of CCK-induced inhibition of gastric emptying (Fig. 3). While there were differences in baseline saline emptying between the groups of rats tested at the

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Fig. 2. Top panel: dose related increases in tension to increasing concentrations of C C K - 8 expressed as a ~o of m a x i m a l response. B o t t o m panel: effects of 3.2 # M C C K - J M V - 1 8 0 or D M S O vehicle on pyloric contraction when administered alone or in combination with 3.2 n M CCK-8.

two CCK-JMV-180 doses, the effects of CCK-8 on saline emptying were similar in that the volume remaining in the stomach at the end of the 5 min emptying period was increased 2 #g/kg CCK-8 by 67% in one group and 48% in the other. ANOVA at the 320 #g/kg dose demonstrated a significant effect of CCK, F(1,5)= 54,436, P<0.001 and no effect of CCK-JMV-180 or no significant interaction. Planned t comparisons demonstrated that the CCK alone condition was significantly different from both the baseline condition and the CCK-JMV-180 alone

T.H. Moran et al. / Regulatory Peptides 52 (1994) 165-172

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tion. At a dose of 1 mg/kg, CCK-JMV-180 produced a complete blockade of the gastric inhibitory effect of 2 #g/kg CCK-8. ANOVA indicated significant effects of both CCK-8 and CCK-JMV-180, as well as a significant interaction (F(1,7)= 7.471, P < 0.05, F(1,7) = 8.997, P < 0 . 0 5 and F(1,7) -- 13.555, P < 0 . 0 1 , respectively). Planned t comparisons indicated that the CCK-8 alone condition was significantly different from all other conditions (P<0.01) and the other conditions did not differ from one another.

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Fig. 3. Top panel: effects of CCK-8 (2 #g/kg) and CCK-JMV180 (320 #g/kg) on gastric emptying of physiological saline over a 5 min test period expressed as volume remaining in the stomach. Emptying is inhibited by CCK-8 and this inhibition is partially blocked by this dose of CCK-JMV-180. Bottom panel: effects of CCK-8 (2 #g/kg) and CCK-JMV-180 (1 mg/kg) on gastric emptying of physiological saline over a 5 min test period expressed as volume remaining in the stomach. Emptying is inhibited by CCK-8 and this inhibition is completely blocked by this dose of CCK-JMV-180.

condition, P < 0.05. 320 gg/kg CCK-JMV-180 produced a partial blockade of the effect of CCK-8, eliminating 50~o of the suppression of gastric emptying. The volume remaining in the stomach following combined administration of CCK-8 and CCKJMV-180 was not significantly different from either the baseline condition or the CCK-8 alone condi-

The results from these experiments demonstrate that CCK-JMV-180 acts as a functional antagonist to CCK-8 for both the inhibition of gastric emptying and for pyloric contraction. In neither paradigm did CCK-JMV-180 alone exert any influences. Pancreatic acinar cells have been demonstrated to contain two distinct classes of C C K A binding sites, both of which interact with CCK [ 10,11]. Data from receptor binding studies have shown that CCK interacts with both a high affinity site and a low affinity site with K d values estimated in the range of 50 pM and 10-20 nM, respectively [14,18]. The dose response curve for CCK stimulated amylase release is biphasic, with the upstroke of the dose response curve mediated through occupancy of the high affinity site and the downstroke mediated through occupancy of the low affinity site. These two sites are functionally differentiated by the CCK analog CCKJMV-180. CCK-JMV-180 produces a monophasic dose response curve for amylase secretion - there is a plateau rather than a downstroke for higher concentration. Thus, CCK-JMV-180 is a full agonist at the high affinity CCK receptor [12,13]. Moreover, occupancy of the CCK low affinity receptors by CCK-JMV-180 inhibits the downstroke of the CCK dose response curve. Thus, CCK-JMV-180 is a full antagonist at the low affinity site [ 12,13 ]. The present data clearly demonstrate that while CCK-JMV-180

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failed to induce pyloric contraction or inhibit gastric emptying, CCK-JMV-180 does block the effects of CCK-8 in these two paradigms. This pattern of resuits is consistent with the interpretation that CCK induces pyloric contraction and inhibits gastric emptying through an interaction with low affinity rather than high affinity CCK receptors. An alternative view of pancreatic CCK biniding sites has recently been proposed [19,20]. In this view pancreatic CCK A receptors exist in 3 affinity states: high, low and very low. The two sites identiffed in binding studies are the high and the low affinity sites. No function has been determined for the high affinity site while it is the low affinity site whose occupation corresponds to the upstroke of the dose response curve for pancreatic amylase secretion. The very low site is not evident in binding studies but its occupation accounts for the downstroke of the dose response curve for amylase secretion. According to this view, CCK-JMV-180 would be an agonist at the low affinity site and an antagonist at the very low affinity site. In this schema, both the pyloric contractile and gastric inhibitory actions of CCK appear to be mediated through activation of the very low affinity site. However, the critical issue in the present studies is not the particular nomenclature for CCK receptor affinity states but that both the pyloric contractile and the gastric inhibitory actions of CCK appear to be mediated by interaction with CCK receptors functionally similar to those that mediate the downstroke of CCK's dose response curve for amylase release [18]. The pylorus of the rat contains a dense band of CCK binding localized to the circular muscle layer [21]. Pharmacological analysis of these receptors has demonstrated them to be C C K A receptors in that in receptor binding studies, they demonstrate a high affinity for sulfated but not desulfated CCK and binding was inhibited by specific C C K A but not CCK 8 antagonists [22]. Previous studies have demonstrated that CCK contracts the isolated rat pylorus [23,24] and that this action is mediated through an interaction with CCK A receptors [25]. The 3.2

nM concentration of CCK used in the present experiments with CCK-JMV180 produced 50~o of maximal pyloric contractile response as in previous reports [23-25]. This concentration of CCK is on the downstroke of the dose response curve for in vitro pancreatic amylase secretion [18]. Concentrations which correspond to those at which amylase secretion is stimulated do not contract the pylorus. Thus, both the dose response characteristics of CCK for pyloric contraction and the antagonism by CCKJMV-190 are consistent with mediation of this action through low affinity CCK receptors. The relationship between CCK's actions in pyloric contraction and the inhibition of gastric emptying is not fully understood. Pyloric contraction is not the primary mechanism through which CCK inhibits gastric emptying as pylorectomy only partially attenuates the ability of CCK to inhibit gastric emptying [25]. The inhibition of gastric emptying by CCK appears to be secondary to reductions in intragastric pressure [26], an action mediated through a vago-vagal reflex [27,28] and dependent upon C C K A receptors [9]. Recent data examining the mediation of CCK induced activation of gastric vagal mechanosensitive afferents has demonstrated that this activity is blocked by CCK-JMV-180 [29]. Thus, the inhibition of gastric emptying by CCK, CCK induced pyloric contractions and CCK induced vagal afferent activation all are mediated through CCK's interactions with low affinity CCK receptors. In some species, the inhibition of gastric emptying by CCK has been proposed to be a physiological endocrine action of the peptide [5,30]. Endogenous CCK clearly plays a physiological role in the control of gastric emptying in the rat as the administration of CCK antagonists results in accelerated emptying [9,31,32]. The present data demonstrating that CCK's actions on gastric emptying are mediated through interactions with a low affinity site make an endocrine mode of action for this inhibition an unlikely possibility in the rat. This interpretation is similar to that which has been made for the satiety actions of CCK [33]. CCK satiety is also mediated

T.H. Moran et al. /Regulatory Peptides 52 (1994) 165-172

through low affinity CCK receptors [15,16] and appears not to correlate with plasma CCK levels. Doses of CCK which produce maximal pancreatic secretion are subthrehold for inhibiting food intake [2] and the ability of intraduodenal nutrient infusions to elevate plasma CCK levels and produce feeding reductions which are blocked by CCK antagonists can be completely dissociated [34,35]. Thus, both inhibitions of gastric emptying and food intake by endogenous CCK in the rat are likely mediated through neurocrine or paracrine actions of the peptide.

[ 9]

[ 10]

[ 11]

[12]

Acknowledgements This study was supported by NIH grant DK 19302.

[ 13]

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[28] McCann, M.J., Verbalis, J.G. and Stricker, E.M., Capsaicin attenuates multiple responses to cholecystokinin in rats, J. Auton. Nerv. Syst., 23 (1988) 265-272. [29] Schwartz, G.J., McHugh, P.R. and Moran, T.H., Pharmacological dissociation of responses to CCK and gastric loads in rat mechanosensitive vagal afferents, Am. J. Physiol. (in press). [30] Debas, H.T., Farooq, O. and Grossman, M.I., Inhibition of gastric emptying is a physiological action of cholecystokinin, Gastroenterology, 68 (1975) 1211-1217. [31] Forster, E.R., Green, T., Elliot, M., Bremner, A. and Dockray, G.J., Gastric emptying in rats: role of afferent neurons and cholecystokinin, Am. J. Physiol., 258 (1990) G552G556. [32] Forster, E.R. and Dockray, G.J., The role of cholecystokinin in inhibition of gastric emptying by peptone in the rat, Exp. Physiol., 77 (1992) 693-699. [33] Smith, G.P. and Gibbs, J., The development and proof of the CCK satiety hypothesis. In C.T. Dourish, S.J. Cooper, S.D. lversen and L.L. Iversen (Eds.), Multiple Cholecystokinin Receptors in the CNS, Oxford University Press, Oxford, 1992, pp. 166-182. [34] Brenner, L., Yox, D.P. and Rotter, R.C., Suppression of sham feeding by intraintestinal nutrients is not correlated with plasma CCK elevation, Am. J. Physiol., 254 (1993) R972-R976. [35] Yox, D.P., Brenner, L. and Ritter, R.C., CCK-receptor antagonists attenuate suppression of sham feeding by intestinal nutrients, Am. J. Physiol., 264 (1992) R554-R561.