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Effects of cholecystokinin-receptor blockade on pancreatic and biliary function in healthy volunteers
GASTROENTEROLOGY
1991;100:1663-1690
Effects of Cholecystokinin-Receptor Blockade on Pancreatic and Biliary Function in Healthy Volunteers JoRG
SCHWARZENDRUBE,
REINHARD Medizinische Diisseldorf,
LUTHEN,
Klinik Germany
und
h4ARKUS
and CLAUS NIEDERAU
Poliklinik,
Abteilung
This study used the specific cholecystokinin (CCK)receptor antagonist loxiglumide to evaluate whether endogenous CCK, which is released after a meal, regulates pancreatic and hiliary functions. Eight healthy volunteers were studied twice on separate days. The subjects received a continuous intraduodenal infusion of a 750-kcal liquid test meal for 2 hours either with or without IV infusion of 5 mg . kg-’ . h-’ of loxiglumide. Loxiglumide at this dose abolishes the actions of CCK at various target organs including gallbladder and pancreas, when given at doses that mimic postprandial plasma concentrations of CCK. Loxiglumide markedly decreased the meal-stimulated outputs of amylase, trypsin, and chymotrypsin by !w%-70% of control values but only slightly decreased duodenal volume (25Ohinhibition of mean integrated secretion). Loxiglumide abolished gallbladder emptying induced by infusion of nutrients and even increased gallbladder volumes when compared with prior fasting values. Correspondingly, loxiglumide almost abolished the output of bilirubin after infusion of nutrients. However, loxiglumide failed to alter the increase in circulating concentrations of glucose, insulin, and C peptide after infusion of nutrients. The present results show that CCK is one of several factors that regulate pancreatic protein secretion after absorption of nutrients. However, CCK is probably not involved in regulation of pancreatic secretion of fluid. In contrast, gallbladder function is mainly regulated by CCK, both in terms of its emptying after intestinal absorption of nutrients and in terms of maintenance of its fasting volume. Cholecystokinin does not play a major physiological role as an insulinotropic factor.
T
NIEDERAU,
he development of new specific cholecystokinin (CCK)-receptor antagonists offers a new approach to evaluate the physiological role of CCK in various
fiir Gastroenterologie,
Heinrich-Heine-Universittit
gastrointestinal functions, avoiding the pitfalls associated with previous studies that administered exogenous CCK. In 1981 proglumide and benzotript were shown to act as specific and competitive CCK-receptor antagonists in vitro (1). However, these compounds had low potencies or were toxic when given in an effective dose in vivo (2). Subsequently, molecules with a proglumidelike structure were synthesized; they were up to 1000 to 5000 times more potent than proglumide in inhibiting the action and binding of CCK in vitro and in vivo (3,~). More recently, several nonpeptide substances originating from the substance asperlicin (5) were shown to act as specific CCK antagonists (6,7). The most potent nonpeptide substance MK-329 (formerly termedL-364,718) is 25,000-l,OOO,OOO times more potent than proglumide in inhibiting CCK’s action and binding in vitro and in vivo (4,6,7). Thus, from the potencies of inhibiting the actions of CCK, both the new amino acid derivatives of proglumide and the new nonpeptide antagonists are well suitable for in vivo studies. The present experiments used the specific CCKreceptor antagonist loxiglumide (formerly termed CR1505) to evaluate the role of endogenous CCK in the regulation of biliary and pancreatic functions in healthy volunteers. Materials
and Methods
Materials The CCK antagonist loxiglumide (CRl505; D,L,+ [3,4-dichloro-benzoylamino]-5-[N-3-methoxy-propyl-pentyl-
Abbreviation used in this paper: ANOVA, analysis of variance. o 1991 by tbe American Gastroenterological Association 0016-5065/91/$3.00
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amino]-5-oxo-pentanoic acid) was kindly provided by Rotta Research Laboratorium (Milano, Italy] as sterile solutions containing 50 mg loxiglumide in 10 mL of solution. The test meal consisted of 500 mL of Fresubin (Fresenius AG, Bad Homburg, Germany), 85 mL Intralipid 20% (Pfrimmer, Erlangen, Germany), 20 g glucose, and 3 g polyethylene glycol (PEG] 4000.
Laboratory Determinations The concentration of PEG in the duodenal aspirates was determined by the turbidimetric method of Malawer and Powell (8). The duodenal concentrations of trypsin, chymotrypsin, and amylase were determined by colorimetric methods using arginine-p-nitroanilide, N-(3-carboxypropionyl]-phenylalanine-p-nitroanilide, and 3-p-nitrophenylmaltose heptaosid as substrates (9-11). For the determination of trypsin, chymotrypsin, and bilirubin, commercially available kits from Boehringer (Mannheim, Germany] were used. For the determination of amylase, the commercially available Phadebas kit (Pharmacia, Piscataway, NJ) was used. The duodenal volume was calculated from the dilution of perfused PEG marker inside the duodenum,
V,=$V,,
GASTROENTEROLOGY
Vol. 100, No. 6
meal with simultaneous IV infusion of 5 mg . kg-’ h-’ loxiglumide. The infusion of loxiglumide was started 30 minutes before the beginning of the intraduodenal infusion and was kept three times higher than the finally desired rate for the first 10 minutes in order to achieve steady state conditions of near continuous plasma concentrations of loxiglumide (14-16). On the second day of the control experiment, the subjects received an IV infusion of saline (0.9% NaCl) instead of loxiglumide. Gallbladder volumes were measured in 20-minute intervals using a Toshiba scanner (Neuss, Germany; Sonolayer SAL 77A) equipped with a linear 3.5-MHz probe. Gallbladder emptying was determined by a method previously reported in detail, assuming an ellipsoid structure of the gallbladder (14,17). In further experiments, parts of which have already been published, gallbladder emptying and pancreatic secretion were studied after IV infusion of increasing doses of cerulein (7.5, 15, 30, and 60 ng . kg-’ h-‘, with each dose given for 15 minutes] (14). In these studies, 10 male healthy volunteers were studied on 2 separate days either with or without infusion of 5 mg . kg-’ . h-’ loxiglumide (14). The experimental techniques for measurements of pancreatic and biliary functions were identical to those already described for the studies with intraduodenal infusion of nutrients.
d
in which C,, and C,, are the concentrations of PEG in the duodenal aspirates and in the perfusion volume [V, = 5 mL/min). The output of pancreatic enzymes corresponds to the product of concentration and the duodenal volume V,. Venous blood samples were taken at 20-minute intervals. Plasma concentrations of insulin and C peptide were measured by commercially available radioimmunoassays (Pharmacia). Plasma concentrations of CCK were measured by a specific and sensitive bioassay, as described previously in detail (I&I 3). Plasma concentrations of glucose were measured by the glucose-oxidase method (Boehringer).
Experimental
Design
Eight healthy volunteers [three men and five women], aged 21-44 years, were studied in the morning after an overnight fasting of at least 12 hours. The subjects were taking no medication and were all within 10% of the ideal body weight. A triplelumen polyvinyl tube was positioned fluoroscopically with its tip located at the ligament of Treitz. The second part of the duodenum was perfused with the test solution at a rate of 5 mL/min, yielding a rate of 375 kcal/h for a total period of 2 hours. Duodenal contents were recovered by a suction pump at the ligament of Treitz. The specimens were collected over ice and pooled in 20-minute fractions. A separate gastric tube was positioned fluoroscopically with its tip lying in the antrum to continuously remove gastric contents. The experiments were performed with the subjects lying in a semirecumbent position. All subjects were studied on separate days with an interval of at least 2 days between the individual experiments, which were done in randomized order. On the first day subjects received the intraduodenal infusion of the test
Approval of the Study Protocol The study was approved by the Ethical Committee of Human Research of the Dusseldorf University. Written informed consent was obtained from each subject.
Statistical Analysis All values are given as means + SEM. In addition to the mean data at the various time intervals after beginning of the intraduodenal infusion, the mean integrated responses to the various stimuli were calculated for glucose, insulin, C peptide, amylase, trypsin, chymotrypsin, and gallbladder volume. The integrated responses were calculated in each subject as the sum of postprandial values after subtracting the prior basal value at each point. The significance of differences between means was determined by analysis of variance (ANOVA) using the method of Duncan (18,19). Pvalues < 0.05 were considered significant.
Results The intraduodenal infusion of the liquid test meal significantly increased plasma concentrations of CCK, which reached maximal levels of 6-12 pmol/L after 20-40 minutes (Figure 1). Basal (fasting) plasma concentrations of CCK ranged from 1 to 2 pmol/L (Figure 1). Plasma concentrations of CCK were not
measurable in experiments in which the CCK antagonist loxiglumide was infused, because even residual amounts of a CCK antagonist in the plasma sample interfere with the CCK bioassay which is based on
June 1991
Figure 1. Plasma concentrations of CCK measured by CCK bioassay (see Materials and Methods). e-0, Studies in which subjects (n = 10) received an N infusion of cerulein at increasing doses as indicated in the figure; O--O, studies in which subjects (n = 8) received an intraduodenal infusion of nutrients (750 kcal/z b). Results are given as mean 2 SEM.
CCK-stimulated amylase release from isolated rat pancreatic acini (12,13,20). However, there was no indication that the same meal when given on a separate day together with the infusion of loxiglumide might have resulted in a lesser increase in plasma concentration of CCK. On the contrary, several recent studies have shown that IV infusion of loxiglumide increases the meal-stimulated increase in plasma CCK when measured by radioimmunoassay (20-22). However, the mechanism of this increase in plasma immunoreactivity of CCK has not yet been fully elucidated. In any case, currently both the bioactivity and radioimmunologic measurement of plasma CCK will not definitely prove or disprove that the same meal given on separate days with and without CCK antagonists will lead to the same release of CCK. Intravenous infusion of cerulein at increasing doses of 7.5, 15.0, 30.0, and 60.0 ng . kg-’ . h-’ (each dose given for 15 minutes) increased plasma bioactivity of CCK in a dose-dependent manner (Figure 1). The two lower doses of 7.5 and 15.0 ng . kg-’ * h-’ resulted in plasma concentrations of CCK that covered the range of CCK concentrations seen after the intraduodenal infusion of nutrients (Figure 1) and were similar to plasma CCK concentrations seen after oral meals (13,23-25). The two higher cerulein doses of 30.0 and 60.0 ng . kg-’ . h-’ resulted in plasma concentrations of CCK exceeding postprandial values. The latter doses of 30.0 and 60.0 ng . kg-’ * h-’ cerulein might, therefore, produce pharmacological effects, whereas the two lower cerulein doses of 7.5 and 15 .O ng . kg-’ . h-’ might mimic the effects caused by the postprandial increase in plasma CCK. Intraduodenal infusion of a liquid test meal markedly decreased gallbladder volume (Figure 2). Similar
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effects were shown previously after oral administration of a test meal or after IV infusion of cerulein. Intravenous infusion of loxiglumide at 5 mg . kg-’ . h-’ completely inhibited cerulein-stimulated gallbladder emptying and even significantly increased gallbladder volumes compared with prior basal fasting values (14). Compared with previous studies evaluating the effect of loxiglumide on gallbladder emptying after IV infusion of the CCK analogue cerulein or after an oral meal, the intraduodenal infusion of the test meal caused a more pronounced decrease in gallbladder volume when compared with the effect of an oral meal but did not reach the effect seen after administration of cerulein at pharmacological doses (14). Intravenous infusion of loxiglumide completely inhibited gallbladder emptying after intraduodenal infusion of the test meal and even increased gallbladder volume compared with prior fasting volumes (Figure 2). Intravenous infusion of 5 mg * kg-’ - h-’ loxiglumide significantly decreased output of bilirubin compared with the infusion of NaCl in the control experiment at all time intervals after intraduodenal infusion of the test meal (Figure 3). The mean integrated secretion of bilirubin was inhibited by loxiglumide by more than 90% when compared with the NaCl control (Figure 3). Loxiglumide did not significantly decrease duodenal volume at the various individual time intervals after intraduodenal infusion of the meal when compared with the corresponding NaCl control (Figure 4). However, the mean integrated secretion of fluid was
1 30
20
10
I I
Figure 2. Gallbladder volume: IV infusion of 5 mg kg-’ b-’ loxiglumide significantly increased gallbladder volumes compared with the infusion of NaCl in the control experiment at all individual time intervals from 20 to 120 minutes after beginning of the intraduodenal infusion (P < 0.05 by ANOVA). Basal values represent measurements performed 30 minutes before beginning of the intraduodenal infusion. The mean integrated gallbladder volumes after intraduodenal infusion of the test meal were decreased by loxiglumide by more than 90% when compared with the NaCl control (leff) (P < 0.001). Data are given as mean f SEM for the individual time intervals after the intraduodenal infusion of nutrients (left) and for the integrated change in gallbladder volume (right). O--O, IV infusion of loxiglumide; O-O, N infusion of NaCl; 0, mean integrated data for experiments with NaCl infusion; ?? , mean integrated data for experiments with loxiglumide infusion.
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Figure 3. Bilirubin output: IV infusion of 5 mg . kg-’ b-’ loxiglumide significantly decreased output of bilirubin compared with the infusion of NaCl in the control experiment at all individual time intervals from 20 to 120 minutes after beginning of the intraduodenal infusion (P < 0.05 by ANOVA). Loxiglumide inhibited the mean integrated secretion of bilirubin by more than 90% when compared with the NaCl control (P < 0.001). Data are given as mean ? SEM for the individual time points (left) and for the integrated data (right). O--O, IV infusion of loxiglumide; O-O, IV infusion of NaCl; 0, mean integrated data for experiments with NaCl infusion; W, mean integrated data for experiments with loxiglumide infusion.
GASTROENTEROLOGY
INTRADUODENAL INFUSION
! NaCl
slightly but significantly decreased by loxiglumide compared with the NaCl control (Figure 4). Loxiglumide significantly decreased meal-stimulated outputs of amylase (Figure 5A-I?), trypsin (Figure 6), and chymotrypsin (data not shown) both in terms of the individual outputs and in terms of the mean integrated secretion. Mean integrated secretion of the proteolytic enzymes trypsin and chymotrypsin was inhibited by loxiglumide to a greater degree of about 70% (Figure 6) than secretion of amylase which
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Figure 5. A. Amylase output: IV infusion of 5 mg kg’ h-’ loxiglumide significantly decreased pancreatic output of amylase compared with the infusion of NaCl in the control experiment at all individual time intervals from 40 to 120 minutes after the beginning of the intraduodenal infusion (P < 0.05 by ANOVA). The mean integrated secretion of amylase after intraduodenal infusion of the test meal was inhibited by loxiglumide by more than 50% when compared with the NaCl control (P < 0.01). Data shown are given as mean ? SEM both for the individual time points (lefr) and for the integrated data (right). O--O, IV infusion of loxiglumide; a-0, IV infusion of NaCl; 0, mean integrated data for experiments with NaCl infusion; ?? , mean integrated data for experiments with loxiglumide infusion. B. Integrated change in amylase output: the mean integrated amylase outputs after intraduodenal infusion of the test meal were decreased by loxiglumide by more than 50% when compared with the NaCl control (left) (P < 0.01). In previous studies with IV infusion of cerulein (right), loxiglumide decreased integrated amylase output to a greater degree (> 90%). Data shown are given as mean ? SEM. 0, Mean integrated data for experiments with NaCl infusion; w, mean integrated data for experiments with loxiglumide infusion.
Figure 4. Duodenal volume: IV infusion of 5 mg kg-’ h-’ loxiglumide did not significantly decrease duodenal volume compared with the infusion of NaCl in the control experiment at any time interval alter the intraduodenal infusion (P > 0.05 by ANOVA). The mean integrated output of volume after intraduodenal infusion of the test meal, however, was significantly inhibited by loxiglumide by about 25%-30% when compared with the NaCl control (P < 0.05). Data are given as mean ? SEM both for the individual time points (lefr) and for the integrated data (right). O--O, IV infusion of loxigluntide; O-O, IV infusion of NaCl; 0, mean integrated data for experiments with NaCl infusion; W, mean integrated data for experiments with loxiglumide infusion.
was inhibited only by about 55% (Figure 5A-B). Loxiglumide at the same dose inhibited ceruleinstimulated pancreatic secretion of various enzymes by more than 90%-W% (data for amylase are given in Figure 5B; data for trypsin and chymotrypsin are not shown). The increase in plasma concentrations of insulin, C peptide, and glucose after the intraduodenal infusion of the test meal was not significantly altered by infusion of loxiglumide when compared with the NaCl control (Figure 7).
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June 1991
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CCK preparation, even considering the dose which best mimics the postprandial increase in plasma CCK, might not reflect the true concentrations of the hormone at its place of action and the kinetics of its release in vivo. The development of new specific CCK-receptor antagonists offers a new approach to evaluate the physiological role of CCK both as a hormone and as a neurotransmitter in various gastrointestinal functions, avoiding the pitfalls associated with previous studies. The present experiments were designed to evaluate whether endogenous CCK regulates the exocrine and the endocrine pancreatic function as well as the biliary function. Loxiglumide was given at a dose that abolishes the actions of endogenous CCK at the gallbladder and the pancreas. The present dose of loxiglumide even abolishes the actions of pharmacological doses of CCK which increase the plasma concentrations of CCK and CCK analogues to levels markedly exceeding those seen under physiological conditions (3,4,14,35-38). Similar to previous experiments with oral administration of food or IV infusion of cerulein (14), loxiglumide completely inhibited gallbladder emptying after intraduodenal infusion of a test meal and even significantly increased gallbladder volumes when compared
I-L Figure 6. Trypsin output: IV infusion of 5 mg kg’ h-’ loxiglumide significantly decreased output of trypsin compared with the infusion of NaCl in the control experiment at all time intervals from 40 to 120 minutes after beginning of the intraduodenal infusion (P < 0.05 by ANOVA). The mean integrated secretion of trypsin after intraduodenal infusion of the test meal was inhibited by loxiglumide by more than 60% when compared with the NaCl control (P < 0.01). Data shown are given as mean ? SEM both for the individual time points (leff) and for the integrated data (right). O--O, IV infusion of loxiglumide; O-O, IV infusion of NaCl; 0, mean integrated data for experiments with NaCl infusion; W, mean integrated data for experiments with loxiglumide infusion.
Discussion Cholecystokinin is a peptide thought to act both as a hormone and as a neurotransmitter in the brain, in the enteric nervous system, and at various gastrointestinal organs. Humoral CCK is released from endocrine mucosal cells of the upper small intestine into the circulation in response to a meal (26). Administration of exogenous CCK or CCK analogues increases gallbladder emptying and pancreatic secretion (14,23,27,28). Exogeneous CCK also increases glucose-induced insulin secretion in vitro and in vivo in the experimental animal (29-32). Recently, it has been shown that CCK, given at “physiological” doses that mimic the postprandial increase in plasma concentration of this peptide, does not increase glucosestimulated insulin secretion in humans but only increases amino acid-stimulated insulin secretion (33). However, in most previous studies, pharmacological doses of CCK or CCK-like analogues that exceeded the physiological postprandial increase in plasma CCK had been administered. Indeed, only in recent years this postprandial increase in plasma CCK has been defined more exactly (12,13,24,25). Recent studies, therefore, tried to administer CCK at doses that mimic the increase in plasma concentrations of CCK seen after a meal (13,25,33,34). However, these studies were still associated with potential pitfalls. There continues to be some controversy about the comparability and quality of radioimmunoassays and bioassays for CCK; also, there is a controversy about which molecular forms of CCK are physiologically most important. In addition, an IV infusion or injection of a
Figure 7. Plasma concentrations of insulin, C peptide, and glucose at various time intervals are given as mean 2 SEM. Plasma insulin (middle), and C concentrations of glucose (bottom), peptide (top) were not significantly altered by IV infusion of loxiglumide compared with the infusion of NaCl in the control experiments at any of the individual time intervals after beginning of the intraduodenal infusion of nutrients (P > 0.2 by ANOVA). O--O, IV infusion of loxiglumide; O-O, IV infusion of NaCl.
1688 SCHWARZENDRUBE ET AL.
to prior fasting values. The results of the imaging studies were corroborated by the secretory studies. Loxiglumide almost completely inhibited meal-stimulated secretion of bilirubin. The present findings show that loxiglumide caused only a minor decrease in duodenal volume after intraduodenal infusion of a test meal. This effect is likely to be caused by inhibition of biliary secretion, because loxiglumide almost abolished bilirubin output. It is interesting that amylase output was decreased to a somewhat lesser degree than trypsin and chymotrypsin output. The lack of the CCK antagonists to completely abolish the exocrine pancreatic secretion of enzymes suggests that other hormones and neural mechanisms are also involved in the regulation of meal-stimulated pancreatic secretion of enzymes. Therefore, CCK is only one of several factors mediating stimulation of pancreatic enzyme secretion after a meal. Cholecystokinin is probably not at all involved in the regulation of pancreatic secretion of fluid. This is probably also true for bicarbonate secretion, which could not be measured in the present experiments because of the intraduodenal infusion of the test meal. Postprandial pancreatic secretion of fluid (and bicarbonate) might be mediated by secretin, other humoral factors, or neural mechanisms. The conclusions drawn from the present results are further supported by a recent study also showing that blockade of the CCK receptor could not completely abolish pancreatic exocrine secretion (39). However, in the latter study CCK blockade decreased mealstimulated bilirubin secretion only by 59% whereas the integrated bilirubin output was almost abolished in the present experiments (39). Correspondingly, in the present study CCK blockade also abolished the gallbladder emptying after the intraduodenal test meal when measured by a sonographic technique and even increased gallbladder volumes compared with prior fasting volumes. These results corroborate our own previous experiments examining the effects of CCK blockade on gallbladder emptying after regular oral meals (14). Recently other investigators have reported almost identical results when evaluating the effects of the CCK antagonists loxiglumide and MK329 (L-364,718) on gallbladder emptying (39,40). Thus, in contrast to the regulation of pancreatic secretion, which is only partly mediated by CCK, gallbladder function is mainly regulated by CCK both in terms of its emptying after absorption of nutrients from the intestine and in terms of maintenance of its fasting volume. More than two decades ago, it was already hypothesized that CCK is one of the gastrointestinal hormones capable of stimulating insulin secretion (29,41). Contrary to these observations, some investigators have suggested that the insulinotropic effect previously
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attributed to CCK may be caused by contaminating gastric inhibitory polypeptide, present in crude preparations of CCK, and not by CCK itself (42,43). Later, however, pure preparations of natural porcine CCK (44), synthetic COOH-terminal octapeptide of CCK (CCK-8) (43,45,46), and synthetic cerulein, which contains a COOH-terminal pentapeptide identical to CCK (44,45,47,48), have also been shown to stimulate insulin and glucagon secretion in vivo and in vitro. Nevertheless, it has been difficult to determine whether the insulinotropic action of these exogenously administered peptides is physiological. Recently, it has been shown that CCK, given at physiological doses that mimic the postprandial increase in plasma concentration of this peptide, does not increase glucosestimulated insulin secretion in humans but only increases amino acid-stimulated insulin secretion (33). In the rat the CCK antagonist MK-329 reduced the meal-stimulated insulin secretion, suggesting that CCK might play a physiological incretin role in this animal (49). It has also been shown that both the new proglumide derivatives as well as the nonpeptide antagonists MK-329 and asperlicin inhibit CCKstimulated insulin release at least as potently, or even more potently, than exocrine secretion from isolated pancreatic islets and acini (50) and from the isolated perfused rat pancreas (51,52), respectively. The latter studies strongly suggest that these CCK antagonists affect the CCK receptor at the endocrine pancreatic tissue at least as effectively than that in the exocrine tissue. The intraduodenal test meal given in the present experiments does not represent a physiological meal. Therefore, a regular oral (physiological) meal might result in different kinetics of the secretion of insulin and CCK, which could, in turn, have a different impact on islet physiology. However, the kinetics of the release of both CCK and insulin in response to the intraduodenal infusion of nutrients is similar to those observed after a meal (13,23-25). Also, recent studies have failed to show an effect of loxiglumide on the increase in glucose, insulin, and C peptide levels after regular oral meals (53). Of course, in the studies with oral meals one cannot exclude that the CCK antagonist might also influence gastric emptying and might thereby alter the release of insulin caused by the nutrients absorbed. The present studies and the conclusion drawn from the results are supported by recent findings from other investigators (54,55). Both the nonpeptide antagonist MK-329 as well as loxiglumide failed to alter the meal-stimulated pancreatic endocrine responses to oral and intraduodenal administration of various meals (54,55). The present results do not support the concept that CCK acts as an important incretin factor in humans. Loxiglumide at a dose that completely inhibits the actions of physiological and even pharmacological
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concentrations of CCK and CCK analogues on the gallbladder and the exocrine pancreas did not alter the increase in circulating concentrations of glucose, insulin, and C peptide after intraduodenal infusion of nutrients. Although the present study does not exclude that during certain conditions CCK may increase insulin secretion also in humans, CCK does not play an important physiological role as an incretin.
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Tacconi M, Rovati L, Setnikar I. Effect of loxiglumide on gallbladder contractile response to caerulein and food in humans. Gastroenterology 1990:98:1307-1310, 17. Gullo L, Bolondi L, Priori P, Casanova P, Labo G. Inhibitory effect of atropine on cholecystokinin-induced gallbladder contraction in man. Digestion 1984;29:209-213. 18. Duncan DB. Multiple range tests for correlated and heteroscedastic means. Biometrics 1957;13:164-176. 19. Byrkit SJ. Elements of statistics. Chapter 10. Analysis of variance. 3. Auflage. New York: van Nostrand, 1980:305-358. 20. Hacker M, Schmidt WE, Creutzfeldt W, Roy Choudhury A, Nustede R, Schafmayer A, Wilms HM, Folsch UR. Determination of plasma CCK concentrations by bioassay and specific radioimmunoassay in man: a critical evaluation (abstr). Pancreas 1989;5:A619. 21. Jansen JBMJ, de Jong AJL, Lamers CBHM. The cholecystokinin receptor antagonist CR1409 increases plasma cholecystokinin in rats. Regul Pept 1989;24:209-213. 22. Jansen JB, Jebbink MC, Douglas BR, Lamers CB. Effect of loxiglumide (CR1505) on bombesinand meal-stimulated plasma cholecystokinin in man. Em J Clin Pharmacol 1990;38: 267-370. 23. Beglinger C, Fried M, Whitehouse I, Jansen JB, Lamers CB, Gyr K. Pancreatic enzyme response to a liquid meal and to hormonal stimulation. Correlation with plasma secretin and cholecystokinin levels. J Clin Invest 1985;75:1471-1476. 24. Chang TM, Chey WY. Radioimmunoassay of cholecystokinin. Dig Dis Sci 1983;28:456-468. 25. Jansen JBMJ, Lamers BHHW. Radioimmunoassay of cholecystokinin in human tissue and plasma. Clin Chim Acta 1983;33: 2197-2205. a hormone and a neurotransmit26. Williams JA. Cholecystokinin: ter. Biomed Res 1982;3:107-115. 27. Anagnostides AA, Chadwik VS, Selden AC, Barr J, Maton PN. Human pancreatic and biliary responses to physiological concentrations of cholecystokinin octapeptide. Clin Sci 1985;69: 259-263. 28. Konturek SJ, Tasler J, Cieszkowski M, Szewczyk K, Hladij M. Effect of cholecystokinin receptor antagonist on pancreatic responses to exogenous gastrin and cholecystokinin and to meal stimuli. Gastroenterology 1988;94:1014-1023. AH. Entero-insular axis. Arch Intern 29. Unger RH, Eisentraud Med 1969;123:261-266. 30. Goldfine ID, Williams JA. Receptors for insulin and CCK in the acinar pancreas: relationship to hormone action. Int Rev Cytol 1983;85:1-38. 31. Verspohl EJ, Ammon HPT, Williams JA, Goldfine ID. Evidence that cholecystokinin interacts with specific receptors and regulates insulin release in isolated rat islets of Langerhans. Diabetes 1986;35:38-43. G, Ammon HPT, Williams JA, Goldfine 32. Verspohl EJ, Wunderle
33.
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ID. Proglumide (gastrin and cholecystokinin receptor antagonist) inhibits insulin secretion in vitro. Naunyn Schmiedebergs’s Arch Pharmacol 1986;332:284-287. Rushakoff RJ, Goldfine ID, Carter JC. Liddle RA. Physiological concentrations of cholecystokinin stimulate amino acidinduced insulin release in humans. J Clin Endocrinol Metab 1987:65:395-401. Wiener I, Inoue K, Fagan CJ, Lilja P. Watson LC, Thompson JC. Release of cholecystokinin in man: correlation of blood levels with gallbladder contraction. Ann Surg 1981;194:321-327. Niederau C, Niederau M, Williams JA, Grendell JH. New proglumide analogue CCK-receptor antagonists: very potent and selective for peripheral tissues. Am J Physiol 1986;251: G856-G860. Niederau C, Liddle RA, Williams JA, Grendell JH. Pancreatic growth. Interaction of exogenous cholecystokinin, a protease
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inhibitor, and a cholecystokinin receptor antagonist in mice. Gut 1987;28:63-69. Niederau C, Karaus M. Effects of cholecystokinin agonists and antagonists on myoelectric and motor activities of ileal colonic muscle in vitro. Int J Gastrointest Motil 1990;2:169-175. Setnikar I, Bani M, Cereda R, Chiste R, Makovec F, Pacini MA, Revel L, Rovati LC, Rovati LA. Pharmacological characterization of a new potent and specific nonpolypeptidic cholecystokinin antagonist. Arzneimittel Forschung 1987;6:703-707. Hildebrandt P, Beglinger C, Gyr K, Jansen JB, Rovati LC, Zuercher M, Lamers CB, Setnikar I, Stalder GA. Effect of a cholecystokinin receptor antagonist on intestinal phase of pancreatic and biliary responses in man. J Clin Invest 1990;85: 640-646. Liddle RA, Gertz BJ, Kanayama S, Beccaria L, Coker LD, Turnbull TA, Morita ET. Effects of a novel cholecystokinin receptor antagonist, MK-329, on gallbladder contraction and gastric emptying in humans. Implications for the physiology of CCK. J Clin Invest 1989;84:1220-1225. Meade RC, Kneubuhler HA, Schulte WJ, Barboiak JJ. Stimulation of insulin secretion by pancreozymin. Diabetes 1967;16:
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Dupre J, Ross SA, Watson D, Brown JD. Stimulation of insulin secretion by gastric inhibitory polypeptide in man. J Clin Endocrinol Metab 1973;37:826-828. Frame CM, Davidson MB, Sturdevant RAL. Effects of octapeptide of cholecystokinin on insulin and glucagon secretion in dogs. Endocrinology 1975;97:549-553. Otsuki M, Sakamoto C, Yuu H, Maeda M, Morita S, Ohki A, Kobayashi N, Terashi K, Okano K, Baba S. Discrepancies between the doses of cholecystokininor caerulein-stimulating exocrine and endocrine responses in perfused isolated rat pancreas. J Clin Invest 1979;63:478-484. Sakamoto C, Otsuki M, Ohki A, Mitsuo M, Yamasaki T, Baba S. Glucose-dependent insulinotropic action of cholecystokinin and caerulein in the isolated perfused rat pancreas. Endocrinology 1982;110:398-402. Fujimoto WY, Williams RH, Ensinck JW. Gastric inhibitory polypeptide, cholecystokinin, and secretin effects on insulin and glucagon secretion by islet cell cultures. Proc Sot Biol Med 1979;160:349-353. Otsuki M, Sakamoto C, Maeda M, Yuu H, Morita S, Baba S. Effect of caerulein on exocrine and endocrine pancreas in the rat. Endocrinology 1979;105:1396-1402. Ohneda A, Horigome K, Ishii S, Kai Y, Chiba M. Effects of
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caerulein upon insulin and glucagon secretion in dogs. Hormon Metab Res 1978;10:7-14. Rossetti L, Shulman GI, Zawalich WS. Physiologic role of cholecystokinin in meal-induced insulin secretion in conscious rats. Studies with L-364,718, a specific inhibitor of CCK-receptor binding. Diabetes 1987;36:1212-1215. Zawalich WS, Diaz VD. Asperlicin antagonizes stimulatory effects of cholecystokinin on isolated islets. Am J Physiol 1987;252:E370-E374. Okabayashi Y, Otsuki M, Nakamura T, Fujii M, Tani S, Fujisawa T, Koide M, Hasegawa H, Baba S. Proglumide analogues CR1409 and CR1392 inhibit cholecystokinin-stimulated insulin release more potently than exocrine secretion from the isolated perfused rat pancreas. Pancreas 1990;5:291297. Nakamura T, Fujii M, Okabayashi Y, Nakamura T, Tani S, Fujisawa T, Koide M, Hasegawa H, Otsuki M. Effects of L-364,718 on pancreatic exocrine and endocrine secretion in the rat. Pancreas 1990;5:216-221. Niederau C, Schwarzendrube J, Niederau M, Luthen R. CCK does not act as a physiologic insulinotropic factor after regular meals in healthy humans (abstr). Eighth International Symposium on Gastrointestinal Hormones, Timmendorf, Germany, September 1990. Digestion 1990;46:A78. Bonato C, Valentini A, Tacconi M, Rovati L, Malesci A. Effect of CCK-receptor blockade on the postprandial release of insulinar hormones (abstr). Gastroenterology 1989;96:A51. Liddle RA, Gerzt BJ, Kanayama S, Rushakoff RJ, Williams VC, Coker LD. Regulation of pancreatic endocrine function by cholecystokinin: studies with MK-329, a nonpeptide cholecystokinin receptor antagonist. J Clin Endocrinol Metab 1990;70: 1312-1318.
Received March 20,199O. Accepted November 2,199O. Address requests for reprints to: Claus Niederau, M.D., Medizinische Klinik und Poliklinik, Abteilung fur Gastroenterologie, Heinrich-Heine-Universitat Dusseldorf, Moorenstrasse 5, 4000 Dusseldorf, Germany. Dr. Niederau was supported by grants from the Deutsche Forschungsgemeinschaft (Ni 224/2-2) and from the Minister fur Wissenschaft und Forschung des Landes Nordrhein-Westfalen. The authors thank Christine Genz and Monika Ebbert for expert technical assistance.
1991;100:1663-1690
Effects of Cholecystokinin-Receptor Blockade on Pancreatic and Biliary Function in Healthy Volunteers JoRG
SCHWARZENDRUBE,
REINHARD Medizinische Diisseldorf,
LUTHEN,
Klinik Germany
und
h4ARKUS
and CLAUS NIEDERAU
Poliklinik,
Abteilung
This study used the specific cholecystokinin (CCK)receptor antagonist loxiglumide to evaluate whether endogenous CCK, which is released after a meal, regulates pancreatic and hiliary functions. Eight healthy volunteers were studied twice on separate days. The subjects received a continuous intraduodenal infusion of a 750-kcal liquid test meal for 2 hours either with or without IV infusion of 5 mg . kg-’ . h-’ of loxiglumide. Loxiglumide at this dose abolishes the actions of CCK at various target organs including gallbladder and pancreas, when given at doses that mimic postprandial plasma concentrations of CCK. Loxiglumide markedly decreased the meal-stimulated outputs of amylase, trypsin, and chymotrypsin by !w%-70% of control values but only slightly decreased duodenal volume (25Ohinhibition of mean integrated secretion). Loxiglumide abolished gallbladder emptying induced by infusion of nutrients and even increased gallbladder volumes when compared with prior fasting values. Correspondingly, loxiglumide almost abolished the output of bilirubin after infusion of nutrients. However, loxiglumide failed to alter the increase in circulating concentrations of glucose, insulin, and C peptide after infusion of nutrients. The present results show that CCK is one of several factors that regulate pancreatic protein secretion after absorption of nutrients. However, CCK is probably not involved in regulation of pancreatic secretion of fluid. In contrast, gallbladder function is mainly regulated by CCK, both in terms of its emptying after intestinal absorption of nutrients and in terms of maintenance of its fasting volume. Cholecystokinin does not play a major physiological role as an insulinotropic factor.
T
NIEDERAU,
he development of new specific cholecystokinin (CCK)-receptor antagonists offers a new approach to evaluate the physiological role of CCK in various
fiir Gastroenterologie,
Heinrich-Heine-Universittit
gastrointestinal functions, avoiding the pitfalls associated with previous studies that administered exogenous CCK. In 1981 proglumide and benzotript were shown to act as specific and competitive CCK-receptor antagonists in vitro (1). However, these compounds had low potencies or were toxic when given in an effective dose in vivo (2). Subsequently, molecules with a proglumidelike structure were synthesized; they were up to 1000 to 5000 times more potent than proglumide in inhibiting the action and binding of CCK in vitro and in vivo (3,~). More recently, several nonpeptide substances originating from the substance asperlicin (5) were shown to act as specific CCK antagonists (6,7). The most potent nonpeptide substance MK-329 (formerly termedL-364,718) is 25,000-l,OOO,OOO times more potent than proglumide in inhibiting CCK’s action and binding in vitro and in vivo (4,6,7). Thus, from the potencies of inhibiting the actions of CCK, both the new amino acid derivatives of proglumide and the new nonpeptide antagonists are well suitable for in vivo studies. The present experiments used the specific CCKreceptor antagonist loxiglumide (formerly termed CR1505) to evaluate the role of endogenous CCK in the regulation of biliary and pancreatic functions in healthy volunteers. Materials
and Methods
Materials The CCK antagonist loxiglumide (CRl505; D,L,+ [3,4-dichloro-benzoylamino]-5-[N-3-methoxy-propyl-pentyl-
Abbreviation used in this paper: ANOVA, analysis of variance. o 1991 by tbe American Gastroenterological Association 0016-5065/91/$3.00
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amino]-5-oxo-pentanoic acid) was kindly provided by Rotta Research Laboratorium (Milano, Italy] as sterile solutions containing 50 mg loxiglumide in 10 mL of solution. The test meal consisted of 500 mL of Fresubin (Fresenius AG, Bad Homburg, Germany), 85 mL Intralipid 20% (Pfrimmer, Erlangen, Germany), 20 g glucose, and 3 g polyethylene glycol (PEG] 4000.
Laboratory Determinations The concentration of PEG in the duodenal aspirates was determined by the turbidimetric method of Malawer and Powell (8). The duodenal concentrations of trypsin, chymotrypsin, and amylase were determined by colorimetric methods using arginine-p-nitroanilide, N-(3-carboxypropionyl]-phenylalanine-p-nitroanilide, and 3-p-nitrophenylmaltose heptaosid as substrates (9-11). For the determination of trypsin, chymotrypsin, and bilirubin, commercially available kits from Boehringer (Mannheim, Germany] were used. For the determination of amylase, the commercially available Phadebas kit (Pharmacia, Piscataway, NJ) was used. The duodenal volume was calculated from the dilution of perfused PEG marker inside the duodenum,
V,=$V,,
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meal with simultaneous IV infusion of 5 mg . kg-’ h-’ loxiglumide. The infusion of loxiglumide was started 30 minutes before the beginning of the intraduodenal infusion and was kept three times higher than the finally desired rate for the first 10 minutes in order to achieve steady state conditions of near continuous plasma concentrations of loxiglumide (14-16). On the second day of the control experiment, the subjects received an IV infusion of saline (0.9% NaCl) instead of loxiglumide. Gallbladder volumes were measured in 20-minute intervals using a Toshiba scanner (Neuss, Germany; Sonolayer SAL 77A) equipped with a linear 3.5-MHz probe. Gallbladder emptying was determined by a method previously reported in detail, assuming an ellipsoid structure of the gallbladder (14,17). In further experiments, parts of which have already been published, gallbladder emptying and pancreatic secretion were studied after IV infusion of increasing doses of cerulein (7.5, 15, 30, and 60 ng . kg-’ h-‘, with each dose given for 15 minutes] (14). In these studies, 10 male healthy volunteers were studied on 2 separate days either with or without infusion of 5 mg . kg-’ . h-’ loxiglumide (14). The experimental techniques for measurements of pancreatic and biliary functions were identical to those already described for the studies with intraduodenal infusion of nutrients.
d
in which C,, and C,, are the concentrations of PEG in the duodenal aspirates and in the perfusion volume [V, = 5 mL/min). The output of pancreatic enzymes corresponds to the product of concentration and the duodenal volume V,. Venous blood samples were taken at 20-minute intervals. Plasma concentrations of insulin and C peptide were measured by commercially available radioimmunoassays (Pharmacia). Plasma concentrations of CCK were measured by a specific and sensitive bioassay, as described previously in detail (I&I 3). Plasma concentrations of glucose were measured by the glucose-oxidase method (Boehringer).
Experimental
Design
Eight healthy volunteers [three men and five women], aged 21-44 years, were studied in the morning after an overnight fasting of at least 12 hours. The subjects were taking no medication and were all within 10% of the ideal body weight. A triplelumen polyvinyl tube was positioned fluoroscopically with its tip located at the ligament of Treitz. The second part of the duodenum was perfused with the test solution at a rate of 5 mL/min, yielding a rate of 375 kcal/h for a total period of 2 hours. Duodenal contents were recovered by a suction pump at the ligament of Treitz. The specimens were collected over ice and pooled in 20-minute fractions. A separate gastric tube was positioned fluoroscopically with its tip lying in the antrum to continuously remove gastric contents. The experiments were performed with the subjects lying in a semirecumbent position. All subjects were studied on separate days with an interval of at least 2 days between the individual experiments, which were done in randomized order. On the first day subjects received the intraduodenal infusion of the test
Approval of the Study Protocol The study was approved by the Ethical Committee of Human Research of the Dusseldorf University. Written informed consent was obtained from each subject.
Statistical Analysis All values are given as means + SEM. In addition to the mean data at the various time intervals after beginning of the intraduodenal infusion, the mean integrated responses to the various stimuli were calculated for glucose, insulin, C peptide, amylase, trypsin, chymotrypsin, and gallbladder volume. The integrated responses were calculated in each subject as the sum of postprandial values after subtracting the prior basal value at each point. The significance of differences between means was determined by analysis of variance (ANOVA) using the method of Duncan (18,19). Pvalues < 0.05 were considered significant.
Results The intraduodenal infusion of the liquid test meal significantly increased plasma concentrations of CCK, which reached maximal levels of 6-12 pmol/L after 20-40 minutes (Figure 1). Basal (fasting) plasma concentrations of CCK ranged from 1 to 2 pmol/L (Figure 1). Plasma concentrations of CCK were not
measurable in experiments in which the CCK antagonist loxiglumide was infused, because even residual amounts of a CCK antagonist in the plasma sample interfere with the CCK bioassay which is based on
June 1991
Figure 1. Plasma concentrations of CCK measured by CCK bioassay (see Materials and Methods). e-0, Studies in which subjects (n = 10) received an N infusion of cerulein at increasing doses as indicated in the figure; O--O, studies in which subjects (n = 8) received an intraduodenal infusion of nutrients (750 kcal/z b). Results are given as mean 2 SEM.
CCK-stimulated amylase release from isolated rat pancreatic acini (12,13,20). However, there was no indication that the same meal when given on a separate day together with the infusion of loxiglumide might have resulted in a lesser increase in plasma concentration of CCK. On the contrary, several recent studies have shown that IV infusion of loxiglumide increases the meal-stimulated increase in plasma CCK when measured by radioimmunoassay (20-22). However, the mechanism of this increase in plasma immunoreactivity of CCK has not yet been fully elucidated. In any case, currently both the bioactivity and radioimmunologic measurement of plasma CCK will not definitely prove or disprove that the same meal given on separate days with and without CCK antagonists will lead to the same release of CCK. Intravenous infusion of cerulein at increasing doses of 7.5, 15.0, 30.0, and 60.0 ng . kg-’ . h-’ (each dose given for 15 minutes) increased plasma bioactivity of CCK in a dose-dependent manner (Figure 1). The two lower doses of 7.5 and 15.0 ng . kg-’ * h-’ resulted in plasma concentrations of CCK that covered the range of CCK concentrations seen after the intraduodenal infusion of nutrients (Figure 1) and were similar to plasma CCK concentrations seen after oral meals (13,23-25). The two higher cerulein doses of 30.0 and 60.0 ng . kg-’ . h-’ resulted in plasma concentrations of CCK exceeding postprandial values. The latter doses of 30.0 and 60.0 ng . kg-’ * h-’ cerulein might, therefore, produce pharmacological effects, whereas the two lower cerulein doses of 7.5 and 15 .O ng . kg-’ . h-’ might mimic the effects caused by the postprandial increase in plasma CCK. Intraduodenal infusion of a liquid test meal markedly decreased gallbladder volume (Figure 2). Similar
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effects were shown previously after oral administration of a test meal or after IV infusion of cerulein. Intravenous infusion of loxiglumide at 5 mg . kg-’ . h-’ completely inhibited cerulein-stimulated gallbladder emptying and even significantly increased gallbladder volumes compared with prior basal fasting values (14). Compared with previous studies evaluating the effect of loxiglumide on gallbladder emptying after IV infusion of the CCK analogue cerulein or after an oral meal, the intraduodenal infusion of the test meal caused a more pronounced decrease in gallbladder volume when compared with the effect of an oral meal but did not reach the effect seen after administration of cerulein at pharmacological doses (14). Intravenous infusion of loxiglumide completely inhibited gallbladder emptying after intraduodenal infusion of the test meal and even increased gallbladder volume compared with prior fasting volumes (Figure 2). Intravenous infusion of 5 mg * kg-’ - h-’ loxiglumide significantly decreased output of bilirubin compared with the infusion of NaCl in the control experiment at all time intervals after intraduodenal infusion of the test meal (Figure 3). The mean integrated secretion of bilirubin was inhibited by loxiglumide by more than 90% when compared with the NaCl control (Figure 3). Loxiglumide did not significantly decrease duodenal volume at the various individual time intervals after intraduodenal infusion of the meal when compared with the corresponding NaCl control (Figure 4). However, the mean integrated secretion of fluid was
1 30
20
10
I I
Figure 2. Gallbladder volume: IV infusion of 5 mg kg-’ b-’ loxiglumide significantly increased gallbladder volumes compared with the infusion of NaCl in the control experiment at all individual time intervals from 20 to 120 minutes after beginning of the intraduodenal infusion (P < 0.05 by ANOVA). Basal values represent measurements performed 30 minutes before beginning of the intraduodenal infusion. The mean integrated gallbladder volumes after intraduodenal infusion of the test meal were decreased by loxiglumide by more than 90% when compared with the NaCl control (leff) (P < 0.001). Data are given as mean f SEM for the individual time intervals after the intraduodenal infusion of nutrients (left) and for the integrated change in gallbladder volume (right). O--O, IV infusion of loxiglumide; O-O, N infusion of NaCl; 0, mean integrated data for experiments with NaCl infusion; ?? , mean integrated data for experiments with loxiglumide infusion.
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Figure 3. Bilirubin output: IV infusion of 5 mg . kg-’ b-’ loxiglumide significantly decreased output of bilirubin compared with the infusion of NaCl in the control experiment at all individual time intervals from 20 to 120 minutes after beginning of the intraduodenal infusion (P < 0.05 by ANOVA). Loxiglumide inhibited the mean integrated secretion of bilirubin by more than 90% when compared with the NaCl control (P < 0.001). Data are given as mean ? SEM for the individual time points (left) and for the integrated data (right). O--O, IV infusion of loxiglumide; O-O, IV infusion of NaCl; 0, mean integrated data for experiments with NaCl infusion; W, mean integrated data for experiments with loxiglumide infusion.
GASTROENTEROLOGY
INTRADUODENAL INFUSION
! NaCl
slightly but significantly decreased by loxiglumide compared with the NaCl control (Figure 4). Loxiglumide significantly decreased meal-stimulated outputs of amylase (Figure 5A-I?), trypsin (Figure 6), and chymotrypsin (data not shown) both in terms of the individual outputs and in terms of the mean integrated secretion. Mean integrated secretion of the proteolytic enzymes trypsin and chymotrypsin was inhibited by loxiglumide to a greater degree of about 70% (Figure 6) than secretion of amylase which
I
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CAERULEIN INFUSION
NH.21
LOX
Figure 5. A. Amylase output: IV infusion of 5 mg kg’ h-’ loxiglumide significantly decreased pancreatic output of amylase compared with the infusion of NaCl in the control experiment at all individual time intervals from 40 to 120 minutes after the beginning of the intraduodenal infusion (P < 0.05 by ANOVA). The mean integrated secretion of amylase after intraduodenal infusion of the test meal was inhibited by loxiglumide by more than 50% when compared with the NaCl control (P < 0.01). Data shown are given as mean ? SEM both for the individual time points (lefr) and for the integrated data (right). O--O, IV infusion of loxiglumide; a-0, IV infusion of NaCl; 0, mean integrated data for experiments with NaCl infusion; ?? , mean integrated data for experiments with loxiglumide infusion. B. Integrated change in amylase output: the mean integrated amylase outputs after intraduodenal infusion of the test meal were decreased by loxiglumide by more than 50% when compared with the NaCl control (left) (P < 0.01). In previous studies with IV infusion of cerulein (right), loxiglumide decreased integrated amylase output to a greater degree (> 90%). Data shown are given as mean ? SEM. 0, Mean integrated data for experiments with NaCl infusion; w, mean integrated data for experiments with loxiglumide infusion.
Figure 4. Duodenal volume: IV infusion of 5 mg kg-’ h-’ loxiglumide did not significantly decrease duodenal volume compared with the infusion of NaCl in the control experiment at any time interval alter the intraduodenal infusion (P > 0.05 by ANOVA). The mean integrated output of volume after intraduodenal infusion of the test meal, however, was significantly inhibited by loxiglumide by about 25%-30% when compared with the NaCl control (P < 0.05). Data are given as mean ? SEM both for the individual time points (lefr) and for the integrated data (right). O--O, IV infusion of loxigluntide; O-O, IV infusion of NaCl; 0, mean integrated data for experiments with NaCl infusion; W, mean integrated data for experiments with loxiglumide infusion.
was inhibited only by about 55% (Figure 5A-B). Loxiglumide at the same dose inhibited ceruleinstimulated pancreatic secretion of various enzymes by more than 90%-W% (data for amylase are given in Figure 5B; data for trypsin and chymotrypsin are not shown). The increase in plasma concentrations of insulin, C peptide, and glucose after the intraduodenal infusion of the test meal was not significantly altered by infusion of loxiglumide when compared with the NaCl control (Figure 7).
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CCK preparation, even considering the dose which best mimics the postprandial increase in plasma CCK, might not reflect the true concentrations of the hormone at its place of action and the kinetics of its release in vivo. The development of new specific CCK-receptor antagonists offers a new approach to evaluate the physiological role of CCK both as a hormone and as a neurotransmitter in various gastrointestinal functions, avoiding the pitfalls associated with previous studies. The present experiments were designed to evaluate whether endogenous CCK regulates the exocrine and the endocrine pancreatic function as well as the biliary function. Loxiglumide was given at a dose that abolishes the actions of endogenous CCK at the gallbladder and the pancreas. The present dose of loxiglumide even abolishes the actions of pharmacological doses of CCK which increase the plasma concentrations of CCK and CCK analogues to levels markedly exceeding those seen under physiological conditions (3,4,14,35-38). Similar to previous experiments with oral administration of food or IV infusion of cerulein (14), loxiglumide completely inhibited gallbladder emptying after intraduodenal infusion of a test meal and even significantly increased gallbladder volumes when compared
I-L Figure 6. Trypsin output: IV infusion of 5 mg kg’ h-’ loxiglumide significantly decreased output of trypsin compared with the infusion of NaCl in the control experiment at all time intervals from 40 to 120 minutes after beginning of the intraduodenal infusion (P < 0.05 by ANOVA). The mean integrated secretion of trypsin after intraduodenal infusion of the test meal was inhibited by loxiglumide by more than 60% when compared with the NaCl control (P < 0.01). Data shown are given as mean ? SEM both for the individual time points (leff) and for the integrated data (right). O--O, IV infusion of loxiglumide; O-O, IV infusion of NaCl; 0, mean integrated data for experiments with NaCl infusion; W, mean integrated data for experiments with loxiglumide infusion.
Discussion Cholecystokinin is a peptide thought to act both as a hormone and as a neurotransmitter in the brain, in the enteric nervous system, and at various gastrointestinal organs. Humoral CCK is released from endocrine mucosal cells of the upper small intestine into the circulation in response to a meal (26). Administration of exogenous CCK or CCK analogues increases gallbladder emptying and pancreatic secretion (14,23,27,28). Exogeneous CCK also increases glucose-induced insulin secretion in vitro and in vivo in the experimental animal (29-32). Recently, it has been shown that CCK, given at “physiological” doses that mimic the postprandial increase in plasma concentration of this peptide, does not increase glucosestimulated insulin secretion in humans but only increases amino acid-stimulated insulin secretion (33). However, in most previous studies, pharmacological doses of CCK or CCK-like analogues that exceeded the physiological postprandial increase in plasma CCK had been administered. Indeed, only in recent years this postprandial increase in plasma CCK has been defined more exactly (12,13,24,25). Recent studies, therefore, tried to administer CCK at doses that mimic the increase in plasma concentrations of CCK seen after a meal (13,25,33,34). However, these studies were still associated with potential pitfalls. There continues to be some controversy about the comparability and quality of radioimmunoassays and bioassays for CCK; also, there is a controversy about which molecular forms of CCK are physiologically most important. In addition, an IV infusion or injection of a
Figure 7. Plasma concentrations of insulin, C peptide, and glucose at various time intervals are given as mean 2 SEM. Plasma insulin (middle), and C concentrations of glucose (bottom), peptide (top) were not significantly altered by IV infusion of loxiglumide compared with the infusion of NaCl in the control experiments at any of the individual time intervals after beginning of the intraduodenal infusion of nutrients (P > 0.2 by ANOVA). O--O, IV infusion of loxiglumide; O-O, IV infusion of NaCl.
1688 SCHWARZENDRUBE ET AL.
to prior fasting values. The results of the imaging studies were corroborated by the secretory studies. Loxiglumide almost completely inhibited meal-stimulated secretion of bilirubin. The present findings show that loxiglumide caused only a minor decrease in duodenal volume after intraduodenal infusion of a test meal. This effect is likely to be caused by inhibition of biliary secretion, because loxiglumide almost abolished bilirubin output. It is interesting that amylase output was decreased to a somewhat lesser degree than trypsin and chymotrypsin output. The lack of the CCK antagonists to completely abolish the exocrine pancreatic secretion of enzymes suggests that other hormones and neural mechanisms are also involved in the regulation of meal-stimulated pancreatic secretion of enzymes. Therefore, CCK is only one of several factors mediating stimulation of pancreatic enzyme secretion after a meal. Cholecystokinin is probably not at all involved in the regulation of pancreatic secretion of fluid. This is probably also true for bicarbonate secretion, which could not be measured in the present experiments because of the intraduodenal infusion of the test meal. Postprandial pancreatic secretion of fluid (and bicarbonate) might be mediated by secretin, other humoral factors, or neural mechanisms. The conclusions drawn from the present results are further supported by a recent study also showing that blockade of the CCK receptor could not completely abolish pancreatic exocrine secretion (39). However, in the latter study CCK blockade decreased mealstimulated bilirubin secretion only by 59% whereas the integrated bilirubin output was almost abolished in the present experiments (39). Correspondingly, in the present study CCK blockade also abolished the gallbladder emptying after the intraduodenal test meal when measured by a sonographic technique and even increased gallbladder volumes compared with prior fasting volumes. These results corroborate our own previous experiments examining the effects of CCK blockade on gallbladder emptying after regular oral meals (14). Recently other investigators have reported almost identical results when evaluating the effects of the CCK antagonists loxiglumide and MK329 (L-364,718) on gallbladder emptying (39,40). Thus, in contrast to the regulation of pancreatic secretion, which is only partly mediated by CCK, gallbladder function is mainly regulated by CCK both in terms of its emptying after absorption of nutrients from the intestine and in terms of maintenance of its fasting volume. More than two decades ago, it was already hypothesized that CCK is one of the gastrointestinal hormones capable of stimulating insulin secretion (29,41). Contrary to these observations, some investigators have suggested that the insulinotropic effect previously
GASTROENTEROLOGY Vol. 100,No. 6
attributed to CCK may be caused by contaminating gastric inhibitory polypeptide, present in crude preparations of CCK, and not by CCK itself (42,43). Later, however, pure preparations of natural porcine CCK (44), synthetic COOH-terminal octapeptide of CCK (CCK-8) (43,45,46), and synthetic cerulein, which contains a COOH-terminal pentapeptide identical to CCK (44,45,47,48), have also been shown to stimulate insulin and glucagon secretion in vivo and in vitro. Nevertheless, it has been difficult to determine whether the insulinotropic action of these exogenously administered peptides is physiological. Recently, it has been shown that CCK, given at physiological doses that mimic the postprandial increase in plasma concentration of this peptide, does not increase glucosestimulated insulin secretion in humans but only increases amino acid-stimulated insulin secretion (33). In the rat the CCK antagonist MK-329 reduced the meal-stimulated insulin secretion, suggesting that CCK might play a physiological incretin role in this animal (49). It has also been shown that both the new proglumide derivatives as well as the nonpeptide antagonists MK-329 and asperlicin inhibit CCKstimulated insulin release at least as potently, or even more potently, than exocrine secretion from isolated pancreatic islets and acini (50) and from the isolated perfused rat pancreas (51,52), respectively. The latter studies strongly suggest that these CCK antagonists affect the CCK receptor at the endocrine pancreatic tissue at least as effectively than that in the exocrine tissue. The intraduodenal test meal given in the present experiments does not represent a physiological meal. Therefore, a regular oral (physiological) meal might result in different kinetics of the secretion of insulin and CCK, which could, in turn, have a different impact on islet physiology. However, the kinetics of the release of both CCK and insulin in response to the intraduodenal infusion of nutrients is similar to those observed after a meal (13,23-25). Also, recent studies have failed to show an effect of loxiglumide on the increase in glucose, insulin, and C peptide levels after regular oral meals (53). Of course, in the studies with oral meals one cannot exclude that the CCK antagonist might also influence gastric emptying and might thereby alter the release of insulin caused by the nutrients absorbed. The present studies and the conclusion drawn from the results are supported by recent findings from other investigators (54,55). Both the nonpeptide antagonist MK-329 as well as loxiglumide failed to alter the meal-stimulated pancreatic endocrine responses to oral and intraduodenal administration of various meals (54,55). The present results do not support the concept that CCK acts as an important incretin factor in humans. Loxiglumide at a dose that completely inhibits the actions of physiological and even pharmacological
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1991
concentrations of CCK and CCK analogues on the gallbladder and the exocrine pancreas did not alter the increase in circulating concentrations of glucose, insulin, and C peptide after intraduodenal infusion of nutrients. Although the present study does not exclude that during certain conditions CCK may increase insulin secretion also in humans, CCK does not play an important physiological role as an incretin.
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Received March 20,199O. Accepted November 2,199O. Address requests for reprints to: Claus Niederau, M.D., Medizinische Klinik und Poliklinik, Abteilung fur Gastroenterologie, Heinrich-Heine-Universitat Dusseldorf, Moorenstrasse 5, 4000 Dusseldorf, Germany. Dr. Niederau was supported by grants from the Deutsche Forschungsgemeinschaft (Ni 224/2-2) and from the Minister fur Wissenschaft und Forschung des Landes Nordrhein-Westfalen. The authors thank Christine Genz and Monika Ebbert for expert technical assistance.