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Effects of a cholecystokinin on intestinal phase of pancreatic and biliary responses in man.

P Hildebrand, … , I Setnikar, G A Stalder

J Clin Invest. 1990;85(3):640-646. https://doi.org/10.1172/JCI114486.

Research Article

The present study was designed (a) to characterize the activity of loxiglumide as a peripheral cholecystokinin (CCK) antagonist in healthy human subjects, and (b) to determine whether CCK is a physiologic regulator of the intestinal phase of meal-stimulated exocrine pancreatic and biliary secretions in man. Intravenous loxiglumide (22 mumol/kg per h) was highly potent in antagonizing CCK8-induced pancreatic and acid secretion as well as release. The potency and selectivity of loxiglumide as an antagonist of CCK provides the tool for evaluating the role of CCK as a physiological mediator of meal-induced pancreatic and biliary responses in humans. Infusion of a liquid test meal into the evoked an immediate response of pancreatic enzyme and bilirubin outputs, respectively. Intravenous loxiglumide significantly inhibited the meal-induced pancreatic amylase output by 63% (P less than 0.05), lipase output by 43% (P less than 0.05), and bilirubin output by 59% (P less than 0.05). These data suggest that CCK is a physiological mediator of the intestinal phase of meal-stimulated pancreatic and biliary responses.

Find the latest version: https://jci.me/114486/pdf Effects of a Antagonist on Intestinal Phase of Pancreatic and Biliary Responses in Man Pius Hildebrand, Christoph Beglinger, Klaus Gyr, Jan B. M. J. Jansen, Lucio C. Rovati, Matthias Zuercher, Comelis B. H. W. Latmers, Ivo. Sbtnikar, and Georg A. Staider Division ofGastroenterology and Department ofResearch, University Hospital, CH-4031 Basel, Switzerland; Department ofInternal Medicine, Kantonsspital, CH-4410 Liestal, Switzerland; Department ofGastroenterology-Hepatology, University Hospital, NL-2333 AA Leiden, The Netherlands; and Rotta Research Laboratorium, I-20050 Monza, Italy

Abstract as a regulator of pancreatic and biliary secretion in man (5-12). The results support the concept that CCK is a major The present study was designed (a) to characterize the activity stimulatory mechanism of intestinal phase-stimulated pancre- of loxiglumide as a peripheral cholecystokinin (CCK) antago- atic enzyme and biliary responses. nist in healthy human subjects, and (b) to determine whether CCK is a physiologic regulator of the intestinal phase of meal- Methods stimulated exocrine pancreatic and biliary secretions in man. Intravenous loxiglumide (22 &mol/kg per h) was highly potent Subjects. 12 healthy male volunteers aged 22-28 yr (mean, 24 yr) in antagonizing CCK8-induced pancreatic enzyme and whose bodyweight was within 10% of their ideal value (mean, 72 kg) secretion as well as pancreatic polypeptide release. The po- participated in the studies. None was taking any medication or had any tency, and selectivity of loxiglumide as an antagonist of CCK history of gastrointestinal or endocrine symptoms and surgery. The provides the tool for evaluating the role of CCK as a physiolog- studies were approved by the University of Basel Human Ethical and Committee on 14 July 1987, and written informed consent was ob- ical mediator of meal-induced pancreatic biliary'responses tained from each subject. in humans. Infusion of a' liquid test meal into the duodenum Experimental procedure. Subjects were studied after an overnight evoked an immediate response of pancreatic enzyme and biliru- fast. The order of experiments was randomized. Pancreatic and biliary bin outputs, respectively. Intravenous loxigiumide significantly secretions were studied with the standard gastroduodenal intubation inhibited the meal-induced pancreatic amylase output by 63% marker peqfusion technique detailed elsewhere (4). In brief, a four- (P < 0.05), lipase output by 43% (P < 0.05), and bilirubin lumen tube was placed under fluoroscopic guidance. The gastric aspi- output by 59% (P < 0.05). These data suggest that CCK is a ration site was situated in the antrum for continuous gastric aspiration. physiological mediator of the intestinal phase of meal-stimu- Normal saline containing polyethylene glycol (PEG4000) as a nonab- lated pancreatic' and biliary responses. (J. Clin. Invest. 1990. sorbable marker was instilled into the second portion ofthe duodenum in a concentration of 2 g/liter and at 2 ml/min. The duodenal contents 85:640-646.) CCK antagonist - were recovered in 15-min aliquots at the distal aspiration site near the ligamentum of Treitz. Introduction Pharmacological characterization ofloxiglumide. In this part ofthe study, was infused intravenously for the duration of each Despite a large number ofphysiological studies on meal-stimu- experiment at a rate of 16.4 pmol/kg per h. After a 75-min period with lated pancreatic enzyme secretion in man, many crucial ques- secretin alone, doubling doses of CCK8 (4.7-37 pmol/kg per h) were tions remain unanswered. For example, the physiological role infused intravenously, each dose for 45 min. These doses have pre- of cholecystokinin (CCK),' one of the classical intestinal hor- viously been'shown to produce threshold to maximal pancreatic en- mones, has remained controversial (1'-3). There appears to be zyme secretion (13, 14). In three subjects, an additional dose of CCK8 no doubt that plasma CCK increases after a meal. Also, by (72 pmol/kg per h) was given to test whether the inhibitory effect of comparing increments in plasma CCK and pancreatic loxiglumide is surmountable with supramaximal'doses of CCK8. On after we have different days and in random order, either saline (control) or two secretion after a meal and exogenous CCK, pre- different doses of loxiglumide (3.3 or 10 mg/kg per h equal to 7 and 22 viously suggested that CCK could be a major stimulatory ;imol/kg per h), dissolved in 0.9% saline, were infused through an mechanism of postprandial pancreatic trypsin secretion (4). indwelling catheter in a forearm vein for the duration of the experi- The availability of potent antagonists of CCK binding to its ments (255 min). membrane receptor enabled us to reexamine the role of CCK Plasma samples were obtained in regular intervals before stimula- tion and during the last 15 min of each dose of CCK8. Blood was collected in ice-chilled EDTA tubes containing 5,000 kallikrein-in- Address correspondence to Dr. Christoph Beglinger, Division of Gas- hibiting units aprotinin/5 ml blood. Samples were immediately centri- troenterology, University Hospital, CH-403 1 Basel, Switzerland. fuged at 4VC and the plasma stored at -20'C until assayed for CCK, Received for publication 1I January 1989 and in revised form 3 pancreatic polypeptide (PP), and loxiglumide plasma concentrations. October 1989. To evaluate the specificity of loxiglumide, dose-response curves were performed in five subjects with graded doses of secretin (5 and 15 1. Abbreviations used in this paper: AUC, area under output curve; pmol/kg per h) and (1-27 pmol/kg per h) given with con- CCK, cholecystokinin; PEG, polyethylene glycol; PP, pancreatic poly- comitant infusions of loxiglumide (22 Atmol/kg per h) or, alternatively, . saline (control). Pancreatic and biliary secretions were collected as described above. Each dose of each stimulant was given for 45 min. J. Clin. Invest. To further test the specificity of loxiglumide as a CCK antagonist X The American Society for Clinical Investigation, Inc. against a structurally related substance such as , the effect of the 0021-9738/90/03/0640/07 $2.00 compound on -stimulated acid secretion was studied in Volume 85, March 1990, 640-646 four healthy subjects. After a 45-min basal period, pentagastrin was

640 Hildebrand et al. given in graded doses (65, 260, and 1,042 pmol/kg per h), each given (millimoles), PP, and CCK (picomolar). Secretory outputs in response for 45 min. The experiments were repeated with loxiglumide (22 to each dose of CCK8 with and without different doses of loxiglumide Amol/kg per h). secretion was measured by continuous were also analyzed by the following routine: For multiple comparisons aspiration using a double-lumen gastric tube and marker perfusion of secretory responses, samples were first tested for normality by technique (PEG4000) (13). Wilk-Shapiro test and for homogeneity of variances (test of Levene). Meal studies. After proper placement of the intestinal four-lumen When normal distribution of data could not be rejected, data were tube, indwelling catheters were inserted into both cubital veins for analyzed by two-way analysis of variance. In case of significant differ- blood sampling and infusion, respectively. After a 60-min basal period ences, this was followed by Newman-Keuls multicomparison test for with saline perfusion to the duodenum, a liquid test meal was perfused pairwise comparisons of AUCs and Dunnett multicomparison test for into the duodenum for 120 min at the rate of 2 ml/min. This perfusion the comparison of the secretory responses during each dose of CCK8 rate was used to simulate the rate of gastric emptying of a liquid test with and without loxiglumide. For nonnormally distributed samples, meal (7). The reflux of duodenal contents into the , measured the nonparametric Friedman test was applied. All statistical tests were by recovery of PEG4000 in the gastric aspirate, averaged 2% (0-9%) of used as routine in RS/ 1, a commercially available software package for the amount infused into the duodenum. On different days, either VAX computers. The nominal level of significance was P = 0.05. saline (control) or an intravenous infusion of the CCK antagonist loxiglumide (22 ,gmol/kg per h) was given throughout the experiment. Results Plasma samples were obtained for the determination of CCK and loxiglumide concentrations during the basal period and at regular in- Pharmacodynamics of loxiglumide tervals after starting the meal perfusion. CCK8-induced pancreatic secretion. As the infusion The meal consisted of 275 ml Ensure@, 85 ml H20, and 0.72 g expected, PEG4000. It had an osmolality of 300 mosM/liter and a pH of 7.4. The of graded doses of CCK8 produced a dose-dependent stimula- total caloric value of the perfused meal was 184 kcal, giving a load of tion of exocrine pancreatic secretion. The maximal amylase 3.9 g /h, 3.1 g /h, and 12.3 g carbohydrates/h. and lipase outputs were observed with 37 pmol/kg per h Determinations. Duodenal fluid output was measured to the next CCK8, whereas 72 pmol/kg per h (n = 3) did not seem to milliliter, bicarbonate concentration ofduodenal juice was determined further increase pancreatic enzyme output, suggesting supra- by the backtitration method (15), amylase using a commercial kit maximal stimulation. (Alpha Amylase PNP; Boehringer Mannheim GmbH, Mannheim, Doses of 3.3 and 10 mg/kg per h of loxiglumide antago- FRG) (16), lipase by the method of Grenner et al. (17) using a com- nized markedly CCK8-stimulated pancreatic enzyme release mercial kit (Behring Werke, Marburg, FRG), bile salts using 3-alpha- (Fig. 1, Table I). The antagonism of pancreatic secretory re- hydroxysteroid dehydrogenase (18), bilirubin spectrophotometrically (19), and PEG turbidimetrically (20). The duodenal fluid secretion for a given period was calculated as described previously (4). The recovery of the duodenal marker averaged 87±3% (mean±SEM), whereas the Loxiglmd iffusiu (71WJ221wM -li)or Salin average percentage of PEG recovered from the stomach was < 2%. - Recoveries were similar in the different experiments and were unaf- Bicarbonate fected by loxiglumide administration. Gastric fluid was measured to T the next milliliter; acid concentration was determined by titration, and PEG concentration was measured turbidimetrically (20). z Assays. Plasma CCK concentrations were estimated as previously E described (21) and all measurements were done with antibody T204. Un This antibody has been extensively characterized (22) and has been shown to bind to all carboxyl-terminal CCK containing the sulfated . The antibody shows < 2% crossreactivity to sulfated .....:...... Figure 1. Effect of lox- and does not bind to unsulfated forms ofgastrin or structurally iglumide (7 and 22 unrelated peptides. Furthermore, loxiglumide does not interfere with ,umol/kg per h) on the assay. The sensitivity of the assay was 0.5 pmol/liter plasma. The CCK8-stimulated pan- intraassay variation was < 10% at 2.6 pmol/liter. All samples were -u- Amylase creatic lipase-, amylase-, assayed in the same run. 40, and bicarbonate out- Coded plasma samples were assayed for PP concentration using puts in healthy subjects. previously described methods (23). The sensitivity of the assay was 2 Secretin was infused pmol/liter plasma. throughout the experi- Loxiglumide concentrations were measured by HPLC according to ments. Data are methods described elsewhere (24). The detection limit was 0.02-0.03 mean±SEM; n = 4 for ytg/ml plasma. Z.--- the lower dose of loxi- Materials. Synthetic CCK8 was purchased from Peninsula Labora- ...... 2 ...... glumide and n = 6 for tories, Inc. (Belmont, CA). The peptide was dissolved in 0.9% saline the upper dose of loxi- containing 0.5% human serum albumin under aseptic conditions by T A I glumide and the control the University of Basel Hospital pharmacy. Vials of 20 tg CCK8 were Lipase experiment. Loxiglu- stored at -20°C. Synthetic human secretin was a kind gift of Prof. L. mide significantly sup- Moroder (Max Planck Institute, Munich, FRG). Loxiglumide was ob- pressed E pancreatic li- tained from Rotta Research Laboratorium (Monza, Italy), and En- 20 - pase, amylase, and bi- sureg from Abbott Co. Ltd., Zug, Switzerland. @ carbonate responses (P Data analysis. Data are expressed as mean±SEM unless stated sy<0.05-P < 0.01, re- otherwise. For each subject and each test, pancreatic and biliary re- ...... ,,,?",,_ spectively). *, NaCI; sponses as well as plasma responses to all doses ofCCK8 or to - o -, 22 ,mol/kg per meal perfusion were calculated as area under the output curve (AUC) 0- -,--.----, h loxiglumide; over basal values for bicarbonate (millimoles), lipase (milligrams), 0 41 9S3 35 3 ... o * * , 7 umol/kg amylase (kallikrein-inhibiting units), bilirubin (milligrams), bile salts CCK I (pxullk-h) per h loxiglumide.

Role of Cholecystokinin in Meal-induced Pancreatic and Biliary Secretions 641 Table L Pancreatic Secretory Responses to Graded Doses 4 -1 -20 ofCCK8 With and Without Concomitant Infusion of Two Different Doses ofLoxiglumide Loxiglumide (,umol/kg per h) I T 0 (Control) 7 22 U

Bicarbonate (mmol) 36.4±3.4 15.7±6.3 10.8±3.6* E Amylase 41.3±8.0 23.3±9.0 12.7±3.8* -10 (MU) E Lipase (mg) 83.6±14.3 32.4±29.2 18.6±15.2* 0- Group means±SEM were calculated from individual AUC above LO- basal values to all doses of CCK8. n = 6, except lower dose of loxiglumide, where n = 4. * < with control (saline). P 0.01 compared K0 sponses to CCK8 produced by the higher dose of loxiglumide Saline Loxigiumide was essentially complete. Both doses of loxiglumide com- Figure 3. Effect of loxiglumide (22 gmol/kg per h) on fasting pancre- pletely antagonized the potentiating effect of CCK8 on the atic enzyme responses in'six healthy human subjects. Data are bicarbonate response to secretin (Fig. 1); In the three subjects mean±SEM. *P < 0.05. who received an additional dose of CCK8 (72 pmol/kg per h), we observed a diminished inhibition of pancreatic enzyme output during the lower dose of loxiglumide (7 pmol/kg per h), CCK8-induced PP release. CCK8 caused a dose-dependent suggesting that the inhibition caused by loxiglumide was sur- increase in plasma PP over basal concentrations (Table II). mountable. Loxiglumide produced a complete antagonization of the PP CCK8-induced bile acid secretion. Basal bile acid output response to CCK8 (P < 0.01). was very low and not affected by loxiglumide. Step-doses of Acid responses to pentagastrin with and without loxiglu- CCK8 induced increasing bile acid output with a maximal mide. Intravenous pentagastrin resulted in a dose-related sig- response obtained at a dose of 9.2 pmol/kg per h (Fig. 2). Both nificant increase in gastric acid output. The administration' of doses of loxiglumide completely blocked CCK8-stimulated 22 ,umol/kg per h loxiglumide did not significantly change the bile acid secretion (P < 0.01). acid response to pentagastrin (Fig. 6). Secretin-stimulated secretion. During infusion of saline Plasma CCK concentrations. Fasting plasma CCK levels (control), mean fasting pancreatic secretion was constant. The were comparable in the different experiments. Increasing infusion of loxiglumide for 75 min caused a marked decrease doses of CCK8 produced a dose-dependent increase in plasma in enzyme output, but had no significant effect on fluid and CCK-like immunoreactivity, which was significant for the bicarbonate secretion (Fig. 3). Graded doses of secretin in- lowest dose already (Table II). Loxiglumide did not affect duced a dose-dependent increase in fluid and bicarbonate se- plasma CCK concentrations in response to graded doses of cretion, which was not affected by loxiglumide (Fig. 4). exogenous CCK. Bombesin-stimulated secretion. Bombesin (1-27 pmol/kg per h) stimulated pancreatic enzyme and bicarbonate secretion Pharmacokinetics ofloxiglumide in a dose-dependent manner (Fig. 5). A dose of 22 ,umol/kg per Infusion of loxiglumide for 4.25 h at the rates of 7 and 22 h of loxiglumide did not affect this response. ,gmol/kg per h produced dose-dependent plasma concentra-

I Lemi.o ( k)w Saline (oI ) I- LszWui(22PmWkO) vSais utdru -I lW00

1I- a NaCI I I i Figure 2. Effect of lox- 50- NaCI\aCI\ iglumide (22 Imol/kg I a 3- M per h) on CCK8-stimu- s lated bile acid output in six healthy subjects. Figure 4. Effect of loxiglu- Data are mean±SE. mide (22 Atmol/kg per h) Mde Loxiglumide signifi- on secretin-stimulated pan- 0o I' cantly suppressed O- creatic bicarbonate secre- 0 41 U as 37 CCK8-stimulated bile tion in six healthy subjects. 0 7' CCK I (pmllkgh) acid output (P < 0.01). Secretinc (pnoiklk b Data are mean+SEM.

642 Hildebrand et al. 12 LBxiklumie (22pwlkj-h)or Salim(cutro) LMighNik(22piulIkgh) or Salims (atreil

12 Bicarbonate 0 6 ma

a

.5

20- -S6-a

Amylase i0m

asJ 0iZ

0 136 543 2179 Peutapstrin (pmdkg-h) Figure 6. Effect of loxiglumide (22 ,mol/kg per h) on gastric acid Lipase output stimulated by graded doses of pentagastrin in four healthy subjects. Data are mean±SEM. 1 Figure 5. Effect of lox- iglumide (22 tmol/kg per h) on incremental edly meal-stimulated pancreatic amylase and lipase outputs. pancreatic secretory re- The inhibition amounted to 63% for amylase and 43% for sponses to graded doses < The time 0- lipase output, respectively (P 0.05) (Table III). of bombesin in six is also in 7. healthy human subjects. course of bilirubin output given Fig. Loxiglumide I II -1 significantly reduced the intraduodenal bilirubin response by To 1 3 99 D Data are mean±SEM. Bombul. (pmulul.h) *, NaCl; o, loxiglumide. Wi s:demiO o~l tions reaching steady-state conditions after 60 min (data not shown). No half-life determinations were done in these 61ribin studies and no side effects were recorded during the experi- 12 - ments. IL I AFT N Meal experiments Bilir"Wiz~~ Pancreatic enzyme and biliary response to test meal. Pancre- 4.- atic and outputs increased immediately after amylase lipase % .9 . starting the meal perfusion (Fig. 7). Loxiglumide (22 >a~~~~v~ Atmol/kg per h), given as an intravenous infusion, antagonized mark- 12-

I3. Table II. Plasma CCK and PP Concentrations in Response to Graded Doses ofCCK8 With and Without a Concomitant Infusion ofLoxiglumide (22 ,.mol/kg per h)

CCK PP 3- Dose of CCK8 Control Loxiglumide Control Loxiglumide ng/kg per h pmol/liter 5. -

0 (Basal) 2.5±0.1 2.0±0.3 15.6±2.6 14.4±2.4 35 - 5 3.8±0.3 3.5±0.6 24.3±6.5 13.2±3.4 />4 Figure 7. Effect of lox-

10 4.5±0.3 4.6±0.5 29.0±3.2 12.6±1.6 -Z a f 4 iglumide (22 gmol/kg 20 7.9±0.6 7.1±0.7 36.1±8.5 11.4±1.8 C t - f T 1 per h) (o) on intraduo- 40 13.2±2.0 12.0±1.7 57.8±7.7 14.5±2.0 I/g denally meal-stimulated pancreatic and biliary responses in six healthy O- The average of two blood samples taken at the end of each infusion I,0 a, 3 ---It.2 subjects. Data are period was used to calculate group means and SEM; n = 6. (Ni) mean±SEM. *, NaCl.

Role ofCholecystokinin in Meal-induced Pancreatic and Biliary Secretions 643 Table III. Pancreatic and Biliary Secretory Responses release, and smooth muscle strip contractions (8-1 1). It is un- to Intraduodenal Test Meal Perfusion likely that loxiglumide acts on postreceptor mechanisms as it With and Without Loxiglumide in Healthy Human Subjects does not inhibit amylase release stimulated by agents such as calcium ionophore A-23187 and 12-0-tetradecanoylphorbol- Control (saline) Loxiglumide (22 ,mol/kg per h) 13-acetate in vitro, which bypass receptor mechanisms (5). In Bilirubin (mg) 39.3±11.9 16.1±7.0* vivo data in dog and demonstrate competitive-like kinetics Amylase (KIU) 40.8±3.4 15.0±3.1* of CCK-stimulated protein secretion (12, 25). Lipase (mg) 200±24 113±29* This is the first study to characterize the activity and selec- tivity of loxiglumide as an antagonist of CCK8 in healthy human volunteers. Here we have shown that CCK8-stimu- AUC (over 120 min) above basal values; n = 6. lated and secretions as well as * pancreatic enzyme biliary P < 0.05 compared with control (saline). plasma pancreatic polypeptide release can be antagonized in a dose-dependent manner, whereas pancreatic bicarbonate out- put stimulated by secretin, pancreatic enzyme responses to 59% (Table III, P < 0.05). Fluid secretion during meal perfu- bombesin, and pentagastrin-stimulated acid secretion were sion reached 308±65 ml/120 min in control experiments and unaffected by the compound. The antagonism was close to was not significantly different with the antagonist (379±127 in the dose range of CCK8. The CCK8 ml/ min). complete physiological 120 dose-response curve was gradually shifted to the right by in- Plasma CCK concentrations. Fasting plasma CCK levels kinetics. pmol/liter for experiments both with and with- creasing loxiglumide, indicating competitive-like were 1.8±0.1 These data suggest that the is a out of the meal into the duodenum compound specific antagonist loxiglumide. Perfusion of CCK in man interacting competitively with CCK receptors evoked a prompt and significant increase of plasma CCK con- in the exocrine and endocrine pancreas and in the Loxiglumide significantly augmented the CCK . centrations. CCK has a variety of biological effects upon the gastroin- response to intraduodenal meal stimulation (AUC with loxi- testinal tract. There is evidence that CCK with control; good circulating glumide was roughly four times higher compared accounts for a major fraction of postprandial pancreatic en- P < 0.05) (Fig. 8). zyme secretion and gallbladder contraction in man and ani- Plasma PP concentrations. Loxiglumide did not affect un- mals (1). Furthermore, physiological effects on gastrointestinal stimulated plasma PP concentrations. Duodenal perfusion of motility have been suggested (1, 6, 7). These conclusions are the test meal caused a significant increase in PP response based on animal data obtained with other CCK = antagonists (AUC 4,431±1,583 pM- 120 min, P < 0.05) during saline and on indirect evidence obtained in humans. We have re- < perfusion. Loxiglumide completely suppressed (P 0.05) this cently demonstrated that administration of loxiglumide com- response (AUC = 614±582, P < 0.05). pletely inhibits postprandial gallbladder contraction in healthy human subjects, thus indicating that endogenously released Discussion CCK is a major regulator of the postprandial gallbladder re- sponse in humans (26). Loxiglumide has been characterized as a new potent antago- The fundamental with regard to the have demonstrated questions physiologi- nist of CCK (8-12). In vitro studies higher cal role of CCK as a regulator of postprandial pancreatic en- affinity of loxiglumide for peripheral CCK receptors than for zyme and biliary secretions have recently been summarized by brain CCK receptors and competitive antagonization of Solomon (1): (a) Are plasma CCK levels increased after a CCK8-stimulated gallbladder contraction, pancreatic enzyme meal? (b) What proportion of the postprandial enzyme secre- tion is due to elevated plasma CCK? (c) Do other factors in- teract with CCK to augment its effect on $31,ei I traduedenal meal perfusion secretion? The present study shows that a significant increase in 15 plasma CCK is found after perfusion of a test meal to the duodenum, confirming several previous reports with oral food of the Loxiglumlde intake (4, 27-29). The magnitude postprandial CCK g - release amounted to 2-3 pmol/liter, which is in the same range I~~~~~~ previously reported for oral meals (4, 27-29). Comparable U plasma CCK-like concentrations were found after infusion of -3 CCK8 in doses < 18.5 pmol/kg per h. These doses of CCK8 E I produced an increase of pancreatic amylase and lipase output, I I J NaCI respectively, therefore indicating that postprandial concentra- 3- II 1 ' tions of CCK-like immunoreactivity are able to stimulate exo- crine pancreatic secretion. Pretreatment with the CCK antago- _I nist loxiglumide inhibited the pancreatic enzyme responses to 0 - food stimulation by 43-63% and bilirubin output by 59%. -30 0 30 60 90 120 Taken together, these findings suggest that the amount ofCCK (min) released postprandially is sufficient to account for a substantial Figure 8. Plasma CCK concentrations after intraduodenal meal per- number 'of the pancreatic enzyme and biliary responses to fusion with and without intravenous loxiglumide (22 gmol/kg per h) intestinal meal stimulation. We interpret these data as direct in six healthy subjects. Data are mean±SEM. proof that circulating CCK is a major stimulatory pathway of

644 Hildebrand et al. the intestinal phase of exocrine pancreatic and biliary re- pancreatic exocrine response to intraluminal amino acids and fat. Am. sponses and thus a true hormone. J. Physiol. 248:G347-G352. Pronounced inhibitory effects have been described after 4. Beglinger, C., M. Fried, I. Whitehouse, J. B. Jansen, C. B. Lamers, and K Gyr. 1985. Pancreatic enzyme response to a liquid on atropine administration or vagotomy exocrine pancreatic meal and to hormonal stimulation. Correlation with plasma secretin secretory responses, but not on plasma CCK release, suggest- and cholecystokinin levels. J. Clin. Invest. 75:1471-1476. ing a major role for direct cholinergic stimulation ofpancreatic 5. Jensen, R. T., R. B. Murphy, M. Trampota, L. H. Schneider, enzyme secretion during meal stimulation (30). The present S. W. Jones, J. M. Howard, and J. D. Gardner. 1985. data are not a contradiction to these observations, but are analogues: potent cholecystokinin receptor antagonists. Am. J. Phys- compatible with these latter results: a sustained secretory re- iol. 249:G214-G220. sponse remained after administration of the CCK antagonist 6. Chang, R. S. L., V. J. Lotti, R. L. Monaghan, J. Birnbaum, E. 0. loxiglumide, although all endogenous CCK should have been Stapley, M. A. Goetz, G. Albers-Schonberg, A. A. Patchett, J. M. blocked in the study by loxiglumide. We take the residual Liesch, 0. D. Hensens, and J. P. Springer. 1985. A potent nonpeptide pancreatic secretory response in response to intestinal meal cholecystokinin antagonist selective for peripheral tissues isolated in the blocker as evidence for from Aspergillus alliaceus. Science (Wash. DC). 230:177-179. stimulation observed experiment 7. Lotti, V. J., R. G. Pendleton, R. J. Gould, H. M. Hanson, R. L. S. the existence of other stimulatory mechanisms such as cholin- Chang, and B. V. Clineschmidt. 1987. In vivo pharmacology of ergic stimulation. L-364,718, a new potent nonpeptide peripheral cholecystokinin antag- This study revealed that plasma CCK levels were increased onist. J. Pharmacol. Exp. Ther. 241:103-109. three- to fourfold in the meal experiment with concomitant 8. Makovec, F., R. Chiste, M. Bani, L. Revel, I. Setnikar, and L. A. loxiglumide administration. Owyang and co-workers have Rovati. 1986. New glutamic and aspartic derivatives with potent shown that CCK release is regulated by intraduodenal trypsin, CCK-antagonist activity. Eur. J. Med. Chem. 21:9-20. suggesting that feedback control of pancreatic enzyme secre- 9. Makovec, F., M. Bani, R. Chist6, L. Revel, L. C. Rovati, and tion is operative in man (30). Thus, the reduced pancreatic L. A. Rovati. 1986. Differentiation of central and peripheral cholecys- enzyme response observed in the blocker experiment could tokinin receptors by new glutaramic acid derivatives with cholecysto- positive feedback con- -antagonistic activity. Arzneim. Forsch. 36:98-102. induce stimulation of CCK through a 10. Jensen, R. T., Z. C. Zhou, R. B. Murphy, S. W. Jones, I. trol. Setnikar, L. A. Rovati, and J. D. Gardner. Structural features of var- The cholinergic mediation of PP release and its inhibition ious proglumide-related cholecystokinin receptor antagonists. Am. J. by atropine are well established (31, 32). Here, we demonstrate Physiol. 251:G839-G846. that loxiglumide nearly abolished intestinal phase-stimulated 11. Setnikar, I., M. Bani, R. Cereda, R. Chist6, F. Makovec, M. A. plasma PP release. These findings extend the previous obser- Pacini, and L. Revel. 1987. Anticholecystokinin activities of loxiglu- vations with atropine (31, 32) and suggest that both the cho- mide. Arzneim. Forsch. 37:1168-1171. linergic and the peptidergic (CCK) system are required for a 12. Hildebrand, P., C. Beglinger, E. Kohler, I. Setnikar, and K. Gyr. regular PP response to intestinal phase stimulation. 1987. Biological effects of a proglumide derivative as cholecystokinin Several questions remain unanswered in this study. What antagonist in conscious dogs. Regul. Pept. 18:213-220. of CCK to the cephalic and gastric phase of 13. Regan, P. T., V. L. W. Go, and E. P. DiMagno. 1980. Compari- is the contribution son of the effects of cholecystokinin and cholecystokinin octapeptide pancreatic secretion? Do other factors interact with CCK to on pancreatic secretion, gallbladder contraction and plasma pancreatic augment its effect on pancreatic enzyme secretion during the polypeptide in man. J. Lab. Clin. Med. 96:743-748. various postprandial phases? Which molecular form ofCCK is 14. Beglinger, C., E. Kohler, G. A. Stalder, J. Jansen, and K. Gyr. predominant after meal stimulation? More information is 1986. What is the maximal effective dose of caerulein in stimulating clearly required to answer these questions. pancreatic secretion in man? . 35:125-128. In conclusion, the results of the present study support the 15. Lagerlof, H. 0. 1942. Pancreatic function and pancreatic dis- concept that cholecystokinin plays a physiological role in the ease studied by means of secretin. Acta Med. Scand. Suppl. 128:1-289. regulation of the intestinal phase of human pancreatic enzyme 16. Rick, W., and H. Stegbauer. 1968. Zur Bestimmung der Amy- and biliary secretions after meal stimulation. We conclude that laseaktivitiat im Ham. Fresenius Z. Anal. Chem. 243:415-416. CCK is an important in human di- 17. Grenner, G., G. Deutsch, R. Schmidtberger, and F. Dati. 1982. Hochempfindlicher Enzymimmunoassay zur Bestimmung von gestive physiology. Human-Pankreas-Lipase. J. Clin. Chem. Clin. Biochem. 20:515-519. 18. Talalay, P. 1960. Enzymatic analysis of steroid . Methods Biochem. Anal. 8:119-143. 19. Schellong, G., and U. Wende. 1960. A technic for standardiza- We thank Miss Sylvia Ketterer for expert technical assistance and Mrs. tion of methods in serum bilirubin determination. Klin. Wochenschr. Carita Frei for editorial assistance and typing of the manuscript. 38:703-707. The study was supported in part by grants from the Swiss National 20. Hyden, S. 1955. A turbidimetric method for the determination Science Foundation (3.866-0.85 and 3.890-0.88). of higher polyethylene glycols in biological materials. Kungliga Lant- brukshogskolans Annaler 22:139-145. 21. Jansen, J. B. M. J., and C. B. H. W. Lamers. 1983. Radioimmu- References noassay of cholecystokinin in human tissue and plasma. Clin. Chim. Acta. 131:305-316. 1. Solomon, T. E. 1987. Control of exocrine pancreatic secretion. 22. Hopman, W. P. M., P. J. S. M. Kerstens, J. B. M. J. Jansen, G. In Physiology of the . L. R. Johnson, editor. 2nd Rosenbusch, and C. B. H. W. Lamers. 1985. Effect of graded physio- ed. Raven Press, New York. 1173-1207. logic doses of cholecystokinin on gallbladder contraction measured by 2. Loewe, C. J., J. R. Grider, J. Gardiner, and Z. R. Vlahcevic. ultrasonography: determination of threshold, dose-response relation- 1985. Selective inhibition of pentagastrin- and cholecystokinin-stimu- ships and comparison with intraduodenal bilirubin output. Gastroen- lated exocrine secretion by proglumide. Gastroenterology. 89:746-751. terology. 89:1242-1247. 3. Stubbs, R. S., and B. E. Stabile. 1985. Role of cholecystokinin in 23. Meyer, F. D., K. Gyr, W. H. HAcki, C. Beglinger, L. Jeker, L.

Role of Cholecystokinin in Meal-induced Pancreatic and Biliary Secretions 645 Varga, L. Kayasseh, D. Gillessen, and G. A. Stalder. 1981. The release P. N. Maton. 1985. Human pancreatic and biliary responses to physio- of pancreatic polypeptide by CCK-octapeptide and some analogues in logical concentrations of cholecystokinin octapeptide. Clin. Sci. the dog. Gastroenterology. 80:742-747. 69:259-263. 24. Setnikar, I., R. Chist6, F. Makovec, L. C. Rovati, and S. J. 29. Liddle, R. A., I. D. Goldfine, M. S. Rosen, R. A. Taplitz, and Warrington. 1988. Pharmacokinetics of loxiglumide after single intra- J. A. Williams. 1985. Cholecystokinin bioactivity in human plasma. venous or oral doses in man. Arzneim. Forsch. 38:716-720. Molecular forms, responses to feeding, and relationship to gallbladder 25. Niederau, M., C. Niederau, G. Strohmeyer, and J. H. Grendell. contraction. J. Clin. Invest. 75:1144-1152. 1989. Comparative effects of CCK receptor antagonists on rat pancre- 30. Owyang, C., D. May, and D. S. Louie. 1986. Trypsin suppres- atic secretion in vivo. Am. J. Physiol. 256:GI50-G 157. sion of pancreatic enzyme secretion. Differential effect on cholecysto- 26. Meyer, B. M., B. A. Werth, C. Beglinger, P. Hildebrand, kinin release and the enteropancreatic reflex. Gastroenterology. J. B. M. J. Jansen, D. Zach, L. Rovati, and G. A. Stalder. 1989. A 91:637-643. physiologic role for cholecystokinin as a regulator of gastrointestinal 31. Adrian, T., H. Besterman, and S. Bloom. 1979. The importance motor functions in man. Lancet. ii: 12-15. of cholinergic tone in the release of pancreatic polypeptide by gut 27. Byrnes, D. J., L. Henderson, T. Borody, and J. F. Rehfeld. hormones in man. Life Sci. 24:1989-1994. 1981. Radioimmunoassay of cholecystokinin in human plasma. Clin. 32. Taylor, I., M. Feldman, C. Richardson, and J. Walsh. 1978. Chim. Acta. 111:81-89. Gastric and cephalic stimulation of human pancreatic polypeptide 28. Anagnostides, A. A., V. S. Chadwick, A. C. Selden, J. Barr, and release. Gastroenterology. 75:432-437.

646 Hildebrand et al.