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Effect of the specific cholecystokinin-receptor antagonist loxiglumide on bombesin stimulated pancreatic enzyme secretion in man
Regulatory Peptides, 32 (1991) 361-368
361
Elsevier REGPEP 01010
Effect of the specific cholecystokinin-receptor antagonist loxiglumide on bombesin stimulated pancreatic enzyme secretion in man M . C . W . Jebbink, J . B , M . J . Jansen, D . M . Mooy, L.C. Rovati t, C. B. H . W . Lamers Department of Gastroenterology and Hepatology. University Hospital, Leiden (The Netherlands) and IRotta Laboratories, Monza (Italy)
(Received 19July 1990; revisedversionreceivedand accepted22 November 1990) K e y words." CCK receptor antagonist; Loxiglumide; Cholecystokinin; Bombesin;
Pancreatic enzyme secretion
Summary We have investigated the effects of the specific cholecystokinin (CCK) receptor antagonist loxiglumide on basal and bombesin stimulated pancreatic enzyme secretion, bilirubin output and plasma CCK release in six healthy subjects. The data were compared with those obtained in control experiments where saline was infused instead of loxiglumide. Basal amylase output (4.7 + 0.8kU/45min), trypsin output (2.9 + 0.8 kU/45 min) and bilirubin output (7.7 + 2.8 mmol/45 min) gradually declined during infusion of loxiglumide to values of 1.3 + 0.3 kU/45 min, 0.5 + 0.1 kU/45 min and 0.4 + 0.0 mmol/45 min, respectively, reaching statistical significance (P < 0.05) in the 30 to 45-min period after the start of the loxiglumide infusion. In the control experiments saline infusion failed to influence basal amylase, trypsine and bilirubin output, while bombesin stimulated amylase output from 4.7 + 0.8 kU/45 min to 25.1 + 5.1kU/45min (P<0.05), trypsin output from 2.9 + 0.8kU/45min to 11.6 + 2.0 kU/45 min (P < 0.05) and bilirubin output from 7.7 + 2.8 mmol/45 min to 68.0 + 16.0 mmol/45 min (P < 0.05). Loxiglumide failed to significantly influence bombesin stimulated amylase output (36.7 + 9.0 kU/45 min) and trypsin output (8.3 + 2.9 kU/45 min), but almpst abolished bilirubin output (9.7 + 3.6 mmol/45 min) (P < 0.05). Basal plasma CCK (2.4 + 0.1 pM) was not significantly influenced by loxiglumide (2.4 + 0.2 pM). Against a background infusion of saline bombesin signifiCorrespondence: J.B.M.J. Jansen, Dept. of Gastroenterology and Hepatology, University Hospital, Building 1, C4-p, Rijnsburgerweg10,12300 RC Leiden,The Netherlands.
0167-0115/91/$03.50 © 1991 Elsevier Science Publishers B.V. (BiomedicalDivision)
362
cantly stimulated plasma CCK levels at all time points (P < 0.01) with a gradual decline after the first 15-min sample. Loxiglumide significantly (P < 0.05) augmented bombesin stimulated plasma CCK at all time intervals without such a decline. These data demonstrate that: (1) basal pancreatic enzyme secretion and gallbladder motility are inhibited by loxiglumide, (2) bombesin-stimulated gallbladder contraction, but not pancreatic enzyme secretion is inhibited by loxiglumide, (3)bombesin-stimulated CCK release is augmented by loxiglumide, which could not be explained by inhibition of pancreatic enzyme secretion.
Introduction
In previous studies, we and others have demonstrated that the specific cholecystokinin receptor antagonist loxiglumide (CR-1505) [ 1], abolishes gallbladder contraction in response to a meal [2-4] and to bombesin (BBS) [5]. In addition, plasma cholecystokinin (CCK) concentrations in response to these stimuli are augmented by loxiglumide [2,4,6]. This CCK-increasing effect of loxiglumide cannot be explained by impaired clearance of CCK during loxiglumide infusion [7]. In the present study, we have investigated the effects of loxiglumide on basal and BB S stimulated pancreatic exocrine secretion, bilirubin output into the duodenum, and plasma CCK release in healthy subjects.
Materials and Methods
Six healthy subjects (4male, 2 female, mean age + S.E.M.: 24 + 3 years) were studied on two occasions separated by at least 1 week. The order of the two experiments was randomized. After an overnight fast of at least 12 h, the volunteers presented at the laboratory, where two indwelling intravenous catheters were placed one into each forearm. The catheters were kept patent by a saline infusion. One catheter was used for collection of blood samples, while bombesin, saline or loxiglumide were infused through the other. The subjects were also intubated with a polyvinyl triple-lumen tube. Aspiration ports were in the stomach and in the duodenum just proximal to the ligament of Treitz, while a perfusion port for infusion of a non-absorbable marker was at the papilla of Vater. After the position of the tube was verified by fluoroscopy, the duodenum at the papilla of Vater was continuously perfused with a volume marker (PEG-4000, 15 mg/ml) dissolved in saline at a perfusion rate of 3 ml/min. Studies were started after a 1 h equilibration period. Gastric contents were continuously aspirated using a suction pump which provided intermittent negative pressure. Duodenal aspiration samples, for determination ofbilirubin, amylase and trypsin outputs, were collected every 15 min as previously described [ 15]. In brief, from the aspiration port at the ligament of Treitz, 3 to 4 ml duodenal contents were recovered by hand every 15 min throughout the study and collected into vials immersed on ice. Before and after duodenal juice was aspirated,
363
the tube was flushed with 3 rnl of air to guarantee fresh duodenal samples. Blood samples were obtained at 15-rain intervals and collected in glass tubes containing EDTA. After an equilibration period and a 45 min basal period during which unstimulated duodenal samples and blood were collected, saline or loxiglumide (15 mg/kg/h for 10 min, followed by 10 mg/kg/h for 110 min) were infused for 2 h. 60 min after the start of saline or loxiglumide infusion, BBS was administered intravenously in a dose of 150 ng/kg/h during the last 60 rain of the experiments. Plasma CCK was determined by radioimmunoassay employing antibody T204, which is directed to the sulfated tyrosine region and binds to CCK 8S and larger bioactive molecular forms [ 8,911. The detection limit of the assay is 0.5 pM. Intra-assay variations are below 8Yo. All samples were measured in duplicate in one assay. Integrated plasma CCK concentrations were calculated as area under the curves (AUC's) after subtraction of basal CCK levels. Bilirubin concentrations, trypsin and amylase activity were measured in duodenal aspirates as previously described [ 10-12]. Concentrations of P E G were measured [ 13 ] and, after correction used to calculate intraduodenal volume flow rates and delivery rates of enzymes and bilirubin to the duodenal aspiration site as described earlier [14]. The studies were approved by the local ethical committee and all subjects gave informed consent. Statistical analysis. Data are expressed as mean + S.E.M. Wilcoxon's test for paired results was used to assess differences between medians. Differences with a probability value of less than 0.05 were considered to be significant.
Results
Basal pancreatic amylase output (4.7 + 0 . 8 k U / 4 5 m i n ) and trypsin output (2.9 + 0.8 kU/45 min) were significantly (P < 0.05) stimulated by bombesin infusion to values of 25.1 + 5.1 kU/45 min and 11.6 + 2.0 kU/45 min, respectively (Table I). Data
TABLE I Integrated bilirubin, amylase, trypsin and C C K output during the last 45 min of intravenous infusion of saline or loxiglumide (CR-1505) under unstimulated and BB S stimulated conditions in six healthy subjects Asterisks denote statistically significar~t differences from basal values during saline infusion (* P < 0.05) and triangles denote significant difference~ between BBS infusion with and without loxiglumide (AP < 0.05). Output
Bilirubin (mmol/45 min) Amylase (kU/45 min) Trypsin (kU/45 min) C C K (pM/45 min)
Basal
Bombesin
Saline
CR- 1505
Saline
7 . 7i+- 2.8 4 . 7 + 0.8 2 . 9 + 0.8 119.0i_+ 8.6
0.4 1.3 0.5 113.3
68.0 25.1 11.6 260.9
+_ 0.0" + 0.3 + 0.1 + 8.3
CR- 1505 + + + +
16.4" 5.1" 2.0* 38.2*
9.7 36.7 8.3 781.3
+ + + +
3.2 A 9.0* 2.9* 46.2 *z~
364
Amylase
(kU/15mm) 2l, 22 20 18 16 14 12 10
I I
Trypsin IkU/1Smln)
Saline ~ ]
I I
Saline
I
A
8itirubin
I mtool/15 32 28
I
rain)
I
Sa(ine
C
2/* 20
zx ,~
16"
86&.
12'
2-
z,.
8" -30
Amytase (kU/15 rain)
0 ~j
60
120 min
Loxiglumide
2/, I 22201816%.
[
Trypsin-30 (kU/15mln)
0
120 rain
60
[
I
_j L°xiglurnide
~Lirubirl -30
Immol/15min } 32:
0
~j
60
120 ~ln
]
Loxi~lumide
28.
I
2420-
z~
~210 1
1612-
rr~..;,~ -30
0
60
8-
o o
z,-
L
120Lmtn
TITIT -30
0
60
120 mm
-30
0
60
120 mln
Fig. 1. Pancreatic amylase output (left panel), pancreatic trypsin output (middle panel) and bilirubin output (right panel) in response to BBS infusion ( 1 5 0 ng/kg/h) against a background infusion of saline (upper panel) or loxiglumide (lower panel) in six healthy volunteers. Data are expressed as mean + S . E . M . For details see Materials and Methods. Loxiglumide significantly (*P < 0 . 0 5 ) suppressed basal amylase, trypsin and bilirubin output. Bombesin significantly ( z x p < 0 . 0 5 ) stimulated pancreatic amylase, trypsin and bilirubin secretion into the duodenum. Loxiglumide failed to suppress BBS-stimulated amylase and trypsin output, but significantly inhibited ( o = P < 0 . 0 5 ) BBS-stimulated bilirubin output.
obtained in divided 15-min samples are shown in Fig. 1. Infusion ofloxiglumide resulted in decreases in both amylase output from 4.7 + 0.8 kU/45 min to 1.3 + 0.3 kU/45 min, and in trypsin output from 2.9 + 0.8kU/45 min to 0.5 + 0.1 kU/45 min, reaching statistical significance in the 30- to 45-min period during loxiglumide infusion under unstimulated conditions (P < 0.05). Bombesin infusion against a background infusion of loxiglumide significantly stimulated basal amylase output from 1.3 + 0.3 kU/45 min to 36.7 + 9.0kU/45 min and trypsin output from 0.5 + 0.1 kU/45 min to 8.3 + 2.9 kU/45 min (Table I and Fig. 1 ; P < 0.05). These values were not significantly different from the corresponding values during bombesin infusion against a saline background. Bombesin infusion against a saline background significantly (P < 0.05) increased duodenal bilirubin output from 7.7 + 2.8 mmol/45 rain to 68.0 + 16.0 mmol/45 rain. Loxiglumide infusion significantly (P < 0.05) decreased basal bilirubin output from 7.7 + 2.8 mmol/45 min to 0.4 + 0.0 mmol/45 min (Table I and Fig. 1), while bombesin infusion during loxiglumide administration significantly (P < 0.05) inhibited bilirubin output from 68.0 + 16.4 mmol/45 min to 9.7 + 3.6 mmol/45 min, values not significantly different from pre-infusion outputs. Basal plasma CCK concentrations before infusion of loxiglumide or saline were 2.4 + 0.1 pM and 2.7 + 0.4 pM, respectively. Loxiglumide did not significantly
365
~j Loxigtumide (o) or Satine ( • ) plasma CCK
I Bombesin
I l
(pM) 20~ IB. 16 % 12 10 8
2- ~
-~s
"0"* ~
-is b Ys 3o ~:s ~ #s ~ Ibs 12o'~io
Fig. 2. Basal and BBS (150 ng/kg/h)-stimulated plasma CCK concentrations (pM; mean _+ S.E.M.) in six healthy subjects during intravenous saline (closed circles) or loxiglumide infusion (open circles). For details see Materials and Methods. Asterisks denote statistically significant differences from basal values (**P < 0.01), triangles denote significant differences between CCK values obtained during BB S stimulation with and without loxiglumide administ~'ation ( A p < 0.05; • A p < 0.01), and circles denote progressive significant (O = P < 0.05; O O = P < 0.01) decrease, compared to the first 15-min sample during BBS infusion alone, which does not occur during concomitant loxiglumide infusion.
influence basal plasma CCK levels (2.4 + 0.2 pM) (Fig. 2). Bombesin infusion significantly (P < 0.05 to P < 0.01) increased plasma CCK values over basal values at all time intervals measured. However, during bombesin infusion there was a significant decline in plasma CCK values in time (P < 0.05), when compared to the first 15-min sample obtained after the bombesin infusion was started (Fig. 2). Loxiglumide significantly (P < 0.05) augmented the plasma CCK response to bombesin at all time intervals measured. In addition, no decrease in CCK response in time was observed during bombesin infusion against a loxiglumide infusion (Fig. 2).
Discussion
At least four interesting findings can be derived from this study. Firstly, the specific CCK receptor antagonist loxiglumide inhibited both basal pancreatic enzyme secretion and bilirubin output. Secondly, loxiglumide almost abolished bilirubin output into the duodenum during BB S infusion into man. Thirdly, loxiglumide had no significant effect on BB S stimulated pancreatic enzyme output into the duodenum. Fourthly, loxiglumide augmented BBS stimulated CCK release, which could not be explained by inhibition of pancreatic enzyme secretioni as suggested before [ 16]. In contrast to previous studies in other species [17,18], we have found that loxiglumide inhibited basal pancreatic enzyme output in man. This suggests that basal
366 plasma CCK levels are involved in maintaining both gall bladder volumes [5] and pancreatic enzyme outputs under unstimulated conditions. In a recent study, we have demonstrated that bilirubin output into the duodenum is highly correlated to gall bladder contraction measured by real-time ultrasonography [19]. Therefore, this study confirms our previous finding that BBS stimulated gallbladder contraction in man is almost abolished by the specific CCK receptor blocker loxiglumide [5]. This suggests that BBS has no major direct effects on gallbladder motility in man and exerts its effects through the release of CCK. Since BBS-receptors have been demonstrated on human pancreatic acinar cells [20], and since previous studies have clearly demonstrated that plasma CCK-levels comparable to those observed during BBS infusion potently stimulate pancreatic enzyme secretion [21], a concerted stimulatory action of direct and indirect (CCK) effects of BBS on pancreatic enzyme output into the duodenum was expected. Surprisingly, no significant inhibition of BBS-stimulated pancreatic amylase and trypsin output during CCK-receptor blockade was observed. This may suggest that loxiglumide, while inhibiting the effects of BBS-stimulated CCK on pancreatic exocrine secretion, augments the direct effects of BB S on pancreatic enzyme output. In vitro experiments in favor of this concept have been published [22,23]. These studies have demonstrated that after incubating dispersed acini from guinea-pig pancreas with CCK, subsequent stimulation of amylase secretion by pancreatic secretagogues whose actions are also mediated by cellular release of calcium, like BBS and carbamylcholine, but also CCK itself, is decreased. If CCK desensitization also occurs in man, its importance in respect to the present study might be that it attenuates the response of pancreatic acinar cells to the direct stimulatory effects of BBS. When this CCK mediated attenuation is blocked by loxiglumide, BBS may augment amylase output to values not significantly different from the values obtained during BBS infusion without CCK-receptor blockade. If so, CCKreceptor blockade cannot be employed to definitively demonstrate the relative contribution of CCK to pancreatic enzyme secretion during BBS infusion. This may probably also relate to meal-stimulation, since CCK also desensitizes the effect of cholinergic stimulation on pancreatic acinar cells [22]. This study also confirms our previous finding that loxiglumide augments BB S-stimulated CCK release [6]. Factors responsible for augmentation of CCK release during BBS infusion could be: (i) interference of loxiglumide in the metabolic clearance rate of CCK [24]. This factor was excluded in a previous study [7], demonstrating that loxiglumide did not interfere with plasma half-life of CCK in man. (ii) A direct effect of CCK on its release from duodenal I-cells is another possibility to explain the augmented CCK release during BBS infusion and loxiglumide administration. Indirect evidence against such an autocrine negative feedback mechanism was obtained from a recent study [25], demonstrating that infusion of synthetic CCK 8S was not able to inhibit endogenous CCK release in response to a meal. (iii) An indirect effect of CCK on its release from duodenal I-cells during BBS infusion is therefore the most likely explanation for the observed effect ofloxiglumide. Such an indirect effect may be related to interference ofloxiglumide with BB S-mediated release ofa CCK inhibiting substance, like somatostatin, or via inhibition of BBS stimulated bile acid secretion into the duodenum [26,27]. Anyhow, the augmented BBS-stimulated CCK release by loxi-
367
glumide observed in this study ~annot be explained by a negative feedback mechanism between CCK release and pancreatic exocrine secretion [ 16], since pancreatic exocrine secretion is not inhibited by loxiglumide during BBS infusion.
References 1 Setnikar, I., ChistG, R., Makovec, F.b Rovati, L. C. and Warrington S.J., Pharmacokinetics of loxiglumide after single intravenous or oral doses in man, Arzneimittelforschung/Drug Res., 38 (1988) 716-720. 2 Meyer, B. M., Beglinger, C., Jansen, J. B. M.J., Rovati, L. C., Werth, B. A., Hildebrand, P., Zach, D. and S talder G. A., Role of cholecystokinin in regulation of gastrointestinal motor functions, Lancet, ii (1989) 12-15. 3 Schmidt, W. E., Creutzfeldt, W., Schlcser, A., Roy Choudhury, A., Nitsche, R., Sostmann, H. and F01sch U. R., Acute and chronic effects of CCK receptor antagonist loxiglumide on pancreatic and biliodigestive functions in man, Digestion, 43 (1989) 170-171. 4 Hildebrand, P., Beglinger, C., Gyr, K., Jansen, J.B.M.J., Rovati, L.C., Zuercher, M., Lamers, C. B. H. W., Setnikar, I. and Stalder~ G. A., Effects of a cholecystokinin receptor antagonist on intestinal phase of pancreatic and biliary responses in man, J. Clin. Invest., 85 (1990) 640-646. 5 Douglas, B. R., Jebbink, M. C. W., Tjon a Tham, R. T. O., Jansen, J. B. M.J. and Lamers C. B. H. W., The effect of loxiglumide (CR-1505) on basal and bombesin stimulated gallbladder volume in man, Eur. J. Pharmacol., 166 (1989) 307-310. 6 Jansen, J.B.M.J., Jebbink, M.C.W., Douglas, B.R. and Lamers, C.B.H.W., Effect of loxiglumide (CR-1505) on bombesin and meal stimulated plasma cholecystokinin in man, Eur. J. Clin. Pharmacol., 38 (1990) 367-370. 7 Jebbink, M.C.W., Jansen, J. B. M.J., Masclee, A. A.M. and Lamers C.B.H.W., Lack of effect of the specific cholecystokinin receptor antagonist loxiglumide on cholecystokinin clearance from plasma in man, Br. J. Clin. Pharmacol., 29 (1990) 770-773. 8 Jansen, J.B.M.J. and Lamers C.B.H.W., Radioimmunoassay of cholecystokinin: production and evaluation of antibodies, J. Clin. Chem. Clin. Biochem., 21 (1983) 387-394. 9 Jansen, J.B.M.J. and Lamers C.B.H.W., Radioimmunoassay of cholecystokinin in human tissue and plasma, Clin. Chim. Acta, 131 (1983) 305-316. 10 Jendrassik, L. and GrofP., Vereinfachte photometrische Methoden zur Bestimmung des Blutbilirubins, Biochem. Z., 297 (1938) 81-89. 11 Hummel B.C., A modified spectrophotometric determination of cbymotrypsin, trypsin and thrombin, Can. J. Biochem., 37 (1955) 1393-1397. 12 Beinfeld P., Alpha amylase, Methods Enzymol., 1 (1955) 149-158. 13 Malawer, S.J. and Powe|l D.W., An improved turbidimetric analysis of polyethylene glycol utilizing an emulsifier, Gastroenterology, 53 (1967) 250-256. 14 Beglinger, C., Fried, M., Whitehouse, I., Jansen, J. B. M.J., Lamers, C. B. H. W. and Gyr, K., Pancreatic enzyme response to a liquid meal and to hormonal stimulation. Correlation with plasma secretin and cholecystokinin levels, J. Clin. Invest., 75 (1985) 1471-1476. 15 Layer, P., Jansen, J.B.M.J., Cherian, L. and Goebell H., Feedback regulation of human pancreatic secretion: Effects of protease inhibition on duodenal delivery and small intestinal transit of pancreatic enzymes, Gastroenterology, 98 (1990) 1311-1319. 16 Liddle, R. A., Goldfine, I. D., Rosen, M. S., Taplitz, R. A. and Williams J. A., Cholecystokinin bioactivity in human plasma. Molecular forms, responses to feeding, and relationship to gallbladder contraction, J. Clin. Invest., 75 (1985) 1142-1152. 17 Niederau, M., Niederau, C., Stroh~neyer, G. and Grendell J.H., Comparative effects of CCK receptor antagonists on rat pancreatic secretion in vivo, Am. J. Physiol., 256 (1989) G150-G157. 18 Lamers, C.B.H.W., De Jong, A.JiL., Singer, M.V., Niebel, W. and Jansen J.B.M.J., Role of cbolecystokinin in bombesin stimulated gastric acid and pancreatic enzyme secretion in dogs, Gastroenterology, 94 (1988) A249.
368 19 Hopman, W.P.M., Kerstens, P.J.S.M., Jansen, J.B.M.J., Rosenbusch, G. and Lamers C.B.H.W., Effect of graded physiologic doses of cholecystokinin on gallbladder contraction measured by ultrasonography. Determination of threshold, dose-response relationships and comparison with intraduodenal bilirubin output, Gastroenterology, 89 (1985) 1242-1247. 20 Scemama, J-L., Zahidi, Z., Fourmy, D., Fagot-Revurat, P., Vaysse, N., Pradayrol, L. and Ribet A., Interaction of (~25I)-Tyra-bombesin with specific receptors on normal human pancreatic membranes, Regal. Pept., 13 (1986) 125-132. 21 Kerstens, P.J.S.M., Lamers, C.B.H.W., Jansen, J.B.M.J., De Jong, A.J.L., Hessels, M. and Hafkenscheid J.C.M., Physiological plasma concentrations of cholecystokinin stimulate pancreatic enzyme secretion and gallbladder contraction in man, Life Sci., 36 (1985) 565-569. 22 Abdelmoumene, S. and Gardner J. D., Cholecystokinin-induced desensitization of enzyme secretion in dispersed acini form guinea-pig pancreas, Am. J. Physiol., 239 (1980) G272-G279. 23 Younes, M., Wank, S. A., Vinayek, R., Jensen, R. T. and Gardner J. D., Regulation ofbombesin receptors on pancreatic acini by cholecystokinin, Am. J. Physiol., 256 (1989) G291-G298. 24 Hosotani, R., Doi, R., Sumi, S., Gu, Y., Inoue, K., Ishikawa, T., Rayford, P.L. and Tobe, T., Effect of a receptor antagonist on the metabolism of cholecystokinin-33, Digestion, 46(S1) (1990) 46. 25 Jebbink, M. C. W., Jansen, J. B. M.J., Schouten, C. and Lamers, C. B. H. W., Effect of exogenous cholecystokinin octapeptide on meal stimulated plasma CCK release in man, Digestion, 46(3) (1990) 147. 26 Gomez, G., Upp, J.R., Lluis, F., Alexander, R.W., Poston, G.J., Greeley, G.H. and Thompson J.C., Regulation of the release of cholecystokinin by bile salts in dogs and humans, Gastroenterology, 94 (1988) 1036-1046. 27 Koop, I., Koop, H., Gerhardt, C., Schafmayer, A. and Arnold R., Do bile acids exert a negative feedback control of plasma cholecystokinin release, Scand. J. Gastroenterol., 24 (1989) 315-320.
361
Elsevier REGPEP 01010
Effect of the specific cholecystokinin-receptor antagonist loxiglumide on bombesin stimulated pancreatic enzyme secretion in man M . C . W . Jebbink, J . B , M . J . Jansen, D . M . Mooy, L.C. Rovati t, C. B. H . W . Lamers Department of Gastroenterology and Hepatology. University Hospital, Leiden (The Netherlands) and IRotta Laboratories, Monza (Italy)
(Received 19July 1990; revisedversionreceivedand accepted22 November 1990) K e y words." CCK receptor antagonist; Loxiglumide; Cholecystokinin; Bombesin;
Pancreatic enzyme secretion
Summary We have investigated the effects of the specific cholecystokinin (CCK) receptor antagonist loxiglumide on basal and bombesin stimulated pancreatic enzyme secretion, bilirubin output and plasma CCK release in six healthy subjects. The data were compared with those obtained in control experiments where saline was infused instead of loxiglumide. Basal amylase output (4.7 + 0.8kU/45min), trypsin output (2.9 + 0.8 kU/45 min) and bilirubin output (7.7 + 2.8 mmol/45 min) gradually declined during infusion of loxiglumide to values of 1.3 + 0.3 kU/45 min, 0.5 + 0.1 kU/45 min and 0.4 + 0.0 mmol/45 min, respectively, reaching statistical significance (P < 0.05) in the 30 to 45-min period after the start of the loxiglumide infusion. In the control experiments saline infusion failed to influence basal amylase, trypsine and bilirubin output, while bombesin stimulated amylase output from 4.7 + 0.8 kU/45 min to 25.1 + 5.1kU/45min (P<0.05), trypsin output from 2.9 + 0.8kU/45min to 11.6 + 2.0 kU/45 min (P < 0.05) and bilirubin output from 7.7 + 2.8 mmol/45 min to 68.0 + 16.0 mmol/45 min (P < 0.05). Loxiglumide failed to significantly influence bombesin stimulated amylase output (36.7 + 9.0 kU/45 min) and trypsin output (8.3 + 2.9 kU/45 min), but almpst abolished bilirubin output (9.7 + 3.6 mmol/45 min) (P < 0.05). Basal plasma CCK (2.4 + 0.1 pM) was not significantly influenced by loxiglumide (2.4 + 0.2 pM). Against a background infusion of saline bombesin signifiCorrespondence: J.B.M.J. Jansen, Dept. of Gastroenterology and Hepatology, University Hospital, Building 1, C4-p, Rijnsburgerweg10,12300 RC Leiden,The Netherlands.
0167-0115/91/$03.50 © 1991 Elsevier Science Publishers B.V. (BiomedicalDivision)
362
cantly stimulated plasma CCK levels at all time points (P < 0.01) with a gradual decline after the first 15-min sample. Loxiglumide significantly (P < 0.05) augmented bombesin stimulated plasma CCK at all time intervals without such a decline. These data demonstrate that: (1) basal pancreatic enzyme secretion and gallbladder motility are inhibited by loxiglumide, (2) bombesin-stimulated gallbladder contraction, but not pancreatic enzyme secretion is inhibited by loxiglumide, (3)bombesin-stimulated CCK release is augmented by loxiglumide, which could not be explained by inhibition of pancreatic enzyme secretion.
Introduction
In previous studies, we and others have demonstrated that the specific cholecystokinin receptor antagonist loxiglumide (CR-1505) [ 1], abolishes gallbladder contraction in response to a meal [2-4] and to bombesin (BBS) [5]. In addition, plasma cholecystokinin (CCK) concentrations in response to these stimuli are augmented by loxiglumide [2,4,6]. This CCK-increasing effect of loxiglumide cannot be explained by impaired clearance of CCK during loxiglumide infusion [7]. In the present study, we have investigated the effects of loxiglumide on basal and BB S stimulated pancreatic exocrine secretion, bilirubin output into the duodenum, and plasma CCK release in healthy subjects.
Materials and Methods
Six healthy subjects (4male, 2 female, mean age + S.E.M.: 24 + 3 years) were studied on two occasions separated by at least 1 week. The order of the two experiments was randomized. After an overnight fast of at least 12 h, the volunteers presented at the laboratory, where two indwelling intravenous catheters were placed one into each forearm. The catheters were kept patent by a saline infusion. One catheter was used for collection of blood samples, while bombesin, saline or loxiglumide were infused through the other. The subjects were also intubated with a polyvinyl triple-lumen tube. Aspiration ports were in the stomach and in the duodenum just proximal to the ligament of Treitz, while a perfusion port for infusion of a non-absorbable marker was at the papilla of Vater. After the position of the tube was verified by fluoroscopy, the duodenum at the papilla of Vater was continuously perfused with a volume marker (PEG-4000, 15 mg/ml) dissolved in saline at a perfusion rate of 3 ml/min. Studies were started after a 1 h equilibration period. Gastric contents were continuously aspirated using a suction pump which provided intermittent negative pressure. Duodenal aspiration samples, for determination ofbilirubin, amylase and trypsin outputs, were collected every 15 min as previously described [ 15]. In brief, from the aspiration port at the ligament of Treitz, 3 to 4 ml duodenal contents were recovered by hand every 15 min throughout the study and collected into vials immersed on ice. Before and after duodenal juice was aspirated,
363
the tube was flushed with 3 rnl of air to guarantee fresh duodenal samples. Blood samples were obtained at 15-rain intervals and collected in glass tubes containing EDTA. After an equilibration period and a 45 min basal period during which unstimulated duodenal samples and blood were collected, saline or loxiglumide (15 mg/kg/h for 10 min, followed by 10 mg/kg/h for 110 min) were infused for 2 h. 60 min after the start of saline or loxiglumide infusion, BBS was administered intravenously in a dose of 150 ng/kg/h during the last 60 rain of the experiments. Plasma CCK was determined by radioimmunoassay employing antibody T204, which is directed to the sulfated tyrosine region and binds to CCK 8S and larger bioactive molecular forms [ 8,911. The detection limit of the assay is 0.5 pM. Intra-assay variations are below 8Yo. All samples were measured in duplicate in one assay. Integrated plasma CCK concentrations were calculated as area under the curves (AUC's) after subtraction of basal CCK levels. Bilirubin concentrations, trypsin and amylase activity were measured in duodenal aspirates as previously described [ 10-12]. Concentrations of P E G were measured [ 13 ] and, after correction used to calculate intraduodenal volume flow rates and delivery rates of enzymes and bilirubin to the duodenal aspiration site as described earlier [14]. The studies were approved by the local ethical committee and all subjects gave informed consent. Statistical analysis. Data are expressed as mean + S.E.M. Wilcoxon's test for paired results was used to assess differences between medians. Differences with a probability value of less than 0.05 were considered to be significant.
Results
Basal pancreatic amylase output (4.7 + 0 . 8 k U / 4 5 m i n ) and trypsin output (2.9 + 0.8 kU/45 min) were significantly (P < 0.05) stimulated by bombesin infusion to values of 25.1 + 5.1 kU/45 min and 11.6 + 2.0 kU/45 min, respectively (Table I). Data
TABLE I Integrated bilirubin, amylase, trypsin and C C K output during the last 45 min of intravenous infusion of saline or loxiglumide (CR-1505) under unstimulated and BB S stimulated conditions in six healthy subjects Asterisks denote statistically significar~t differences from basal values during saline infusion (* P < 0.05) and triangles denote significant difference~ between BBS infusion with and without loxiglumide (AP < 0.05). Output
Bilirubin (mmol/45 min) Amylase (kU/45 min) Trypsin (kU/45 min) C C K (pM/45 min)
Basal
Bombesin
Saline
CR- 1505
Saline
7 . 7i+- 2.8 4 . 7 + 0.8 2 . 9 + 0.8 119.0i_+ 8.6
0.4 1.3 0.5 113.3
68.0 25.1 11.6 260.9
+_ 0.0" + 0.3 + 0.1 + 8.3
CR- 1505 + + + +
16.4" 5.1" 2.0* 38.2*
9.7 36.7 8.3 781.3
+ + + +
3.2 A 9.0* 2.9* 46.2 *z~
364
Amylase
(kU/15mm) 2l, 22 20 18 16 14 12 10
I I
Trypsin IkU/1Smln)
Saline ~ ]
I I
Saline
I
A
8itirubin
I mtool/15 32 28
I
rain)
I
Sa(ine
C
2/* 20
zx ,~
16"
86&.
12'
2-
z,.
8" -30
Amytase (kU/15 rain)
0 ~j
60
120 min
Loxiglumide
2/, I 22201816%.
[
Trypsin-30 (kU/15mln)
0
120 rain
60
[
I
_j L°xiglurnide
~Lirubirl -30
Immol/15min } 32:
0
~j
60
120 ~ln
]
Loxi~lumide
28.
I
2420-
z~
~210 1
1612-
rr~..;,~ -30
0
60
8-
o o
z,-
L
120Lmtn
TITIT -30
0
60
120 mm
-30
0
60
120 mln
Fig. 1. Pancreatic amylase output (left panel), pancreatic trypsin output (middle panel) and bilirubin output (right panel) in response to BBS infusion ( 1 5 0 ng/kg/h) against a background infusion of saline (upper panel) or loxiglumide (lower panel) in six healthy volunteers. Data are expressed as mean + S . E . M . For details see Materials and Methods. Loxiglumide significantly (*P < 0 . 0 5 ) suppressed basal amylase, trypsin and bilirubin output. Bombesin significantly ( z x p < 0 . 0 5 ) stimulated pancreatic amylase, trypsin and bilirubin secretion into the duodenum. Loxiglumide failed to suppress BBS-stimulated amylase and trypsin output, but significantly inhibited ( o = P < 0 . 0 5 ) BBS-stimulated bilirubin output.
obtained in divided 15-min samples are shown in Fig. 1. Infusion ofloxiglumide resulted in decreases in both amylase output from 4.7 + 0.8 kU/45 min to 1.3 + 0.3 kU/45 min, and in trypsin output from 2.9 + 0.8kU/45 min to 0.5 + 0.1 kU/45 min, reaching statistical significance in the 30- to 45-min period during loxiglumide infusion under unstimulated conditions (P < 0.05). Bombesin infusion against a background infusion of loxiglumide significantly stimulated basal amylase output from 1.3 + 0.3 kU/45 min to 36.7 + 9.0kU/45 min and trypsin output from 0.5 + 0.1 kU/45 min to 8.3 + 2.9 kU/45 min (Table I and Fig. 1 ; P < 0.05). These values were not significantly different from the corresponding values during bombesin infusion against a saline background. Bombesin infusion against a saline background significantly (P < 0.05) increased duodenal bilirubin output from 7.7 + 2.8 mmol/45 rain to 68.0 + 16.0 mmol/45 rain. Loxiglumide infusion significantly (P < 0.05) decreased basal bilirubin output from 7.7 + 2.8 mmol/45 min to 0.4 + 0.0 mmol/45 min (Table I and Fig. 1), while bombesin infusion during loxiglumide administration significantly (P < 0.05) inhibited bilirubin output from 68.0 + 16.4 mmol/45 min to 9.7 + 3.6 mmol/45 min, values not significantly different from pre-infusion outputs. Basal plasma CCK concentrations before infusion of loxiglumide or saline were 2.4 + 0.1 pM and 2.7 + 0.4 pM, respectively. Loxiglumide did not significantly
365
~j Loxigtumide (o) or Satine ( • ) plasma CCK
I Bombesin
I l
(pM) 20~ IB. 16 % 12 10 8
2- ~
-~s
"0"* ~
-is b Ys 3o ~:s ~ #s ~ Ibs 12o'~io
Fig. 2. Basal and BBS (150 ng/kg/h)-stimulated plasma CCK concentrations (pM; mean _+ S.E.M.) in six healthy subjects during intravenous saline (closed circles) or loxiglumide infusion (open circles). For details see Materials and Methods. Asterisks denote statistically significant differences from basal values (**P < 0.01), triangles denote significant differences between CCK values obtained during BB S stimulation with and without loxiglumide administ~'ation ( A p < 0.05; • A p < 0.01), and circles denote progressive significant (O = P < 0.05; O O = P < 0.01) decrease, compared to the first 15-min sample during BBS infusion alone, which does not occur during concomitant loxiglumide infusion.
influence basal plasma CCK levels (2.4 + 0.2 pM) (Fig. 2). Bombesin infusion significantly (P < 0.05 to P < 0.01) increased plasma CCK values over basal values at all time intervals measured. However, during bombesin infusion there was a significant decline in plasma CCK values in time (P < 0.05), when compared to the first 15-min sample obtained after the bombesin infusion was started (Fig. 2). Loxiglumide significantly (P < 0.05) augmented the plasma CCK response to bombesin at all time intervals measured. In addition, no decrease in CCK response in time was observed during bombesin infusion against a loxiglumide infusion (Fig. 2).
Discussion
At least four interesting findings can be derived from this study. Firstly, the specific CCK receptor antagonist loxiglumide inhibited both basal pancreatic enzyme secretion and bilirubin output. Secondly, loxiglumide almost abolished bilirubin output into the duodenum during BB S infusion into man. Thirdly, loxiglumide had no significant effect on BB S stimulated pancreatic enzyme output into the duodenum. Fourthly, loxiglumide augmented BBS stimulated CCK release, which could not be explained by inhibition of pancreatic enzyme secretioni as suggested before [ 16]. In contrast to previous studies in other species [17,18], we have found that loxiglumide inhibited basal pancreatic enzyme output in man. This suggests that basal
366 plasma CCK levels are involved in maintaining both gall bladder volumes [5] and pancreatic enzyme outputs under unstimulated conditions. In a recent study, we have demonstrated that bilirubin output into the duodenum is highly correlated to gall bladder contraction measured by real-time ultrasonography [19]. Therefore, this study confirms our previous finding that BBS stimulated gallbladder contraction in man is almost abolished by the specific CCK receptor blocker loxiglumide [5]. This suggests that BBS has no major direct effects on gallbladder motility in man and exerts its effects through the release of CCK. Since BBS-receptors have been demonstrated on human pancreatic acinar cells [20], and since previous studies have clearly demonstrated that plasma CCK-levels comparable to those observed during BBS infusion potently stimulate pancreatic enzyme secretion [21], a concerted stimulatory action of direct and indirect (CCK) effects of BBS on pancreatic enzyme output into the duodenum was expected. Surprisingly, no significant inhibition of BBS-stimulated pancreatic amylase and trypsin output during CCK-receptor blockade was observed. This may suggest that loxiglumide, while inhibiting the effects of BBS-stimulated CCK on pancreatic exocrine secretion, augments the direct effects of BB S on pancreatic enzyme output. In vitro experiments in favor of this concept have been published [22,23]. These studies have demonstrated that after incubating dispersed acini from guinea-pig pancreas with CCK, subsequent stimulation of amylase secretion by pancreatic secretagogues whose actions are also mediated by cellular release of calcium, like BBS and carbamylcholine, but also CCK itself, is decreased. If CCK desensitization also occurs in man, its importance in respect to the present study might be that it attenuates the response of pancreatic acinar cells to the direct stimulatory effects of BBS. When this CCK mediated attenuation is blocked by loxiglumide, BBS may augment amylase output to values not significantly different from the values obtained during BBS infusion without CCK-receptor blockade. If so, CCKreceptor blockade cannot be employed to definitively demonstrate the relative contribution of CCK to pancreatic enzyme secretion during BBS infusion. This may probably also relate to meal-stimulation, since CCK also desensitizes the effect of cholinergic stimulation on pancreatic acinar cells [22]. This study also confirms our previous finding that loxiglumide augments BB S-stimulated CCK release [6]. Factors responsible for augmentation of CCK release during BBS infusion could be: (i) interference of loxiglumide in the metabolic clearance rate of CCK [24]. This factor was excluded in a previous study [7], demonstrating that loxiglumide did not interfere with plasma half-life of CCK in man. (ii) A direct effect of CCK on its release from duodenal I-cells is another possibility to explain the augmented CCK release during BBS infusion and loxiglumide administration. Indirect evidence against such an autocrine negative feedback mechanism was obtained from a recent study [25], demonstrating that infusion of synthetic CCK 8S was not able to inhibit endogenous CCK release in response to a meal. (iii) An indirect effect of CCK on its release from duodenal I-cells during BBS infusion is therefore the most likely explanation for the observed effect ofloxiglumide. Such an indirect effect may be related to interference ofloxiglumide with BB S-mediated release ofa CCK inhibiting substance, like somatostatin, or via inhibition of BBS stimulated bile acid secretion into the duodenum [26,27]. Anyhow, the augmented BBS-stimulated CCK release by loxi-
367
glumide observed in this study ~annot be explained by a negative feedback mechanism between CCK release and pancreatic exocrine secretion [ 16], since pancreatic exocrine secretion is not inhibited by loxiglumide during BBS infusion.
References 1 Setnikar, I., ChistG, R., Makovec, F.b Rovati, L. C. and Warrington S.J., Pharmacokinetics of loxiglumide after single intravenous or oral doses in man, Arzneimittelforschung/Drug Res., 38 (1988) 716-720. 2 Meyer, B. M., Beglinger, C., Jansen, J. B. M.J., Rovati, L. C., Werth, B. A., Hildebrand, P., Zach, D. and S talder G. A., Role of cholecystokinin in regulation of gastrointestinal motor functions, Lancet, ii (1989) 12-15. 3 Schmidt, W. E., Creutzfeldt, W., Schlcser, A., Roy Choudhury, A., Nitsche, R., Sostmann, H. and F01sch U. R., Acute and chronic effects of CCK receptor antagonist loxiglumide on pancreatic and biliodigestive functions in man, Digestion, 43 (1989) 170-171. 4 Hildebrand, P., Beglinger, C., Gyr, K., Jansen, J.B.M.J., Rovati, L.C., Zuercher, M., Lamers, C. B. H. W., Setnikar, I. and Stalder~ G. A., Effects of a cholecystokinin receptor antagonist on intestinal phase of pancreatic and biliary responses in man, J. Clin. Invest., 85 (1990) 640-646. 5 Douglas, B. R., Jebbink, M. C. W., Tjon a Tham, R. T. O., Jansen, J. B. M.J. and Lamers C. B. H. W., The effect of loxiglumide (CR-1505) on basal and bombesin stimulated gallbladder volume in man, Eur. J. Pharmacol., 166 (1989) 307-310. 6 Jansen, J.B.M.J., Jebbink, M.C.W., Douglas, B.R. and Lamers, C.B.H.W., Effect of loxiglumide (CR-1505) on bombesin and meal stimulated plasma cholecystokinin in man, Eur. J. Clin. Pharmacol., 38 (1990) 367-370. 7 Jebbink, M.C.W., Jansen, J. B. M.J., Masclee, A. A.M. and Lamers C.B.H.W., Lack of effect of the specific cholecystokinin receptor antagonist loxiglumide on cholecystokinin clearance from plasma in man, Br. J. Clin. Pharmacol., 29 (1990) 770-773. 8 Jansen, J.B.M.J. and Lamers C.B.H.W., Radioimmunoassay of cholecystokinin: production and evaluation of antibodies, J. Clin. Chem. Clin. Biochem., 21 (1983) 387-394. 9 Jansen, J.B.M.J. and Lamers C.B.H.W., Radioimmunoassay of cholecystokinin in human tissue and plasma, Clin. Chim. Acta, 131 (1983) 305-316. 10 Jendrassik, L. and GrofP., Vereinfachte photometrische Methoden zur Bestimmung des Blutbilirubins, Biochem. Z., 297 (1938) 81-89. 11 Hummel B.C., A modified spectrophotometric determination of cbymotrypsin, trypsin and thrombin, Can. J. Biochem., 37 (1955) 1393-1397. 12 Beinfeld P., Alpha amylase, Methods Enzymol., 1 (1955) 149-158. 13 Malawer, S.J. and Powe|l D.W., An improved turbidimetric analysis of polyethylene glycol utilizing an emulsifier, Gastroenterology, 53 (1967) 250-256. 14 Beglinger, C., Fried, M., Whitehouse, I., Jansen, J. B. M.J., Lamers, C. B. H. W. and Gyr, K., Pancreatic enzyme response to a liquid meal and to hormonal stimulation. Correlation with plasma secretin and cholecystokinin levels, J. Clin. Invest., 75 (1985) 1471-1476. 15 Layer, P., Jansen, J.B.M.J., Cherian, L. and Goebell H., Feedback regulation of human pancreatic secretion: Effects of protease inhibition on duodenal delivery and small intestinal transit of pancreatic enzymes, Gastroenterology, 98 (1990) 1311-1319. 16 Liddle, R. A., Goldfine, I. D., Rosen, M. S., Taplitz, R. A. and Williams J. A., Cholecystokinin bioactivity in human plasma. Molecular forms, responses to feeding, and relationship to gallbladder contraction, J. Clin. Invest., 75 (1985) 1142-1152. 17 Niederau, M., Niederau, C., Stroh~neyer, G. and Grendell J.H., Comparative effects of CCK receptor antagonists on rat pancreatic secretion in vivo, Am. J. Physiol., 256 (1989) G150-G157. 18 Lamers, C.B.H.W., De Jong, A.JiL., Singer, M.V., Niebel, W. and Jansen J.B.M.J., Role of cbolecystokinin in bombesin stimulated gastric acid and pancreatic enzyme secretion in dogs, Gastroenterology, 94 (1988) A249.
368 19 Hopman, W.P.M., Kerstens, P.J.S.M., Jansen, J.B.M.J., Rosenbusch, G. and Lamers C.B.H.W., Effect of graded physiologic doses of cholecystokinin on gallbladder contraction measured by ultrasonography. Determination of threshold, dose-response relationships and comparison with intraduodenal bilirubin output, Gastroenterology, 89 (1985) 1242-1247. 20 Scemama, J-L., Zahidi, Z., Fourmy, D., Fagot-Revurat, P., Vaysse, N., Pradayrol, L. and Ribet A., Interaction of (~25I)-Tyra-bombesin with specific receptors on normal human pancreatic membranes, Regal. Pept., 13 (1986) 125-132. 21 Kerstens, P.J.S.M., Lamers, C.B.H.W., Jansen, J.B.M.J., De Jong, A.J.L., Hessels, M. and Hafkenscheid J.C.M., Physiological plasma concentrations of cholecystokinin stimulate pancreatic enzyme secretion and gallbladder contraction in man, Life Sci., 36 (1985) 565-569. 22 Abdelmoumene, S. and Gardner J. D., Cholecystokinin-induced desensitization of enzyme secretion in dispersed acini form guinea-pig pancreas, Am. J. Physiol., 239 (1980) G272-G279. 23 Younes, M., Wank, S. A., Vinayek, R., Jensen, R. T. and Gardner J. D., Regulation ofbombesin receptors on pancreatic acini by cholecystokinin, Am. J. Physiol., 256 (1989) G291-G298. 24 Hosotani, R., Doi, R., Sumi, S., Gu, Y., Inoue, K., Ishikawa, T., Rayford, P.L. and Tobe, T., Effect of a receptor antagonist on the metabolism of cholecystokinin-33, Digestion, 46(S1) (1990) 46. 25 Jebbink, M. C. W., Jansen, J. B. M.J., Schouten, C. and Lamers, C. B. H. W., Effect of exogenous cholecystokinin octapeptide on meal stimulated plasma CCK release in man, Digestion, 46(3) (1990) 147. 26 Gomez, G., Upp, J.R., Lluis, F., Alexander, R.W., Poston, G.J., Greeley, G.H. and Thompson J.C., Regulation of the release of cholecystokinin by bile salts in dogs and humans, Gastroenterology, 94 (1988) 1036-1046. 27 Koop, I., Koop, H., Gerhardt, C., Schafmayer, A. and Arnold R., Do bile acids exert a negative feedback control of plasma cholecystokinin release, Scand. J. Gastroenterol., 24 (1989) 315-320.