Evidence against autocrine feedback regulation of cholecystokinin secretion in man

Peptides,Vol. 13, pp. 287-290, 1992

0196-9781/92 $5.00 + .00 Copyright © 1992PergamonPressLtd.

Printed in the USA.

Evidence Against Autocrine Feedback Regulation of Cholecystokinin Secretion in Man M A X C. W. JEBBINK,* J A N B. M. J. J A N S E N , t I D I A N A M. M O O Y , * C A R O L I E N M. S C H O U T E N * A N D C O R N E L I S B. H. W. L A M E R S *

Departments of Gastroenterology and Hepatology, University Hospitals of *Leiden and JNijmegen, The Netherlands" Received 3 S e p t e m b e r 1991 JEBBINK, M. C. W., J. B. M. J. JANSEN, D. M. MOOY, C. M. SCHOUTEN AND C. B. H. W. LAMERS. Evidence against autocrinefeedback regulation of cholecystokinin secretion in man. PEPTIDES 13(2) 287-290, 1992.--To determine whether exogenous cholecystokinin (CCK) inhibits endogenous CCK release, cholecystokinin-8S (CCK-8S) was infused intravenously during continuous intraduodenal stimulation of endogenous CCK by a meal. CCK was measured in plasma by 2 region-specific radioimmunoassays employing antibodies T204 and 1703. AB T204 binds to carboxy-terminal CCK peptides containing the sulphated tyrosyl region, including CCK-8S, and AB 1703 to carboxy-terminal CCK peptides containing at least 14 amino acid residues. Meal-stimulated plasma CCK concentrations remained elevated during the entire infusion period. CCK-8S infusion further increased meal-stimulated plasma CCK concentrations, when measured with AB T204, while meal-stimulated plasma CCK concentrations were not suppressedby CCK-8S infusion, when measured with AB 1703. It is concluded that meal-stimulated endogenous CCK release is not affected by the effects of intravenously administered CCK-8S. These data suggest that autocrine feedback regulation of CCK release is not operative in man. Cholecystokinin

Feeding

Autocrine feedback mechanism

TOGETHER with gastrin and secretin, cholecystokinin (CCK) comprises the classical triad of gut hormones. CCK shares with gastrin the biological active pentapeptide in the carboxy-terminal part of the molecule. Like gastrin from antral G-cells, CCK release from duodenal I-cells is believed to be dependent on a negative feedback system (22,24). In rats, this feedback system is mediated by protease activity in the lumen of the small intestine (7). The situation in man, however, is probably far more complicated. Some investigators have shown that intraluminal protease activity is involved in CCK release in man (9,23,25), but others have failed to demonstrate such an effect (1,2,4,11). Recent studies with powerful CCK receptor antagonists, like CR-1505 and MK-329, have demonstrated an augmented plasma CCK release in response to a meal and to bombesin (3,8,12,20). This augmented CCK response, however, is not related to a decreased activity of proteases in the lumen of the gastrointestinal tract (17). Other mediators, like bile acids, may therefore be involved in the regulation of CCK secretion (5,6,19). Since other hormones like insulin and somatostatin have been demonstrated to exert inhibitory effects on their own secretion (10,21), it can be postulated that CCK inhibits its own release from duodenal I-cells via an autocrine negative feedback mechanism. In order to test this hypothesis, we have investigated the effect of intravenously administered CCK-8S on meal-stimulated CCK release in healthy volunteers. The problem of distinguishing between exogenously administered CCK-8S and endogenously

Radioimmunoassay

Humans

secreted CCK was circumvented by using 2 different radioimmunoassay systems. In one assay, employing antibody 1703, CCK-8S was not detectable, while CCK-8S fully cross-reacted with the antibody (T204) in the other assay. METHOD Six healthy volunteers (mean age _+ SE: 24 _+ 1 years) were studied on 2 days separated by at least one week. After an overnight fast, the volunteers presented at the laboratory on the morning of the next day, where 2 indwelling intravenous catheters were placed, one into each forearm. One catheter was used for collection of blood samples, while CCK-8S or saline was infused through the other. The subjects were also intubated with a polyvinyl single-lumen feeding tube, which was placed into the proximal part of the duodenum approximately 10 cm distal to the pylorus. The position of the tip of the tube was verified by fluoroscopy. After the collection of 2 blood samples at a 10rain interval under unstimulated conditions, a liquid test meal was infused intraduodenally for 3 hours at a perfusion rate of 2.5 ml/min. The 450 ml liquid meal consisted of 29.4 g fat, 22.5 g protein, and 80.7 g carbohydrates, with an osmolality of 320 mOsm/l, and the total caloric value of the perfused meal amounted to 675 kcal. During the second hour of meal stimulation, CCK-SS (70 pmol/kg/h) or saline was infused intravenously for one hour in random order. Blood samples were drawn

Requests for reprints should be addressed to Jan B. M. J. Jansen, M.D., Ph.D., Department of Gastroenterology and Hepatology, University Hospital, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands.

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at 10-min intervals during the entire study period. These samples were collected into ice-chilled glass tubes containing EDTA. Within 1 hour after collection of the blood, the tubes were centrifuged and the plasma was stored at - 2 0 ° C until assayed for CCK. Plasma CCK was determined by 2 specific radioimmunoassays. In one assay antibody T204 was employed. This antibody recognizes all biologically active circulating molecular forms of CCK, including CCK-8S. In the other assay antibody 1703 was used. This antibody only recognizes biologically active molecular forms of CCK containing at least 14 amino acid residues. Validation of both assays has been reported previously (13,14). All volunteers gave informed consent and the experiments were approved by the local ethical committee.

(Fig. 1). However, meal-stimulated plasma CCK concentrations were not suppressed during or after the infusion of CCK-8S, when measured with antibody 1703 (Fig. 2). DISCUSSION In the present study, we have demonstrated that meal-stimulated CCK release is not suppressed by intravenous infusion o f a supraphysiologic dose of synthetic CCK-8S. These data suggest that endogenous CCK release is not inhibited by direct effects of the peptide on its own secretion from I-cells in the mucosa of the small intestine in man. The possibility of the existence of such an autocrine negative feedback mechanism arose from recent observations, which have demonstrated that specific CCK receptor blockers, like loxiglumide (CR-1505), are able to augment plasma CCK release in response to meal administration and bombesin infusion (3,8,12,20). In previous studies, two possible explanations for this phenomenon could be excluded. Firstly, loxiglumide did not interfere with the distribution and metabolism of CCK (18), and secondly, CCK receptor blockade failed to affect BBS-stimulated exocrine pancreatic secretion while inducing increased plasma CCK levels (17). This last finding suggests that protease activity in the lumen of the proximal small intestine is not the major feedback regulator of CCK release in man, leaving scope for other feedback systems to explain the augmented BBS- and meal-stimulated CCK release during CCK receptor blockade. One of the possibilities for this observed effect is the existence of an autocrine feedback system by which mealor BBS-stimulated CCK, directly or indirectly, inhibits its own release from I-cells in the small intestinal mucosa. Since the existence of such a feedback system cannot be studied during CCK receptor blockade, we have examined the effect of intravenously administered synthetic CCK-8S on meal-stimulated CCK release by using two region-specific radioimmunoassays employing antibodies T204 and 1703 (13,14). Antibody T204 is directed towards the region containing the sulphated tyrosyl residue and binds to all known biologically active molecular forms of CCK, including CCK-8S, while antibody 1703 recognizes biologically active forms of CCK containing at least 14

Statistical Analysis Data are expressed as mean + SE. Statistical inference of differences between means was performed by analyses of variance (ANOVA) on repeated measures. The tests comprised the time during CCK-8S or saline infusion. Post hoc multiple range tests were done by the Newman-Keuls procedure. Differences with a probability of less than 0.01 were considered to be significant. RESULTS Ten minutes after the start of the intraduodenal infusion of the liquid test meal, basal plasma CCK concentrations were stimulated in the two groups, from 3.9 + 0.5 pM (control) and 4.1 + 0.5 pM to 10.4 + 1.5 pM (p < 0.01) and 9.8 ± 1.8 pM (p < 0.01), respectively, when measured with antibody T204 (Fig. 1) and from 3.7 ± 0.7 pM (control) and 4.0 + 0 . 4 pM to 10.1 + 2.1 pM (p < 0.01) and 10.3 + 1.6 pM (p < 0.01), respectively, when measured with antibody 1703 (Fig. 2). During the test meal infusion, CCK values remained in this elevated range without significant differences in the 10-rain time intervals from 10 to 180 minutes after the start of the meal infusion (Figs. 1 and 2). When measured with antibody T204, administration of CCK-8S significantly (p < 0.01) elevated meal-stimulated plasma CCK concentrations from approximately 8.0 ___ 1.1 pM to a range of 16.0 ± 1.3 to 19.6 ± 1.2 pM during the infusion period A b T204 Plasma CCK(pM)

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FIG. 1. Effect of exogenous CCK-8S (O = endogenous CCK and © = endogenous CCK and CCK-8S) or saline (D) on meal-stimulated plasma CCK release in 6 normal subjects as measured by RIA using antibody T204. *p < 0.01, compared to saline study (ANOVA).

EVIDENCE AGAINST AUTOREGULATION OF CCK

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a m i n o acid residues. In previous studies, we have demonstrated that Sephadex G50 SF elution profiles of CCK-like i m m u n o reactivity released after a meal or during BBS infusion were very similar when measured with both radioimmunoassays (15,16). Furthermore, it has been demonstrated that the biological activity of CCK-8S and CCK-33 are comparable (26). Therefore, infusion of CCK-8S should suppress meal-stimulated release of larger molecular forms of CCK, detected by antibody 1703, in order to fulfill the hypothesis that an autocrine feedback mechanism between C C K release and C C K suppression is operative in man.

Since we were unable to demonstrate an autocrine feedback regulation of C C K release, other mechanisms are probably involved in the augumented bombesin- and meal-stimulated CCK release during C C K receptor blockade. These possibilities include inhibition of C C K release by bile acids in the lumen of the small intestine or inhibition via a mediator, i.e., somatostatin, coreleased with C C K during meal or bombesin stimulation. In conclusion, the present study has failed to demonstrate inhibitory effects of intravenously administered CCK-8S on meal-stimulated release of larger molecular forms of CCK.

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