- Email: [email protected]
Loxiglumide inhibits cholecystokinin stimulated somatostatin secretion and simultaneously enhances gastric acid secretion in humans
ELSEVIER
Regulatory Peptides 53 (1994) 185-193
Loxiglumide inhibits cholecystokinin stimulated somatostatin secretion and simultaneously enhances gastric acid secretion in humans Marie Louise Verhulst a, Hugo A.J. Gielkens a, Wim P.M. Hopman a, Annie van Schaik a, Albert Tangerman a, Lucio C. Rovati b, Jan B.M.J. Jansen a,, aDepartment of Gastroenterology and Hepatology, University Hospital St. Radboud, Geert Grooteplein 8, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands b ROTTA research laboratories, Monza, Italy Received 30 July 1992; revised version received 27 June 1994; accepted 27 June 1994
Abstract In vitro studies have demonstrated that cholecystokinin releases somatostatin from the gastric mucosa. To date, there is no information about the in vivo significance of this finding in man. Therefore, we have studied the effect of infusion of cholecystokinin resulting in plasma concentrations within the range found after meal-stimulation, on somatostatin release and on gastric acid secretion. In addition we have studied these functions during infusion of the type A cholecystokinin receptor antagonist loxiglumide. In eight healthy subjects, basal gastric acid secretion was distinctly stimulated by cholecystokinin. The effect of cholecystokinin on gastric acid secretion was markedly enhanced by loxiglumide. Cholecystokinin also significantly stimulated somatostatin output into the gastric lumen, but not into the systemic circulation. Somatostatin output into the gastric lumen during infusion of cholecystokinin was abolished by loxiglumide. The data indicate that on the one hand circulating cholecystokinin, like gastrin, stimulates gastric acid secretion probably by binding to less specific type B receptors on parietal cells that are not blocked by loxiglumide, but on the other hand that cholecystokinin, in contrast to gastrin, also inhibits gastric acid secretion probably by binding to specific type A receptors present on somatostatin producing D-cells in the gastric mucosa, that are blocked by loxiglumide.
Keywords." Loxiglumide; Somatostatin; Cholecystokinin; Gastric acid secretion; CCK-receptor
1. Introduction Immunochemical studies have demonstrated that somatostatin (SST) is present in D-cells in the gas* Corresponding author. 0167-0115/94/$7.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 1 6 7 - 0 1 1 5 ( 9 4 ) 0 0 0 6 3 - 8
tric m u c o s a of several species, including man [ 1 ]. It has been suggested that S S T in the gastric m u c o s a acts as a paracrine substance, since D-cells were demonstrated to possess offshoots that were in close contact with adjacent cells, like parietal cells and gastrin producing cells [2,3]. In vitro, it has been
186
M.L. Verhulst et al. / Regulator), Peptides 53 (1994) 185-193
demonstrated that specific CCK type A-receptors are present on SST containing D-cells in the gastric mucosa [4-6], and that CCK is able to stimulate SST-release from the gastric mucosa [4,6]. Whether CCK, circulating in concentrations similar to those found after meals, is able to stimulate SST release in vivo, has not been demonstrated yet, although indirect evidence for a stimulating role of CCK on S ST-secretion was obtained from several studies [7-10]. Stimulation of SST by circulating CCK in concentrations as observed as after a meal, may have important consequences, since SST is a powerful inhibitor of several gastro-intestinal functions [10-12], and since SST is present in many organs [13], allowing local release by CCK. In the present study, we have infused CCK to plasma concentrations as observed as after a meal, and we have studied the effects on gastric acid output, on local release of SST into the gastric lumen, and on release of SST into the systemic circulation. In addition, we have studied the effects of the specific CCK type A-receptor antagonist loxiglumide on these parameters during infusion of CCK. It has also been shown in vivo, that loxiglumide is by far more specific for CCK type A receptors than for CCK type B/gastrin receptors, since loxiglumide is not able to influence the effects of gastrin, infused to plasma concentrations as observed as after a meal, on gastric acid secretion in humans [8], and since loxiglumide abolishes the effects of CCK on gallbladder contraction [9]. 2. Materials and methods
2.1. Experimental protocol Eight healthy subjects (6 F, 2 M; mean age 29 + 5 years) participated in two studies which were performed in random order on different days separated by at least one week. After an overnight fast, the subjects presented at the laboratory. During the studies, the subjects where seated in a comfortable chair in a recumbent position. A gastric suction tube
with a small bore perfusion catheter attached to it, was placed in the stomach. The position of the tube was checked by the water recovery technique [14]. The perfusion catheter was then pulled back about 10 cm disconnecting this tube from the suction tube. 15 min before the start of the experiments, the small bore tube was perfused with a recovery marker (phenol red-saline solution 200 ml/15 min; 3 mg/1 of phenol red) as previously described [14]. Subsequently two indwelling intravenous catheters were placed one in each fore-arm. The catheters were kept patent by a heparine-saline solution. From one catheter blood samples were drawn for determination of CCK at 15 min intervals, while saline or synthetic loxiglumide (Rotta Laboratories, Monza, Italy) dissolved in saline, was infused through the other in a dose of 7.5 mg/kg/h during the entire 150 min study period, followed by 12.5 pmol/kg/h of synthetic human CCK33 (Peninsula laboratories, San Carlos, CA, USA) dissolved in saline containing 0.5~o human serum albumin, for the last 90 min in each experiment. Gastric contents were aspirated continuously by a suction pump providing intermittent negative presure, and collected in 15 min portions. The experiments were approved by the local ethical committee and all subjects gave informed consent.
2.2. Gastric acid Gastric juice was collected into ice chilled glass bottles. Gastric acid concentrations were measured by titration to pH 7.0 with 0.1 M NaOH. After filtration and alkalinisation with 2.5 M NaOH, the concentration of phenol red was measured in the perfusion fluid and the aspirates by spectrophotometry at 560 nm. Results of acid output and SSTsecretion were corrected for losses using this recovery marker as previously described [14]. For determination of SST, gastric juice was handled according to a method that was described by Wisen et al. [15] with modifications by us. In brief, after the addition of 1 mg/ml of camostate (a
M.L. Verhulst et al. / Regulatory Peptides 53 (1994) 185-193
gift from professor Guido Adler, Ulm, Germany) to gastric juice immediately after its collection, 10 ml of each 15 min gastric aspirate was adjusted to pH 5.0 by adding 1 M NaOH or 1 M HCL, depending on the original pH of the sample, and boiled in a beaker. Within 15 min, the volume was reduced by more than 80 ~o. Distilled water was subsequently added to the boiled aspirate to obtain a total volume of 2.0 ml. These samples were centrifuged and the clear supernatants were stored at -20 °C until assayed for SST.
2.3. Radioimmunoassays Plasma CCK was measured by radioimmunoassay employing antibody T204 as previously described [16,17]. In brief, the antibody was raised in a rabbit against partly purified porcine CCK kindly provided by Prof. V. Mutt (Karolinska Institutet, Stockholm, Sweden). Synthetic CCK33 was labelled with [125I]-Bolton-Hunter reagent and used as tracer. Synthetic CCK33 was alSO used as standard. The antibody was used in a final dilution of 1:80,000 and binds equally to all carboxyl-terminal CCKpeptides containing the sulphated tyrosyl region. The antibody shows very little cross-reactivity with sulphated gastrins (<2~o), and does not bind to unsulphated gastrins or structurally unrelated peptides. Antibody bound CCK was separated from free CCK by a second antibody technique. The detection limit of the assay was 1 pmol/1 plasma. The intra-assay precision ranged from 4.6~o to 11.5~o in the working range of the assay, whereas inter-assay precision ranged from 11.3~o to 26.1~o. CCK was extracted from plasma by two volumes of ethanol, centrifuged (4000 g, 10 min), the supernatant was evaporated to dryness at 37 °C and reconstituted in the same volume of assay buffer. All samples were measured in duplicate in one assay. SST was also measured by radioimmunoassay. The antibody was harvested in a rabbit after the seventh monthly immunization with synthetic SST28 (Peninsula laboratories, San Carlos, CA, USA)
187
coupled to bovine serum albumin by the carbodiimide technique [ 16]. 33 Bq of commercially available [125I]-SST14 (DuPont-New England Nuclear, Boston, MA, USA) was used as tracer and synthetic SST14 (Peninsula laboratories, San Carlos, CA, USA) as standard. Antibody, in a final dilution of 1:500,000, [125I]-SST14 and standard SST14 or gastric sample in a total volume of 1.0 ml were incubated for 24 h at 4 ° C, after which antibody bound SST was separated from free SST by a second antibody technique. The detection limit for SST14 of the assay was 5 pmol/1. On a molar base, SST28 and SST~4 cross-reacted equally in the assay and no cross-reactivity was found with other gastro-intestinal peptides, like CCK, gastrin, VIP, substance P, secretin, bombesin, GRP, insulin, glucagon, pancreatic polypeptide, PHI, PYY, calcitonin, C-GRP and GIP. Intra- and inter-assay variations ranged from 4.6~o to 13.2~o and from 5.9~o to 20.1~o, respectively, in the working range of the assay. SST-like immunoreactivity in plasma was measured after extraction of plasma by Sep-Pak C18 cartridges [ 18]. 10 ml of gastric aspirates collected during CCK and loxiglumide infusion, that were found to have low endogenous SST concentrations, were spiked with SST14 or SST28 to 2 (n = 12), 10 (n = 12) and 250 pM (n = 1) and left for 15 min at room temperature after addition of 1 mg/ml of camostate. Subsequently the pH was brought to 5.0 before boiling. By boiling, the volume was reduced to 2.0 ml. Estimated SST-immunoreactivity after subtraction of endogenous values in the boiled samples are thus 10, 50 or 1250 pM of SSTI4 or SST28. These samples were measured by RIA and recoveries were calculated. Of the sample that was spiked to 250 pM and subsequently concentrated to 1250 pM, 0.4 ml was applied to a Sephadex G-50 SF (100 x 1 cm) column for calibration and recovery experiments. Before Sephadex G-50 SF was applied, the 100 x 1 cm glass tube was filled with 2~o dichlorodimethylsilane for 5 min and subsequently washed with methanol, in order to avoid adhesion of peptides.
188
M.L. Verhulst et al. /Regulatory Peptides 53 (1994) 185-193 15
SALINE OR LOXIGLUMIDE
[
12
CItOLECYSTOKININ
;J rj
T
r
i
I
9
6
3
0
_
i
i
i
I
I
Gastric juice from three subjects during infusion of CCK was pooled, treated according to the modified method of Wisen by which the total volume was reduced to 2 ml and fractionated by gel chromatography. The column was previously calibrated with synthetic SST28 and SST14 dissolved in elution buffer, with albumin to indicate the void volume and with 125I to indicate the total volume of the column. 0.05 M sodium phosphate pH 7.4 was used as elution buffer [ 19,20].
i
2.4. Statistical analysis
5
Results are presented as mean_+ S.E.M. Differences between data obtained in the CCK- and saline-infusion experiments and the CCK- and loxiglumide-infusion experiments were analysed for statistical significances by Wilcoxon's Rank Sumtest for integrated values and by the StudentNewman-Keuls test for individual 15 min samples. Statistical significance was defined as a two-tailed probability of less than 0.05. Integrated gastric acidand SST-outputs (mmol H ÷/h and pmol/h, respectively) were calculated by summation of the results from the last four 15-min samples during infusion of saline, during infusion of loxigtumide, during infusion of CCK and saline and during infusion of the combination of loxiglumide and CCK, respectively.
12
-k
9 -k
6 3. Results 3
30
60
90
120
150
Basal gastric acid output during saline infusion (2.1 + 0.7 mmol/h) was not significantly different (P = 0.08) from gastric acid secretion during infusion
TIME Fig. 1. (Upper panel) Plasma cholecystokinin concentrations (pM) during infusion of saline with and without 12.5 pmol/kg/h of cholecystokinin-33 (squares) and during infusion of 7.5 mg/ kg/h of loxiglumide with and without 12.5 pmol/kg/h of cholecystokinin-33 (circles) in eight healthy subjects. (Middle panel) Gastric acid output (mmol/15 min) during infusion of saline with and without cholecystokinin (open blocks) and during infusion of loxiglumide with and without cholecystokinin (closed
blocks). Asterisks indicate significant differences (P< 0.05) from corresponding samples during infusion of saline. (Lower panel) Gastric somatostatin output (pmol/15 min) during infusion of saline with and without cholecystokinin (open blocks) and during infusion of loxiglumide with and without cholecystokinin (closed blocks). Asterisks indicate significant differences (P< 0.05) from corresponding samples during infusion of loxiglumide.
M.L. Verhulst et al. / Regulatory Peptides 53 (1994) 185-193
of loxiglumide (2.7 + 0.8 mmol/h). Infusion of CCK resulting in plasma CCK-concentrations similar to those found after a meal ([21,22]; Fig. 1, upper panel), markedly stimulated gastric acid secretion from 2.1 + 0.7 mmol/h to 5.8 + 1.5 mmol/h (P< 0.01) (Fig. 1, middle panel). Loxiglumide significantly enhanced CCK-stimulated gastric acid secretion from 5.8+1.5 to 10.8+2.1 mmol/h (P<0.01) (Fig. 1, middle panel). Basal SST-output into the gastric juice (5.5 + 0.6 pmol/h) was not significantly influenced by loxiglumide (5.4 + 0.5 pmol/h) (Fig. 1, lower panel). CCK markedly stimulated SST-output into the gastric juice from 5.5 + 0.6 to 18.5 + 5.7 pmol/h (P<0.01). SST-concentrations were significantly (P<0.05) higher in all individual 15-min samples during CCK-infusion when compared to prestimulated fractions, without significant differences between the mutual stimulated fractions. This effect
70 60
Vo
28
14 vt
4.
4"
4.4.
50
30
10
•
•
/..\
189
was abolished by loxiglumide, since CCK did not significantly stimulate basal S ST-output into gastric juice during administration of loxiglumide (from 5.4 + 0.5 to 6.1 + 0.8 pmol/h) (Fig. 1, lower panel). Recovery of SST from gastric juice spiked to 250 pM with SST28 or SSTs4 and subsequently boiled by which the volume was reduced to 2 ml with an estimated SST concentration of 1250 pM, was 84Fo (1050 pM) and 78~o (980 pM), respectively. 80~o ( S S T 2 8 ) and 81~o ( S S T I 4 ) of this immunoreactivity was recovered in the positions of synthetic S ST28 and synthetic S STs4 after fractionation by gel chromatography. Recoveries of SST from gastric juice spiked with S ST28 or S ST~4 and subsequently concentrated by boiling to estimated concentrations of 10 and 50 pM, were 78 + 8~o and 88 + 5~o, respectively for SST28 and 83 _+5~o and 92 + 3~o, respectively for S S T I 4 (n = 12). 61 ~o of endogenous SST-like immunoreactivity secreted into gastric juice during infusion of CCK eluted in the position of S S T 1 4 , the remaining immunoreactivity eluted in the void volume (8 ~ ) and in the position of SST2s (31Yo) (Fig. 2). Plasma SST-like immunoreactivity was not significantly influenced by CCK-infusion (Fig. 3).
4. Discussion •. e .
•
"IIOOOOoql
0 "'" ''''~ ~' ' ' ' " 0 10 20 30 40 50 60 70 80 90 Fraction Number Fig. 2. Elution profile of pooled gastric fractions from 3 healthy subjects adjusted to pH 5 after addition ofcamostate ! mg/ml and concentrated by boiling. The fractions were collected during infusion of cholecystokinin. The extract was applied to a 100 cm Sephadex (3-50 SF column with a diameter of 1.0 cm, that was previously calibrated with SST28 [28], SST14 [14], albumin to indicate the void volume (Vo) and t25I to indicate the total volume (Vt) of the column. Fractions of 1 ml were collected and measured by radioinamunoassay for somatostatin-like immunoreactivity. The recovery of the SST-immunoreaetivity applied to the column was 80~/o.
Cholecystokinin and gastrin share a common pentapeptide in the biologically active carboxyl-terminal domain of the molecules. In vitro, CCKs and gastrin are equipotent in stimulating canine parietal cell function [23]. In an in vivo rat model, CCK s and gastrin also seem to be equipotent in stimulating gastric acid secretion [25]. However, species differences in the effects of CCK and gastrin on gastric acid secretion may exist as well as differences in acid secreting potency of different molecular forms of CCK, since in humans C C K 3 3 is much less potent in stimulating gastric acid secretion than gastrin [24]. The weak stimulation of gastric acid secretion by
190
M.L. Verhulst et al. / Regulatory Peptides 53 (1994) 185-193 30
27
LOXIGLUMIDE OR SALINE J
24 21 18 Ill
FCHOLECYSTOKININ
15
J
r~T]-TT
12
T
9 6
3 0 -30
i
0
i
i
30
i
i
i
~
60 90 Time (rain)
i
i
120
i
i
150
Fig. 3. Plasma somatostatin concentrations (pM) during infusion of saline (squares) or loxiglumide (circles) with and without cholecystokinin in eight healthy subjects. Somatostatin was extracted from plasma by SepPak cartridges according to the method described by Ensinck [19].
CCK33-infusion when compared to gastrin-infusion in humans, may also be explained by simultaneous activation of parietal cells and of cells that release an inhibitor of gastric acid secretion by CCK, and not solely from a direct diminished effect of CCK on parietal cells. However, it may also be possible that differences in the effects of CCK and gastrin on gastric acid secretion in previous in vivo experiments, are explained by differences in circulating concentrations of CCK and gastrin. Under physiologic conditions, circulating CCK concentrations are considerably lower than circulating gastrin concentrations in humans. Therefore, the effects of infusion of CCK and gastrin on gastric acid secretion can only be compared by infusing supraphysiological doses of CCK in order to obtain plasma levels that can be compared on a molar base to plasma gastrin levels in response to infusion of gastrin to concentrations as observed as after a meal. Some years ago, it was demonstrated that CCK was able to stimulate SST-release from gastric D-cells in vitro by binding to a specific type A CCKreceptor [4]. Since SST is a potent inhibitor of gas-
trin release and gastric acid secretion [26], it was hypothesized that the poor stimulation of gastric acid secretion by CCK in vivo, was related to this effect on gastric SST-release. In healthy volunteers, we have recently demonstrated that Loxiglumide is by far more selective for type A CCK-receptors, than for type B CCK/gastrin receptors, since loxiglumide does not affect gastric acid secretion stimulated by infusion of gastrin, but augments gastrin- and gastric acid responses to CCK stimulated by bombesin-infusion [8]. Indirectly, it was concluded from these studies that this augmented gastrin- and gastric acid response to bombesin was induced by specific inhibition of the effect of bombesin-stimulated CCK on SST-producing D-cells in the gastric mucosa. To date, there is no information about the effect of CCK, circulating in concentrations as observed as after a meal, on gastric SST-release in humans. One study has demonstrated that CCK, infused to plasma concentrations in the physiological range, did not stimulate the release of prosomatostatin-derived peptides into the systemic circulation [18]. Previous studies have demonstrated that SST in the systemic circulation mainly originates from the small intestinal mucosa and represents SST28 [27,28]. SST in the gastric mucosa, however, is mainly present as SST14 [13]. Furthermore, it has been demonstrated that S ST is also released into the gastric lumen [ 15]. We therefore have measured S ST both in plasma and in gastric juice, and we have demonstrated that infusion of CCK to plasma concentrations in the same range as found after a meal, potently stimulates the release of SST into the gastric lumen, but not into the systemic circulation. CCK-stimulated SST-release into the gastric lumen was abolished by loxiglumide. Our recovery experiments and chromatography data suggest that the increase of SST-like immunoreactivity in gastric juice during CCK-infusion, is indeed SST, since most of the immunoreactivity co-elutes with synthetic SSTI4. This confirms previous studies on the nature of molecular forms of SST in the gastric mucosa [ 13].
M.L. Verhulst et aL/ Regulatory Peptides 53 (1994) 185-193
Loxiglumide also markedly enhanced gastric acid output in response to CCK-infusion, but did not affect gastrin-stimulated gastric acid secretion [8]. The findings therefore suggest that stimulation of gastric acid secretion by binding of CCK to parietal cells is augmented by inhibition of concomitant CCK-stimulated SST-release from gastric D-cells by loxiglumide. At first glance, the time course of S ST-release into the gastric lumen during CCK-infusion suggests that SST might not be responsible for the augmented CCK-stimulated gastric acid secretion during infusion of loxiglumide, since there is a decline in the CCK-stimulated SST-output after an initial rise, while the enhanced gastric acid output does not show this pattern. One of the possibilities for this effect might be that, SST-immunoreactivity decreases in gastric juice due to an increased activity of peptic digestion of S ST by the augmented gastric acid output. This hypothesis is not very likely, since incubation of synthetic SST28 and SSTl4 with gastric juice collected during infusion of CCK and loxiglumide does not result in rapid degradation of the peptides. Statistical analysis, however, showed that the impression that SST in gastric juice decreases despite continuous infusion of CCK is not of significance. Therefore, the presented data are not in conflict with the hypothesis that S ST is involved in the augmented BBS-stimulated gastric acid secretion during loxiglumide infusion. Previously, we have demonstrated that loxiglumide also enhanced basal gastric acid output [8]. This effect was not confirmed in the present study. Most likely, the failure to observe a statistically significant difference in basal gastric acid output between the saline and loxiglumide experiments in the present study, is due the smaller amount of loxiglumide that was infused for a shorter period of time. Alternatively, spontaneous day to day variation in gastric acid secretion within one subject, may have attenuated the effect of loxiglumide on basal gastric acid secretion in the present study, since the experiments were performed on different days.
191
In rats, the acid secretory actions of gastrin and CCK might also be exerted at two different sites [25]. However, instead of augmentation, inhibition of CCK stimulated gastric acid secretion was observed after administration of the specific type A CCK receptor antagonist L-364,718 to anaesthetized animals, while no effect on gastrin stimulated gastric acid secretion was found [25]. On the other hand, we and others have recently found that CCK type A receptor antagonists, like lorglumide or L-364,718 also inhibit gastrin or pentagastrin stimulated gastric acid secretion in consious or anaesthetized rats [29,30]. In dogs, lorglumide has also been demonstrated to inhibit gastrin stimulated gastric acid secretion [31]. These conflicting data, may result from methodological differences, anaesthesia, differences in the doses of gastrin and CCK used, differences in actions between the various type A CCK receptor antogonists, or point to species differences in the regulation of gastric acid secretion by gastrin and CCK. From this study it cannot be concluded whether SST acts as a luminal active peptide [32] or whether the release of SST into the gastric lumen is simply a reflection of paracrine active SST [2,3]. Anyhow, the release of S ST into the gastric lumen during infusion of CCK is not simply explained by spill over of circulating SST into the gastric lumen, since our study confirms that circulating CCK in concentrations within the physiological range does not stimulate the release of SST into the systemic circulation [18]. In conclusion, the present study demonstrates that on the one hand circulating CCK, like gastrin, stimulates gastric acid secretion in humans, probably by binding to type B CCK/gastrin receptors on parietal cells, but on the other hand that cholecystokinin, in contrast with gastrin, also inhibits gastric acid secretion, probably by binding to specific type A CCK receptors present on SST producing D-cells in the gastric mucosa.
192
M.L. Verhulst et al. / Regulator), Peptides 53 H994) 185-193
References [ 1] Chayvialle, J.A.P., Descos, F., Bernard, C., Martin, A., Barbe, C., Partensky, C., Somatostatin in mucosa of stomach and duo denum in gastroduodenal disease, Gastroenterology, 75 (1978) 13-19. [ 2] Larsson, L.I., Golterman, N., De Magistris, L., Rehfeld, J.F., Schwartz, T.W., Somatostatin cell processes as pathway for paracrine secretion, Science, 205 (1979) 13931395. [ 3] Saffouri, B., Weir, G., Bitar, K., Makhlouf, G., Stimulation of gastrin secretion from the perfused rat stomach by soma tostatin antiserum, Life Sci., 25 (1979) 1749-1754. [ 4] So11, A.H., Amirian, D.A., Park, J., Cholecystokinin potently releases somatostatin from canine fundic mucosal cells in short term culture, Am. J. Physiol., 11 (1985) G569G573. [ 5] Park, J., Chiba, T., Yokotani, K., Delvalle, J., Yamada, T., Soma tostatin receptors on canine fundic D-cells. Evidence for autocrine regulation of gastric secretion, Am. J. Physiol., 257 (1989) G235-G241. [ 6] Buchan, A.M.J., Curtis, S.B., Meloche, R.M., Release of somatostatin immunoreactivity from human antral D-cells in culture, Gastroenterology, 99 (1990) 690-696. [ 7] Schmidt, W., Creutzfeldt, W., Hocken, M., Nustede, R., Choudhury, A.R., Schleser, A., Rovati, L.C., Folsch, U.R., Cholecystokinin receptor antagonist loxiglumide modulates plasma levels ofgastro-entero-pancreatic hormones in man. Feedback control of cholecystokinin and gastrin, Eur. J. Clin. Invest., 21 (1991) 501-511. [ 8] Jebbink, M.C.W., Lamers, C.B.H.W., Mooy, D.M., Rovati, L.C., Jansen JBMJ. Effect of loxiglumide on basal, gastrin and bombesin stimulated gastric acid and serum gastrin, Gastroenterology, 103 (1992) 1215-1220. [ 9] Hildebrand, P., Beglinger, C., Gyr, K., Jansen, J.B.M.J., Rovati, L.C., Zuercher, M., Lamers, C.B.H.W., Setnikar, I., Stalder, G.A., Effects of a cholecystokinin receptor antagonist on intestinal phase of pancreatic and biliary responses in man, J. Clin. Invest., 85 (1990) 640-642. [10] Meyer, B.M., Werth, B.A., Beglinger, C., Hildebrand, P., Jansen, J.B.M.J., Zach, D., Rovati, L.C., Stalder, G.A., Role of cholecysto kinin in regulation of gastrointestinal motor functions, Lancet, ii (1989) 12-15. [ 11] Jansen, J.B.M.J., Lamers, C.B.H.W., The effect of somatostatin on bombesin-stimulated serum gastrin and gastric acid in man, Digestion, 21 (1981) 193-197. [12] Reichlin, S., Somatostatin, New Engl. J. Med., 309 (1983) 1495-1501 and 1556-1563. [13] Baldiserra, F.G.A., Holst, J.J., Jensen, S.L., Krarup, T., Distribution and molecular forms of peptides containing somatostatin immunodeterminants in extracts from thc en-
[14]
[15]
[ 16]
[17]
[ 18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
tire gastrointestinal tract from man and pig, Biochim. Biophys. Acta, 838 (1985) 132-143. Jansen, J.B,M.J., Lundborg, P., Baak, L.C., Greve, J., Ohman, M., Stover, C., Rohss, K., Lamers, C.B.H.W., Effect of single and repeated dosis of omeprazole on pentagastrin stimulated gastric acid secretion and pharmacokinetics in man, Gut, 29 (1988) 75-81. Wisen, O., Uvnas-Wallensten, K., Efendic, S., Johansson, C., Release of somatostatin into the gastric lumen of healthy subjects and patients with duodenal ulcer and achlorhy dria, Acta Physiol. Scand., 108 (1980) 297-300. Jansen, J.B.M.J., Lamers, C.B.H.W., Radioimmunoassay of cholecystokinin: Production and evaluation of antibodies, J. Clin. Chem. Clin. Biochem., 21 (1983) 387-394. Jansen, J.B.M.J., Lamers, C.B.H.W., Radioimmunoassay of cholecystokinin in human tissue and plasma, Clin. Chim. Acta, 131 (1983) 305-316. Liddle, R.A., Ensinck, J.W., Cholecystokinin does not stimulate prosomatostatin-derived peptides in man, J. Clin. Endocrinol. Metab., 70 (1990) 1403-1407. Conlon, J.M., Bridgeman, M., George, K., Alberti, M.M., The nature of big plasma somatostatin: Implications for the measure merit of somatostatin-like immunoreactivity in human plasma, Anal. Biochem., 125 (1982) 243-252. Patel, Y.C., Wheatley, T., In vivo and in vitro plasma disappearance and metabolism of somatostatin-28 and somatostatin-14 in the rat, Endocrinology, 112 (1983) 220-225. Jansen, J.B.M.J., Hopman, W.P.M., Rosenbusch, G., Lamers, C.B.H.W., Plasma cholecystokinin and gallbladder responses to intraduodenal fat and intravenous cholecystokinin in patients with coeliac disease, Eur. J. Gastroenterol. Hepatol., 4 (1992) 349-353. Jansen, J.B.M.J., Jebbink, M.C.W., Mulders, H.J.A., Lamers, C.B.H.W., Effect of pancreatic enzyme supplementation on postprandial plasma cholecystokinin secretion in patients with pancreatic insufficiency, Regul. Pept., 25 (1989) 333-342. Soil, A.H., Amirian, D.A., Thomas, L.P., Reedy, T.J., Elashoff, J.D., Gastrin receptors on isolated canine parietal cells, J. Clin. Invest., 73 (1984) 1434-1447. Corazziari, E., Solomon, T.E., Grossman, M.I., Effect of ninety-five percent pure cholecystokinin on gastrin stimulated acid secretion in man and dog, Gastroenterology, 77 (1979) 91-95. Dockray, G.J., Garner, A., Hampson, S.E., Moros, S., Action ofgastrin-cholecystokinin antagonists on acid secretion in the anaesthetized rat, J. Physiol., 422 (1990) 81p. Loud, F.B., Hoist, J.J., Egense, E. et al., Is somatostatin a hormonal regulator of the endocrine pancreas and gastric acid secretion in man?, Gut, 26 (1985) 445-449. Ensinck, J.W., Vogel, R.E., Laschansky, E.C., Francis,
M.L. Verhulst et al. / Regulatory Peptides 53 (1994) 185-193
B.H., Effect of ingested carbohydrates, fat and protein on the release of somatostatin-28 in humans, Gastroenterology, 98 (1990) 633-638. [28] Francis, B.H., Baskin, D.G., Saunders, D.R., Ensinck, J.W., Distribution of somatostatin-14 and somatostatin-28 in gastrointestinal-pancreatic cells of rats and humans, Gastroenterology, 99 (1990) 1283-1289. [29] Van de Brug, F.J., Jansen, J.B.M.J., Kuijpers, l.J., Lamers, C.B.H.W., Effects of lorglumide on gastrin- and peptonestimulated gastric acid secretion in rats, Life Sci., 47 (1990) 2459-2464. [30] Hirst, B.H., Elliot, K.J., Ryder, H., Szelke, M., Inhibition of gastrin- and histamine-stimulated gastric acid secretion
193
by gastrin and cholecystokinin antagonists in the rat, Aliment. Pharmacol. Ther., 5 (1991) 31-39. [311 Konturek, S.J., 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, 94 (1988) 1014-1023. [321 Johansson, C., Wisen, O., Kollberg, B., Efendic, S., Uvnas-Wal lensten, K., Effects of intragastrically administered somatostatin on basal and pentagastrin stimulated gastric secretion in man, Acta Physiol. Scand., 104 (1979) 232-234.
Regulatory Peptides 53 (1994) 185-193
Loxiglumide inhibits cholecystokinin stimulated somatostatin secretion and simultaneously enhances gastric acid secretion in humans Marie Louise Verhulst a, Hugo A.J. Gielkens a, Wim P.M. Hopman a, Annie van Schaik a, Albert Tangerman a, Lucio C. Rovati b, Jan B.M.J. Jansen a,, aDepartment of Gastroenterology and Hepatology, University Hospital St. Radboud, Geert Grooteplein 8, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands b ROTTA research laboratories, Monza, Italy Received 30 July 1992; revised version received 27 June 1994; accepted 27 June 1994
Abstract In vitro studies have demonstrated that cholecystokinin releases somatostatin from the gastric mucosa. To date, there is no information about the in vivo significance of this finding in man. Therefore, we have studied the effect of infusion of cholecystokinin resulting in plasma concentrations within the range found after meal-stimulation, on somatostatin release and on gastric acid secretion. In addition we have studied these functions during infusion of the type A cholecystokinin receptor antagonist loxiglumide. In eight healthy subjects, basal gastric acid secretion was distinctly stimulated by cholecystokinin. The effect of cholecystokinin on gastric acid secretion was markedly enhanced by loxiglumide. Cholecystokinin also significantly stimulated somatostatin output into the gastric lumen, but not into the systemic circulation. Somatostatin output into the gastric lumen during infusion of cholecystokinin was abolished by loxiglumide. The data indicate that on the one hand circulating cholecystokinin, like gastrin, stimulates gastric acid secretion probably by binding to less specific type B receptors on parietal cells that are not blocked by loxiglumide, but on the other hand that cholecystokinin, in contrast to gastrin, also inhibits gastric acid secretion probably by binding to specific type A receptors present on somatostatin producing D-cells in the gastric mucosa, that are blocked by loxiglumide.
Keywords." Loxiglumide; Somatostatin; Cholecystokinin; Gastric acid secretion; CCK-receptor
1. Introduction Immunochemical studies have demonstrated that somatostatin (SST) is present in D-cells in the gas* Corresponding author. 0167-0115/94/$7.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 1 6 7 - 0 1 1 5 ( 9 4 ) 0 0 0 6 3 - 8
tric m u c o s a of several species, including man [ 1 ]. It has been suggested that S S T in the gastric m u c o s a acts as a paracrine substance, since D-cells were demonstrated to possess offshoots that were in close contact with adjacent cells, like parietal cells and gastrin producing cells [2,3]. In vitro, it has been
186
M.L. Verhulst et al. / Regulator), Peptides 53 (1994) 185-193
demonstrated that specific CCK type A-receptors are present on SST containing D-cells in the gastric mucosa [4-6], and that CCK is able to stimulate SST-release from the gastric mucosa [4,6]. Whether CCK, circulating in concentrations similar to those found after meals, is able to stimulate SST release in vivo, has not been demonstrated yet, although indirect evidence for a stimulating role of CCK on S ST-secretion was obtained from several studies [7-10]. Stimulation of SST by circulating CCK in concentrations as observed as after a meal, may have important consequences, since SST is a powerful inhibitor of several gastro-intestinal functions [10-12], and since SST is present in many organs [13], allowing local release by CCK. In the present study, we have infused CCK to plasma concentrations as observed as after a meal, and we have studied the effects on gastric acid output, on local release of SST into the gastric lumen, and on release of SST into the systemic circulation. In addition, we have studied the effects of the specific CCK type A-receptor antagonist loxiglumide on these parameters during infusion of CCK. It has also been shown in vivo, that loxiglumide is by far more specific for CCK type A receptors than for CCK type B/gastrin receptors, since loxiglumide is not able to influence the effects of gastrin, infused to plasma concentrations as observed as after a meal, on gastric acid secretion in humans [8], and since loxiglumide abolishes the effects of CCK on gallbladder contraction [9]. 2. Materials and methods
2.1. Experimental protocol Eight healthy subjects (6 F, 2 M; mean age 29 + 5 years) participated in two studies which were performed in random order on different days separated by at least one week. After an overnight fast, the subjects presented at the laboratory. During the studies, the subjects where seated in a comfortable chair in a recumbent position. A gastric suction tube
with a small bore perfusion catheter attached to it, was placed in the stomach. The position of the tube was checked by the water recovery technique [14]. The perfusion catheter was then pulled back about 10 cm disconnecting this tube from the suction tube. 15 min before the start of the experiments, the small bore tube was perfused with a recovery marker (phenol red-saline solution 200 ml/15 min; 3 mg/1 of phenol red) as previously described [14]. Subsequently two indwelling intravenous catheters were placed one in each fore-arm. The catheters were kept patent by a heparine-saline solution. From one catheter blood samples were drawn for determination of CCK at 15 min intervals, while saline or synthetic loxiglumide (Rotta Laboratories, Monza, Italy) dissolved in saline, was infused through the other in a dose of 7.5 mg/kg/h during the entire 150 min study period, followed by 12.5 pmol/kg/h of synthetic human CCK33 (Peninsula laboratories, San Carlos, CA, USA) dissolved in saline containing 0.5~o human serum albumin, for the last 90 min in each experiment. Gastric contents were aspirated continuously by a suction pump providing intermittent negative presure, and collected in 15 min portions. The experiments were approved by the local ethical committee and all subjects gave informed consent.
2.2. Gastric acid Gastric juice was collected into ice chilled glass bottles. Gastric acid concentrations were measured by titration to pH 7.0 with 0.1 M NaOH. After filtration and alkalinisation with 2.5 M NaOH, the concentration of phenol red was measured in the perfusion fluid and the aspirates by spectrophotometry at 560 nm. Results of acid output and SSTsecretion were corrected for losses using this recovery marker as previously described [14]. For determination of SST, gastric juice was handled according to a method that was described by Wisen et al. [15] with modifications by us. In brief, after the addition of 1 mg/ml of camostate (a
M.L. Verhulst et al. / Regulatory Peptides 53 (1994) 185-193
gift from professor Guido Adler, Ulm, Germany) to gastric juice immediately after its collection, 10 ml of each 15 min gastric aspirate was adjusted to pH 5.0 by adding 1 M NaOH or 1 M HCL, depending on the original pH of the sample, and boiled in a beaker. Within 15 min, the volume was reduced by more than 80 ~o. Distilled water was subsequently added to the boiled aspirate to obtain a total volume of 2.0 ml. These samples were centrifuged and the clear supernatants were stored at -20 °C until assayed for SST.
2.3. Radioimmunoassays Plasma CCK was measured by radioimmunoassay employing antibody T204 as previously described [16,17]. In brief, the antibody was raised in a rabbit against partly purified porcine CCK kindly provided by Prof. V. Mutt (Karolinska Institutet, Stockholm, Sweden). Synthetic CCK33 was labelled with [125I]-Bolton-Hunter reagent and used as tracer. Synthetic CCK33 was alSO used as standard. The antibody was used in a final dilution of 1:80,000 and binds equally to all carboxyl-terminal CCKpeptides containing the sulphated tyrosyl region. The antibody shows very little cross-reactivity with sulphated gastrins (<2~o), and does not bind to unsulphated gastrins or structurally unrelated peptides. Antibody bound CCK was separated from free CCK by a second antibody technique. The detection limit of the assay was 1 pmol/1 plasma. The intra-assay precision ranged from 4.6~o to 11.5~o in the working range of the assay, whereas inter-assay precision ranged from 11.3~o to 26.1~o. CCK was extracted from plasma by two volumes of ethanol, centrifuged (4000 g, 10 min), the supernatant was evaporated to dryness at 37 °C and reconstituted in the same volume of assay buffer. All samples were measured in duplicate in one assay. SST was also measured by radioimmunoassay. The antibody was harvested in a rabbit after the seventh monthly immunization with synthetic SST28 (Peninsula laboratories, San Carlos, CA, USA)
187
coupled to bovine serum albumin by the carbodiimide technique [ 16]. 33 Bq of commercially available [125I]-SST14 (DuPont-New England Nuclear, Boston, MA, USA) was used as tracer and synthetic SST14 (Peninsula laboratories, San Carlos, CA, USA) as standard. Antibody, in a final dilution of 1:500,000, [125I]-SST14 and standard SST14 or gastric sample in a total volume of 1.0 ml were incubated for 24 h at 4 ° C, after which antibody bound SST was separated from free SST by a second antibody technique. The detection limit for SST14 of the assay was 5 pmol/1. On a molar base, SST28 and SST~4 cross-reacted equally in the assay and no cross-reactivity was found with other gastro-intestinal peptides, like CCK, gastrin, VIP, substance P, secretin, bombesin, GRP, insulin, glucagon, pancreatic polypeptide, PHI, PYY, calcitonin, C-GRP and GIP. Intra- and inter-assay variations ranged from 4.6~o to 13.2~o and from 5.9~o to 20.1~o, respectively, in the working range of the assay. SST-like immunoreactivity in plasma was measured after extraction of plasma by Sep-Pak C18 cartridges [ 18]. 10 ml of gastric aspirates collected during CCK and loxiglumide infusion, that were found to have low endogenous SST concentrations, were spiked with SST14 or SST28 to 2 (n = 12), 10 (n = 12) and 250 pM (n = 1) and left for 15 min at room temperature after addition of 1 mg/ml of camostate. Subsequently the pH was brought to 5.0 before boiling. By boiling, the volume was reduced to 2.0 ml. Estimated SST-immunoreactivity after subtraction of endogenous values in the boiled samples are thus 10, 50 or 1250 pM of SSTI4 or SST28. These samples were measured by RIA and recoveries were calculated. Of the sample that was spiked to 250 pM and subsequently concentrated to 1250 pM, 0.4 ml was applied to a Sephadex G-50 SF (100 x 1 cm) column for calibration and recovery experiments. Before Sephadex G-50 SF was applied, the 100 x 1 cm glass tube was filled with 2~o dichlorodimethylsilane for 5 min and subsequently washed with methanol, in order to avoid adhesion of peptides.
188
M.L. Verhulst et al. /Regulatory Peptides 53 (1994) 185-193 15
SALINE OR LOXIGLUMIDE
[
12
CItOLECYSTOKININ
;J rj
T
r
i
I
9
6
3
0
_
i
i
i
I
I
Gastric juice from three subjects during infusion of CCK was pooled, treated according to the modified method of Wisen by which the total volume was reduced to 2 ml and fractionated by gel chromatography. The column was previously calibrated with synthetic SST28 and SST14 dissolved in elution buffer, with albumin to indicate the void volume and with 125I to indicate the total volume of the column. 0.05 M sodium phosphate pH 7.4 was used as elution buffer [ 19,20].
i
2.4. Statistical analysis
5
Results are presented as mean_+ S.E.M. Differences between data obtained in the CCK- and saline-infusion experiments and the CCK- and loxiglumide-infusion experiments were analysed for statistical significances by Wilcoxon's Rank Sumtest for integrated values and by the StudentNewman-Keuls test for individual 15 min samples. Statistical significance was defined as a two-tailed probability of less than 0.05. Integrated gastric acidand SST-outputs (mmol H ÷/h and pmol/h, respectively) were calculated by summation of the results from the last four 15-min samples during infusion of saline, during infusion of loxigtumide, during infusion of CCK and saline and during infusion of the combination of loxiglumide and CCK, respectively.
12
-k
9 -k
6 3. Results 3
30
60
90
120
150
Basal gastric acid output during saline infusion (2.1 + 0.7 mmol/h) was not significantly different (P = 0.08) from gastric acid secretion during infusion
TIME Fig. 1. (Upper panel) Plasma cholecystokinin concentrations (pM) during infusion of saline with and without 12.5 pmol/kg/h of cholecystokinin-33 (squares) and during infusion of 7.5 mg/ kg/h of loxiglumide with and without 12.5 pmol/kg/h of cholecystokinin-33 (circles) in eight healthy subjects. (Middle panel) Gastric acid output (mmol/15 min) during infusion of saline with and without cholecystokinin (open blocks) and during infusion of loxiglumide with and without cholecystokinin (closed
blocks). Asterisks indicate significant differences (P< 0.05) from corresponding samples during infusion of saline. (Lower panel) Gastric somatostatin output (pmol/15 min) during infusion of saline with and without cholecystokinin (open blocks) and during infusion of loxiglumide with and without cholecystokinin (closed blocks). Asterisks indicate significant differences (P< 0.05) from corresponding samples during infusion of loxiglumide.
M.L. Verhulst et al. / Regulatory Peptides 53 (1994) 185-193
of loxiglumide (2.7 + 0.8 mmol/h). Infusion of CCK resulting in plasma CCK-concentrations similar to those found after a meal ([21,22]; Fig. 1, upper panel), markedly stimulated gastric acid secretion from 2.1 + 0.7 mmol/h to 5.8 + 1.5 mmol/h (P< 0.01) (Fig. 1, middle panel). Loxiglumide significantly enhanced CCK-stimulated gastric acid secretion from 5.8+1.5 to 10.8+2.1 mmol/h (P<0.01) (Fig. 1, middle panel). Basal SST-output into the gastric juice (5.5 + 0.6 pmol/h) was not significantly influenced by loxiglumide (5.4 + 0.5 pmol/h) (Fig. 1, lower panel). CCK markedly stimulated SST-output into the gastric juice from 5.5 + 0.6 to 18.5 + 5.7 pmol/h (P<0.01). SST-concentrations were significantly (P<0.05) higher in all individual 15-min samples during CCK-infusion when compared to prestimulated fractions, without significant differences between the mutual stimulated fractions. This effect
70 60
Vo
28
14 vt
4.
4"
4.4.
50
30
10
•
•
/..\
189
was abolished by loxiglumide, since CCK did not significantly stimulate basal S ST-output into gastric juice during administration of loxiglumide (from 5.4 + 0.5 to 6.1 + 0.8 pmol/h) (Fig. 1, lower panel). Recovery of SST from gastric juice spiked to 250 pM with SST28 or SSTs4 and subsequently boiled by which the volume was reduced to 2 ml with an estimated SST concentration of 1250 pM, was 84Fo (1050 pM) and 78~o (980 pM), respectively. 80~o ( S S T 2 8 ) and 81~o ( S S T I 4 ) of this immunoreactivity was recovered in the positions of synthetic S ST28 and synthetic S STs4 after fractionation by gel chromatography. Recoveries of SST from gastric juice spiked with S ST28 or S ST~4 and subsequently concentrated by boiling to estimated concentrations of 10 and 50 pM, were 78 + 8~o and 88 + 5~o, respectively for SST28 and 83 _+5~o and 92 + 3~o, respectively for S S T I 4 (n = 12). 61 ~o of endogenous SST-like immunoreactivity secreted into gastric juice during infusion of CCK eluted in the position of S S T 1 4 , the remaining immunoreactivity eluted in the void volume (8 ~ ) and in the position of SST2s (31Yo) (Fig. 2). Plasma SST-like immunoreactivity was not significantly influenced by CCK-infusion (Fig. 3).
4. Discussion •. e .
•
"IIOOOOoql
0 "'" ''''~ ~' ' ' ' " 0 10 20 30 40 50 60 70 80 90 Fraction Number Fig. 2. Elution profile of pooled gastric fractions from 3 healthy subjects adjusted to pH 5 after addition ofcamostate ! mg/ml and concentrated by boiling. The fractions were collected during infusion of cholecystokinin. The extract was applied to a 100 cm Sephadex (3-50 SF column with a diameter of 1.0 cm, that was previously calibrated with SST28 [28], SST14 [14], albumin to indicate the void volume (Vo) and t25I to indicate the total volume (Vt) of the column. Fractions of 1 ml were collected and measured by radioinamunoassay for somatostatin-like immunoreactivity. The recovery of the SST-immunoreaetivity applied to the column was 80~/o.
Cholecystokinin and gastrin share a common pentapeptide in the biologically active carboxyl-terminal domain of the molecules. In vitro, CCKs and gastrin are equipotent in stimulating canine parietal cell function [23]. In an in vivo rat model, CCK s and gastrin also seem to be equipotent in stimulating gastric acid secretion [25]. However, species differences in the effects of CCK and gastrin on gastric acid secretion may exist as well as differences in acid secreting potency of different molecular forms of CCK, since in humans C C K 3 3 is much less potent in stimulating gastric acid secretion than gastrin [24]. The weak stimulation of gastric acid secretion by
190
M.L. Verhulst et al. / Regulatory Peptides 53 (1994) 185-193 30
27
LOXIGLUMIDE OR SALINE J
24 21 18 Ill
FCHOLECYSTOKININ
15
J
r~T]-TT
12
T
9 6
3 0 -30
i
0
i
i
30
i
i
i
~
60 90 Time (rain)
i
i
120
i
i
150
Fig. 3. Plasma somatostatin concentrations (pM) during infusion of saline (squares) or loxiglumide (circles) with and without cholecystokinin in eight healthy subjects. Somatostatin was extracted from plasma by SepPak cartridges according to the method described by Ensinck [19].
CCK33-infusion when compared to gastrin-infusion in humans, may also be explained by simultaneous activation of parietal cells and of cells that release an inhibitor of gastric acid secretion by CCK, and not solely from a direct diminished effect of CCK on parietal cells. However, it may also be possible that differences in the effects of CCK and gastrin on gastric acid secretion in previous in vivo experiments, are explained by differences in circulating concentrations of CCK and gastrin. Under physiologic conditions, circulating CCK concentrations are considerably lower than circulating gastrin concentrations in humans. Therefore, the effects of infusion of CCK and gastrin on gastric acid secretion can only be compared by infusing supraphysiological doses of CCK in order to obtain plasma levels that can be compared on a molar base to plasma gastrin levels in response to infusion of gastrin to concentrations as observed as after a meal. Some years ago, it was demonstrated that CCK was able to stimulate SST-release from gastric D-cells in vitro by binding to a specific type A CCKreceptor [4]. Since SST is a potent inhibitor of gas-
trin release and gastric acid secretion [26], it was hypothesized that the poor stimulation of gastric acid secretion by CCK in vivo, was related to this effect on gastric SST-release. In healthy volunteers, we have recently demonstrated that Loxiglumide is by far more selective for type A CCK-receptors, than for type B CCK/gastrin receptors, since loxiglumide does not affect gastric acid secretion stimulated by infusion of gastrin, but augments gastrin- and gastric acid responses to CCK stimulated by bombesin-infusion [8]. Indirectly, it was concluded from these studies that this augmented gastrin- and gastric acid response to bombesin was induced by specific inhibition of the effect of bombesin-stimulated CCK on SST-producing D-cells in the gastric mucosa. To date, there is no information about the effect of CCK, circulating in concentrations as observed as after a meal, on gastric SST-release in humans. One study has demonstrated that CCK, infused to plasma concentrations in the physiological range, did not stimulate the release of prosomatostatin-derived peptides into the systemic circulation [18]. Previous studies have demonstrated that SST in the systemic circulation mainly originates from the small intestinal mucosa and represents SST28 [27,28]. SST in the gastric mucosa, however, is mainly present as SST14 [13]. Furthermore, it has been demonstrated that S ST is also released into the gastric lumen [ 15]. We therefore have measured S ST both in plasma and in gastric juice, and we have demonstrated that infusion of CCK to plasma concentrations in the same range as found after a meal, potently stimulates the release of SST into the gastric lumen, but not into the systemic circulation. CCK-stimulated SST-release into the gastric lumen was abolished by loxiglumide. Our recovery experiments and chromatography data suggest that the increase of SST-like immunoreactivity in gastric juice during CCK-infusion, is indeed SST, since most of the immunoreactivity co-elutes with synthetic SSTI4. This confirms previous studies on the nature of molecular forms of SST in the gastric mucosa [ 13].
M.L. Verhulst et aL/ Regulatory Peptides 53 (1994) 185-193
Loxiglumide also markedly enhanced gastric acid output in response to CCK-infusion, but did not affect gastrin-stimulated gastric acid secretion [8]. The findings therefore suggest that stimulation of gastric acid secretion by binding of CCK to parietal cells is augmented by inhibition of concomitant CCK-stimulated SST-release from gastric D-cells by loxiglumide. At first glance, the time course of S ST-release into the gastric lumen during CCK-infusion suggests that SST might not be responsible for the augmented CCK-stimulated gastric acid secretion during infusion of loxiglumide, since there is a decline in the CCK-stimulated SST-output after an initial rise, while the enhanced gastric acid output does not show this pattern. One of the possibilities for this effect might be that, SST-immunoreactivity decreases in gastric juice due to an increased activity of peptic digestion of S ST by the augmented gastric acid output. This hypothesis is not very likely, since incubation of synthetic SST28 and SSTl4 with gastric juice collected during infusion of CCK and loxiglumide does not result in rapid degradation of the peptides. Statistical analysis, however, showed that the impression that SST in gastric juice decreases despite continuous infusion of CCK is not of significance. Therefore, the presented data are not in conflict with the hypothesis that S ST is involved in the augmented BBS-stimulated gastric acid secretion during loxiglumide infusion. Previously, we have demonstrated that loxiglumide also enhanced basal gastric acid output [8]. This effect was not confirmed in the present study. Most likely, the failure to observe a statistically significant difference in basal gastric acid output between the saline and loxiglumide experiments in the present study, is due the smaller amount of loxiglumide that was infused for a shorter period of time. Alternatively, spontaneous day to day variation in gastric acid secretion within one subject, may have attenuated the effect of loxiglumide on basal gastric acid secretion in the present study, since the experiments were performed on different days.
191
In rats, the acid secretory actions of gastrin and CCK might also be exerted at two different sites [25]. However, instead of augmentation, inhibition of CCK stimulated gastric acid secretion was observed after administration of the specific type A CCK receptor antagonist L-364,718 to anaesthetized animals, while no effect on gastrin stimulated gastric acid secretion was found [25]. On the other hand, we and others have recently found that CCK type A receptor antagonists, like lorglumide or L-364,718 also inhibit gastrin or pentagastrin stimulated gastric acid secretion in consious or anaesthetized rats [29,30]. In dogs, lorglumide has also been demonstrated to inhibit gastrin stimulated gastric acid secretion [31]. These conflicting data, may result from methodological differences, anaesthesia, differences in the doses of gastrin and CCK used, differences in actions between the various type A CCK receptor antogonists, or point to species differences in the regulation of gastric acid secretion by gastrin and CCK. From this study it cannot be concluded whether SST acts as a luminal active peptide [32] or whether the release of SST into the gastric lumen is simply a reflection of paracrine active SST [2,3]. Anyhow, the release of S ST into the gastric lumen during infusion of CCK is not simply explained by spill over of circulating SST into the gastric lumen, since our study confirms that circulating CCK in concentrations within the physiological range does not stimulate the release of SST into the systemic circulation [18]. In conclusion, the present study demonstrates that on the one hand circulating CCK, like gastrin, stimulates gastric acid secretion in humans, probably by binding to type B CCK/gastrin receptors on parietal cells, but on the other hand that cholecystokinin, in contrast with gastrin, also inhibits gastric acid secretion, probably by binding to specific type A CCK receptors present on SST producing D-cells in the gastric mucosa.
192
M.L. Verhulst et al. / Regulator), Peptides 53 H994) 185-193
References [ 1] Chayvialle, J.A.P., Descos, F., Bernard, C., Martin, A., Barbe, C., Partensky, C., Somatostatin in mucosa of stomach and duo denum in gastroduodenal disease, Gastroenterology, 75 (1978) 13-19. [ 2] Larsson, L.I., Golterman, N., De Magistris, L., Rehfeld, J.F., Schwartz, T.W., Somatostatin cell processes as pathway for paracrine secretion, Science, 205 (1979) 13931395. [ 3] Saffouri, B., Weir, G., Bitar, K., Makhlouf, G., Stimulation of gastrin secretion from the perfused rat stomach by soma tostatin antiserum, Life Sci., 25 (1979) 1749-1754. [ 4] So11, A.H., Amirian, D.A., Park, J., Cholecystokinin potently releases somatostatin from canine fundic mucosal cells in short term culture, Am. J. Physiol., 11 (1985) G569G573. [ 5] Park, J., Chiba, T., Yokotani, K., Delvalle, J., Yamada, T., Soma tostatin receptors on canine fundic D-cells. Evidence for autocrine regulation of gastric secretion, Am. J. Physiol., 257 (1989) G235-G241. [ 6] Buchan, A.M.J., Curtis, S.B., Meloche, R.M., Release of somatostatin immunoreactivity from human antral D-cells in culture, Gastroenterology, 99 (1990) 690-696. [ 7] Schmidt, W., Creutzfeldt, W., Hocken, M., Nustede, R., Choudhury, A.R., Schleser, A., Rovati, L.C., Folsch, U.R., Cholecystokinin receptor antagonist loxiglumide modulates plasma levels ofgastro-entero-pancreatic hormones in man. Feedback control of cholecystokinin and gastrin, Eur. J. Clin. Invest., 21 (1991) 501-511. [ 8] Jebbink, M.C.W., Lamers, C.B.H.W., Mooy, D.M., Rovati, L.C., Jansen JBMJ. Effect of loxiglumide on basal, gastrin and bombesin stimulated gastric acid and serum gastrin, Gastroenterology, 103 (1992) 1215-1220. [ 9] Hildebrand, P., Beglinger, C., Gyr, K., Jansen, J.B.M.J., Rovati, L.C., Zuercher, M., Lamers, C.B.H.W., Setnikar, I., Stalder, G.A., Effects of a cholecystokinin receptor antagonist on intestinal phase of pancreatic and biliary responses in man, J. Clin. Invest., 85 (1990) 640-642. [10] Meyer, B.M., Werth, B.A., Beglinger, C., Hildebrand, P., Jansen, J.B.M.J., Zach, D., Rovati, L.C., Stalder, G.A., Role of cholecysto kinin in regulation of gastrointestinal motor functions, Lancet, ii (1989) 12-15. [ 11] Jansen, J.B.M.J., Lamers, C.B.H.W., The effect of somatostatin on bombesin-stimulated serum gastrin and gastric acid in man, Digestion, 21 (1981) 193-197. [12] Reichlin, S., Somatostatin, New Engl. J. Med., 309 (1983) 1495-1501 and 1556-1563. [13] Baldiserra, F.G.A., Holst, J.J., Jensen, S.L., Krarup, T., Distribution and molecular forms of peptides containing somatostatin immunodeterminants in extracts from thc en-
[14]
[15]
[ 16]
[17]
[ 18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
tire gastrointestinal tract from man and pig, Biochim. Biophys. Acta, 838 (1985) 132-143. Jansen, J.B,M.J., Lundborg, P., Baak, L.C., Greve, J., Ohman, M., Stover, C., Rohss, K., Lamers, C.B.H.W., Effect of single and repeated dosis of omeprazole on pentagastrin stimulated gastric acid secretion and pharmacokinetics in man, Gut, 29 (1988) 75-81. Wisen, O., Uvnas-Wallensten, K., Efendic, S., Johansson, C., Release of somatostatin into the gastric lumen of healthy subjects and patients with duodenal ulcer and achlorhy dria, Acta Physiol. Scand., 108 (1980) 297-300. Jansen, J.B.M.J., Lamers, C.B.H.W., Radioimmunoassay of cholecystokinin: Production and evaluation of antibodies, J. Clin. Chem. Clin. Biochem., 21 (1983) 387-394. Jansen, J.B.M.J., Lamers, C.B.H.W., Radioimmunoassay of cholecystokinin in human tissue and plasma, Clin. Chim. Acta, 131 (1983) 305-316. Liddle, R.A., Ensinck, J.W., Cholecystokinin does not stimulate prosomatostatin-derived peptides in man, J. Clin. Endocrinol. Metab., 70 (1990) 1403-1407. Conlon, J.M., Bridgeman, M., George, K., Alberti, M.M., The nature of big plasma somatostatin: Implications for the measure merit of somatostatin-like immunoreactivity in human plasma, Anal. Biochem., 125 (1982) 243-252. Patel, Y.C., Wheatley, T., In vivo and in vitro plasma disappearance and metabolism of somatostatin-28 and somatostatin-14 in the rat, Endocrinology, 112 (1983) 220-225. Jansen, J.B.M.J., Hopman, W.P.M., Rosenbusch, G., Lamers, C.B.H.W., Plasma cholecystokinin and gallbladder responses to intraduodenal fat and intravenous cholecystokinin in patients with coeliac disease, Eur. J. Gastroenterol. Hepatol., 4 (1992) 349-353. Jansen, J.B.M.J., Jebbink, M.C.W., Mulders, H.J.A., Lamers, C.B.H.W., Effect of pancreatic enzyme supplementation on postprandial plasma cholecystokinin secretion in patients with pancreatic insufficiency, Regul. Pept., 25 (1989) 333-342. Soil, A.H., Amirian, D.A., Thomas, L.P., Reedy, T.J., Elashoff, J.D., Gastrin receptors on isolated canine parietal cells, J. Clin. Invest., 73 (1984) 1434-1447. Corazziari, E., Solomon, T.E., Grossman, M.I., Effect of ninety-five percent pure cholecystokinin on gastrin stimulated acid secretion in man and dog, Gastroenterology, 77 (1979) 91-95. Dockray, G.J., Garner, A., Hampson, S.E., Moros, S., Action ofgastrin-cholecystokinin antagonists on acid secretion in the anaesthetized rat, J. Physiol., 422 (1990) 81p. Loud, F.B., Hoist, J.J., Egense, E. et al., Is somatostatin a hormonal regulator of the endocrine pancreas and gastric acid secretion in man?, Gut, 26 (1985) 445-449. Ensinck, J.W., Vogel, R.E., Laschansky, E.C., Francis,
M.L. Verhulst et al. / Regulatory Peptides 53 (1994) 185-193
B.H., Effect of ingested carbohydrates, fat and protein on the release of somatostatin-28 in humans, Gastroenterology, 98 (1990) 633-638. [28] Francis, B.H., Baskin, D.G., Saunders, D.R., Ensinck, J.W., Distribution of somatostatin-14 and somatostatin-28 in gastrointestinal-pancreatic cells of rats and humans, Gastroenterology, 99 (1990) 1283-1289. [29] Van de Brug, F.J., Jansen, J.B.M.J., Kuijpers, l.J., Lamers, C.B.H.W., Effects of lorglumide on gastrin- and peptonestimulated gastric acid secretion in rats, Life Sci., 47 (1990) 2459-2464. [30] Hirst, B.H., Elliot, K.J., Ryder, H., Szelke, M., Inhibition of gastrin- and histamine-stimulated gastric acid secretion
193
by gastrin and cholecystokinin antagonists in the rat, Aliment. Pharmacol. Ther., 5 (1991) 31-39. [311 Konturek, S.J., 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, 94 (1988) 1014-1023. [321 Johansson, C., Wisen, O., Kollberg, B., Efendic, S., Uvnas-Wal lensten, K., Effects of intragastrically administered somatostatin on basal and pentagastrin stimulated gastric secretion in man, Acta Physiol. Scand., 104 (1979) 232-234.