Gastrin Receptor Antagonist Research Articles

Gastrin and gastrin receptor antagonists bind to both N- and C-terminal halves of the 78 kDa gastrin-binding protein

A 78 kDa gastrin-binding protein (GBP) has previously been identified as the target of the anti-proliferative effects of non-selective gastrin/cholecystokinin receptor antagonists on colorectal carcinoma cell lines. The GBP was related in sequence to a family of fatty acid oxidation enzymes possessing enoyl CoA hydratase and 3-hydroxyacyl CoA dehydrogenase activity. This study aims to define the binding site for gastrin and gastrin antagonists in greater detail. The N- and C-terminal halves of the porcine GBP were expressed independently as glutathione S-transferase fusion proteins in E. coli. Affinities of gastrin and gastrin antagonists for the fusion proteins were measured by competition for 125I-[Nle 15]-gastrin 2,17 binding in a covalent cross-linking assay. The N- and C-terminal fusion proteins bound gastrin 17 with affinities of 9.9 ± 6.1 and 71 ± 48 μM, respectively ( n = 3). These values were 40-fold and 300-fold lower than the affinity of the full-length GBP for gastrin 17 (0.23 ± 0.15 μM). In contrast, the affinities of the N- and C-terminal halves for the antagonists proglumide (22 ± 13 and 10 ± 4 mM, respectively) and benzotript (350 ± 90 and 400 ± 160 μM, respectively) were similar to each other and to the affinities of proglumide and benzotript for the full-length GBP (5.1 ± 3.6 mM and 200 ± 120 μM, respectively). It is concluded that proglumide and benzotript bind independently to both the hydratase and dehydrogenase active sites of the GBP, while a single molecule of gastrin 17 may bind simultaneously to both active sites. A model is proposed which is consistent with these data, and which will assist in the development of more potent and selective GBP antagonists.

Visualization and characterization of CCK receptors in exocrine pancreas of rat with storage phosphor autoradiography.

Studies on cholecystokinin (CCK) receptors in rat pancreas are usually performed on homogenates. Using storage phosphor autoradiography, a new imaging technique with a high sensitivity and large linear dynamic range, we visualized and characterized CCK receptors in tissue sections of normal rat pancreas. The density of CCK receptors in pancreatic tissue sections from 10 normal rats appeared to be unevenly distributed and variable in serial sections. The binding of labeled CCK-8 was markedly inhibited by CCK-8 and CCK-A receptor antagonists, but it was only weakly affected by gastrin and CCK-B receptor antagonists. At room temperature the CCK-8 dose-inhibition curve was fitted by a two-site model: one with a high-affinity but low-capacity site and another with a low-affinity but high-capacity site. The CCK-8 dose-inhibition curve showed that the inhibition of the variable high-density receptors took place at a low concentration of CCK-8, while the diffuse low-density receptors were inhibited at the high concentration of 1 microM CCK-8. Binding of labeled CCK-8 at 37 degrees C was homogeneous with a low affinity and comprised only 4% of that found at room temperature. In summary, an uneven density of CCK receptors in the rat exocrine pancreas was observed and attributed to the variable expression of high-affinity CCK receptors in pancreatic acini.

Evaluation of the specificity and potency of a series of cholecystokinin-B/gastrin receptor antagonists in vivo.

The potency and specificity of five proposed cholecystokinin-B receptor antagonists, YM022, RP73870, L-740,093, L-365,260 and LY288513, were studied in rats and mice. Gastrin activates rat stomach histidine decarboxylase via cholecystokinin-B/gastrin receptors. To examine cholecystokinin-B receptor-mediated effects of the five drugs, they were infused intravenously to fasted rats and the histidine decarboxylase activity in the oxyntic mucosa was determined. While YM022, RP73870, L-740,093 and L-365,260 failed to activate histidine decarboxylase, they dose-dependently antagonized the gastrin-induced histidine decarboxylase activation. LY288513 had no effect in the doses tested. The maximal inhibitory effect of L-365,260, L-740,093, RP73870 and YM022 on histidine decarboxylase, activated by the intravenous infusion of an ED50 does of gastrin (0.4 nmoles/kg/hr), was seen at doses of 3, 0.3, 0.1 and 0.1 mumoles/kg/hr, respectively; the corresponding ID50 values were 0.4, 0.02, 0.007 and 0.004 mumoles/kg/h. In a follow-up study, YM022 and RP73870 were found to produce a rightward shift of the gastrin dose-response curve, which is consistent with competitive inhibition. The effect of the five drugs on a cholecystokinin-A receptor-mediated response was examined by studying gastric emptying in mice. Cholecystokinin-8s, given by a subcutaneous bolus injection, dose-dependently inhibits gastric emptying. The specific cholecystokinin-A receptor antagonist devazepide (given intravenously as a bolus injection) antagonized the effect of cholecystokinin-8s in a dose-dependent manner, with an ID50 value of 28 nmoles/kg. None of the drugs inhibited the gastric emptying or prevented the cholecystokinin-8s-induced effect at the doses tested. The results indicate that YM022, RP73870, L-740,093 and L-365,260 act as cholecystokinin-B receptor antagonists in vivo, being without measurable agonistic activity. Furthermore, they do not interact with cholecystokinin-A receptors at te doses tested. Among the cholecystokinin-B receptor antagonists studied YM022 and RP73870 are superior, the rank order of potency being YM022 > or = RP73870 > L-740,093 > L-365,260.

Pentagastrin selectively modulates levels of mRNAs encoding apical H/K adenosine triphosphatase and basolateral Na−K−Cl cotransporter in rat gastric fundic mucosa

Gastrin regulates gastric acid secretion and gastric mucosal cell proliferation. We hypothesized that pentagastrin administration would affect mRNA levels of two membrane proteins that are important during stimulated states of HCl secretion, the basolateral Na-K-Cl cotransporter (BSC) and the apical H/K adenosine triphosphatase (H/K). Two groups of Fischer rats received intraperitoneal injections of pentagastrin (2.5 or 25 micrograms/kg) every 8 hours for three doses. A third group served as controls. An additional group received pentagastrin plus the gastrin receptor antagonist (GRA) L740,093. Fundic mucosae were subjected to semiquantitative Northern analysis of mRNAs encoding H/K and BSC. The mRNA for Na/K adenosine triphosphatase (Na/K), a transport protein not involved directly in acid secretion, also was evaluated. Administration of pentagastrin caused dose-dependent increases in levels of mRNAs encoding H/K and BSC but had no significant effect on levels of Na/K mRNA. Administration of GRA prevented the pentagastrin-induced changes in mRNA levels for these transporters. Pentagastrin administration selectively up-regulates levels of mRNA encoding membrane proteins involved in acid secretion. The up-regulation of the mRNAs encoding BSC during pentagastrin stimulation indicates that regulation of basolateral Cl- movement may be as important as the regulation of apical H+ movement under stimulated states.

Synthesis and pharmacological evaluation of highly potent dual histamine H 2 and gastrin receptor antagonists

The chemical modification of the dual histamine H 2 and gastrin receptor antagonists described in our preceding paper, particularly the modification of spacers as well as the alteration of their connecting bonds at the gastrin receptor antagonist site (GA) from the amide bond to the carbamate bond, significantly improved not only their dual activity but also the GA versus CCK-A receptor selectivity.

Design, synthesis, and pharmacological evaluation of dual histamine H 2 and gastrin receptor antagonists

The joint type of hybrid molecules composed of two pharmacophore moieties taken from histamine H 2 and gastrin receptor antagonists have been designed and synthesized to exhibit dual histamine H 2 and gastrin receptor antagonistic activities. Here we report the importance of spacers as well as binding sites of both pharmacophores for the dual activity.

Antiproliferative effect of a novel cholecystokinin-B/gastrin receptor antagonist, YM022.

Cholecystokinin (CCK)‐B and gastrin receptors are expressed on a variety of human tumor cells. Recently, we have demonstrated that the human brain CCK‐B receptors are identical to the gastrin receptors derived from the stomach mucosa, and that the brain‐gut peptides, CCK‐8 and gastrin I are mitogenic for mouse NIH 3T3 fibroblasts expressing human CCK‐B/gastrin receptors (N‐hCCKBR). In this report, we evaluated the antiproliferative potency of CCK‐B/gastrin receptor antagonists by using N‐hCCKBR cells. Among several antagonists, a benzodiazepine derivative, YM022 had the most potent activities in competing with [125I]CCK‐8 or [125I]gastrin I binding, inhibition of CCK‐8‐ or gastrin I‐induced phosphoinositide hydrolysis and increasing cytoplasmic free calcium. Interestingly, a potent antagonist for rat CCK‐B/gastrin receptors did not have such activities in N‐hCCKBR cells. YM022 inhibited the CCK‐8‐ or gastrin I‐induced [methyl‐3H]thymidine incorporation of N‐hCCKBR cells in a dose‐dependent manner. In the absence of exogenous peptide ligands, YM022 also inhibited the proliferation of several human cancer cell lines expressing the genes for both gastrin and its receptor. These results suggest that YM022 could intervene in the autocrine stimulation of human tumor cell lines through CCK‐B/gastrin receptors. N‐hCCKBR cells are an excellent tool to screen for novel human CCK‐B/gastrin receptor antagonists possessing antiproliferative activity for human cancer cells.

Cytosolic Ca 2+ evaluation in rabbit parietal cells: a novel method to screen gastrin receptor antagonists

We have evaluated the application of the fura-2 method to detect cytosolic Ca 2+ increase in gastric cells expressing CCK B/gastrin receptors, in order to screen gastrin receptor antagonists, as an alternative to functional studies. We have characterized the receptors on parietal cell suspension from rabbit gastric mucosa and validated the method using both the CCK B and CCK A receptor agonists and antagonists. Human gastrin I (gastrin) (0.1 nM–4 μM) and sulfated cholecystokinin 26–33 (CCK-8) (0.01 nM–2 μM) dose-dependently augmented cytosolic Ca 2+. The efficacies of the two agonists were similar, but the potency of CCK-8 (EC 50 1.03 nM) was about 10-fold greater than that of gastrin (11 nM). Response to a submaximal dose of gastrin (50 nM) was dose-dependently blocked by the CCK B-receptor antagonists CAM-1028 (4-[[2-[[3-(1 H-indol-3-yl)-2-methyl-1-oxo-2-[[[1,7,7-trimethylbicyclo[2.2.1]hept-2-yl)oxy]carbonyl]amino]propyl]amino]-1-phenylethyl]amino-4-oxo-[1 (3 R(+)- N-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1 H-1,4-benzodiazepin-3-yl)- N′-(3-methylphenyl)urea) (IC 50 10 nM) and spiroglumide (( R)-4-(3,5-dichlorobenzamido)-5-(8-azaspiro[4.5]decan-8-yl)-5-oxopentanoic acid) (IC 50 2 μM). The results were in agreement with those obtained from binding studies in guinea-pig cortical membranes. The model was employed to optimize the synthesis of a new class of spiroglumide analogues which led to a new molecule, ( S)-4-{( R)-4′-(3,5-dichlorobenzoylamino)-5′-(8-azaspiro[4.5]decan-8-yl)-5′-oxo}-pentanoylamino-5-(1-naphthylamino)-5-oxopentanoic acid (CR 2622), whose potency was about 100-fold greater than that of spiroglumide. CR 2622, as well as the other CCK B receptor antagonists tested, exhibited no effect on basal [Ca 2+] i. The simplicity and the reproducibility of this method suggest that it is a useful model to screen gastrin and antigastrin activity in parallel or as an alternative to binding studies.

Gastrin receptor antagonist CI-988 inhibits growth of human colon cancer in vivo and in vitro.

Whilst gastrin has been found to be trophic for some colorectal cancer cell lines, and gastrin receptor antagonists are able to block this phenomenon, their potency has been modest. The effect of a new, potent and selective CCK B receptor antagonist, CI-988 on the growth of LoVo, a human colon cancer cell line both in vitro and in vivo was instigated. Basal growth of LoVo in vitro was inhibited by up to 58.93 +/- 7.30% with concentrations of CI-988 as low as 1 X 10(-11) mol/L whereas the addition of gastrin (G17) at 0.5 nmol/L had no effect. LoVo was also grown in vivo for 10 days in nude mice subsequently treated with CI-988 at 10 mg/kg per day orally for 20 days. CI-988 inhibited the growth of xenografts by 53%. This was the first study in cancer with this potent gastrin receptor antagonist, CI-988. The results suggest that CI-988 may be of use in inhibiting the growth of colorectal cancer.

Regulation of peptide YY homeostasis by gastric acid and gastrin.

Peptide YY (PYY) is a gut hormone localized primarily in the distal bowel. Because circulating PYY inhibits gastric acid secretion, we investigated the effects of gastric acid secretion and gastrin on gene expression and secretion of PYY. In conscious dogs, PYY release in response to oral food was inhibited (P < 0.05) by pharmacologic inhibition of gastric acid secretion (omeprazole, famotidine). In rats, omeprazole treatment resulted in a significant elevation in serum gastrin concentrations and a simultaneous decrease in PYY messenger RNA (mRNA) and peptide levels in the colon; administration of a gastrin receptor antagonist (L365, 260) prevented the inhibitory actions of omeprazole on colonic PYY mRNA levels. In athymic-nude mice, implantation of a human gastrinoma resulted in an elevation of serum gastrin concentrations and a concomitant depression of colonic PYY mRNA levels. We conclude that endogenous gastric acid secretion up-regulates PYY release and PYY mRNA expression. Circulating gastrin acts to down-regulate PYY release and PYY mRNA expression. This study provides evidence that foregut functions (i.e., gastric acid secretion and gastrin release) exert control over an antiacid signal (e.g. PYY release) emanating from the hindgut.

ChemInform Abstract: Improving the Affinity and Selectivity of a Nonpeptide Series of Cholecystokinin‐B/Gastrin Receptor Antagonists Based on the Dibenzobicyclo(2.2.2)octane Skeleton.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Effect of endogenous hypergastrinemia on gastrin receptor expressing human colon carcinoma transplanted to athymic rats

Background & Aims The effect of endogenous hypergastrinemia on growth of human colon carcinoma is not known. Our aim was to study the growth of human colon carcinoma in an animal model with endogenous hypergastrinemia. Methods Human colon carcinoma was transplanted to the colon of 40 athymic rats. Of these, 25 underwent gastric fundectomy to accomplish endogenous hypergastrinemia, and 15 were sham operated to serve as controls. The duration of the study was 8 weeks. During the last week, 12 fundectomized animals received a gastrin (cholecystokinin B) receptor antagonist. Metaphase arrest index, local invasion, and distant spread of the tumor were investigated. Expression of gastrin and cholecystokinin B receptor messenger RNA was examined by reverse-transcription polymerase chain reaction. Results Tumor spread by direct extension outside the colon was observed in all animals, and liver metastases were observed in 10 of the 25 fundectomized animals. Sham-operated animals showed none of these features. The metaphase arrest index of the tumor did not differ between fundectomized animals given the cholecystokinin B receptor antagonist and sham-operated animals, whereas it was significantly increased in fundectomized animals not given the antagonist. The tumor expressed both gastrin and cholecystokinin B receptor messenger RNA. Conclusions The results indicate that endogenous hypergastrinemia may promote proliferation and spread of human colon carcinoma expressing cholecystokinin B receptor.

Gastrin sensitivity of primary human colorectal cancer: the effect of gastrin receptor antagonism.

The purpose of this study was to determine the effect of the gastrin receptor antagonist, CR2093, on basal and gastrin-stimulated growth of primary human colorectal adenocarcinomas and to relate this to gastrin receptor expression. Tumour cells, derived from surgical specimens by enzymatic disaggregation, were grown on matrices of type I collagen and irradiated fibroblasts. Gastrin receptor expression was measured by using a mouse monoclonal antibody directed against the gastrin receptor and an avidin-biotin immunocytochemical method. Increased growth in the presence of gastrin-17 (used at physiological concentrations and as assessed by [3H] thymidine uptake) was shown in 16/34 (47%) tumours. CR2093 significantly reversed this stimulated growth (P < 0.05, one way analysis of variance) in 9/16 (56.3%) of the tumours and inhibited the basal growth of 11/34 (32.4%). Basal growth inhibition was reversed by gastrin-17 in 82% (9/11) of tumours. Gastrin receptor expression was widespread, but was not related to the degree of growth response to gastrin, and there was no significant correlation between intensity of receptor expression and inhibition of basal growth by CR2093. In conclusion, both gastrin-stimulated and basal growth of primary human colorectal can be inhibited by gastrin receptor antagonism, but gastrin receptor expression does not predict the sensitivity of tumours to (i) the proliferative effects of gastrin or (ii) the inhibitory effects of a gastrin receptor antagonist on basal growth. Antigastrin agents may have clinical value in the treatment of gastrin-sensitive colorectal tumours, and gastrin receptor expression may be related to endogenous gastrin production by colorectal tumour cells.

Early signalling mechanism in colonic epithelial cell response to gastrin.

The hormone gastrin exerts a growth-promoting effect on gastrointestinal cells. The molecular mechanisms by which colonic epithelial cells respond to gastrin are still poorly understood. In this study, we demonstrate a novel feature of the action of gastrin on normal colonic cells, namely the rapid phosphorylation on tyrosine of phospholipase C gamma 1 (PLC gamma 1). Tyrosine phosphorylation of PLC gamma 1, elicited by gastrin, was transient, concentration-dependent, and was abrogated by pretreating the colonic cells with the gastrin-receptor antagonist proglumide, the tyrosine kinase inhibitor genistein, and by removal of the tyrosine phosphatase inhibitor orthovanadate from the isolation buffer. Tyrosine phosphorylation of PLC gamma 1 correlated with the time- and concentration-dependent decrease in the mass of membrane phosphatidylinositol 4,5-bisphosphate (PIP2) and the increase in the epithelial concentration of inositol 1,4,5-trisphosphate (IP3). Likewise, the stimulated increase in IP3 was also prevented by proglumide and genistein. Gastrin induced a definite but transient increase in the intracellular concentration of free Ca2+ [Ca2+]i, and increased membrane-translocation of immunoreactive alpha- and beta-protein kinase C. The data thus indicate that gastrin elicits at least one signalling cascade, through rapid tyrosine phosphorylation of PLC gamma 1, leading to the activation of a PIP2-specific PLC pathway.

Effect of gastrointestinal hormones and synthetic analogues on the growth of pancreatic cancer.

The effects of hormones and synthetic analogues have been examined on the growth of 2 human pancreatic cancer cell lines, MiaPaCa2 a well-established cell line and PANI which was derived in our own laboratories from a tumour specimen. The hormones/growth factors included gastrin (G-17), epidermal growth factor (EGF) and bombesin, while the synthetic analogues used were a gastrin receptor antagonist (CR 1718), a somatostatin analogue (RC-160) and a bombesin receptor antagonist (ICI 216,140). Cell proliferation was assessed by the [75Se]selenomethionine uptake method which has been shown to correlate with cell counts. The effect of each hormone or growth factor on growth was expressed as a percentage of the untreated control. There were 5 replicates in each experiment, and each one was repeated at least 3 times. In vitro growth of both cell lines was unaffected by gastrin, bombesin or the respective antagonists (CR1718 and ICI 216140). The somatostatin analogue RC-160 also had no effect on basal growth. Significant growth stimulation of both MiaPaCa2 and PANI was seen with epidermal growth factor. We tested the hypothesis that somatostatin analogues may inhibit EGF-stimulated growth on both MiaPaCa2, a somatostatin receptor positive cell line, and on PANI which is negative for somatostatin receptors. RC-160 did not inhibit EGF-stimulated growth of either MiaPaCA2 or PANI. Both cell lines were established in vivo as xenografts in nude mice. The effect of RC-160 on tumour growth was measured. RC-160 inhibited the growth of MiaPaCa2, the somatostatin receptor-positive cell line, but not of PANI.

Mobilization of gastric histamine during repeated administration of a proton potassium adenosine triphosphatase inhibitor in intact and antrectomized rats

Intact and antrectomized female rats were treated with the potent proton pump inhibitor, E3810 (daily 40 mg/kg weight, s.c.) for 4 weeks. Plasma gastrin concentration and urinary excretion of N-terminal big gastrin increased until day 14 and persisted at a high level in intact rats treated with E3810, but did not increase in antrectomized rats. Urinary excretion of histamine increased progressively and reached 7 times the control value following 4 weeks of treatment with E3810 in intact rats, but not in antrectomized rats. At the termination of the treatment, the endocrine cell density in the oxyntic mucosa of intact rats had increased by 85% with increased histamine content and elevated histidine decarboxylase activity, while antrectomized rats showed a low histamine level and low histidine decarboxylase activity. Administration of gastrin-17 I (10 μg/kg weight, sc) itself caused a significant increase in urinary excretion of histamine, which was inhibited by the specific gastrin receptor antagonist, L-365,260. These results suggests that the massive urinary excretion of histamine caused by the treatment with E3810 reflects gastrin-induced mobilization of gastric histamine and that neither E3810 itself nor E3810-induced luminal pH elevation has direct effects on mobilization of oxyntic mucosal histamine.

Stimulatory effect of muscimol on gastric acid secretion stimulated by secretagogues in vagotomized rats under anesthesia

The effect of intravenous administration of muscimol, a GABA A receptor agonist, on gastric acid secretion from perfused stomach was studied in vagotomized rats anesthetized with urethane. Muscimol did not stimulate acid secretion by itself. In contrast, muscimol dose dependently potentiated acid secretion induced by pentagastrin, bethanechol and direct vagal stimulation, but not histamine. Muscimol-potentiated acid secretion induced by pentagastrin and bethanechol was not influenced by pretreatment with atropine or cimetidine, respectively. Muscimol-potentiated acid secretion evoked by direct vagal stimulation was prevented by pretreatment with proglumide, a gastrin receptor antagonist. Muscimol-potentiated acid secretion evoked by bethanechol was dose dependently prevented by bicuculline methiodide, suggesting an involvement of peripheral GABA A receptors. These results suggest that muscimol stimulates acid secretion under certain conditions, and that two mechanisms are involved in this effect. The effects of muscimol on acid secretion may be mediated by increasing the release of histamine by pentagastrin, bethanechol and direct vagal stimulation. In addition, muscimol would also be effective if muscarinic agents were already occupying muscarinic acetylcholine receptors on parietal cells.

Effect of CCK-B Gastrin Receptor Antagonist on Pepsinogen-Producing Cells During Omeprazole Treatment

The present study investigated the effect of long-term treatment with omeprazole on pepsinogen-producing cells and examined whether the selective CCK-B/gastrin receptor antagonist was able to prevent the omeprazole-induced changes, if occurred, in rat stomach. Rats were treated with omeprazole and/or the CCK-B/gastrin receptor antagonist for 28 days. As a result, omeprazole markedly reduced mucosal pepsinogen activity and its mRNA concentration in rat stomach. Morphologically, in fundic glands omeprazole drastically decreased the proportion of mature chief cells and reciprocally increased that of immature chief cells which were positive for class III mucin. These effects of omeprazole were attenuated by an addition of the CCK-B/gastrin receptor antagonist. Our results suggest that omeprazole retards the differentiation of chief cells in fundic mucosa probably through hypergastrinemia in adult rat.

Characterization of Cholecystokinin Receptors and Messenger RNA Expression in Rat Pancreas: Evidence for Expression of Cholecystokinin-A Receptors but Not Cholecystokinin-B (Gastrin) Receptors

It has been previously demonstrated that guinea pig pancreas possesses both cholecystokinin-A (CCK-A) receptors and CCK-B (gastrin) receptors. In contrast to guinea pig pancreas, it is not known whether CCK receptors in rat pancreas are CCK-A receptors, CCK-B (gastrin) receptors, or both. Thus, in the present study, we characterized CCK receptors in rat pancreas at the receptor and mRNA level. 125I-Bolton-Hunter-labeled CCK octapeptide ( 125I-BH-CCK-8), the specific CCK-A and CCK-B (gastrin) receptor antagonists L364,718 and L365,260, and 125I-labeled gastrin-I were utilized to characterize CCK receptors in normal rat pancreas. Additionally, we utilized 32P-labeled cDNA probes of the CCK-A receptor and CCK-B (gastrin) receptor coding regions in order to examine the expression of CCK receptor subtypes in normal rat pancreas at the mRNA level. The dose-inhibition curve of CCK-8 inhibiting binding of 125I-BH-CCK-8 was significantly best fit by a two-site model with a high-affinity site ( K d = 0.68 ± 0.13 n M) and a low-affinity site ( K d = 656 ± 289 n M). L364,718 inhibited binding of 125 I-BH-CCK-8 with high affinity, whereas no high-affinity inhibition for L365,260 to inhibit binding of 125I-BH-CCK-8 was detected. L364,718 was 627 times as potent as L365,260 in inhibiting binding of 125 I-BH-CCK-8. No saturable binding was present for 125 I-labeled gastrin-I. Gastrin-17-I did not inhibit binding of 125 I-BH-CCK-8. The CCK-A receptor mRNA was identified in normal rat pancreas using Northern blot analysis and reverse transcription-polymerase chain reaction. However, no CCK-B (gastrin) receptor mRNA was detected by these techniques. In conclusion, normal rat pancreas expresses two classes of CCK receptors, i.e., high-affinity receptor and low-affinity receptor. All of the CCK receptors are CCK-A receptors and no CCK-B (gastrin) receptors appear to be present. This study suggests that the rat is a pure in vivo model for studying the biological activity of the CCK-A receptor in the pancreas.

Inhibitory effects of the gastrin receptor antagonist CR2093 on basal, gastrin-stimulated and growth factor-stimulated growth of the rat pancreatic cell line AR42J

AR42J, a rat pancreatic cell line expressing receptors for both gastrin and epidermal growth factor (EGF), has been used to examine the effect of the gastrin receptor antagonist CR2093 on basal, gastrin-17 (G17), EGF and transforming growth factor (TGF)-alpha stimulated growth in vitro. In serum-free conditions, CR2093 reduced the basal growth of AR42J at a concentration known to displace physiological levels of G17 from gastrin receptors and this effect was reversed by G17 at 1 x 10(-9) M. Alone, G17 had little effect on growth, but EGF and TGF-alpha stimulated growth to 181 and 176% of control values, respectively, and marked synergy was observed when G17 was used in combination with both EGF (212%) and TGF-alpha (259%). When CR2093 was added, the synergistic effects of the G17/EGF and G17/TGF-alpha combinations were reduced to basal levels. In addition, CR2093 inhibited the growth stimulation induced by EGF and TGF-alpha alone. When the ability of CR2093 to bind to EGF receptors was assessed in a ligand binding assay, it was found that the antagonist displaced up to 23% of labeled EGF. Thus CR2093 has potent inhibitory effects on the basal growth of AR42J which can be reversed by G17. It can also inhibit type 1 growth factor-stimulated growth, but although this action may in part be related to the antagonist's ability to inhibit binding to the EGF receptor, other mechanisms may be involved.