Cholecystokinin Receptor Subtype Research Articles

Elucidating the Structure-Activity Relationship of the Pentaglutamic Acid Sequence of Minigastrin with Cholecystokinin Receptor Subtype 2.

Derivatized minigastrin analogues make up a promising class of candidates for targeting cholecystokinin receptor subtype 2 (CCK2R), which is overexpressed on cancer cells of various neuroendocrine tumors. The pentaglutamic acid sequence of minigastrin influences its biological properties. In particular, it plays a crucial role in the kidney reuptake mechanism. However, the importance of the binding affinity and interaction of this region with the receptor on a molecular level remains unclear. To elucidate its structure-activity relationship with CCK2R, we replaced this sequence with various linkers differing in their amount of anionic charge, structural characteristics, and flexibility. Specifically, a flexible aliphatic linker, a linker with only three d-Glu residues, and a structured linker with four adjacent β3-glutamic acid residues were evaluated and compared to the lead compound PP-F11N (DOTA-[d-Glu1-6,Nle11]gastrin-13). 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was conjugated to the minigastrin derivatives, which allowed radiolabeling with Lutetium-177. The levels of In vitro internalization into MZ-CRC1 cells and in vivo tumor uptake as well as human blood plasma stability increased in the following order: aliphatic linker < three d-Glu < (β3-Glu)4 < (d-Glu)6. The in vitro and in vivo behavior was therefore significantly improved with anionic charges. Computational modeling of a CCK2 receptor-ligand complex revealed ionic interactions between cationic residues (Arg and His) of the receptor and anionic residues of the ligand in the linker.

Structural studies on radiopharmaceutical DOTA-minigastrin analogue (CP04) complexes and their interaction with CCK2 receptor

BackgroundThe cholecystokinin receptor subtype 2 (CCK-2R) is an important target for diagnostic imaging and targeted radionuclide therapy (TRNT) due to its overexpression in certain cancers (e.g., medullary thyroid carcinoma (MTC)), thus matching with a theranostic principle. Several peptide conjugates suitable for the TRNT of MTC have been synthesized, including a very promising minigastrin analogue DOTA-(DGlu)6-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2 (CP04). In this contribution, we wanted to see whether CP04 binding affinity for CCK-2R is sensitive to the type of the complexed radiometal, as well as to get insights into the structure of CP04-CCK2R complex by molecular modeling.ResultsIn vitro studies demonstrated that there is no significant difference in CCK-2R binding affinity and specific cellular uptake between the CP04 conjugates complexed with [68Ga]Ga3+ or [177Lu]Lu3+. In order to investigate the background of this observation, we proposed a binding model of CP04 with CCK-2R based on homology modeling and molecular docking. In this model, the C-terminal part of the molecule enters the cavity formed between the receptor helices, while the N-terminus (including DOTA and the metal) is located at the binding site outlet, exposed in large extent to the solvent. The radiometals do not influence the conformation of the molecule except for the direct neighborhood of the chelating moiety.ConclusionsThe model seems to be in agreement with much of structure-activity relationship (SAR) studies reported for cholecystokinin and for CCK-2R-targeting radiopharmaceuticals. It also explains relative insensitivity of CCK-2R affinity for the change of the metal. The proposed model partially fits the reported site-directed mutagenesis data.

Two cholecystokinin receptor subtypes are identified in goldfish, being the CCKAR involved in the regulation of intestinal motility

Cholecystokinin (CCK) plays a key role in the digestive physiology of vertebrates. However, very little is known about the role of CCK on intestinal functions in fish. The present study identifies two CCK receptor subtypes in a stomachless teleost, the goldfish (Carassius auratus), and investigates by using an in vitro system their involvement mediating the effects of the sulfated octapeptide of CCK (CCK-8S) on the motility of isolated proximal intestine. Partial-length mRNAs encoding two CCK receptor isoforms (CCKAR and CCKBR.I) were sequenced and the structural analysis showed that both receptors belong to the G-protein coupled receptor superfamily. Both goldfish CCK receptor sequences were more closely related to zebrafish sequences, sharing the lowest similarities with cavefish and tilapia. The highest expression of goldfish CCKAR was observed along the whole intestine whereas the CCKBR gen was predominantly expressed in the hypothalamus, vagal lobe and posterior intestine. Application of CCK-8S to the organ bath evoked a concentration-dependent contractile response in intestine strips. The contractions were not blocked by either tetrodotoxin or atropine, suggesting that CCK-8S acts on the gut smooth muscle directly. Preincubations of intestine strips with devazepide and L365,260 (CCKAR and CCKBR receptor selective antagonists) showed that the CCK-8S-induced contraction could be partially mediated by the CCKAR receptor subtype, which is also the most abundant CCK receptor found in gastrointestinal tissues. In conclusion, two CCK receptors with a differential distribution pattern has been identified in goldfish, and the CCKAR subtype is mainly involved in the regulation of intestinal motility by the CCK-8S.

Preclinical evaluation of gastrin derivatives labelled with &lt;sup&gt;111&lt;/sup&gt;In: Radiolabelling, affinity profile and pharmacokinetics in rats

Cholecystokinin receptor subtype 2 (CCK-2) is overexpressed in various tumours like medullary thyroid carcinomas and small cell lung cancer. Radiolabelled peptides that bind with high affinity and specificity to CCK-2 receptors, thus hold great potential for visualizing such tumours. We compared four 111In labelled gastrin analogues, called minigastrins (MG), namely MG11, MG45, MG47 and MG48 linked to metal chelating DOTA in preclinical experiments. The radiolabelled peptides were tested for peptide binding in CCK-2 receptor-bearing cell line AR42J and for their pharmacokinetics in normal rats. The experiments suggest that all gastrin analogues had similar and relatively rapid internalization into AR42J cells. Binding to CCK-2 receptors in AR42J cells was saturable for all agents but there were some differences in receptor affinity. This biodistribution study in rats showed a rapid decrease in blood radioactivity, predominantly renal clearance and saturable uptake of the radiopharmaceutical and/or its metabolites in the CCK-2 receptor-positive stomach. Higher kidney accumulation of radioactivity was only found for 111In-DOTA-minigastrin 48. The data suggest that the 111In-DOTA-minigastrin analogues studied are promising candidates for the scintigraphy of CCK-2 receptor-expressing tumours; 111In-DOTA-MG47 and 111In-DOTA-MG11 are the most promising.

Preclinical Evaluation of Radiolabeled DOTA-Derivatized Cyclic Minigastrin Analogs for Targeting Cholecystokinin Receptor Expressing Malignancies

Targeting of cholecystokinin receptor expressing malignancies such as medullary thyroid carcinoma is currently limited by low in vivo stability of radioligands. To increase the stability, we have developed and preclinically evaluated two cyclic 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-minigastrin analogs radiolabeled with (111)In and (68)Ga. Radiolabeling efficiency, in vitro characterization, cholecystokinin receptor subtype 2 (CCK-2) binding in human tumor tissues, and cell internalization on CCK-2 receptor expressing AR42J cells, as well as biodistribution and small animal imaging in two different mouse xenograft models were studied. High receptor affinity and receptor-mediated uptake of the radioligands in AR42J cells was confirmed in vitro. (111)In-labeled cyclic DOTA-peptides showed a specific tumor uptake of ~1% ID/g in vivo, (68)Ga-labeled analogs of ~3% ID/g. Small animal SPECT imaging resulted to be superior with (111)In-DOTA-cyclo-MG2 in comparison with (111)In-DOTA-cyclo-MG1. Cyclic DOTA-minigastrin analogs are promising candidates for gastrin receptor scintigraphy and targeted radionuclide therapy.

Development of a new radioligand for cholecystokinin receptor subtype 2 scintigraphy: From molecular modeling to in vivo evaluation

To improve the targeting to tumors expressing the cholecystokinin receptor subtype 2 (CCK2R) with limited kidney uptake, we synthesized a novel cholecystokinin C-terminal tetrapeptide (CCK4)-based derivative conjugated to an original bipyridine-chelator (BPCA), 111In-BPCA-(Ahx)2-CCK4. To our knowledge this is the first CCK4-based radioligand that presents a high affinity for the CCK2R, a high and specific tumor uptake, a low renal accumulation and a very good visualization of tumors in vivo compared with an internal control, 111Indium-trans-cyclohexyldiethylenetriaminepenta-acetic acid-cholecystokinin octapeptide (111In-CHX-A″-DTPA-CCK8). These properties make 111In-BPCA-(Ahx)2-CCK4, a promising candidate for imaging and peptide receptor radionuclide therapy of CCK2R positive tumors.

Anthranilic sulfonamide CCK1/CCK2 dual receptor antagonists I: Discovery of CCKR1 selectivity in a previously CCKR2-selective lead series

A series of CCK2R-selective anthranilic amides is shown to derive CCK1R affinity via selective substitution of the amide side chain. Thus, extending the length of the original benzamide side chain by a single methylene unit imparts CCK1R affinity to the series, and further fine tuning of the affinity results in CCK1R selectivity of greater than 100-fold.

Intestinal Cholecystokinin Controls Glucose Production through a Neuronal Network

Cholecystokinin (CCK) is a peptide hormone that is released from the gut in response to nutrients such as lipids to lower food intake. Here we report that a primary increase of CCK-8, the biologically active form of CCK, in the duodenum lowers glucose production independent of changes in circulating insulin levels. Furthermore, we show that duodenal CCK-8 requires the activation of the gut CCK-A receptor and a gut-brain-liver neuronal axis to lower glucose production. Finally, duodenal CCK-8 fails to lower glucose production in the early onset of high-fat diet-induced insulin resistance. These findings reveal a role for gut CCK that lowers glucose production through a neuronal network and suggest that intestinal CCK resistance may contribute to hyperglycemia in response to high-fat feeding.

CCK 2 receptors mediate inhibitory effects of cholecystokinin on the motor activity of guinea-pig distal colon

Cholecystokinin and related peptides are involved in the control of intestinal motility and cholecystokinin receptor ligands might represent new pharmacological tools for the treatment of symptoms associated with functional bowel disorders. However, the respective roles played by cholecystokinin receptor subtypes and the mechanisms underlying these regulatory actions remain undetermined. This study was designed to examine the influence of cholecystokinin receptor subtypes on the motor activity of guinea-pig distal colon. The effects of drugs acting on CCK 1 and CCK 2 receptors were assessed in vitro on the contractile activity of longitudinal smooth muscle, both under basal conditions and in the presence of transmural electrical stimulation or KCl-induced contractions. The application of cholecystokinin octapeptide sulphate (cholecystokinin-8S) to colonic preparations induced concentration-dependent contractions which were prevented by devazepide (CCK 1 receptor antagonist), enhanced by GV150013 (CCK 2 receptor antagonist) or N ω -nitro- l-arginine methylester ( l-NAME, nitric oxide synthase inhibitor), and unaffected by tetrodotoxin. The application of gastrin-17 to colonic preparations resulted in relaxant responses which were insensitive to devazepide, and prevented by GV150013, l-NAME or tetrodotoxin. l-NAME, N ω -propyl- l-arginine (NPA, neuronal nitric oxide synthase inhibitor) or GV150013 enhanced electrically evoked contractile responses, whereas devazepide did not. When tested in the presence of l-NAME or NPA the enhancing effect of GV150013 on electrically induced contractions no longer occurred. In the presence of KCl-induced pre-contractions, cholecystokinin-8S or gastrin-17 evoked concentration-dependent relaxations, which were unaffected by devazepide and were counteracted by GV150013, l-NAME, NPA or tetrodotoxin. In conclusion, the present results indicate that, at level of distal colon, CCK 1 receptors mediate direct contractile effects on smooth muscle, whereas CCK 2 receptors on enteric neurons mediate relaxant responses via nitric oxide release.

Haemodynamic and Exocrine Effects of Caerulein at Submaximal and Supramaximal Levels on the Rat Pancreas: Role of Cholecystokinin Receptor Subtypes

Background: We have investigated the involvement of cholecystokinin (CCK) receptor subtypes in haemodynamic changes in the pancreas of anaesthetized rats during submaximal and supramaximal stimulation with the CCK analogue, caerulein. Methods: For submaximal stimulation, caerulein (0.4 nmol/kg/h) was infused intravenously, while acute pancreatitis was induced by intraperitoneal injections of high doses of caerulein (3 × 25 nmol/kg). Pancreatic blood flow was measured by hydrogen clearance. Results: Low caerulein doses increased pancreatic blood flow by 26 ± 8% and vascular conductance by 24 ± 4%. This effect was mimicked by the CCK₂ agonist gastrin-17. All effects were abolished by a CCK₂ antagonist while a CCK₁ antagonist remained inactive. Conversely, amylase output by caerulein was abolished by CCK₁ receptor blockade, but not by inhibition of CCK₂ receptors. During caerulein-induced pancreatitis, vascular conductance increased by 109 ± 26% and remained elevated throughout the experiment; vascular flow initially increased by 62 ± 27% and then returned to baseline. The vascular effects were prevented by a CCK₂ receptor antagonist, while the induction of pancreatitis was due to CCK₁ receptor stimulation. Conclusions: Caerulein increases pancreatic vascular flow via activation of CCK₂ receptors. This effect occurs both at submaximal and at supramaximal levels of exocrine stimulation.

Cross-interactions of Two p38 Mitogen-activated Protein (MAP) Kinase Inhibitors and Two Cholecystokinin (CCK) Receptor Antagonists with the CCK1 Receptor and P38 MAP Kinase

Although SB202190 and SB203580 are described as specific p38 MAP kinase inhibitors, several reports have indicated that other enzymes are also sensitive to SB203580. Using a pharmacological approach, we report for the first time that compounds SB202190 and SB203580 were able to directly and selectively interact with a G-protein-coupled receptor, namely the cholecystokinin receptor subtype CCK1, but not with the CCK2 receptor. We demonstrated that these compounds were non-competitive antagonists of the CCK1 receptor at concentrations typically used to inhibit protein kinases. By chimeric construction of the CCK2 receptor, we determined the involvement of two CCK1 receptor intracellular loops in the binding of SB202190 and SB203580. We also showed that two CCK antagonists, L364,718 and L365,260, were able to regulate p38 mitogen-activated protein (MAP) kinase activity. Using a reporter gene strategy and immunoblotting experiments, we demonstrated that both CCK antagonists inhibited selectively the enzymatic activity of p38 MAP kinase. Kinase assays suggested that this inhibition resulted from a direct interaction with both CCK antagonists. Molecular modeling simulations suggested that this interaction occurs in the ATP binding pocket of p38 MAP kinase. These results suggest that SB202190 and SB203580 bind to the CCK1 receptor and, as such, these compounds should be used with caution in models that express this receptor. We also found that L364,718 and L365,260, two CCK receptor antagonists, directly interacted with p38 MAP kinase and inhibited its activity. These findings suggest that the CCK1 receptor shares structural analogies with the p38 MAP kinase ATP binding site. They open the way to potential design of either a new family of MAP kinase inhibitors from CCK1 receptor ligand structures or new CCK1 receptor ligands based on p38 MAP kinase inhibitor structures.

Library Generation via Postcondensation Modifications of Isocyanide‐Based Multicomponent Reactions

AbstractFor Abstract see ChemInform Abstract in Full Text.

Library Generation via Postcondensation Modifications of Isocyanide-Based Multicomponent Reactions

Publisher Summary This chapter focuses on a particular branch of multicomponent condensation reaction (MCR) methodologies— namely, the postcondensation modifications (or secondary reactions) of isocyanide-based multicomponent reactions (IMCRs). One attractive feature of the multicomponent reaction is the relative ease of its automation. Discrete reactions may be run in parallel by either solution- or solid-phase protocols in a standard 96-well format. One of the methodologies discussed in the chapter is the UDC (Ugi/De-Boc/Cyclize) methodology. The biological utility of 1,4-benzodiazepine-2,5-diones (BDPs) appears in many areas, including the applications as antagonists of the platelet glycoprotein IIb-IIIa, anticonvulsant agents, antihypnotic agents, reverse transcriptase inhibitors, and selective cholecystokinin (CCK) receptor subtype A or B antagonists. The chapter presents three miscellaneous postcondensation modification reactions: (1) TMSN 3 modified Ugi Reactions, (2) postcondensation Passerini reactions, and (3) TMSN 3 -modified Passerini reaction. The TMSN 3 -modified Ugi reaction involves the condensation of an appropriately substituted aldehyde or ketone with a primary or secondary amine.

Stimulation of either cholecystokinin receptor subtype reduces while antagonists potentiate or sensitize a morphine-induced excitatory response

Cholecystokinin peptides (CCK) have been shown to antagonize many opioid-mediated effects. The present study was undertaken to determine whether peripheral injections of cholecystokinin sulphated octapeptide (CCK8), cholecystokinin tetrapeptide (CCK4), the CCK 1 (lorglumide) and the CCK 2 (PD-135,158 and LY-225910) receptor antagonists can influence a classic morphine excitatory effect, i.e. the display of Straub tail reaction in mice (STR). A total of 570 female Balb/C mice were tested. Experiment 1 was undertaken to determine whether i.p. injections of CCK8 or CCK4 can influence STR. Each animal was treated with i.p. injections of saline or CCK8 (10 and 20 nmol/kg) or CCK4 (20 and 40 nmol/kg). After 30 min all animals received an i.p. injection of morphine hydrochloride (10.0 mg/kg). The highest doses of both CCK8 (35% STR) and CCK4 (40% STR) significantly reduced STR as compared to saline (85% STR) treated mice (Fisher test; P < 0.01). In experiment 2 each animal was treated with ip injections of saline or 1.0 mg/kg lorglumide or PD-135,158 fifteen minutes before an injection of morphine at doses ranging from 1.0 to 50.0 mg/kg. In experiment 3 animals were treated with injections of saline, 0.1 or 10.0 mg/kg lorglumide or LY-225910 before an injection of a fixed MC dose (2.0 mg/kg). Both lorglumide and PD-135,158 induced a significant shift to the left in the morphine dose-response curves as well as a significant decrease in ED50 of the STR. ED50 for lorglumide was significantly lower than ED50 for PD-135,158. Both doses of lorglumide and the highest dose of LY-225910 significantly increased the percent of animals displaying STR. Experiment 4 was undertaken to determine whether repeated peripheral injections of morphine or the morphine-potentiating agents CCK 1 (lorglumide) and the CCK 2 (LY-225910) receptor antagonists can induce morphine sensitization. Each animal was treated with 5 daily i.p. injections of saline (control group), 1.5 mg/Kg morphine hydrochloride (group morphine), and 1.0 mg/Kg lorglumide (group LOR) or LY-225910 (group LY). One, two, three and four weeks after the last treatment day, all animals were challenged with one i.p. injection of morphine (1.5 mg/Kg). The morphine, LOR groups and group LY showed a significant increase in percentage of animals displaying STR. These data demonstrate that the blockade of endogenous CCK actions leads to morphine sensitization probably through both CCK receptors. The present data are consistent with the antagonistic effects of CCK and opioids in the control of morphine-induced STR. In addition, these results suggest that both CCK receptors are involved in the modulatory effects of CCK on this morphine effect.

Activation of phospholipase C by cholecystokinin receptor subtypes with different G-protein-coupling specificities in hormone-secreting pancreatic cell lines

Phospholipase C (PLC) activity was investigated by stimulation of membrane preparations obtained from insulin (β-TC3)-, somatostatin (Rin 1027-B2)-, and glucagon (INR1-G9)-producing pancreatic cell lines using the non-hydrolyzable GTP analogue GTPγS alone, the C-terminal octapeptide cholecystokinin (CCK-8), or gastrin. All compounds caused a significant 2- to 4.4-fold stimulation of PLC activity in the different cell lines, which was diminished by the non-hydrolyzable GDP analogue GDPβS. CCK receptor subtypes were characterized by radioligand binding experiments. High-affinity binding sites for tritiated CCK A receptor antagonist L-364,718 ( K d = 0.24 nM) and tritiated CCK B receptor antagonist L-365,260 ( K d = 0.13 nM) were only present in Rin 1027-B2 cells. High-affinity binding sites for both ligands were not found in β-TC3 or INR1-G9 cells. Competition binding experiments with non-labeled CCK receptor antagonists CR 1505 (CCK A receptor-selective) and CR 2945 (CCK B receptor-selective), as well as microphysiometry experiments, resulted in the same receptor distribution. Reverse transcriptase–polymerase chain reaction confirmed the CCK receptor distribution pattern for Rin 1027-B2 cells, but in addition showed the existence of CCK B receptors in β-TC3 cells. Immunoblocking experiments with C-terminal antibodies against different G-protein α-subunits demonstrated inhibition of CCK-stimulated PLC activity in β-TC3 cells by G q/11α antiserum (70%), in Rin 1027-B2 cells by G q/11α antiserum (70%) and G i-3α antiserum (23%), and in INR1-G9 cells by G q/11α antiserum (60%) and G oα antiserum (45%). We conclude that CCK receptor subtypes with different G-protein-coupling specificities to PLC are present in the different hormone-secreting cells of the endocrine pancreas.

Potentiation of opioid-induced conditioned place preference by the selective serotonin reuptake inhibitor fluoxetine

The ability of the selective serotonin reuptake inhibitor, fluoxetine, to modify the effects of morphine, N-(( S)-2-benzyl-3[( S) 2-amino-4-methylthio)butyldithio-]-1-oxopropyl)- l-alanine benzylester (RB 120; mixed inhibitor of enkephalin metabolism), and 4-{[2-[[3-(1 H-indol-3-yl))-2-methyl-1-oxo-2-[[(tricyclo[3,3,1,1] dec-2-yloxy) carbonyl] amino} propyl] amino]-1-phenylethyl] amino}-4-oxo-[ R-( R*, R*)]-butanoate N-methyl- d-glucamine (CI 988; cholecystokinin receptor subtype [CCK 2] antagonist), was assessed using conditioned place preference. RB 120 and morphine both induced significant, dose-dependent conditioned place preference, whilst CI 988 failed to elicit conditioned place preference. A subthreshold dose of fluoxetine (2.5 mg/kg) potentiated the morphine submaximal response. Notably, the combination of a subthreshold dose of fluoxetine (2.5 mg/kg) with RB 120 (5 mg/kg) or CI 988 (3 mg/kg) was devoid of any significant conditioned place preference properties. Fluoxetine may act via enhanced serotonergic activity to modulate enkephalinergic tone. Agents that increase enkephalinergic tone more directly such as RB 120 and CI 988, at submaximal doses, did not induce conditioned place preference when co-administered with fluoxetine. These data suggest that fluoxetine, in combination with CI 988 or RB 120, might prove to be a beneficial treatment strategy for opioid drug addiction, though further studies are necessary.

Differential Role of Cholecystokinin Receptor Subtypes in Opioid Modulation of Ongoing Maternal Behavior

MIRANDA-PAIVA, C. M. AND L. F. FELICIO. Differential role of cholecystokinin receptor subtypes in opioid modulation of ongoing maternal behavior. PHARMACOL BIOCHEM BEHAV. 64(1) 165–169, 1999.—Cholecystokinin (CCK) can have effects opposite those of opioids. The present study was undertaken to determine whether peripheral injections of antagonists of the CCK 1 receptor (lorglumide) and the CCK 2 receptor (L-365,260) can influence the effects of morphine on maternal behavior during lactation. A total of 110 female Wistar rats were tested on days 5 and 6 postpartum. Groups were randomly assigned to morphine vehicle (MV—SC) + saline (S—IP), MV + lorglumide (LOR: 1.0 or 10.0 mg/kg), MV + L-365,260 (10 mg/kg), morphine chlorhydrate (MC: 7.0 mg/kg) + S, MC + LOR (1.0 or 10.0 mg/kg), and MC + L-365,260 (1.0 or 10 mg/kg). Maternal behavior testing was started 30 min after the injections, at which time pups were placed in the home cage of their mother. Latencies for retrieval, grouping, and crouching responses were scored. The results show that both lorglumide and L-365,260 potentiated the MC-induced inhibition of maternal behavior. In addition L-365,260 treatment alone inhibited maternal behavior. Blockade of both the CCK 1 and CCK 2 receptors potentiated the morphine-induced disruption of maternal behavior, while CCK 2 antagonism alone also inhibited this behavior. The results suggest that CCK antagonism of opioid-induced disruption of maternal behavior occurs due to the action of CCK on both CCK 1 and CCK 2 receptor subtypes.

Expression of cholecystokinin-B/gastrin receptors in medullary thyroid cancer.

To characterise the cholecystokinin (CCK) receptor subtypes in medullary thyroid cancer by measuring the expression of CCK-A and CCK-B/gastrin receptor mRNA. Open study. Teaching hospital, Sweden. 6 patients with medullary thyroid cancer. Pentagastrin stimulation test and measurement of calcitonin concentration. Biopsy specimens were analysed using reverse transcription polymerase chain reaction (RT-PCR). Presence of CCK-A and CCK-B/gastrin receptors. All 6 patients with medullary thyroid cancer had positive pentagastrin tests preoperatively. CCK-B/gastrin receptors but not CCK-A receptors were detected by RT-PCR in all six biopsy specimens. By contrast, no CCK receptors were found in normal thyroid tissues or in other thyroid tumours (follicular adenoma, papillary carcinoma, or anaplastic carcinoma). The presence of CCK-B/gastrin receptors in medullary thyroid tumours may have important clinical implications.

CCK 1 and CCK 2 receptors regulate gastric pepsinogen secretion

The present study investigated (1) the pharmacological profile of cholecystokinin (CCK) receptor subtypes involved in the regulation of gastric pepsinogen secretion, (2) the influence of gastric acidity on peptic responses induced by CCK-8-sulfate (CCK-8S) or gastrin-I; and (3) the mechanisms accounting for the effects of CCK-like peptides on pepsinogen secretion. In anaesthetized rats, i.v. injection of CCK-8S or gastrin-I increased both pepsinogen and acid secretion. The pepsigogue effect of CCK-8S was higher than that of gastrin-I, whereas acid hypersecretion after CCK-8S was lower than that induced by gastrin-I. Peptic output following CCK-8S was partly blocked by i.v. injection of the CCK 1 receptor antagonist, devazepide (−75.3%), or the CCK 2 receptor antagonist, l-365,260 [3 R(+)- N-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1 H-1,4-benzodiazepine-3yl)- N′-(3-methyl-phenyl)urea; −27.9%], but was fully prevented by combined administration of devazepide and l-365,260. The gastric acid hypersecretory effect of CCK-8S was enhanced by devazepide (+84.5%) and blocked by l-365,260. In contrast, the gastric secretory actions of gastrin-I were insensitive to devazepide, but abolished by l-365,260. Excitatory effects of CCK-8S and gastrin-I were not modified by vagotomy or atropine, whereas cimetidine or α-fluoromethylhistidine (irreversible blocker of histidine decarboxylase) partly prevented acid hypersecretion induced by both peptides without affecting their pepsigogue effects. After pretreatment with omeprazole, both CCK-8S and gastrin-I failed to stimulate acid secretion, while they increased pepsinogen output. In rats with gastric perfusion of acid solutions, CCK-8S or gastrin-I increased peptic output in a pH-independent manner either with or without pretreatment with omeprazole. Ablation of capsaicin-sensitive sensory nerves as well as application of lidocaine to the gastric mucosa failed to modify the excitatory effects of CCK-8S or gastrin-I on pepsinogen and acid secretion. Blockade of the nitric oxide (NO) synthase pathway by N G-nitro- l-arginine-methyl ester prevented the pepsigogue actions of both CCK-8S and gastrin-I (−61.8% and −71.7%, respectively), without affecting the concomitant increase in acid output. In addition, both these peptides significantly increased the release of NO breakdown products into the gastric lumen. The present results suggest that: (1) both CCK 1 and CCK 2 receptors mediate the peptic secretory responses induced by CCK-like peptides; (2) the excitatory inputs of CCK-8S and gastrin-I to chief cells are not driven through acid-dependent mechanisms or capsaicin-sensitive afferent sensory nerves; and (3) under in vivo conditions, the stimulant actions of CCK-like peptides on pepsinogen secretion are mediated, at least in part, by an increase in NO generation.

The cholecystokinin-A receptor mediates inhibition of food intake yet is not essential for the maintenance of body weight.

Food intake and body weight are determined by a complex interaction of regulatory pathways. To elucidate the contribution of the endogenous peptide cholecystokinin, mice lacking functional cholecystokinin-A receptors were generated by targeted gene disruption. To explore the role of the cholecystokinin-A receptor in mediating satiety, food intake of cholecystokinin-A receptor-/- mice was compared with the corresponding intakes of wild-type animals and mice lacking the other known cholecystokinin receptor subtype, cholecystokinin-B/gastrin. Intraperitoneal administration of cholecystokinin failed to decrease food intake in mice lacking cholecystokinin-A receptors. In contrast, cholecystokinin diminished food intake by up to 90% in wild-type and cholecystokinin-B/gastrin receptor-/- mice. Together, these findings indicate that cholecystokinin-induced inhibition of food intake is mediated by the cholecystokinin-A receptor. To explore the long-term consequences of either cholecystokinin-A or cholecystokinin-B/gastrin receptor absence, body weight as a function of age was compared between freely fed wild-type and mutant animals. Both cholecystokinin-A and cholecystokinin-B/gastrin receptor-/- mice maintained normal body weight well into adult life. In addition, each of the two receptor-/- strains had normal pancreatic morphology and were normoglycemic. Our results suggest that although cholecystokinin plays a role in the short-term inhibition of food intake, this pathway is not essential for the long-term maintenance of body weight.