Cholecystokinin-Induced Duodenogastric Bile Reflux Increases the Severity of Indomethacin-Induced Gastric Antral Ulcers in Re-fed Mice.

BACKGROUND/AIMS: We examined the involvement of cholecystokinin (CCK) in the exacerbation of indomethacin (IND)-induced gastric antral ulcers by gastroparesis caused by atropine or dopamine in mice. METHODS: Male mice were fed for 2 h (re-feeding) following a 22-h fast. Indomethacin (IND; 10 mg/kg, s.c.) was administered after re-feeding; gastric lesions were examined 24 h after IND treatment. In another experiment, mice were fed for 2 h after a 22-h fast, after which the stomachs were removed 1.5 h after the end of the feeding period. Antral lesions, the amount of gastric contents, and the gastric luminal bile acids concentration were measured with or without the administration of the pro- and antimotility drugs CCK-octapeptide (CCK-8), atropine, dopamine, SR57227 (5-HT3 receptor agonist), apomorphine, lorglumide (CCK1 receptor antagonist), ondansetron, and haloperidol alone and in combination. RESULTS: IND produced severe lesions only in the gastric antrum in re-fed mice. CCK-8, atropine, dopamine, SR57227 and apomorphine administered just after re-feeding increased bile reflux and worsened IND-induced antral lesions. These effects were significantly prevented by pretreatment with lorglumide. Although atropine and dopamine also increased the amount of gastric content, lorglumide had no effect on the delayed gastric emptying provoked by atropine and dopamine. Both ondansetron and haloperidol significantly inhibited the increase of bile reflux and the exacerbation of antral lesions induced by atropine and dopamine, respectively, but did not affect the effects of CCK-8. CONCLUSIONS: These results suggest that CCK-CCK1 receptor signal increases bile reflux during gastroparesis induced by atropine and dopamine, exacerbating IND-induced antral ulcers.

Ligand recognition and G-protein coupling selectivity of cholecystokinin A receptor.

Cholecystokinin A receptor (CCKAR) belongs to family A G-protein-coupled receptors and regulates nutrient homeostasis upon stimulation by cholecystokinin (CCK). It is an attractive drug target for gastrointestinal and metabolic diseases. One distinguishing feature of CCKAR is its ability to interact with a sulfated ligand and to couple with divergent G-protein subtypes, including Gs, Gi and Gq. However, the basis for G-protein coupling promiscuity and ligand recognition by CCKAR remains unknown. Here, we present three cryo-electron microscopy structures of sulfated CCK-8-activated CCKAR in complex with Gs, Gi and Gq heterotrimers, respectively. CCKAR presents a similar conformation in the three structures, whereas conformational differences in the 'wavy hook' of the Gα subunits and ICL3 of the receptor serve as determinants in G-protein coupling selectivity. Our findings provide a framework for understanding G-protein coupling promiscuity by CCKAR and uncover the mechanism of receptor recognition by sulfated CCK-8.

Molecular cloning of cholecystokinin (CCK) and CCK-A receptor and mechanism of CCK-induced gastrointestinal motility in Suncus murinus.

Cholecystokinin (CCK) is a peptide hormone mainly secreted by small intestinal endocrine I-cells and functions as a regulator of gallbladder contraction, gastric emptying, gastrointestinal (GI) motility, and satiety. The cellular effects of CCK in these peripheral tissues are predominantly mediated via CCK-A receptors which are found in smooth muscles, enteric neurons, and vagal afferent neurons in humans and animal models. Although various functions of CCK have been reported to be neurally mediated, it can also stimulate contraction via the CCK receptor on the smooth muscle. However, the entire underlying neural and cellular mechanisms involved in CCK-induced GI contractions are not clearly understood. Here, we first determined the cDNA and amino acid sequences of CCK and CCK-A receptor along with the distributions of cck mRNA and CCK-producing cells in house musk shrew (Suncus murinus, the laboratory strain named as suncus) and examined the mechanism of CCK-induced contraction in the GI tract. Mature suncus CCK-8 was identical to other mammalian species tested here, and suncus CCK-A receptor presented high nucleotide and amino acid homology with that of human, dog, mouse, and rat, respectively. Suncus CCK mRNA and CCK-producing cells were found mainly in small intestine and colon. In the organ bath study, CCK-8 induced dose-dependent contractions in the suncus stomach, duodenum, and jejunum, and these contractions were inhibited by atropine and CCK-A receptor antagonist. These results suggest that CCK-8-induced contraction is mediated in the myenteric cholinergic neural network and that CCK-A receptor is partly responsible for CCK-8-induced contractions. This study indicates that suncus is a useful animal model to study the functions of CCK involved in GI motility.

Effects of TAK-639, a novel topical C-type natriuretic peptide analog, on intraocular pressure and aqueous humor dynamics in mice.

Primary open-angle glaucoma (POAG) is a leading cause of irreversible blindness, and individuals with ocular hypertension are at risk to develop POAG. Currently, the only modifiable risk factor for glaucoma progression is lowering of intraocular pressure (IOP). A novel mechanism for lowering IOP involves activation of the type B natriuretic peptide receptor (NPR-B), the naturally occurring agonist of which is C-type natriuretic peptide (CNP). Being a cyclic peptide of 22 amino acids, CNP does not readily penetrate the cornea and its ocular hypotensive effect requires intraocular injection. TAK-639 is a synthetic, cornea-permeable, 9-amino acid CNP analog has been studied for the treatment of ocular hypertension and POAG. We assessed TAK-639 in a receptor binding profile and the effects of TAK-639 on NPR-B-mediated cyclic GMP production in cultured transformed human trabecular meshwork (TM) cells (GTM-3). We also evaluated the effects of topical ocular administration of TAK-639 on mouse IOP and aqueous humor dynamics. Among 89 non-natriuretic peptide receptors, transporters, and channels evaluated, TAK-639 at 10 µM displaced ligand binding by more than 50% to only two receptors: the type 2 angiotensin receptor (IC50 = 8.2 µM) and the cholecystokinin A receptor (IC50 = 25.8 µM). In vitro, TAK-639 selectively activates NPR-B (EC50 = 61 ±â€¯11 nM; GTM-3 cells) relative to NPR-A (EC50 = 2179 ±â€¯670 nM; 293T cells). In vivo, TAK-639 lowered mouse IOP by three mechanisms: increase in aqueous humor outflow facility (C), reduction in the aqueous humor formation rate (Fin), and reduction in episcleral venous pressure (Pe). The maximum mean IOP decreases from baseline were -12.1%, -21.0%, and -36.1% for 0.1%, 0.3%, and 0.6% doses of TAK-639, respectively. Maximum IOP-lowering effect was seen at 2 h, and the duration of action was >6 h. With TAK-639 0.6%, at 2 h post-dose, aqueous outflow facility (C) increased by 155.8%, Fin decreased by 41.0%, the uveoscleral outflow rate (Fu) decreased by 52.6%, and Pe decreased by 31.5% (all p < 0.05). No ocular adverse effects were observed. TAK-639 is an efficacious IOP-lowering agent, with a unique combination of mechanisms of action on both aqueous formation and aqueous outflow facility. Further study of this mechanism of treatment may optimize pharmacologic outcomes and provide disease management in patients with POAG and ocular hypertension.

Dual-agonist occupancy of orexin receptor 1 and cholecystokinin A receptor heterodimers decreases G-protein-dependent signaling and migration in the human colon cancer cell line HT-29.

The orexin (OX1R) and cholecystokinin A (CCK1R) receptors play opposing roles in the migration of the human colon cancer cell line HT-29, and may be involved in the pathogenesis and pathophysiology of cancer cell invasion and metastasis. OX1R and CCK1R belong to family A of the G-protein-coupled receptors (GPCRs), but the detailed mechanisms underlying their functions in solid tumor development remain unclear. In this study, we investigated whether these two receptors heterodimerize, and the results revealed novel signal transduction mechanisms. Bioluminescence and Förster resonance energy transfer, as well as proximity ligation assays, demonstrated that OX1R and CCK1R heterodimerize in HEK293 and HT-29 cells, and that peptides corresponding to transmembrane domain 5 of OX1R impaired heterodimer formation. Stimulation of OX1R and CCK1R heterodimers with both orexin-A and CCK decreased the activation of Gαq, Gαi2, Gα12, and Gα13 and the migration of HT-29 cells in comparison with stimulation with orexin-A or CCK alone, but did not alter GPCR interactions with ß-arrestins. These results suggest that OX1R and CCK1R heterodimerization plays an anti-migratory role in human colon cancer cells.

Intragastric nutrient infusion reduces motivation for food in male and female rats.

The idea that gut-derived satiation signals influence food reward has recently gained traction, but this hypothesis is largely based on studies focused on neural circuitry, not the peripherally released signals. Here, we directly tested the hypothesis that intragastric (IG) nutrient infusion can suppress motivation for food. In a series of experiments, IG sucrose infusion (15 kcal) significantly and reliably reduced operant responding for a sucrose reward on a progressive ratio (PR) schedule. Moreover, food deprivation for 24 h before the test session did not prevent the suppressive effect of nutrients. The suppressive effect of IG sucrose on fixed ratio 5 (FR5) operant responding was also assessed as a comparison. The effect of IG nutrients to reduce motivation was not limited to sucrose; IG Ensure infusion (9.3 kcal) also significantly reduced PR operant responding for sucrose pellets. To verify that these effects were not secondary to the osmotic challenge of concentrated nutrients, we tested IG infusion of noncaloric saline solutions equiosmolar to 40% sucrose or Ensure and found no effect. Finally, we focused on glucagon-like peptide-1 (GLP-1) and cholecystokinin (CCK) as candidate mediators for the effect of IG nutrients. Pretreatment with exendin-9, a GLP-1 receptor antagonist, delivered intraperitoneally, significantly attenuated the ability of IG nutrients to suppress PR responding and breakpoint in males, but not in females, whereas pretreatment with devazepide, a CCKA receptor antagonist, failed to do so in both sexes. Together, these data support the idea that nutrient-induced satiation signals influence food reward and may implicate GLP-1 in this process.

A GLP-1:CCK fusion peptide harnesses the synergistic effects on metabolism of CCK-1 and GLP-1 receptor agonism in mice.

Combination approaches for the treatment of metabolic diseases such as obesity and diabetes are becoming increasingly relevant. Co-administration of a glucagon-like peptide-1 receptor (GLP-1R) agonist with a cholecystokinin receptor-1 (CCKR1) agonist exert synergistic effects on weight loss in obese rodents. Here, we report on the effects of a novel fusion peptide (C2816) comprised of a stabilized GLP-1R agonist, AC3174, and a CCKR1-selective agonist, AC170222. C2816 was constructed such that AC3174 was linked to the N-terminus of AC170222, thus preserving the C-terminal amide of the CCK moiety. In functional in vitro assays C2816 retained full agonism at GLP-1R and CCKR1 at lower potency compared to parent molecules, whereas a previously reported fusion peptide in the opposite orientation, (pGlu-Gln)-CCK-8/exendin-4, exhibited no activity at either receptor. Acutely, in vivo, C2816 increased cFos in key central nuclei relevant to feeding behavior, and reduced food intake in wildtype (WT), but less so in GLP-1R-deficient (GLP-1RKO), mice. In sub-chronic studies in diet-induced obese (DIO) mice, C2816 exerted superior reduction in body weight compared to co-administration of AC3174 and AC170222 albeit at a higher molar dose. These data suggest that the synergistic pharmacological effects of GLP-1 and CCK pathways can be harnessed in a single therapeutic peptide.

A Chinese Perspective on Receptors and Receptor Regulation.

A receptor is a protein molecule that receives chemical signals from outside a cell, which enables the cell to respond to the signal molecule. Receptors mediate numerous important physiologic effects upon binding extracellular agonists. However, sustained activation of the receptor may lead to pathologic effects. Cells can regulate the number and function of receptors to alter their sensitivity to different molecules by a feedback mechanism, such as change in the receptor conformation, uncoupling of the receptor effector molecules, receptor sequestration, etc. In this special issue, some Chinese scientists were invited to contribute impactful discoveries and insightful reviews in the field of molecular pharmacology, especially receptor and receptor regulation.

Intrarenal signaling mediated by CCK plays a role in salt intake-induced natriuresis.

The natriuretic hormone CCK exhibits its gene transcripts in total kidney extracts. To test the possibility of CCK acting as an intrarenal mediator of sodium excretion, we examined mouse kidneys by 1) an in situ hybridization technique for CCK mRNA in animals fed a normal- or a high-sodium diet; 2) immuno-electron microscopy for the CCK peptide, 3) an in situ hybridization method and immunohistochemistry for the CCK-specific receptor CCKAR; 4) confocal image analysis of receptor-mediated Ca2+ responses in isolated renal tubules; and 5) metabolic cage experiments for the measurement of urinary sodium excretion in high-salt-fed mice either treated or untreated with the CCKAR antagonist lorglumide. Results showed the CCK gene to be expressed intensely in the inner medulla and moderately in the inner stripe of the outer medulla, with the expression in the latter being enhanced by high sodium intake. Immunoreactivity for the CCK peptide was localized to the rough endoplasmic reticulum of the medullary interstitial cells in corresponding renal regions, confirming it to be a secretory protein. Gene transcripts, protein products, and the functional activity for CCKAR were consistently localized to the late proximal tubule segments (S2 and S3) in the medullary rays, and the outer stripe of the outer medulla. Lorglumide significantly diminished natriuretic responses of mice to a dietary sodium load without altering the glomerular filtration rate. These findings suggest that the medullary interstitial cells respond to body fluid expansion by CCK release for feedback regulation of the late proximal tubular reabsorption.

The cholecystokinin-1 receptor antagonist devazepide increases cholesterol cholelithogenesis in mice.

BACKGROUND: A defect in gallbladder contraction function plays a key role in the pathogenesis of gallstones. The cholecystokinin-1 receptor (CCK-1R) antagonists have been extensively investigated for their therapeutic effects on gastrointestinal and metabolic diseases in animal studies and clinical trials. However, it is still unknown whether they have a potential effect on gallstone formation. DESIGN: To study whether the CCK-1R antagonists enhance cholelithogenesis, we investigated cholesterol crystallization, gallstone formation, hepatic lipid secretion, gallbladder emptying function and intestinal cholesterol absorption in male C57BL/6J mice treated by gavage with devazepide (4 mg/day/kg) or vehicle (as controls) twice per day and fed the lithogenic diet for 21 days. RESULTS: During 21 days of feeding, oral administration of devazepide significantly accelerated cholesterol crystallization and crystal growth to microlithiasis, with 40% of mice forming gallstones, whereas only agglomerated cholesterol monohydrate crystals were found in mice receiving vehicle. Compared to the vehicle group, fasting and postprandial residual gallbladder volumes in response to the high-fat meal were significantly larger in the devazepide group during cholelithogenesis, showing reduced gallbladder emptying and bile stasis. Moreover, devazepide significantly increased hepatic secretion of biliary cholesterol, but not phospholipids or bile salts. The percentage of intestinal cholesterol absorption was higher in devazepide-treated mice, increasing the bioavailability of chylomicron-derived cholesterol in the liver for biliary hypersecretion into bile. These abnormalities induced supersaturated bile and rapid cholesterol crystallization. CONCLUSIONS: The potent CCK-1R antagonist devazepide increases susceptibility to gallstone formation by impairing gallbladder emptying function, disrupting biliary cholesterol metabolism and enhancing intestinal cholesterol absorption in mice.

Expression of cholecystokinin receptors in colon cancer and the clinical correlation in Taiwan.

Cholecystokinin and gastrin receptors are upregulated in many human digestive malignancies; however, the correlation of their expressions with severity of colon carcinoma remains sketchy. Here, we determined the expression of cholecystokinin-1 and cholecystokinin-2 receptor, CCK1R and CCK2R, in colon carcinomas and investigated their correlations with clinicopathological characteristics and 1-year survival rate. Expression of CCK1R and CCK2R was determined by immunohistochemical assay in tissue samples obtained from 97 surgical specimens. Clinicopathological character analysis revealed that higher expression of cytoplasmic CCK1R and CCK2R was significantly associated with several variables including the depth of tumor invasion (P = 0.001), venous invasion (P = 0.023), and progression stage (P = 0.013). In addition, immunohistochemical staining revealed statistically significant associations of nuclear CCK1R expression with higher lymphatic invasion (P = 0.042), progression stage (P = 0.025), and unfavorable survival (P = 0.025). Interestingly, we found no link between nuclear CCK2R expression and all the clinicopathological characteristics examined. Taken these, our findings indicate that nuclear CCK1R represents a potential biomarker for poor prognosis, and CCK1R may play a role differing from CCK2R in colon carcinogenesis.

The feeding responses evoked by endogenous cholecystokinin are regulated by different gastrointestinal sites.

The current study tested the hypothesis that cholecystokinin (CCK) A receptor (CCKAR) in areas supplied by the celiac artery (CA), stomach and upper duodenum, and the cranial mesenteric artery (CMA), small and parts of the large intestine, is necessary for reduction of meal size, prolongation of the intermeal interval (time between first and second meal) and increased satiety ratio (intermeal interval/meal size or amount of food consumed during any given unit of time) by the non-nutrient stimulator of endogenous CCK release camostat. Consistent with our previous findings camostat reduced meal size, prolonged the intermeal interval and increased the satiety ratio. Here, we report that blocking CCKAR in the area supplied by the celiac artery attenuated reduction of meal size by camostat more so than the cranial mesenteric artery route. Blocking CCKAR in the area supplied by the cranial mesenteric artery attenuated prolongation of the intermeal interval length and increased satiety ratio by camostat more so than the celiac artery route. Blocking CCKAR in the areas supplied by the femoral artery (control) failed to alter the feeding responses evoked by camostat. These results support the hypothesis that CCKAR in the area supplied by the CA is necessary for reduction of meal size by camostat whereas CCKAR in the area supplied by the CMA is necessary for prolongation of the intermeal interval and increased satiety ratio by this substance. Our results demonstrate that meal size and intermeal interval length by camostat are regulated through different gastrointestinal sites.

Polymorphism and Expression Profile of Cholecystokinin Type A Receptor in Relation to Gallstone Disease Susceptibility.

In the present study, we investigated expression pattern of Cholecystokinin type A receptor (CCKAR) in relation to its commonly studied polymorphism (rs1800857, T/C) in gallstone disease (GSD) patients and controls. A total of 502 subjects (272 GSD and 230 controls) were enrolled, and genotyping was performed by evaluating restriction fragments of PstI digested DNA. For analyzing expression pattern of CCKAR in relation to polymorphism, gallbladder tissue samples from 80 subjects (GSD-55; control-25) were studied. Expression of CCKAR mRNA was evaluated by reverse transcriptase-PCR and confirmed using real-time PCR. Protein expression was evaluated by enzyme-linked immunosorbent assay. We observed significantly (p < 0.0001) lower expression of CCKAR mRNA and protein in GSD tissues as compared with control. Significantly higher frequency of A1/A1 genotype (C/T transition) (p = 0.0005) was observed for GSD as compared with control. Expression of CCKAR protein was found to be significantly lower (p < 0.0001) in A1/A1 genotype as compared with other genotypes for GSD patients. Perhaps, this is the first report providing evidence of alteration in CCKAR expression in relation to its polymorphism elucidating the molecular pathway of the disease. Additional investigations with lager sample size are needed to confirm these findings.

Development of a highly selective allosteric antagonist radioligand for the type 1 cholecystokinin receptor and elucidation of its molecular basis of binding.

Understanding the molecular basis of ligand binding to receptors provides insights useful for rational drug design. This work describes development of a new antagonist radioligand of the type 1 cholecystokinin receptor (CCK1R), (2-fluorophenyl)-2,3-dihydro-3-[(3-isoquinolinylcarbonyl)amino]-6-methoxy-2-oxo-l-H-indole-3-propanoate (T-0632), and exploration of the molecular basis of its binding. This radioligand bound specifically with high affinity within an allosteric pocket of CCK1R. T-0632 fully inhibited binding and action of CCK at this receptor, while exhibiting no saturable binding to the closely related type 2 cholecystokinin receptor (CCK2R). Chimeric CCK1R/CCK2R constructs were used to explore the molecular basis of T-0632 binding. Exchanging exonic regions revealed the functional importance of CCK1R exon 3, extending from the bottom of transmembrane segment (TM) 3 to the top of TM5, including portions of the intramembranous pocket as well as the second extracellular loop region (ECL2). However, CCK1R mutants in which each residue facing the pocket was changed to that present in CCK2R had no negative impact on T-0632 binding. Extending the chimeric approach to ECL2 established the importance of its C-terminal region, and site-directed mutagenesis of each nonconserved residue in this region revealed the importance of Ser(208) at the top of TM5. A molecular model of T-0632-occupied CCK1R was consistent with these experimental determinants, also identifying Met(121) in TM3 and Arg(336) in TM6 as important. Although these residues are conserved in CCK2R, mutating them had a distinct impact on the two closely related receptors, suggesting differential orientation. This establishes the molecular basis of binding of a highly selective nonpeptidyl allosteric antagonist of CCK1R, illustrating differences in docking that extend beyond determinants attributable to distinct residues lining the intramembranous pocket in the two receptor subtypes.

A type 1 cholecystokinin receptor mutant that mimics the dysfunction observed for wild type receptor in a high cholesterol environment.

Cholecystokinin (CCK) stimulates the type 1 CCK receptor (CCK1R) to elicit satiety after a meal. Agonists with this activity, although potentially useful for treatment of obesity, can also have side effects and toxicities of concern, making the development of an intrinsically inactive positive allosteric modulator quite attractive. Positive allosteric modulators also have the potential to correct the defective receptor-G protein coupling observed in the high membrane cholesterol environment described in metabolic syndrome. Current model systems to study CCK1R in such an environment are unstable and expensive to maintain. We now report that the Y140A mutation within a cholesterol-binding motif and the conserved, class A G protein-coupled receptor-specific (E/D)RY signature sequence results in ligand binding and activity characteristics similar to wild type CCK1R in a high cholesterol environment. This is true for natural CCK, as well as ligands with distinct chemistries and activity profiles. Additionally, the Y140A construct also behaved like CCK1R in high cholesterol in regard to its internalization, sensitivity to a nonhydrolyzable GTP analog, and anisotropy of a bound fluorescent CCK analog. Chimeric CCK1R/CCK2R constructs that systematically changed the residues in the allosteric ligand-binding pocket were studied in the presence of Y140A. This established increased importance of unique residues within TM3 and reduced the importance of TM2 for binding in the presence of this mutation, with the agonist trigger likely pulled away from its Leu(356) target on TM7. The distinct conformation of this intramembranous pocket within Y140A CCK1R provides an opportunity to normalize this by using a small molecule allosteric ligand, thereby providing safe and effective correction of the coupling defect in metabolic syndrome.

Reference genome of wild goat (capra aegagrus) and sequencing of goat breeds provide insight into genic basis of goat domestication.

BACKGROUND: Domestic goats (Capra hircus) have been selected to play an essential role in agricultural production systems, since being domesticated from their wild progenitor, bezoar (Capra aegagrus). A detailed understanding of the genetic consequences imparted by the domestication process remains a key goal of evolutionary genomics. RESULTS: We constructed the reference genome of bezoar and sequenced representative breeds of domestic goats to search for genomic changes that likely have accompanied goat domestication and breed formation. Thirteen copy number variation genes associated with coat color were identified in domestic goats, among which ASIP gene duplication contributes to the generation of light coat-color phenotype in domestic goats. Analysis of rapidly evolving genes identified genic changes underlying behavior-related traits, immune response and production-related traits. CONCLUSION: Based on the comparison studies of copy number variation genes and rapidly evolving genes between wild and domestic goat, our findings and methodology shed light on the genetic mechanism of animal domestication and will facilitate future goat breeding.

Impact of ursodeoxycholic acid on a CCK1R cholesterol-binding site may contribute to its positive effects in digestive function.

Dysfunction of the type 1 cholecystokinin (CCK) receptor (CCK1R) as a result of increased gallbladder muscularis membrane cholesterol has been implicated in the pathogenesis of cholesterol gallstones. Administration of ursodeoxycholic acid, which is structurally related to cholesterol, has been shown to have beneficial effects on gallstone formation. Our aims were to explore the possible direct effects and mechanism of action of bile acids on CCK receptor function. We studied the effects of structurally related hydrophobic chenodeoxycholic acid and hydrophilic ursodeoxycholic acid in vitro on CCK receptor function in the setting of normal and elevated membrane cholesterol. We also examined their effects on a cholesterol-insensitive CCK1R mutant (Y140A) disrupting a key site of cholesterol action. The results show that, similar to the impact of cholesterol on CCK receptors, bile acid effects were limited to CCK1R, with no effects on CCK2R. Chenodeoxycholic acid had a negative impact on CCK1R function, while ursodeoxycholic acid had no effect on CCK1R function in normal membranes but was protective against the negative impact of elevated cholesterol on this receptor. The cholesterol-insensitive CCK1R mutant Y140A was resistant to effects of both bile acids. These data suggest that bile acids compete with the action of cholesterol on CCK1R, probably by interacting at the same site, although the conformational impact of each bile acid appears to be different, with ursodeoxycholic acid capable of correcting the abnormal conformation of CCK1R in a high-cholesterol environment. This mechanism may contribute to the beneficial effect of ursodeoxycholic acid in reducing cholesterol gallstone formation.

CCK8 negatively regulates the TLR9-induced activation of human peripheral blood pDCs by targeting TRAF6 signaling.

Plasmacytoid dendritic cells (pDCs) are specialized in rapid and massive secretion of type I interferon in response to foreign nuclei acids. Combined with their antigen presentation capacity, this powerful functionality enables pDCs to orchestrate innate and adaptive immune responses. Cholecystokinin octapeptide (CCK8) is a potent immunomodulator, whose role in pDCs function is unknown. In this study, we found that two different cholecystokinin receptors, CCK1R and CCK2R, are expressed on human peripheral blood pDCs. Exogenous CCK8 was able to modulate the TLR-induced activation of pDCs, including phenotypic maturation, IFN-α synthesis and secretion, and could also regulate the potential of pDCs to induce adaptive immune responses in vitro. CCK8 inhibited TLR9-induced activation of tumor-necrosis factor receptor-associated factor 6, which is an important adapter protein in activation of interferon-regulatory factor (IRF)5 and IRF7, possibly through CCK2R, by evoking the activity of protein kinase (PK)A and reducing the activity of PKC. All these results indicate that CCK8 can inhibit the TLR9-induced phenotypic maturation and activation of pDCs, acting through CCK2R by modulating the tumor-necrosis factor receptor-associated factor 6 signaling pathways.

Cellular and subcellular localization of cholecystokinin (CCK)-1 receptors in the pancreas, gallbladder, and stomach of mice.

Information concerning the cellular localization of cholecystokinin (CCK)-1 receptors has been discrepant and remained scanty at ultrastructural levels. The present immunohistochemical study at light and electron microscopic levels revealed the distinct localization of CCK1 receptors in visceral organs. Immunohistochemistry by use of a purified antibody against mouse CCK1 receptor was applied to fixed tissue sections of the pancreas, gallbladder, stomach, and intestine of mice. A silver-intensified immunogold method revealed the subcellular localization under electron microscope. The immunoreactivity for CCK1 receptors was selectively found in the basolateral membrane of pancreatic acinar cells and gastric chief cells but was absent in pancreatic islets and gastric D cells. Another intense expression in the gut was seen in the myenteric nerve plexus of the antro-duodenal region and some populations of c-Kit-expressing pacemaker cells in the duodenal musculature. The gallbladder contained smooth muscle fibers with an intense immunoreactivity of CCK1 receptors on cell surfaces. The restricted localization of CCK1 receptors on the basolateral membrane of pancreatic acinar cells and gastric chief cells, along with their absence in the islets of Langerhans and gastric D cells, provides definitive information concerning the regulatory mechanism by circulating CCK. Especially, the subcellular localization in the acinar cells completes the investigation for the detection of circulating CCK by the basolateral membrane.

Synergistic metabolic benefits of an exenatide analogue and cholecystokinin in diet-induced obese and leptin-deficient rodents.

AIM: To test the impact of cholecystokinin (CCK) plus either amylin or a glucagon-like peptide-1 receptor (GLP-1R) agonist on metabolic variables in diet-induced obese (DIO) rodents. METHODS: A stabilized acetylated version of CCK-8 (Ac-Y*-CCK-8), selective CCK1 receptor (CCK1R) or CCK2 receptor (CCK2R) agonists, amylin or the GLP-1R agonist and exenatide analogue AC3174 were administered in select combinations via continuous subcutaneous infusion to DIO rats for 14 days, or Lep(ob) /Lep(ob) mice for 28 days, and metabolic variables were assessed. RESULTS: Combined administration of Ac-Y*-CCK-8 with either amylin or AC3174 induced greater than additive weight loss in DIO rats, with the overall magnitude of effect being greater with AC3174 + Ac-Y*-CCK-8 treatment. Co-infusion of AC3174 with a specific CCK1R agonist, but not a CCK2R agonist, recapitulated the weight loss mediated by AC3174 + Ac-Y*-CCK-8 in DIO rats, suggesting that synergy is mediated by CCK1R activation. In a 4 × 4 full-factorial response surface methodology study in DIO rats, a synergistic interaction between AC3174 and the CCK1R-selective agonist on body weight and food intake was noted. Co-administration of AC3174 and the CCK1R-selective agonist to obese diabetic Lep(ob) /Lep(ob) mice elicited a significantly greater reduction in percentage of glycated haemoglobin and food intake relative to the sum effects of monotherapy groups. CONCLUSIONS: The anti-obesity and antidiabetic potential of combined GLP-1R and CCK1R agonism is an approach that warrants further investigation.