Development of a CCK1R-membrane nanoparticle as a fish-out tool for bioactive peptides.

The cholecystokinin receptor type 1 (CCK1R) is a G protein-coupled receptor (GPCR) that is involved in several biological processes including the regulation of the secretion of digestive enzymes. The peptide hormone cholecystokinin (CCK) binds to CCK1R, which is an important pharmacological target for several diseases, including obesity. Interestingly, nutritional dietary peptides also appear to activate CCK1R, and may play a role in CCK1R signaling in the gut. In this study, a novel technique to screen for CCK1R ligands based on affinity-selection is described. Functional expressed CCK1R is reconstituted into membrane nanoparticles called NABBs (nanoscale apo-lipoprotein bound bilayers). NABBs are native-like bilayer membrane systems for incorporation of GPCRs. CCK1R-NABBs were characterized using a fluorescently labeled CCK analog and can be used as a cutting-edge technology to screen for CCK1R ligands using affinity-selection mass spectrometry.

Effect of Chaiqin Chengqi Decoction on cholecystokinin receptor 1-mediated signal transduction of pancreatic acinar cells in acute necrotizing pancreatitis rats.

OBJECTIVE: To investigate the effect of Chaiqin Chengqi Decoction (,CQCQD) on cholecystokinin receptor 1 (CCKR1)-mediated signal transduction of pancreatic acinar cell in rats with acute necrotic pancreatitis (ANP). METHODS: Twenty-seven Sprague-Dawley rats were randomized into three groups: the control group, the ANP group, and the CQCQD group (9 in each group). ANP rats were induced by two intraperitoneal injections of 8% L-arginine (pH=7.0, 4.4 g/kg) over a 2-h period. Rats were treated with 1.5 mL/100 g body weight of CQCQD (CQCQD group) or physiological saline (control and ANP groups) at 2 h interval. And 6 h after induction, pancreatic tissues were collected for histopathological examination. Pancreatic acinar cells were isolated for determination of CCKR1 mRNA and protein expression, phospholipase C (PLC) and inositol-1,4,5-triphosphate (IP3), and determination of fluorescence intensity (FI) as a measure of intracellular calcium ion concentration [Ca(2+)]i. RESULTS: The pancreatic histopathological score (6.2 ± 1.1) and the levels of PLC (1,187.2 ± 228.2 µg/mL) and IP3 (872.2 ± 88.4 µg/mL) of acinar cells in the ANP group were higher than those in the control (2.8 ± 0.4, 682.5 ± 121.8 µg/mL, 518.4 ± 115.8 µg/mL) and the CQCQD (3.8 ± 0.8, 905.3 ± 78.5 µg/mL, 611.0 ± 42.5 µg/mL) groups (P<0.05). [Ca(2+)]i FI for the ANP group (34.8±27.0) was higher than that in the control (5.1 ± 2.2) and CQCQD (12.6 ± 2.5) groups (P<0.05). The expression of pancreatic acinar cell CCKR1 mRNA in the ANP group was up-regulated (expression ratio=1.761; P=0.024) compared with the control group. The expression of pancreatic acinar cell CCKR1 mRNA in the CQCQD group was down-regulated (expression ratio=0.311; P=0.035) compared with the ANP group. The ratio of gray values of the CCKR1 and ß-actin in the ANP group (1.43 ± 0.17) was higher than those in the control (0.70 ± 0.15) and CQCQD (0.79 ± 0.11) groups (P<0.05). CONCLUSIONS: Pancreatic acinar cell calcium overload of ANP induced by L-arginine was related to the up-regulated expressions of pancreatic acinar cell CCKR1 mRNA and protein. CQCQD can down-regulate expressions of pancreatic acinar cell CCKR1 mRNA and protein to reduce the PLC and IP3 of pancreatic acinar cells, relieving the calcium overload and reducing the pathological changes in rats with ANP.

Duodenal enteroendocrine I-cells contain mRNA transcripts encoding key endocannabinoid and fatty acid receptors.

Enteroendocrine cells have a critical role in regulation of appetite and energy balance. I-cells are a subtype of enteroendocrine cells localized in duodenum that release cholecystokinin in response to ingested fat and amino-acids. Despite their potentially pivotal role in nutrient sensing and feeding behaviour, native I-cells have previously been difficult to isolate and study. Here we describe a robust protocol for the isolation and characterization of native duodenal I-cells and additionally, using semi-quantitative RT-PCR we determined that mouse duodenal I-cells contain mRNA transcripts encoding key fatty acid and endocannabinoid receptors including the long chain fatty acid receptors GPR40/FFAR1, GPR120/O3FAR1; short chain fatty acid receptors GPR41/FFAR3 and GPR43/FFAR2; the oleoylethanolamide receptor GPR119 and the classic endocannabinoid receptor CB1. These data suggest that I-cells sense a wide range of gut lumen nutrients and also have the capacity to respond to signals of fatty-acid derivatives or endocannabinoid peptides.

Time-resolved quantitative analysis of CCK1 receptor-induced intracellular calcium increase.

Cholecystokinin (CCK) is a gastrointestinal hormone, which regulates many physiological functions such as satiety by binding to the CCK receptor (CCKR). Molecules, which recognize this receptor can mimic or block CCK signaling and thereby influence CCKR-mediated processes. We have set up a quantitative heterologous assay with CHO cells over-expressing the rat CCK1 receptor to screen for such candidate molecules. Receptor activation, induced by agonist binding, is followed by an intracellular calcium increase, which was monitored using a fluorescent sensor dye. For quantification of the calcium increase, a population average technique using a fluorescence plate reader was optimized and subsequently compared with a single-cell approach using confocal microscopy. With both strategies, dose-response curves were generated for the natural agonist CCK-8S, the partial agonist JMV-180 as well as the antagonist lorglumide. Significant differences were found between the ligands and a strong correspondence was observed between both methods in terms of maximum response and median effect concentrations. Both highly sensitive methods proved complementary: whereas the plate reader assay allowed faster, high throughput screening, the confocal microscopy identified single-cell variations and revealed factors that reduce specificity and sensitivity.

Screening a plant extract library for inhibitors of cholecystokinin receptor CCK1 pathways.

This study describes the screening of a plant extract library for inhibitors of signal transduction pathways mediated by the cholecystokinin receptor, CCK1. CCK1 receptors are coupled to Galpha(q/11)-proteins, localized mainly in the gastrointestinal tract, and implicated in the regulation of various digestive functions. A primary screen was performed using a cell-based assay that used the beta-lactamase gene reporter controlled by the transcriptional activator NFAT. The assay was validated with the CCK1 receptor antagonist, lorglumide, and automated by the use of a liquid-handling robot MultiProbe II. Off-target hits were triaged by counterscreening against gene reporter cells activated by a combination of thapsigargin and phorbol ester. Purification of active compounds was guided by the beta-lactamase gene reporter and Ca2+ mobilization assays. Pure compounds were characterized by Ca2+ mobilization, radioligand binding, inositol-1 phosphate formation, and Eu-GTP binding assays. The selectivity of inhibition was tested against a panel of Galpha(q/11), Galpha(s), and Galpha(i/0)-coupled receptors. These studies led to the identification of a novel Galpha(q/11)-selective inhibitor.

Role of lysine187 within the second extracellular loop of the type A cholecystokinin receptor in agonist-induced activation. Use of complementary charge-reversal mutagenesis to define a functionally important interdomain interaction.

Activation of guanine nucleotide-binding protein (G protein)-coupled receptors is believed to involve conformational change that exposes a domain for G protein coupling at the cytosolic surface of the helical confluence, although the mechanisms for achieving this are not well understood. This conformational change can be achieved by docking a diverse variety of agonist ligands, known to occur by interacting with different regions of these receptors. In this study, we focus on the importance of a specific basic residue (Lys187) within the second extracellular loop of the receptor for the peptide hormone, cholecystokinin. Alanine-replacement and charge-reversal mutagenesis of this residue showed that it had no effect on the binding of natural peptide and nonpeptidyl ligands of this receptor but markedly interfered with agonist-stimulated signaling. It was demonstrated that this negative effect on biological activity could be eliminated with the truncation of the first 30 residues of the amino-terminal tail of this receptor. Complementary charge-reversal mutagenesis of each of the five conserved acidic residues within this region of the receptor in the presence of the charge-reversed Lys187 revealed that only the Asp5 mutant fully reversed the negative functional impact of the Lys187 charge reversal. Thus, we have demonstrated that a basic residue within the second extracellular loop of the cholecystokinin receptor interacts with a specific acidic residue within the amino terminus of this receptor. This residue-residue interaction is nicely accommodated within a new molecular model of the agonist-occupied cholecystokinin receptor.

Mechanism of exocrine pancreatic insufficiency in streptozotocin-induced type 1 diabetes mellitus.

Diabetes mellitus (DM) is a major health problem at present affecting about 180 million people worldwide. DM is associated with many metabolic abnormalities in the body including the indigestion of carbohydrates leading to malnutrition and weight loss. In this article we investigate the cellular and molecular mechanisms of exocrine pancreatic insufficiency in streptozotocin (STZ, 60 mg kg(-1), i.p.)-induced DM in male rats compared to healthy age-matched controls. Either electrical field stimulation (EFS) or cholecystokinin octapeptide (CCK-8, 10(-8) M) can elicit large and significant (P < 0.05) increases in amylase output from pancreatic segments compared to basal secretion. Insulin (10(-6) M) alone has no significant effect on amylase output compared to basal but it enhanced the secretory responses to either EFS or CCK-8. When rats were rendered diabetic with STZ, either EFS or CCK-8-evoked amylase output was significantly (P < 0.01) decreased compared to the responses obtained with either EFS or CCK-8 alone in healthy age-matched control pancreas. In addition, CCK-8 can elicit large dose-dependent release of amylase in age-matched control and diabetic acinar cells with significantly (P < 0.05) reduced responses in diabetic acinar cells. CCK-8 evoked a large rapid increase in peak cytosolic free calcium concentration ([Ca2+]c) followed by a decrease to a plateau phase in age-matched control fura-2-loaded pancreatic acinar cells. These responses were significantly (P < 0.05) decreased in STZ-induced diabetic acinar cells. In the presence of 10(-6) M insulin, CCK-8 evoked a much larger increase in the Ca2+ transient compared to the response obtained with CCK-8 alone. These effects were significantly (P < 0.01) inhibited in STZ-induced diabetic acinar cells. Similarly, in zero extracellular Ca2+ [Ca2+]c, the CCK-8-evoked [Ca2+]c was significantly (P < 0.05) reduced in both diabetic and age-matched control acinar cells, but with more pronounced reduction in diabetic acinar cells. CCK(A) receptor mRNA levels remained unchanged in diabetic rat acinar cells compared to age-matched healthy control. In contrast, amylase mRNA was significantly (P < 0.05) reduced in diabetic acinar cells compared to control. The results indicate that reduced amylase secretion in response to either EFS or CCK-8 in the diabetic pancreas may be due to reduced [Ca2+]c and gene expression for amylase and not to the gene expression of CCK(A) receptor in pancreatic acinar cells.

Gastrin activates nuclear factor kappaB (NFkappaB) through a protein kinase C dependent pathway involving NFkappaB inducing kinase, inhibitor kappaB (IkappaB) kinase, and tumour necrosis factor receptor associated factor 6 (TRAF6) in MKN-28 cells transfected with gastrin receptor.

BACKGROUND: We previously reported that gastrin induces expression of CXC chemokines through activation of nuclear factor kappaB (NFkappaB) in gastric epithelial cells that express gastrin receptor. AIMS: To clarify gastrin receptor mediated signals leading to activation of NFkappaB. METHODS: MKGR26 cells were created by transfecting gastrin receptor cDNA into MKN-28 cells. Degradation of inhibitor kappaB (IkappaB) and phosphorylation of protein kinase C (PKC)-delta were both detected by western blot analysis. NFkappaB activation was determined by luciferase assay and electrophoretic mobility shift analysis. RESULTS: Gastrin induced degradation of IkappaB-alpha and activation of NFkappaB, which was abolished by the selective gastrin receptor antagonist L-740,093 and the general PKC inhibitor GF109203X. Gastrin induced phosphorylation of PKC-delta, and its inhibitor rottlerin partially suppressed NFkappaB activation. However, the mitogen activated protein kinase (MAPK) kinase inhibitor PD98059, p38 MAPK inhibitor SB203580, and tyrphostin AG1478 had no effect on NFkappaB activation. Introduction of the dominant negative mutant of IkappaB kinase, of NFkappaB inducing kinase, and of tumour necrosis factor receptor associated factor 6 (TRAF6), but not that of TRAF2, inhibited gastrin induced activation of NFkappaB. CONCLUSIONS: Gastrin activates NFkappaB via a PKC dependent pathway which involves IkappaB kinase, NFkappaB inducing kinase, and TRAF6.

Actions of CCK in the controls of food intake and body weight: lessons from the CCK-A receptor deficient OLETF rat.

The OLETF rat, lacking CCK-A receptors, provides an important model for identifying roles for CCK in the controls of food intake and body weight. OLETF rats are obese and diabetic and express deficits in the control of the size of individual meals. Meal size in OLETF rats is doubled and although meal number is decreased, the decrease is not sufficient to prevent hyperphagia. Analyses of patterns of hypothalamic gene expression in OLETF rats indicate the presence of a primary deficit in DMH NPY signaling. These data suggest an important role for CCK in controlling NPY expression in a population of non-leptin regulated hypothalamic neurons. In the absence of this control, NPY is overexpressed, contributing to hyperphagia and obesity. Thus, the obesity in the OLETF rats may be the outcome of two regulatory disruptions, one depending upon a peripheral within meal satiety pathway and the other depending upon a central pathway critical to overall energy balance.

The association of cholecystokinin-A receptor expression with the responsiveness of electroacupuncture analgesic effects in rat.

The purpose of this study is to determine whether the level of cholecystokinin (CCK) receptor expression causes the differences between the responder and non-responder to electroacupuncture mediated analgesic effects. Male Sprague-Dawley rats were stimulated at the Zusanli (ST36) acupoint in the absence of any anesthetics and holders. The tail flick latency test was performed to quantify analgesic effects and then the responder and non-responder groups were classified. The hypothalamus of each group was dissected and RNA was purified. The amount of mRNA expression of CCK-A and CCK-B receptors was determined by reverse transcription-polymerase chain reaction. The results show that CCK-A receptors are significantly more expressed in non-responders than responders, whereas CCK-B receptor expression is similar in both groups.

Refinement of the conformation of a critical region of charge-charge interaction between cholecystokinin and its receptor.

Insight into the molecular basis of cholecystokinin (CCK) binding to its receptor has come from receptor mutagenesis and photoaffinity labeling studies, with both contributing to the current hypothesis that the acidic Tyr-sulfate-27 residue within the peptide is situated adjacent to basic Arg(197) in the second loop of the receptor. Here, we refine our understanding of this region of interaction by examining a structure-activity series of these positions within both ligand and receptor and by performing three-dimensional molecular modeling of key pairs of modified ligand and receptor constructs. The important roles of Arg(197) and Tyr-sulfate-27 were supported by the marked negative impact on binding and biological response with their natural partner molecule when the receptor residue was replaced by acidic Asp or Glu and when the peptide residue was replaced by basic Arg, Lys, p-amino-Phe, p-guanidino-Phe, or p-methylamino-Phe. Complementary ligand-receptor charge-exchange experiments were unable to regain the lost function. This was supported by the molecular modeling, which demonstrated that the charge-reversed double mutants could not form a good interaction without extensive rearrangement of receptor conformation. The models further predicted that R197D and R197E mutations would lead to conformational changes in the extracellular domain, and this was experimentally supported by data showing that these mutations decreased peptide agonist and antagonist binding and increased nonpeptidyl antagonist binding. These receptor constructs also had increased susceptibility to trypsin degradation relative to the wild-type receptor. In contrast, the relatively conservative R197K mutation had modest negative impact on peptide agonist binding, again consistent with the modeling demonstration of loss of a series of stabilizing inter- and intramolecular bonds. The strong correlation between predicted and experimental results support the reported refinement in the three-dimensional structure of the CCK-occupied receptor.

Startle and sensorimotor gating in rats lacking CCK-A receptors.

Otsuka Long Evans Tokushima Fatty (OLETF) rats lack CCK-A receptors because of a genetic mutation. Previous studies have shown that CCK-A receptors seem to play a role in the regulation of prepulse inhibition (PPI) of the startle reflex, an operational measure of sensorimotor gating. This study investigated baseline and drug-disrupted PPI in OLETF rats and their non-mutant counterparts, Long Evans Tokushima Otsuka (LETO) rats. Baseline PPI did not differ significantly between the two rat genotypes but OLETF rats exhibited a higher acoustic startle response compared to LETO rats. Amphetamine (2 mg/kg), and the non-competitive NMDA antagonist, dizocilpine (0.1 mg/kg), disrupted PPI in LETO rats but not in the OLETF rats. Apomorphine (0.5 mg/kg) failed to disrupt PPI in both LETO and OLETF rats, and haloperidol (0.5 mg/kg) produced a comparable facilitation of PPI in both groups. In a separate study, OLETF rats were found to be less sensitive to the locomotor stimulating effects of amphetamine. These results suggest that CCK-A receptors play a significant role in the behavioral effects of amphetamine and dizocilpine. The PPI response of OLETF rats to amphetamine and dizocilpine is similar to normal rats pretreated with atypical antipsychotics, suggesting that CCK-A receptors may play an important role in the restoration of drug-disrupted PPI by antipsychotics.

A cholecystokinin-mediated pathway to the paraventricular thalamus is recruited in chronically stressed rats and regulates hypothalamic-pituitary-adrenal function.

Chronic stress alters hypothalamic-pituitary-adrenal (HPA) responses to acute, novel stress. After acute restraint, the posterior division of the paraventricular thalamic nucleus (pPVTh) exhibits increased numbers of Fos-expressing neurons in chronically cold-stressed rats compared with stress-naive controls. Furthermore, lesions of the PVTh augment HPA activity in response to novel restraint only in previously stressed rats, suggesting that the PVTh is inhibitory to HPA activity but that inhibition occurs only in chronically stressed rats. In this study, we further examined pPVTh functions in chronically stressed rats. We identified afferent projections to the pPVTh using injection of the retrograde tracer fluorogold. Of the sites containing fluorogold-labeled cells, neurons in the lateral parabrachial, periaqueductal gray, and dorsal raphe containing fluorogold also expressed cholecystokinin (CCK) mRNA. We then examined whether these CCKergic inputs to the pPVTh were involved in HPA responses to acute, novel restraint after chronic stress. We injected the CCK-B receptor antagonist PD 135,158 into the PVTh before restraint in control and chronically cold-stressed rats. ACTH responses to restraint stress were augmented by PD 135,158 only in chronically stressed rats but not in controls. In addition, CCK-B receptor mRNA expression in the pPVTh was not altered by chronic cold stress. We conclude that previous chronic stress specifically facilitates the release of CCK into the pPVTh in response to acute, novel stress. The CCK is probably secreted from neurons in the lateral parabrachial, the periaqueductal gray, and/or the dorsal raphe nuclei. Acting via CCK-B receptors in pPVTh, CCK then constrains facilitated ACTH responses to novel stress in chronically stressed but not naive rats. These results demonstrate clearly that chronic stress recruits a new set of pathways that modulate HPA responsiveness to a novel stress.

Gastrin induces heparin-binding epidermal growth factor-like growth factor in rat gastric epithelial cells transfected with gastrin receptor.

BACKGROUND & AIMS: Parietal cells express heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF). However, it is unknown whether HB-EGF mediates the trophic action of gastrin. The purpose of this study was to determine whether gastrin modulates the expression of HB-EGF, which mediates the proliferative effects of gastrin on gastric epithelial cells. METHODS: RGM1 cells, a rat gastric epithelial cell line, were transfected with a human gastrin receptor complementary DNA. Gastrin induction of messenger RNAs (mRNAs) for EGF-related polypeptides was assayed by Northern blotting. Processing of cell surface-associated proHB-EGF and secretion of HB-EGF were determined by flow cytometry and Western blotting, respectively. Tyrosine phosphorylation of the EGF receptor was assayed by immunoprecipitation and Western blotting with an antiphosphotyrosine antibody. Cell growth was evaluated by [3H]thymidine incorporation. RESULTS: Gastrin induced expression of HB-EGF mRNA, processing of proHB-EGF, release of HB-EGF into the medium, and tyrosine phosphorylation of the EGF receptor. The growth-stimulatory effects of gastrin were partly inhibited by anti-rat HB-EGF serum and completely blocked by AG1478, an EGF receptor-specific tyrphostin. CONCLUSIONS: The findings suggest that HB-EGF at least partially mediates the proliferative effects of gastrin on gastric epithelial cells.

Mutations within the cholecystokinin-B/gastrin receptor ligand 'pocket' interconvert the functions of nonpeptide agonists and antagonists.

We have reported previously that the transmembrane domains of the cholecystokinin-B/gastrin receptor (CCK-BR) comprise a putative ligand binding pocket. In the present study, we examined whether amino acid substitutions within the CCK-BR pocket altered the affinities and/or functional activities of L-365,260 (the prototypical nonpeptide CCK-BR antagonist) and two structural derivatives, YM022 (a higher affinity antagonist) and L-740,093S (a partial agonist). Eight amino acids that project into the CCK-BR pocket were individually replaced by alanine, using site-directed mutagenesis. Affinities for the nonpeptide molecules, as well as ligand-induced inositol phosphate production, were assessed with the wild-type and mutant receptors. For each of the nonpeptide ligands examined, a distinct series of mutations altered the affinity, suggesting that each ligand possessed a characteristic pattern of interactions within the CCK-BR pocket. Basal signaling levels and inositol phosphate formation induced by the full agonist CCK octapeptide were comparable for the wild-type receptor and all of the mutant CCK-BR forms. In contrast to the peptide agonist CCK octapeptide, the functional activities of the nonpeptide molecules were selectively altered by single point mutations within the CCK-BR pocket, resulting in interconversion of agonists and antagonists. These findings suggest that interactions between nonpeptide molecules and transmembrane domain amino acids of the CCK-BR can determine the functional activity and affinity of the ligands.

Mutational analysis of the putative devazepide binding site of the CCK(A) receptor.

Recently a molecular model was proposed for the binding site of the antagonist 3S(-)-N-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepine-3-yl) -1H-indole-2-carboxamide (devazepide) on the cholecystokinin-A (CCK(A)) receptor (Van der Bent et al., 1994. Drug Design Discov. 12, 129-148). Fifteen amino acids were identified, including hydrophilic ones such as Ser139, Asn349 and Ser379, that might interact with the carboxamide moiety in devazepide. To provide mutational evidence for this model, wild-type and mutant receptors (S139A, N349A and S379A) were transiently expressed and compared with respect to the ability of devazepide to inhibit binding of radiolabelled cholecystokinin-(26-33)-peptide amide (CCK-8) and CCK-8-evoked Ca2+ mobilization. The data presented suggest the involvement of the three residues in antagonist binding, although to a different extent. However, it does not seem likely that hydrogen bonds are the driving force in view of the relatively minor changes in receptor affinity and activity.

Basic amino acids at the C-terminus of the third intracellular loop are required for the activation of phospholipase C by cholecystokinin-B receptors.

In common with other Gq protein-coupled receptors, the third intracellular loop of the cholecystokinin-B (CCK-B) receptor contains three basic amino acids (K333/K334/R335) at the C-terminal segment. To determine the importance of these conserved basic residues in Gq-protein activation and stimulation of phospholipase C, these basic amino acids were mutated. Subsequently, the ability of resulting mutant receptors to activate phospholipase C was investigated by measuring inositol phosphate formation in COS-7 cells and recording Ca(2+)-activated Cl- currents from Xenopus oocytes. Site-directed mutagenesis was performed to mutate the three basic amino acids, K333/K334/R335, to neutral amino acids, M333/T334/L335. When the resulting mutant CCK-B receptors were expressed in COS-7 cells and Xenopus oocytes, sulfated cholecystokinin octapeptide (CCK-8) failed to induce inositol phosphate formation in COS-7 cells and evoke Ca(2+)-activated Cl- currents from oocytes. Each basic amino acid was also mutated (K333M, K334T, and R335L). All three single-point mutations resulted in a significant reduction in CCK-8-induced inositol phosphate formation and CCK-8-activated Ca(2+)-dependent Cl- currents. It is interesting that substituting the basic amino acids, K333/K334/R335, with three other basic residues, R333/R334/K335, did not change the maximal CCK-8-simulated inositol phosphate formation and the amplitude of CCK-8-evoked Ca(2+)-dependent Cl- currents. Radioligand-binding studies showed that the above-mentioned mutations did not affect the affinity for CCK-8 and receptor expression level in COS-7 cells. These findings suggest that basic amino acids at the C-terminus of the third cytoplasmic loop are required for the signal transduction by CCK-B receptors.

Abnormal processing of the human cholecystokinin receptor gene in association with gallstones and obesity.

BACKGROUND & AIMS: Cholesterol gallstone disease and obesity are often associated and share the potential, yet unreported, common etiology of cholecystokinin (CCK) dysfunction. While cloning the human CCK-A receptor complementary DNA (cDNA), we found predominance of a 262-base pair coding region deletion in a cDNA library prepared from a patient with this phenotype. The aim of this study was to determine the abundance, functional significance, and mechanism for generating this gene product. METHODS: Relative abundance of CCK receptor gene products was determined using polymerase chain reaction and hybridization analysis. Constructs were expressed in COS cells and studied for radioligand binding and intracellular calcium responses. A human genomic clone for this receptor was sequenced, and the critical regions were compared with those of the patient. RESULTS: Ninety-three percent of the patient's CCK receptor transcripts contained the 262-base pair deletion, whereas only 1.5% +/- 0.9% of control patients had the deletion. This encoded a receptor that did not bind or signal. The deletion corresponded with the third exon; however, this sequence and flanking introns were normal in the patient. CONCLUSIONS: Abnormality of processing an apparently normal CCK receptor gene yields the predominant product with an absent third exon and encoding a nonfunctional receptor, probably reflecting a defective trans-acting splicing factor. An atypical lariat region in the third intron may explain the presence of small amounts of this product in control patients.

Activity-linked alterations in cholecystokininB receptor messenger RNA levels in magnocellular hypothalamic neurones by food and water deprivation in the rat.

CholecystokininB receptors in the hypothalamic supraoptic and paraventricular nuclei may be involved in the regulation of appetite and neuroendocrine function. In situ hybridisation was used to determine levels of mRNA encoding cholecystokininB receptors in these nuclei in normal rats and rats deprived of food, water or both food and water for 4 days. Food deprivation produced no significant change in the level of cholecystokininB mRNA in these hypothalamic nuclei. Water deprivation increased cholecystokininB mRNA levels (412 +/- 16% and 1009 +/- 69% of control for supraoptic and paraventricular nuclei, respectively) while combined food and water deprivation resulted in significantly smaller increases in these nuclei (193 +/- 20% and 303 +/- 44% of control). Increases in the paraventricular nucleus were most prominent in magnocellular (especially oxytocin-rich) subdivisions of this nucleus. These selective alterations are consistent with similar changes in cholecystokinin receptor density in these hypothalamic nuclei, and support an autocrine/paracrine role for cholecystokinin in the regulation of appetite and endocrine function via effects on hypothalamic oxytocinergic activity.

Cholecystokinin B receptor gene expression in hypothalamic neurosecretory neurons after experimental manipulations.

Magnocellular neurons of the supraoptic (SON) and paraventricular (PVN) nuclei projecting to the neurohypophysis produce, in addition to the classical hormones vasopressin and oxytocin, a large number of other peptides, one of which is cholecystokinin (CCK). Binding sites for CCK have been identified in the posterior pituitary. Recently the cDNAs for CCKA and CCKB receptors were isolated and characterized, and CCKA and CCKB receptor mRNAs were localized in the SON and PVN. We have used complementary oligonucleotides and in situ hybridization histochemistry to study CCKB receptor mRNA in hypothalamic neurons. Changes in the expression of CCKB receptor mRNA in the SON and PVN were analysed in salt-loaded as well as in hypophysectomized animals. Levels of CCKB receptor mRNA in the PVN and SON increased markedly in salt-loaded animals as compared to controls. An increase in CCKB receptor mRNA levels was seen in the SON and PVN after 3 days of salt loading, with high levels continuing through 5 and 7 days. At 14 days, the levels of CCKB receptor mRNA in the PVN were significantly lower as compared to 7 days. Hypophysectomy 5 days prior to sacrifice, resulting in a nerve lesion in the neurohypophysial pathway and removal of the anterior pituitary hormones, induced a significant increase in CCKB receptor mRNA levels in neurons of the PVN. The increase in CCKB receptor mRNA labelling after salt loading was mainly observed in the ventrolateral part of the PVN and in the dorsolateral part of the SON, corresponding to oxytocin-containing neurons, whereas the increase after hypophysectomy was mainly seen in the central part of the PVN and in the ventral part of the SON, corresponding to vasopressin-containing neurons. The results suggest that the synthesis of CCKB receptors in magnocellular neurons is increased upon osmotic challenge and hypophysectomy.