Cholecystokinin B receptor antagonists for the treatment of depression via blocking long-term potentiation in the basolateral amygdala.

Depression is a common and severe mental disorder. Evidence suggested a substantial causal relationship between stressful life events and the onset of episodes of major depression. However, the stress-induced pathogenesis of depression and the related neural circuitry is poorly understood. Here, we investigated how cholecystokinin (CCK) and CCKBR in the basolateral amygdala (BLA) are implicated in stress-mediated depressive-like behavior. The BLA mediates emotional memories, and long-term potentiation (LTP) is widely considered a trace of memory. We identified that the cholecystokinin knockout (CCK-KO) mice impaired LTP in the BLA, while the application of CCK4 induced LTP after low-frequency stimulation (LFS). The entorhinal cortex (EC) CCK neurons project to the BLA and optogenetic activation of EC CCK afferents to BLA-promoted stress susceptibility through the release of CCK. We demonstrated that EC CCK neurons innervate CCKBR cells in the BLA and CCK-B receptor knockout (CCKBR-KO) mice impaired LTP in the BLA. Moreover, the CCKBR antagonists also blocked high-frequency stimulation (HFS) induced LTP formation in the BLA. Notably, CCKBR antagonists infusion into the BLA displayed an antidepressant-like effect in the chronic social defeat stress model. Together, these results indicate that CCKBR could be a potential target to treat depression.

Cholecystokinin is involved in triglyceride fatty acid uptake by rat adipose tissue.

The incorporation of plasma triglyceride (TG) fatty acids to white adipose tissue (WAT) depends on lipoprotein lipase (LPL), which is regulated by angiopoietin-like protein-4 (ANGPTL-4), an unfolding molecular chaperone that converts active LPL dimers into inactive monomers. The production of ANGPTL-4 is promoted by fasting and repressed by feeding. We hypothesized that the postprandial hormone cholecystokinin (CCK) facilitates the storage of dietary TG fatty acids in WAT by regulating the activity of the LPL/ANGPTL-4 axis and that it does so by acting directly on CCK receptors in adipocytes. We report that administration of CCK-8 (a bioactive fragment of CCK) to rats: (i) reduces plasma ANGTPL-4 levels; (ii) represses Angptl-4 expression in WAT and (iii) simultaneously enhances LPL activity in this tissue without inducing Lpl expression. In vivo CCK-8 effects are specifically antagonized by the CCK-2 receptor (CCK-2R) antagonist, L-365,260. Moreover, CCK-8 downregulates Angptl-4 expression in wild-type pre-adipocytes, an effect that is not observed in engineered pre-adipocytes lacking CCK-2R. These effects have functional consequences as CCK-8 was found to promote the uptake of dietary fatty acids by WAT, as demonstrated by means of proton nuclear magnetic resonance (1H-NMR). The efficacy of acute CCK-8 administration was not reduced after chronic CCK-8 treatment. Moreover, the effects of CCK-8 on WAT were not associated to the increase of circulating insulin. Our results show that cholecystokinin promotes lipid storage in WAT by acting on adipocyte CCK-2R, suggesting a pivotal role for CCK in TG homeostasis.

Identification of Candidate Genes for Generalized Tonic-Clonic Seizures in Noda Epileptic Rat.

The Noda epileptic rat (NER) exhibits generalized tonic-clonic seizures (GTCS). A genetic linkage analysis identified two GTCS-associated loci, Ner1 on Chr 1 and Ner3 on Chr 5. The wild-type Ner1 and Ner3 alleles suppressed GTCS when combined in double-locus congenic lines, but not when present in single-locus congenic lines. Global expression analysis revealed that cholecystokinin B receptor (Cckbr) and suppressor of tumorigenicity 5 (St5), which map within Ner1, and PHD finger protein 24 (Phf24), which maps within Ner3, were significantly downregulated in NER. De novo BAC sequencing detected an insertion of an endogenous retrovirus sequence in intron 2 of the Phf24 gene in the NER genome, and PHF24 protein was almost absent in the NER brain. Phf24 encodes a Gαi-interacting protein involved in GABAB receptor signaling pathway. Based on these findings, we conclude that Cckbr, St5, and Phf24 are strong candidate genes for GTCS in NER.

Stereochemistry of amino acid spacers determines the pharmacokinetics of (111)In-DOTA-minigastrin analogues for targeting the CCK2/gastrin receptor.

The metabolic instability and high kidney retention of minigastrin (MG) analogues hamper their suitability for use in peptide-receptor radionuclide therapy of CCK2/gastrin receptor-expressing tumors. High kidney retention has been related to N-terminal glutamic acids and can be substantially reduced by coinjection of polyglutamic acids or gelofusine. The aim of the present study was to investigate the influence of the stereochemistry of the N-terminal amino acid spacer on the enzymatic stability and pharmacokinetics of (111)In-DOTA-(d-Glu)6-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2 ((111)In-PP11-D) and (111)In-DOTA-(l-Glu)6-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2 ((111)In-PP11-L). Using circular dichroism measurements, we demonstrate the important role of secondary structure on the pharmacokinetics of the two MG analogues. The higher in vitro serum stability together with the improved tumor-to-kidney ratio of the (d-Glu)6 congener indicates that this MG analogue might be a good candidate for further clinical study.

CCK2R identifies and regulates gastric antral stem cell states and carcinogenesis.

OBJECTIVE: Progastrin is the incompletely cleaved precursor of gastrin that is secreted by G-cells in the gastric antrum. Both gastrin and progastrin bind to the CCK2 receptor (Cckbr or CCK2R) expressed on a subset of gastric epithelial cells. Little is known about how gastrin peptides and CCK2R regulate gastric stem cells and carcinogenesis. Interconversion among progenitors in the intestine is documented, but the mechanisms by which this occurs are poorly defined. DESIGN: We generated CCK2R-CreERT mice and performed inducible lineage tracing experiments. CCK2R+ antral cells and Lgr5+ antral stem cells were cultured in a three-dimensional in vitro system. We crossed progastrin-overexpressing mice with Lgr5-GFP-CreERT mice and examined the role of progastrin and CCK2R in Lgr5+ stem cells during MNU-induced carcinogenesis. RESULTS: Through lineage tracing experiments, we found that CCK2R defines antral stem cells at position +4, which overlapped with an Lgr5(neg or low) cell population but was distinct from typical antral Lgr5(high) stem cells. Treatment with progastrin interconverts Lgr5(neg or low) CCK2R+ cells into Lgr5(high) cells, increases CCK2R+ cell numbers and promotes gland fission and carcinogenesis in response to the chemical carcinogen MNU. Pharmacological inhibition or genetic ablation of CCK2R attenuated progastrin-dependent stem cell expansion and carcinogenesis. CONCLUSIONS: CCK2R labels +4 antral stem cells that can be activated and expanded by progastrin, thus identifying one hormonal trigger for gastric stem cell interconversion and a potential target for gastric cancer chemoprevention and therapy.

Interaction between the cholecystokinin and endogenous cannabinoid systems in cued fear expression and extinction retention.

Post-traumatic stress disorder (PTSD) is thought to develop, in part, from improper inhibition of fear. Accordingly, one of the most effective treatment strategies for PTSD is exposure-based psychotherapy. Ideally, neuroscience would inform adjunct therapies that target the neurotransmitter systems involved in extinction processes. Separate studies have implicated the cholecystokinin (CCK) and endocannabinoid systems in fear; however, there is a high degree of anatomical colocalization between the cannabinoid 1 receptor (Cnr1) and CCK in the basolateral amygdala (BLA), a brain region critical for emotion regulation. Although most research has focused on GABA and GABAergic plasticity as the mechanism by which Cnr1 mediates fear inhibition, we hypothesize that a functional interaction between Cnr1 and CCKB receptor (CCKBR) is critical for fear extinction processes. In this study, systemic pharmacological manipulation of the cannabinoid system modulated cued fear expression in C57BL/6J mice after consolidation of auditory fear conditioning. Knockout of the CCKBR, however, had no effect on fear- or anxiety-like behaviors. Nonetheless, administration of a Cnr1 antagonist increased freezing behavior during a cued fear expression test in wild-type subjects, but had no effect on freezing behavior in CCKBR knockout littermates. In addition, we found that Cnr1-positive fibers form perisomatic clusters around CCKBR-positive cell bodies in the BLA. These CCKBR-positive cells comprise a molecularly heterogenous population of excitatory and inhibitory neurons. These findings provide novel evidence that Cnr1 contributes to cued fear expression via an interaction with the CCK system. Dysfunctional Cnr1-CCKBR interactions might contribute to the etiology of, or result from, fear-related psychiatric disease.

Prefrontal cortical circuit for depression- and anxiety-related behaviors mediated by cholecystokinin: role of ΔFosB.

Decreased medial prefrontal cortex (mPFC) neuronal activity is associated with social defeat-induced depression- and anxiety-like behaviors in mice. However, the molecular mechanisms underlying the decreased mPFC activity and its prodepressant role remain unknown. We show here that induction of the transcription factor ΔFosB in mPFC, specifically in the prelimbic (PrL) area, mediates susceptibility to stress. ΔFosB induction in PrL occurred selectively in susceptible mice after chronic social defeat stress, and overexpression of ΔFosB in this region, but not in the nearby infralimbic (IL) area, enhanced stress susceptibility. ΔFosB produced these effects partly through induction of the cholecystokinin (CCK)-B receptor: CCKB blockade in mPFC induces a resilient phenotype, whereas CCK administration into mPFC mimics the anxiogenic- and depressant-like effects of social stress. We previously found that optogenetic stimulation of mPFC neurons in susceptible mice reverses several behavioral abnormalities seen after chronic social defeat stress. Therefore, we hypothesized that optogenetic stimulation of cortical projections would rescue the pathological effects of CCK in mPFC. After CCK infusion in mPFC, we optogenetically stimulated mPFC projections to basolateral amygdala or nucleus accumbens, two subcortical structures involved in mood regulation. Stimulation of corticoamygdala projections blocked the anxiogenic effect of CCK, although no effect was observed on other symptoms of social defeat. Conversely, stimulation of corticoaccumbens projections reversed CCK-induced social avoidance and sucrose preference deficits but not anxiogenic-like effects. Together, these results indicate that social stress-induced behavioral deficits are mediated partly by molecular adaptations in mPFC involving ΔFosB and CCK through cortical projections to distinct subcortical targets.

Progastrin stimulates colonic cell proliferation via CCK2R- and ß-arrestin-dependent suppression of BMP2.

BACKGROUND & AIMS: Progastrin stimulates colonic mucosal proliferation and carcinogenesis through the cholecystokinin 2 receptor (CCK2R)-partly by increasing the number of colonic progenitor cells. However, little is known about the mechanisms by which progastrin stimulates colonic cell proliferation. We investigated the role of bone morphogenetic proteins (BMPs) in progastrin induction of colonic cell proliferation via CCK2R. METHODS: We performed microarray analysis to compare changes in gene expression in the colonic mucosa of mice that express a human progastrin transgene, gastrin knockout mice, and C57BL/6 mice (controls); the effects of progastrin were also determined on in vitro colonic crypt cultures from cholecystokinin 2 receptor knockout and wild-type mice. Human colorectal and gastric cancer cells that expressed CCK2R were incubated with progastrin or Bmp2; levels of ß-arrestin 1 and 2 were knocked down using small interfering RNAs. Cells were analyzed for progastrin binding, proliferation, changes in gene expression, and symmetric cell division. RESULTS: The BMP pathway was down-regulated in the colons of human progastrin mice compared with controls. Progastrin suppressed transcription of Bmp2 through a pathway that required CCK2R and was mediated by ß-arrestin 1 and 2. In mouse colonic epithelial cells, down-regulation of Bmp2 led to decreased phosphorylation of Smads1/5/8 and suppression of inhibitor of DNA binding 4. In human gastric and colorectal cancer cell lines, CCK2R was necessary and sufficient for progastrin binding and induction of proliferation; these effects were blocked when cells were incubated with recombinant Bmp2. Incubation with progastrin increased the number of CD44(+), bromodeoxyuridine+, and NUMB(+) cells, indicating an increase in symmetric divisions of putative cancer stem cells. CONCLUSIONS: Progastrin stimulates proliferation in colons of mice and cultured human cells via CCK2R- and ß-arrestin 1 and 2-dependent suppression of Bmp2 signaling. This process promotes symmetric cell division.

Modulation of acetylcholine release by cholecystokinin in striatum: receptor specificity; role of dopaminergic neuronal activity.

Cholecystokinin, a neuroactive peptide functioning as a neurotransmitter and neuromodulator in the central nervous system, mediates a number of processes and is implicated in neurological and psychiatric disorders such as Parkinson's disease, anxiety and schizophrenia. Striatum is one of the brain structures with the highest concentrations of CCK in the brain, rich in CCK receptors as well. The physiological effect of CCK on cholinergic interneurons, which are the major interneurons in striatum and the modulatory interactions which exist between dopamine, acetylcholine and cholecystokinin in this brain structure are still unclear. We studied the effect of cholecystokinin octapeptide (CCK-8) on the release of acetylcholine (ACh) from striatal slices of the rat brain. CCK-8 (0.01-0.1µM) showed no statistically significant effect on the basal but enhanced dose-dependently the electrically (2Hz)-evoked release of [(3)H]ACh. When slices were preperfused with 100µM sulpiride, a selective dopamine D(2) receptor antagonist, the CCK-8 (0.01µM) effect on electrically stimulated ACh release was increased nearly 2-fold. A similar increase was observed after depletion of endogenous dopamine (DA) from nigro-striatal dopaminergic neurons with 6-hydroxydopamine (6-OHDA) (2× 250µg/animal, i.c.v.). Furthermore in the presence of dopamine (100µM) or apomorphine (10µM), the prototypical DA receptor agonist, CCK-8 (0.01µM) failed to enhance the stimulation-evoked release of [(3)H]ACh. The D(2) receptor agonist quinpirol (1µM) abolished the CCK-8 effect on electrically stimulated ACh release as well. The increase in electrically induced [(3)H]ACh release produced by 0.01µM CCK-8 was antagonized by d,l loxiglumide (CR 1505), 10µM, a non-peptide CCK-A receptor antagonist and by Suc-Tyr-(OSO3)-Met-Gly-Trp-Met-Asp-ß-phenethyl-amide (GE-410), 1µM, a peptide CCK-A receptor antagonist. The antagonistic effect of GE-410 on the CCK-8-potentiated, electrically induced release of [(3)H]ACh was studied in striatum for the first time. CAM 1028 (10µM), a CCK-B receptor antagonist, also prevented the potentiating effect of CCK-8 (0.01µM) on electrically stimulated release of [(3)H]ACh. The presented results indicate that (i) CCK-8 is capable of increasing ACh elicited by field electrical stimulation in striatum; (ii) CCK-8 is more effective in its ACh-stimulating effect when dopaminergic activity in striatum is blocked i.e. CCK-8-facilitated release of electrically induced ACh from cholinergic interneurons in the striatum is under the inhibitory control of the tonic activity of dopamine from the nigrostriatal pathway; (iii) the enhancing effect of CCK-8 on electrically evoked ACh release is mediated through both CCK-A and CCK-B cholecystokinin receptors located most likely on the cell bodies of cholinergic interneurons in striatum.

Somatic mutations in CCK2R alter receptor activity that promote oncogenic phenotypes.

The roles of cholecystokinin 2 receptor (CCK2R) in numerous physiologic processes in the gastrointestinal tract and central nervous system are well documented. There has been some evidence that CCK2R alterations play a role in cancers, but the functional significance of these alterations for tumorigenesis is unknown. We have identified six mutations in CCK2R among a panel of 140 colorectal cancers and 44 gastric cancers. We show that these mutations increase receptor activity, activate multiple downstream signaling pathways, increase cell migration, and promote angiogenesis. Our findings suggest that somatic mutations in CCK2R may promote tumorigenesis through deregulated receptor activity and highlight the importance of evaluating CCK2R inhibitors to block both the normal and mutant forms of the receptor.

Lipid sensing in the gut, brain and liver.

Elevation of lipid levels affects energy and glucose homeostasis. Organs such as the gut, brain and liver detect a rise in lipids and orchestrate a biochemical, molecular, neuronal and physiological network of responses that alters appetite and the rate of hepatic glucose production. The factors involved in these responses are unclear but the formation of esterified lipids (long-chain fatty acyl-CoAs) and subsequent activation of protein kinase Cδ remain a common sensing mechanism in all three organs. In this paper, we discuss the mechanisms underlying lipid sensing within the gut, brain and liver and their physiological impact on the regulation of glucose and energy homeostasis.

Gastrin-deficient mice have disturbed hematopoiesis in response to iron deficiency.

Gastrins are peptide hormones important for gastric acid secretion and growth of the gastrointestinal mucosa. We have previously demonstrated that ferric ions bind to gastrins, that the gastrin-ferric ion complex interacts with the iron transport protein transferrin in vitro, and that circulating gastrin concentrations positively correlate with transferrin saturation in vivo. Here we report the effect of long-term dietary iron modification on gastrin-deficient (Gas(-/-)) and hypergastrinemic cholecystokinin receptor 2-deficient (Cck2r(-/-)) mice, both of which have reduced basal gastric acid secretion. Iron homeostasis in both strains appeared normal unless the animals were challenged by iron deficiency. When fed an iron-deficient diet, Gas(-/-) mice, but not Cck2r(-/-) mice, developed severe anemia. In iron-deficient Gas(-/-) mice, massive splenomegaly was also apparent with an increased number of splenic megakaryocytes accompanied by thrombocytosis. The expression of the mRNA encoding the iron-regulatory peptide hepcidin, Hamp, was down-regulated in both Cck2r(-/-) and Gas(-/-) mice on a low-iron diet, but, interestingly, the reduction was greater in Cck2r(-/-) mice and smaller in Gas(-/-) mice than in the corresponding wild-type strains. These data suggest that gastrins play an important direct role, unrelated to their ability to stimulate acid secretion, in hematopoiesis under conditions of iron deficiency.

Z-360, a novel cholecystokinin-2/gastrin receptor antagonist, inhibits gemcitabine-induced expression of the vascular endothelial growth factor gene in human pancreatic cancer cells.

Z-360 is a novel cholecystokinin (CCK)-2/gastrin receptor antagonist that is being developed for the treatment of pancreatic adenocarcinoma in combination with gemcitabine. A previous study shows that the co-administration of Z-360 with gemcitabine significantly prolonged the survival of mice with orthotopically implanted human pancreatic adenocarcinoma cell lines. To clarify the therapeutic effects of Z-360 in combined with gemcitabine, we analyzed gene expression. When gemcitabine was administered, CCK-2/gastrin receptor expression was induced in an orthotropic xenograft model; the result indicating that Z-360 could act on gemcitabine-sensitive cells. Both in vitro and in vivo studies showed that gemcitabine increased the expression of vascular endothelial growth factor A (VEGFA), a prognostic factor for survival in pancreatic cancer, while Z-360 suppressed this induction of VEGFA gene expression. These results help to explain how Z-360 prolongs survival when used in combination with gemcitabine.

Pharmacological evaluation of analgesic effects of the cholecystokinin2 receptor antagonist Z-360 in mouse models of formalin- and cancer-induced pain.

Z-360, a novel cholecystokinin(2) (CCK(2)) receptor antagonist, has been developed as a therapeutic drug for pancreatic cancer and showed pain relief action in phase Ib/IIa clinical trial. This study was attempted to elucidate the analgesic efficacy of Z-360 in mice. Oral administration of Z-360 (30-300 mg/kg) showed a dose-dependent inhibitory effect on the late phase of nociceptive responses to formalin. YF476, another CCK(2) receptor antagonist, was without effects at 1 and 10 mg/kg. In contrast, the CCK(1) receptor antagonist devazepide inhibited the nociceptive responses to formalin. In a mouse model of cancer pain, significant anti-allodynic effect of Z-360 was observed after single and repeated oral administration of 100 and 300 mg/kg doses. Anti-allodynic effect was also observed after repeated administration of devazepide. Combined single treatment with morphine and Z-360 caused an increase inhibition of pain-related responses in the pain models produced by formalin and cancer. Although Z-360 has lower affinity for CCK(1) receptor than for CCK(2) receptor, Z-360 exhibited an inhibitory effect on sulfated CCK-8-induced gallbladder emptying, a CCK(1) receptor-mediated effect, at a dose of 100 mg/kg. These results suggest that Z-360 inhibits inflammatory and cancer pain probably through the blockade of CCK(1) receptors. Z-360 is expected to become a useful drug for the pancreatic cancer with analgesic effects as well as the prolongation of survival.

Gastrin, inflammation, and carcinogenesis.

PURPOSE OF REVIEW: Chronic infection of the gastric mucosa with Helicobacter pylori has long been recognized as a significant risk factor for gastric cancer, and indeed, this model represents the prototypical inflammation-associated cancer. In this review, we present the latest clinical and experimental evidence showing that gastrin peptides and their receptors [the cholecystokinin (CCK2) receptors] potentiate the progression of gastric cancer and other gastrointestinal malignancies in the presence of inflammation. RECENT FINDINGS: We highlight the feed-forward mechanisms by which gastrin and CCK2 receptor expression are upregulated during inflammation and in gastrointestinal cancers, summarize gastrin's proinflammatory role by inducing the production of cyclooxgenase-2 (COX-2) and interleukin-8 (IL-8), and relate evidence suggesting that gastrin and their receptors modulate the function of immune cells and fibroblasts following cellular stress, injury, repair, as well as during cancer progression. SUMMARY: We discuss trends for future studies directed toward the elucidation of gastrin peptides' role in regulating intercellular molecular signaling mechanisms between local and circulating immune cells, fibroblasts, epithelial cells, and other cell types in the microenvironments of inflammation-related cancers. Elucidation of the molecular and cellular pathways that relate inflammation with cancer may provide additional opportunities to develop complementary therapies that target the inflammatory microenvironment of the cancer.

Cannabinoid CB1 and cholecystokinin CCK2 receptors modulate, in an opposing way, electrically evoked [3H]GABA efflux from rat cerebral cortex cell cultures: possible relevance for cortical GABA transmission and anxiety.

The effects of treatments with cannabinoid (CB)(1) and cholecystokinin (CCK)(2) receptor agonists and antagonists, as well as compounds that enhance endocannabinoid signaling by inhibiting degradation, e.g., the fatty acid amide hydrolase inhibitor 3'-carbamoyl-biphenyl-3-yl-cyclohexylcarbamate (URB597) or the endocannabinoid reuptake inhibitor (5Z,8Z,11Z,14Z)-N-(3-furanylmethyl)-5,8,11,14-eicosatetraenamide (UCM707), were studied both on spontaneous and electrically evoked [(3)H]GABA efflux from rat cerebral cortex cell cultures. The CCK(2) receptor agonist CCK-8S, the CB(1) receptor agonist (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone (WIN55,212-2), URB597, UCM707, the CB(1) receptor antagonist N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazole-carboxamide (SR141716A), and the CCK(2) receptor antagonist 2-[2-(5-Br-1H-indol-3-yl)ethyl]-3-[3-(1-methylethoxy)phenyl]-4-(3H)-quinazolinone (LY225910) did not affect spontaneous [(3)H]GABA efflux. CCK-8S concentration-dependently increased electrically evoked [(3)H]GABA overflow, and this effect was prevented by LY225910. WIN55,212-2, URB597, and UCM707 induced a reduction of electrically evoked [(3)H]GABA overflow. This reduction was counteracted by SR141716A. When CCK-8S and one of cannabinoid-interfering compounds were simultaneously added, at concentrations by themselves ineffective, to the superfusion medium, an enhancement in electrically evoked [(3)H]GABA efflux was observed. This increase was counteracted by either SR141716A or LY225910 as well as by the inhibitor of protein kinase C, (1R)-2-[12-[(2R)-2-(benzoyloxy)propyl]-3,10-dihydro-4,9-dihydroxy-2,6,7,11-tetramethoxy-3,10-dioxo-1-perylenyl]-1-methylethylcarbonic acid 4-hydroxyphenyl ester (calphostin C). These results indicate that CB(1) and CCK(2) receptors modulate, in an opposing way, electrically evoked [(3)H]GABA efflux from rat cerebral cortex cell cultures. The existence of a CB(1)/CCK(2) receptor heteromer on cortical GABA terminals, with a possible relevance for cortical GABA transmission and anxiety, is postulated.

Evidence for a direct and functional interaction between the regulators of G protein signaling-2 and phosphorylated C terminus of cholecystokinin-2 receptor.

Signaling of G protein-coupled receptors (GPCRs) is regulated by different mechanisms. One of these involves regulators of G protein signaling (RGS), which are diverse and multifunctional proteins that bind to active Galpha subunits of G proteins and act as GTPase-activating proteins. Little is known about the molecular mechanisms that govern the selective use of RGS proteins in living cells. We first demonstrated that CCK2R-mediated inositol phosphate production, known to be G(q)-dependent, is more sensitive to RGS2 than to RGS4 and is insensitive to RGS8. Both basal and agonist-stimulated activities of the CCK2R are regulated by RGS2. By combining biochemical, functional, and in silico structural approaches, we demonstrate that a direct and functional interaction occurs between RGS2 and agonist-stimulated cholecystokinin receptor-2 (CCK2R) and identified the precise residues involved: phosphorylated Ser434 and Thr439 located in the C-terminal tail of CCK2R and Lys62, Lys63, and Gln67, located in the N-terminal domain of RGS2. These findings confirm previous reports that RGS proteins can interact with GPCRs to modulate their signaling and provide a molecular basis for RGS2 recognition by the CCK2R.

Glycine-extended gastrin stimulates proliferation via JAK2- and Akt-dependent NF-kappaB activation in Barrett's oesophageal adenocarcinoma cells.

Glycine-extended gastrin (G-Gly) is a mitogen for several gastrointestinal tissues although the mechanisms responsible are ill-defined and it is unknown if G-Gly can influence signalling in Barrett's oesophagus. G-Gly stimulated proliferation in OE19 and OE33 cells in a dose-dependant manner. This was unaffected by a CCK2 receptor antagonist but abolished by COX-2 inhibitors. G-Gly induced proliferation, COX-2 mRNA abundance, and PGE2 secretion, were all abolished by inhibition of JAK2, PI3-kinase, Akt or NF-kappaB. G-Gly stimulated phosphorylation of JAK2 and increased PI3-kinase activity in JAK2 immunoprecipitates. G-Gly increased Akt phosphorylation and kinase activity and NF-kappaB reporter activity in a JAK2-, PI3-kinase- and Akt-sensitive manner. G-Gly increased COX-2 promoter transcription in an Akt and NF-kappaB-dependent manner and also reduced COX-2 mRNA degradation in an Akt-insensitive manner. We conclude that G-Gly induced signalling involves a JAK2/PI3-kinase/Akt/NF-kappaB sequence leading to COX-2 transcription. G-Gly also seems to stabilise COX-2 mRNA via a separate pathway.

Interrelationships between circulating gastrin and iron status in mice and humans.

The observations that the peptide hormone gastrin interacts with transferrin in vitro and that circulating gastrin concentrations are increased in the iron-loading disorder hemochromatosis suggest a possible link between gastrin and iron homeostasis. This study tested the hypothesis that gastrin and iron status are interrelated by measurement of iron homeostasis in mice and humans with abnormal circulating gastrin concentrations. Intestinal iron absorption was determined by (59)Fe uptake following oral gavage, and concentrations of duodenal divalent metal transporter-1 (DMT-1) and hepatic hepcidin mRNAs were determined by quantitative real-time PCR in agastrinemic (GasKO), hypergastrinemic cholecystokinin 2 receptor-deficient (CCK2RKO), or wild-type mice. Iron status was measured by standard methods in the same mice and in hypergastrinemic humans with multiple endocrine neoplasia type 1 (MEN-1). Iron absorption was increased sixfold and DMT-1 mRNA concentration fourfold, and transferrin saturation was reduced 0.8-fold and hepcidin mRNA expression 0.5-fold in juvenile GasKO mice compared with age-matched wild-type mice. In mature mice, few differences were observed between the strains. Juvenile CCK2RKO mice were hypergastrinemic and had a 5.4-fold higher DMT-1 mRNA concentration than wild-type mice without any increase in iron absorption. In contrast to juvenile GasKO mice, juvenile CCK2RKO mice had a 1.5-fold greater transferrin saturation, which was reflected in a twofold increase in liver iron deposition at maturity compared with wild-type mice. The correlation between transferrin saturation and circulating gastrin concentration observed in mutant mice was also observed in human patients with MEN, in whom hypergastrinemia correlated positively (P = 0.004) with an increased transferrin saturation. Our data indicate that, in juvenile animals when iron demand is high, circulating gastrin concentrations may alter iron status by a CCK2R-independent mechanism.