Otsuka Long-Evans Tokushima Fatty (OLETF) rats lacking CCK-A receptors are hyperphagic, obese, and diabetic. We have previously demonstrated that these rats have a peripheral satiety deficit resulting in increased meal size. To examine the potential role of hypothalamic pathways in the hyperphagia and obesity of OLETF rats, we compared patterns of hypothalamic neuropeptide Y (NPY), proopiomelanocortin (POMC), and leptin receptor mRNA expression in ad libitum-fed Long-Evans Tokushima (LETO) and OLETF rats and food-restricted OLETF rats that were pair-fed to the intake of LETO controls. Pair feeding OLETF rats prevented their increased body weight and elevated levels of plasma insulin and leptin and normalized their elevated POMC and decreased NPY mRNA expression in the arcuate nucleus. In contrast, NPY expression was upregulated in the dorsomedial hypothalamus (DMH) in pair-fed OLETF rats. A similar DMH NPY overexpression was evident in 5-wk-old preobese OLETF rats. These findings suggest a role for DMH NPY upregulation in the etiology of OLETF hyperphagia and obesity.

OBJECTIVE

We aimed to evaluate the role of fat and cholecystokinin (CCK) in the pathophysiology of functional dyspepsia (FD) by investigating symptoms and plasma CCK levels following increasing doses of duodenal lipid during gastric distension, and the effect of CCK-A receptor blockade.

METHODS

In study A, six FD patients were studied on three occasions during duodenal infusion of saline or lipid (1.1 (L-1) or 2 kcal/min (L-2)) and proximal gastric distensions. Six healthy subjects were also studied as controls during L-2 only. In study B, the effect of the CCK-A antagonist dexloxiglumide (5 mg/kg/h) on L-2 induced symptoms was studied in 12 FD patients. Changes in gastric volume at minimal distending pressure and plasma CCK (study A) were assessed, gastric distensions were performed using a barostat, and dyspeptic symptoms were monitored.

RESULTS

Lipid increased gastric volume compared with saline (DeltaV (ml): saline 15 (20), L-1 122 (42), L-2 114 (28)) in patients and even more so in controls (221 (37); p<0.05). During distensions, symptoms were greater during L-2 than during saline or L-1, and greater in patients than in controls, while gastric compliance was smaller in patients than in controls (p<0.05). Lipid increased plasma CCK levels in patients and controls (p>0.05). Dexloxiglumide abolished the increase in gastric volume (DeltaV (ml): dexloxiglumide 17 (9), placebo 186 (49)) and dyspeptic symptoms (sum of scores: dexloxiglumide 24 (7), placebo 44 (19)) during duodenal lipid infusion. Dexloxiglumide also reduced gastric compliance (ml/mm Hg: dexloxiglumide 51 (7), placebo 72 (11)) and symptoms (sum of scores: dexloxiglumide 101 (17), placebo 154 (21)) during gastric distension.

CONCLUSIONS

CCK-A receptors are involved in the generation of dyspeptic symptoms by duodenal lipid during gastric distension.

17beta-estradiol (E2), acting via estrogen receptor (ER)-alpha, inhibits feeding in animals. One mechanism apparently involves an increase in the satiating potency of cholecystokinin (CCK) released from the small intestine by ingested food. For example, the satiating potency of intraduodenal lipid infusions is increased by E2 in ovariectomized rats; this increased satiation is dependent on CCK, and it is accompanied by increases in the numbers of ERalpha-positive cells that express c-Fos in a subregion of the caudal nucleus tractus solitarius (cNTS) that receives abdominal vagal afferent projections. To test whether direct administration of E2 to this area of the hindbrain is sufficient to inhibit food intake, we first implanted 0.2 microg estradiol benzoate (EB) in cholesterol or cholesterol alone either sc or onto the surface of the hindbrain over the cNTS. Food intake was significantly reduced after hindbrain EB implants but not after sc EB implants. Next we verified that equimolar hindbrain implants of E2 and EB had similar feeding-inhibitory effects and determined that only small amounts of E2 reached brain areas outside the dorsal caudal hindbrain after hindbrain implants of (3)H-labeled E2. Neither plasma estradiol concentration nor plasma inflammatory cytokine concentration was increased by either hindbrain or sc EB implants. Finally, hindbrain EB implants, but not sc implants, increased c-Fos in ERalpha-positive cells in the cNTS after ip injection of 4 microg/kg CCK-8. We conclude that E2, acting via ERalpha in cNTS neurons, including neurons stimulated by ip CCK, is sufficient to inhibit feeding.

We studied the role of gastrin in regulating cholangiocyte proliferation induced by bile duct ligation (BDL). In purified cholangiocytes, we evaluated (1) for the presence of cholecystokinin-B (CCK-B)/gastrin receptors, (2) the effect of gastrin on D-myo-Inositol 1,4,5-triphosphate (IP(3)) levels, and (3) the effect of gastrin on DNA synthesis and adenosine 3', 5'-monophosphate (cAMP) levels in the absence or presence of CCK-A (L-364,718) and CCK-B/gastrin (L-365,260) receptor inhibitors, 1, 2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetrakis(acetxymethyl ester) (BAPTA/AM; an intracellular Ca(2+) chelator), and 2 protein kinase C (PKC) inhibitors, 1-(5-Isoquinolinylsulfonyl)-2-methylpiperazine (H7) and staurosporin. To evaluate if gastrin effects on cholangiocyte proliferation are mediated by the isoform PKCalpha, we evaluated (1) for the presence of PKCalpha in cholangiocytes and (2) the effect of gastrin on the PKCalpha protein expression in a triton-soluble (containing cytoplasm + membrane) and a triton-insoluble (containing cytoskeleton) fraction. To evaluate the effects of gastrin in vivo, immediately following BDL, gastrin or bovine serum albumin (BSA) was infused by minipumps for 7 days to rats and we measured cholangiocyte growth and cAMP levels. We found CCK-B/gastrin receptors on cholangiocytes. Gastrin increased IP(3) levels. Gastrin inhibited DNA synthesis and cAMP synthesis in cholangiocytes. Gastrin effects on cholangiocyte functions were blocked by L-365,260, BAPTA/AM, H7, and staurosporin but not by L-364,718. Gastrin induced translocation of PKCalpha from cholangiocyte cytoskeleton to membrane. In vivo, gastrin decreased cholangiocyte growth and cAMP synthesis compared with controls. We concluded that gastrin inhibits cholangiocyte growth in BDL rats by interacting with CCK-B/gastrin receptors through a signal transduction pathway involving IP(3), Ca(2+), and PKCalpha.

OBJECTIVE

Fatty acids induce cholecystokinin (CCK) secretion and modify gastric motility, but the chain length requirements for these effects are not known. Nor is it clear whether the effects of fatty acids on gastric motility in humans are CCK mediated or directly exerted. The aim of this study was to determine the role of fatty acyl chain length in CCK secretion and in influencing gastric motility.

METHODS

Fatty acids were infused into the upper gut in healthy volunteers; plasma CCK was determined by radioimmunoassay. Effects of fatty acids on antral contractility were determined by percutaneous ultrasonography; effects on proximal gastric tone were studied during fundal distention.

RESULTS

Plasma CCK concentration was consistently and similarly elevated by fatty acids with a chain of 12 carbon atoms or longer, whereas those of 11 or fewer carbon atoms failed to increase plasma CCK. A 12-carbon but not a 10-carbon-long chain fatty acid reduced antral contractile amplitude, an effect that was abolished by loxiglumide (a specific CCK-A receptor antagonist). The 12-carbon fatty acid also reduced proximal gastric tone more than the 10-carbon fatty acid.

CONCLUSIONS

A highly specific, chain length-sensitive fatty acid recognition system exists in the proximal gut mediating CCK secretion and gastric motility. An additional, probably CCK-independent, effect of fatty acid also regulates proximal gastric tone.

Receptor targeting with radiolabeled peptides has become very important in nuclear oncology in the past few years. The most frequently used peptides in the clinic are analogs of somatostatin (SRIF), e.g. OctreoScan, which contain chelators for the radioisotopes 111In, 86Y, 90Y, 67Ga, 68Ga and 64Cu or for 99mTc and 188Re. and were labelled with the halogens 123I and 18F. Radiolabeled analogs of &alpha-melanocyte-stimulating hormone (&alpha-MSH), neurotensin, vasoactive intestinal peptide (VIP), bombesin (BN), substance P (SP) and gastrin/cholecystokinin (CCK) are also being developed, evaluated in vitro and in vivo and tested for clinical application. This review focuses on the expression in tumors and the regulation of receptors for these neuropeptides as well as the development of novel chelator-peptide conjugates suitable for in vivo scintigraphy or internal radiotherapy. The state of the art of radiopeptide pharmaceuticals is illustrated with four SRIF analogs, modified with the macrocyclic chelator 1, 4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-tetraacetic acid (DOTA): [D-Phe1]-octreotide (DOTAOC), [D-Phe1, Tyr3]-octreotide (DOTATOC), vapreotide (DOTAVAP) and lanreotide (DOTALAN). DOTA is almost a universal chelator capable of strongly encapsulating hard metals such as 111In and 67Ga for Single Photon Emission Tomography (SPET), 68Ga, 86Y and 64Cu for Positron Emission Tomography (PET) as well as 90Y for receptor-mediated radionuclide therapy and radiolanthanides which exhibit different interesting decay schemes. From biodistribution studies in experimental animals and from clinical data it is concluded that DOTATOC is currently the most suitable SRIF radiopeptide with the best potential in the clinic.

Amatoxins are the main poison of the green death cap (Amanita phalloides) and among the most dangerous natural toxins causing hepatic failure. A possible therapeutic approach is the inhibition of the transporting systems mediating the uptake of amatoxins into human hepatocytes, which, however, have yet to be identified. In the current study we tested whether members of the organic anion-transporting polypeptide (OATP) family, localized in the sinusoidal membranes of human hepatocytes, are involved in amatoxin uptake. For this, Madin Darby canine kidney strain II (MDCKII) cells stably expressing human OATP1B3, OATP2B1, or OATP1B1, were assayed for the uptake of 3H-labeled O-methyl-dehydroxymethyl-alpha-amanitin. Under our conditions, only OATP1B3 was able to transport amanitin with a K(m) value of 3.7 microM +/- 0.6 microM. Accordingly, toxin uptake was inhibited by OATP1B3 substrates and inhibitors (cyclosporin A, rifampicin, the quinoline derivatives MK571 ([(3-(3-(2-(7-chloro-2-quinolinyl)ethenyl)phenyl)((3-dimethylamino-3-oxopropyl)thio)methyl)thiopropanoic acid]) and montelukast, the cholecystokinin octapeptide (CCK-8), paclitaxel, and bromosulfophthalein), as well as by some antidotes used in the past for the treatment of human amatoxin poisoning (silibinin dihemisuccinate, penicillin G, prednisolone phosphate, and antamanide). These transport studies are in line with viability assays monitoring the toxic effect of amanitin on the transfected MDCKII cells. Further support for amatoxin transport was found in primary human hepatocytes, expressing OATP1B3, OATP2B1, and OATP1B1, where CCK-8, a substrate specific for OATP1B3, prevented the fragmentation of nucleoli, a lesion typical for amanitin action. In conclusion, we have identified OATP1B3 as the human hepatic uptake transporter for amatoxins; moreover, substrates and inhibitors of OATP1B3, among others rifampicin, may be useful for the treatment of human amatoxin poisoning.

BACKGROUND

The role of genetic predisposition to the development of dyspepsia is unclear. Recently, a significant association was reported with CC genotype of GNbeta3.

OBJECTIVE

To explore the association of candidate genotypes altering adrenergic, serotonergic, CCKergic, and G protein functions, and dyspepsia in a sample from a U.S. community.

METHODS

Dyspeptics and healthy controls were identified among community respondents who had been randomly selected to complete validated questionnaires. Other diseases were excluded by face-to-face history and physical examination. Polymorphisms of candidate genes for alpha(2A), alpha(2C), 5-HT(1A), 5-HT(2A), 5-HT(2C), CCK-1 receptors and CCK promoter, GNbeta3 protein, and SERT-promoter (SERT-P) were studied. The association between polymorphisms and meal-related or meal-unrelated dyspepsia, high somatic symptom scores, and somatization were evaluated using Fisher's exact test.

RESULTS

DNA was available from 41 dyspeptics and 47 healthy controls from Olmsted County. Community dyspepsia unrelated to meals was associated with both homozygous GNbeta3 protein 825T and C alleles. There were no significant associations with meal-related dyspepsia. Using Rome II subgroups, the same genotype was associated with dysmotility-like and other dyspepsia. Higher somatization scores were not significantly associated with any of the candidate genes when considered as single factors.

CONCLUSIONS

Meal-unrelated dyspepsia in a U.S. community study is associated with the homozygous 825T or C alleles of GNbeta3 protein. Candidate genes controlling adrenergic, serotonergic, and CCKergic functions do not appear to be associated with dyspepsia.

The CCK and gastrin families of peptides act as hormones and neuropeptides on central and peripheral receptors to mediate secretion and motility in the gastrointestinal tract in the physiological response to a normal meal. Thus far, two CCK receptors have been molecularly identified to mediate the actions of CCK and gastrin, CCK-A and CCK-B receptors (CCK-AR and CCK-BR, respectively). The regulation of CCK-AR and CCK-BR affinity by guanine nucleotides and the receptor activation of G protein-dependent stimulation of phospholipase C and adenylyl cyclase suggested that they were guanine nucleotide-binding protein-coupled receptors [G protein-coupled receptors (GPCRs)]; however, the eventual cloning of their cDNAs revealed their heptahelical structure and confirmed their membership in the GPCR superfamily. The gastrointestinal system is a rich source of neuroendocrine hormones that interact with a large number of GPCRs to regulate the complex tasks of digestion, absorption, and excretion of a meal. This article focuses on the CCK family of GPCRs, and its activities in the gastrointestinal system.

Intraperitoneal (i.p.) administration of sulfated CCK octapeptide (CCK-8S) has been shown to induce changes in neuronal activity in the nucleus of the solitary tract (NTS) and area postrema (AP), sensory parts of the dorsal vagal complex (DVC), and in the paraventricular nucleus of the hypothalamus (PVN), as determined by activation of c-fos expression. Whether peripheral CCK influences neuronal activity in the locus coeruleus (LC)/subcoeruleus nucleus (SC) was investigated in awake rats at intraperitoneal (i.p.) injection of CCK-8S by c-Fos immunohistochemistry. CCK-8S i.p. (25, 50, and 100 micrograms/kg, respectively) dose-dependently increased the average number of c-Fos-LI-positive cells/section in the LC/SC by the factor 5.9, 8.2, and 11.7, respectively. Pretreatment with the CCK-A receptor antagonist MK-329 (devazepide; 1 mg/kg and 2 mg/kg i.p.) reduced the CCK-induced increase in c-fos expression in the LC/SC by 54% and 75%, respectively; the CCK-B receptor antagonist L-365,260 had no effect. Perivagal capsaicin pretreatment diminished the CCK-induced increase in the number of c-Fos-LI-positive cells in the LC/SC by 65%. In comparison, the CCK-A antagonist devazepide (1 mg/kg and 2 mg/kg i.p.) reduced the increase in c-fos expression by 76% and 88% in the PVN, 69% and 88% in the NTS, 86% and 83%, respectively, in the AP. Capsaicin diminished the CCK-induced increase in c-Fos-LI-positive cells in the PVN by 64%, in the NTS by 60%, but in the AP only by 25%. Immunostaining against the nuclear antigen c-Fos and the cytoplasmatic antigen tyrosine hydroxylase (TH) showed that 40% of all c-Fos-LI-positive cells in the LC/SC were TH-LI positive at 25 micrograms CCK/kg. The data indicate that CCK-8S i.p. induces modulation of neuronal activity in the LC/SC, DVC and PVN predominantly by peripheral action of CCK-A receptors and capsaicin-sensitive vagal afferents. These findings suggest that the LC/SC is involved in CNS-mediated regulatory influences of peripheral CCK.

Cholecystokinin (CCK) and gastrin together constitute a family of homologous peptide hormones, which are both physiological ligands for the gastrin/CCK-B receptor, whereas the CCK-A receptor binds only sulfated CCK-peptides. CCK peptides are mainly produced in small intestinal endocrine I-cells and in cerebral neurons. CCK peptides regulate pancreatic enzyme secretion and growth, gallbladder contraction, intestinal motility, satiety and inhibit gastric acid secretion. Moreover, they are potent neurotransmitters in the brain and the periphery. CCK peptides are derived from proCCK and have the bioactive heptasequence -Tyr(SO4)-Met-Gly-Trp-Met-Asp-Phe-NH2 as their C-terminus. The dominant forms in plasma are CCK-58, CCK-33, CCK-22 and CCK-8, whereas CCK-8 is the major transmitterform. Due to scarcity of specific assays, knowledge about CCK in disease is still limited. Gastrin peptides are mainly synthetized in antroduodenal G-cells, from where they are released to blood to regulate gastric acid secretion and mucosal growth. Small amounts are synthetized further down the intestinal tract, in the foetal pancreas, in a few cerebral and peripheral neurons, in the pituitary gland and in spermatozoes. Gastrin peptides are derived from progastrin and all have the C-terminal bioactive hexasequence -Tyr (SO4)-Gly-Trp-Met-Asp-Phe-NH2. The major gastrin forms in tissue and plasma are gastrin-34 and gastrin-17, but also gastrin-71, -14 and -6 have been identified. Gastrin peptides are secreted in excessive amounts from gastrinomas and are expressed at lower levels in bronchogenic, colorectal, gastric, ovarian and pancreatic cancers. A carcinogenetic significance of gastrin peptides remains, however, to be proven.

We report the synthesis of a new reagent for the introduction of an oxidative iodination site into the amino terminus of acid-labile peptides, and the use of this reagent to synthesize a novel affinity-labeling probe for the cholecystokinin (CCK) receptor. The acylation reagent, N,O-bis-fluorenylmethyloxycarbonyl-D-tyrosine hydroxysuccinimide ester, utilizes base-labile protection of both the alpha amino group and the aromatic ring hydroxyl. This can be safely removed to expose a cross-linkable free amino group on the aminopeptidase-resistant D-enantiomer of tyrosine. The synthetic probe, D-Tyr-Gly-Asp-Tyr(OSO3H)-Nle-Gly-Trp-Nle-Asp-Phe-NH2, was fully biologically active, could be radioiodinated to high-specific radioactivity (2000 Ci/mmol), bound with high affinity to the pancreatic CCK receptor, and covalently labeled the hormone-binding site. This reagent should be useful for the synthesis of a wide variety of analogues of CCK and other acid-labile peptides.

Although cholecystokinin A (CCK-A) receptors (CCK-AR) mediate the feeding inhibitory actions of CCK in both rats and mice, the absence of CCK-AR results in species-specific phenotypes. The lack of CCK-AR in Otsuka Long-Evans Tokushima fatty (OLETF) rats results in hyperphagia and obesity. We have suggested that demonstrated increases in meal size and elevated levels of dorsomedial hypothalamic (DMH) neuropeptide Y (NPY) gene expression may contribute to this phenotype. In contrast to OLETF rats, CCK-AR(-/-) mice have normal total daily food intake and do not develop obesity. To assess the basis underlying the different phenotypes in rats and mice lacking CCK-AR, we characterized meal patterns in CCK-AR(-/-) mice and determined whether CCK-AR(-/-) mice exhibited an alteration in DMH NPY gene expression. We demonstrate that although CCK-AR(-/-) mice show a similar dysregulation in meal size as OLETF rats, they do not have an elevation in DMH NPY mRNA expression levels. In fact, intact mice have no CCK-AR in the DMH. Furthermore, in intact rats, NPY and CCK-AR are colocalized in DMH neurons, and parenchymal injection of CCK into the DMH reduces food intake and down-regulates DMH NPY mRNA expression. These results suggest that although CCK-AR plays a role in the mediation of CCK actions in the control of meal size in both rats and mice, CCK-AR seems to contribute to modulating DMH NPY levels only in rats. The deficit in CCK's action in the control of DMH NPY gene expression may play a major role in the obese phenotype in OLETF rats.

A novel hybrid hydrogel system based on N-(2-hydroxypropyl)methacrylamide copolymers was proposed. It consisted of the hydrophilic polymer backbone and a pair of oppositely charged peptide grafts. Two distinct pentaheptad peptides (CCE and CCK) were anticipated to create a dimerization motif and serve as physical cross-linkers. Consequently, the graft copolymers CCE-P and CCK-P self-assembled into hybrid hydrogels in situ; the process was modulated by the formation of antiparallel heterodimeric coiled-coils. This approach possesses an advantage to decrease the steric hindrance of the polymer backbone on the "in-register" alignment of peptide grafts. Indeed, equimolar mixtures of the graft copolymers, CCE-P/CCK-P, have been observed to self-assemble into hydrogels in PBS solution at neutral pH at concentrations as low as 0.1 wt %. Circular dichroism spectroscopy, sedimentation equilibrium experiments, and microrheology revealed that the self-assembly process corresponded to the two-stranded alpha-helical coiled-coil formation between CCE and CCK. Moreover, the formation of hybrid hydrogels was reversible. Denaturation of the coiled-coil domains with guanidine hydrochloride (GdnHCl) solutions resulted in disassembly of the hydrogels. Removal of GdnHCl by dialysis caused coiled-coil refolding and hydrogel reassembly. Scanning electron microscopy results demonstrated that the concentration of the graft copolymers had a significant impact on the structure and morphology of self-assembled hydrogels.

Exogenous cholecystokinin (CCK) induces early satiety when infused into humans. Whether alimentary CCK (CCK-A) receptor blockade stimulates food intake in humans is, however, uncertain. The aim of the present investigation was, therefore, to establish the effect of CCK-A receptor blockade on satiety and eating behavior in healthy volunteers. To further explore the role of endogenous CCK, the effects of the specific CCK-A receptor antagonist loxiglumide (Lox; 22 micromol. kg(-1). h(-1)) on satiety and eating behavior were investigated in healthy men and compared with saline infusions (as placebo) in a series of randomized, double-blind, placebo-controlled, crossover studies. Lox produced a slight (7%), but not significant (P = 0.104), increase in food intake that was accompanied by a modest (10%), but significant (P < 0.004), increase in calorie intake. Fluid ingestion was not affected by Lox. Subjects experienced more hunger and delayed fullness during Lox infusion than during saline infusion (P < 0.05). This study provides further evidence that CCK is an endogenous physiological satiety signal acting through CCK-A receptor-mediated mechanisms. Repeated-dose studies comparing hunger and satiety responses after CCK-A receptor blockade in healthy subjects and patients with eating disorders may help clarify the possible involvement of endogenous CCK in these conditions.

BACKGROUND

Exosomes secreted by human mesenchymal stem cells (hMSCs) have been shown to promote cartilage regeneration. This study aimed to explore whether exosomal lncRNA-KLF3-AS1 derived from hMSCs can promote chondrocyte proliferation via miR-206/GIT1 axis in osteoarthritis (OA).

METHODS

hMSCs and MSC-derived exosomes (MSC-exo) were prepared for morphological observation and identification by transmission electron microscopy (TEM) and flow cytometry. IL-1β-induced OA chondrocytes and collagenase-induced mouse OA model were established for the further experiments. Luciferase activity assay was performed to test whether miR-206 could bind to KLF3-AS1 or GIT1. Cell proliferation and apoptosis were evaluated by CCK-8 assay and flow cytometry, respectively.

RESULTS

MSC-Exos increased chondrogenic genes Col2a1 (type II collagen alpha 1) and aggrecan, decreased hondrocyte hypertrophy markers MMP-13 (matrix metalloproteinase-13) and Runx2 (runt-related transcription factor 2) in chondrocytes isolated from OA model mice. Furthermore, MSC-Exos attenuated IL-1β-induced chondrocyte proliferation inhibition and apoptosis induction. Moreover, MSCKLF3-AS1-Exos (exosomes derived from KLF3-AS1-overexpressing-MSCs) ameliorated IL-1β-induced chondrocyte injury. Results also demonstrated that KLF3-AS1 acted as a competitive endogenous RNA (ceRNA) by sponging miR-206 to facilitate GIT1 expression. In addition, miR-206 overexpression and GIT1 knockdown reversed MSCKLF3-AS1-Exos-mediated attenuation of chondrocyte injury.

CONCLUSIONS

Exosomal KLF3-AS1 derived from MSCs involved in MSC-Exos-mediated chondrocyte proliferation induction and chondrocyte apoptosis inhibition via miR-206/GIT1 axis. Abbreviation: G-protein-coupled receptor kinase interacting protein-1 (GIT1).

In the almost 30 years since the ability of peripheral administration of the brain/gut peptide cholecystokinin (CCK) to inhibit food intake was first demonstrated, significant progress in our overall understanding of the role of CCK in ingestive behavior has been made. A physiologic role for endogenous CCK in the control of meal size has been demonstrated and sites and mechanisms of action for CCK in food intake have been investigated. Recent work has uncovered roles for the CCK satiety pathway in the mediation of the feeding modulatory actions of estradiol, insulin, and leptin. The availability of the Otsuka Long Evans Tokushima Fatty (OLETF) rat, a strain lacking CCK(A) receptors, provides a unique model for the study of how deficits in a within-meals satiety signaling pathway may result in long-term changes in food intake and body weight.

OBJECTIVE

The mechanism of intraduodenal fat induced inhibition of food intake is still unclear. Therefore, we tested the ability of duodenal fatty acids to suppress food intake at a lunchtime meal; in addition, we were interested to test if these effects were mediated by cholecystokinin (CCK) A receptors.

METHODS

Three sequential double blind, three period crossover studies were performed in 12 healthy males each: (1) subjects received intraduodenal fat with or without 120 mg of tetrahydrolipstatin, an inhibitor of gastrointestinal lipases, or saline; (2) volunteers received intraduodenal long chain fatty acids, medium chain fatty acids, or saline; (3) subjects received long chain fatty acids or saline together with concomitant intravenous infusions of saline or loxiglumide, a specific CCK-A receptor antagonist. The effect of these treatments on food intake and feelings of hunger was quantified.

RESULTS

Intraduodenal fat perfusion significantly (p<0.05) reduced calorie intake. Inhibition of fat hydrolysis abolished this effect. Only long chain fatty acids significantly (p<0.05) decreased calorie intake, whereas medium chain fatty acids were ineffective. Infusion of loxiglumide abolished the effect of long chain fatty acids.

CONCLUSIONS

Generation of long chain fatty acids through hydrolysis of fat is a critical step for fat induced inhibition of food intake; the signal is mediated via CCK-A receptors.

Targeting of dorsal root ganglia by diabetes could account for the selective sensory abnormalities that patients with early diabetic polyneuropathy develop. In this work, we addressed survival, phenotype and gene expression in sensory neurones in lumbar dorsal root ganglia in a long-term model of experimental streptozotocin-induced diabetes in rats, designed to reflect human disease. Motor and sensory conduction slowing developed early, by the 2-month time point. At 2 months, sensory neurones had no detectable alterations in their calibre or gene expression, assessed using quantitative in situ hybridization studies for mRNA markers that included alpha CGRP, beta CGRP, NFM, t alpha 1-tubulin, SP, VIP, B50 (GAP43), galanin, somatostatin, PACAP, HSP27, c-jun, SNAP 25, p75, TrkA, TrkB and TrkC. By 12 months, however, diabetics had developed neurone perikaryal and distal axon atrophy, accompanied by generalized downregulation of mRNA expression, particularly of CGRP transcripts, PACAP, SP, NFM, p75, trkA and trkC. With the exception of HSP-27, no elevation in mRNAs that increase after injury, such as VIP, galanin, CCK, PACAP, B50 and t alpha 1-tubulin, was observed and constitutive levels, when detectable, trended towards lower rather than increased levels. There was relative preservation of neurone numbers at 12 months; only a non-significant trend towards fewer diabetic neurones was detected using a rigorous and systematic physical dissector counting approach through the entire L5 ganglia. There was no change in the relative populations of CGRP- and SP-immunoreactive neurones. Our findings indicate that even long-term experimental diabetes is associated with relative preservation of sensory neurone populations, but the neurones are atrophic and their gene expression is altered. This pattern of change differs from that following axotomy, implies a degenerative rather than an injury phenotype and has important implications for how such neurones might be rescued.

Human gallbladders with cholesterol stones exhibit impaired muscle contraction induced by agonists that act on transmembrane receptors, increased membrane cholesterol content, and abnormal cholesterol-to-phospholipid ratio compared with those with pigment stones. The present study was designed to investigate the functions of the CCK receptor of gallbladder muscle membranes by radioreceptor assay and cross-linking. 125I-labeled CCK-8 binding was time-dependent, competitive, and specific. Scatchard analysis showed that the maximum specific binding (Bmax) was significantly decreased in cholesterol compared with pigment stone gallbladders (0.18 +/- 0. 07 vs. 0.38 +/- 0.05 pmol/mg protein, P < 0.05). In contrast, the affinity for CCK was higher in cholesterol than pigment stone gallbladders (0.18 +/- 0.06 vs. 1.2 +/- 0.23 nM). Similar results were observed in binding studies with the CCK-A receptor antagonist [3H]L-364,718. Cross-linking and saturation binding studies also showed significantly less CCK binding in gallbladders with cholesterol stones. These abnormalities were reversible after incubation with cholesterol-free liposomes. The Bmax increased (P < 0.01) and the dissociation constant decreased (P < 0.001) after incubation with cholesterol-free liposomes. In conclusion, human gallbladders with cholesterol stones have impaired CCK receptor binding compared with those with pigment stones. These changes are reversed by removal of the excess membrane cholesterol. These receptor alterations may contribute to the defective contractility of the gallbladder muscle in patients with cholesterol stones.

Gustatory perception arises not only from intracellular transduction cascades within taste receptor cells but also from cell-to-cell communication among the cells of the taste bud. This study presents novel data demonstrating that the brain-gut peptide cholecystokinin (CCK) is expressed in subsets of taste receptor cells, and that it may play a signaling role unknown previously within the taste bud. Immunocytochemistry revealed positively stained subsets of cells within taste buds throughout the oral cavity. These cells typically displayed round nuclei with full processes, similar to those classified as light cells. Peptide expression was verified using nested PCR on template cDNA derived from mRNA extracted from isolated posterior taste buds. Multiple physiological actions of cholecystokinin on taste receptor cells were observed. An outward potassium current, recorded with the patch-clamp technique, was inhibited by exogenous application of sulfated cholecystokinin octapeptide in a reversible and concentration-dependent manner. Pharmacological analysis suggests that this inhibition is mediated by CCK-A receptors and involves PKC phosphorylation. An inwardly rectifying potassium current, typically invariant to stimulation, was also inhibited by cholecystokinin. Additionally, exogenous cholecystokinin was effective in elevating intracellular calcium as measured by ratiometric techniques with the calcium-sensitive dye fura-2. Pharmacology similarly demonstrated that these calcium elevations were mediated by CCK-A receptors and were dependent on intracellular calcium stores. Collectively, these observations suggest a newly discovered role for peptide neuromodulation in the peripheral processing of taste information.

In indirect immunofluorescence tests, antibodies against pure porcine cholecystokinin (CCK) have detected specific CCK cells in the duodenal and jejunal mucosa of the dog and man. The CCK cells were scattered in the epithelium of the crypts, although some were in the villi. No CCK cells were found in the stomach, pancreas, terminal ileum, or colon. Some pyloric G cells also showed some reactivity with CCK antiserum, but absorption of CCK antiserum with gastrin C terminal pentapeptide prevented the staining of pyloric cells and provided specific staining of intestinal CCK cells. Anti-human gastrin I serum stained some intestinal cells too. Most of such cells did not react when gastrin antiserum was absorbed with pure CCK (a treatment that did not prevent the staining of pyloric gastrin cells); they were interpreted as cross-reacting CCK cells rather than as intestinal gastrin cells.

Taste receptor cells are primary sensory receptors utilized by the nervous system to detect the presence of gustatory stimuli in the oral cavity. These cells are particularly heterogeneous and may be divided into various subtypes based on morphological, histochemical, or physiological criteria. One example is the heterogeneous expression of neuropeptides, such as cholecystokinin and vasoactive intestinal polypeptide. These peptides are hypothesized to participate in the transduction processes. To pursue examination of this hypothesis, this study explored the relationship of peptide expression with two important and mostly non-overlapping transductive elements--the taste-specific G protein gustducin, involved in bitter and sweet transduction cascades, and the seven transmembrane taste receptor T1R2, hypothesized to respond to sweet compounds. Double labeling experiments were performed on taste buds of the posterior rat tongue combining immunocytochemistry for peptide expression and in situ hybridization experiments for either gustducin or T1R2 expression. Additionally, vasoactive intestinal peptide (VIP)-expression in posterior taste receptor cells was confirmed using the technique of RT-PCR. More than half (56%) of the CCK-expressing taste receptor cells co-expressed alpha-gustducin mRNA whereas far fewer (15%) co-expressed T1R2 mRNA. A majority of VIP-expressing taste receptor cells co-expressed alpha-gustducin mRNA (60%) whereas only 19% of these cells co-expressed T1R2 mRNA. More remarkable was the observation that these two peptides displayed almost identical expression patterns with these signal transduction molecules, suggesting that peptides are not randomly expressed with relation to signal transduction molecules. This observation supports the hypothesis that peptides may play roles in transduction. Further physiological exploration will be required to elucidate the nature of these roles.

OBJECTIVE

We determined the role of gastrin in the regulation of cholangiocarcinoma growth.

METHODS

We evaluated for the functional presence of cholecystokinin (CCK)-B/gastrin receptors in the cholangiocarcinoma cell lines, Mz-ChA-1, HuH-28 and TFK-1. We determined the effect of gastrin on the growth of Mz-ChA-1, HuH-28 and TFK-1 cells. We evaluated the effect of gastrin on growth and apoptosis of Mz-ChA-1 in the absence or presence of inhibitors for CCK-A (L-364, 718) and CCK-B/gastrin (L-365, 260) receptors, the intracellular Ca2+ chelator (BAPTA/AM), and the protein kinase C (PKC)-alpha inhibitor, H7. We evaluated if gastrin effects on Mz-ChA-1 growth and apoptosis are associated with membrane translocation of PKC-alpha.

RESULTS

Gastrin inhibited DNA synthesis of Mz-ChA-1, HuH-28 and TFK-1 cells in a dose- and time-dependent fashion. The antiproliferative effect of gastrin on Mz-ChA-1 cells was inhibited by L-365, 260, H7 and BAPTA/AM but not L-364, 718. Gastrin induced membrane translocation of PKC-alpha. The inhibition of growth of Mz-ChA-1 cells by gastrin was associated with increased apoptosis through a PKC-dependent mechanism.

CONCLUSIONS

Gastrin inhibits the growth of Mz-ChA-1, HuH-28 and TFK-1 cells. Gastrin inhibits growth and induces apoptosis in Mz-ChA-1 cells through the Ca2+-dependent PKC-alpha. The data suggest a therapeutic role for gastrin in the modulation of cholangiocarcinoma growth.