Evidence is accumulating that gastrin precursors may act as growth factors for the colonic mucosa in vivo. The aims of this study were to prepare recombinant human progastrin(6-80) and to investigate its structure and biological activities in vitro. Human progastrin(6-80) was expressed in Escherichia coli as a glutathione S-transferase fusion protein. After thrombin cleavage progastrin(6-80) was purified by reverse phase high pressure liquid chromatography and characterized by radioimmunoassay, amino acid sequencing, and mass spectrometry. Assays for metal ions by atomic emission spectroscopy revealed the presence of a single tightly bound calcium ion. Progastrin(6-80) at concentrations in the pm to nm range stimulated proliferation of the conditionally transformed mouse colon cell line YAMC. The observations that progastrin(6-80) did not bind to either the cholecystokinin (CCK)-A or the gastrin/CCK-B receptor expressed in COS cells and that antagonists selective for either receptor did not reverse the proliferative effects of progastrin(6-80) suggested that progastrin(6-80) stimulated proliferation independently of either the CCK-A or the gastrin/CCK-B receptor. We conclude that recombinant human progastrin(6-80) is biologically active and contains a single calcium ion. With the exception of the well known zinc-dependent polymerization of insulin and proinsulin, this is the first report of selective, high affinity binding of metal ions to a prohormone.

Anterograde tracing studies were conducted in order to identify efferents from the arcuate nucleus, which contains the hypothalamic opiocortin neuronal pool. Phaseolus vulgaris leucoagglutinin (PHA-L) was stereotaxically iontophoresed into the arcuate nucleus and the terminal fields emanating from the labelled perikarya were identified immunocytochemically. PHA-L-immunoreactive (-ir) fibers were identified in nucleus accumbens, lateral septal nucleus, bed nucleus of the stria terminalis, medial and lateral preoptic areas, anterior hypothalamus, amygdaloid complex, lateral hypothalamus, paraventricular nucleus, zona incerta, dorsal hypothalamus, periventricular gray, medial thalamus and medial habenula. In the brainstem, arcuate terminals were identified in the periaqueductal gray (PAG), dorsal raphe nucleus (DRN), nucleus raphe magnus (NRM), nucleus raphe pallidus, locus coeruleus, parabrachial nucleus, nucleus reticularis gigantocellularis pars alpha, nucleus tractus solitarius and dorsal motor nucleus of the vagus nerve. Dual immunostaining was used to identify the neurochemical content of neurons in arcuate terminal fields in the brainstem. Arcuate fiber terminals established putative contacts with serotonergic neurons in the ventrolateral PAG, DRN and NRM and with noradrenergic neurons in periventricular gray, PAG and locus coeruleus. In the PAG, arcuate fibers terminated in areas with neurons immunoreactive to substance P, neurotensin, enkephalin and cholecystokinin (CCK) and putative contacts were identified with CCK-ir cells. This study provides neuroanatomical evidence that putative opiocortin neurons in the arcuate nucleus influence a descending system which modulates nociception.

Adult Otsuka Long-Evans Tokushima fatty (OLETF) rats lack functional cholecystokinin A (CCK-A) receptors, are diabetic, hyperphagic, and obese, and have patterns of ingestion consistent with a satiety deficit secondary to CCK insensitivity. Because dietary fat potently stimulates CCK release, we examined how dietary fat modulates feeding in adult male OLETF rats and their lean [Long-Evans Tokushima (LETO)] controls. High-fat feeding produced sustained overconsumption of high-fat diet (30% corn oil in powdered chow) over a 3-wk period in OLETF but not LETO rats. We then assessed the ability of gastric gavage (5 ml, 1-2 kcal/ml x 15 s) or duodenal preloads (1 kcal/ml, 0.44 ml/min x 10 min) of liquid carbohydrate (glucose), protein (peptone), or fat (Intralipid) to suppress subsequent 30-min 12.5% glucose intake in both strains. In OLETF rats, gastric and duodenal fat preloads were significantly less effective in suppressing subsequent intake than were equicaloric peptone or glucose. These results demonstrate that OLETF rats fail to compensate for fat calories and suggest that their hyperphagia and obesity may stem from a reduced ability to process nutrient-elicited gastrointestinal satiety signals.

The present study was designed to explore whether exosomal lncRNA-KLF3-AS1 derived from human mesenchymal stem cells (hMSCs) can serve as a positive treatment for osteoarthritis (OA). hMSCs and MSC-derived exosomes (MSC-exo) were prepared for morphological observation and identification by transmission electron microscopy and flow cytometry. IL-1β-induced OA chondrocytes and collagenase-induced rat model of OA were established for the further experiments. Lentivirus-mediated siRNA targeting KLF3-AS1 was transfected into MSCs for silencing KLF3-AS1. The real-time quantitative PCR and western blotting analysis were performed to examine the mRNA and protein levels of type II collagen alpha 1 (Col2a1), aggrecan, matrix metalloproteinase 13 and runt-related transcription factor 2. Cell proliferation, apoptosis and migration were evaluated by CCK-8 assay, flow cytometry and transwell assay. HE (hematoxylin and eosin) staining and immunohistochemistry were used for histopathological studies. MSC-exo ameliorated IL-1β-induced cartilage injury. Furthermore, lncRNA KLF3-AS1 was markedly enriched in MSC-exo, and exosomal KLF3-AS1 suppressed IL-1β-induced apoptosis of chondrocytes. Further in vivo investigation indicated that exosomal KLF3-AS1 promoted cartilage repair in a rat model of OA. Exosomal KLF3-AS1 promoted cartilage repair and chondrocyte proliferation in a rat model of OA, which might be an underlying therapeutic target for OA.

Cholecystokinin (CCK) plays an important role in pancreatic carcinogenesis. While human CCK-A and -B receptors have been fully characterized, their relative roles in human pancreatic adenocarcinoma remain unclear. Thus, expression of CCK-A and -B receptors in normal human pancreas, pancreatic adenocarcinomas, and other human extrapancreatic tissues and malignancies was examined, using reverse transcription followed by the polymerase chain reaction (RT-PCR). mRNA isolated from 15 normal pancreas specimens, 22 pancreatic adenocarcinomas, and 58 extrapancreatic tissues and tumors was subjected to RT-PCR using primers specific for human CCK-A and -B receptors. Expression of CCK-B receptors was detected in all tissues arising from pancreas and in most extrapancreatic tissues and tumors. In contrast, CCK-A receptors exhibited a more selective pattern of expression in gall bladder, intestine, brain, ovary, spleen, and thymus. Of significance, CCK-A receptors were expressed selectively in all pancreatic adenocarcinomas, but not in any normal pancreas specimens. In situ hybridization, using receptor-specific riboprobes, localized CCK-A receptor expression to ductal cells, the presumed origin of most human pancreatic adenocarcinomas. Southern blot analysis revealed no evidence of CCK-A receptor gene amplification or rearrangement in pancreatic adenocarcinomas. Because of its selective expression, the CCK-A receptor may serve as selective biomarker for pancreatic adenocarcinoma.

OBJECTIVE

Transient lower esophageal sphincter relaxations have been found to be involved in gastroesophageal reflux. The purpose of this study was to determine whether cholecystokinin (CCK) and nitric oxide are involved in the occurrence of these relaxations.

METHODS

Pharyngeal, esophageal, lower esophageal sphincter, and gastric pressures were monitored in five dogs through a cervical esophagostomy. Gastric distentions with air, at a constant pressure of 1.56 kPa, were performed for 30-minute sessions using a barostat.

RESULTS

During gastric distention at 1.56 kPa, transient relaxations occurred at a mean rate of 7.2 +/- 0.6 every 30 minutes. CCK-8 infused intravenously (0.1-1 microgram.kg-1.h-1) dose dependently increased the occurrence of relaxations while it was reduced by the CCK-A receptor antagonist devazepide but not the CCK-B antagonist L365260, both administered intravenously in a dose range of 0.1-100 micrograms/kg. The two antagonists administered intracerebroventricularly (1 microgram/kg) did not modify the occurrence of relaxations. Both devazepide and L365260 (10 micrograms/kg) reduced the CCK-induced relaxations, but devazepide was more potent. The nitric oxide synthase inhibitor NG-nitro-L-arginine-methyl ester (20 mg/kg) reduced the number of relaxations during gastric distention in the presence or absence of CCK infusion. This effect was reversed by L-arginine but not D-arginine (200 mg/kg).

CONCLUSIONS

CCK is involved in the occurrence of transient lower esophageal sphincter relaxations through peripheral CCK-A receptors and an L-arginine nitric oxide pathway.

OBJECTIVE

Gastrointestinal inflammation reduces food intake but the biological mechanisms explaining suppressed feeding during inflammation are unknown. We have used a model of upper gut infection (Trichinella spiralis in the mouse) to study the effect of inflammation on food intake, and explored the role of a key enteroendocrine cell (EEC) in the regulation of feeding by the immune response.

METHODS

Food intake of NIH mice infected with the intestinal nematode Trichinella spiralis was measured. Duodenal cholecystokinin (CCK) cells were counted. Plasma CCK was measured. Infected mice were treated with a specific CCKCCK was mechanistically examined by treating mice with CD4 or mast cell neutralising antibodies. The role of the T helper 2 response was further explored in mice genetically deficient for interleukin (IL)-4, IL-13, or IL-4Ralpha (receptor alpha subunit).

RESULTS

Food intake of infected mice was significantly reduced at the temporal peak of intestinal inflammation. CCK expressing EEC were upregulated in infected mice, and plasma CCK levels were increased. A CCKalpha.

CONCLUSIONS

The data show for the first time that intestinal inflammation results in reduced food intake due to upregulation of CCK. Moreover, following infection, food intake and CCK expressing cells are under the specific control of CD4+ T-cells, via release of IL-4 and IL-13.

The 2 gastrointestinal peptides cholecystokinin (CCK) and gastrin, which act through CCK-A receptors (having high affinity for CCK) or CCK-B/gastrin receptors (having high affinity for CCK and gastrin), are considered to be important tumor growth factors. We have evaluated CCK-A and CCK-B/gastrin receptors in 34 human thyroid cancers using in vitro receptor autoradiography with 2 different radioligands. We demonstrate high-affinity CCK-B/gastrin receptors in medullary thyroid carcinomas, present at very high incidence (92%) but the absence of these receptors in non-medullary thyroid carcinomas or in normal thyroid glands. CCK-B/gastrin receptors are therefore likely to be the molecular substrate for the pentagastrin-stimulation test, widely used in medullary thyroid carcinomas; moreover, they represent the targets for physiologically secreted gastrin or CCK which, as growth factors, may stimulate the growth of medullary thyroid carcinomas. Furthermore, these results have diagnostic as well as therapeutic implications: radiolabeled gastrin and CCK analogs may be used for scintigraphic tumor localization in vivo, whereas CCK-B-selective antagonists may be of therapeutic value.

Food intake and meal size are reduced in female Long-Evans rats during estrus. To investigate the contribution of the satiating action of endogenous cholecystokinin (CCK) to this, rats were injected with 1 mg/kg of the potent, selective CCK(A) receptor antagonist, devazepide, during diestrus, when meal size is maximal, and during estrus, when it is minimal. Devazepide increased spontaneous food intake and meal size during estrus, but not during diestrus. Meal frequency was not affected by devazepide treatment. These results indicate that the potency of the CCK satiety-signaling system increases during estrus.

Supraphysiological concentrations of cholecystokinin (CCK) induce chemokine expression in rat pancreatic acini through the activation of the transcription factor NF-kappaB. In the current study, the intracellular signals involved in these pathophysiological effects of CCK were investigated. CCK induction of mob-1 expression in isolated rat pancreatic acini was blocked by the protein kinase C (PKC) inhibitors GF-109203X and Ro-32-0432 and by the intracellular Ca(2+) chelator BAPTA. CCK induced NF-kappaB nuclear translocation, and DNA binding was also blocked by GF-109203X and BAPTA. Direct activation of PKC with TPA induced mob-1 chemokine expression and activated NF-kappaB DNA binding to a similar extent as did CCK. Increasing intracellular Ca(2+) using ionomycin had no effect on mob-1 mRNA levels or NF-kappaB activity. Both CCK and TPA treatments decreased inhibitory kappaB-alpha (IkappaB-alpha) levels, whereas ionomycin had no effect. However, the effects of TPA on IkappaB-alpha degradation were less complete than for CCK. In combination, TPA and ionomycin degraded IkappaB-alpha to a similar extent as CCK. Therefore, activation of NF-kappaB and mob-1 expression by supraphysiological CCK is likely mediated by both PKC activation and elevated intracellular Ca(2+).

The anteroventral periventricular nucleus (AVPv), which lies in the periventricular zone of the preoptic region, is critical for normal phasic gonadotropin secretion since lesions of this nucleus abolish the progesterone-induced surge of luteinizing hormone secretion from the anterior pituitary, block ovulation, and induce persistent vaginal estrus in female rats. However, very little is known about the neurotransmitter-specific pathways associated with this nucleus. In the present study we evaluated the distribution of biochemically specific cells and fibers within the AVPv and adjacent regions by using an indirect immunohistochemical method with antisera to serotonin (5-HT), dopamine beta-hydroxylase (DBH), tyrosine hydroxylase (TH), neuropeptide Y (NPY), cholecystokinin-8 (CCK), vasoactive intestinal polypeptide (VIP), substance P (SP), neurotensin (NT), corticotropin-releasing factor (CRF), luteotropin-releasing hormone (LRH), somatostatin (SS), thyrotropin-releasing hormone (TRH), oxytocin (OXY), vasopressin (VAS), adrenocorticotropic hormone (ACTH1-24), alpha-melanocyte-stimulating hormone (alpha-MSH), leucine-enkephalin (L-ENK), and calcitonin gene-related peptide (CGRP). Our findings indicate that both cells and fibers containing these putative neurotransmitters are differentially distributed in and around the AVPv in accordance with the cytoarchitectonic organization of this part of the preoptic region. The AVPv itself appears to receive strong inputs from SP-, VAS-, CCK-, and SS-containing pathways, whereas the highest densities of L-ENK-, NT-, 5-HT-, NPY-, and DBH-immunoreactive fibers were found in the cell-sparse zone just lateral to the AVPv. The suprachiasmatic preoptic nucleus (PSCh), a small group of cells located ventral to the AVPv just dorsal to the optic chiasm, contained high densities of alpha-MSH- and ACTH-immunoreactive fibers, as well as substantial numbers of fibers containing catecholamines or NPY. In contrast, a dense plexus of VAS-stained fibers was distributed fairly evenly throughout the AVPv and PSCh. Numerous L-ENK-immunoreactive cell bodies, and moderate numbers of CCK-, NT-, and CRF-stained cell bodies were found in the AVPv. The PSCh contained many TH-stained cells (presumably dopaminergic), in addition to a moderate number of CCK-containing cell bodies, while a high density of NT- and CRF-stained cells were found in the cell-sparse zone lateral to the AVPv, in addition to several CCK-, SP-, VIP-, and TH-containing cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Five rhesus monkeys were infused intravenously with partially purified cholecystokinin (CCK) Just prior to a test meal of solid food after overnight food deprivation; CCK produced large, rapid, dose related suppressions of feeding. The lowest dose tested (5 Ivy U/kg body wt) produced a significant inhibition of food intake (26% suppression, P less than 0.05). Equivalent infusions of partially purified CCK or the synthetic COOH-terminal octapeptide of CCK (a pure fragment with all the biological activity of the full molecule) produced equivalent suppressions. In a second experiment, gastric preloads of a potent releaser of endogenous CCK, L-phenylalanine (L-Phe), and a weak releaser, D-phenylalanine (D-Phe) were compared for their relative abilities to suppress food intake at a test meal in nine rhesus monkeys after overnight deprivation. L-Phenylalanine produced large, rapid, dose-related suppressions of feeding, but D-Phe did not. The threshold dose of L-Phe was 0.5 g/kg (32% suppression, P less than 0.01). Neither CCK nor L-Phe caused signs of illness in these experiments. The results demonstrate that intravenous exogenous CCK suppresses feeding in rhesus monkeys and suggest that endogenous CCK has the same effect; they are consistent with the hypothesis that CCK is a satiety signal.

Cholecystokinin (CCK), a peptide originally discovered in the gastrointestinal tract, is one of the most abundant and widely distributed neuropeptides in the brain. In spite of its abundance, recent data indicate that CCK modulates intrinsic neuronal excitability and synaptic transmission in a surprisingly cell-type specific manner, acting as a key molecular switch to regulate the functional output of neuronal circuits. The central importance of CCK in neuronal networks is also reflected in its involvement in a variety of neuropsychiatric and neurological disorders including panic attacks and epilepsy.

Through binding to cholecystokinin (CCK) A receptors, CCK is an important physiologic regulator of both gallbladder contraction and pancreatic enzyme secretion. In this work, we have used a combination of hybridization screening of a cDNA library and polymerase chain reaction to clone a 2.1 kb cDNA which encodes the human gallbladder CCKA receptor. Nucleotide sequence analysis revealed an open reading frame encoding a 428 amino acid protein, with seven putative transmembrane domains and a high degree of homology with the rat CCKA receptor. COS cells transfected with this cDNA clone bound CCK-8 and L-364,718 with high affinities appropriate for the CCKA receptor, and exhibited a transient increase in intracellular calcium in response to CCK. This should provide an important resource for the analysis of the role of this receptor in human physiology and pathophysiology.

Cholecystokinin (CCK) interacts with two types of G protein-coupled receptors in the brain: CCK-A and CCK-B receptors. Both CCK and CCK-B receptors are widely distributed in the hippocampal formation, but the functions of CCK there have been poorly understood. In the present study, we initially examined the effects of CCK on GABA(A) receptor-mediated synaptic transmission in the hippocampal formation and then explored the underlying cellular mechanisms by focusing on the dentate gyrus region, where the highest levels of CCK-binding sites have been detected. Our results indicate that activation of CCK-B receptors initially and transiently increased spontaneous IPSC (sIPSC) frequency, followed by a persistent reduction. The effects of CCK were more evident in juvenile rats, suggesting that they are developmentally regulated. Cholecystokinin failed to modulate the miniature IPSCs recorded in the presence of TTX and the amplitude of the evoked IPSCs, but produced a transient increase followed by a reduction in action potential firing frequency recorded from GABAergic interneurons, suggesting that CCK acts by modulating the excitability of the interneurons to regulate GABA release. Cholecystokinin reduced the amplitude of the after-hyperpolarization of the action potentials, and application of paxilline or charybdotoxin considerably reduced CCK-mediated modulation of sIPSC frequency, suggesting that the effects of CCK are related to the inhibition of Ca(2+)-activated K(+) currents (I(K(Ca))). The effects of CCK were independent of the functions of phospholipase C, intracellular Ca(2+) release, protein kinase C or phospholipase A(2), suggesting a direct coupling between the G proteins of CCK-B receptors and I(K(Ca)). Our results provide a novel mechanism underlying CCK-mediated modulation of GABA release.

PKC is known to be activated by pancreatic secretagogues such as CCK and carbachol and to participate along with calcium in amylase release. Four PKC isoforms, alpha, delta, epsilon, and zeta, have been identified in acinar cells, but which isoforms participate in amylase release are unknown. To identify the responsible isoforms, we used translocation assays, chemical inhibitors, and overexpression of individual isoforms and their dominant-negative variants by means of adenoviral vectors. CCK stimulation caused translocation of PKC-alpha, -delta, and -epsilon, but not -zeta from soluble to membrane fraction. CCK-induced amylase release was inhibited approximately 30% by GF109203X, a broad spectrum PKC inhibitor, and by rottlerin, a PKC-delta inhibitor, but not by Gö6976, a PKC-alpha inhibitor, at concentrations from 1 to 5 microM. Neither overexpression of wild-type or dominant-negative PKC-alpha affected CCK-induced amylase release. Overexpression of PKC-delta and -epsilon enhanced amylase release, whereas only dominant-negative PKC-delta inhibited amylase release by 25%. PKC-delta overexpression increased amylase release at all concentrations of CCK, but dominant-negative PKC-delta only inhibited the maximal concentration; both similarly affected carbachol and JMV-180-induced amylase release. Overexpression of both PKC-delta and its dominant-negative variant affected the late but not the early phase of amylase release. GF109203X totally blocked the enhancement of amylase release by PKC-delta but had no further effect in the presence of dominant-negative PKC-delta. These results indicate that PKC-delta is the PKC isoform involved with amylase secretion.

Cholecystokinin (CCK) is an important satiety factor, acting via the vagus nerve to influence central feeding centers. CCK binding sites have been demonstrated in the vagal sensory nodose ganglion and within the nerve proper. Using in situ hybridization, expression of the CCK(A) and (B) receptors (Rs), as well as of CCK itself, was studied in the normal nodose ganglion (NG), and after vagotomy, starvation and high-fat diet. CCK(A)-R mRNA expression in dorsal root ganglia (DRGs) was also explored. In the NG, 33% of the neuron profiles (NPs) contained CCK(A)-R mRNA and in 9% we observed CCK(B)-R mRNA. CCK mRNA was not found in normal NGs. Peripheral vagotomy decreased the number of CCK(A)-R mRNA-expressing NPs, dramatically increased the number of CCK(B)-R mRNA, and induced CCK mRNA and preproCCK-like immunoreactivity in nodose NPs. No significant differences in the number of NPs labelled for either mRNA species were detected following 48 h food deprivation or in rats fed a high-fat content diet. In DRGs, 10% of the NPs expressed CCK(A)-R mRNA, a number that was not affected by either axotomy or inflammation. This cell population was distinct from neurons expressing calcitonin gene-related peptide mRNA. These results demonstrate that the CCK(A)-R is expressed by both viscero- and somatosensory primary sensory neurons, supporting a role for this receptor as a mediator both of CCK-induced satiety and in sensory processing at the spinal level. The stimulation of CCK and CCK(B)-R gene expression following vagotomy suggests a possible involvement in the response to injury for these molecules.

Pharmacological experiments suggest that satiation associated with intestinal infusion of several nutrients is mediated by CCK-A receptors. Otsuka Long-Evans Tokushima Fatty, (OLETF), rats do not express CCK-A receptors and are insensitive to the satiation-producing effects of exogenous CCK. To further evaluate the role of CCK-A receptors in satiation by intestinal nutrient infusion, we examined intake of solid (pelleted rat chow) or liquid (12.5% glucose) food intake, following intestinal infusions of fats (oleic acid or fat emulsion), sugars (maltotriose or glucose), or peptone in OLETF rats and Long Evans Tokushima Otsuka control rats (LETO). Intestinal infusion of glucose or maltotriose reduced solid food intake more in LETO than in OLETF rats from 30 min through 4 h post infusion. Reduction of solid food intake by intestinal infusions of fat or peptone did not differ between OLETF and LETO rats during the first 30 min post infusion, but reduction of intake by these infusates was attenuated in OLETF rats over the ensuing 4h post infusion. Intestinal infusion of glucose, oleate, fat emulsion and peptone reduced 30-min intake of 12.5% glucose more in LETO than OLETF rats. Furthermore, pretreatment with the CCK-A receptor antagonist, devazepide, attenuated intestinal nutrient-induced reduction of food intake only in LETO, but not OLETF rats. Our results confirm pharmacological results, indicating that CCK-A receptors participate in satiation by nutrients that elevate plasma CCK concentrations, as well as by nutrients that do not stimulate secretion of endocrine CCK. In addition, our results indicate: 1) that OLETF rats have deficits in the satiation response to a variety of intestinal nutrient infusions; 2) that the temporal pattern for CCK-A receptor participation in satiation by intestinal nutrients is different during ingestion of liquid and solid foods and 3) that intestinal nutrients provide some satiation signals that are CCK-A receptor mediated and some that are not.

The gastrointestinal peptide cholecystokinin (CCK) is known to stimulate growth of human pancreatic cancer in a receptor-mediated fashion. The purpose of this study was to characterize the receptor responsible for the trophic effects of CCK in cancer cells. With the use of homogenates of PANC-1 human pancreatic cancer cells grown in vitro, the binding characteristics and optimal conditions of radiolabeled selective CCK-receptor antagonists ([3H]L-365,260 and [3H]L-364,718) were examined. Specific and saturable binding was detected with [3H]L-365,260, and Scatchard analysis revealed that the data were consistent for a single site of binding with a binding affinity of 4.3 +/- 0.6 nM and a binding capacity (Bmax) of 283 +/- 68 fmol/mg protein in log phase cells. Binding was dependent on protein concentration, time, temperature, and pH and was sensitive to Na+, K+, Mg2+, and ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. In contrast to log phase cells, Bmax decreased by 80 and 92% in confluent and postconfluent cultures, respectively. Subcellular fractionation studies revealed that binding was in the membrane fraction. Competition experiments indicated that L-365,260 and gastrin were more effective at displacing the radiolabeled L-365,260 than CCK. No binding was detected with the CCK-A antagonist [3H]L-364,718. Assays performed with [3H]L-365,260 on five additional human pancreatic cancer cell lines in vitro and tumor tissue from xenografts in nude mice also revealed specific and saturable binding. These results provide the first identification of a CCK-B/gastrin receptor in human pancreatic cancer cells and tumors and explain the effects of CCK on the growth of this malignancy.

Exogenous cholecystokinin (CCK) decreases food intake and causes satiety in animals and man. However, it has not been established that endogenous CCK causes satiety or whether the response is mediated by peripheral-type (CCK-A) or brain-type (CCK-B) receptors. The development of potent and selective antagonists for CCK-A (MK-329) and CCK-B (L-365,260) receptors now allows these issues to be addressed. The CCK-A antagonist MK-329 and the CCK-B antagonist L-365,260 increased food intake in partially satiated rats and postponed the onset of satiety; however, L-365,260 was 100 times more potent than MK-329 in increasing feeding and preventing satiety. These results suggest that endogenous CCK causes satiety by an agonist action on CCK-B receptors in the brain.

This study was aimed at an assessment of the role of oxygen-derived free radicals, cytokines and endogenous cholecystokinin (CCK) in the pathogenesis of L-arginine (Arg)-induced acute pancreatitis in rat. We measured the levels of malonyl dialdehyde (MDA), glutathione peroxidase (GPx), catalase and superoxide dismutase (Mn- and Cu, Zn-SOD) in pancreatic tissue, the serum levels of tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and CCK, and evaluated the protective effect of the xanthine oxidase inhibitor allopurinol and a novel CCK receptor antagonist KSG-504. Acute pancreatitis was induced in male Wistar rats by injecting 2x 250 mg/100 g body weight of Arg intraperitoneally in an 1-h interval, as a 20% solution in 0.15 M NaCl. Control rats received the same quantity of glycine. 200 mg x kg(-1) allopurinol 30 min before the first Arg treatment or 50 mg x kg(-1) KSG-504 30 min before and 6, 18 and 36 h after the first Arg injection was administered subcutaneously. Rats were killed at 6, 12, 24 and 48 h following Arg administration, and acute pancreatitis was confirmed by a serum amylase level elevation and typical inflammatory features observed microscopically. The serum level of amylase reached the peak level at 24 h after the Arg injection (30,800 +/- 3,813 versus 6,382 +/- 184 U x L(-1) in the control) and normalized at 48 h. The tissue concentration of MDA was significantly elevated at 24 h, and reached the peak value at 48 h (5.00 +/- 1.75 versus 0.28 +/- 0.05 nM x mg(-1) protein in the control). The catalase and Mn-SOD activities were significantly decreased throughout the study, while the GPx activity was significantly reduced at 6 and 12 h, and the Cu, Zn-SOD activity was significantly lower at 12 h after the Arg injection as compared with the controls. Both the TNF-alpha and the IL-6 levels were already elevated significantly at 12 h and peak at 24 h versus the controls (19.1 +/- 7.9 U x mL(-1) and 57.6 +/- 11.2 pg x mL(-1) versus 3.1 +/- 0.8 U x mL(-1) and 15.2 +/- 3.1 pg x mL(-1), respectively). No significant changes in plasma CCK levels were observed. Allopurinol treatment markedly reduced the serum amylase elevation (12.631 +/- 2.257 U x L(-1) at 24 h), prevented the increase in tissue MDA concentration (0.55 +/- 0.09 nM x mg(-1) protein at 48 h) and significantly ameliorated the pancreatic edema, necrosis and inflammation at 48 h after Arg administration. KSG-504 administration did not exert any beneficial effect on the development of histopathological changes neither modified the serum amylase or cytokine levels. Oxygen-derived free radicals and cytokines are involved, while endogenous CCK does not seem to play a role in the pathogenesis of Arg-induced acute pancreatitis.

OBJECTIVE

Gastrin and cholecystokinin (CCK) are gut-brain peptides, with multiple functions in the gastrointestinal tract mediated through CCK-B-gastrin and CCK-A receptors. The receptor localization is, however, not well established in humans. The aim of this study was to investigate the distribution and pharmacological characteristics of CCK-A and CCK-B receptors in the human upper gastrointestinal tract and compare them with those in the rat and dog.

METHODS

CCK receptors were localized by in vitro receptor autoradiography with 125I-[Leu15]gastrin-I and 125I-[D-Tyr-Gly, Nle(28,31)]-CCK 26-33 in stomach and gallbladder.

RESULTS

High concentrations of CCK-B-gastrin receptors were detected in the midglandular region of the human fundic mucosa. CCK-A receptors were found in the basal region of the antral and fundic mucosa. CCK-A receptors were also located in the muscularis propria of antrum, fundus, and gallbladder, whereas CCK-B-gastrin receptors were only detected in gastric fundic circular muscle. Two nonpeptide CCK receptor antagonists distinguish both receptor subtypes. Comparisons among humans, dogs, and rats suggest that localization and density of CCK receptor subtypes in the upper gastrointestinal tract are species-dependent.

CONCLUSIONS

Localization of CCK receptor subtypes in human upper gastrointestinal tract provides a biochemical and morphological basis for the separate regulatory roles of gastrin and CCK.

OBJECTIVE

To study the effect of cholecystokinin-octapeptide (CCK-8) on systemic hypotension and cytokine production in lipopolysaccharide (LPS)-induced endotoxic shock (ES) rats.

METHODS

The changes of blood pressure were observed using physiological record instrument in four groups of rats: LPS (8mg.kg(-1),iv) induced ES; CCK-8 (40 microg.kg(-1), iv) pretreatment 10 min before LPS (8mg.kg(-1)); CCK-8 (40 micro.kg(-1), iv) or normal saline (control) groups. Differences in tissue and circulating specificity of the proinflammatory cytokines (TNF-alpha, IL-1beta and IL-6) were assayed with ELISA kits.

RESULTS

CCK-8 reversed LPS-induced decrease of mean artery blood pressure (MABP) in rats. Compared with control, LPS elevated the serum level of IL-6 significantly (3567 +/- 687 ng.L(-1) vs 128 +/- 22 ng.L(-1), P<0.01), while contents of TNF-alpha and IL-1beta elevated significantly (277 +/- 86 ng.L(-1) vs not detectable and 43 +/- 9 ng.L(-1) vsnot detectable, P<0.01) but less extent than IL-6. CCK-8 significantly inhibited the LPS-induced increase in serum TNF-alpha IL-1beta and IL-6. LPS elevated spleen and lung content of IL-1beta significantly (5184 +/- 85 ng.L(-1) vs 1047 +/- 21 ng.L(-1) and 4050 +/- 614 ng.L(-1) vs not detectable, P<0.01), while levels of TNF-alpha and IL-6 also rose significantly but in less extent than IL-1beta. CCK-8 inhibited the LPS-induced increase of the cytokines in spleen and lung. In the heart, CCK-8 significantly inhibited LPS-induced increase of TNF-alpha (864 +/- 123 ng.L(-1) in CCK-8+LPS group vs 1599 +/- 227 ng.L(-1) in LPS group, P < 0.01), and IL-1beta (282 +/- 93 ng.L(-1) in CCK-8+LPS group vs 621 +/- 145ng.L(-1) in LPS group, P < 0.01).

CONCLUSIONS

CCK-8 reverses ES, which may be related to its inhibitory effect on the overproduction of cytokines.

OBJECTIVE

Orexin-A and -B are brain-gut peptides that stimulate food intake via orexin-R1 and -R2 receptors. Cholecystokinin (CCK) inhibits food intake via CCK(A) receptors expressed on vagal afferent neurons. The purpose of the study was to determine whether vagal afferent neurons express OX-R1 and OX-R2 and whether orexin-A inhibits responses to CCK.

METHODS

OX-R1 and -R2 expression by rat and human nodose ganglia was examined by reverse-transcriptase polymerase chain reaction (RT-PCR). Receptor localization was determined by immunohistochemistry. Responses of rat jejunal afferent fibers were examined by electrophysiology.

RESULTS

Both rat and human nodose ganglia expressed OX-R1 as detected by RT-PCR, and humans also expressed OX-R2. The identity of the products was confirmed by sequencing. Immunohistochemistry indicated expression of OX-R1 in both species in neurons that also expressed CCK(A) and leptin receptors. In human ganglia there was also expression in glial cells that was absent in rats. Orexin-A had no effect on the resting discharge of afferent nerve fibers but inhibited responses to CCK.

CONCLUSIONS

OX-R1 and CCK(A) receptors are expressed by human and rat vagal afferent neurons. Orexin inhibits responses to CCK suggesting a role in modulation of gut to brain signaling.