Lanthanide-based luminescent ligand binding assays are superior to traditional radiolabel assays due to improving sensitivity and affordability in high-throughput screening while eliminating the use of radioactivity. Despite significant progress using lanthanide(III)-coordinated chelators such as diethylenetriaminepentaacetic acid (DTPA) derivatives, dissociation-enhanced lanthanide fluoroimmunoassays (DELFIAs) have not yet been successfully used with more stable chelators (e.g., tetraazacyclododecyltetraacetic acid [DOTA] derivatives) due to the incomplete release of lanthanide(III) ions from the complex. Here a modified and optimized DELFIA procedure incorporating an acid treatment protocol is introduced for use with Eu(III)-DOTA-labeled peptides. Complete release of Eu(III) ions from DOTA-labeled ligands was observed using hydrochloric acid (2.0M) prior to the luminescent enhancement step. [Nle(4),d-Phe(7)]-alpha-melanocyte-stimulating hormone (NDP-alpha-MSH) labeled with Eu(III)-DOTA was synthesized, and the binding affinity to cells overexpressing the human melanocortin-4 (hMC4) receptor was evaluated using the modified protocol. Binding data indicate that the Eu(III)-DOTA-linked peptide bound to these cells with an affinity similar to its DTPA analogue. The modified DELFIA procedure was further used to monitor the binding of an Eu(III)-DOTA-labeled heterobivalent peptide to the cells expressing both hMC4 and cholecystokinin-2 (CCK-2) receptors. The modified assay provides superior results and is appropriate for high-throughput screening of ligand libraries.

We transfected COS cells with cDNA for rat cholecystokinin-A (CCK-A) and different CCK-B receptors and measured binding of 125I-CCK-8, [3H]L-364,718 and [3H]L-365,260 to characterize the different affinity states for each type of CCK receptor. Rat CCK-A and CCK-B receptors, canine CCK-B receptors and canine mutant CCK-B (M-CCK-B) receptors in which the leucine in position 355 was replaced by valine each existed in three different affinity states for CCK-8, high affinity, low affinity, and very low affinity. In rat CCK-A and probably CCK-B receptors, most were in the very low affinity state, whereas with canine CCK-B and M-CCK-B receptors, most were in the low affinity state. Studies with CCK receptor agonists, CCK-8, gastrin, and CCK-JMV-180, in conjunction with CCK receptor antagonists, L-364,718 and L-365,260, showed a different pattern of affinities for these ligands at the different CCK receptors. Thus, each transfected CCK receptor can exist in three different affinity states for CCK-8 and has a characteristic pattern of interaction with different ligands. This ability to exist in multiple affinity states is an intrinsic property of the CCK receptor molecule itself.

OBJECTIVE

Exogenous cholecystokinin (CCK) decreases lower esophageal sphincter (LES) pressure and increases transient LES relaxations (TLESRs) in humans. The aims of this study were to determine whether endogenous CCK increases gastroesophageal reflux in humans and whether this is a direct effect on the LES.

METHODS

Esophageal pH, LES pressure, and gallbladder volume were measured in 8 healthy volunteers after ingestion of a 181-kcal meal alone or adding 12 g cholestyramine to increase endogenous CCK release. In 7 additional volunteers, the effect of cholestyramine was studied during intravenous perfusion of saline or the CCK-A receptor antagonist loxiglumide. In circular LES strips from 9 transplant donors, we measured the effect of CCK-8 (10(-11) to 3 x 10(-8) mol/L) on basal tension and on electrical field-induced relaxation.

RESULTS

Cholestyramine increased gallbladder emptying, reflux episodes, TLESRs, and time of esophageal pH of <4. Loxiglumide inhibited postprandial gallbladder emptying, reflux episodes, TLESRs, and time of pH of <4 and prevented the decrease in LES pressure induced by cholestyramine. In vitro, CCK-8 contracted LES strips through a tetrodotoxin-insensitive pathway but did not modify electrical field-induced LES relaxations.

CONCLUSIONS

Endogenous CCK enhances postprandial gastroesophageal reflux in humans by increasing the rate of TLESRs and reduces postprandial LES pressure. These actions seem mediated by extrasphincteric CCK-A receptors that override a direct contractile effect of CCK on the LES muscle.

OBJECTIVE

To study the effect of sulfated cholecystokinin-octapeptide (CCK-8) on systemic hypotension, gene and protein expression of TNF-alpha in the spleen of lipopolysaccharide (LPS)-induced endotoxic shock (ES) rats, and further investigate the signal transduction mechanism of p38 mitogen-activated protein kinase (MAPK).

METHODS

The changes of blood pressure were observed using physiological record instrument in the four groups of rats: LPS (8 mg x kg(-1), iv), CCK-8 (40 microg x kg(-1), iv) pretreatment 10 min before LPS (8 mg x kg(-1)), CCK-8 (40 microg x kg(-1), iv) or normal saline (control) group. The content of TNF-alpha in the spleen was assayed 2 h after LPS administration using ELISA kit and the expression of TNF-alpha mRNA was examined 30 min, 2 h and 6 h after LPS administration by reverse transcribed polymerase chain reaction (RT-PCR). Activation of p38 MAPK was detected with Western blot 30 min after LPS administration.

RESULTS

CCK-8 reversed LPS-induced decrease of mean arterial pressure (MAP) in rats. The content of TNF-alpha in the spleen was (282+/-30) ng x L(-1) in control group, while it increased to (941+/-149) ng x L(-1) in LPS group, P<0.01. CCK-8 significantly inhibited the LPS-induced increase of TNF-alpha content in spleen. It decreased to (462 +/-87) ng x L(-1) in CCK-8+LPS group, P<0.01. The expression of TNF-alpha mRNA 30 min and 2 h after treatment was stronger in LPS group, while it was lowered after CCK-8 pretreatment. The p38 MAPK expression increased significantly in LPS group (5.84 times of control) and CCK-8 increased the activation of p38 MAPK in ES rats (10.74 times of control).

CONCLUSIONS

CCK-8 reverses the decrease of MAP in ES rats and has inhibitory effect on the gene and protein expression of TNF-alpha in spleen, and p38 MAPK may be involved in its signal transduction mechanisms.

OBJECTIVE

To investigate the expression of feeding-related peptide receptors mRNA in GT1-7 cell line and roles of leptin and orexins in the control of GnRH secretion.

METHODS

Receptors of bombesin3, cholecystokinin (CCK)-A, CCK-B, glucagon-like peptide (GLP)1, melanin-concentrating hormone (MCH)1, orexin1, orexin2, neuromedin-B, neuropeptide Y (NPY)1 and NPY5, neurotensin (NT)1, NT2, NT3, and leptin receptor long form mRNA in GT1-7 cells were detected by reversed transcriptase-polymerase chain reaction. GT1-7 cells were treated with leptin, orexin A and orexin B at a cohort of concentrations for different lengths of time, and GnRH in medium was determined by radioimmunoassay (RIA).

RESULTS

Receptors of bombesin 3, CCK-B, GLP1, MCH1, orexin1, neuromedin-B, NPY1, NPY5, NT1, NT3, and leptin receptor long form mRNA were expressed in GT1-7 cells, of which, receptors of GLP1, neuromedin-B, NPY1, and NT3 were highly expressed. No amplified fragments of orexin2, NT2, and CCK-A receptor cDNA were generated with GT1-7 RNA, indicating that the GT1-7 cells did not express mRNA of them. Leptin induced a significant stimulation of GnRH release, the results being most significant at 0.1 nmol/L for 15 min. In contrast to other studies in hypothalamic explants, neither orexin A nor orexin B affected basal GnRH secretion over a wide range of concentrations ranging from 1 nmol/L to 500 nmol/Lat 15, 30, and 60 min.

CONCLUSIONS

Feeding and reproductive function are closely linked. Many orexigenic and anorexigenic signals may control feeding behavior as well as alter GnRH secretion through their receptors on GnRH neurons.

The effects of sulfated (CCK-8-SE) and non-sulfated (CCK-8-NS) cholecystokinin octapeptide on striatal dopamine (DA) metabolism have been investigated on mice. CCK-8-NS facilitated the disappearance of striatal DA, measured after synthesis inhibition with 350 mg/kg of alpha-methyl-p-tyrosine. CCK-8-SE did not affect DA disappearance. In vitro uptake of [3H]DA by striatal slices was affected by neither CCK-8-SE, nor CCK-8-NS (10(-5) M). Potassium-induced in vitro release of [3H]DA from striatal slices was significantly increased by 10(-5) M CCK-8-NS: however, CCK-8-SE likewise increased DA release in this model system. Apomorphine-induced (1.0 mg/kg) stereotyped cage-climbing behavior was not affected by CCK-8-SE but was enhanced by CCK-8-NS. This effect could be antagonized by haloperidol, but not by naloxone. The data suggest that CCK-8-NS affects striatal DA release, disappearance and receptor sensitivity in the mouse. Dopaminergic mechanisms should therefore be regarded as a possible mode of action of CCK-8-NS on brain functions.

Modulation of learning and memory is one of the physiological roles that the neuropeptide cholecystokinin (CCK-8) may play. We have used a behavioural model of olfactory recognition among rats to test this hypothesis and to explore the relationship between CCK-A and CCK-B receptors and memory retention. Adult male rats form a transient memory of a juvenile congenere as indicated by a reduction in the duration of investigatory behaviour upon re-exposure 30 min after an initial exposure, but not when re-exposure is delayed until 120 min afterwards. In the present study, rats were treated after the first contact with various compounds; inhibition and facilitation of olfactory recognition were evaluated as the persistence in investigation 30 min and the decrease in investigation 120 min after pharmacological manipulations, respectively. Systemic injection of CCK-8, of a selective CCK-A agonist, or of non-peptide CCK-B antagonists (CI-988 and LY-262691) enhanced olfactory recognition. In contrast, the CCK-B selective agonist BC 264 and the tetrapeptide CCK-4 both disrupted it. Taken together with previous evidence of the detrimental effect of the nonpeptide. CCK-A antagonist devazepide on olfactory recognition, these results confirm and extend the hypothesis that there is a balance between CCK-A-mediated facilitative effects and CCK-B-mediated inhibitory effects on memory retention.

Dietary proteins and trypsin inhibitors are known to stimulate the secretion of the satiety hormone cholecystokinin (CCK). A potato extract (Potein) contains 60% carbohydrate and 20% protein including trypsin inhibitory proteins. In this study, we examined whether Potein suppresses food intake in rats and whether it directly stimulates CCK secretion in enteroendocrine cells. In fasted rats, food consumption was measured up to 6 h after the oral administration of Potein or soybean trypsin inhibitor (SBTI). CCK-releasing activities of Potein and SBTI were examined in the murine CCK-producing cell line STC-1. Potein inhibited the trypsin activity in vitro with a potency 20-fold lower than that of SBTI. Oral administration of Potein dose-dependently suppressed food intake for 1-6 h. Potein, but not the SBTI, dose-dependently induced CCK secretion in STC-1 cells. These results suggest that Potein suppresses food intake through the CCK secretion induced by direct stimulation on enteroendocrine cells and through inhibition of luminal trypsin.

BACKGROUND

SV40 DNA replication system is a very useful tool to understand the mechanism of replication, which is a tightly regulated process. Many environmental and cellular factors can induce cell cycle arrest or apoptosis by inhibiting DNA replication. In the course of our search for bioactive metabolites from the marine sponges, psammaplin A was found to have some anticancer properties, the possible mechanism of which was studied.

METHODS

Cell viability was determined by Cell Counting Kit-8 (CCK-8) to count living RAW264.7 cells by combining 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-8) and 1-methoxy-phenazine methosulfate (1-methoxy-PMS). The effect of psammaplin A on DNA replication was carried out in SV40 DNA replication system in vitro. The activities of topoisomerase I and polymerase alpha-primase were measured by the relaxation of superhelical plasmid DNA and the incorporation of [3H]dTTP to the template respectively. The ssDNA binding activity of RPA was assessed by Gel Mobility Shift Assay (GMSA).

RESULTS

We have found that psammaplin A delivers significant cytotoxic activity against the RAW264.7 cell line. It was also found that psammaplin A could substantially inhibit SV40 DNA replication in vitro, in which polymerase alpha-primase is one of its main targets.

CONCLUSIONS

Taken together, we suggest that psammaplin A-induced cytotoxicity may correlate with its inhibition on DNA replication. Psammaplin A has the potential to be developed as an anticancer drug.

The effects of pretreatment with the selective cholecystokinin (CCK) B antagonists (3R-(+)-N-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1, 4-benzodiazepin-3-yl)-N1-(3-methylphenyl urea (L-365,260), 4-([2-[[3-(1H-indol-3-yl)-2-methyl-1-oxo-2-[[tricyclo[3.3, 1.1(3.7)]dec-2-yloxy)carbonyl]amino]propyl]amino]-1-phenylethyl] amin)-4-oxo-[R-(R*,R*)]butanoate-N-methyl-D-glucamine (PD-134,308) and N-(2-adamantyloxycarbonyl)-D-alpha-methyltryptophanyl-[N-(2- (4-chlorophenyl)ethyl)]glycine (RB 211), on the naloxone-reversible, antinociceptive responses induced by systemic (i.v.) administration of the complete inhibitor of the enkephalin-catabolizing enzymes, N-((R,S,)-2-benzyl-3[(S)-(2-amino-4-methylthio)butyldithio]-1- oxopropyl)-L-phenylalanine benzyl ester (RB 101), were determined in rat tail-flick and mouse hot-plate tests. L-365,260 (0.12, 0.25 and 0.5 mg/kg s.c.), PD-134,308 (0.3, 1 and 3 mg/kg i.p.) and RB 211 (0.5, 1 and 1.5 mg/kg i.p.) strongly potentiated the antinociceptive effects induced by RB 101 in the rat tail-flick test, in which spinal control of nociception is predominant. Thus, the antinociception observed after the association of L-365,260 (0.5 mg/kg), RB 211 (1.5 mg/kg) or PD-134,308 (3 mg/kg) with RB 101 (5 mg/kg) was, respectively, 300, 500 and 800% higher than that observed with RB 101 given alone. This facilitatory effect was partially blocked by the administration of naloxone (1 mg/kg s.c.). Under the same conditions the potentiation of the antinociceptive response produced by morphine (0.1-4 mg/kg s.c.) was inferior to 250%. In the mouse hot-plate test, L-365,260 (0.02 and 0.1 mg/kg i.p.) and PD-134,308 (0.3, 1 and 3 mg/kg i.p.) also enhanced endogenous enkephalin induced antiociception, but this potentiating effect, completely reversed by administration of naloxone (0.1 mg/kg s.c.), was about 2 times less effective than in the tail-flick assay. The present findings demonstrate an opposing physiological role of endogenous CCK, acting on CCK B receptors, and opioid peptides in the control of pain perception at both spinal and supraspinal levels. These results could have important clinical applications because a combination of a CCK B antagonist and RB 101, which has been showed to be almost devoid of morphine side effects, would increase the overall antinociceptive efficacy into a range that will be more clinically useful.

Agonists and antagonists selective for the brain-type [cholecystokinin (CCK)-B] and the peripheral-type (CCK-A) CCK receptor were used to localize the site(s) of action at which CCK inhibits food consumption. BC 264, a highly selective CCK-B receptor agonist, did not decrease consumption of a palatable meal when administered either i.p. or into the lateral ventricles of the brain, whereas CCK decreased feeding when administered i.p. at the same doses. CCK decreased feeding when administered i.v.t. at a high dose, 5 micrograms. L-364,718, an antagonist selective for the CCK-A receptor, blocked completely the action of centrally administered CCK, whereas L-365,260, a selective CCK-B receptor antagonist, had no effect on the ability of centrally administered CCK to inhibit feeding. To estimate the quantity of i.v.t. administered CCK which reached the periphery, a tracer of radiolabeled [3H]p-CCKCCK octapeptide sulfate), combined with unlabeled pCCKCCKCCK octapeptide sulfate reported previously to be present in the plasma after a meal. Intraventricularly administered CCK thus appears to reduce feeding in the rat through a mechanism involving a CCK-A receptor subtype in the periphery.

Most peptide hormone genes are, in addition to endocrine cells, also expressed in neurons. The peptide hormone cholecystokinin (CCK) is expressed in different molecular forms in cerebral neurons and intestinal endocrine cells. To understand this difference, we examined the roles of the neuroendocrine prohormone convertases (PC) 1/3, PC2, and PC5/6 by measurement of proCCK, processing intermediates and bioactive, alpha-amidated, and O-sulfated CCK peptides in cerebral and jejunal extracts of null mice, controls, and in the PC5/6-expressing SK-N-MC cell-line. In PC1/3 null mice, the synthesis of bioactive CCK peptide in the gut was reduced to 3% of the translational product, all of which was in the form of alpha-amidated and tyrosine O-sulfated CCK-22, whereas the neuronal synthesis in the brain was largely unaffected. This is opposite to the PC2 null mice in which only the cerebral synthesis was affected. SK-N-MC cells, which express neither PC1/3 nor PC2, synthesized alone the processing intermediate, glycine-extended CCK-22. Immunocytochemistry confirmed that intestinal endocrine CCK cells in wild-type mice express PC1/3 but not PC2. In contrast, cerebral CCK neurons contain PC2 and only little, if any, PC1/3. Taken together, the data indicate that PC1/3 governs the endocrine and PC2 the neuronal processing of proCCK, whereas PC5/6 contributes only to a modest endocrine synthesis of CCK-22. The results suggest that the different peptide patterns in the brain and the gut are due to different expression of PCs.

Recent data on the immunolocalization of regulatory peptides and related propeptide sequences in endocrine cells and tumors of the gastrointestinal tract, pancreas, lung, thyroid, pituitary (ACTH and opioids), adrenals and paraganglia have been revised and discussed. Gastrin, xenopsin, cholecystokinin (CCK), somatostatin, motilin, secretin, GIP (gastric inhibitory polypeptide), neurotensin, glicentin/glucagon-37 and PYY (peptide tyrosine tyrosine) are the main products of gastrointestinal endocrine cells; glucagon, CRF (corticotropin releasing factor), somatostatin, PP (pancreatic polypeptide) and GRF (growth hormone releasing factor), in addition to insulin, are produced in pancreatic islet cells; bombesin-related peptides are the main markers of pulmonary endocrine cells; calcitonin and CGRP (calcitonin gene-related peptide) occur in thyroid and extrathyroid C cells; ACTH and endorphins in anterior and intermediate lobe pituitary cells, alpha-MSH and CLIP (corticotropin-like intermediate lobe peptide) in intermediate lobe cells; met- and leu-enkephalins and related peptides in adrenal medullary and paraganglionic cells as well as in some gut (enterochromaffin) cells; NPY (neuropeptide Y) in adrenaline-type adrenal medullary cells, etc.. Both tissue-appropriate and tissue-inappropriate regulatory peptides are produced by endocrine tumours, with inappropriate peptides mostly produced by malignant tumours.

Gastric emptying after food ingestion is regulated by neural and hormonal factors. However, the relative contributions of each pathway is not yet clearly defined. The classic gut hormone CCK seems to be involved in the regulation of gastric emptying in humans. Experimental evidence is best for gastric emptying of liquid meals that release CCK from the duodenum: (1) CCK infused at postprandial plasma concentrations inhibits gastric emptying of a liquid and a semisolid meal. (2) Administration of the CCK antagonist loxiglumide significantly accelerated gastric emptying of a liquid mixed meal and a glucose meal. Discrepant results with the antagonist MK329 are difficult to explain considering the marked acceleration of gastric emptying rates by the specific and potent antagonist MK329 shown in several animal studies. Taken together, current information favors the conclusion, however, that CCK mainly controls gastric emptying of the liquid but not the solid components. Thus, CCK is involved in the physiologic regulation of gastric emptying and gastric motility in man. Blocking CCK-A receptors accelerates gastric emptying of liquid meals and abolishes the gastrocolonic reflex. Therefore, CCK may play a role as a common regulator of postprandial gallbladder contraction and pancreatic enzyme secretion as well as of gastric emptying rates under certain conditions. Such common control would optimize the nutrient-to-digestive juices concentration ratio. The importance of endogenous CCK on gastric emptying of solid meals, however, is poorly understood and remains to be defined. Only very limited information is available on gastric motility. Much more work has to be done before a clear concept can be developed.

Cholecystokinin (CCK) and gastrin exert their effects through two receptors, the CCK-A and CCK-B receptors. We have cloned the mouse CCK-B receptor gene (Cckbr) and determined its complete genomic structure, nucleotide sequence, and tissue-specific expression pattern. Cckbr is divided into five exons spanning 11 kb. A primer extension assay was used to map the transcription initiation site to 199 bp upstream of the translational start site. Rapid amplification of cDNA ends was used to localize the 3' end downstream of an atypical polyadenylation site (GATAAA). Mouse Cckbr transcripts were most abundant in brain and stomach, but were also detected in colon, kidney, ovary, and pancreas. Prenatal expression of both CCK-A and CCK-B receptors in various tissues was analyzed by RT-PCR. The expression pattern was similar to the adult pattern, suggesting that receptor transcription is an early event in gastrointestinal development.

OBJECTIVE

Periodontal ligament stem cells (PDLSCs) can be used in periodontal regeneration. Tumour necrosis factor-alpha (TNF-α) participates in the regulation of cell proliferation, apoptosis, differentiation, and migration. However, whether TNF-α can affect the biological features of PDLSCs is still unclear. The objective of this study was to illustrate the biological effects (proliferation, apoptosis, osteogenesis and migration) of TNF-α on human CD146 positive periodontal ligament cells (CD146+PLDCs) and CD146 negative periodontal ligament cells (CD146-PDLCs).

METHODS

CD146±PDLCs were isolated from human PDLCs and analyzed using a fluorescence-activated cell sorter. The biological effects of TNF-α on CD146±PDLCs were evaluated by CCK-8 assay (proliferation), DAPI staining (apoptosis), alizarin red staining and alkaline phosphatase activities assay (osteogenesis), and wounding assay and transwell assay (migration).

RESULTS

CD146+PDLCs, which expressed MSC surface markers CD105, CD90, CD73, CD44, and Stro-1, showed higher proliferative and osteogenic potential than CD146-PDLCs. TNF-α at a dose of 2.5ng/ml was found to enhance both proliferation and osteogenesis in CD146+PDLCs. At 5ng/ml, TNF-α promoted proliferation, osteogenesis, and apoptosis in CD146+PDLCs and enhanced osteogenesis in CD146-PDLCs. At 10ng/ml, TNF-α only aggravated apoptosis in CD146+PDLCs. The migratory ability of both CD146+PDLCs and CD146-PDLCs was not altered by TNF-α.

CONCLUSIONS

CD146+PDLCs were subpopulation of MSC. It showed greater proliferative and osteogenic potential than CD146-PDLCs. At low concentration, TNF-α was beneficial to CD146+PDLCs on proliferation and osteogenesis, and at high concentration it was detrimental. CD146-PDLCs were found to be less sensitive to TNF-α.

We previously demonstrated that soybean beta-conglycinin peptone suppresses food intake and gastric emptying by direct action on rat small intestinal mucosal cells to stimulate cholecystokinin (CCK) release. The aim of the present study was to define the active fragment in beta-conglycinin by using synthetic peptides chosen from the sequence of three beta-conglycinin subunits. We selected the fragments that had multiple nonadjacent arginine residues, and investigated their ability to bind to components of the rat intestinal brush border membrane as well as to stimulate CCK release and appetite suppression. The fragment from 51 to 63 of the beta subunit (beta 51-63) had the strongest binding activity. Intraduodenal infusion of beta 51-63 inhibited food intake and markedly increased portal CCK concentration. The threshold concentration of beta 51-63 to affect food intake was 3 micro mol/L. The CCK-A receptor antagonist abolished the beta 51-63-induced suppression of food intake. Three types of smaller fragments of beta 51-63 (beta 51-59, beta 53-63 and beta 53-59) and two types of fragments similar to beta 51-63 in the beta-conglycinin alpha and alpha' subunits (alpha 212-224 and alpha' 230-240) had less binding ability than did beta 51-63. Model peptides constructed with arginine (R) and glycine (G), such as GRGRGRG, had strong binding affinity, but peptides containing a single R or RR did not. These results indicate that the beta-conglycinin beta 51-63 fragment is the bioactive appetite suppressant in beta-conglycinin, and multiple arginine residues in the fragment may be involved in this effect.

BACKGROUND

The physiological relevance of duodenal bile acids in the control of cholecystokinin release and pancreatic enzyme secretion is still unknown.

OBJECTIVE

To provide a near physiological situation by perfusing a bile acid mixture mimicking the individual endogenous bile acid composition of the person under investigation. For maximal reduction of endogenous bile output the CCK-A receptor antagonist loxiglumide was infused intravenously.

METHODS

Seven healthy volunteers were studied on four different days by a duodenal marker perfusion technique. The individual bile acid composition in duodenal juice and test meal stimulated bile acid output was assessed on day 1. Bile acids were perfused at an amount of 30 or 100% as determined on day 1 in combination with the test meal in the presence or absence of loxiglumide. Pancreatic enzymes, bilirubin, and bile acid output were determined in duodenal juice. Plasma cholecystokinin (CCK) and plasma pancreatic polypeptide (PP) were measured radioimmunologically.

RESULTS

Bile acid perfusion did not significantly alter stimulated pancreatic enzyme, bilirubin or bile acid output or plasma CCK. Loxiglumide did not alter basal CCK release but increased test meal stimulated CCK output fourfold (p < 0.05). The addition of bile acids to the test meal at a dose resembling 30% of bile acid output as determined on day 1 prevented this increase. Plasma PP concentration remained unchanged by bile acids and were mostly undetectable during loxiglumide infusion.

CONCLUSIONS

The CCK producing cell is under constant suppression by intraduodenal bile acids which cannot be further enhanced by a physiological bile acid mixture. However, removal of duodenal bile acids by inhibition of gall bladder contraction unmasks this suppression leading to a dramatic increase in plasma CCK levels. As little as one third of postprandially released bile acids completely reverse this effect. Bile acids are the most important luminal regulator of CCK release in humans.

Systemic administration of CCK-8 increased plasma oxytocin (OXT) level in rats anesthetized with a mixture of urethane and alpha-chloralose. Extracellular recordings were made from magnocellular neurosecretory neurons in the paraventricular nucleus (PVN) of the hypothalamus in anesthetized rats to examine the effects of CCK-8 on the firing of PVN neurons. Thirteen out of 16 nonphasic neurons (putative OXT-secreting neurons) were excited by intravenous and/or intraperitoneal administration of CCK-8. By contrast, 8 out of 10 phasic cells, vasopressin(AVP)-secreting cells, were inhibited by systemic administration of CCK-8. Four out of five nonneurosecretory neurons in the PVN were excited by the administration of CCK-8. Moreover, microiontophoretically applied phentolamine blocked the excitatory responses induced by CCK-8 in nonphasic neurons. We measured extracellular noradrenaline (NA) level in the PVN, using in vivo microdialysis. Intravenous administration of CCK-8 induced NA release in the PVN. These results suggest that CCK-8 activates the excitatory afferent pathway to putative OXT-secreting neurons in the PVN which may, at least in part, be involved in the central noradrenergic projection.

Cholecystokinin (CCK) was first described as a gastrointestinal hormone, but its receptors have been located in cardiac and vascular tissues, as well as in immune cells. Our aims were to investigate the role of CCK on lipopolysaccharide (LPS)-induced hypotension and its ability to modulate previously reported inflammatory mediators, therefore affecting cardiovascular function. To conduct these experiments, rats had their jugular vein cannulated for drug administration, and also, the femoral artery cannulated for mean arterial pressure (MAP) and heart rate records. Endotoxemia induced by LPS from Escherichia coli (1.5 mg/kg; i.v.) stimulated the release of CCK, a progressive drop in MAP, and increase in heart rate. Plasma tumor necrosis factor α (TNF-α), interleukin 10 (IL-10), nitrate, vasopressin, and lactate levels were elevated in the endotoxemic rats. The pretreatment with proglumide (nonselective CCK antagonist; 30 mg/kg; i.p.) aggravated the hypotension and also increased plasma TNF-α and lactate levels. On the other hand, CCK (0.4 μg/kg; i.v.) administered before LPS significantly restored MAP, reduced aortic and hepatic inducible nitric oxide synthase (iNOS) production, and elevated plasma vasopressin and IL-10 concentrations; it did not affect TNF-α. Physiological CCK concentration reduced nitrite and iNOS synthesis by peritoneal macrophages, possibly through a self-regulatory IL-10-dependent mechanism. Together, these data suggest a new role for the peptide CCK in modulating MAP, possibly controlling the inflammatory response, stimulating the anti-inflammatory cytokine, IL-10, and reducing vascular and macrophage iNOS-derived nitric oxide production. Based on these findings, CCK could be used as an adjuvant therapeutic agent to improve cardiovascular function.

Endogenous cholecystokinin (CCK) is thought to participate in the satiating action of foods, and some data suggest that it may also mediate their postingestive reinforcing effects. This was investigated by determining if the CCK(A) receptor antagonist, devazepide, attenuates flavor preference conditioning by intraduodenal (I.D.) carbohydrate infusions. In Experiment 1, food-restricted female rats were trained 30 min/day to associate a cue flavor (CS+) with I.D. infusions of 8% Polycose and a different flavor (CS ) with I.D. water infusions. Half of the rats (DEV group) were pretreated with devazepide (300 microg/kg body weight) and the other half (CON group) with vehicle, 30 min prior to CS training sessions and choice tests. Both groups displayed similar CS+ preferences (CON: 68%; DEV: 69%). In contrast, devazepide blocked the feeding inhibitory effects of I.D. Polycose infusion and cholecystokinin octapeptide injection in Experiment 2. A higher dose of devazepide (1200 microg/kg) also failed to inhibit preference conditioning by I.D. Polycose in Experiment 3. These results indicate that, although CCK(A) mechanisms play a role in the satiating effect of I.D. carbohydrates, they do not mediate their reinforcing effect. The present study, along with other recent reports, indicate that different mechanisms mediate the satiating and reinforcing actions of nutrients.

Helicobacter pylori infection may be associated with duodenal ulcer (DU) and accompanied by enhanced gastrin release but the mechanism of this H pylori related hypergastrinaemia in DU patients is unclear. Cholecystokinin (CCK) has been implicated in the feedback control of gastrin release and gastric acid secretion in healthy subjects. This study therefore investigated if CCK participates in the impairment of postprandial gastrin release and gastric secretion in six DU patients. Tests were undertaken with and without elimination of endogenous CCK by loxiglumide, a selective CCK-A receptors antagonist, before and after eradication of H pylori with triple therapy (omeprazole, amoxicyllin, bismuth). In H pylori positive DU patients, the post-prandial decline in pH (with median pH 3.5) was accompanied by a pronounced increment in plasma gastrin but the administration of loxiglumide did not affect significantly this postprandial rise in plasma gastrin and gastric pH profile. After eradication of H pylori, the plasma gastrin concentration was reduced while the median postprandial pH was significantly increased (median pH 4.3). The administration of loxiglumide resulted in significantly greater increase in postprandial plasma gastrin and greater decrease in pH (median pH 3.1) in these patients. This study shows that (a) infection with H pylori is accompanied by an enhanced gastrin release and gastric acidity in DU patients, (b) the failure of loxiglumide to affect plasma gastrin or gastric acid secretion in H pylori infected DU patients could be attributed, at least in part, to the failure of endogenous CCK to control gastrin release and gastric secretion by releasing somatostatin, and (c) the test with loxiglumide may be useful in the identification of patients with impaired feedback control of gastrin release and gastric secretion resulting from infection with H pylori.

To examine the mechanism of the satiety-producing effect of cholecystokinin (CCK) in the central nervous system, we compared the potency of intraperitoneally (ip) or intracerebroventricularly (icv) administered CCK-8 and its analogues on food intake in fasted mice. The icv administration of a small dose of CCK-8 (0.03 nmol/brain) or of Suc-(Thr28, Leu29, MePhe33)-CCK-7 (0.001 nmol/brain) suppressed food intake for 20 min, whereas CCK-8 (1 nmol/kg, which is equivalent to 0.03 nmol/brain) or Suc-(Thr28, Leu29, MePhe33)-CCK-7 (1 nmol/kg) had satiety effect after ip administration. Dose-response studies indicated the following rank order of potency: Suc-CCK-7>> or = Suc-(Thr28, Leu29, MePhe33)-CCK-7>> or = CCK-8>> or = (Nle28,31)-CCK-8>>) desulfated CCK-8 = CCK-4 = 0 in the case of ip administration and Suc-(Thr28, Leu29, MePhe33)-CCK-7>>) Suc-CCK-7>> or = CCK-8>> or = (Nle28,31)-CCK-8>>) desulfated CCK-8 = CCK-4 = 0 in the case of icv administration. The selective CCK-A receptor antagonist MK-329 reversed the inhibitory effect of the centrally as well as peripherally administered CCK-8, or of Suc-(Thr28, Leu29, MePhe33)-CCK-7, whereas the selective CCK-B receptor antagonist L-365260 did not. The icv administered CCK-8 did not appear in the peripheral circulation. These findings suggest the participation of CCK-A receptors in the brain in mediating the satiety effect of CCK and the difference in CCK-A receptors in the brain and peripheral tissues.

Cholecystokinin (CCK) is one of the most abundant neurotransmitter peptides in the brain. As OLETF rats lack CCK-A receptor because of a genetic abnormality, we examined whether learning and memory were impaired in these animals using an elevated eight-arm radial maze. After the completion of a radial maze study, the animals were sacrificed for histological examination of the brain. In some animals, long-term potentiation (LTP) in the hippocampus was measured. In the radial maze, the level of activity (seconds/entry) and the time remaining in the arms were significantly longer in OLETF rats. The number of errors was also significantly higher, and that of the correct choices was significantly lower in OLETF rats compared to the controls (LETO rats). The LTP of the population spike was significantly lower in the OLETF than in the LETO rats. No histological abnormalities were observed. From these observations, we concluded that learning and memory functions were impaired in the OLETF rats.