The transcription factor NF-kappaB plays a critical role in inflammatory and cell death responses during acute pancreatitis. Previous studies in our laboratory demonstrated that protein kinase C (PKC) isoforms PKCdelta and epsilon are key regulators of NF-kappaB activation induced by cholecystokinin-8 (CCK-8), tumor necrosis factor-alpha, and ethanol. However, the downstream participants in regulating NF-kappaB activation in exocrine pancreas remain poorly understood. Here, we demonstrate that protein kinase D1 (PKD1) is a key downstream target of PKCdelta and PKCepsilon in pancreatic acinar cells stimulated by two major secretagogues, CCK-8 and the cholinergic agonist carbachol (CCh), and that PKD1 is necessary for NF-kappaB activation induced by CCK-8 and CCh. Both CCK-8 and CCh dose dependently induced a rapid and striking activation of PKD1 in rat pancreatic acinar cells, as measured by in vitro kinase assay and by phosphorylation at PKD1 activation loop (Ser744/748) or autophosphorylation site (Ser916). The phosphorylation and activation of PKD1 correlated with NF-kappaB activity stimulated by CCK-8 or CCh, as measured by NF-kappaB DNA binding. Either inhibition of PKCdelta or epsilon by isoform-specific inhibitory peptides, genetic deletion of PKCdelta and epsilon in pancreatic acinar cells, or knockdown of PKD1 by using small interfering RNAs in AR42J cells resulted in a marked decrease in PKD1 and NF-kappaB activation stimulated by CCK-8 or CCh. Conversely, overexpression of PKD1 resulted in augmentation of CCK-8- and CCh-stimulated NF-kappaB activation. Finally, the kinetics of PKD1 and NF-kappaB activation during cerulein-induced rat pancreatitis showed that both PKD1 and NF-kappaB activation were early events during acute pancreatitis and that their time courses of response were similar. Our results identify PKD1 as a novel early convergent point for PKCdelta and epsilon in the signaling pathways mediating NF-kappaB activation in pancreatitis.

In the brain of adult specimens of the tobacco hornworm moth, Manduca sexta (L), cells immunoreactive for several kinds of neuropeptides were localized by means of the PAP procedure, by use of antisera raised against mammalian hormones or hormonal peptides. In contrast, no such neurosecretory cells were found in the corpora cardiaca and corpora allata (CC/CA); in the CC/CA, however, immunoreactive nerve fibres were observed, reaching these organs from the brain. The neurosecretory cells found in the brain were immunoreactive with at least one of the following mammalian antisera, namely those raised against the insulin B-chain, somatostatin, glucagon C-terminal, glucagon N-terminal, pancreatic polypeptide (PP), secretin, vasoactive intestinal polypeptide (VIP), glucose-dependent insulinotropic peptide (GIP), gastrin C-terminus, enkephalin, alpha- and beta-endorphin, Substance P, and calcitonin. No cells were immunoreactive with antisera specific for detecting neurons containing the insulin A-chain, nerve growth factor, epidermal growth factor, insulin connecting peptide (C-peptide), polypeptide YY (PYY), gastrin mid-portion (sequence 6-13), cholecystokinin (CCK) mid-portion (sequences 9-20 and 9-25), neurotensin C-terminus, bombesin, motilin, ACTH, or serotonin. All the neuropeptide-immunoreactive cells observed emitted nerve fibers passing through the brain to the CC and in some cases also to the CA. In CC these immunoreactive nerve fibers tended to accumulate near the aorta. It was speculated that neuropeptides are released into the circulating haemolymph and act as neurohormones.

Oleoylethanolamide (OEA), a lipid synthesized in the intestine, reduces food intake and stimulates lipolysis through peroxisome proliferator-activated receptor-alpha. OEA also activates transient receptor potential vanilloid type 1 (TRPV1) in vitro. Because the anorexigenic effect of OEA is associated with delayed feeding onset and reduced locomotion, we examined whether intraperitoneal administration of OEA results in nonspecific behavioral effects that contribute to the anorexia in rats. Moreover, we determined whether circulating levels of other gut hormones are modulated by OEA and whether CCK is involved in OEA-induced anorexia. Our results indicate that OEA reduces food intake without causing a conditioned taste aversion or reducing sodium appetite. It also failed to induce a conditioned place aversion. However, OEA induced changes in posture and reduced spontaneous activity in the open field. This likely underlies the reduced heat expenditure and sodium consumption observed after OEA injection, which disappeared within 1 h. The effects of OEA on motor activity were similar to those of the TRPV1 agonist capsaicin and were also observed with the peroxisome proliferator-activated receptor-alpha agonist Wy-14643. Plasma levels of ghrelin, peptide YY, glucagon-like peptide 1, and apolipoprotein A-IV were not changed by OEA. Finally, antagonism of CCK-1 receptors did not affect OEA-induced anorexia. These results suggest that OEA suppresses feeding without causing visceral illness and that neither ghrelin, peptide YY, glucagon-like peptide 1, apolipoprotein A-IV, nor CCK plays a critical role in this effect. Despite that OEA-induced anorexia is unlikely to be due to impaired motor activity, our data raise a cautionary note in how specific behavioral and metabolic effects of OEA should be interpreted.

Cholecystokinin (CCK) decreases meal size through activation of CCK-A receptors on vagal afferents. We tested the hypothesis that the selective CCK-A agonist GI181771X induces weight loss in obese patients. Patients with body mass index>> or = 30 or>> or = 27 kg/m2 with concomitant risk factors were randomized to 24-week, double-blind treatment with different GI181771X doses or matching placebo together with a hypocaloric diet. The primary efficacy end point was the absolute change in body weight. To monitor pancreatic and gallbladder effects, patients underwent abdominal ultrasound and magnetic resonance imaging before and after treatment. We randomized 701 patients to double-blind treatment. GI181771X did not reduce body weight and had no effect on waist circumference or other cardiometabolic risk markers. Gastrointestinal side effects were more common with GI181771X than with placebo treatment, whereas hepatobiliary or pancreatic abnormalities did not occur. CCK-A by itself does not have a central role in long-term energy balance.

G-protein-coupled receptors signaling bitter taste (T2Rs) in the oral gustatory system and the alpha-subunit of the taste-specific G-protein gustducin are expressed in the gastrointestinal (GI) tract. alpha-Subunit of the taste-specific G-protein gustducin colocalizes with markers of enteroendocrine cells in human and mouse GI mucosa, including peptide YY. Activation of T2Rs increases cholecystokinin (CCK) release from the enteroendocrine cell line, STC-1. The aim of this study was to determine whether T2R agonists in the GI tract activate neurons in the nucleus of the solitary tract (NTS) and whether this activation is mediated by CCK and peptide YY acting at CCK(1) and Y(2) receptors. Immunocytochemistry for the protooncogene c-Fos protein, a marker for neuronal activation, was used to determine activation of neurons in the midregion of the NTS, the region where vagal afferents from the GI tract terminate. Intragastric administration of the T2R agonist denatonium benzoate (DB), or phenylthiocarbamide (PTC), or a combination of T2R agonists significantly increased the number of Fos-positive neurons in the mid-NTS; subdiaphragmatic vagotomy abolished the NTS response to the mixture of T2R agonists. Deletion of CCK(1) receptor gene or blockade of CCK(1) receptors with devazepide abolishes the activation of NTS neurons in response to DB, but had no effect on the response to PTC. Administration of the Y(2) receptor antagonist BIIE0246 blocks the activation of NTS neurons to DB, but not PTC. These findings suggest that activation of neurons in the NTS following administration of T2R agonists to the GI tract involves CCK(1) and Y(2) receptors located on vagal afferent terminals in the gut wall. T2Rs may regulate GI function via release of regulatory peptides and activation of the vagal reflex pathway.

Leptin administered (either intracerebroventricularly, icv, or intraperitoneally, ip) acts in synergy with CCK to suppress food intake and body weight in lean mice or rats. The potentiating effect induced by the co-injection of ip CCK and leptin to inhibit food consumption in mice is mediated by the CCK-A receptor and capsaicin sensitive afferents. In vitro, studies in rats showed that a subset of gastric vagal afferent fibers responded to leptin injected directly into the gastric artery only after a prior intra-arterial CCK injection. Moreover, the tonic activity of gastric-related neurons in the nucleus tractus solitarius (NTS) increased when leptin was delivered into the gastric chamber of an in vitro stomach-brainstem preparation. CCK co-injected with leptin potentiated Fos expression selectively in the area postrema, NTS and paraventricular nucleus of the hypothalamus (PVN), which points to the PVN as part of the afferent and efferent limbs of the circuitry involved in the synergistic interaction between leptin and CCK. The dampening of CCK or leptin inhibitory action on ingestive behavior when either factor is not present or their receptors are non functional supports the notion that such leptin-CCK interaction may have a physiological relevance. These observations provide a mean through which leptin and CCK integrate short- and mid-term meal-related input signals into long-term control of energy balance.

Cholecystokinin (CCK), a neuropeptide originally discovered in the gastrointestinal tract, is abundantly distributed in the mammalian brains including the hippocampus. Whereas CCK has been shown to increase glutamate concentration in the perfusate of hippocampal slices and in purified rat hippocampal synaptosomes, the cellular and molecular mechanisms whereby CCK modulates glutamatergic function remain unexplored. Here, we examined the effects of CCK on glutamatergic transmission in the hippocampus using whole-cell recordings from hippocampal slices. Application of CCK increased AMPA receptor-mediated EPSCs at perforant path-dentate gyrus granule cell, CA3-CA3 and Schaffer collateral-CA1 synapses without effects at mossy fiber-CA3 synapses. CCK-induced increases in AMPA EPSCs were mediated by CCK-2 receptors and were not modulated developmentally and transcriptionally. CCK reduced the coefficient of variation and paired-pulse ratio of AMPA EPSCs suggesting that CCK facilitates presynaptic glutamate release. CCK increased the release probability and the number of readily releasable vesicles with no effects on the rate of recovery from vesicle depletion. CCK-mediated increases in glutamate release required the functions of phospholipase C, intracellular Ca(2+) release and protein kinase Cgamma. CCK released endogenously from hippocampal interneurons facilitated glutamatergic transmission. Our results provide a cellular and molecular mechanism to explain the roles of CCK in the brain.

Because the pancreas undergoes involutional changes during total parenteral nutrition (TPN) and because pancreatico-biliary secretions are trophic to the intestine, we studied jejunal and ileal structure and function and exocrine pancreatic function before and after 6 weeks of TPN in two groups of beagle dogs, one of which had TPN alone, the other having TPN plus daily stimulation of pancreatico-biliary secretions with intravenous infusions of cholecystokinin (CCK) and secretin. The injections of 1 U each per kg of body weight per day of CCK and secretin completely prevented the proximal and distal small bowel mucosal hypoplasia which developed in the TPN alone group. They also resulted in significant increases in in vivo galactose absorption (64 mM) per unit length of jejunum and ileum. However, there was no significant change in mucosal alpha-glucosidase and catalase activity or in in vitro mucosal uptake of 1 mM [14C]leucine when expressed per unit weight of intestinal mucosa. The capacity of the pancreas to respond to CCK and secretin was unaffected by excluding food from the intestine with 6 weeks of TPN in terms of pH, volume, and peak secretion rates of bicarbonate and protein, but maximum amylase output (units per 15 min per kg of body weight) fell significantly (P less than 0.05) from a mean of 1022 +/- 155 to 874 +/- 426 in TPN alone group and to 472 +/- 79 in the TPN dogs given CCK and secretin. These results show that daily CCK and secretin is trophic to the intestine of dogs nourished by TPN but do not indicate whether this trophic effect is attributable to CCK alone, secretin alone, the combination of the two hormones, or to the resultant stimulation of pancreatico-biliary secretions.

Cholecystokinin (CCK) is released from enteroendocrine cells after ingestion of nutrients and induces multiple effects along the gastrointestinal tract, including gastric relaxation and short-term satiety. We used whole cell patch-clamp and immunohistochemical techniques in rat brain stem slices to characterize the effects of CCK. In 45% of the neurons of nucleus tractus solitarius subnucleus centralis (cNTS), perfusion with the sulfated form of CCK (CCK-8s) increased the frequency of spontaneous excitatory currents (sEPSCs) in a concentration-dependent manner (1-300 nM). The threshold for the CCK-8s excitatory effect was 1 nM, the EC(50) was 20 nM, and E(max) was 100 nM. The excitatory effects of CCK-8s were still present when the slices were preincubated with tetrodotoxin or bicuculline or when the recordings were conducted with Cs(+) electrodes. Pretreatment with the CCK-A receptor antagonist, lorglumide (1 microM), antagonized the effects of CCK-8s, whereas perfusion with the CCK-B preferring agonist CCK-8 nonsulfated (CCK-ns, 1 microM) did not affect the frequency of sEPSCs. Similarly, pretreatment with the CCK-B receptor antagonist, triglumide (1 microM), did not prevent the actions of CCK-8s. Although the majority (i.e., 76%) of CCK-8s unresponsive cNTS neurons had a bipolar somata shape and were TH-IR negative, no differences were found in either the morphological or the neurochemical phenotype of cNTS neurons responsive to CCK-8s. Our results suggest that the excitatory effects of CCK-8s on terminals impinging on a subpopulation of cNTS neurons are mediated by CCK-A receptors; these responsive neurons, however, do not have morphological or neurochemical characteristics that automatically distinguish them from nonresponsive neurons.

The hormone gastrin stimulates acid secretion by releasing histamine from gastric enterochromaffin-like (ECL) cells and induces ECL cell proliferation in vivo. This study uses a>> 90% pure ECL cell preparation in culture to compare gastrin effects on histamine release, histidine decarboxylase (HDC) activity, and DNA synthesis. Gastrin and the cholecystokinin octapeptide (CCK-8, nonsulfated) induced histamine release from ECL cells (24-96 h of primary culture) within 5 min of incubation [concentration eliciting 50% of maximal response (EC50), 4 and 2 x 10(-11) M, respectively]. The CCK-B antagonist L-365,260 inhibited this effect [concentration inhibiting 50% of maximal response (IC50), 2 x 10(-8) M], whereas the CCK-A antagonist L-364,718 (10(-8) M) and the tyrosine kinase inhibitor genistein (10(-4) M) had no effect. Histamine release was associated with a biphasic elevation of intracellular Ca2+. Gastrin stimulated HDC activity two- to threefold after 60 min of incubation (EC50, 10(-10) M). Gastrin also increased DNA synthesis in ECL cells, with an EC50 of 1.7 x 10(-12) M as measured by the incorporation of 5-bromo-2'-deoxyuridine (BrdU). Positive nuclear immunostaining increased two- to threefold in up to 20% of ECL cells after 48-96 h of incubation. This effect was inhibited by L-365,260 (IC50, 5 x 10(-9) M) and by genistein (10(-4) M) but was not altered by L-364,718 (10(-8) M). The antisecretory drugs omeprazole, lansoprazole, and pantoprazole did not affect BrdU incorporation in isolated ECL cells. In conclusion, acute and chronic gastrin effects on the ECL cell are mediated via CCK-B receptors but differ in apparent receptor affinity and signal transduction pathways.

We investigated the effect of acarbose, an alpha-glucosidase and pancreatic alpha-amylase inhibitor, on gastric emptying of solid meals of varying nutrient composition and plasma responses of gut hormones. Gastric emptying was determined with scintigraphy in healthy subjects, and all studies were performed with and without 100 mg of acarbose, in random order, at least 1 wk apart. Acarbose did not alter the emptying of a carbohydrate-free meal, but it delayed emptying of a mixed meal and a carbohydrate-free meal given 2 h after sucrose ingestion. In meal groups with carbohydrates, acarbose attenuated responses of plasma insulin and glucose-dependent insulinotropic polypeptide (GIP) while augmenting responses of CCK, glucagon-like peptide-1 (GLP-1), and peptide YY (PYY). With mixed meal + acarbose, area under the curve (AUC) of gastric emptying was positively correlated with integrated plasma response of GLP-1 (r = 0.68, P < 0.02). With the carbohydrate-free meal after sucrose and acarbose ingestion, AUC of gastric emptying was negatively correlated with integrated plasma response of GIP, implying that prior alteration of carbohydrate absorption modifies gastric emptying of a meal. The results demonstrate that acarbose delays gastric emptying of solid meals and augments release of CCK, GLP-1, and PYY mainly by retarding/inhibiting carbohydrate absorption. Augmented GLP-1 release by acarbose appears to play a major role in the inhibition of gastric emptying of a mixed meal, whereas CCK and PYY may have contributory roles.

BACKGROUND

Alpha-amidated gastrin promotes the growth of nontransfected pancreatic cell lines expressing the gastrin/cholecystokinin (CCK)-B receptor. Gastrin/CCK-B and CCK-A receptors recently were demonstrated in human pancreatic adenocarcinomas, but to the authors' knowledge expression of their ligands to date have not been adequately investigated. As a prerequisite for making suggestions regarding local growth stimulation, the authors examined whether gastrin and the homologous CCK peptides as well as their specific receptors were expressed in consecutively collected solid human pancreatic adenocarcinomas.

METHODS

Using a library of radioimmunoassays specific for different epitopes on proCCK, progastrin, their processing intermediates, and bioactive end products, CCK and gastrin gene expression was measured in extracts of solid human pancreatic adenocarcinomas (n = 19), resection margins (n = 15), and normal pancreatic tissue (n = 8). Moreover, CCK, CCK-A receptor, and gastrin/CCK-B receptor mRNA were measured by reverse transcriptase-polymerase chain reaction.

RESULTS

Amidated gastrins were synthetized in 14 of 19 carcinomas (median, 0.4 pmol/g; range, < 0.1-84.0 pmol/g) and in 12 of 15 resection margin samples (median, 0.3 pmol/g; range, < 0.1-6.1 pmol/g). In contrast, normal human pancreatic tissue expressed only traces of poorly processed progastrin. Gastrin/CCK-B receptor mRNA was present in all carcinomas, resection margins, and normal pancreatic tissue. CCK-A receptor mRNA was detected in most tumors, but neither the mature ligands (alpha-amidated and O-sulfated CCK peptides) nor their precursors were expressed in carcinoma and normal pancreatic tissue.

CONCLUSIONS

The results of the current study demonstrate that alpha-amidated gastrin peptides and their receptor invariably are coexpressed in pancreatic adenocarcinoma. Therefore these findings support the contention of a role for local gastrin regulatory mechanisms, but no CCK mechanisms, in pancreatic carcinoma.

We have reported previously mitogenic effects of gastrin on several immortalized and neoplastic cell lines, including Swiss 3T3 fibroblasts. Receptor subtypes, cholecystokinin (CCK)-A and CCK-B, for a closely related peptide, cholecystokinin, were recently cloned. These studies were undertaken to investigate if CCK-A- and CCK-B receptors were perhaps mediating the mitogenic effects of gastrin on Swiss 3T3 cells. Receptor antagonists that inhibit the biological effects and binding of peptides to the CCK-A (L-364,718 (L18)) and CCK-B (L-365,260 (L60)) receptors were ineffective toward inhibiting the binding and proliferative effects of gastrin on Swiss 3T3 cells. Radiolabeled L18 and L60 demonstrated no binding to the cells, indicating that CCK-A and CCK-B receptors may be absent on Swiss 3T3 cells. Radiolabeled CCK-8, gastrin, L18, and L60, on the other hand, demonstrated specific binding to a pancreatic cancer cell line (AR42J cells) (used as a positive control). In cross-linking studies the molecular mass of the major band of gastrin receptors (GR) on Swiss 3T3 cells was determined to be approximately 45 kDa. The mitogenic potency of 0.1-1.0 nM gastrin-like peptides on Swiss 3T3 cells was in the order of G1-17>> or = G1-17-Gly>> G5-17>> or = G5-17-Gly>> G2-17>> CCK-8-Gly>> or = G1-17-Lys>> or = CCK-8. The relative binding affinity of the peptides (based on the dose-dependent inhibition of binding of 125I-G1-17 to Swiss 3T3 cells) was similar to the relative mitogenic potency of the peptides as given above. Furthermore, G1-17-Gly was equally effective as G1-17 in displacing the binding of 125I-G1-17 to the 45-kDa GR from the Swiss 3T3 cells. Based on these studies it became evident that the novel gastrin preferring GR, expressed by Swiss 3T3 cells, binds and mediates the mitogenic effects of not only the mature (amidated) forms of gastrin-like peptides but also binds and mediates the mitogenic effects of glycine-extended forms of gastrin-like peptides. Possible mRNA expression of CCK-A and CCK-B receptor subtypes by gastrin-responsive rodent intestinal and fibroblast cell lines (Swiss 3T3, IEC-6, CA) was measured by the methods of Northern blot analysis and reverse transcriptase-polymerase chain reaction. mRNA from rat pancreas, AR42J cells, and rat antrum served as positive controls.(ABSTRACT TRUNCATED AT 400 WORDS)

Lipid in the intestine initiates feedback inhibition of proximal gastrointestinal function and food intake. In rats and humans, inhibition of gastric emptying is mediated, at least in part, by cholecystokinin (CCK)-A receptors, and in rats there is evidence for involvement of an intestinal vagal afferent pathway. The mechanism by which luminal lipid acts to release CCK or activate vagal afferent nerve terminals is unclear. The role of chylomicron formation in this sensory transduction pathway has been investigated using the hydrophobic surfactant Pluronic L-81 that inhibits chylomicron formation. Gastric emptying of liquids was measured in awake rats fitted with a Thomas gastric fistula and a duodenal cannula. Intestinal perfusion of lipid induced a dose-dependent inhibition of gastric emptying (6, 12, and 39% inhibition for 25, 50, and 100 mg lipid, respectively). Perfusion of lipid with Pluronic L-81 (2.8% wt/vol) reversed the lipid-induced inhibition of gastric emptying. Pluronic L-63, a chemically similar surfactant that has no effect on chylomicron formation, had no effect on lipid-induced inhibition of gastric emptying. Perfusion of the intestine with lipid (100 mg) increased plasma levels of CCK from 1.9 +/- 0.8 to 6. 5 +/- 1 pM. This increase was blocked by Pluronic L-81 but unaffected by L-63. These results provide evidence that chylomicron formation is important in the signaling of lipid in the intestinal lumen to CCK endocrine cells and to producing feedback inhibition of gastric emptying.

Cholecystokinin (CCK) is one of the most abundant peptides in the central nervous system (CNS). Radioligand-binding studies have identified and localized both central (CCK(B)-R) and peripheral (CCK(A)-R) receptor subtypes in the CNS. However, these studies have been limited by the relative specificities of agonists and antagonists for receptor subtypes and their inability to distinguish cell bodies from dendritic projection. Recently, we isolated and cloned the cDNAs for both CCK(A) and CCK(B) receptors. In the present study, (35)S-labeled cRNA antisense probes were synthesized for both receptors and in situ hybridization studies were performed in the rat brain, allowing a direct and independent comparison of the distinct distribution of expression of CCK(A) and CCK(B) receptor mRNAs in the rat brain for the first time. mRNAs for both CCK(A) and CCK(B) receptors were expressed mainly in the cortex, olfactory regions, hippocampal formation, septum, and interpeduncular nucleus. Only CCK(A) receptor mRNAs were expressed in some of the hypothalamic nuclei (paraventricular nucleus, arcuate nucleus, and medial preoptic area). In most of amygdaloid nuclei only CCK(B) receptor mRNAs were expressed. Although the presence of receptor mRNAs does not necessarily imply the presence of functional receptor proteins in the same location, this study showed the regional distribution of mRNAs for CCK(A) and CCK(B) receptors and provides important information about the distribution of CCK receptor subtypes in the CNS which will allow better resolution of their functional roles in the brain.

In a previous study (Comings DE et al. Comparison of the role of dopamine, serotonin, and noradrenergic genes in ADHD, ODD and conduct disorder. Multivariate regression analysis of 20 genes. Clin Genet 2000: 57: 178-196) we examined the role of 20 dopamine, serotonin and norepinephrine genes in attention deficit hyperactivity disorder (ADHD), oppositional defiant disorder (ODD), and conduct disorder (CD), using a multivariate analysis of associations (MAA) technique. We have now brought the total number of genes examined to 42 by adding an additional 22 candidate genes. These results indicate that even with the inclusion of these additional genes the noradrenergic genes still played a greater role in ADHD than any other group. Six other neurotransmitter genes were included in the regression equation - cholinergic, nicotinic, alpha 4 receptor (CHNRA4), adenosine A2A receptor (ADOA2A), nitric oxide synthase (NOS3), NMDAR1, GRIN2B, and GABRB3. In contrast to ADHD and ODD, CD preferentially utilized hormone and neuropeptide genes These included CCK, CYP19 (aromatase cytochrome P-450), ESR1, and INS (p = 0.005). This is consistent with our prior studies indicating a role of the androgen receptor (AR) gene in a range of externalizing behavors. We propose that the MAA technique, by focusing on the additive effect of multiple genes and on the cummulative effect of functionally related groups of genes, provides a powerful approach to the dissection of the genetic basis of polygenic disorders.

BACKGROUND

Overexpression of regulatory peptide receptors in selected human tumours is of diagnostic and therapeutic relevance.

OBJECTIVE

To evaluate the expression of somatostatin, vasoactive intestinal peptide (VIP), substance P, cholecystokinin (CCK) A and B, and neurotensin receptors in hepatocellular carcinoma (HCC).

METHODS

In vitro receptor autoradiography for the various peptide receptors using selective iodinated radioligands on tissue sections in 59 cases of HCC.

RESULTS

41% of HCC expressed somatostatin receptors; 47% expressed VIP receptors. VIP receptors were always identified in non-neoplastic liver tissue. Substance P receptors were only identified in 5% of HCC but in the majority of their peritumorous and intratumorous vessels. CCK-A and -B and neurotensin receptors were not detected in HCC. The somatostatin receptors showed high affinity for somatostatin and octreotide. The VIP receptors had high affinity for VIP, pituitary adenylate cyclase activating peptide (PACAP) 27, and a VIP1 selective analogue, suggesting the presence of VIP1/PACAP II type receptors. PACAP I receptors were identified in two cases. Substance P receptors were all of the NK1 subtype. The density of somatostatin receptors in HCC was low compared with the density found in liver metastases of neuroendocrine tumours. The VIP receptor density was always lower in HCC than in adjacent liver tissue.

CONCLUSIONS

Somatostatin, VIP, and substance P may have a receptor mediated role in HCC. Substance P receptors may be involved in regulation of tumour associated blood flow; somatostatin receptors and VIP receptors may mediate tumour growth. Diagnostic and therapeutic evaluation of somatostatin and VIP analogues may be of interest in receptor positive HCC.

The mechanism by which lipid in the duodenum inhibits gastric emptying was investigated in awake rats fitted with chronic gastric and duodenal cannulas. Perfusion of the duodenum with lipid (Intralipid, 5 and 10%; total amount 50 and 100 mg) caused a significant inhibition (26 and 78%, respectively) of gastric emptying of a nonnutrient liquid (0.9% saline). Functional ablation of the capsaicin-sensitive vagal, but not the spinal, sensory innervation to the upper gastrointestinal tract significantly attenuated by 57% lipid-induced inhibition of gastric emptying. In intact rats, administration of a specific cholecystokinin (CCK)-A receptor antagonist, devazepide, significantly attenuated by 66% the response to lipid. Administration of devazepide in perivagal capsaicin-treated rats did not further reduce the response to lipid. These results suggest that lipid in the duodenum inhibits gastric emptying via a mechanism involving an action of CCK at type A receptors and capsaicin-sensitive vagal afferents.

In transfected Chinese hamster ovary (CHO-<em>A</em>1) cells the human adenosine <em>A</em>1 receptor directly stimulates pertussis toxin-sensitive increases in inositol phosphate production and potentiates (synergistically) the inositol phosphate responses mediated by Gq-coupled P2Y2 purinoceptor and <em>CCK</em>(<em>A</em>) receptors. In the present study we have investigated the role of Gbetagamma subunits in mediating adenosine <em>A</em>1 receptor effects on phospholipase C activation (both direct and synergistic) by transiently transfecting CHO-<em>A</em>1 cells with a scavenger of Gbetagamma subunits: the C-terminus of beta-adrenoceptor kinase 1 (beta ark1 residues 495-689). [3H]inositol phosphate responses to the selective adenosine <em>A</em>1 receptor agonist N6-cyclopentyladenosine (CP<em>A</em>; 1 microM) were inhibited (41 +/- 1%) in CHO-<em>A</em>1 cells transiently transfected with the Gbetagamma scavenger, beta ark1 (495-689). Expression of beta ark1 (495-689) protein was confirmed by Western blotting. In contrast, adenosine <em>A</em>1 receptor-mediated inhibition of forskolin stimulated [3H]cyclic <em>A</em>MP accumulation was unaffected by transient expression of beta ark1 (495-689). Beta ark1 (495-689) expression had no significant effect on the [3H]inositol phosphate responses produced by activation of the endogenous P2Y2 purinoceptor (100 microM UTP; 92 +/- 0.8% of control). [3H]inositol phosphate accumulation in response to adenosine <em>A</em> receptor activation was also attenuated in CHO-K1 cells co-transfected with the beta ark1 (495-689) minigene (59 +/- 4% inhibition of control response to 1 microM CP<em>A</em>). Finally, transient expression of beta ark1 (495-689) in CHO-<em>A</em>1 cells inhibited the augmentation of [3H]inositol phosphate responses resulting from co-activation of adenosine <em>A</em>1 receptors and P2Y2 purinoceptors. These experiments indicate that Gbetagamma subunits are involved in the direct coupling the adenosine <em>A</em>1 receptor to phospholipase C and that they also participate in the augmentation of P2Y2 purinoceptor-mediated [3H]inositol phosphate responses by the adenosine <em>A</em>1 receptor.

Transient lower esophageal sphincter (LES) relaxations (TLESRs) are the main underlying mechanism of gastroesophageal reflux. Although CCK acts through CCK-A receptors to increase the TLESRs induced by gastric distension, the respective roles of endogenous CCK and fundic tone in triggering postprandial TLESRs remain unknown. The aim of this study was to determine the effect of the CCK-A receptor antagonist, loxiglumide, on postprandial LES function and fundic tone in humans. LES motor events and fundic tone were simultaneously monitored in two groups of healthy volunteers. Recordings were performed during fasting and for 3 h after a liquid meal (200 ml/200 kcal) administered either orally or intraduodenally at a rate mimicking gastric emptying. Each subject received loxiglumide (10 mg. kg-1. h-1) or saline (control) in randomized order, which was started 40 min before the meal and maintained for 3 h thereafter. After the oral meal, loxiglumide significantly reduced TLESRs (P = 0.002) without significantly affecting LES pressure and fundic tone. After duodenal infusion of the meal, loxiglumide totally abolished the increase in TLESRs, reduced LES pressure fall (P < 0.02), and strongly inhibited fundic relaxation (P = 0.0001). We concluded that endogenous CCK is involved in the postprandial control of both LES function and fundic tone through activation of CCK-A receptors.

Cholecystokinin (CCK) is an abundant and widely distributed neuropeptide that plays a modulatory role in a variety of behaviours. This paper focuses on the role of CCK in modulating anxiety-related and reward-related behaviours in key brain regions of the amygdala and mesolimbic dopamine system, respectively. The role of CCK in mediating aspects of these behaviours has been studied in a variety of behavioural paradigms, but inconsistent results have led to confusion regarding the precise role of the receptor subtypes in mediating behaviour. The confusion in the literature may come in part from the diverse behavioural paradigms that are used, the differences in regional effects of CCK manipulations in different areas and at different receptor subtypes in these areas and the dependence of the behavioural outcome on the baseline state of arousal of the animal. Evidence on the role of CCK in anxiety-related and reward-related behaviours in various animal models indicates that CCK-B receptors in the basolateral amygdala are important mediators of anxiety-related behaviours and that CCK-A and CCK-B receptors in the nucleus accumbens are important in mediating different aspects of reward-related behaviour. Emphasis is placed upon the role of CCK as a neuromodulator that is recruited only under conditions of high frequency neuronal firing.

We previously reported a synergistic interaction between leptin and cholecystokinin (CCK) to reduce food intake through CCK-A receptors in lean mice fasted for 24 h. To identify the activated neuronal pathways, we investigated changes in Fos expression in brain nuclei 2 h after single or combined intraperitoneal (i.p.) injections of leptin (120 microg/kg) and sulfated CCK-8 (3.5 microg/kg) in male lean mice (C57BL/6) fasted for 24 h using immunohistochemistry for Fos, the protein product of the early gene, c-fos. Leptin did not increase Fos expression in the brain compared with vehicle-treated mice. CCK increased the numbers of Fos-positive neurons in the nucleus of the solitary tract (NTS)/area postrema (AP), central nucleus of the amygdala (CeA) and, to a smaller extent, in the paraventricular nucleus of the hypothalamus (PVN) (5.2-, 2.3- and 0. 3-fold respectively). Injections of leptin-CCK further enhanced Fos expression by 40% in the PVN compared with that induced by CCK alone, but not in the other nuclei. Devazepide (a CCK-A receptor antagonist, 1 mg/kg, i.p.) prevented the increase in Fos expression induced by leptin-CCK in the PVN and by CCK alone in the PVN, CeA and NTS/AP. These results indicate that in fasted mice, i.p. injection of CCK increases Fos expression in specific brain nuclei through CCK-A receptors while leptin alone had no effect. Leptin in conjunction with CCK selectively enhanced Fos expression in the PVN. The PVN may be an important site mediating the synergistic effect of leptin-CCK to regulate food intake.

OBJECTIVE

Gastric emptying plays an important role in regulating food intake. This study was designed to investigate whether intraperitoneally injected urocortin reduces gastric emptying, feeding, and body weight in lean and ob/ob obese mice.

METHODS

Food intake and body weight were measured after intraperitoneal injections of one of the following: urocortin, deamidated form of urocortin (urocortin OH), corticotropin-releasing factor (CRF), CRF6-33, cholecystokinin octapeptide (CCK-8), and leptin in 16-hour food-deprived animals. Gastric emptying was assessed 2, 4, or 8 hours after intraperitoneal injection. Repeated injections of urocortin were continued for 5 days in ob/ob mice.

RESULTS

Urocortin (0.003-3 nmol) dose-dependently and potently decreased food intake and body weight gain in lean mice. The ranking order of potency was urocortin>> urocortin OH>>/= CRF>> CCK-8>> CRF6-33>> leptin. Gastric emptying was also potently reduced by urocortin with a similar ranking order of potency of urocortin>> CRF>> urocortin OH>> CCK-8. Simultaneous administration of urocortin and CRF receptor antagonist, alpha-helical CRF9-41, blocked the effects of urocortin. Urocortin reduced food intake and body weight gain, as well as the rate of gastric emptying, in ob/ob mice, which was significantly faster than that of lean mice. Five daily injections of urocortin significantly lowered body weight and improved glycemic control in ob/ob mice.

CONCLUSIONS

The urocortin-induced decrease in food intake and body weight in lean and ob/ob mice is closely related to gastric emptying and opens new possibilities for the treatment of obesity.

Bacterial infection can trigger the development of functional GI disease. Here, we investigate the role of the gut-brain axis in gastric dysfunction during and after chronic H. pylori infection. Control and chronically H. pylori-infected Balb/c mice were studied before and 2 mo after bacterial eradication. Gastric motility and emptying were investigated using videofluoroscopy image analysis. Gastric mechanical viscerosensitivity was assessed by cardioautonomic responses to distension. Feeding patterns were recorded by a computer-assisted system. Plasma leptin, ghrelin, and CCK levels were measured using ELISA. IL-1beta, TNF-alpha, proopiomelanocortin (POMC), and neuropeptide Y mRNAs were assessed by in situ hybridizations on frozen brain sections. Gastric inflammation was assessed by histology and immunohistochemistry. As shown previously, H. pylori-infected mice ate more frequently than controls but consumed less food per bout, maintaining normal body weight. Abnormal feeding behavior was accompanied by elevated plasma ghrelin and postprandial CCK, higher TNF-alpha (median eminence), and lower POMC (arcuate nucleus) mRNA. Infected mice displayed delayed gastric emptying and visceral hypersensitivity. Eradication therapy normalized gastric emptying and improved gastric sensitivity but had no effect on eating behavior. This was accompanied by persistently increased TNF-alpha in the brain and gastric CD3(+) T-cell counts. In conclusion, chronic H. pylori infection in mice alters gastric emptying and mechanosensitivity, which improve after bacterial eradication. A feeding pattern reminiscent of early satiety persists after H. pylori eradication and is accompanied by increased TNF-alpha in the brain. The results support a role for altered gut-brain pathways in the maintenance of postinfective gut dysfunction.